Dosing regimens for mobilizing hematopoietic stem and progenitor cells

文档序号：1102040 发布日期：2020-09-25 浏览：6次中文

阅读说明：本技术 用于动员造血干细胞和祖细胞的给药方案 (Dosing regimens for mobilizing hematopoietic stem and progenitor cells ) 是由德怀特·莫罗帕特里克·C·法拉赫安东尼·博伊坦诺迈克尔·P·库克凯文·A·冈卡夫斯于 2018-12-06 设计创作，主要内容包括：本发明提供了可用于动员供体中的造血干细胞和祖细胞(hematopoietic stem and progenitor cells)的群体,以及可用于确定动员的细胞的样品是否适于释放用于离体扩增和/或治疗用途的组合物和方法。根据本文描述的组合物和方法,可以从供体抽取动员的造血干细胞和祖细胞并将其施用至患者,用于治疗多种干细胞紊乱,尤其包括造血系统疾病(hematopoietic diseases)、代谢紊乱、癌症和自身免疫性疾病。在某些实施方案中,本文描述的组合物和方法导致具有免疫抑制作用并能够降低移植物抗宿主病发生率的CD34<Sup>dim</Sup>细胞的群体的动员。(The present invention provides compositions and methods useful for mobilizing populations of hematopoietic stem and progenitor cells (hematopoietic stem and promoter cells) in donors, and for determining whether a sample of mobilized cells is suitable for release for ex vivo expansion and/or therapeutic use. According to the compositions and methods described herein, mobilized hematopoietic stem and progenitor cells can be drawn from a donor and administered to a patient for the treatment of a variety of stem cell disorders, including hematopoietic disorders (hematopoietic disorders), metabolic disorders, cancer, and autoimmune diseases, among others. In certain embodiments, the compositions and methods described herein result in CD34 having immunosuppressive effects and being capable of reducing the incidence of graft versus host disease dim Mobilization of a population of cells.)

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, and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 300 mg/kg.

2. The method of claim 1, wherein said CXCR2 agonist is Gro-beta T.

3. The method of claim 1 or 2, wherein said CXCR2 agonist is administered to said donor at a dose of from about 100 μ g/kg to about 250 μ g/kg.

4. The method of claim 3, wherein said CXCR2 agonist is administered to said donor at a dose of from about 125 μ g/kg to about 225 μ g/kg.

5. The method of claim 4, wherein said CXCR2 agonist is administered to said donor at a dose of about 150 μ g/kg.

6. The method of any one of claims 1-5, wherein said CXCR2 agonist is administered intravenously to said donor.

7. The method of any one of claims 1-6, wherein said CXCR4 antagonist is administered subcutaneously to said donor.

8. The method of any one of claims 1-7, wherein said 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 μ g/kg to about 500 μ g/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 μ g/kg to about 300 μ g/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 μ g/kg.

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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being 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 peripheral blood sample of the donor following administration of the CXCR2 agonist and the 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 peripheral blood of the donor with CD34+ cells relative to white blood cells in a ratio of from about 3.4:1 to about 6.9:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

15. The method of claim 14, wherein the peripheral blood of the donor is enriched for CD34+ cells relative to leukocytes in 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ cells to neutrophils in a peripheral blood sample of the donor of from about 0.0018 to about 0.0058 following administration of the CXCR2 agonist and the 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 peripheral blood of the donor with CD34+ cells relative to neutrophils in a ratio from about 2.1:1 to about 8.1:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

19. The method of claim 18, wherein the peripheral blood of the donor is enriched for CD34+ cells relative to neutrophils in 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ cells to lymphocytes from about 0.0021 to about 0.0094 in a peripheral blood sample of the donor following administration of the CXCR2 agonist and the 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 peripheral blood of the donor with CD34+ cells relative to lymphocytes in a ratio from about 4.8:1 to about 8.4 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

23. The method of claim 22, wherein the peripheral blood of the donor is enriched for CD34+ cells relative to lymphocytes in a ratio 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being 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 peripheral blood sample of the donor following administration of the CXCR2 agonist.

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 peripheral blood of the donor for CD34+ cells relative to monocytes in a ratio of from about 1.1:1 to about 2.3:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

27. The method of claim 26, wherein the peripheral blood of the donor is enriched for CD34+ cells relative to monocytes in 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, the amount of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a frequency of CD34+ cells from about 0.051% to about 0.140% in a peripheral blood sample of the donor following administration of the CXCR2 agonist and the 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 from about 3.4-fold to about 7.1-fold as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

31. The method of claim 30, wherein the frequency of CD34+ cells in the peripheral blood of the donor is increased from about 4.0-fold to about 6.0-fold following administration of the CXCR2 agonist and the 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ CD90+ CD45 RA-cells to leukocytes of from about 0.0003 to about 0.0016 in a peripheral blood sample of the donor following administration of the CXCR2 agonist and the 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 peripheral blood of the donor with CD34+ CD90+ CD 45-cells relative to leukocytes at a ratio of from about 5.5:1 to about 26.9:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

35. The method of claim 34, wherein the peripheral blood of the donor is enriched for CD34+ CD90+ CD45RA "cells relative to leukocytes in 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ CD90+ CD45 RA-cells to neutrophils in a peripheral blood sample of the donor from about 0.0007 to about 0.0043 following administration of the CXCR2 agonist and the 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.

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 peripheral blood of the donor with CD34+ CD90+ CD 45-cells relative to neutrophils at a ratio of from about 3.5:1 to about 22.0:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

39. The method of claim 38, wherein the donor's peripheral blood is enriched for CD34+ CD90+ CD45RA "cells relative to neutrophils in 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ CD90+ CD45 RA-cells to lymphocytes from about 0.0008 to about 0.0069 in a peripheral blood sample of the donor following administration of the CXCR2 agonist and the 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 peripheral blood of the donor with CD34+ CD90+ CD 45-cells relative to lymphocytes in a ratio of from about 5.6:1 to about 37.0:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

43. The method of claim 42, wherein the donor's peripheral blood is enriched for CD34+ CD90+ CD45 RA-cells relative to lymphocytes in a ratio from about 8.0:1 to about 10.0: 1.

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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ CD90+ CD45 RA-cells to monocytes from about 0.0028 to about 0.0130 in a peripheral blood sample 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 peripheral blood of the donor with CD34+ CD90+ CD45 RA-cells relative to monocytes at a ratio of from about 1.5:1 to about 8.5:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

47. The method of claim 46, wherein the peripheral blood of the donor is enriched for CD34+ CD90+ CD45 RA-cells relative to monocytes at 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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a ratio of CD34+ CD90+ CD45 RA-cells to CD34+ cells in a peripheral blood sample of the donor of from about 0.393 to about 0.745 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 peripheral blood of the donor with CD34+ CD90+ CD 45-cells relative to CD34+ cells at a ratio of from about 1.1:1 to about 4.8:1 as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist.

51. The method of claim 50, wherein the peripheral blood of the donor is enriched for CD34+ CD90+ CD45RA "cells relative to CD34+ cells in 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, the amount of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce a population of cells having a frequency of from about 0.020% to about 0.110% CD34+ CD90+ CD45 RA-cells in a peripheral blood sample of the donor following administration of the CXCR2 agonist and the 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+ CD45 RA-cells in the peripheral blood of the donor from about 5.1-fold to about 25.7-fold as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the 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 from about 5.1-fold to about 7.1-fold following administration of the CXCR2 agonist and the CXCR4 antagonist.

56. A method of mobilizing a population of hematopoietic stem cells from bone marrow of a mammalian donor into peripheral blood, the method comprising:

a. administering to the donor a mobilizing amount of a CXCR2 agonist and a CXCR4 antagonist;

b. obtaining input values for each of one or more parameters characterizing a peripheral blood sample of the donor listed in table 2; and

c. releasing the sample for ex vivo expansion of the hematopoietic stem cells or for treating 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 criteria for each of the one or more parameters.

57. The method of claim 56, wherein the one or more reference parameters is a set of parameters listed in any 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 treatment of the mammalian patient.

60. The method of any one of claims 12-59, wherein the sample is isolated from the donor from about 3 hours to about 5 hours after administration of the CXCR2 agonist and the CXCR4 antagonist.

61. The method of claim 60, wherein said sample is isolated from said donor about 4 hours after administration of said CXCR2 agonist and said CXCR4 antagonist.

62. The method of any one of claims 12-61, wherein said CXCR2 agonist is Gro- β T or a variant thereof.

63. The method of claim 62, wherein said 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 said CXCR2 agonist differs from the amino acid sequence of SEQ ID NO 2 only by one or more conservative amino acid substitutions.

65. The method of claim 63, wherein said CXCR2 agonist is Gro- β T.

66. The method of any one of claims 12-61, wherein said CXCR2 agonist is Gro- β or a variant thereof.

67. The method of claim 66, wherein said 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 said CXCR2 agonist differs from the amino acid sequence of SEQ ID NO 1 only by one or more conservative amino acid substitutions.

69. The method of claim 67, wherein said CXCR2 agonist is Gro- β.

70. The method of any one of claims 12-69, wherein said CXCR2 agonist is administered to said donor at a dose of from about 50 μ g/kg to about 1 mg/kg.

71. The method of claim 70, wherein said CXCR2 agonist is administered to said donor at a dose of from about 50 μ g/kg to about 300 μ g/kg.

72. The method of claim 71, wherein said CXCR2 agonist is administered to said donor at a dose of from about 100 μ g/kg to about 250 μ g/kg.

73. The method of claim 72, wherein said CXCR2 agonist is administered to said donor at a dose of about 150 μ g/kg.

74. The method of any one of claims 1-73, wherein said CXCR2 agonist is administered intravenously to said donor.

75. The method of any one of claims 12-74, wherein the CXCR2 agonist and the CXCR4 antagonist are administered concurrently to the donor.

76. The method of any one of claims 12-75, wherein said CXCR4 antagonist is a compound represented by formula (I)

Z-joint-Z' (I)

Or a pharmaceutically acceptable salt thereof, wherein Z is:

(i) cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 of said ring members are nitrogen atoms separated from each other by 2 or more carbon atoms; or

(ii) An amine represented by the formula (IA)

Wherein a comprises a monocyclic or bicyclic fused ring system containing at least one nitrogen atom, and B is H or a substituent having 1 to 20 atoms;

and wherein Z' is:

(i) cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 of said ring members are nitrogen atoms separated from each other by 2 or more carbon atoms;

(ii) an amine represented by the formula (IB)

Wherein a 'comprises a monocyclic or bicyclic fused ring system containing at least one nitrogen atom, and B' is H or a substituent having 1 to 20 atoms; or

(iii) A substituent represented by the formula (IC)

–N(R)–(CR₂)_n–X (IC)

Wherein each R is independently H or C₁-C₆Alkyl, n is 1 or 2, and X is an aryl group or a heteroaryl group or a thiol;

wherein the linker is a bond, optionally substituted C₁-C₆Alkylene, optionally substituted C₁-C₆Heteroalkylene, optionally substituted C₂-C₆Alkenylene, optionally substituted C₂-C₆Heteroalkenylene, optionally substituted C₂-C₆Alkynylene, optionally substituted C₂-C₆Heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene or optionally substitutedSubstituted heteroarylene.

77. The method according to claim 76 wherein Z and Z' are each independently a cyclic polyamine containing 9 to 32 ring members, wherein 2 to 8 of the ring members are nitrogen atoms separated from each other by 2 or more carbon atoms.

78. The method of claim 76 or 77, wherein Z and Z' are the same substituent.

79. The method according to any one of claims 76-78, wherein Z is a cyclic polyamine containing 10 to 24 ring members.

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 according to any one of claims 76-81, wherein Z is 1,4,8, 11-tetraazacyclotetradecane.

83. The method of any of claims 76-82, wherein the joint is represented by a formula (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 is

X and Y are each independently optionally substituted C₁-C₆Alkylene, optionally substituted C₁-C₆Heteroalkylene, optionally substituted C₂-C₆Alkenylene, optionally substituted C₂-C₆Heteroalkenylene, optionally substituted C₂-C₆Alkynylene or optionally substituted C₂-C₆Heteroalkynylene.

84. The method of claim 83, wherein the joint is represented by the formula (IE)

X and Y are each independently optionally substituted C₁-C₆Alkylene, optionally substituted C₁-C₆Heteroalkylene, optionally substituted C₂-C₆Alkenylene, optionally substituted C₂-C₆Heteroalkenylene, optionally substituted C ₂-C₆Alkynylene or optionally substituted C₂-C₆Heteroalkynylene.

85. The method of claim 83 or 84, wherein X and Y are each independently optionally substituted C₁-C₆An alkylene group.

86. The method of any one of claims 83-85, wherein X and Y are the same substituent.

87. The method of claim 86, wherein X and Y are each a methylene group.

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 μ g/kg to about 500 μ g/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 μ g/kg to about 300 μ g/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 μ g/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.

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 said 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+ CD45 RA-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+ CD45 RA-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.

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+ CD45 RA-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+ CD45 RA-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+ CD45 RA-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 according to 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 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 a 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 hemoglobinopathic disorder.

119. The method of claim 118, wherein the hemoglobinopathic disorder is selected from the group consisting of: sickle cell anemia, thalassemia, fanconi's 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 congenital immunodeficiency.

123. The method of claim 121, wherein the immunodeficiency disorder is acquired immunodeficiency.

124. The method of claim 123, wherein the acquired immunodeficiency is a human immunodeficiency virus or an acquired immunodeficiency 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 disease, mucopolysaccharidosis, gaucher's disease, herler's disease, sphingolipid storage disease 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.

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, eastern scission disease, hereditary lymphocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, 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, treatment of psoriasis, type 1 diabetes, 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, Barlow disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas 'disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac-herpetiform dermatitis, cold agglutinin disease, CREST syndrome, malignant atrophic papulosis, discoid lupus, autonomic nerve dysfunction, endometriosis, primary mixed cryoglobulinemia, psoriasis, diabetes mellitus, chronic lymphocytic leukemia, human immunodeficiency syndrome, chronic lymphocytic leukemia, fibromyalgia-fibromyositis, goodpasture's syndrome, graves ' 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 disease, lichen planus, lyme disease, meniere disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, strabismus myoclonus syndrome, optic neuritis, alder's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polymyalgia rheumatica, primary agammaglobulinemia, raynaud's syndrome, rheumatic fever, sarcoidosis, scleroderma, and/or scleroderma, Sjogren'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 to the patient.

135. The method of any one of claims 115-133, wherein the hematopoietic stem cells are allogeneic to the patient.

136. The method of claim 135, wherein the hematopoietic stem cells are HLA-matched 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 into the patient.

140. The method of any one of claims 115-139, wherein the hematopoietic stem cells or progeny thereof are localized to hematopoietic tissue and/or repopulate hematopoiesis following infusion of the hematopoietic stem cells or progeny 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 cause recovery of a population of cells selected from the group consisting of: megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, 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.

146. The method of any one of the above claims, further comprising collecting the hematopoietic stem cells or progeny thereof from the donor using apheresis.

147. The method according to any one of the preceding claims, wherein said Gro- β, Gro- β T and variants thereof have a purity of at least about 95% relative to deamidated forms of the peptides.

148. An enriched human blood cell preparation 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. An enriched human blood cell preparation comprising a population of hematopoietic stem cells or progeny thereof, wherein the ratio of CD34+ CD90+ CD45 RA-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 of claims 1-92.

151. CD34^dimA method of mobilizing 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- β, Gro- β T, and variants thereof, and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

152. A method of allogeneic hematopoietic stem cell transplantation 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 are mobilized from the bone marrow of a human donor into the 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- β, Gro- β T, and variants thereof and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

153. A method of preventing, reducing the risk of, or reducing the severity of a post-transplant infection in a patient in need thereof, comprising infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from the bone marrow of a human donor into the 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- β, Gro- β T, and variants thereof and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

154. A method of preventing, reducing the risk of, or reducing the severity of Graft Versus Host Disease (GVHD) in a patient in need thereof, said method comprising infusing into said patient a therapeutically effective amount of hematopoietic stem cells, wherein said hematopoietic stem cells are mobilized from the bone marrow of a human donor into the peripheral blood of said human donor by a method comprising: administering to the human donor (i) a CXCR2 agonist selected from the group consisting of Gro- β, Gro- β T, and variants thereof and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

155. The method as recited in any one of claims 151-154, wherein the CD34 is^dimCells are present in the peripheral blood in an amount of at least 2-fold to 10-fold more than if the CXCR4 antagonist alone were used to mobilize the hematopoietic stem cells.

156. The method as set forth in any one of claims 151-155 wherein the CD34 is^dimThe cells are capable of inhibiting the proliferation of alloreactive T lymphocytes 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 μ g/kg to about 250 μ g/kg.

159. The method of claim 158, wherein said CXCR2 agonist is administered to said donor at a dose of from about 125 μ g/kg to about 225 μ g/kg.

160. The method of claim 159, wherein said CXCR2 agonist is administered to said donor at a dose of about 150 μ g/kg.

161. The method of any one of claims 151-160, wherein the CXCR2 agonist is administered to the donor intravenously.

162. The method of any one of claims 151-161 wherein the CXCR4 antagonist is administered subcutaneously to the donor.

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 μ g/kg to about 500 μ g/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 μ g/kg to about 300 μ g/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 μ g/kg.

167.CD34^dimA population of cells, said CD34^dimThe population of cells is derived from the method of any one of claims 151-165.

Technical Field

The present invention relates to the mobilization of hematopoietic stem and progenitor cells (hematotoietic stem and promoter cells) from donors, such as human donors, and the treatment of patients suffering from various pathologies, such as inter alia hematological diseases, metabolic disorders, cancer and autoimmune diseases.

Background

Despite advances in the medical field, there remains a need for the treatment of hematopoietic pathologies, such as diseases, especially of specific blood cells, metabolic disorders, cancer and autoimmune conditions. Although hematopoietic stem cells have significant therapeutic potential, one limitation that has prevented their clinical use is the difficulty associated with releasing hematopoietic stem cells from the bone marrow of a donor from which they can be isolated for infusion into a patient to peripheral blood.

An additional limitation is that up to 80% of mobilized peripheral blood (mPB) allorecipients will experience Graft Versus Host Disease (GVHD). Although the levels of CD3+ T cells were higher in the mPB grafts compared to bone marrow grafts, the levels of acute GVHD observed after transplantation of HLA-matched mPB were comparable to HLA-matched bone marrow. One explanation is that G-CSF mobilized grafts contain Myeloid Derived Suppressor Cells (MDSCs), which have potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro. The percentage of MDSCs in G-CSF mobilized grafts was variable, and clinical data indicated that patients transplanted mPB grafts containing higher numbers of MDSCs may have better outcomes, including lower rates of acute GVHD (Venndramin et al, (2014) BBMT20(12): 2049-.

Thus, there is a need for compositions and methods for promoting mobilization of hematopoietic stem and progenitor cells, and in particular for methods of identifying populations of mobilized cells suitable for therapeutic use. There is also a need for compositions and methods for promoting mobilization of hematopoietic stem and progenitor cells that consistently produce higher numbers of MDSCs than prior art methods.

Summary of The Invention

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 cell and progenitor cell donor (i.e., donor), such as a mammalian donor, and particularly a human donor. The invention additionally provides compositions and methods for treating 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- β or a variant thereof, such as a truncated form of Gro- β (e.g., Gro- β T), as described herein, optionally in combination with a C-X-C chemokine receptor type 4 (CXCR4) antagonist, such as 1, 1' - [1, 4-phenylenebis (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane, or a variant thereof, can be administered to a subject in an amount sufficient to mobilize hematopoietic stem and progenitor cells. Importantly, the compositions and methods described herein can be used to mobilize hematopoietic stem and progenitor cells from the 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 leukocytes, neutrophils, lymphocytes, and monocytes). Thus, the compositions and methods described herein are capable of selectively mobilizing hematopoietic stem and progenitor cells in a donor, which can then be isolated from the donor for therapeutic use.

In some embodiments, hematopoietic stem or progenitor cells can be mobilized from the bone marrow of a donor to peripheral blood from which they can be collected and/or isolated. After the mobilized cells are collected, the drawn hematopoietic stem or progenitor cells can then be infused into a patient, which can be a donor or another subject, such as a subject HLA-matched to the donor, for treatment of one or more pathologies of the hematopoietic system. In some embodiments, the extracted hematopoietic stem or progenitor cells are first expanded ex vivo and then these cells and/or their progeny are infused into the patient. The compositions and methods described herein provide important clinical benefits, namely enabling the generation of cell populations enriched for 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 suited for hematopoietic stem cell transplantation therapy, optionally first expanded ex vivo to increase the number of hematopoietic stem and progenitor cells available for infusion into a patient.

Furthermore, the methods described herein provide the advantage of inhibiting leukocytosis in a donor. Leukocytosis can lead to adverse effects such as spleen rupture, renal dysfunction, acute febrile non-contagious pneumonia (i.e. pulmonary toxicity), cardiovascular toxicity (e.g. hypercoagulable state, heart attack, dyspnea, angina, arrhythmia, rupture of atherosclerotic plaques due to pro-inflammatory effects associated with high neutrophil counts), neurological disorders (such as blurred vision, headache and retinal hemorrhage) and sickle cell crisis. See, for example, D' Souza et al (2008) Transfusion Medicine Reviews 22(4):280- > 290.

As described herein, hematopoietic stem cells are capable of differentiating into numerous cell types in the hematopoietic lineage, and thus can be administered to a patient in order to fill (deposit) or refill (re-deposit) defective or deficient cell types in the patient. The patient may be, for example, a patient suffering from one or more blood disorders such as autoimmune diseases, cancer, hemoglobinopathies or other hematopoietic pathologies and thus requiring hematopoietic stem cell transplantation. Accordingly, the present invention provides methods of treating various hematopoietic conditions such as, inter alia, sickle cell anemia, thalassemia, Fanconi anemia (Fanconi anemia), Wiskott-Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection and acquired immunodeficiency syndrome, as well as cancer and autoimmune diseases.

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 comprising administering to the donor a CXCR2 agonist and a CXCR4 antagonist, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce, in a peripheral blood sample of the donor, a CD34 having a blood flow from about 0.0008 to about 0.0021 following administration of a CXCR2 agonist and a CXCR4 antagonist⁺A ratio of cells to leukocytes. In some embodiments, the CD34 in the sample⁺The ratio of 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, 36 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, 0.00122, 3600131, 3600172, 0.00122, 360010, 0.00122, 3600172, 0.00122, 360010.00172, 0.00122, 3600172, 0.00122, 360010, 0.00122, 3600172, 0.00122, 360010.72, 0.00122, 3600172, 0.00122, 360010, 0.00122, 3600172, 0.00122, 360010.0010.0010, 36 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 CD34 in the sample⁺The ratio of cells to leukocytes 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 CD34 in the sample⁺The ratio of cells to leukocytes is from about 0.0010 to about 0.0018, e.g., the ratio of hematopoietic stem cells to leukocytes in the sample is about 0.00100, 0.00106, 0.00108, 0.00109, 0.00112, 0.00115, 0.00119, 0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00131, 0.00135, 0.00139, 0.00142, 0.00152, 0.00155, 0.00156, 0.00159, 0.00162. In some embodiments, the CD34 in the sample ⁺The ratio of cells to leukocytes is from about 0.0012 to about 0.0016, e.g., CD34 in the sample⁺The ratio of cells to leukocytes is 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, 0.0.00123, 0.001250157. 0.00158, 0.00159, or 0.00160. In some embodiments, the CD34 in the sample⁺The ratio of cells to leukocytes was about 0.0014.

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 sufficient to produce CD34 having at least about 38,000 cells/ml ⁺Cell densities such as from about 38,000 to about 100,000, about 40,000 to about 90,000, about 50,000 to about 80,000, or about 60,000 to about 70,000 cells/ml (e.g., about 38,00, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 55,000, 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) of CD 34) ⁺Cell density and has a cell density of no more than about 5.3 × 10⁷Leukocyte density of individual cells/ml, such as about 2.3 × 10⁷Individual cells/ml to about 5.3 × 10⁷Individual cell/ml, about 2.5 × 10⁷Individual cells/ml to about 5.1 × 10⁷Individual cell/ml, 2.9 × 10⁷Individual cells/ml to about 4.5 × 10⁷Individual cell/ml, about 3 × 10⁷Individual cell/ml to about 4 × 10⁷Individual cell/ml (e.g., 5.3 × 10)⁷Individual cell/ml, 5.2 × 10⁷Individual cell/ml, 5.1 × 10⁷Individual cells/ml, 5 × 10⁷Individual cell/ml, 4.9 × 10⁷Individual cell/ml, 4.8 × 10⁷Individual cell/ml, 4.7 × 10 ⁷Individual cell/ml, 4.6 × 10⁷Individual cell/ml, 4.5 × 10⁷Individual cell/ml, 4.4 × 10⁷Individual cell/ml, 4.3 × 10⁷Individual cell/ml, 4.2 × 10⁷Individual cell/ml, 4.1 × 10⁷Individual cells/ml, 4 × 10⁷Individual cell/ml, 3.9 × 10⁷Individual cell/ml, 3.8 × 10⁷Individual cell/ml, 3.7 × 10⁷Individual cell/ml, 3.6 × 10⁷Individual cell/ml, 3.5 × 10⁷Individual cell/ml, 3.4 × 10⁷Individual cell/ml, 3.3 × 10⁷Individual cell/ml, 3.2 × 10⁷Individual cell/ml, 3.1 × 10⁷Individual cells/ml, 3 × 10 ⁷Individual cell/ml, 2.9 × 10⁷Individual cell/ml, 2.8 × 10⁷Individual cell/ml, 2.7 × 10⁷Individual cell/ml, 2.6 × 10⁷Individual cell/ml, 2.5 × 10⁷Individual cell/ml, 2.4 × 10⁷Individual cell/ml, 2.3 × 10⁷Individual cells/ml or less) of leukocytesA cell population at cellular density of an amount of a CXCR2 agonist and a CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 38,000 cells/ml to about 100,000 cells/ml⁺Cell density and has a density of from about 2.3 × 10⁷Individual cells/ml to about 5.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of leukocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 40,000 cells/ml to about 80,000 cells/ml ⁺Cell density and has a density of from about 2.5 × 10⁷Individual cells/ml to about 5 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of leukocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 50,000 cells/ml to about 90,000 cells/ml⁺Cell density and has a density of from about 3 × 10⁷Individual cell/ml to about 4 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of leukocyte density.

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 comprising administering to the donor a CXCR2 agonist and a CXCR4 antagonist, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce, in a peripheral blood sample of the donor, a CD34 having from about 0.0021 to about 0.0094 following administration of a CXCR2 agonist and a CXCR4 antagonist ⁺A cell population of a ratio of cells to lymphocytes. In some embodiments, the CD34 in the sample⁺The ratio of cells to lymphocytes can be about 0.00210, 0.00218, 0.00241, 0.00244, 0.00245, 0.00247, 0.00264, 0.00266, 0.00267, 0.00268, 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、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, 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 CD34 in the sample ⁺The ratio of cells to lymphocytes 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 CD34 in the sample⁺The ratio of cells to lymphocytes is from about 0.0025 to about 0.0035, e.g., CD34 in a sample⁺The ratio of cells to lymphocytes is 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, 0.00287, 0.00276. In some embodiments, the CD34 in the sample ⁺The ratio of cells to lymphocytes was about 0.0031.

