WO2019113375A2 - Dosing regimens for the mobilization of hematopoietic stem and progenitor cells - Google Patents
Dosing regimens for the mobilization of hematopoietic stem and progenitor cells Download PDFInfo
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Definitions
- the invention relates to the mobilization of hematopoietic stem and progenitor cells from a donor, such as a human donor, and to the treatment of patients suffering from various pathologies, such as blood diseases, metabolic disorders, cancers, and autoimmune diseases, among others.
- hematopoietic stem cells have significant therapeutic potential
- a limitation that has hindered their use in the clinic has been the difficulty associated with releasing hematopoietic stem cells from the bone marrow into the peripheral blood of a donor, from which the hematopoietic stem cells may be isolated for infusion into a patient.
- a further limitation is that up to 80% of mobilized peripheral blood (mPB) allogeneic recipients will experience graft-versus-host disease (GVHD).
- GVHD graft-versus-host disease
- G-CSF mobilized grafts contain myeloid-derived suppressor cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro.
- MDSCs myeloid-derived suppressor cells
- the percentage of MDSCs is variable in grafts mobilized with G-CSF, and clinical data suggest that patients transplanted with mPB grafts that contain higher numbers of MDSCs may have better outcomes including lower rates of acute GVHD
- compositions and methods for promoting the mobilization of hematopoietic stem and progenitor cells are currently a need for compositions and methods for promoting the mobilization of hematopoietic stem and progenitor cells, and particularly for methods of identifying populations of mobilized cells that are suitable for therapeutic use.
- compositions and methods for promoting the mobilization of hematopoietic stem and progenitor cells that consistently produce higher numbers of MDSCs than do prior art methods.
- the present invention provides compositions and methods for mobilizing
- the subject may be a hematopoietic stem and progenitor cell donor (z.e., a donor), such as a mammalian donor, and particularly a human donor.
- a donor hematopoietic stem and progenitor cell donor
- the invention additionally provides compositions and methods for the treatment of disorders, such as stem cell