In another aspect, the invention features a kitA method of mobilizing a population of blood stem cells from the bone marrow of a mammalian donor (e.g., a human donor) into peripheral blood, the method comprising administering to the donor a CXCR2 agonist and a CXCR4 antagonist, the amounts of the CXCR2 agonist and the CXCR4 antagonist administered being sufficient to enrich the peripheral blood of the donor with CD34 25 relative to lymphocytes in a ratio from about 4.8:1 to 8.4:1, as assessed by comparing a peripheral blood sample of the donor after administration of the CXCR2 agonist and the CXCR4 antagonist to a peripheral blood sample of the donor prior to administration of the CXCR2 agonist and the CXCR4 antagonist⁺A cell. In some embodiments, the peripheral blood of the donor may be at 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, 6.70:1, 7.5.55: 1, 7.5.5.75: 1, 7.5.80: 1, 7.5.5: 1, 6.5.5.5.5.5.5: 1, 6.5.5.5: 1, 6.5.5.5.5.5.5.5: 1, 6.5.5: 1, 6.5.5.5: 1, 6.5: 1, 6.5.5.5.5: 1, 6.5.5.5.5.5: 1, 6., Ratios of 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 enriched with CD34 relative to lymphocytes ⁺A cell. In some embodiments, the peripheral blood of the donor is enriched for CD34 relative to lymphocytes in a ratio 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⁺A cell. In some embodiments, the peripheral blood of the donor is enriched for CD34 relative to lymphocytes in a ratio of from about 5.0:1 to about 6.5:1, such as 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⁺A cell. In some embodiments, the peripheral blood of the donor is enriched for CD34 relative to lymphocytes in a ratio of about 5.7:1⁺A cell.

In another aspect, the inventionCharacterized by 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 comprising administering to the donor a density of CD34+ cells sufficient to produce a population of hematopoietic stem cells having at least about 38,000 cells/ml, such as 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, 74,000,000 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) of CD34 ⁺Cell density and has a cell density of no more than about 2.4 × 10⁷Lymphocyte density per cell/ml, such as about 1 × 10⁷Individual cells/ml to about 2.3 × 10⁷Individual cell/ml, about 1.3 × 10⁷Individual cell/ml to about 2.1 × 10⁷Individual cell/ml or 1.5 × 10⁷Individual cell/ml to about 1.9 × 10⁷Individual cell/ml (e.g., about 2.4 × 10⁷Individual cell/ml, 2.3 × 10⁷Individual cell/ml, 2.2 × 10⁷Individual cell/ml, 2.1 × 10⁷Individual cells/ml, 2 × 10⁷Individual cell/ml, 1.9 × 10⁷Individual cell/ml, 1.8 × 10⁷Individual cell/ml, 1.7 × 10⁷Individual cell/ml, 1.6 × 10 ⁷Individual cell/ml, 1.5 × 10⁷Individual cell/ml, 1.4 × 10⁷Individual cell/ml, 1.3 × 10⁷Individual cell/ml, 1.2 × 10⁷Individual cell/ml, 1.1 × 10⁷Individual cells/ml, 1 × 10⁷0.9 × 10 cell/ml or less⁷Individual cell/ml, 0.8 × 10⁷Individual cells/ml or less) of a population of cells at lymphocyte density, a CXCR2 agonist and a CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 38,000 cells/ml to about 100,000 cells/ml⁺Cell density and has a density of from about 1 × 10⁷Individual cells/ml to about 2.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of lymphocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 40,000 cells/ml to about 80,000 cells/ml ⁺Cell density and has a density of from about 1.3 × 10⁷Individual cells/ml to about 2.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of lymphocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 50,000 cells/ml to about 90,000 cells/ml⁺Cell density and has a density of from about 1.5 × 10⁷Individual cell/ml to about 2 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of lymphocyte density.

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 comprising administering to the donor sufficient to produce CD34 having at least about 38,000 cells/ml⁺Cell densities such as from about 38,000 to about 100,000, about 40,000 to about 90,000, about 50,000 to about 80,000, or about 60,000 to about 70,000 cells/ml (e.g., about 38,00, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50,000, 51,000, 52,000, 53,000, 54,000, 55,000, 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 One cell/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) of CD34⁺Cell density and has a cell density of no more than about 6 × 10⁶Monocyte density per cell/ml, such as from 3.4 × 10⁶Individual cells/ml to about 5.9 × 10⁶Individual cell/ml, about 3.5 × 10⁶Individual cells/ml to about 5.7 × 10⁶Individual cells/ml or about 4 × 10⁶Individual cells/ml to about 5 × 10⁶Individual cell/ml (e.g., 5.9 × 10)⁶Individual cell/ml, 5.8 × 10⁶Individual cell/ml, 5.7 × 10⁶Individual cell/ml, 5.6 × 10 ⁶Individual cell/ml, 5.5 × 10⁶Individual cell/ml, 5.4 × 10⁶Individual cell/ml, 5.3 × 10⁶Individual cell/ml, 5.2 × 10⁶Individual cell/ml, 5.1 × 10⁶Individual cells/ml, 5 × 10⁶Individual cell/ml, 4.9 × 10⁶Individual cell/ml, 4.8 × 10⁶Individual cell/ml, 4.7 × 10⁶Individual cell/ml, 4.6 × 10⁶Individual cell/ml, 4.5 × 10⁶Individual cell/ml, 4.4 × 10⁶Individual cell/ml, 4.3 × 10⁶Individual cell/ml, 4.2 × 10⁶Individual cell/ml, 4.1 × 10⁶Individual cells/ml, 4 × 10⁶Individual cell/ml, 3.9 × 10 ⁶Individual cell/ml, 3.8 × 10⁶Individual cell/ml, 3.7 × 10⁶Individual cell/ml, 3.6 × 10⁶Individual cell/ml, 3.5 × 10⁶Individual cell/ml, 3.4 × 10⁶Individual cells/ml or less) of a cell population at a monocyte density of CXCR2 agonist and CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 38,000 cells/ml to about 100,000 cells/ml⁺Cell density and has a density of from about 3.4 × 10⁶Individual cells/ml to about 6 × 10⁶A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population at monocyte density. In some embodiments, the method comprises administering to the donor a composition sufficient to produce a cell population having from about 40,000 cells/ml to about 80,000 cells/mlCD34⁺Cell density and has a density of from about 4 × 10⁶Individual cells/ml to about 5.5 × 10 ⁶A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population at monocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 50,000 cells/ml to about 90,000 cells/ml⁺Cell density and has a density of from about 4 × 10⁶Individual cells/ml to about 5 × 10⁶A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population at monocyte density.

In another aspect, the invention features a method of enriching a population of hematopoietic stem cellsA method of mobilizing bone marrow of a dairy animal donor (e.g., a human donor) into peripheral blood, the method comprising administering to the donor sufficient to produce CD34 having at least about 16,000 cells/ml⁺CD90⁺CD45RA^-Cell density, such as from about 20,000 to about 75,000, about 25,000 to about 70,000, about 30,000 to about 65,000, about 35,000 to about 60,000, about 40,000 to about 55,000 or about 45,000 to about 50,000 (e.g., about 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29, 65, 75, 52, 75, 52, 65, 52, 75, 52, 65, 52, 65, 52, 75, 52, 75, 52, 65, 52, 75, 52, 53, 52, 65, 52, 75, 53, 52, 53, 75, 53, 52, 75, 52, 53, 52, 75, 65, 53, 52, 75, 52, 65, 52, 75, 40, 65, 95, 40, 95, 000, 95, 40, 95, 000, 95, 8, 95, 8, 95, 8, 95, 000, 95, or more cells/ml of the ⁷Leukocyte density of individual cells/ml, such as from about 2.3 × 10⁷Individual cells/ml to about 5.3 × 10⁷Individual cell/ml, about 2.5 × 10⁷Individual cell/ml to about5.1×10⁷Individual cell/ml, 2.9 × 10⁷Individual cells/ml to about 4.5 × 10⁷Individual cell/ml, about 3 × 10⁷Individual cell/ml to about 4 × 10⁷Individual cell/ml (e.g., 5.3 × 10)⁷Individual cell/ml, 5.2 × 10⁷Individual cell/ml, 5.1 × 10⁷Individual cells/ml, 5 × 10⁷Individual cell/ml, 4.9 × 10⁷Individual cell/ml, 4.8 × 10⁷Individual cell/ml, 4.7 × 10⁷Individual cell/ml, 4.6 × 10⁷Individual cell/ml, 4.5 × 10 ⁷Individual cell/ml, 4.4 × 10⁷Individual cell/ml, 4.3 × 10⁷Individual cell/ml, 4.2 × 10⁷Individual cell/ml, 4.1 × 10⁷Individual cells/ml, 4 × 10⁷Individual cell/ml, 3.9 × 10⁷Individual cell/ml, 3.8 × 10⁷Individual cell/ml, 3.7 × 10⁷Individual cell/ml, 3.6 × 10⁷Individual cell/ml, 3.5 × 10⁷Individual cell/ml, 3.4 × 10⁷Individual cell/ml, 3.3 × 10⁷Individual cell/ml, 3.2 × 10⁷Individual cell/ml, 3.1 × 10⁷Individual cells/ml, 3 × 10⁷Individual cell/ml, 2.9 × 10⁷Individual cell/ml, 2.8 × 10 ⁷Individual cell/ml, 2.7 × 10⁷Individual cell/ml, 2.6 × 10⁷Individual cell/ml, 2.5 × 10⁷Individual cell/ml, 2.4 × 10⁷Individual cell/ml, 2.3 × 10⁷Individual cells/ml or less) of a cell population of leukocyte density of CXCR2 agonist and CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 20,000 cells/ml to about 75,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 2.3 × 10⁷Individual cells/ml to about 5.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of leukocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 30,000 cells/ml to about 60,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 2.5 × 10⁷Individual cells/ml to about 5 × 10 ⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of leukocyte density. In some embodiments, the method comprisesComprising administering to the donor sufficient to produce CD34 having from about 40,000 cells/ml to about 50,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 3 × 10⁷Individual cell/ml to about 4 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of leukocyte density.

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 sufficient to produce CD34 having at least about 16,000 cells/ml⁺CD90⁺CD45RA^-Cell densities such as from about 20,000 to about 75,000, about 25,000 to about 70,000, about 30,000 to about 65,000, about 35,000 to about 60,000, about 40,000 to about 55,000 or about 45,000 to about 50,000 (e.g., about 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 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 has a density of no more than about 2.5 × 10⁷Neutrophil density per cell/ml, such as from about 1 × 10⁷Individual cells/ml to about 2.5 × 10⁷Individual cell/ml, about 1.3 × 10⁷Individual cell/ml to about 2 × 10⁷Individual cell/ml or 1.5 × 10⁷Individual cell/ml to about 1.9 × 10⁷Individual cell/ml (e.g., about 2.5 × 10⁷Individual cell/ml, 2.4 × 10⁷Individual cell/ml, 2.3 × 10⁷Individual cell/ml, 2.2 × 10⁷Individual cell/ml, 2.1 × 10⁷Individual cells/ml, 2 × 10⁷Individual cell/ml, 1.9 × 10⁷Individual cell/ml, 1.8 × 10 ⁷Individual cell/ml, 1.7 × 10⁷Individual cell/ml, 1.6 × 10⁷Individual cell/ml, 1.5 × 10⁷Individual cell/ml, 1.4 × 10⁷Individual cell/ml, 1.3 × 10⁷Individual cell/ml, 1.2 × 10⁷Individual cell/ml, 1.1 × 10⁷Individual cells/ml, 1 × 10⁷Individual cells/ml or less) of a cell population of neutrophil density, a CXCR2 agonist and a CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 20,000 cells/ml to about 75,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 1 × 10⁷Individual cells/ml to about 2.5 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell/ml of a cell population of neutrophil density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 30,000 cells/ml to about 60,000 cells/ml ⁺CD90⁺CD45RA^-Cell density and has a density of from about 1.3 × 10⁷Individual cells/ml to about 2.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell/ml of a cell population of neutrophil density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 40,000 cells/ml to about 50,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 1.5 × 10⁷Individual cell/ml to about 2 × 10⁷CXCR2 agonist and C in an amount per cell/ml of a cell population of neutrophil densityAn XCR4 antagonist.

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 comprising administering to the donor a CXCR2 agonist and a CXCR4 antagonist, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce, in a peripheral blood sample of the donor, a CD34 having from about 0.0008 to about 0.0069 following administration of a CXCR2 agonist and a CXCR4 antagonist⁺CD90⁺CD45RA^-A cell population of a ratio of cells to lymphocytes. In some embodiments, the CD34 in the sample ⁺CD90⁺CD45RA^-The ratio of cells to lymphocytes may be about 0.00080, 0.00081, 0.00083, 0.00084, 0.00085, 0.00086, 0.00093, 0.00094, 0.00096, 0.00098, 0.00100, 0.00112, 0.00115, 0.00119, 0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00131, 0.00135, 0.00139, 0.00131, 0.00159, 0.00139, 0, 0.00142, 0, e, 0, e, 0, e.00159, 0, e, 0, e.00175, 0, e, 0, e, 0, e, 0, 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, 0.002 18、0.00219、0.00220、0.00221、0.00222、0.00223、0.00224、0.00225、0.00226、0.00227、0.00228、0.00229、0.00230、0.00231、0.00232、0.00233、0.00234、0.00235、0.00236、0.00237、0.00238、0.00239、0.00240、0.00241、0.00242、0.00243、0.00244、0.00245、0.00246、0.00247、0.00248、0.00249、0.00250、0.00251、0.00252、0.00253、0.00254、0.00255、0.00256、0.00257、0.00258、0.00259、0.00260、0.00261、0.00262、0.00263、0.00264、0.00265、0.00266、0.00267、0.00268、0.00269、0.00270、0.00271、0.00272、0.00273、0.00274、0.00275、0.00276、0.00278、0.00279、0.00280、0.00281、0.00282、0.00283、0.00284、0.00285、0.00286、0.00287、0.00288、0.00289、0.00290、0.00291、0.00292、0.00293、0.00294、0.00295、0.00296、0.00297、0.00298、0.00299、0.00300、0.00301、0.00302、0.00303、0.00304、0.00305、0.00306、0.00307、0.00308、0.00309、0.00310、0.00311、0.00312、0.00313、0.00314、0.00315、0.00316、0.00317、0.00318、0.00319、0.00320、0.00321、0.00322、0.00323、0.00324、0.00325、0.00326、0.00327、0.00328、0.00329、0.00330、0.00331、0.00332、0.00333、0.00334、0.00335、0.00336、0.00337、0.00338、0.00339、0.00340、0.00341、0.00342、0.00343、0.00344、0.00345、0.00346、0.00347、0.00348、0.00349、0.00350、0.00351、0.00352、0.00353、0.00354、0.00355、0.00356、0.00357、0.00358、0.00359、0.00360、0.00361、0.00362、0.00363、0.00364、0.00365、0.00366、0.00367、0.00368、0.00369、0.00370、0.00371、0.00372、0.00373、0.00374、0.00375、0.00376、0.00378、0.00379、0.00380、0.00381、0.00382、0.00383、0.00384、0.00385、0.00386、0.00387、0.00388、0.00389、0.00390、0.00391、0.00392、0.00393、0.00394、0.00395、0.00396、0.00397、0.00398、0.00399、0.00401、0.00402、0.00403、0.00404、0.00405、0.00406、0.00407、0.00408、0.00409、0.00410、0.00411、0.00412、0.00413、0.00414、0.00415、0.00416、0.00417、0.00418、0.00419、0.00420、0.00421、0.00422、0.00423、0.00424、0.00425、0.00426、0.00427、0.00428、0.00429、0.00430、0.00431、0.00432、0.00433、0.00434、0.00435、0.00436、0.00437、0.00438、0.00439、0.00440、0.00441、0.00442、0.00443、0.00444、0.00445、0.00446、0.00447、0.00448、0.00449、0.00450、0.00451、0.00452、0.00453、0.00454、0.00455、0.00456、0.00457、0.00458、0.00459、0.00460、0.00461、0.00462、0.00463、0.00464、0.00465、0.00466、0.00467、0.00468、0.00469、0.00470、0.00471、0.00472、0.00473、0.00474、0.00475、0.00476、0.00478、0.00479、0.00480、0.00481、0.00482、0.00483、0.00484、0.00485、0.00486、0.00487、0.00488、0.00489、0.00490、0.00491、0.00492、0.00493、0.00494、0.00495、0.00496、0.00497、0.00498、0.00499、0.00500、0.00501、0.00502、0.00503、0.00504、0.00505、0.00506、0.00507、0.00508、0.00509、0.00510、0.00511、0.00512、0.00513、0.00514、0.00515、0.00516、0.00517、0.00518、0.00519、0.00520、0.00521、0.00522、0.00523、0.00524、0.00525、0.00526、0.00527、0.00528、0.00529、0.00530、0.00531、0.00532、0.00533、0.00534、0.00535、0.00536、0.00537、0.00538、0.00539、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、000633, 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 CD34 in the sample⁺CD90⁺CD45RA^-The ratio of cells to lymphocytes is from about 0.0011 to about 0.0031, e.g., CD34 in the sample⁺CD90⁺CD45RA^-The ratio of cells to lymphocytes is about 0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00123, 0.00124, 0.00125, 0.00126, 0.00127, 0.00128, 0.00120, 0.00131, 0.00120, 0.00135, 0.00120, 3600172, 0.00120, 3600152, 0.00120, 0.00155, 0.00156, 0.00120, 3659, 0.00120, 3600172, 0.00120, 36, 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. In some embodiments, the CD34 in the sample⁺CD90⁺CD45RA^-The ratio of cells to lymphocytes is about 0.0021.

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 comprising administering to the donor a CXCR2 agonist and a CXCR4 antagonist, as assessed by comparing a peripheral blood sample of the donor after administration of a CXCR2 agonist and a CXCR4 antagonist to a peripheral blood sample of the donor before administration of a CXCR2 agonist and a CXCR4 antagonist, the amount of CXCR2 agonist and CXCR4 antagonist administered being sufficient to enrich the peripheral blood of the donor with CD34 25 relative to lymphocytes at a ratio from about 5.6:1 to about 37.0:1 ⁺CD90⁺CD45RA^-A cell. In some embodiments, the peripheral blood of the donor may be at 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, 7.90:1, 7.95:1, 7.00:1, 7.5: 1, 7.15:1, 7.20:1, 7.25:1, 7.90:1, 7.5: 1, 8.5: 1, 7.5: 1, 7、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、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, 30.35:1, 30.75:1, 30.40:1, 30.45:1, 31.31: 1, 31.5: 1, 31.31: 1, 31.30.30.30.65: 1, 31: 1, 31.31: 1, 31: 1, 31.30.30.30.5: 1, 31: 1, 31.31: 1, 31: 1, 31.31: 1, 31.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30.30: 1, 31: 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, 34.34: 1, 34: 1, 34.35:1, 34: 1, 34.34: 1, 34: 1, 34.35:1, 34: 1, 34.35:1, 34: 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 relative to lymphocyte enriched CD34 ⁺CD90⁺CD45RA^-A cell. In some embodiments, the peripheral blood of the donor is in a ratio of from about 8.0:1 to about 10.0:1, such as 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, 9.3:1CD34 enriched for lymphocytes in a ratio of 5:1, 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⁺CD90⁺CD45RA^-A cell. In some embodiments, the peripheral blood of the donor is enriched for CD34 relative to lymphocytes in a ratio of about 9.3:1⁺CD90⁺CD45RA^-A cell.

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 sufficient to produce CD34 having at least about 16,000 cells/ml⁺CD90⁺CD45RA^-Cell densities such as from about 20,000 to about 75,000, about 25,000 to about 70,000, about 30,000 to about 65,000, about 35,000 to about 60,000, about 40,000 to about 55,000 or about 45,000 to about 50,000 (e.g., about 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 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 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 has a density of no more than about 2.4 × 10⁷Lymphocyte density per cell/ml, such as from about 1 × 10⁷Individual cells/ml to about 2.3 × 10⁷Individual cell/ml, about 1.3 × 10⁷Individual cell/ml to about 2.1 × 10⁷Individual cells/ml or about 1.5 × 10⁷Individual cell/ml to about 1.9 × 10⁷Individual cell/ml (e.g., about 2.4 × 10⁷Individual cell/ml, 2.3 × 10⁷Individual cell/ml, 2.2 × 10⁷Individual cell/ml, 2.1 × 10 ⁷Individual cells/ml, 2 × 10⁷Individual cell/ml, 1.9 × 10⁷Individual cell/ml, 1.8 × 10⁷Individual cell/ml, 1.7 × 10⁷Individual cell/ml, 1.6 × 10⁷Individual cell/ml, 1.5 × 10⁷Individual cell/ml, 1.4 × 10⁷Individual cell/ml, 1.3 × 10⁷Individual cell/ml, 1.2 × 10⁷Individual cell/ml, 1.1 × 10⁷Individual cells/ml, 1 × 10⁷0.9 × 10 cell/ml or less⁷Individual cell/ml, 0.8 × 10⁷Individual cells/ml or less) of a population of cells at lymphocyte density, a CXCR2 agonist and a CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 20,000 cells/ml to about 75,000 cells/ml ⁺CD90⁺CD45RA^-Cell density and has a density of from about 1 × 10⁷Individual cells/ml to about 2.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of lymphocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 30,000 cells/ml to about 60,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 1.3 × 10⁷Individual cells/ml to about 2.3 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of lymphocyte density. In some embodiments, the method comprises administering to the donor an amount sufficient to result in a composition havingFrom about 40,000 cells/ml to about 50,000 cells/ml CD34⁺CD90⁺CD45RA^-Cell density and has a density of from about 1.5 × 10⁷Individual cell/ml to about 2 × 10⁷A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population of lymphocyte density.

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, the amounts of CXCR2 agonist and CXCR4 antagonist administered being sufficient to produce, in a peripheral blood sample of the donor, CD34 having a blood flow from about 0.0028 to about 0.0130 following administration of the CXCR2 agonist ⁺CD90⁺CD45RA^-A cell population of a ratio of cells to monocytes. In some embodiments, the CD34 in the sample⁺CD90⁺CD45RA^-The ratio of cells to monocytes may be about 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00287, 0.00284, 0.00291, 0.00284, 0.00293, 0.00284, 0.00307, 0.00284, 0.00311, 0.00284, 0.00315, 0.00284, 0.00318, 0.00319, 0.00320, 0.00284, 0.00323, 0.00324, 0.00284 94、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、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、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 CD34 in the sample⁺CD90⁺CD45RA^-The ratio of cells to monocytes is from about 0.0063 to about 0.0083, e.g., CD34 in the sample⁺CD90⁺CD45RA^-The ratio of the cells to the mononuclear cells is about, in (b), (c), (d), (c), (d), (, 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, 0.00794, 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 CD34 in the sample⁺CD90⁺CD45RA^-The ratio of cells to monocytes was about 0.0073.

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 sufficient to produce CD34 having at least about 16,000 cells/ml⁺CD90⁺CD45RA^-Cell densities such as from about 20,000 to about 75,000, about 25,000 to about 70,000, about 30,000 to about 65,000, about 35,000 to about 60,000, about 40,000 to about 55,000 or about 45,000 to about 50,000 (e.g., about 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 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, 53,00 cells/ml 0 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 has a density of no more than about 6 × 10 cells/ml, 55,000 cells/ml, 56,000 cells/ml, 61,000 cells/ml or more), and has a density of no more than about 6 ×⁶Monocyte density per cell/ml, such as from 3.4 × 10⁶Individual cells/ml to about 5.9 × 10⁶Individual cell/ml, about 3.5 × 10⁶Individual cells/ml to about 5.7 × 10⁶Individual cells/ml or about 4 × 10⁶Individual cells/ml to about 5 × 10⁶Individual cell/ml (e.g., 5.9 × 10) ⁶Individual cell/ml, 5.8 × 10⁶Individual cell/ml, 5.7 × 10⁶Individual cell/ml, 5.6 × 10⁶Individual cell/ml, 5.5 × 10⁶Individual cell/ml, 5.4 × 10⁶Individual cell/ml, 5.3 × 10⁶Individual cell/ml, 5.2 × 10⁶Individual cell/ml, 5.1 × 10⁶Individual cells/ml, 5 × 10⁶Individual cell/ml, 4.9 × 10⁶Individual cell/ml, 4.8 × 10⁶Individual cell/ml, 4.7 × 10⁶Individual cell/ml, 4.6 × 10⁶Individual cell/ml, 4.5 × 10⁶Individual cell/ml, 4.4 × 10⁶Individual cell/ml, 4.3 × 10⁶Individual cell/ml, 4.2 × 10 ⁶Individual cell/ml, 4.1 × 10⁶Individual cells/ml, 4 × 10⁶Individual cell/ml, 3.9 × 10⁶Individual cell/ml, 3.8 × 10⁶Individual cell/ml, 3.7 × 10⁶Individual cell/ml, 3.6 × 10⁶Individual cell/ml, 3.5 × 10⁶Individual cell/ml, 3.4 × 10⁶Individual cells/ml or less) of a cell population at a monocyte density of CXCR2 agonist and CXCR4 antagonist. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 20,000 cells/ml to about 75,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 3.4 × 10⁶Individual cell or cellml to about 6 × 10⁶A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population at monocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 30,000 cells/ml to about 60,000 cells/ml ⁺CD90⁺CD45RA^-Cell density and has a density of from about 4 × 10⁶Individual cells/ml to about 5.5 × 10⁶A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population at monocyte density. In some embodiments, the method comprises administering to the donor CD34 sufficient to produce a cell population having from about 40,000 cells/ml to about 50,000 cells/ml⁺CD90⁺CD45RA^-Cell density and has a density of from about 4 × 10⁶Individual cells/ml to about 5 × 10⁶A CXCR2 agonist and a CXCR4 antagonist in an amount per cell per ml of a cell population at monocyte density.

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 a mobilizing amount of a CXCR2 agonist and a CXCR4 antagonist; obtaining an input value for each of one or more parameters characterizing a peripheral blood sample of the donor in table 2, and releasing the sample for ex vivo expansion of hematopoietic stem cells or for treating 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 standard for each of the one or more parameters. In some embodiments, the one or more reference parameters is a set of parameters listed in any of tables 3-6 herein.

In some embodiments of any of the above aspects of the invention, the sample is isolated from the donor about 3 hours to about 5 hours after administration of the CXCR2 agonist and CXCR4 antagonist (e.g., 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 after administration of the CXCR2 agonist and CXCR4 antagonist). In some embodiments, the sample is isolated from the donor about 4 hours after administration of the CXCR2 agonist and the CXCR4 antagonist.

In some embodiments of any of the above aspects of the invention, the CXCR2 agonist is Gro-beta T or a variant thereof. In some embodiments, a CXCR2 agonist can be a peptide having at least about 85% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, a 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, a CXCR2 agonist is a peptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID No. 2 only by one or more conservative amino acid substitutions (e.g., only by 1 to 10 conservative amino acid substitutions, 1 to 5 conservative amino acid substitutions, or 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- β T. In some embodiments, Gro- β T is not covalently modified. In some embodiments, Gro-beta T is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene glycol moiety.

In some embodiments of any of the above aspects of the invention, the CXCR2 agonist is Gro- β or a variant thereof. In some embodiments, a CXCR2 agonist can 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, a 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, a CXCR2 agonist is a peptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID No. 1 only by one or more conservative amino acid substitutions (e.g., only by 1 to 10 conservative amino acid substitutions, 1 to 5 conservative amino acid substitutions, or 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- β. In some embodiments, Gro- β T is not covalently modified. In some embodiments, Gro- β is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene glycol moiety.

In some embodiments, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered to a donor at a dose of from about 50 μ g/kg to about 1mg/kg, such as a dose of: about 50. mu.g/kg, 55. mu.g/kg, 60. mu.g/kg, 65. mu.g/kg, 70. mu.g/kg, 75. mu.g/kg, 80. mu.g/kg, 85. mu.g/kg, 90. mu.g/kg, 95. mu.g/kg, 100. mu.g/kg, 105. mu.g/kg, 110. mu.g/kg, 115. mu.g/kg, 120. mu.g/kg, 125. mu.g/kg, 130. mu.g/kg, 135. mu.g/kg, 140. mu.g/kg, 145. mu.g/kg, 150. mu.g/kg, 155. mu.g/kg, 160. mu.g/kg, 165. mu.g/kg, 170. mu.g/kg, 175. mu.g/kg, 180. mu.g/kg, 185. mu.g/kg, 190. mu.g/kg, 195. mu.g/kg, 200. mu, 210. mu.g/kg, 215. mu.g/kg, 220. mu.g/kg, 225. mu.g/kg, 230. mu.g/kg, 235. mu.g/kg, 240. mu.g/kg, 245. mu.g/kg, 250. mu.g/kg, 255. mu.g/kg, 260. mu.g/kg, 265. mu.g/kg, 270. mu.g/kg, 275. mu.g/kg, 280. mu.g/kg, 285. mu.g/kg, 290. mu.g/kg, 295. mu.g/kg, 300. mu.g/kg, 305. mu.g/kg, 310. mu.g/kg, 315. mu.g/kg, 320. mu.g/kg, 325. mu.g/kg, 330. mu.g/kg, 335. mu.g/kg, 340. mu.g/kg, 345. mu.g/kg, 350. mu.g/kg, 355. mu.g/kg, 360. mu.g/kg, 365, 375. mu.g/kg, 380. mu.g/kg, 400. mu.g/kg, 405. mu.g/kg, 410. mu.g/kg, 415. mu.g/kg, 425. mu.g/kg, 430. mu.g/kg, 435. mu.g/kg, 440. mu.g/kg, 445. mu.g/kg, 450. mu.g/kg, 210. mu.g/kg, 300. mu.g/kg, 400. mu.g/kg, 405. mu.g/kg, 410. mu.g/kg, 415. mu.g/kg, 420. mu.g/kg, 425. mu.g/kg, 430. mu.g/kg, 435. mu.g/kg, 440. mu.g/kg, 445. mu.g/kg, 450. mu.g/kg, 455. mu.g/kg, 460. mu.g/kg, 465. mu.g/kg, 470. mu.g/kg, 475. mu.g/kg, 480. mu.g/kg, 485, 495. mu.g/kg, 500. mu.g/kg, 505. mu.g/kg, 510. mu.g/kg, 505. mu.g/kg, 515. mu.g/kg, 520. mu.g/kg, 525. mu.g/kg, 530. mu.g/kg, 545. mu.g/kg, 550. mu.g/kg, 555. mu.g/kg, 560. mu.g/kg, 565. mu.g/kg, 570. mu.g/kg, 575. mu.g/kg, 580. mu.g/kg, 585. mu.g/kg, 590. mu.g/kg, 595. mu.g/kg, 600. mu.g/kg, 605. mu.g/kg, 610. mu.g/kg, 615. mu.g/kg, 620. mu.g/kg, 625. mu.g/kg, 630. mu.g/kg, 635. mu.g/kg, 640. mu.g/kg, 645. mu.g/kg, 650. mu.g/kg, 655, 665, 670, 675, 680, 685, 730, 735, 695, 700, 705, 710, 715, 720, 725, 730, 735, 745, 750, 755, 760, 765, 770, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 675, 830. mu.g/kg, 835. mu.g/kg, 840. mu.g/kg, 845. mu.g/kg, 850. mu.g/kg, 855. mu.g/kg, 860. mu.g/kg, 865. mu.g/kg, 870. mu.g/kg, 875. mu.g/kg, 880. mu.g/kg, 885. mu.g/kg, 890. mu.g/kg, 895. mu.g/kg, 900. mu.g/kg, 905. mu.g/kg, 910. mu.g/kg, 915. mu.g/kg, 920. mu.g/kg, 925. mu.g/kg, 930. mu.g/kg, 935. mu.g/kg, 940. mu.g/kg, 945. mu.g/kg, 950. mu.g/kg, 955. mu.g/kg, 960. mu.g/kg, 965. mu.g/kg, 970. mu.g/kg, 975. mu.g/kg, 980. mu.g/kg, 990 995. mu.g/kg or 1,000. mu.g/kg. In some embodiments, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered to a donor at a dose of from about 50 μ g/kg to about 300 μ g/kg, such as from about 100 μ g/kg to about 250 μ g/kg or from about 125 μ g/kg to about 225 μ g/kg. In some embodiments, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered to a donor at a dose of about 150 μ g/kg.

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 dose from about 50 μ g/kg to about 1mg/kg (e.g., 50 μ g/kg, 55 μ g/kg, 60 μ g/kg, 65 μ g/kg, 70 μ g/kg, 75 μ g/kg, 80 μ g/kg, 85 μ g/kg, 90 μ g/kg, 95 μ g/kg, 100 μ g/kg, 105 μ g/kg, 110 μ g/kg, 115 μ g/kg, 120 μ g/kg, 125 μ g/kg, 130 μ g/kg, 135 μ g/kg, 140 μ g/kg, 145 μ g/kg, 150 μ g/kg, 155. mu.g/kg, 160. mu.g/kg, 165. mu.g/kg, 170. mu.g/kg, 175. mu.g/kg, 180. mu.g/kg, 185. mu.g/kg, 190. mu.g/kg, 195. mu.g/kg, 200. mu.g/kg, 205. mu.g/kg, 210. mu.g/kg, 215. mu.g/kg, 220. mu.g/kg, 225. mu.g/kg, 230. mu.g/kg, 235. mu.g/kg, 240. mu.g/kg, 245. mu.g/kg, 250. mu.g/kg, 255. mu.g/kg, 260. mu.g/kg, 265. mu.g/kg, 270. mu.g/kg, 275. mu.g/kg, 280. mu.g/kg, 285. mu.g/kg, 290. mu.g/kg, 295. mu.g/kg, 300. mu.g/kg, 305. mu.g/kg, 310, 320. mu.g/kg, 325. mu.g/kg, 330. mu.g/kg, 335. mu.g/kg, 340. mu.g/kg, 345. mu.g/kg, 350. mu.g/kg, 355. mu.g/kg, 360. mu.g/kg, 365. mu.g/kg, 370. mu.g/kg, 375. mu.g/kg, 380. mu.g/kg, 400. mu.g/kg, 405. mu.g/kg, 410. mu.g/kg, 415. mu.g/kg, 425. mu.g/kg, 430. mu.g/kg, 435. mu.g/kg, 440. mu.g/kg, 445. mu.g/kg, 450. mu.g/kg, 210. mu.g/kg, 300. mu.g/kg, 400. mu.g/kg, 405. mu.g/kg, 410. mu.g/kg, 415. mu.g/kg, 420. mu.g/kg, 425. mu.g/kg, 430, 440. mu.g/kg, 445. mu.g/kg, 450. mu.g/kg, 455. mu.g/kg, 460. mu.g/kg, 465. mu.g/kg, 470. mu.g/kg, 475. mu.g/kg, 480. mu.g/kg, 485. mu.g/kg, 490. mu.g/kg, 495. mu.g/kg, 500. mu.g/kg, 505. mu.g/kg, 510. mu.g/kg, 505. mu.g/kg, 515. mu.g/kg, 520. mu.g/kg, 525. mu.g/kg, 530. mu.g/kg, 545. mu.g/kg, 550. mu.g/kg, 555. mu.g/kg, 560. mu.g/kg, 565. mu.g/kg, 570. mu.g/kg, 575. mu.g/kg, 580. mu.g/kg, 585. mu.g/kg, 590. mu.g/kg, 595. mu.g/kg, 600, 610. mu.g/kg, 615. mu.g/kg, 620. mu.g/kg, 625. mu.g/kg, 630. mu.g/kg, 635. mu.g/kg, 640. mu.g/kg, 645. mu.g/kg, 650. mu.g/kg, 655. mu.g/kg, 660. mu.g/kg, 665. mu.g/kg, 670. mu.g/kg, 675. mu.g/kg, 680. mu.g/kg, 685. mu.g/kg, 690. mu.g/kg, 695. mu.g/kg, 700. mu.g/kg, 705. mu.g/kg, 710. mu.g/kg, 715. mu.g/kg, 720. mu.g/kg, 725. mu.g/kg, 730. mu.g/kg, 735. mu.g/kg, 740. mu.g/kg, 745. mu.g/kg, 750. mu.g/kg, 755. mu.g/kg, 760. mu.g/kg, 765, 775 μ g/kg, 780 μ g/kg, 785 μ g/kg, 790 μ g/kg, 795 μ g/kg, 800 μ g/kg, 805 μ g/kg, 810 μ g/kg, 815 μ g/kg, 820 μ g/kg, 825 μ g/kg, 830 μ g/kg, 835 μ g/kg, 840 μ g/kg, 845 μ g/kg, 850 μ g/kg, 855 μ g/kg, 860 μ g/kg, 865 μ g/kg, 870 μ g/kg, 875 μ g/kg, 880 μ g/kg, 885 μ g/kg, 890 μ g/kg, 895 μ g/kg, 900 μ g/kg, 905 μ g/kg, 910 μ g/kg, 915 μ g/kg, 920 μ g/kg, 925 μ g/kg, 930 μ g/kg, 935 μ g/kg, 940 μ g/kg, 945 μ g/kg, 950 μ g/kg, 955 μ g/kg, 960 μ g/kg, 965 μ g/kg, 970 μ g/kg, 975 μ g/kg, 980 μ g/kg, 985 μ g/kg, 990 μ g/kg, 995 μ g/kg or 1,000 μ g/kg) of a CXCR2 agonist selected from the group consisting of Gro- β, Gro- β T and variants thereof. In some embodiments, the method further comprises administering to the donor a CXCR4 antagonist.

In some embodiments of any of the above aspects of the invention, the CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered at a dose of from about 50 μ g/kg to about 300 μ g/kg, such as about 50 μ g/kg, 55 μ g/kg, 60 μ g/kg, 65 μ g/kg, 70 μ g/kg, 75 μ g/kg, 80 μ g/kg, 85 μ g/kg, 90 μ g/kg, 95 μ g/kg, 100 μ g/kg, 105 μ g/kg, 110 μ g/kg, 115 μ g/kg, 120 μ g/kg, 125 μ g/kg, 130 μ g/kg, 135 μ g/kg, 140 μ g/kg, 145 μ g/kg, 150 μ g/kg, or, 155, 160, 165, 170, 175, 225, 230, 235, 240, 245, 255, 260, 265, 270, 275, 280, 285, 290, 295 or 300 μ g/kg are administered to the donor.

In some embodiments of any of the above aspects of the invention, the CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered at a dose of from about 100 μ g/kg to about 250 μ g/kg, such as about 100 μ g/kg, 105 μ g/kg, 110 μ g/kg, 115 μ g/kg, 120 μ g/kg, 125 μ g/kg, 130 μ g/kg, 135 μ g/kg, 140 μ g/kg, 145 μ g/kg, 150 μ g/kg, 155 μ g/kg, 160 μ g/kg, 165 μ g/kg, 170 μ g/kg, 175 μ g/kg, 180 μ g/kg, 185 μ g/kg, 190 μ g/kg, 195 μ g/kg, 200 μ g/kg, or, 205, 210, 215, 220, 225, 230, 235, 240, 245 or 250 μ g/kg of the drug is administered to the donor.

In some embodiments of any of the above aspects of the invention, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered to the donor at a dose of about 150 μ g/kg. For example, in some embodiments, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered at a dosage of from about 50 μ g/kg/day to about 1 mg/kg/day, such as about 50 μ g/kg/day, 55 μ g/kg/day, 60 μ g/kg/day, 65 μ g/kg/day, 70 μ g/kg/day, 75 μ g/kg/day, 80 μ g/kg/day, 85 μ g/kg/day, 90 μ g/kg/day, 95 μ g/kg/day, 100 μ g/kg/day, 105 μ g/kg/day, 110 μ g/kg/day, 115 μ g/kg/day, 120 μ g/kg/day, or a pharmaceutically acceptable salt thereof, 125. mu.g/kg/day, 130. mu.g/kg/day, 135. mu.g/kg/day, 140. mu.g/kg/day, 145. mu.g/kg/day, 150. mu.g/kg/day, 155. mu.g/kg/day, 160. mu.g/kg/day, 165. mu.g/kg/day, 170. mu.g/kg/day, 175. mu.g/kg/day, 180. mu.g/kg/day, 185. mu.g/kg/day, 190. mu.g/kg/day, 195. mu.g/kg/day, 200. mu.g/kg/day, 205. mu.g/kg/day, 210. mu.g/kg/day, 215. mu.g/kg/day, 220. mu.g/kg/day, 225. mu.g/kg/day, 230. mu.g/kg/day, 235. mu.g/kg/day, 240. mu.g/kg/day, 245. mu.g/kg/day, 250. mu.g/kg/day, 255. mu.g/kg/day, 260. mu.g/kg/day, 265. mu.g/kg/day, 270. mu.g/kg/day, 275. mu.g/kg/day, 280. mu.g/kg/day, 285. mu.g/kg/day, 290. mu.g/kg/day, 295. mu.g/kg/day, 300. mu.g/kg/day, 305. mu.g/kg/day, 310. mu.g/kg/day, 315. mu.g/kg/day, 320. mu.g/kg/day, 325. mu.g/kg/day, 330. mu.g/kg/day, 335. mu.g/kg/day, 340. mu.g/kg, 345. mu.g/kg/day, 350. mu.g/kg/day, 355. mu.g/kg/day, 360. mu.g/kg/day, 365. mu.g/kg/day, 370. mu.g/kg/day, 375. mu.g/kg/day, 380. mu.g/kg/day, 400. mu.g/kg/day, 405. mu.g/kg/day, 410. mu.g/kg/day, 415. mu.g/kg/day, 425. mu.g/kg/day, 430. mu.g/kg/day, 435. mu.g/kg/day, 440. mu.g/kg/day, 445. mu.g/kg/day, 450. mu.g/kg/day, 210. mu.g/kg/day, 300. mu.g/kg/day, 400. mu.g/kg/day, 405. mu.g/kg, 410. mu.g/kg/day, 415. mu.g/kg/day, 420. mu.g/kg/day, 425. mu.g/kg/day, 430. mu.g/kg/day, 435. mu.g/kg/day, 440. mu.g/kg/day, 445. mu.g/kg/day, 450. mu.g/kg/day, 455. mu.g/kg/day, 460. mu.g/kg/day, 465. mu.g/kg/day, 470. mu.g/kg/day, 475. mu.g/kg/day, 480. mu.g/kg/day, 485. mu.g/kg/day, 490. mu.g/kg/day, 495. mu.g/kg/day, 500. mu.g/kg/day, 505. mu.g/kg/day, 510. mu.g/kg/day, 505. mu.g/day, 515. mu.g/kg/day, 520. mu.g/kg/day, 525. mu.g/kg/day, 530. mu.g/kg/day, 545. mu.g/kg/day, 550. mu.g/kg/day, 555. mu.g/kg/day, 560. mu.g/kg/day, 565. mu.g/kg/day, 570. mu.g/kg/day, 575. mu.g/kg/day, 580. mu.g/kg/day, 585. mu.g/kg/day, 590. mu.g/kg/day, 595. mu.g/kg/day, 600. mu.g/kg/day, 605. mu.g/kg/day, 610. mu.g/kg/day, 615. mu.g/kg/day, 620. mu.g/kg/day, 625. mu.g/kg/day, 630. mu.g/kg/day, beta.g/kg/day, 635. mu.g/kg/day, 640. mu.g/kg/day, 645. mu.g/kg/day, 650. mu.g/kg/day, 655. mu.g/kg/day, 660. mu.g/kg/day, 665. mu.g/kg/day, 670. mu.g/kg/day, 675. mu.g/kg/day, 680. mu.g/kg/day, 685. mu.g/kg/day, 690. mu.g/kg/day, 695. mu.g/kg/day, 700. mu.g/kg/day, 705. mu.g/kg/day, 710. mu.g/kg/day, 715. mu.g/kg/day, 720. mu.g/kg/day, 725. mu.g/kg/day, 730. mu.g/kg/day, 735. mu.g/kg/day, 740. mu.g/day, 745. mu.g/kg/day, 750. mu.g/kg/day, 755. mu.g/kg/day, 760. mu.g/kg/day, 765. mu.g/kg/day, 770. mu.g/kg/day, 775. mu.g/kg/day, 780. mu.g/kg/day, 785. mu.g/kg/day, 790. mu.g/kg/day, 795. mu.g/kg/day, 800. mu.g/kg/day, 805. mu.g/kg/day, 810. mu.g/kg/day, 815. mu.g/kg/day, 820. mu.g/kg/day, 825. mu.g/kg/day, 830. mu.g/kg/day, 835. mu.g/kg/day, 840. mu.g/kg/day, 845. mu.g/kg/day, 850. mu.g/day, 855 μ g/kg/day, 860 μ g/kg/day, 865 μ g/kg/day, 870 μ g/kg/day, 875 μ g/kg/day, 880 μ g/kg/day, 885 μ g/kg/day, 890 μ g/kg/day, 895 μ g/kg/day, 900 μ g/kg/day, 905 μ g/kg/day, 910 μ g/kg/day, 915 μ g/kg/day, 920 μ g/kg/day, 925 μ g/kg/day, 930 μ g/kg/day, 935 μ g/kg/day, 940 μ g/kg/day, 945 μ g/kg/day, 950 μ g/kg/day, 955 μ g/kg/day, 960 μ g/kg/day, A dose of 965. mu.g/kg/day, 970. mu.g/kg/day, 975. mu.g/kg/day, 980. mu.g/kg/day, 985. mu.g/kg/day, 990. mu.g/kg/day, 995. mu.g/kg/day, or 1,000. mu.g/kg/day is administered to the donor. In some embodiments, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered to a donor at a dose of from about 50 μ g/kg/day to about 300 μ g/kg/day, such as from about 100 μ g/kg/day to about 250 μ g/kg/day or from about 125 μ g/kg/day to about 225 μ g/kg/day or from about 125 μ g/kg/day to about 175 μ g/kg/day. In some embodiments, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered to a donor at a dose of about 150 μ g/kg/day. In some embodiments, a CXCR2 agonist can be administered as a single dose. In other embodiments, the CXCR2 agonist can be administered as two or more doses.

In some embodiments, a Human Equivalent Dose (HED) can be derived from animal dose data using a conversion factor. For example, Nair and Jacob, J.basic Clin.Pharma. (2016)7:27-31 disclose methods for inter-species dose extrapolation. For example, in one non-limiting example, HED can be derived from a rhesus monkey (rhesus monkey) dose by multiplying the rhesus monkey dose by about 0.324.

In some embodiments of any of the above aspects of the invention, a CXCR2 agonist (e.g., Gro- β or Gro- β T, such as unmodified Gro- β or Gro- β T) is administered intravenously to the donor.

In some embodiments of any of the above aspects of the invention, the CXCR4 antagonist is a compound represented by formula (I)

Z-joint-Z' (I)

Or a pharmaceutically acceptable salt thereof, wherein Z is:

(i) cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 ring members are nitrogen atoms separated from each other by 2 or more carbon atoms; or

(ii) An amine represented by the formula (IA)

Wherein a comprises a monocyclic or bicyclic fused ring system containing at least one nitrogen atom, and B is H or a substituent having 1 to 20 atoms;

and wherein Z' is:

(i) cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 ring members are nitrogen atoms separated from each other by 2 or more carbon atoms;

(ii) An amine represented by the formula (IB)

Wherein a 'comprises a monocyclic or bicyclic fused ring system containing at least one nitrogen atom, and B' is H or a substituent having 1 to 20 atoms; or

(iii) A substituent represented by the formula (IC)

–N(R)–(CR₂)_n–X (IC)

Wherein each R is independently H or C₁-C₆Alkyl, n is 1 or 2, and X is an aryl group or a heteroaryl group or a thiol;

wherein the linker is a bond, optionally substituted C₁-C₆Alkylene (alkylene), optionally substituted C₁-C₆Heteroalkylene, optionally substituted C₂-C₆Alkenylene (alkenny)lens), optionally substituted C₂-C₆Heteroalkenylene, optionally substituted C₂-C₆Alkynylene (alkynylene), optionally substituted C₂-C₆Heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene or optionally substituted heteroarylene.

In some embodiments, Z and Z' are each independently cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 ring members are nitrogen atoms separated from each other by 2 or more carbon atoms. Z and Z' may be the same substituent. In some embodiments, Z and/or Z' is a cyclic polyamine containing 10 to 24 ring members, such as a cyclic polyamine containing 14 ring members. In some embodiments, Z includes 4 nitrogen atoms. Z and/or Z' may be, for example, 1,4,8, 11-tetraazacyclotetradecane.

In some embodiments, the linker is represented by formula (ID)

In some embodiments, the linker is represented by formula (IE)

In some embodiments, X and Y are each independently optionally substituted C₁-C₆An alkylene group. In some embodiments, X and Y are the same substituents, such as the same alkylene substituents (e.g., methylene substituents, ethylene substituents, propylene substituents, or butylene substituents).

In some embodiments, the CXCR4 antagonist is plerixafor or a pharmaceutically acceptable salt thereof. In some embodiments, a CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered subcutaneously to a donor. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is present at a dose of from about 50 μ g/kg to about 500 μ g/kg, such as about 50 μ g/kg, 55 μ g/kg, 60 μ g/kg, 65 μ g/kg, 70 μ g/kg, 75 μ g/kg, 80 μ g/kg, 85 μ g/kg, 90 μ g/kg, 95 μ g/kg, 100 μ g/kg, 105 μ g/kg, 110 μ g/kg, 115 μ g/kg, 120 μ g/kg, 125 μ g/kg, 130 μ g/kg, 135 μ g/kg, 140 μ g/kg, 145 μ g/kg, 150 μ g/kg, 155 μ g/kg, 160 μ g/kg, 165 μ g/kg, 170 μ g/kg, or a pharmaceutically acceptable salt thereof, 175. mu.g/kg, 180. mu.g/kg, 185. mu.g/kg, 190. mu.g/kg, 195. mu.g/kg, 200. mu.g/kg, 205. mu.g/kg, 210. mu.g/kg, 215. mu.g/kg, 220. mu.g/kg, 225. mu.g/kg, 230. mu.g/kg, 235. mu.g/kg, 240. mu.g/kg, 245. mu.g/kg, 250. mu.g/kg, 255. mu.g/kg, 260. mu.g/kg, 265. mu.g/kg, 270. mu.g/kg, 275. mu.g/kg, 280. mu.g/kg, 285. mu.g/kg, 290. mu.g/kg, 295. mu.g/kg, 300. mu.g/kg, 305. mu.g/kg, 310. mu.g/kg, 315. mu.g/kg, 320. mu.g/kg, 325. mu.g/kg, 330, 340. mu.g/kg, 345. mu.g/kg, 350. mu.g/kg, 355. mu.g/kg, 360. mu.g/kg, 365. mu.g/kg, 370. mu.g/kg, 375. mu.g/kg, 380. mu.g/kg, 385. mu.g/kg, 390. mu.g/kg, 395. mu.g/kg, 400. mu.g/kg, 405. mu.g/kg, 410. mu.g/kg, 415. mu.g/kg, doses of 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495 or 500 μ g/kg are administered to the donor. In some embodiments, a CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to a donor at a dose of from about 200 μ g/kg to about 300 μ g/kg, such as a dose of about 240 μ g/kg.

For example, in some embodiments, a CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is at a dose of from about 50 μ g/kg/day to about 500 μ g/kg/day, such as about 50 μ g/kg/day, 55 μ g/kg/day, 60 μ g/kg/day, 65 μ g/kg/day, 70 μ g/kg/day, 75 μ g/kg/day, 80 μ g/kg/day, 85 μ g/kg/day, 90 μ g/kg/day, 95 μ g/kg/day, 100 μ g/kg/day, 105 μ g/kg/day, 110 μ g/kg/day, 115 μ g/kg/day, 120 μ g/kg/day, 125 μ g/kg/day, 100 μ g/kg/day, or, 130. mu.g/kg/day, 135. mu.g/kg/day, 140. mu.g/kg/day, 145. mu.g/kg/day, 150. mu.g/kg/day, 155. mu.g/kg/day, 160. mu.g/kg/day, 165. mu.g/kg/day, 170. mu.g/kg/day, 175. mu.g/kg/day, 180. mu.g/kg/day, 185. mu.g/kg/day, 190. mu.g/kg/day, 195. mu.g/kg/day, 200. mu.g/kg/day, 205. mu.g/kg/day, 210. mu.g/kg/day, 215. mu.g/kg/day, 220. mu.g/kg/day, 225. mu.g/kg/day, 230. mu.g/kg/day, 235. mu.g/kg/day, 240. mu.g/kg/day, 245. mu.g/kg/day, 250. mu.g/kg/day, 255. mu.g/kg/day, 260. mu.g/kg/day, 265. mu.g/kg/day, 270. mu.g/kg/day, 275. mu.g/kg/day, 280. mu.g/kg/day, 285. mu.g/kg/day, 290. mu.g/kg/day, 295. mu.g/kg/day, 300. mu.g/kg/day, 305. mu.g/kg/day, 310. mu.g/kg/day, 315. mu.g/kg/day, 320. mu.g/kg/day, 325. mu.g/kg/day, 330. mu.g/kg/day, 335. mu.g/kg/day, 340. mu.g/kg/day, 345. mu.g/day, 350. mu.g/kg/day, 355. mu.g/kg/day, 360. mu.g/kg/day, 365. mu.g/kg/day, 370. mu.g/kg/day, 375. mu.g/kg/day, 380. mu.g/kg/day, 385. mu.g/kg/day, 390. mu.g/kg/day, 395. mu.g/kg/day, 400. mu.g/kg/day, 405. mu.g/kg/day, 410. mu.g/kg/day, 415. mu.g/kg/day, 420. mu.g/kg/day, 425. mu.g/kg/day, 430. mu.g/kg/day, 435. mu.g/kg/day, 440. mu.g/kg/day, 445. mu.g/kg/day, 450. mu.g/kg/day, 455. mu.g/day, Doses of 460. mu.g/kg/day, 465. mu.g/kg/day, 470. mu.g/kg/day, 475. mu.g/kg/day, 480. mu.g/kg/day, 485. mu.g/kg/day, 490. mu.g/kg/day, 495. mu.g/kg/day or 500. mu.g/kg/day were administered to the donor. In some embodiments, a CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to a donor at a dose of from about 200 μ g/kg/day to about 300 μ g/kg/day, such as a dose of about 240 μ g/kg/day. In some embodiments, the CXCR4 antagonist can be administered as a single dose. In other embodiments, the CXCR4 antagonist can be administered as two or more doses.

In some embodiments of any of the above aspects of the invention, the CXCR2 agonist and the CXCR4 antagonist are administered concurrently to the donor. In some embodiments, the CXCR4 antagonist is administered to the donor prior to administration of the CXCR2 agonist. In some embodiments, the CXCR4 antagonist can be about 1 minute to about 180 minutes, such as 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 prior to the 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, 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 30 minutes, about 60 minutes, or about 80 minutes prior to the administration of the CXCR2 agonist, About 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about 170 minutes, about 175 minutes, or about 180 minutes) is administered to the donor. In some embodiments, the CXCR4 antagonist is administered to the donor 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, a CXCR4 antagonist can be administered to a donor about 45 minutes prior to the administration of a CXCR2 agonist.

In another aspect, the invention features a pharmaceutical composition that includes a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g., a human donor), wherein CD34 is in the population⁺The ratio of cells to lymphocytes is from about 0.0021 to about 0.0094. In some embodiments, CD34⁺The ratio of cells to lymphocytes may be about 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219, 0.00220, 0.00221, 0.00222, 0.00223, 0.00224, 0.00225, 0.00226, 0.00227, 0.00228, 0.00229, 0.00230, 0.00231, 0.00232, 0.00233, 0.00234、0.00235、0.00236、0.00237、0.00238、0.00239、0.00240、0.00241、0.00242、0.00243、0.00244、0.00245、0.00246、0.00247、0.00248、0.00249、0.00250、0.00251、0.00252、0.00253、0.00254、0.00255、0.00256、0.00257、0.00258、0.00259、0.00260、0.00261、0.00262、0.00263、0.00264、0.00265、0.00266、0.00267、0.00268、0.00269、0.00270、0.00271、0.00272、0.00273、0.00274、0.00275、0.00276、0.00277、0.00278、0.00279、0.00280、0.00281、0.00282、0.00283、0.00284、0.00285、0.00286、0.00287、0.00288、0.00289、0.00290、0.00291、0.00292、0.00293、0.00294、0.00295、0.00296、0.00297、0.00298、0.00299、0.00300、0.00300、0.00301、0.00302、0.00303、0.00304、0.00305、0.00306、0.00307、0.00308、0.00309、0.00310、0.00311、0.00312、0.00313、0.00314、0.00315、0.00316、0.00317、0.00318、0.00319、0.00320、0.00321、0.00322、0.00323、0.00324、0.00325、0.00326、0.00327、0.00328、0.00329、0.00330、0.00331、0.00332、0.00333、0.00334、0.00335、0.00336、0.00337、0.00338、0.00339、0.00340、0.00341、0.00342、0.00343、0.00344、0.00345、0.00346、0.00347、0.00348、0.00349、0.00350、0.00351、0.00352、0.00353、0.00354、0.00355、0.00356、0.00357、0.00358、0.00359、0.00360、0.00361、0.00362、0.00363、0.00364、0.00365、0.00366、0.00367、0.00368、0.00369、0.00370、0.00371、0.00372、0.00373、0.00374、0.00375、0.00376、0.00377、0.00378、0.00379、0.00380、0.00381、0.00382、0.00383、0.00384、0.00385、0.00386、0.00387、0.00388、0.00389、0.00390、0.00391、0.00392、0.00393、0.00394、0.00395、0.00396、0.00397、0.00398、0.00399、0.00400、0.00401、0.00402、0.00403、0.00404、0.00405、0.00406、0.00407、0.00408、0.00409、0.00410、0.00411、0.00412、0.00413、0.00414、0.00415、0.00416、0.00417、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、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, 0.00871, 0.00861, 0.00875, 0.00861, 36. In some embodiments, CD34 ⁺The ratio of cells to lymphocytes is from about 0.0025 to about 0.0035, e.g., CD34⁺The ratio of cells to lymphocytes is 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, 0.00287, 0.00281, 0.00291, 0.00281, 0.00293, 0.00281, 3600372, 0.00281, 36 0.00345, 0.00346, 0.00347, 0.00348, 0.00349 or 0.00350. In some embodiments, CD34⁺The ratio of cells to lymphocytes was about 0.0031.

In a further aspect, the invention features a pharmaceutical composition comprising a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g., a human donor), wherein CD34 is in the population⁺CD90⁺CD45RA^-The ratio of cells to monocytes is from about 0.0028 to about 0.0130. In some embodiments, CD34⁺CD90⁺CD45RA^-The ratio of the cells to the mononuclear cells may be, for example, about, by (i) each of (i) to (ii) to (iii) to (iv) to (iv), 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、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、The composition comprises a plurality of components, such as a metal, a, 0.00953, 0.00954, 0.00955, 0.00956, 0.00957, 0.00958, 0.00959, 0.00960, 0.00984, 0.00960, 3600987, 0.00960, 0.00992, 0.00960, 0.0100, 0.0101, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.00960, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0120.0120.0125, 0.0120.0120.0120, 0120.0120, 0125, 0.0120, 0.0128, 0.0120, 0, 0.0120, 0, 0.1. In some embodiments In this case, CD34⁺CD90⁺CD45RA^-The ratio of cells to monocytes is from about 0.0063 to about 0.0083, e.g., CD34⁺CD90⁺CD45RA^-The ratio of the cells to the mononuclear cells is about, in (b), (c), (d), (c), (d), (, 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, 36808, 0.00804, 0.00814, 0.00804, 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 0.00830. In some embodiments, CD34⁺CD90⁺CD45RA^-The ratio of cells to monocytes was about 0.0073.

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 comprising mobilizing a population of hematopoietic stem cells in a mammalian donor (e.g., a human donor) according to any of the above methods, and infusing into the patient a therapeutically effective amount of the hematopoietic stem cells or progeny thereof.

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 comprising infusing into the patient a therapeutically effective amount of hematopoietic stem cells mobilized by any of the above methods, or progeny thereof.

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 pharmaceutical compositions.

In some embodiments of any of the preceding three 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 an innate immunodeficiency or an acquired immunodeficiency, e.g., a human immunodeficiency virus or an acquired immunodeficiency syndrome. In some embodiments, the stem cell disorder is a metabolic disorder, such as a metabolic disorder selected from: glycogen storage Disease, mucopolysaccharidosis, Gaucher's Disease, hurler Disease, sphingolipid storage Disease and metachromatic leukodystrophy.

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 hematologic 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.

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 lymphocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

In some embodiments, the stem cell disorder is an autoimmune disorder, such as an autoimmune disorder selected from the group consisting of: multiple sclerosis, human systemic lupus, rheumatoid arthritis, inflammatory bowel disease, treatment of psoriasis, type 1 diabetes, 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, Barlo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas ' disease, chronic fatigue immunity -dysfunctional disease syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, malignant atrophic papulopathy (Degos disease), discoid lupus, autonomic dysfunction, endometriosis, primary mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome (Guillain-Barre syndrome), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, cystitis, juvenile arthritis, Kawasaki's disease, Lichen planus, Lyme disease, Meniere disease, mixed connective tissue disease, myasthenia gravis, neuromuscular rigidity, strabismus myoclonus syndrome, optic neuritis, alder's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, glandular syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, sjogren's syndrome, mixed connective tissue disease, myasthenia gravis, nervousness Stiff person syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, and Wegener's granulomatosis.

In some embodiments, the hematopoietic stem cells are autologous to the patient. In some embodiments, the hematopoietic stem cells are allogeneic to the patient, and may be HLA-matched, for example, to the patient.

In some embodiments, hematopoietic stem cells have been genetically modified to disrupt an endogenous gene, such as a gene encoding a major histocompatibility complex protein. Hematopoietic stem cells can be genetically modified to disrupt an endogenous gene by, for example, a CRISPR-associated protein (such as caspase 9) or another nuclease described herein (such as a transcription activator-like effector nuclease, meganuclease, or zinc finger nuclease).

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 are localized to (lineage to) hematopoietic tissue and/or reconstituted hematopoiesis upon infusion of the hematopoietic stem cells or progeny into a patient. In some embodiments, upon infusion into a patient, hematopoietic stem cells or progeny thereof cause the restoration of a population of cells selected from the group consisting of: megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells.

In another aspect, the present disclosure is directed to a method of converting CD34^dima method of mobilizing 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- β, Gro- β T, and variants thereof, and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

In another aspect, the present disclosure relates to a method of allogeneic hematopoietic stem cell transplantation 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 are mobilized from the bone marrow of the human donor into the 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- β, Gro- β T, and variants thereof and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

In another aspect, the present 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, comprising infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from bone marrow of the human donor into peripheral blood of the human donor by a method comprising: administering to a human donor (i) a CXCR2 agonist selected from the group consisting of Gro- β, Gro- β T, and variants thereof and (ii) a CXCR4 antagonist at a dose from about 50 μ g/kg to about 1,000 μ g/kg.

In another aspect, the present disclosure relates to a method of preventing, reducing the risk of, or reducing the severity of Graft Versus Host Disease (GVHD) in a patient in need thereof, comprising infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from the bone marrow of the human donor into the peripheral blood of the human donor by a method comprising: administering to a human donor (i) a CXCR2 agonist selected from the group consisting of Gro- β, Gro- β T, and variants thereof and (ii) a CXCR4 antagonist at a dose from about 50 μ g/kg to about 1,000 μ g/kg.

In certain embodiments, CD34^dimCells are present in peripheral blood in higher amounts than if CXCR4 antagonist alone was used to mobilize hematopoietic stem cells. In certain embodiments, CD34^dimThe cells are capable of inhibiting the proliferation of alloreactive T lymphocytes when administered to a recipient.

In certain embodiments, the CXCR2 agonist is Gro- β T. In certain embodiments, a CXCR2 agonist is administered to a donor at a dose of from about 100 μ g/kg to about 250 μ g/kg. In certain embodiments, a CXCR2 agonist is administered to a donor at a dose of from about 125 μ g/kg to about 225 μ g/kg. In certain embodiments, a CXCR2 agonist is administered to a donor at a dose of about 150 μ g/kg. In certain embodiments, a CXCR2 agonist is administered intravenously to a donor.

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, plerixafor or a pharmaceutically acceptable salt thereof is administered to the donor at a dose of from about 50 μ g/kg to about 500 μ g/kg. In certain embodiments, plerixafor or a pharmaceutically acceptable salt thereof is administered to the donor at a dose of from about 200 μ g/kg to about 300 μ g/kg. In certain embodiments, plerixafor or a pharmaceutically acceptable salt thereof is administered to the donor at a dose of about 240 μ g/kg.

In certain embodiments, the method further comprises testing the peripheral blood sample for CD34^dimThe presence of cells and release of the sample for CD34^dimEx vivo expansion of cells.

In certain embodiments, the present disclosure relates to CD34 derived from any one of the above methods^dimA population of cells, or comprising said CD34^dimA composition of a population of cells.

Brief Description of Drawings

FIG. 1A provides a graph showing that when Gro- β T is co-administered with plerixafor (AMD3100) in mice, mobilization outcomes are synergistically increased and grafts are enriched with highly implantable long-term hematopoietic stem cells (LT-HSC ═ Lin-c-kit) ⁺Sca-1⁺CD150⁺CD48⁺) FIG. 1B provides a graph showing that transplants containing Gro- β T and plerixafor mobilized cells resulted in a higher relative number of Competitive Refill Units (CRUs) at week 16 than transplants containing G-CSF mobilized cells alone.

Figure 2A is a graph showing the pharmacokinetic profile of different doses of Gro-beta T when administered intravenously to rhesus monkeys. Figure 2B is a graph showing the pharmacokinetic profile of different doses of Gro-beta T when administered subcutaneously to rhesus monkeys. In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Fig. 3A shows a series of graphs showing the mobilization response of white blood cells ("WBCs") to different doses of Gro-beta T following intravenous administration to rhesus monkeys. The leukocyte response is shown as both the number of cells that are mobilized (upper panel) and the fold change in leukocyte density relative to baseline leukocyte density before administration (lower panel). Fig. 3B shows a series of graphs showing the mobilization response of white blood cells ("WBCs") to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. The leukocyte response is shown as both the number of cells that are mobilized (upper panel) and the fold change in leukocyte density relative to baseline leukocyte density before administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Fig. 4A shows a series of graphs showing the mobilization response of neutrophils to different doses of Gro-beta T following intravenous administration to rhesus monkeys. Neutrophil response is shown as both the number of cells mobilized (upper panel) and fold change in neutrophil density from baseline prior to administration (lower panel). Fig. 4B shows a series of graphs showing the mobilization response of neutrophils to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. Neutrophil response is shown as both the number of cells mobilized (upper panel) and fold change in neutrophil density from baseline prior to administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Figure 5A shows a series of graphs showing the mobilization response of lymphocytes to different doses of Gro-beta T following intravenous administration to rhesus monkeys. Lymphocyte responses are shown as both the number of cells that are mobilized (upper panel) and fold change in lymphocyte density relative to baseline lymphocyte density prior to administration (lower panel). Figure 5B shows a series of graphs showing the mobilization response of lymphocytes to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. Lymphocyte responses are shown as both the number of cells that are mobilized (upper panel) and fold change in lymphocyte density relative to baseline lymphocyte density prior to administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Figure 6A shows a series of graphs showing the mobilization response of monocytes to different doses of Gro-beta T following intravenous administration to rhesus monkeys. Monocyte response is shown as both the number of cells that were mobilized (upper panel) and the fold change in monocyte density relative to baseline monocyte density before administration (lower panel). Figure 6B shows a series of graphs showing the mobilization response of monocytes to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. Monocyte response is shown as both the number of cells that were mobilized (upper panel) and the fold change in monocyte density relative to baseline monocyte density before administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

FIG. 7A shows CD34 following intravenous administration to rhesus monkeys⁺series of graphs of the mobilization response of cells to different doses of Gro- β T CD34⁺Cellular responses are shown as CD34 in samples obtained from peripheral blood of subjects⁺Frequency of cells (upper panel) and CD34⁺Cellular frequency versus pre-administration baseline CD34⁺Fold change in cell frequency (lower panel). FIG. 7B shows CD34 after subcutaneous administration to rhesus monkeys ⁺series of graphs of the mobilization response of cells to different doses of Gro- β T CD34⁺Cellular responses are shown as CD34 in samples obtained from peripheral blood of subjects⁺Frequency of cells (upper panel) and CD34⁺Cellular frequency versus pre-administration baseline CD34⁺in all experiments Gro- β T was administered to subjects in parallel with plerixafor.

FIG. 8A shows CD34 following intravenous administration to rhesus monkeys⁺series of graphs of the mobilization response of cells to different doses of Gro- β T CD34⁺Cellular responses are shown as the number of cells mobilized (upper panel) and CD34⁺Cell density relative to pre-administration baseline CD34⁺Fold change in cell density (lower panel). FIG. 8B shows CD34 after subcutaneous administration to rhesus monkeys⁺series of graphs of the mobilization response of cells to different doses of Gro- β T CD34⁺Cellular responses are shown as the number of cells mobilized (upper panel) and CD34⁺Cell density relative to pre-administration baseline CD34⁺in all experiments Gro- β T was administered in parallel with plerixafor A subject.

FIG. 9A shows a graph showing hematopoietic stem cells (CD 34) following intravenous administration to rhesus monkeys⁺CD90⁺CD45RA^-cells) a series of charts of mobilization response to different doses of Gro- β T CD34⁺CD90⁺CD45RA^-Cellular responses are shown as CD34 in samples obtained from peripheral blood of subjects⁺CD90⁺CD45RA^-Frequency of cells (upper panel) and CD34⁺CD90⁺CD45RA^-Cellular frequency versus pre-administration baseline CD34⁺CD90⁺CD45RA^-Fold change in cell frequency (lower panel). FIG. 9B shows a graph showing hematopoietic stem cells (CD 34) following subcutaneous administration to rhesus monkeys (see FIGS.)⁺CD90⁺CD45RA^-cells) a series of charts of mobilization response to different doses of Gro- β T CD34⁺CD90⁺CD45RA^-Cellular responses are shown as CD34 in samples obtained from peripheral blood of subjects⁺CD90⁺CD45RA^-Frequency of cells (upper panel) and CD34⁺CD90⁺CD45RA^-Cellular frequency versus pre-administration baseline CD34⁺CD90⁺CD45RA^-in all experiments Gro- β T was administered to subjects in parallel with plerixafor.

FIG. 10A shows a graph showing hematopoietic stem cells (CD 34) following intravenous administration to rhesus monkeys⁺CD90⁺CD45RA^-cells) a series of charts of mobilization response to different doses of Gro- β T CD34 ⁺CD90⁺CD45RA^-Cellular responses are shown as the number of cells mobilized (upper panel) and CD34⁺CD90⁺CD45RA^-Cell density relative to pre-administration baseline CD34⁺CD90⁺CD45RA^-Fold change in cell density (lower panel). FIG. 10B shows a graph showing hematopoietic stem cells (CD 34) following subcutaneous administration to rhesus monkeys (see FIGS.)⁺CD90⁺CD45RA^-cells) a series of charts of mobilization response to different doses of Gro- β T CD34⁺Cellular responses are shown as the number of cells mobilized (upper panel) and CD34⁺CD90⁺CD45RA^-Cell density relative to pre-administration baseline CD34⁺CD90⁺CD45RA^-Gro- β T was administered to subjects in parallel with plerixafor in all experiments.

Figure 11 shows a series of graphs showing the increase in the amount of Colony Forming Units (CFU) of hematopoietic stem cells achieved by intravenous administration of different doses of Gro-beta T to rhesus monkeys. CFU response is shown as both concentration of CFU (upper panel) and fold change in CFU concentration relative to baseline CFU concentration prior to administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Figure 12A shows a series of graphs showing the response of plasma matrix metalloproteinase 9(MMP9) to different doses of Gro-beta T following intravenous administration to rhesus monkeys. Plasma MMP9 response was shown as both absolute concentration (upper panel) and fold change in plasma MMP9 concentration relative to baseline MMP9 concentration prior to administration (lower panel). Figure 12B shows a series of graphs showing the response of plasma MMP9 to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. Plasma MMP9 response was shown as both absolute concentration (upper panel) and fold change in plasma MMP9 concentration relative to baseline MMP9 concentration prior to administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Figure 13A shows a series of graphs showing the response of tissue inhibitor of plasma matrix metalloproteinase 1 (TIMP-1) to different doses of Gro-beta T following intravenous administration to rhesus monkeys. Plasma TIMP-1 response is shown as both an absolute concentration (upper panel) and a fold change in plasma TIMP-1 concentration relative to baseline TIMP-1 concentration prior to administration (lower panel). Figure 13B shows a series of graphs showing the response of plasma TIMP-1 to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. Plasma TIMP-1 response is shown as both an absolute concentration (upper panel) and a fold change in plasma TIMP-1 concentration relative to baseline TIMP-1 concentration prior to administration (lower panel). In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

Figure 14A is a graph showing the molar ratio of plasma MMP9 to plasma TIMP-1 versus response to different doses of Gro-beta T following intravenous administration to rhesus monkeys. Figure 14B is a graph showing the molar ratio of plasma MMP9 to plasma TIMP-1 versus response to different doses of Gro-beta T following subcutaneous administration to rhesus monkeys. In all experiments, Gro- β T was administered to subjects concurrently with plerixafor.

FIG. 15 provides a representative flow chart of blood samples taken from rhesus monkeys 4 hours after automatic mobilization intravenous administration of 450 μ g/kg Gro- β T and subcutaneous administration of 1mg/kg plerixafor (AMD3100) resulted in CD34 ^dimMobilization of the cell population.

FIG. 16 provides representative flow charts from blood samples taken from rhesus monkeys at baseline and 4 hours post-mobilization the mobilization was induced by (1) intravenous administration of 450 μ g/kg Gro- β T and subcutaneous administration of 1mg/kg plerixafor (AMD3100) or (2) subcutaneous administration of 1mg/kg plerixafor (AMD 3100). the combination of Gro- β T and plerixafor (as compared to plerixafor alone) resulted in CD34^dimThe mobilization of the cells is enhanced.

FIG. 17 is a graph of CD34 in peripheral blood from untreated rhesus monkeys ("unmoved"), rhesus monkeys treated with intravenous administration of 450 μ G/kg Gro- β T and subcutaneous administration of 1mg/kg plerixafor ("Gro- β T + plerixafor"), rhesus monkeys treated with subcutaneous administration of 1mg/kg plerixafor ("plerixafor"), and rhesus monkeys treated with subcutaneous administration of 50 μ G/kg (q.d.x 5) G-CSF ("G-CSF")^dimgraph quantifying the concentration of cells CD34 in blood mobilized with Gro- β T-Calla Saffer^dimCells are present in significantly higher concentrations.

FIG. 18 is a graph depicting the composition of non-mobilized cells and grafts mobilized by G-CSF, Gro- β T and AMD3100, and AMD3100 alone^dim3-fold increase in cells, and 3-fold increase in T cells.

FIG. 19 provides a graph showing Gro- β T and AMD3100 mobilized CD34 as measured by carboxyfluorescein succinimidyl ester (CFSE) staining after 4 days of culture^dimThe cells inhibit T cell proliferation. Bead indicationT cells were stimulated using anti-CD 2/CD3/CD28 coated microbeads.

FIG. 20 provides survival curves showing that by day 24, all mice transplanted with unmoved PBMC (13/13) die from aGVHD compared to 5/16 mice transplanted with AMD3100 mobilized peripheral blood die from aGVHD, 3/16 mice transplanted with G-CSF mobilized PBMC die from aGVHD, and none of the mice transplanted with Gro-. beta.T and AMD3100 mobilized PBMC die from aGVHD. On day 60 post-transplantation, 15/16 mice transplanted with Gro- β T and AMD3100 remained alive, while only 10/16 mice transplanted with AMD3100 mobilized PBMC and only 11/16 mice transplanted with G-CSF mobilized PBMC remained alive. p <0.0001 (compare Gro-beta T and AMD3100 to unarmed) and p <0.05 (compare Gro-beta T and AMD3100 to AMD3100 alone).

FIG. 21A shows the number of rhesus monkey (rhesus) CD45+ CD3+ T cells in mice at day 14 after transplantation of unmoved PBMC, Gro- β T and plerixafor (i.e., AMD3100), PBMC mobilized with plerixafor alone and PBMC mobilized with G-CSF alone FIG. 21B shows PBMC mobilized with unmoved PBMC, Gro- β T and plerixafor (i.e., AMD3100) and depleted CD34 mobilized with Gro- β T and plerixafor^dimT cell number in mice on day 14 post PBMC of cells FIG. 21C provides transplanted non-mobilized PBMC, Gro- β T and AMD3100 mobilized PBMC and Gro- β T and AMD3100 mobilized depleted CD34^dimMouse survival curves of PBMC of cells.

Detailed Description

The present invention provides compositions and methods for mobilizing hematopoietic stem and progenitor cells in a subject. For example, the subject can be a hematopoietic stem cell and progenitor cell donor (i.e., donor), such as a mammalian donor (e.g., a human donor). The compositions and methods described herein may additionally be used to treat 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- β or a variant thereof, such as a truncated form of Gro- β (e.g., Gro- β T), as described herein, optionally in combination with a C-X-C chemokine receptor type 4 (CXCR4) antagonist, such as 1, 1' - [1, 4-phenylenebis (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane, or a variant thereof, can be administered to a donor, as described herein, in an amount sufficient to mobilize hematopoietic stem and progenitor cells. The compositions and methods described herein are capable of mobilizing hematopoietic stem and progenitor cells from the stem cell niche within the donor into the circulating peripheral blood, while reducing the mobilization of other cells of the hematopoietic lineage (such as leukocytes, neutrophils, lymphocytes, and monocytes). Thus, the compositions and methods described herein enable selective mobilization of hematopoietic stem and progenitor cells in a donor, which can then be isolated from the donor for therapeutic use.

The present invention is based, in part, on the discovery that administration of a CXCR2 agonist, such as Gro-beta, Gro-beta T, or variants thereof, optionally in combination with a CXCR4 antagonist, such as plerixafor, or a pharmaceutically acceptable salt thereof, at a specific dose, can provide important clinical benefits, namely, mobilizing a cell population that is enriched for hematopoietic stem cells relative to other cell types, such as leukocytes, neutrophils, and monocytes. This ability is advantageous because these other cell types may be undesirable for administration to a human patient undergoing hematopoietic stem cell transplantation therapy. Thus, the populations of mobilized hematopoietic stem and progenitor cells produced using the compositions and methods described herein are particularly suited for hematopoietic stem cell transplantation therapies.

Following mobilization, hematopoietic stem or progenitor cells can be isolated for ex vivo expansion and/or therapeutic use. In some embodiments, following collection of mobilized hematopoietic stem and/or progenitor cells, the drawn cells can be infused into a patient, such as a donor or another subject (e.g., a subject HLA matched to a donor), for treatment of one or more pathologies of the hematopoietic system. Additionally or alternatively, mobilized cells can be drawn and then expanded ex vivo, such as by contacting the cells with an aromatic hydrocarbon receptor antagonist, in order to generate a population of hematopoietic stem cells with sufficient numbers of cells for transplantation.

As described herein, hematopoietic stem cells are capable of differentiating into numerous cell types in the hematopoietic lineage, and thus can be administered to a patient in order to fill or refill a defective or deficient cell type in the patient. The patient may be, for example, a patient suffering from one or more blood disorders such as autoimmune diseases, cancer, hemoglobinopathies or other hematopoietic pathologies and thus requiring hematopoietic stem cell transplantation. Accordingly, the present invention provides methods of treating various hematopoietic system conditions such as, inter alia, sickle cell anemia, thalassemia, fanconi anemia, wiskott-aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infections and acquired immunodeficiency syndrome, as well as cancer and autoimmune diseases.

The following sections provide descriptions of CXCR4 antagonists and CXCR2 agonists that can be administered to a donor in order to induce mobilization of a population of hematopoietic stem or progenitor cells from stem cell niches into peripheral blood, from which cells can then be isolated and infused into a patient for the treatment of, for example, one or more stem cell disorders described herein, such as cancer, autoimmune diseases, or metabolic disorders. The following section additionally describes methods of determining whether a population of cells mobilized with a CXCR2 agonist and/or a CXCR antagonist are suitable for release for ex vivo expansion and/or therapeutic applications.

Definition of

As used herein, the term "about" refers to a value within 10% above or below the value described. For example, the term "about 5 nM" indicates a range from 4.5nM to 5.5 nM.

As used herein, the terms "obtain" and "obtaining" mean obtaining possession of, directly obtaining, or indirectly obtaining a physical entity or value, such as a numerical value. By "directly obtaining" is meant performing a process to obtain a physical entity or value (e.g., assaying or testing a sample or analyzing a sample). "indirectly obtaining" refers to receiving a physical entity or value from another party or source (e.g., a third party laboratory that directly obtains the physical entity or value). Directly obtaining a physical entity includes performing, for example, a process of analyzing a sample, such as a sample of hematopoietic cells isolated from a donor that has undergone or is undergoing the hematopoietic stem cell mobilization protocol described herein. Directly obtaining a value includes performing a process such as an assay on the sample or another substance, for example, performing an analytical process that includes determining the number of hematopoietic stem cells in the sample, the ratio of hematopoietic stem cells to another type of cell in the hematopoietic lineage, or the frequency of hematopoietic stem cells in the total number of cells in the sample.

As used herein, the term "affinity" refers to the strength of a non-covalent interaction between two or more molecules, such as two or more proteins (e.g., a metalloprotease and an endogenous inhibitor thereof as described herein). Affinity can be quantitatively expressed, for example, as the equilibrium dissociation constant (K)_d) Or, in the case where one of the binding partners is an enzyme, expressed as the inhibition constant (K)_i). Binding affinities can be determined using standard techniques such as enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance assays, and isothermal titration calorimetric assays (isotermal ligation assays), among others.

As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a particular antigen and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including but not limited to chimeric, humanized, heteroconjugate antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies) and antigen-binding fragments of antibodies, including, for example, Fab ', F (ab')₂Fab, Fv, rlgG and scFv fragments. Unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact molecules as well as antibody fragments (including, e.g., Fab and F (ab') ₂Fragments) are added. As used herein, Fab and F (ab')₂Fragments refer to antibody fragments lacking the Fc fragment of an intact antibody. Examples of such antibody fragments are described herein。

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 a fragment of a full-length antibody. The antibody fragment may be, for example, Fab, F (ab')₂scFv, diabody, triabody, affibody (affibody), nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) fab fragment from V_LDomain, V_HDomain, C_LDomains and C _H1 domain; (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) from V_HDomains and C _H1 domain; (iv) v from one arm of an antibody_LDomains and V_H(iv) an Fv fragment consisting of the domain (V) comprising V_HDomains and V_LA dAb of a domain; (vi) from V_HdAb fragments consisting of domains (see, e.g., Ward et al (1989) Nature 341: 544-546); (vii) from V _HDomain or V_LA domain constituting dAb; (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, despite the two domains V of the Fv fragment_LAnd V_HEncoded by separate genes, but they can be joined using recombinant methods by linkers that enable them to be single protein chains, where V_LRegion and V_HThe regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science 242:423-426 and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be cleaved enzymatically or chemically by recombinant DNA techniques, intact immunoglobulins, or in some cases, by techniques already known in the artKnown chemical peptide synthesis procedures.

As used herein, the term "bispecific antibody" refers to, for example, a monoclonal antibody, typically a human or humanized antibody, which is capable of binding to at least two different antigens.

As used herein, the term "CD 34^dimBy cell "is meant a population of cells, wherein 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 relatively low levels. Such cell populations exhibit characteristics of monocytes, e.g., the ability to inhibit the proliferation of alloreactive T lymphocytes. (D' Aveni et al (2015) Science relative Medicine 7(281): 1-12. in some embodiments, CD34^dimThe population of cells may be CD14 +.

One skilled in the art can readily visualize CD34 when viewing flow cytometry images^dimThe cell population was identified as CD34⁺But with a basal level of fluorescence (e.g. autofluorescence) between the cell populations examined and CD34⁺Bright cells (e.g., hematopoietic stem cells and/or CD 34)⁺CD90⁺Cells) a population of cells having a brightness between the brightness of the population. For example, in certain embodiments, CD34^dimThe cell population showed CD34⁺Brightness between 5% and 95% of a population of bright cells (e.g., hematopoietic stem cells), but greater than CD34^-The cell population is brighter.

In certain embodiments, CD34^dimCells appear to be intermediate to CD34⁺10% and 90%, 10% and 80%, 10% and 70%, 10% and 60%, 10% and 50%, 10% and 40%, 10% and 30%, 10% and 20%, 20% and 90%, 20% and 80%, 20% and 70%, 20% and 60%, 20% and 50%, 20% and 40%, 20% and 30%, 30% and 90%, 30% and 80%, 30% and 60%, 30% and 50%, 30% and 40%, 40% and 90%, 40% and 80%, 40% and 70%, 40% and 60%, 40% and 50%, 50% and 90%, 50% and 80%, 50% and 70%, 50% and 60%, 60% and 90%, 60% and 80%, of bright cells (e.g., hematopoietic stem cells) are present in the sample at 10% and 90%, 10% and 60%, 10% and 70%, 10% and 50%, 20% and 20%, 20% and 60%, 20% and 50%, 20% and 40%, 40% and 80%, 40% and, Brightness between 60% and 70%, 70% and 90%, 70% and 80%, or 80% and 90%, but over CD34^-The cells are brighter. In certain embodiments, CD34^dimCell ratio CD34^-The cells are at least 5% bright, at least 10% bright, at least 20% bright, or at least 30% bright, but not as bright as CD34⁺Bright cells (e.g., hematopoietic stem cells) are bright.

In certain embodiments, CD34 is identified in a cell sample by flow cytometry using magnetic beads rather than fluorescence^dimA cell. The magnetic beads will selectively pull down (pull down) the CD34⁺Bright cells, leaving CD34 in the cell sample^dimA cell.

In certain embodiments, CD34 is identified by measuring the copy number of CD34 expressed by the cell^dimA cell. For example, CD34^dimThe cells may exhibit CD34⁺Between 5% and 95% of the copy number of CD34 compared to bright cells (e.g., hematopoietic stem cells), or CD34⁺Bright cells (e.g., hematopoietic stem cells) are between 10% and 90%, 10% and 80%, 10% and 70%, 10% and 60%, 10% and 50%, 10% and 40%, 10% and 30%, 10% and 20%, 20% and 90%, 20% and 80%, 20% and 70%, 20% and 60%, 20% and 50%, 20% and 40%, 20% and 30%, 30% and 90%, 30% and 80%, 30% and 70%, 30% and 60%, 30% and 50%, 30% and 40%, 40% and 90%, 40% and 80%, 40% and 70%, 40% and 60%, 40% and 50%, 50% and 90%, 50% and 80%, 50% and 70%, 50% and 60%, 60% and 90%, 60% and 80%, 60% and 70%, 70% and 90%, 70% and 80%, or 80% and 90% of the copy number of CD 34.

As used herein, the term "complementarity determining region" (CDR) refers to a hypervariable region found in both the light chain and heavy chain variable domains of an antibody. The more highly conserved portions of the variable domains are called Framework Regions (FR). The amino acid positions depicting the hypervariable regions of an antibody can vary according to the background and various definitions known in the art. Some positions within a variable domain may be considered mixed hypervariable positions in that these positions may be considered within a hypervariable region according to one set of criteria, while being considered outside the hypervariable region according to a different set of criteria. One or more of these positions may also be found in an extended hypervariable region. The antibodies described herein may contain modifications in these mixed hypervariable positions. The variable domains of native heavy and light chains each contain four framework regions, largely adopting a β -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β -sheet structure. The CDRs in each chain are in close proximity in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 through the framework regions and contribute, together with the CDRs from the other antibody chains, to the formation of the target binding site for the antibody (see Kabat et al, Sequences of Proteins of immunologicalcalest, National Institute of Health, Bethesda, MD., 1987). As used herein, the numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated.

As used herein in the context of administering one or more agents to a subject, the term "complete administration" refers to the point in time at which the one or more agents are administered to the subject in its entirety (in the third entirrety). 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-beta or a variant or truncate thereof (truncation), such as Gro-beta T) may be administered to a subject over a period of time, e.g., by intravenous injection or subcutaneous injection. After the prescribed dose of the agent is administered to the subject as a whole, the agent is considered to have been "administered completed". Where multiple agents are administered to a subject, such as both a CXCR4 antagonist (e.g., plerixafor or a variant thereof) and a CXCR2 agonist (e.g., Gro-beta or a variant or truncate thereof, such as Gro-beta T), the agents are considered to have been "administered complete" after the prescribed dose of all agents is administered to the subject as a whole in a particular regimen.

As used herein, the terms "conservative mutation," "conservative substitution," or "conservative amino acid substitution" refer to the substitution of one or more amino acids to one or more different amino acids that exhibit similar physicochemical properties (such as polarity, electrostatic charge, and steric bulk). Table 1 below summarizes these properties for each of the 20 naturally occurring amino acids.

TABLE 1. representative physicochemical characteristics of naturally occurring amino acids

Based on the volume in a 3: 50-100 are small, 100-150 are medium, 150-200 are large, and>200 are bulky

As will be appreciated from this table, the conserved amino acid family includes, for example, (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. Thus, a conservative mutation or substitution is one that replaces an amino acid as a member of the same amino acid family (e.g., Ser for Thr or Lys for Arg).

As used herein, "CRU (competitive repopulating unit)" refers to a unit of measure of chronically implanted stem cells that can be detected after in vivo transplantation.

As used herein, the term "donor" refers to a subject, such as a mammalian subject (e.g., a human subject), from which cells are isolated prior to administration of one or more cells or progeny thereof to a recipient. The one or more cells may be, for example, a population of hematopoietic stem or progenitor cells.

The term "diabody" as used herein refers to a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises a V connected by a linker _HDomains and V_L(ii) a domain whose linker is too short (e.g., a linker comprising five amino acids) to allow V on the same peptide chain_HDomains and V_LIntramolecular of the DomainAnd (4) associating. This configuration forces each domain to pair with a complementary domain on the other polypeptide chain to form a homodimeric structure. Thus, the term "trisomy" refers to a trivalent antibody comprising three peptide chains, each of which contains one V linked by a linker_HDomains and a V_L(ii) a domain which linker is very short (e.g. a linker comprising 1-2 amino acids) without allowing V within the same peptide chain_HDomains and V_LIntramolecular association of domains. Peptides configured in this manner are typically trimerized in order to fold into their native structure, so as to convert V of adjacent peptide chains_HDomains and V_LThe domains are located in close spatial proximity to each other (see, e.g., Holliger et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-48).

As used herein, the term "disruption" with respect to a gene refers to the prevention of the formation of a functional gene product. A gene product is functional only when it fulfills its normal (wild-type) function. Disruption of a gene prevents expression of a functional factor encoded by the gene and includes insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or operator gene necessary for expression of the gene in an animal. A disrupted gene can be disrupted by, for example, removing at least a portion of the gene from the genome of the animal, altering the gene to prevent expression of a functional factor encoded by the gene, interfering RNA, or expression of a dominant negative factor of a foreign gene. US 8,518,701; US 2010/0251395; and US 2012/0222143, the disclosure of each of which is incorporated herein by reference in its entirety (in case of conflict, the present specification controls).

Various techniques known in the art can be used to inactivate genes to create knock-out animals and/or introduce nucleic acid constructs into animals to create creater animals (founder animals) and lines of manufacturing animals in which the knock-out or nucleic acid construct is integrated into the genome. These include, but are not limited to, prokaryotic microinjection (U.S. Pat. No. 4,873,191), retrovirus-mediated gene transfer to germ Cell lines (germ lines) (Van der Putten et al (1985) Proc. Natl.Acad.Sci.USA,82:6148-6152), gene-targeted embryonic stem cells (Thompson et al (1989) Cell,56:313-321), embryo electroporation (Lo (1983) mol.cell. biol.,3:1803-1814), sperm-mediated gene transfer (Lavitrano et al (2002) Proc. Natl.Acad.Sci.USA,99: 14230-14235; Lavino et al (2006) reprod.Fert.device. op.,18:19-23), and somatic cells such as cumulus ovale or mammary cells, or in vitro transformation of adult, foetus or embryonic stem cells, followed by nuclear transfer (Wilmut et al (1997) Nature et al: 394; Nature et al: 374) Nature et al (1998). Prokaryotic microinjection, sperm-mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques. A genomically modified animal is one in which all of its cells, including its germ line, have been genetically modified. When using a method for producing an animal that is chimeric (mosaic) in its genetic modification, the animal may be inbred and a progeny of the genomic modification (progeny) may be selected. For example, cloning can be used to make chimeric animals if their cells are modified in the blastocyst state, or genome modification can occur when a single cell is modified. Animals modified to lack sexual maturity may be homozygous or heterozygous, depending on the particular method used. If a particular gene is inactivated by a knockout modification, homozygosity will generally be required. Heterozygosity is usually sufficient if a particular gene is inactivated by RNA interference or dominant negative strategies.

As used herein, "dual variable domain immunoglobulin" ("DVD-Ig") refers to combining the target binding variable domains of two monoclonal antibodies via a linker to produce a tetravalent dual single dose targeting antibody (see, e.g., Gu et al (2012) meth.enzymol.,502: 25-41).

As used herein, the term "endogenous" describes a substance, such as a molecule, cell, tissue, or organ, that naturally occurs in a particular organism, such as a human patient (e.g., a hematopoietic stem cell or hematopoietic lineage cell, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglia, granulocyte, monocyte, osteoclast, antigen presenting cell, macrophage, dendritic cell, natural killer cell, T lymphocyte, or B lymphocyte).

As used herein, the term "engraftment potential" is used to refer to the ability of hematopoietic stem and progenitor cells to refill into tissue, whether such cells are naturally circulating or provided by transplantation. The term encompasses all events surrounding or resulting in implantation, such as tissue homing of cells and colonization of cells within the tissue of interest. Using any clinically acceptable parameter as known to those skilled in the art or evaluating the progression of the subject by disease progression, survival of hematopoietic stem and progenitor cells or survival of the recipient, engraftment efficiency or engraftment rate can be evaluated or quantified and can include, for example, assessing the incorporation or expression of Competitive Refill Units (CRUs), markers in one or more tissues into which stem cells home, engraft or become engrafted (tissue (s)). Implantation can also be determined by measuring the white blood cell count in the peripheral blood during the later stages of transplantation. Engraftment can also be assessed by measuring the recovery of bone marrow cells by donor cells in a sample of bone marrow aspirate.

As used herein, the term "exogenous" describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or hematopoietic lineage cell, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglia, granulocyte, monocyte, osteoclast, antigen presenting cell, macrophage, dendritic cell, natural killer cell, T lymphocyte, or B lymphocyte) that does not naturally occur in a particular organism, such as a human patient. Exogenous materials include those provided to the organism from an external source or cultured materials extracted from an external source.

As used herein, the term "framework region" or "FW region" includes amino acid residues adjacent to the CDRs of an antibody or antigen-binding fragment thereof. The 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.

As used herein, the term "hematopoietic progenitor cell" includes pluripotent (pluripotent) cells capable of differentiating into several cell types of the hematopoietic system, including but not limited to granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-cells, among others. Hematopoietic progenitor cells belong to the hematopoietic lineage and are generally not self-renewing. Hematopoietic progenitor cells can be identified, for example, by the expression pattern of cell surface antigens, and include cells with the following immunophenotypes: lin ^-KLS⁺Flk2^-CD34⁺. Hematopoietic progenitor cells include short-term hematopoietic stem cells, multipotent (multi-potential) 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 an intact methylcellulose assay, or phenotypically by detecting cell surface markers using flow cytometry and cell sorting assays described herein and known in the art.

As used herein, the term "hematopoietic stem cell" ("HSC") refers to an immature blood cell that has the ability to self-renew and differentiate into mature blood cells containing cells of different lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, 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 ⁺A cell. CD34⁺The cells are immature cells expressing CD34 cell surface markers. In humans, CD34⁺Cells are considered to comprise a subpopulation of cells having the characteristics of stem cells as defined above, whereas in mice the HSC is CD 34-. Furthermore, HSC also refers to long term refilled HSC (LT-HSC) and short term refilled HSC (ST-HSC). LT-HSC and ST-HSC are differentiated based on functional potential and cell surface marker expression. For example, the human HSC is CD34⁺、CD38^-、CD45RA^-、CD90⁺、CD49F⁺And lin^-(negative for markers of mature lineage (including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD 235A)). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit +, CD135-, Slamfl/CD150+, CD48-, and lin- (negative for mature lineage markers (including Ter119, CD11B, Gr1, CD3, CD4, CD8, B220, IL7 ra)), while ST-HSCs are CD34⁺、SCA-1⁺、C-kit⁺、CD135^-、Slamfl/CD150⁺And lin^-(negative for markers of mature lineage (including Ter119, CD11B, Gr1, CD3, CD4, CD8, B220, IL7 ra)). Furthermore, ST-HSCs are less quiescent and proliferate more than LT-HSCs under homeostatic conditions. However, LT-HSCs have greater self-renewal potential (i.e., they survive throughout adulthood and can be transplanted continuously by continuous recipients), whereas ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time and do not have continuous 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 therefore can produce differentiated progeny more quickly.

As used herein, the term "hematopoietic stem cell functional potential" refers to the functional properties of hematopoietic stem cells, including 1) multipotentiality (which refers to the ability to differentiate into a variety of different blood lineage cells, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, 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 produce progeny cells with equivalent potential to the mother cell, and furthermore this ability can occur repeatedly throughout the life of the individual without failure), and 3) the ability of hematopoietic stem cells or their progeny to be reintroduced into the transplant recipient, at which time they home to the hematopoietic stem cell niche and reconstitute productive and sustained hematopoiesis.

As used herein, the term "major histocompatibility complex antigen" ("MHC," also referred to in the human context as "human leukocyte antigen" ("HLA")) refers to a protein expressed on the surface of a cell that confers a unique antigenic identity (identity) to the 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 ("self") as immune effector cells or from another source of hematopoietic reconstituting cells ("non-self"). HLA antigens fall into two broad categories: HLA class I and HLA class II. HLA class I antigens (A, B and C in humans) allow each cell to be recognized as "self", while HLA class II antigens (DR, DP, and DQ in humans) are involved in the reaction between lymphocytes and antigen presenting cells. Both are associated with rejection of the transplanted organ. An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR), is present in a different allele. For example, two unrelated individuals may carry HLA class I-B genes B5 and Bw41, respectively. The allele products differ in one or more amino acids of the alpha and/or beta domains. Individuals are typed for HLA haplotypes using leukocytes expressing class I and class II molecules using large amounts of specific antibodies or nucleic acid reagents. The genes commonly used for HLA typing are six MHC class I and II proteins, with two alleles for each of HLA-A, HLA-B and HLA-DR. HLA genes are clustered in a "super locus" present on chromosome position 6p21, which encodes 6 classical transplantation HLA genes and at least 132 protein-encoding genes that play important roles in the regulation of the immune system and in some other fundamental molecular and cellular processes. The complete locus measures about 3.6Mb, with a locus of at least 224 genes. One effect of such clustering is "haplotyping", i.e., the allelic genome that exists on a single chromosome and is inherited from one parent tends to be inherited as a group (group). Haplotypes are formed from the allelic genomes inherited from each parent, with some alleles tending to associate together. Identifying the haplotype of a patient can help predict the probability of finding a matching donor and help develop search strategies because some alleles and haplotypes are more common than others and their distribution frequency differs among different ethnic and ethnic groups.

As used herein, the term "HLA-matched" refers to a donor-recipient pair in which there is no HLA-antigen mismatch between the donor and recipient, such as where the donor provides a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplantation therapy. Since endogenous T cells and NK cells are unlikely to recognize an incoming graft as foreign and thus unlikely to generate an immune response against the graft, HLA-matched (i.e. all 6 alleles matched) donor-recipient pairs have a reduced risk of graft rejection.

As used herein, the term "HLA-mismatched" refers to a donor-recipient pair wherein at least one HLA antigen between the donor and the recipient, particularly with respect to HLA-A, HLA-B and HLA-DR, is mismatched, such as where the donor provides a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplantation therapy. In some embodiments, one haplotype is matched and the other is unmatched. HLA-mismatched donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched donor-recipient pairs because, in the case of HLA-mismatched donor-recipient pairs, endogenous T cells and NK cells are more likely to identify the incoming graft as foreign, and thus such T cells and NK cells are more likely to generate an immune response against the graft.

As used herein, the term "human antibody" refers to an antibody in which substantially every portion of a protein (e.g., all CDRs, framework regions, C)_LDomain, C_HDomains (e.g., C)_H1、C _H2、C_H3) Hinge and V_LDomains and V_HDomains) are substantially non-immunogenic in humans with only minor sequence changes or variations. Human antibodies can be produced in human cells (e.g., by recombinant expression) or from non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (such as heavy and/or light chain) genes. When the human antibody is a single chain antibody, it may include a linker peptide that is not present in the native human antibody. For example, the Fv can contain a linker peptide, such as two to about eight glycine or other amino acid residues, that links 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 prepared 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 do not express functional endogenous immunoglobulins but express human immunoglobulin genes (see, e.g., PCT publication Nos. WO 1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Pat. No. 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).

As used herein, the term "humanized" antibody refers to a non-human antibody that contains minimal sequence derived from a non-human immunoglobulin. Typically, 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 human immunoglobulin sequences. The humanized antibody may 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 in the following: for example, Riechmann et al (1988) Nature 332: 323-7; U.S. Pat. nos. 5,530,101; U.S. Pat. No. 5,585,089; nos. 5,693,761; nos. 5,693,762; and U.S. Pat. No. 6,180,370.

As used herein, patients "in need of" a hematopoietic stem cell graft include patients who exhibit a deficiency or defect in one or more blood cell types, as well as patients who have stem cell disorders, autoimmune diseases, cancer, or other pathologies described herein. Hematopoietic stem cells typically exhibit: 1) multipotent, and thus can differentiate into a variety of different blood lineage cells, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, 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 and thus can produce daughter cells with equivalent potential to mother cells, and 3) the ability to be reintroduced into the transplant recipient, at which time they home to the hematopoietic stem cell niche and reconstitute productive and sustained hematopoiesis. Thus, hematopoietic stem cells can be administered to a patient having one or more cell types deficient or lacking in hematopoietic lineage in one or more cell types of hematopoietic lineage in order to reconstitute a population of deficient or lacking cells in vivo. For example, a patient may suffer from cancer, and the defect may result from administration of a chemotherapeutic agent or other drug that selectively or non-specifically depletes the population of cancerous cells. Additionally or alternatively, the patient may suffer 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 a subject suffering from: adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. The subject may have or be affected by: hereditary blood disorders (e.g., sickle cell anemia) or autoimmune disorders. Additionally or alternatively, the subject may have or be affected by: malignant tumors, such as neuroblastoma or hematological cancers. In some embodiments, the subject may have 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 from or otherwise be affected by a metabolic disorder selected from the group consisting of: glycogen storage disease, mucopolysaccharide storage disease, gaucher's disease, Heller disease, sphingolipid storage disease, metachromatic leukodystrophy, or any other Disease or disorder that may benefit from the treatments and therapies disclosed herein, including, but not limited to, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper-immunoglobulin M (IgM) syndrome, Cheers' Disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage Disease, thalassemia major, sickle cell Disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and the diseases or disorders described in "Bonemorow Transplantation for Non-Malignant Disease," ASH efficacy Book,1:319-338(2000) ", the disclosure of which is incorporated herein by reference in its entirety as it relates to pathologies that can be treated by administration of hematopoietic stem cell transplantation therapy. Additionally or alternatively, a patient "in need of" hematopoietic stem cell transplantation may be a patient who has suffered from one of the aforementioned pathologies or does not suffer from it but still exhibits: reduced levels of one or more endogenous cell types within the hematopoietic lineage (e.g., compared to levels in other healthy subjects), such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. For example, one skilled in the art can readily determine whether the levels of one or more of the foregoing cell types or other blood cell types in a person are reduced relative to other healthy subjects by, among other procedures, flow cytometry and Fluorescence Activated Cell Sorting (FACS) methods known in the art.

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 broad categories: polymorphonuclear cells, which include neutrophils, eosinophils, and basophils, and monocytes, which include lymphocytes and monocytes. Polymorphonuclear cells contain many cytoplasmic granules and multilobal nuclei (multilobal nuclei) and include the following: neutrophils, which are generally proteid, phagocytic, and stained with both basic and acid dyes; and eosinophils and basophils, which contain cytoplasmic granules stained with an acid dye and a basic dye, respectively.

As used herein, the term "lymphocyte" refers to a mononuclear leukocyte involved in the generation of an immune response. Generally, lymphocytes include B lymphocytes, T lymphocytes, and NK cells.

As used herein, the terms "mobilization" and "mobilization" refer to the process by which a population of hematopoietic stem or progenitor cells is released from a stem cell niche, such as a subject's bone marrow, into peripheral blood circulation. Mobilization of hematopoietic stem and progenitor cells can be monitored, for example, by assessing the number or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from the subject. For example, after administering a hematopoietic stem cell or progenitor cell mobilization protocol to a subject, a peripheral blood sample can be drawn from the subject, and the number or concentration of hematopoietic stem cells or progenitor cells in the peripheral blood sample can then be assessed. The mobilization protocol can include any of a number of, for example, an antagonist of 4, 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- β or a variant thereof, a truncate such as Gro- β, e.g., Gro- β T) can be compared to the number or concentration of hematopoietic stem cells or progenitor cells in a peripheral blood sample isolated from the subject after administration of the mobilization protocol to the number or concentration of hematopoietic stem cells or progenitor cells in a peripheral blood sample isolated from the subject prior to administration of the mobilization protocol, an observation that the number or concentration of hematopoietic stem cells or progenitor cells in the peripheral blood of the subject increases after administration of the mobilization protocol is an indication that the subject has responded to the mobilization protocol and that the hematopoietic stem cells and progenitor cells have been released into the peripheral blood circulation from one or more stem cell niches such as bone marrow in some embodiments, such as an indication that the number or concentration of hematopoietic stem cells or progenitor cells in the peripheral blood of the subject has increased by 1%, 100%, 1%, 1,000% or more% or a 5% or more (e.g., a CXCR 4% or a CXCR4 antagonist such as a CXCR4 antagonist (e.g., a variant thereof) and a CXCR 3975% or CXCR 6% or progenitor cell based on the methods described herein include a method in a method described in the following administration of a 20% or a method in the following administration of a 20% mobilization protocol, such as a 20% of a method of a human hematopoietic stem cell 20% or 10% or a human hematopoietic stem cell 20% or a 20% or 10% or 20% of the same, and a human hematopoietic stem cell 20% or 10% of the present field, and a method, including a method, such ⁺、CD38^-、CD45RA^-、CD90⁺、CD49F⁺And lin- (negative for markers of mature lineage (including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD 235A)). For determining hematopoietic stem cells or in a peripheral blood sample isolated from a subjectAdditional methods of determining the number or concentration of progenitor cells include the quantitative determination of the number of Colony Forming Units (CFU) in a sample, a measure of the number of viable hematopoietic stem or progenitor cells (a population of individuals that produce hematopoietic stem or progenitor cells upon incubation with an appropriate medium).

as used herein, the term "mobilizing amount" refers to the amount of one or more agents, such as the amount of CXCR4 antagonist and/or CXCR2 agonist described herein (in some embodiments, plerixafor or a variant thereof, and/or Gro- β or a variant thereof, such as a truncate of Gro- β, e.g., Gro- β T), that mobilize a population of hematopoietic stem or progenitor cells upon administration to a subject, such as a mammalian subject (e.g., a human subject) ⁺Cells/. mu.L peripheral blood, such as from about 21 to about 39 CD34⁺Cells/. mu.L peripheral blood, about 22 to about 38 CD34⁺Cells/. mu.L peripheral blood, about 23 to about 37 CD34⁺Cells/. mu.L peripheral blood, about 24 to about 36 CD34⁺Cells/. mu.L peripheral blood, about 25 to about 35 CD34⁺Cells/. mu.L peripheral blood, about 26 to about 34 CD34⁺Cells/. mu.L peripheral blood, about 27 to about 33 CD34⁺Cells/. mu.L peripheral blood, about 28 to about 32 CD34⁺Cells/. mu.L peripheral blood or about 29 to about 31 CD34⁺Cells/. mu.L peripheral blood (e.g., about 20 CD 34)⁺Cell/. mu.L peripheral blood, 21 CD34 cells⁺Cell/. mu.L peripheral blood, 22 CD34⁺Cell/. mu.L peripheral blood, 23 CD34⁺Cell/. mu.L peripheral blood, 24 CD34⁺Cell/. mu.L peripheral blood, 25 CD34⁺Cell/. mu.L peripheral blood, 26 CD34 cells⁺Cell/. mu.L peripheral blood, 27 CD34 cells⁺Cell/. mu.L peripheral blood, 28 CD34 cells⁺Cell/. mu.L peripheral blood, 29 CD34 cells⁺Cell/. mu.L peripheral blood, 30 CD34⁺Cell/. mu.L peripheral blood, 31 CD34⁺Cell/. mu.L peripheral blood, 32 CD34⁺Cell/. mu.L peripheral blood, 33 CD34⁺Cell/. mu.L peripheral blood, 34 CD34⁺Cell/. mu.L peripheral blood, 35 CD34 cells⁺Cell/. mu.L peripheral blood, 36 CDs 34⁺Cell/. mu.L peripheral blood, 37 CD34⁺Cells/. mu.L peripheral blood, 38 CD34 cells⁺Cell/. mu.L peripheral blood, 39 CD34 cells ⁺Cell/. mu.L peripheral blood, 40 CD34 cells⁺cells/μ L peripheral blood or more) for example, the amount of a CXCR2 agonist, such as Gro- β T, includes from about 50 μ g/kg recipient to about 1mg/kg recipient, such as from about 50 μ g/kg to about 300 μ g/kg, 100 μ g/kg to about 250 μ g/kg, or about 150 μ g/kg. the amount of a cxcr4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, includes from about 50 μ g/kg recipient to about 500 μ g/kg recipient, such as from about 200 μ g/kg to about 300 μ g/kg or about 240 μ g/kg.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), rather than a method of producing an antibody.

As used herein, the term "monocyte" refers to CD14⁺And CD 34-Peripheral Blood Mononuclear Cells (PBMCs), which are generally capable of differentiating into macrophages and/or dendritic cells upon activation by one or more foreign substances such as microbial products. In particular, monocytes 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 bound to Ig-like lectin 7 or Siglec7) and CD329 (sialic acid bound to Ig-like lectin 9 or Siglec9) and peanut lectin Protein (PNA).

As used herein, "peptide" refers to a single-chain polyamide containing more than one amino acid residue, such as naturally occurring and/or non-natural amino acid residues, bound consecutively through amide bonds. Examples of peptides include full-length proteins, such as shorter fragments of full-length naturally occurring proteins.

As used herein, the term "recipient" refers to a patient who receives a transplant (such as a transplant containing a population of hematopoietic stem cells). The transplanted cells administered to the recipient may be, for example, autologous cells, syngeneic cells, or allogeneic cells.

As used herein, the term "sample" refers to a sample (e.g., blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus sample, and cells) taken from a subject. For example, the sample may be peripheral blood drawn from a donor who is undergoing or has undergone a hematopoietic stem cell or progenitor cell mobilization protocol described herein.

As used herein, the term "scFv" refers to a single chain Fv antibody in which the variable domains from the heavy and light chains of the antibody have joined to form one chain. scFv fragments comprise variable regions of the antibody light chain (V) comprising a spacer _L) (e.g., CDR-L1, CDR-L2 and/or CDR-L3) and antibody heavy chain variable region (V)_H) (e.g., CDR-H1, CDR-H2, and/or CDR-H3). V linking scFv fragments_LRegion and V_HThe linker of the region may be a peptide linker comprising proteinogenic (proteinogenic) amino acids. Alternative linkers can be used in order to increase the resistance of the scFv fragment to proteolytic degradation (e.g., a linker comprising a D-amino acid), to enhance the solubility of the scFv fragment (e.g., a hydrophilic linker such as a linker comprising polyethylene glycol or a polypeptide comprising repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker comprising cysteine residues that form an intramolecular or intermolecular disulfide bond), or to reduce the immunogenicity of the scFv fragment (e.g., a linker comprising a glycosylation site). One of ordinary skill in the art will also appreciate that the variable regions of the scFv molecules described herein can be modified such that their amino acid sequences differ from the amino acid sequence of the antibody molecule from which they are derived. For example, nucleotide or amino acid substitutions that result in conservative substitutions or changes may be made at amino acid residues (e.g., in CDRs and/or framework residues) in order to preserve or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.

As used herein, the phrase "stem cell disorder" broadly refers to any disease, disorder or condition that can be treated or cured by engraftment or transplantation of a population of hematopoietic stem or progenitor cells into a target tissue in a patient. For example, type 1 diabetes and a variety of other disorders have been shown to be curable by hematopoietic stem cell transplantation. Exemplary diseases that can be treated by infusion of hematopoietic stem or progenitor cells into a patient are sickle cell anemia, thalassemia, fanconi's anemia, aplastic anemia, wiskott-aldrich syndrome, ADA SCID, HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. Additional diseases that can 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 cell and progenitor cell transplantation therapies include cancers, such as those described herein. Exemplary stem cell disorders are malignancies, such as neuroblastoma or hematological 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 transplantation 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 from or otherwise be affected by a metabolic disorder selected from the group consisting of: glycogen storage disease, mucopolysaccharide storage disease, gaucher's disease, Heller disease, sphingolipid storage disease, metachromatic leukodystrophy, or any other Disease or disorder that may benefit from the treatments and therapies disclosed herein, and which include, but are not limited to, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper-immunoglobulin M (IgM) syndrome, Cheers' Disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage Disease, thalassemia major, sickle cell Disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and those described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Educationbook,1:319-338(2000), the disclosure of which is incorporated herein by reference in its entirety as it relates to pathologies that can be treated by administration of hematopoietic stem cell or progenitor cell transplantation therapies.

As used herein in the context of hematopoietic stem cell mobilization, the term "stem cell niche" refers to a microenvironment in which endogenous hematopoietic stem cells or progenitor cells are present within a donor, such as a mammalian donor (e.g., a human donor). An exemplary stem cell niche is bone marrow tissue.

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, in need of hematopoietic stem cell transplantation may be treated with a population comprising hematopoietic stem cells in order to treat a stem cell disorder, such as a cancer, an autoimmune disease, or a metabolic disorder described herein. The hematopoietic stem cells transplanted into a patient may be, for example, a population of hematopoietic stem cells mobilized and drawn from a donor according to the compositions and methods described herein. In some embodiments, hematopoietic stem cells transplanted into a patient may be mobilized within the donor by administering a CXCR4 antagonist and/or a CXCR2 agonist to the donor.

As used herein, the term "transfection" refers to any of a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

As used herein, the term "treatment" or "treatment" refers to a therapeutic treatment wherein the objective is to prevent or slow down (lessen) an undesired physiological change or disorder, or promote a beneficial phenotype in the treated patient. Beneficial or desired clinical results include, but are not limited to, facilitating the engraftment of exogenous hematopoietic cells in a patient following hematopoietic stem cell or progenitor cell transplantation therapy. In certain embodiments, benefits include faster 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, within about 5-15 days post-transplant, within about 5-10 days post-transplant, within about 7-12 days post-transplant, within about 8-12 days post-transplant, within about 9-15 days post-transplant, within about 10-15 days post-transplant, or within about 10 days post-transplant. In certain embodiments, using the methods described herein, platelet recovery occurs within about 10-20 days post-transplant, within about 10-15 days post-transplant, within about 15-20 days post-transplant, within about 12-18 days post-transplant, within about 12-17 days post-transplant, within about 13-18 days post-transplant, within about 12-17 days post-transplant, or within 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 hematopoietic stem cell or progenitor cell transplantation following administration of an exogenous hematopoietic stem cell or progenitor cell transplant to the patient. Beneficial results of the therapies described herein may also include an increase in cell count or relative concentration of one or more cells of the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes, after a subsequent hematopoietic stem cell transplantation therapy. Additional beneficial results may include a reduction in the number of pathogenic cell populations, such as populations of cancer cells or autoimmune cells.

As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic and semi-synthetic analogs of the compounds, peptides, proteins or other substances described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original material.

As used herein, the term "vector" includes nucleic acid vectors, such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein may contain polynucleotide sequences as well as additional sequence elements, e.g., for expressing proteins and/or integrating these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used to express peptides and proteins, such as those described herein, include plasmids that contain regulatory sequences (such as promoter and enhancer regions) that direct transcription of genes. Other useful vectors for expressing the 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 mRNA produced by transcription of the genes. These sequence elements may include, for example, 5 'and 3' untranslated regions and polyadenylation signal sites to direct the efficient transcription of genes carried on expression vectors. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

As used herein, the term "alkyl" refers to a straight or branched alkyl group having, for example, 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.

As used herein, the term "alkylene" refers to a straight or branched chain divalent alkyl group. The divalent sites may be on the same or different atoms within the alkyl chain. Examples of alkylene groups include methylene, ethylene, propylene, isopropylene, and the like.

As used herein, the term "heteroalkyl" refers to a straight or branched alkyl group having, for example, from 1 to 20 carbon atoms in the chain and also containing one or more heteroatoms (such as, inter alia, oxygen, nitrogen, or sulfur atoms) in the chain.

As used herein, the term "heteroalkylene" refers to a straight or branched chain divalent heteroalkyl group. The divalent sites may be on the same or different atoms within the heteroalkyl chain. The divalent position may be one or more heteroatoms.

As used herein, the term "alkenyl" refers to a straight or branched chain alkenyl group having, for example, 2 to 20 carbon atoms in the chain. Examples of alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, tert-butenyl, hexenyl, and the like.

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 vinylene, propenylene, isopropenylene, butenylene, and the like.

As used herein, the term "heteroalkenyl" refers to a straight or branched chain alkenyl group having, for example, 2 to 20 carbon atoms in the chain and also containing one or more heteroatoms (such as, inter alia, oxygen, nitrogen, or sulfur atoms) in the chain.

As used herein, the term "heteroalkenylene" refers to a straight or branched divalent heteroalkenylgroup. The divalent positions may be on the same or different atoms within the heteroalkenyl chain. The divalent position may be one or more heteroatoms.

As used herein, the term "alkynyl" refers to straight or branched chain alkynyl groups having, for example, 2 to 20 carbon atoms in the chain. Examples of alkynyl groups include propargyl, butynyl, pentynyl, hexynyl, and the like.

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.

As used herein, the term "heteroalkynyl" refers to a straight or branched chain alkynyl group having, for example, 2 to 20 carbon atoms in the chain and also containing one or more heteroatoms (such as, inter alia, oxygen, nitrogen or sulfur atoms) in the chain.

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 position may be one or more heteroatoms.

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, 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.

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 of cycloalkylene include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and the like.

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, 3 to 12 ring atoms per ring structure selected from carbon atoms and heteroatoms selected from, for example, nitrogen atoms, oxygen atoms, and sulfur atoms, among others. The ring structure may contain one or more oxo groups, for example on a carbon, nitrogen or sulphur ring member.

As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkyl group. The divalent positions may be on the same or different atoms within the ring structure.

As used herein, the term "aryl" refers to a monocyclic or polycyclic 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 position may be one or more heteroatoms.

As used herein, the term "arylene" refers to a divalent aryl group. The divalent positions may be on the same or different atoms.

As used herein, the term "heteroaryl" refers to a monocyclic heteroaromatic or a bicyclic or 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-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-triazinyl, 1,2, 3-triazinyl, benzofuranyl, [2, 3-dihydro ] benzofuranyl, isobenzofuranyl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo [1,2-a ] pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, pyrazolyl, 1,2, 4-triazinyl, 1-triazinyl, and the like, Phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, 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.

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 position may be one or more heteroatoms.

Unless otherwise limited by the definition of an individual substituent, the aforementioned chemical moieties, such as "alkyl", "alkylene", "heteroalkyl", "heteroalkylene", "alkenyl", "alkenylene", "heteroalkenylene", "alkynyl", "alkynylene", "heteroalkynyl", "heteroalkynylene", "cycloalkyl", "cycloalkylene", "heterocycloalkyl", "heterocycloalkylene", "aryl", "arylene", "heteroaryl", and "heteroarylene" groups may be optionally substituted. As used herein, the term "optionally substituted" means that a compound or moiety contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substituents as permitted by the valency of the compound or moiety or position thereof, such as substituents selected from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, mercapto, nitro, and the like. Substitution may include situations where adjacent substituents undergo ring closure, such as ring closure of ortho-functional substituents to form lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals formed by, for example, ring closure to provide, for example, a protecting group.

Methods of mobilizing hematopoietic stem and progenitor cells and releasing cells for expansion and therapeutic use

The present invention is based, in part, on the discovery that hematopoietic stem and progenitor cells can be mobilized while reducing the mobilization of other cell types, such as leukocytes, neutrophils, lymphocytes, and monocytes, by administering to a mammalian donor (e.g., a human donor) a specific dose of a CXCR2 agonist, such as Gro-beta, Gro-beta T, or variants thereof, optionally in combination with a CXCR4 antagonist. This property is particularly beneficial in the context of hematopoietic stem cell transplantation therapies, as hematopoietic stem cells mobilized and isolated from donors using the compositions and methods described herein have a reduced number of cell types that are undesirable for administration to human patients suffering from stem cell disorders.

In particular, it has been found that CXCR2 agonists such as Gro-beta, Gro-beta T, or variants thereof, when administered intravenously at a dose of from about 50 μ g/kg to about 1mg/kg, preferably from about 100 μ g/kg to about 250 μ g/kg, and even more preferably about 150 μ g/kg, exhibit the ability to rapidly mobilize hematopoietic stem and progenitor cells in a donor (e.g., a mammalian donor, such as a human donor) while reducing the ability to mobilize other cells of the hematopoietic lineage that may be undesirable for infusion to a patient undergoing hematopoietic stem cell transplantation therapy (e.g., a mammalian patient, such as a human patient). CXCR2 agonists such as Gro-beta, Gro-beta T, or variants thereof, when administered to donors at the doses described above, exhibit the ability to selectively mobilize hematopoietic stem cells, as described in detail in example 1 below.

When determining whether hematopoietic stem cells mobilized in a donor by administration of a CXCR2 agonist (such as Gro-beta, Gro-beta T, or variants 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 therapeutic use, one can obtain the input values for each of the one or more parameters listed in table 2 that characterize the peripheral blood sample of the donor. One or more parameters can be compared to a corresponding reference standard for each parameter, and if a sample of hematopoietic stem cells meets the reference standard, cells isolated from the donor can be released for ex vivo expansion and/or infusion into a patient for therapeutic use (e.g., for treating one or more stem cell disorders described herein).

Exemplary hematopoietic stem cell parameters and corresponding reference standards that can be used in conjunction with the compositions and methods described herein are listed in table 2 below.

TABLE 2 hematopoietic stem cell population parameters and corresponding reference standards

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 of the input parameters listed in table 2. In some embodiments, one may select individual parameters from parameter numbers 1-21. Alternatively, one may select a combination of parameters, such as CD34 ⁺A combination of a 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 to determine 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 the parameters listed in any of tables 3-6 below.

TABLE 3 Bi-directional combination of hematopoietic stem cell population parameters for evaluation

TABLE 4 three-way combinations of hematopoietic stem cell population parameters used for evaluation

TABLE 5 quadriversal combinations of hematopoietic stem cell population parameters used for evaluation

TABLE 6 five-way combinations of hematopoietic stem cell population parameters for evaluation

CXCR2 agonists

Gro-beta, Gro-beta T and variants thereof

Exemplary CXCR2 agonists that can be used with the compositions and methods described herein are Gro-beta and variants thereof. Gro-beta (also known as growth regulatory protein beta, chemokine (C-X-C motif) ligand 2(CXCL2) and macrophage inflammatory protein 2-alpha (MIP 2-alpha)) is a cytokine that is capable of mobilizing hematopoietic stem and progenitor cells by, for example, stimulating the release of proteases from peripheral neutrophils and in particular MMP 9. Without being limited by mechanism, MMP9 can induce mobilization of hematopoietic stem and progenitor cells from stem cell niches such as bone marrow to circulating peripheral blood by stimulating the degradation of proteins such as stem cell factors, their corresponding receptors CD117 and CXCL12 (all of which generally maintain the hematopoietic stem and progenitor cells fixed in the bone marrow).

In addition to Gro-beta, exemplary CXCR2 agonists that can be used with the compositions and methods described herein are truncated forms of Gro-beta, such as those characterized by deletions of 1 to 8 amino acids at the N-terminus of Gro-beta (e.g., peptides characterized by deletions of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, or 8 amino acids at the N-terminus). In some embodiments, CXCR2 agonists that can be used with the compositions and methods described herein include Gro- β T, which is characterized by the deletion of the first four amino acids from the N-terminus of Gro- β. Gro-beta T exhibits particularly advantageous biological properties, such as the ability to induce mobilization of hematopoietic stem and progenitor cells, with efficiencies several orders of magnitude higher than those of Gro-beta. Gro-beta and Gro-beta T are described, for example, in U.S. Pat. No. 6,080,398, the disclosure of which is incorporated herein by reference in its entirety.

Furthermore, exemplary CXCR2 agonists that can be used with the compositions and methods described herein are Gro- β variants 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- β N69D, retains Gro- β's hematopoietic stem and progenitor cell mobilization function, but induces this effect with excellent efficacy. Similarly, CXCR2 agonists that can be used with the compositions and methods described herein include Gro- β T variants comprising 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- β TN65D, not only retains hematopoietic stem and progenitor cell mobilization ability, but also exhibits much greater efficacy than Gro- β T. Gro- β N69D and Gro- β T N65D are described, for example, in U.S. patent No. 6,447,766, the disclosures of which are incorporated herein by reference in their entirety.

The amino acid sequences of Gro-beta, Gro-beta T, Gro-beta N69D, and Gro-beta T N65D are listed in Table 7 below.

TABLE 7 amino acid sequences of Gro-beta and selected variants thereof

Additional CXCR2 agonists that can be used with the compositions and methods described herein include other variants of Gro-beta, such as peptides having one or more amino acid substitutions, insertions, and/or deletions relative to Gro-beta. In some embodiments, CXCR2 agonists that can be used 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., peptides 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: 1). In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence of SEQ id no:1 only by one or more conservative amino acid substitutions. In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence 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 non-conservative amino acid substitution.

Further examples of CXCR2 agonists that can be used with the compositions and methods described herein are variants of Gro-beta T, such as peptides having one or more amino acid substitutions, insertions, and/or deletions relative to Gro-beta T. In some embodiments, a CXCR2 agonist can 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 a CXCR2 agonist differs from the amino acid sequence of SEQ ID No. 2 only by one or more conservative amino acid substitutions. In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence 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 non-conservative amino acid substitution.

Further examples of CXCR2 agonists that can be used with the compositions and methods described herein are variants of Gro- β N69D, such as peptides having one or more amino acid substitutions, insertions, and/or deletions relative to Gro- β N69D. In some embodiments, a CXCR2 agonist can 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%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3). In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence of SEQ ID No. 3 only by one or more conservative amino acid substitutions. In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence 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 non-conservative amino acid substitution.

Further examples of CXCR2 agonists that can be used with the compositions and methods described herein are variants of Gro- β TN65D, such as peptides having one or more amino acid substitutions, insertions, and/or deletions relative to Gro- β T N65D. In some embodiments, a CXCR2 agonist can be a peptide having at least 85% sequence 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%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4). In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence of SEQ ID No. 4 only by one or more conservative amino acid substitutions. In some embodiments, the amino acid sequence of a CXCR2 agonist differs from the amino acid sequence 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 non-conservative amino acid substitution.

Agonistic anti-CXCR 2 antibodies and antigen binding fragments thereof

In some embodiments, a CXCR2 agonist is an antibody or antigen binding fragment thereof that binds to CXCR2 and activates CXCR2 signaling. In some embodiments, a CXCR2 agonist may be an antibody or antigen binding fragment thereof that binds to the same epitope (e.g., as assessed by a competitive CXCR2 binding assay) on CXCR2 as Gro-beta or variants or truncations thereof such as Gro-beta T. In some embodiments, a CXCR2 agonist is an antibody or antigen binding fragment thereof that competes with Gro-beta or variants or truncations thereof, such as Gro-beta T, for binding to CXCR 2.

In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof is selected from the group consisting of: monoclonal antibodies or antigen-binding fragments thereof, polyclonal antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, bispecific antibodies or antigen-binding fragments thereof, dual variable immunoglobulin domains, single chain Fv molecules (scFv), diabodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F (ab')₂Molecules and tandem di-scFv (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

Peptide CXCR2 agonists described herein, such as Gro-beta, Gro-beta T, and variants thereof, can be prepared synthetically, e.g., using solid phase peptide synthesis techniques. Systems and methods for performing solid phase peptide synthesis include those known in the art and have been described in, for example, U.S. patent nos. 9,169,287; 9,388,212 No; 9,206,222 No; 6,028,172 and 5,233,044, the disclosure of each of which is incorporated herein by reference in its entirety as it relates to protocols and techniques for synthesizing peptides on solid supports. Solid phase peptide synthesis is a process of adding amino acid residues to peptides immobilized on a solid support such as a polymeric resin (e.g., a hydrophilic resin, such as a polyethylene glycol-containing resin, or a hydrophobic resin, such as a polystyrene-based resin).

Peptides, such as those containing protecting groups at amino, hydroxyl, thiol, and carboxyl substituents, among others, can 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 a solid support via their C-termini, thereby immobilizing the peptides for subsequent reaction at the resin-liquid interface.

The process of adding an amino acid residue to the immobilized peptide can include exposing a deprotection reagent to the immobilized peptide to remove at least a portion of the protecting group from at least a portion of the immobilized peptide. The deprotection reagent exposure step may be configured, for example, such that the side chain protecting group is retained, while the N-terminal protecting group is removed. For example, an exemplary amino protection contains a fluorenylmethoxycarbonyl (Fmoc) substituent. A deprotection reagent comprising a strongly basic substance such as piperidine (e.g., a solution of piperidine in a suitable organic solvent such as Dimethylformamide (DMF)) may be exposed to the immobilized peptide such that the Fmoc protecting group is removed from at least a portion of the immobilized peptide. Other protecting groups suitable for protecting amino substituents include, for example, tert-butoxycarbonyl (Boc) moieties. A deprotection reagent comprising a strong acid such as trifluoroacetic acid (TFA) can be exposed to the immobilized peptide containing the Boc-protected amino substituent to remove the Boc protecting group by an ionization process. In this way, the peptide may be protected and deprotected at specific sites, such as at one or more side chains of the immobilized peptide or at the N-terminus or C-terminus, so as to regioselectively append chemical functionality at one or more of these positions. This can be used, for example, to derivatize the side chains of the immobilized peptide, or to synthesize the peptide, for example, from the C-terminus to the N-terminus.

The process of adding amino acid residues to the immobilized peptide can include, for example, exposing a protected activated amino acid to the immobilized peptide such that at least a portion of the activated amino acid bonds to the immobilized peptide to form a newly bonded amino acid residue. For example, the peptide may be exposed to an activated amino acid that reacts with the deprotected N-terminus of the peptide to extend the peptide chain by one amino acid. The amino acid may be activated for reaction with the deprotected peptide 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 convert protected amino acids into activated species (e.g., BOP, PyBOP, HBTU, and TBTU all produce HOBt esters) in the presence of tertiary bases (e.g., Diisopropylethylamine (DIPEA) and Triethylamine (TEA), among others). Other agents may be used to help prevent racemization that may be induced in the presence of a base. These agents include carbodiimides (e.g., DCC or WSCDI) or derivatives thereof with the addition of an auxiliary nucleophile (e.g., 1-hydroxy-benzotriazole (HOBt), 1-hydroxy-azabenzotriazole (HOAt), or HOSu). Another reagent that can be used to prevent racemization is TBTU. Mixed anhydride methods (using isobutyl chloroformate with or without the addition of an auxiliary nucleophile) and azide methods can also be used due to the low racemization associated with this reagent. These types of compounds can also increase the carbodiimide-mediated coupling rate, as well as prevent dehydration of Asn and gin residues. Typical additional reagents also include bases such as N, N-Diisopropylethylamine (DIPEA), Triethylamine (TEA) or N-methylmorpholine (NMM). These agents are described in detail, for example, in U.S. patent No. 8,546,350, the disclosure of which is incorporated herein in its entirety.

During recombinant expression and folding of Gro- β and Gro- β T in aqueous solution, certain C-terminal asparagine residues (Asn 69 within Gro- β and Asn65 within Gro- β T) are susceptible to deamidation. This process results in the conversion of asparagine residues to aspartic acid. Without wishing to be bound by any theory, the chemical synthesis of Gro- β and Gro- β T may overcome this problem by, for example, providing conditions that reduce the exposure of the asparagine residue to nucleophilic solvents. When prepared synthetically (i.e., chemically synthesized), e.g., using solid phase peptide synthesis techniques such as those described above, the synthetic Gro- β, Gro- β T, and variants thereof that can be used with the compositions and methods described herein can have a purity of, e.g., at least about 95% relative to deamidated forms of these peptides (i.e., peptides containing less than 5% of the corresponding deamidation). For example, synthetic Gro- β, Gro- β T, and variants thereof that can be used with the compositions and methods described herein can have a purity of about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or more, relative to the deamidated form of these peptides (e.g., the Asn69 deamidated form of SEQ ID NO:1 or the Asn65 deamidated form of SEQ ID NO: 2). For example, synthetic Gro- β, Gro- β T, and variants thereof can have a purity, e.g., from about 95% to about 99.99% relative to deamidated forms of these peptides (e.g., Asn69 deamidated form of SEQ ID NO:1 or Asn65 deamidated form of SEQ ID NO: 2), such as from about 95% to about 99.99%, about 96% to about 99.99%, about 97% to about 99.99%, about 98% to about 99.99%, about 99% to about 99.99%, about 99.9% to about 99.99%, about 95% to about 99.5%, about 96% to about 99.5%, about 95% to about 99%, or about 97% to about 99%.

CXCR4 antagonists

Exemplary CXCR4 antagonists for use with the compositions and methods described herein are compounds represented by formula (I)

Z-joint-Z' (I)

Or a pharmaceutically acceptable salt thereof, wherein Z is:

(i) cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 ring members are nitrogen atoms separated from each other by 2 or more carbon atoms; or

(ii) An amine represented by the formula (IA)

Wherein a comprises a monocyclic or bicyclic fused ring system containing at least one nitrogen atom, and B is H or a substituent having 1 to 20 atoms;

and wherein Z' is:

(i) cyclic polyamines containing 9 to 32 ring members, wherein 2 to 8 ring members are nitrogen atoms separated from each other by 2 or more carbon atoms;

(ii) an amine represented by the formula (IB)

Wherein a 'comprises a monocyclic or bicyclic fused ring system containing at least one nitrogen atom, and B' is H or a substituent having 1 to 20 atoms; or

(iii) A substituent represented by the formula (IC)

–N(R)–(CR₂)_n–X (IC)

Wherein each R is independently H or C₁-C₆Alkyl, n is 1 or 2, and X is an aryl group or a heteroaryl group or a thiol;

wherein the linker is a bond, optionally substituted alkylene (e.g., optionally substituted C₁-C₆Alkylene), optionally substituted heteroalkylene (e.g., optionally substituted C) ₁-C₆Heteroalkylene), optionally substituted alkenylene (e.g., optionally substituted C)₂-C₆Alkenylene), optionally substituted heteroalkenylene (e.g., optionally substituted C)₂-C₆Heteroalkenylene), optionally substituted alkynylene (e.g., optionally substituted C₂-C₆Alkynylene), optionally substituted heteroalkynylene (e.g., optionally substituted C)₂-C₆Heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene.

In some embodiments, Z and Z' may each independently be a cyclic polyamine containing 9 to 32 ring members, wherein 2 to 8 ring members are nitrogen atoms separated from each other by 2 or more carbon atoms. In some embodiments, Z and Z' are the same substituents. As an example, Z may be a cyclic polyamine containing 10 to 24 ring members. In some embodiments, Z may be a cyclic polyamine containing 14 ring members. In some embodiments, Z includes 4 nitrogen atoms. In some embodiments, Z is 1,4,8, 11-tetraazacyclotetradecane.

In some embodiments, the linker is represented by formula (ID)

As an example, the joint may be represented by the formula (IE)

X and Y are each independently optionally substituted alkylene (e.g., optionally substituted C)₁-C₆Alkylene), optionally substituted heteroalkylene (e.g., optionally substituted C) ₁-C₆Heteroalkylene), optionally substituted C₂-C₆Alkenylene (e.g., optionally substituted C₂-C₆Ene (II)Yl), optionally substituted heteroalkenylene (e.g., optionally substituted C₂-C₆Heteroalkenylene), optionally substituted alkynylene (e.g., optionally substituted C₂-C₆Alkynylene) or optionally substituted heteroalkynylene (e.g., optionally substituted C₂-C₆Heteroalkynylene). In some embodiments, X and Y are each independently optionally substituted C₁-C₆An alkylene group. In some embodiments, X and Y are the same substituent. In some embodiments, X and Y may each be a methylene group, an ethylene group, a n-propylene group, a n-butylene group, a n-pentylene group, or a n-hexylene group. In some embodiments, X and Y are each a methylene group.

The linker may be, for example, 1, 3-phenylene, 2, 6-pyridine, 3, 5-pyridine, 2, 5-thiophene, 4 '- (2, 2' -bipyrimidine), 2,9- (1, 10-phenanthroline), or the like. In some embodiments, the linker is 1, 4-phenylene-bis- (methylene).

CXCR4 antagonists that can be used with the compositions and methods described herein include plerixafor (also referred to herein as "AMD 3100" and "Mozibil"), or a pharmaceutically acceptable salt thereof, represented by formula (II), 1, 1' - [1, 4-phenylenebis (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane.

Additional CXCR4 antagonists that can be used with the compositions and methods described herein include variants of plerixafor, such as the compounds described in U.S. patent No. 5,583,131, the disclosure of which is incorporated herein by reference as it relates to CXCR4 antagonists. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: 1, 1' - [1, 3-phenylenebis (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; 1, 1' - [1, 4-phenylene-bis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; bis-zinc or bis-copper complexes of 1, 1' - [1, 4-phenylene-bis- (methylene) ] -bis-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, 1' - [2, 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-thiophene-bis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; 1,1 ' - [4,4 ' - (2,2 ' -bipyridine) -bis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; 1, 1' - [2,9- (1, 10-phenanthroline) -bis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; 1, 1' - [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-phenylenebis (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-naphthylene-bis- (methylene) ] bis-1, 4,8, 11-tetraazacyclotetradecane; 1, 1' - [1, 3-phenylenebis- (methylene) ] bis-1, 5, 9-triazacyclododecane; 1, 1' - [1, 4-phenylene-bis- (methylene) ] -1,5, 9-triazacyclododecane; 1, 1' - [2, 5-dimethyl-1, 4-phenylenebis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; 1, 1' - [2, 5-dichloro-1, 4-phenylenebis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; 1, 1' - [ 2-bromo-1, 4-phenylenebis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane; and 1, 1' - [ 6-phenyl-2, 4-pyridinebis- (methylene) ] -bis-1, 4,8, 11-tetraazacyclotetradecane.

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 relates to a CXCR4 antagonist. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: 3,7,11, 17-tetraazabicyclo (13.3.1) heptad-1- (17),13, 15-triene; 4,7,10, 17-tetraazabicyclo (13.3.1) heptad-1- (17),13, 15-triene; 1,4,7, 10-tetraazacyclotetradecane; 1,4, 7-triazacyclotetradecane; and 4,7, 10-triazabicyclo (13.3.1) heptad-1 (17),13, 15-triene.

The CXCR4 antagonist may be a compound described in WO 2001/044229, the disclosure of which is incorporated herein by reference as it relates to a CXCR4 antagonist. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: n- [4- (11-fluoro-1, 4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- (11, 11-difluoro-1, 4, 7-triazacyclonetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- (1,4, 7-triazacyclotetradecane-2-acyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [12- (5-oxo-1, 9-diazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- (11-oxo-1, 4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- (11-thia-1, 4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- (11-thia (foxo) -1,4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- (11-sulfonyl-1, 4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; and N- [4- (3-carboxyhetero (carboxo) -1,4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine.

Additional CXCR4 antagonists that can be used in conjunction with the compositions and methods described herein include the compounds described in WO 2000/002870, the disclosure of which is incorporated herein by reference as it relates to CXCR4 antagonists. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis- (methylene) ] -2- (aminomethyl) pyridine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -N-methyl-2- (aminomethyl) pyridine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -4- (aminomethyl) pyridine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -3- (aminomethyl) pyridine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] - (2-aminomethyl-5-methyl) pyrazine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -2- (aminoethyl) pyridine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -2- (aminomethyl) thiophene; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -2- (aminomethyl) thiol; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -2-aminobenzylamine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -4-aminobenzylamine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -4- (aminoethyl) imidazole; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -benzylamine; n- [4- (1,4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [7- (4,7,10, 17-tetraazabicyclo [13.3.1] heptad-1 (17),13, 15-trienyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [7- (4,7, 10-triazabicyclo [13.3.1] heptad-1 (17),13, 15-trienyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [1- (1,4, 7-triazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- [4,7,10, 17-tetraazabicyclo [13.3.1] heptad-1 (17),13, 15-trienyl ] -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [4- [4,7, 10-triazabicyclo [13.3.1] heptad-1 (17),13, 15-trienyl ] -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; n- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -purine; 1- [1,4,8, 11-tetraazacyclotetradecyl-1, 4-phenylenebis (methylene) ] -4-phenylpiperazine; n- [4- (1, 7-diazacyclotetradecyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine; and N- [7- (4, 10-diazabicyclo [13.3.1] heptad-1- (17),13, 15-trienyl) -1, 4-phenylenebis (methylene) ] -2- (aminomethyl) pyridine.

In some embodiments, the CXCR4 antagonist is a compound selected from the group consisting of: 1- [2, 6-dimethoxypyridin-4-yl (methylene) ] -1,4,8, 11-tetraazacyclotetradecane; 1- [ 2-chloropyridin-4-yl (methylene) ] -1,4,8, 11-tetraazacyclotetradecane; 1- [2, 6-dimethylpyridin-4-yl (methylene) ] -1,4,8, 11-tetraazacyclotetradecane; 1- [ 2-methylpyridin-4-yl (methylene) ] -1,4,8, 11-tetraazacyclotetradecane; 1- [2, 6-dichloropyridin-4-yl (methylene) ] -1,4,8, 11-tetraazacyclotetradecane; 1- [ 2-chloropyridin-5-yl (methylene) ] -1,4,8, 11-tetraazacyclotetradecane; and 7- [ 4-methylphenyl (methylene) ] -4,7,10, 17-tetraazabicyclo [13.3.1] heptad-1 (17),13, 15-triene.

In some embodiments, the CXCR4 antagonist is a compound described in U.S. patent No. 5,698,546, the disclosure of which is incorporated herein by reference as it relates to a CXCR4 antagonist. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: 7, 7' - [1, 4-phenylene-bis (methylene)]Bis-3, 7,11, 17-tetraazabicyclo [13.3.1]]Seventeen-1 (17),13, 15-triene; 7, 7' - [1, 4-phenylene-bis (methylene)]Bis [ 15-chloro-3, 7,11, 17-tetraazabicyclo [13.3.1] ]Seventeen-1 (17),13, 15-triene](ii) a 7, 7' - [1, 4-phenylene-bis (methylene)]Bis [ 15-methoxy-3, 7,11, 17-tetraazabicyclo [13.3.1 ]]Seventeen-1 (17),13, 15-triene](ii) a 7, 7' - [1, 4-phenylene-bis (methylene)]Bis-3, 7,11, 17-tetraazabicyclo [13.3.1 ]]-heptadeca-13, 16-trien-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, 8' - [1, 4-phenylene-bis (methylene)]Bis-4, 8,12, 19-tetraazabicyclo [15.3.1 ]]Nineteen-1 (19),15, 17-triene; 6, 6' - [1, 4-phenylene-bis (methylene)]Bis-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 [17.3.1.1^8,12]Twenty-four-1 (23),8,10,12(24),19, 21-hexene.

In some embodiments, the CXCR4 antagonist is a compound described in U.S. patent No. 5,021,409, the disclosure of which is incorporated herein by reference as it relates to a CXCR4 antagonist. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: 2,2 '-bicycloamine (bicyclam), 6' -bicycloamine; 3, 3' - (bis-1, 5,9, 13-tetraazacyclohexadecane); 3, 3' - (bis-1, 5,8,11, 14-pentaazacyclohexadecane); methylene (or polymethylene) bis-1-N-1, 4,8, 11-tetraazacyclotetradecane; 3, 3' -bis-1, 5,9, 13-tetraazacyclohexadecane; 3, 3' -bis-1, 5,8,11, 14-pentaazacyclohexadecane; 5, 5' -bis-1, 4,8, 11-tetraazacyclotetradecane; 2, 5' -bis-1, 4,8, 11-tetraazacyclotetradecane; 2, 6' -bis-1, 4,8, 11-tetraazacyclotetradecane; 11, 11' - (1, 2-ethanediyl) bis-1, 4,8, 11-tetraazacyclotetradecane; 11, 11' - (1, 2-propanediyl) bis-1, 4,8, 11-tetraazacyclotetradecane; 11, 11' - (1, 2-butanediyl) bis-1, 4,8, 11-tetraazacyclotetradecane; 11, 11' - (1, 2-pentanediyl) bis-1, 4,8, 11-tetraazacyclotetradecane; and 11, 11' - (1, 2-hexanediyl) bis-1, 4,8, 11-tetraazacyclotetradecane.

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 relates to a CXCR4 antagonist. In some embodiments, a CXCR4 antagonist can be a compound selected from the group consisting of: n- (2-pyridylmethyl) -N' - (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (6, 7-dihydro-5H-cyclopenta [ b ] pyridin-7-yl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (1,2,3, 4-tetrahydro-1-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (1-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (2-pyridylmethyl) amino ] ethyl ] -N' -1-methyl-1, 2,3, 4-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (1H-imidazol-2-ylmethyl) amino ] ethyl ] -N' - (1-methyl-1, 2,3, 4-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (1,2,3, 4-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (1H-imidazol-2-ylmethyl) amino ] ethyl ] -N' - (1,2,3, 4-tetrahydro-1-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (2-phenyl-5, 6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n, N '-bis (2-pyridylmethyl) -N' - (2-phenyl-5, 6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (5,6,7, 8-tetrahydro-5-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-imidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-5-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-imidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ (2-amino-3-phenyl) propyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-imidazol-4-ylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (2-quinolinylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (2- (2-naphthoyl) aminoethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ (S) - (2-acetylamino-3-phenyl) propyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ (S) - (2-acetylamino-3-phenyl) propyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [3- ((2-naphthylmethyl) amino) propyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- (S) -pyrrolidinylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- (R) -pyrrolidinylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ 3-pyrazolylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ 2-pyrrolylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ 2-thienylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ 2-thiazolylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ 2-furylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (phenylmethyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (2-aminoethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '-3-pyrrolidinyl-N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '-4-piperidinyl-N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (phenyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (7-methoxy-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (6-methoxy-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (1-methyl-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (7-methoxy-3, 4-dihydronaphthyl) -1- (aminomethyl) -4-benzamide; n- (2-pyridylmethyl) -N' - (6-methoxy-3, 4-dihydronaphthyl) -1- (aminomethyl) -4-benzamide; n- (2-pyridylmethyl) -N '- (1H-imidazol-2-ylmethyl) -N' - (7-methoxy-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (8-hydroxy-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-imidazol-2-ylmethyl) -N' - (8-hydroxy-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (8-fluoro-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-imidazol-2-ylmethyl) -N' - (8-fluoro-1, 2,3, 4-tetrahydro-2-naphthyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N' - (5,6,7, 8-tetrahydro-7-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-imidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-7-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (2-naphthylmethyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- (isobutylamino) ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (2-pyridylmethyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (2-furylmethyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (2-guanidinoethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ bis- [ (2-methoxy) phenylmethyl ] amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (1H-imidazol-4-ylmethyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- [ (1H-imidazol-2-ylmethyl) amino ] ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- (phenylureido) ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ [ N "- (N-butyl) carboxamido ] methyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (formylaminomethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ (N "-phenyl) formylaminomethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (carboxymethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (phenylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (1H-benzoimidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (5, 6-dimethyl-1H-benzoimidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine (hydrobromide); n- (2-pyridylmethyl) -N '- (5-nitro-1H-benzoimidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [ (1H) -5-azabenzimidazol-2-ylmethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N- (4-phenyl-1H-imidazol-2-ylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- [2- (2-pyridyl) ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (2-benzoxazolyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (trans-2-aminocyclohexyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (2-phenylethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (3-phenylpropyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N '- (trans-2-aminocyclopentyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -glycinamide; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) - (L) -alaninamide; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) - (L) -asparagine; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -pyrazinamide; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) - (L) -prolinamide; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) - (L) -lysinamide; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -benzamide; n- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -pyridinecarboxamide; n' -benzyl-N- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -urea; n' -phenyl-N- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -urea; n- (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -4- [ [ (2-pyridylmethyl) amino ] methyl ] benzamide; n- (5,6,7, 8-tetrahydro-8-quinolinyl) -4- [ [ (2-pyridylmethyl) amino ] methyl ] benzamide; n, N '-bis (2-pyridylmethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n, N '-bis (2-pyridylmethyl) -N' - (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -1, 4-xylylenediamine; n, N '-bis (2-pyridylmethyl) -N' - (6, 7-dihydro-5H-cyclopenta [ bacteri-pyridin-7-yl) -1, 4-xylylene diamine; n, N '-bis (2-pyridylmethyl) -N' - (1,2,3, 4-tetrahydro-1-naphthyl) -1, 4-xylylenediamine; n, N '-bis (2-pyridylmethyl) -N' - [ (5,6,7, 8-tetrahydro-8-quinolinyl) methyl ] -1, 4-xylylenediamine; n, N '-bis (2-pyridylmethyl) -N' [ (6, 7-dihydro-5H-cyclopenta [ bacteri-pyridin-7-yl) methyl ] -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N- (2-methoxyethyl) -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (2-pyridylmethyl) -N- [2- (4-methoxyphenyl) ethyl ] -N' - (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n, N' -bis (2-pyridylmethyl) -1,4- (5,6,7, 8-tetrahydro-8-quinolinyl) xylylenediamine; n- [ (2, 3-dimethoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n, N' -bis (2-pyridylmethyl) -N- [1- (N "-phenyl-N" -methylureido) -4-piperidinyl ] -1, 3-xylylenediamine; n, N' -bis (2-pyridylmethyl) -N- [ N "-p-toluenesulfonphenylalanyl) -4-piperidinyl ] -1, 3-xylylenediamine; n, N' -bis (2-pyridylmethyl) -N- [1- [3- (2-chlorophenyl) -5-methyl-isoxazol-4-yl ] -4-piperidinyl ] -1, 3-xylylenediamine; n- [ (2-hydroxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -1, 4-xylylenediamine; n- [ (4-cyanophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -1, 4-xylylenediamine; n- [ (4-cyanophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (4-acetamidophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (4-phenoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -1, 4-xylylenediamine; n- [ (1-methyl-2-carboxamido) ethyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (4-benzyloxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -1, 4-xylylenediamine; n- [ (thiophen-2-yl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ bacterial pyridin-9-yl) -1, 4-xylylenediamine; n- [1- (benzyl) -3-pyrrolidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ [ 1-methyl-3- (pyrazol-3-yl) ] propyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [1- (phenyl) ethyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (3, 4-methylenedioxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ 1-benzyl-3-carboxymethyl-4-piperidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (3, 4-methylenedioxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (3-pyridylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ [ 1-methyl-2- (2-tolyl) carboxamido ] ethyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (1, 5-dimethyl-2-phenyl-3-pyrazolon-4-yl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (4-propoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (1-phenyl-3, 5-dimethylpyrazolin-4-ylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ H-imidazol-4-ylmethyl ] -N, N' -bis (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (3-methoxy-4, 5-methylenedioxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (3-cyanophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (3-cyanophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (5-ethylthiophen-2-ylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (5-ethylthiophen-2-ylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (2, 6-difluorophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (2, 6-difluorophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (2-difluoromethoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (2-difluoromethoxyphenylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (1, 4-benzodioxohex-6-ylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n, N' -bis (2-pyridylmethyl) -N- [1- (N "-phenyl-N" -methylureido) -4-piperidinyl ] -1, 4-xylylenediamine; n, N' -bis (2-pyridylmethyl) -N- [ N "-p-toluenesulfonylphenylaminoacyl) -4-piperidinyl ] -1, 4-xylylenediamine; n- [1- (3-pyridinecarboxamido) -4-piperidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [1- (cyclopropylcarboxamido) -4-piperidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [1- (1-phenylcyclopropylcarboxamido) -4-piperidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- (1, 4-benzodioxohex-6-ylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [1- [3- (2-chlorophenyl) -5-methyl-isoxazole-4-carboxamido ] -4-piperidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [1- (2-thiomethylpyridine-3-carboxamido) -4-piperidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ (2, 4-difluorophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (1-methylpyrrol-2-ylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (2-hydroxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (3-methoxy-4, 5-methylenedioxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (3-pyridylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [2- (N "-morpholinylmethyl) -1-cyclopentyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ (1-methyl-3-piperidinyl) propyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- (1-methylbenzimidazol-2-ylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [1- (benzyl) -3-pyrrolidinyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ [ (1-phenyl-3- (N ' -morpholine) ] propyl ] -N, N ' -bis (2-pyridylmethyl) -1, 4-xylylenediamine, N- [1- (iso-propyl) -4-piperidinyl ] -N, N ' -bis (2-pyridylmethyl) -1, 4-xylylenediamine, N- [1- (ethoxycarbonyl) -4-piperidinyl ] -N ' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine, N- [ (1-methyl-3-pyrazolyl) propyl ] -N ' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ 1-methyl-2- (N ', N ' -diethylcarboxamido) ethyl ] -N, N ' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ (1-methyl-2-benzenesulfonyl) ethyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (2-chloro-4, 5-methylenedioxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ 1-methyl-2- [ N '- (4-chlorophenyl) carboxamido ] ethyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (1-acetoxyindol-3-ylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (3-benzyloxy-4-methoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (3-quinolinylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- [ (8-hydroxy) -2-quinolinylmethyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (2-quinolinylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (4-acetamidophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ 1H-imidazol-2-ylmethyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- (3-quinolinylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (2-thiazolylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (4-pyridylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (5-benzyloxy) benzo [ b ] pyrrol-3-ylmethyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- (1-methylpyrazol-2-ylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (4-methyl) -1H-imidazol-5-ylmethyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ [ (4-dimethylamino) -1-naphthyl ] methyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [1, 5-dimethyl-2-phenyl-3-pyrazolon-4-ylmethyl ] -N, N' -bis (2-pyridylmethyl) -1, 4-xylylenediamine; n- [1- [ (1-acetyl-2- (R) -prolyl ] -4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N ' - (2-pyridylmethyl) -1, 3-xylylenediamine, N- [1- [ 2-acetamidobenzoyl-4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N ' - (2-pyridylmethyl) -1, 3-xylylenediamine, N- [ (2-cyano-2-phenyl) ethyl ] -N ' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ (N "-acetyltryptophanyl) -4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (N "-benzoylvalyl) -4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (4-dimethylaminophenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- (4-pyridylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (1-methylbenzimidazol-2-ylmethyl) -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 4-xylylenediamine; n- [ 1-butyl-4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ 1-benzoyl-4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [1- (benzyl) -3-pyrrolidinyl ] -N- [2- (2-pyridyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ (1-methyl) benzo [ b ] pyrrol-3-ylmethyl ] -N- [2- (2-pyridyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [ 1H-imidazol-4-ylmethyl ] -N- [2- (2-pyridyl) ethyl ] -N' - (2-pyridylmethyl) -1, 3-xylylenediamine; n- [1- (benzyl) -4-piperidinyl ] -N- [2- (2-pyridinyl) ethyl ] -N' - (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ 1-methylbenzimidazol-2-ylmethyl ] -N- [2- (2-pyridyl) ethyl ] -N' - (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ (2-phenyl) benzo [ b ] pyrrol-3-ylmethyl ] -N- [2- (2-pyridyl) ethyl ] -N' - (2-pyridylmethyl) -1, 4-xylylenediamine; n- [ (6-methylpyridin-2-yl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 4-xylylenediamine; n- (3-methyl-1H-pyrazol-5-ylmethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 3-xylylenediamine; n- [ (2-methoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 3-xylylenediamine; n- [ (2-ethoxyphenyl) methyl ] -N' - (2-pyridylmethyl) -N- (6,7,8, 9-tetrahydro-5H-cyclohepta [ b ] pyridin-9-yl) -1, 3-xylylenediamine; n- (benzyloxyethyl) -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 3-xylylenediamine; n- [ (2-ethoxy-1-naphthyl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 3-xylylenediamine; n- [ (6-methylpyridin-2-yl) methyl ] -N' - (2-pyridylmethyl) -N- (5,6,7, 8-tetrahydro-8-quinolinyl) -1, 3-xylylenediamine; 1- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] guanidine; n- (2-pyridylmethyl) -N- (8-methyl-8-azabicyclo [3.2.1] oct-3-yl) -1, 4-xylylenediamine; 1- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] homopiperazine; 1- [ [3- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] homopiperazine; trans-and cis-1- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -3, 5-piperidinediamine; n, N' - [1, 4-phenylenebis (methylene) ] bis-4- (2-pyrimidinyl) piperazine; 1- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -1- (2-pyridyl) methylamine; 2- (2-pyridyl) -5- [ [ (2-pyridylmethyl) amino ] methyl ] -1,2,3, 4-tetrahydroisoquinoline; 1- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -3, 4-diaminopyrrolidine; 1- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -3, 4-diacetylaminopyrrolidine; 8- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -2,5, 8-triaza-3-oxabicyclo [4.3.0] nonane; and 8- [ [4- [ [ (2-pyridylmethyl) amino ] methyl ] phenyl ] methyl ] -2,5, 8-triazabicyclo [4.3.0] nonane.

Additional CXCR4 antagonists that may be used with the compositions and methods described herein include those described in WO2001/085196, WO 1999/050461, WO 2001/094420, and WO2003/090512, the disclosure of each of which is incorporated herein by reference as it relates to compounds that inhibit CXCR4 activity or expression.

Expansion of hematopoietic stem and progenitor cells

The hematopoietic cells and progenitor cells may be expanded ex vivo prior to infusion into a patient, for example, by contacting the cells with an aromatic hydrocarbon receptor antagonist. Aromatic hydrocarbon receptor antagonists that may be used with the compositions and methods described herein include those described in U.S. patent No. 9,580,426, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the aromatic hydrocarbon receptor antagonists include those represented by formula (III) or salts thereof

Wherein:

l is selected from-NR_5a(CH₂)_2-3-、-NR_5a(CH₂)₂NR_5b-、-NR_5a(CH₂)₂S-、-NR_5aCH₂CH (OH) -and-NR_5aCH(CH₃)CH₂-; wherein R is_5aAnd R_5bIndependently selected from hydrogen and C_1-4An alkyl group;

R₁selected from the group consisting of thiophenyl, 1H-benzimidazolyl, isoquinolinyl, 1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, and thiazolyl; in some embodiments, wherein R is₁The thiophenyl, 1H-benzimidazolyl, isoquinolinyl, 1H-imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, or thiazolyl group of (a) may be optionally substituted with 1 to 3 groups independently selected from: cyano, hydroxy, C _1-4Alkyl radical, C_1-4Alkoxy, halogen substituted-C_1-4Alkyl, halogen substituted-C_1-4Alkoxy, amino, -C (O) R_8a、-S(O)_0-2R_8a、-C(O)OR_8aand-C (O) NR_8aR_8b(ii) a Wherein R is_8aAnd R_8bIndependently selected from hydrogen and C_1-4An alkyl group;

R₂selected from-S (O)₂NR_6aR_6b-、-NR_6aC(O)R_6b-、-NR_6aC(O)NR_6bR_6cPhenyl, 1H-pyrrolopyridin-3-yl, 1H-pyrrolopyridin-5-yl, 1H-indolylthiophenyl, pyridyl, 1H-1,2, 4-triazolyl, 2-oxoimidazolidinyl, 1H-pyrazolyl, 2-oxo-2, 3-dihydro-1H-benzimidazolyl and 1H-indazolyl; wherein R is_6a、R_6bAnd R_6cIndependently selected from hydrogen and C_1-4An alkyl group; and R is₂Phenyl, 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-benzimidazolyl, or 1H-indazolyl are optionally substituted with 1 to 3 groups independently selected from: hydroxy, halogen, methyl, methoxyAmino, -O (CH)₂)₂NR_7aR_7b、-S(O)₂NR_7aR_7b、-OS(O)₂NR_7aR_7band-NR_7aS(O)₂R_7b(ii) a Wherein R is_7aAnd R_7bIndependently selected from hydrogen and C_1-4An alkyl group;

R₃selected from hydrogen, C_1-4Alkyl and biphenyl radicals; and is

R₄Is selected from C_1-10Alkyl, prop-1-en-2-yl, cyclohexyl, cyclopropyl, 2- (2-oxopyrrolidin-1-yl) ethyl, oxetan-2-yl, oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, phenyl, tetrahydrofuran-3-yl and benzyl, (4-pentylphenyl) (phenyl) methyl and 1- (1- (2-oxo-6, 9, 12-trioxa-3-azatetradecan-14-yl) -1H-1,2, 3-triazol-4-yl) ethyl, wherein said alkyl, cyclopropyl, cyclohex-1-en-2-yl, cyclohexyl, cyclopropyl, tetrahydro-2H-pyran-1-yl) ethyl, Cyclohexyl, 2- (2-oxopyrrolidin-1-yl) ethyl, oxetan-3-yl, oxetan-2-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H-pyran-3-yl, phenyl, tetrahydrofuran-3-yl, benzyl, (4-pentylphenyl) (phenyl) methyl or 1- (1- (2-oxo-6, 9, 12-trioxa-3-azatetradecan-14-yl) -1H-1,2, 3-triazol-4-yl) ethyl may optionally be substituted with 1 to 3 groups independently selected from: hydroxy, C _1-4Alkyl and halogen substituted C_1-4An alkyl group.

In some embodiments, the aromatic hydrocarbon receptor antagonists that may be used with the compositions and methods described herein include SR-1, represented by the following formula (1).

Methods for recombinant expression of peptides and proteins

The peptides and proteins described herein (e.g., CXCR2 agonists, such as Gro-beta, Gro-beta T, Gro-beta N69D, Gro-beta TN65D, and variants thereof) can be expressed in a host cell by, for example, delivering to the host cell a nucleic acid encoding the corresponding peptide or protein. The following sections describe various techniques that may be used for the purpose of introducing nucleic acids encoding the peptides and proteins described herein into host cells for the purpose of recombinant expression.

Transfection technique

Techniques that can be used to introduce polynucleotides, such as nucleic acids encoding CXCR2 agonists (such as Gro- β, Gro- β T, Gro- β N69D, Gro- β T N65D, or variants thereof) into cells (e.g., mammalian cells, such as human cells) are known in the art in some embodiments, electroporation can be used to permeabilize mammalian cells (e.g., human cells) by applying an electrostatic potential to the cells of interest, mammalian cells, such as human cells, that are subjected to an external electric field in this manner are then susceptible to uptake of exogenous nucleic acids ^TMThe application of an electric field is used to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. Nucleofection^TMAnd protocols that can be used to carry out this technique are described in detail, for example, in Distler et al (2005) Experimental Dermatology 14:315, and US2010/0317114, the disclosures of each of which are incorporated by reference.

Additional techniques that may be used to transfect host cells for recombinant peptide and protein expression purposes include extrusion-perforation (s-perforation). This technique induces rapid mechanical deformation of the cell to stimulate uptake of exogenous DNA through the membrane pores formed in response to applied pressure. An advantage of this technique is that no vector is required to deliver the nucleic acid into a cell, such as a human cell. Squeeze perforation is described in detail, for example, in Sharei et al (2013) Journal of visualized experiments 81: e50980, the disclosure of which is incorporated herein by reference.

Lipofectation represents another technique that can be used for cell transfection. The method comprises loading the nucleic acid into liposomes, which typically display cationic functional groups, such as quaternary ammonium or protonated amines, to the exterior of the liposomes. Due to the anionic nature of the cell membrane, this promotes electrostatic interactions between the liposome and the cell, which ultimately leads to the uptake of exogenous nucleic acids, either by direct fusion of the liposome with the cell membrane or endocytosis of the complex, for example. Lipofectins are described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with cell membranes to facilitate uptake of foreign nucleic acids include contacting the cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides in order to impart a positive charge that facilitates interaction with cell membranes are activated dendrimers (e.g., as described 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 in, for example, gulck et al (1997) Current Protocols in Molecular Biology 40: I:9.2:9.2.1, the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technique is described in detail in, for example, US 2010/0227406, the disclosure of which is incorporated herein by reference.

Another useful tool for inducing uptake of exogenous nucleic acids by cells is laser transfection, a technique which involves exposing cells to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow the polynucleotides to penetrate the cell membrane. This technique is described in detail in Rhodes et al (2007) Methods in Cell Biology82:309, the disclosure of which is incorporated herein by reference.

Microvesicles represent another potential vehicle that can be used to introduce nucleic acids encoding the peptides or proteins described herein into host cells for the purpose of recombinant expression. In some embodiments, microvesicles induced by co-overexpression of the glycoprotein VSV-G with, for example, a genome modification protein, such as a nuclease, can be used to efficiently deliver proteins into cells that subsequently catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the cellular genome for covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles (also known as Gesicles) for the Genetic Modification of eukaryotic Cells is in the Quinn et al Genetic Modification of Target Cells by Direct deletion of active Protein [ Abstract ], in the Methylation changes in early organizing genes in the Abstract ], in the Proceedings of the 18th annular Meeting of the American society of Gene and Cell Therapy; 2015May 13, abstract No. 122.

Viral vectors for nucleic acid delivery

The viral genome provides an abundant source of vectors that can be used to efficiently deliver exogenous nucleic acids encoding the peptides and proteins described herein, such as CXCR2 agonists, including Gro-beta, Gro-beta T, Gro-beta N69D, Gro-beta T N65D and variants thereof, into host cells for recombinant expression purposes. Viral genomes are particularly useful vectors for gene delivery, as polynucleotides contained in such genomes can be incorporated into the genome of a cell by, for example, generalized or specialized transduction. These processes may occur as part of the natural replication cycle of the viral vector and may not require the addition of proteins or agents that induce gene integration. Examples of viral vectors that can be used to introduce nucleic acid molecules encoding the peptides or proteins described herein into host cells for recombinant expression include parvoviruses (paraviruses) such as adeno-associated viruses (AAV), retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), coronaviruses (coronavirus), negative strand RNA viruses such as orthomyxoviruses (such as influenza viruses), rhabdoviruses (e.g., rabies viruses and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and sendai viruses), positive strand RNA viruses such as picornaviruses (picornaviruses) and alphaviruses (alphavirus), and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex viruses type 1 and 2, MVA, cytomegalovirus) and epstein barr (e.g., vaccinia virus, modified vaccinia virus (MVA), Chicken pox virus and canarypox virus (canarypox)). Other viruses that may be used to deliver polynucleotides encoding the peptides and proteins described herein to a host cell for recombinant expression purposes include, for example, norwalk virus, togavirus, flavivirus, reovirus, papova virus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma virus, mammalian type C virus, type B virus, type D virus, HTLV-BLV group, lentivirus, spumavirus (springin, J.M., Retroviridae: The viruses and The replication, In Fundamental Virology, third edition, B.N.fields, et al, Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine (murine) leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine (bovine) leukemia virus, feline (feline) leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon (baboon) endogenous virus, Gibbon (Gibbon ape) leukemia virus, Mason Pfizer monkey virus, simian (simian) immunodeficiency virus, simian sarcoma virus, rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it relates to viral vectors for gene delivery and expression of recombinant proteins and peptides.

Method of treatment

As described herein, hematopoietic stem cell transplantation therapy can be administered to a subject in need of treatment in order to fill or refill one or more blood cell types (such as a blood cell lineage deficient or defective in a patient suffering from a stem cell disorder). Hematopoietic stem and progenitor cells exhibit multipotency, and thus can differentiate into a variety of different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., promegakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Additionally, hematopoietic stem cells are capable of self-renewal and thus can produce progeny cells with equivalent potential to the parent cells, and also feature the ability to be reintroduced into the transplant recipient, at which time they home to the hematopoietic stem cell niche and reconstitute productive and sustained hematopoiesis. Thus, hematopoietic stem and progenitor cells represent a useful therapeutic modality for the treatment of a wide range of disorders, where patients have a deficiency or lack of a cell type of the hematopoietic lineage. The defect or deficiency may result from, for example, depletion of a population of hematopoietic system endogenous cells as a result of administration of a chemotherapeutic agent (e.g., where the patient suffers from a cancer, such as a hematologic cancer described herein). The defect or deficiency may result from, for example, depletion of a population of endogenous hematopoietic cells due to autoreactive 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 that described herein). Additionally or alternatively, a defect or lack of cellular activity may result from abnormal expression of the enzyme (e.g., where a patient suffers from a variety of metabolic disorders, such as the metabolic disorders described herein).

Thus, hematopoietic stem cells can be administered to patients having one or more cell types deficient or lacking in hematopoietic lineage in one or more cell types of hematopoietic lineage in order to reconstitute the population of deficient or deficient cells in vivo, thereby treating pathologies associated with the deficiency or depletion of endogenous blood cell populations. Hematopoietic stem and progenitor cells can be used to treat, for example, 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 can 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 bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells so mobilized can then be drawn from the donor and administered to a patient where the cells can home to the hematopoietic stem cell niche and reconstitute a population of damaged or deficient cells in the patient.

Additionally or alternatively, hematopoietic stem and progenitor cells may be used to treat immunodeficiency, such as congenital immunodeficiency. Additionally or alternatively, the compositions and methods described herein may be used to treat acquired immunodeficiency (e.g., acquired immunodeficiency selected from the group consisting of HIV and AIDS). In these cases, for example, a CXCR4 antagonist and/or a CXCR2 agonist can 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 bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells so mobilized can then be drawn from the donor and administered to a patient where the cells can home to the hematopoietic stem cell niche and reconstitute a population of damaged or deficient cells (e.g., T lymphocytes, B lymphocytes, NK cells, or other immune cells) in the patient.

Hematopoietic stem and progenitor cells can also be used to treat metabolic disorders (e.g., a metabolic disorder selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, gaucher's disease, herler's disease, sphingolipid storage disease, and metachromatic leukodystrophy). In these cases, for example, a CXCR4 antagonist and/or a CXCR2 agonist can 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 bone marrow, into circulating peripheral blood in response to such treatment. The hematopoietic stem and progenitor cells so mobilized may then be drawn from the donor and administered to a patient where the cells can home to the hematopoietic stem cell niche and reconstitute a population of damaged or deficient hematopoietic cells in the patient.

Additionally or alternatively, the hematopoietic stem or progenitor cells may be used to treat a malignant tumor or a proliferative disorder, such as a hematologic cancer or a myeloproliferative disease. In the context of cancer therapy, for example, CXCR4 antagonists and/or CXCR2 agonists 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 bone marrow, into circulating peripheral blood in response to such therapy. The hematopoietic stem and progenitor cells so mobilized may then be drawn from the donor and administered to a patient where the cells may home to the hematopoietic stem cell niche and reconstitute a population of damaged or deficient cells in the patient, such as a population of hematopoietic cells damaged or deficient as a result of administration of one or more chemotherapeutic agents to the patient. In some embodiments, hematopoietic stem or progenitor cells can be infused into a patient in order to refill the population of cells depleted during cancer cell elimination, such as during systemic chemotherapy. Exemplary hematologic cancers that can be treated by administration of hematopoietic stem and progenitor cells according to the compositions and methods described herein are acute myelogenous leukemia, acute lymphatic leukemia, chronic myelogenous leukemia, chronic lymphatic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-hodgkin's lymphoma, as well as other cancerous conditions, including neuroblastoma.

Hematopoietic stem or progenitor cells mobilized into the peripheral blood of a subject can be drawn (e.g., harvested or collected) from the subject by any suitable technique. For example, hematopoietic stem or progenitor cells can be drawn by blood withdrawal. In some embodiments, as contemplated herein, hematopoietic stem or progenitor cells mobilized into the peripheral blood of a subject can be harvested (i.e., collected) using apheresis. In some embodiments, apheresis may be used to enrich the donor's blood for mobilized hematopoietic stem or progenitor cells.

Additional diseases that may be treated by administering hematopoietic stem and progenitor cells to a patient include, but are not limited to, adenosine deaminase deficiency and severe combined immunodeficiency, hyper-immunoglobulin M syndrome, east-cutting disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

In addition, the administration of hematopoietic stem and progenitor cells can be used to treat autoimmune disorders. In some embodiments, following infusion into a patient, the transplanted hematopoietic stem and progenitor cells can home to a stem cell niche such as bone marrow and establish productive hematopoiesis. This in turn can reconstitute the population of cells depleted during the depletion of autoimmune cells, which may occur due to the activity of autoreactive lymphocytes (e.g., autoreactive T lymphocytes and/or autoreactive B lymphocytes). Autoimmune diseases that can be treated by administering hematopoietic stem and progenitor cells to a patient include, but are not limited to, psoriasis, psoriatic arthritis, Type 1diabetes mellitus (Type 1diabetes mellitus), Rheumatoid Arthritis (RA), human Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), Inflammatory Bowel Disease (IBD), lymphocytic colitis, Acute Disseminated Encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, Autoimmune Inner Ear Disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, baliosis, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas ' disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, crohn's disease, cicatricial pemphigoid, celiac-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, malignant atrophic papulopathy, discoid lupus, autonomic dysfunction, endometriosis, primary mixed cryoglobulinemia, fibromyalgia-fibromyositis, goodpasture's syndrome, graves 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 disease, lichen planus, lyme disease, meniere disease, Mixed Connective Tissue Disease (MCTD), myasthenia gravis, neuromyotonia, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (crsychiasm), sclerosing disease (MCTD), autoimmune diseases (ecs), autoimmune diseases, Strabismus myoclonus syndrome (OMS), optic neuritis, alder's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, primary agammaglobulinemia, raynaud's phenomenon, reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, sjogren's syndrome, stiff man's 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.

In some embodiments, a method of harvesting hematopoietic stem cells from a human subject is provided. The method comprises administering to a human subject a CXCR2 agonist and a CXCR4 antagonist, and harvesting hematopoietic stem cells from peripheral blood of the human subject.

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 hematopoietic stem cells from peripheral blood of the donor, and transplanting the harvested hematopoietic stem cells into a patient.

Selection of donors and patients

In some embodiments, the patient is a donor. In such cases, the drawn hematopoietic stem or progenitor cells may be reinfused into the patient so that these cells can subsequently home to the hematopoietic tissue and establish productive hematopoiesis, filling or refilling the deficient or deficient cell line in the patient (e.g., populations of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes). In this case, the transplanted hematopoietic stem or progenitor cells are least likely to undergo graft rejection because the infused cells are derived from the patient and express the same HLA class I and HLA class II antigens as the patient expresses.

Alternatively, the patient and donor may be different. In some embodiments, the patient and donor are related, and may, for example, be HLA matched. As described herein, HLA-matched donor-recipient pairs have a reduced risk of graft rejection because endogenous T cells and NK cells in the graft recipient are less likely to recognize the incoming hematopoietic stem cell or progenitor cell graft as foreign and, therefore, less likely to generate an immune response against the graft. Exemplary HLA-matched donor-recipient pairs are genetically related donors and recipients, such as familial donor-recipient pairs (e.g., sibling donor-recipient pairs).

In some embodiments, the patient and donor are HLA mismatched, which occurs when there is a mismatch of at least one HLA antigen between the donor and recipient, particularly antigens related to HLA-A, HLA-B and HLA-DR. For example, to reduce the likelihood of transplant rejection, one haplotype may be matched and the other haplotype may not be matched between the donor and recipient.

CD34^dimCells

Use of a composition comprising CD34 when the donor and patient are different^dimMethods of treatment of cellular hematopoietic stem cells are particularly useful, in part, because of CD34 ^dimThe cells are capable of inhibiting the proliferation of alloreactive T lymphocytes when administered to a recipient, thereby reducing the risk of Graft Versus Host Disease (GVHD). (D' Aveni et al (2015), supra). In certain embodiments, administration of a CXCR2 agonist and a CXCR4 antagonist will comprise CD34 according to the methods disclosed herein^dimHematopoietic stem cells of the cells are mobilized from the bone marrow of the donor into the peripheral blood. CD34 compared to peripheral blood from an unmoved mammal^dimCells are present in higher amounts in peripheral blood. In certain embodiments, CD34^dimCells are present in peripheral blood in higher amounts than if hematopoietic stem cells were mobilized using CXCR4 antagonist alone.

Thus, the methods disclosed herein can be used to perform allogeneic hematopoietic stem cell transplantation in a patient in need thereof. For example, the method can comprise infusing into the patient a therapeutically effective amount of allogeneic hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from the bone marrow of the human donor into the peripheral blood of the human donor using the methods herein. In certain embodiments, the method comprises administering to the donor (i) a CXCR2 agonist selected from the group consisting of Gro-beta, Gro-beta T, and variants thereof, and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg.

Furthermore, CD34 was found to be present in patients with standard risk disease who received allogeneic hematopoietic cell transplantation^dimCells have been shown to increase Overall Survival (OS), reduce non-recurring mortality (NRM, i.e. time to death without recurrence (relapse)/recurrence (recurrence), and reduce the risk of infection (e.g. Cytomegalovirus (CMV) infection). (Nakasone et al "CD 34⁺monocytes mobilized by G-CSF in donor PB and clinical outcomes afterall-HCT from related donors,”Poster presented at 44^thAnnual Meeting of theEuropean Society for Blood and Marrow Transplantation,March 18-21,2018,Lisbon,Portugal.)。

Thus, in certain embodiments, the methods of treating a stem cell disorder in a human patient disclosed herein may comprise infusing into the patient a therapeutically effective amount of a hematopoietic stem cell mobilized by any of the methods disclosed herein, wherein the mobilized hematopoietic stem cell comprises CD34^dimA cell, and wherein the treatment results in increased OS, decreased NRM, and/or decreased risk of infection (e.g., CMV infection).

Furthermore, the methods described herein may be used to prevent, reduce the risk of developing, or reduce the severity of post-transplant infection in a patient in need thereof. The method can comprise infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from the bone marrow of the human donor into the peripheral blood of the human donor according to the methods described herein, e.g., administering to the human donor (i) a CXCR2 agonist selected from the group consisting of Gro-beta, Gro-beta T, and variants thereof, and (ii) a CXCR4 antagonist at a dose of from about 50 μ g/kg to about 1,000 μ g/kg. In certain embodiments, the infection is a CMV infection.

Furthermore, the present disclosure relates to a method of preventing, reducing the risk of, or reducing the severity of Graft Versus Host Disease (GVHD) in a patient in need thereof, wherein the method comprises infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells are mobilized from the bone marrow of the mammalian donor into the peripheral blood by the methods described herein, e.g., comprising administering to the mammalian donor a CXCR2 agonist and a CXCR4 antagonist.

In certain embodiments, hematopoietic stem and progenitor cells mobilized from the bone marrow of the 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 of CD34 as compared to peripheral blood from an unmoved mammal^dimA cell. In certain embodiments, the hematopoietic stem and progenitor cells mobilized from the bone marrow of the donor into peripheral blood comprise 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% of CD34 as compared to peripheral blood from an unmoved mammal ^dimA cell.

In certain embodiments, hematopoietic stem and progenitor cells mobilized from the bone marrow of the 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 CD34 as compared to peripheral blood from an unmoved mammal^dimA cell. In certain embodiments, hematopoietic stem and progenitor cells mobilized from the bone marrow of the 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 compared to peripheral blood from an unmoved mammalCD34 (1)^dimA cell.

In certain embodiments, hematopoietic stem and progenitor cells mobilized from the bone marrow of the 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 of CD34 as compared to hematopoietic stem cells mobilized if a CXCR4 antagonist alone was used ^dimA cell. In certain embodiments, the hematopoietic stem and progenitor cells mobilized from the bone marrow of the donor into peripheral blood comprise 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%, from about 34%, from about 15% to about 25% of CD34, as compared to hematopoietic stem cells mobilized if a CXCR4 antagonist alone was used^dimA cell.

In certain embodiments, hematopoietic stem and progenitor cells mobilized from the bone marrow of the 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 CD34 than hematopoietic stem cells mobilized if the CXCR4 antagonist alone was used ^dimA cell. In certain embodiments, hematopoietic stem and progenitor cells mobilized from the bone marrow of the 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 CD34 than hematopoietic stem cells mobilized if a CXCR4 antagonist alone is used^dimA cell.

Methods for genetic modification of hematopoietic stem and progenitor cells

Hematopoietic stem cells (or progeny thereof) obtained from a donor may be genetically modified by, for example, disruption of an endogenous gene prior to infusion into a patient, such as a patient having one or more of the stem cell disorders described herein. For example, the strategy may be used to silence the expression of one or more major histocompatibility complex genes in hematopoietic stem cells that are allogeneic to the patient, thereby reducing the likelihood of graft rejection following transplantation.

A wide variety of methods for disrupting target genes in a cell population have been established. In some embodiments, one such method is through the use of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system that originally evolved into an adaptive defense mechanism against viral infections in bacteria and archaea. The CRISPR/Cas system includes a palindromic repeat within the plasmid DNA and an associated Cas9 nuclease. This integration of DNA and protein directs site-specific DNA cleavage of the target sequence by first incorporating foreign DNA into the CRISPR locus. Polynucleotides containing these foreign sequences and CRISPR locus repeat spacer elements are then transcribed in the host cell to produce a guide RNA that can then anneal to the target sequence and localize the Cas9 nuclease at that site. In this way, highly site-specific cas 9-mediated DNA cleavage can be generated in foreign polynucleotides, since the interaction of cas9 in close proximity to the target DNA molecule is controlled by RNA-DNA hybridization. Thus, one can theoretically design a CRISPR/Cas system that cleaves any target DNA molecule of interest. This technique has been used to edit the genome of eukaryotes (Hwang et al (2013) nature biotechnology 31:227, the disclosure of which is incorporated herein by reference), and can be used as an effective means to site-specifically edit the genome of hematopoietic stem cells, for example to cleave DNA prior to incorporation of the gene encoding the target protein. The use of CRISPR/Cas for modulating gene expression has been described, for example, in US 8,697,359, the disclosure of which is incorporated herein by reference. Alternative methods for site-specific cleavage of genomic DNA prior to incorporation of the gene of interest into hematopoietic stem cells include the use of Zinc Finger Nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike CRISPR/Cas systems, these enzymes do not contain a guide polynucleotide that is targeted to a specific target sequence. Rather, target specificity is controlled by the DNA binding domain within these enzymes. Uses of ZFNs and TALENs in genome editing applications are described, for example, in Urnov et al (2010) nature reviews Genetics 11: 636; and in journal et al (2013) Nature Reviews Molecular biology 14:49, the disclosures of both of which are incorporated herein by reference.

Additional genome editing techniques that can be used to incorporate a polynucleotide encoding a target gene into the genome of hematopoietic stem cells include the use of ARCUS^TMMeganucleases that can be rationally designed to site-specifically cleave genomic DNA. In view of the established structure-activity relationships that have been established for such enzymes, it is advantageous to use these enzymes to incorporate a gene encoding a target gene into the genome of a mammalian cell. Single-stranded meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at desired positions, thereby enabling site-specific incorporation of target genes into the nuclear DNA of hematopoietic stem cells. These single-stranded nucleases have been widely described in, for example, US8,021,867 and US8,445,251, the disclosures of each of which are incorporated herein by reference.

Kinetics of CXCR2 agonist and CXCR4 antagonist administration

For the case where both a CXCR4 antagonist and a CXCR2 agonist are administered to the donor, the two agents may be administered concurrently to the donor. In some embodiments, a CXCR4 antagonist and a CXCR2 agonist can be co-formulated with each other and administered in the same pharmaceutical composition. Alternatively, CXCR4 antagonists and CXCR2 agonists may be formulated as different pharmaceutical compositions and administered separately but simultaneously to the donor.

In some embodiments, a CXCR4 antagonist is administered to the donor prior to administration of a CXCR2 agonist. In some embodiments, the CXCR4 antagonist can be about 30 minutes to about 180 minutes, such as 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 prior to the 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, 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 60 minutes, or about 80 minutes prior to the administration of the CXCR2 agonist, About 170 minutes, about 175 minutes, or about 180 minutes) is administered to the donor. In some embodiments, the CXCR4 antagonist is administered to the donor 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, a CXCR4 antagonist can be administered to a donor about 45 minutes prior to the administration of a CXCR2 agonist.

The isolation of the population of hematopoietic stem or progenitor cells may begin about 10 minutes to about 60 minutes (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 after the administration of the CXCR4 antagonist and CXCR2 agonist is complete) after the administration of the CXCR4 antagonist and CXCR2 agonist is complete About 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes). In some embodiments, isolation of the population of hematopoietic stem or progenitor cells begins about 15 minutes after completion of administration of the CXCR4 antagonist and CXCR2 agonist.

In some embodiments, the population of hematopoietic stem or progenitor cells is isolated from the donor over a period of time 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 can be isolated from the donor over a period of time 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).

In some embodiments, hematopoietic stem or progenitor cells can be harvested by apheresis. In some embodiments, hematopoietic stem or progenitor cells can be harvested by drawing peripheral blood from a donor (i.e., a subject).

Routes of administration of CXCR2 agonists and CXCR4 antagonists

In any given case, the most suitable route of administration will depend on the particular agent being administered, the patient, the pharmaceutical formulation, the method of administration (e.g., time of administration and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the diet of the patient, and the excretion rate of the patient⁺CD90⁺CD45RA^-A cell population of cells (hematopoietic stem cells), and reduce mobilization of other cell types, such as leukocytes, neutrophils, lymphocytes, and monocytes. This property is described in further detail in example 1 below.

Pharmaceutical composition

The CXCR2 agonist and CXCR4 antagonist contemplated herein can each be formulated into a pharmaceutical composition for administration to a subject, such as a mammalian subject (e.g., a human subject). For example, pharmaceutical compositions comprising a CXCR2 agonist and/or a CXCR4 antagonist in admixture with one or more suitable diluents, carriers and/or excipients are contemplated herein. The pharmaceutical composition may comprise a sterile aqueous suspension. Conventional procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington, The Science and Practice of Pharmacy (2012, 22 nd edition), and in The united states Pharmacopeia, The National Formulary (2015, USP 38NF 33), The disclosures of which are incorporated herein by reference in their entirety.

The pharmaceutical compositions may be administered to a subject, such as a human subject, alone or in combination with a pharmaceutically acceptable carrier, the proportions of which may be determined by the amount of active pharmaceutical ingredient (i.e., CXCR2 agonist and/or CXCR4 antagonist), the chosen route of administration, and standard pharmaceutical practice.

Administration and administration of CXCR2 agonists and/or CXCR4 antagonists

Contemplated CXCR2 agonists and CXCR4 antagonists can be administered to a subject, such as a mammalian subject (e.g., a human subject), by one or more routes of administration. For example, contemplated CXCR2 agonists and CXCR4 antagonists may be administered to a subject by intravenous infusion, intraperitoneal infusion, intramuscular infusion, intra-arterial infusion, or subcutaneous infusion, among others.

Contemplated CXCR2 agonists and CXCR4 antagonists can be administered to a subject in one or more doses. For example, a CXCR2 agonist and/or a CXCR4 antagonist can be administered in a single dose or in two, three, four, five or more doses. When multiple doses are administered, subsequent doses can be provided on the same day or during one or more days, weeks, months or years after the initial dose. For example, contemplated CXCR2 agonists and CXCR4 antagonists described herein may be administered to a subject, such as a human subject, once or more per day, weekly, monthly, or yearly depending on factors such as, for example, the subject's age, weight, sex, subject's diet, and subject's rate of excretion. In certain embodiments, contemplated CXCR2 agonists and CXCR4 antagonists are each administered in a single dose once daily.

The hematopoietic stem or progenitor cells and pharmaceutical compositions described herein can be administered to a subject in one or more doses. When multiple doses are administered, subsequent doses can be provided one or more days, weeks, months, or years after the initial dose. For example, depending on factors such as, for example, the age, weight, sex, severity of the disease being treated, diet of the subject, and excretion rate of the subject, hematopoietic stem cells and pharmaceutical compositions described herein may be administered to a subject, such as a human subject suffering from one or more of the diseases, conditions, or disorders described herein, one or more times daily, weekly, monthly, or yearly.

Examples

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 can 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.

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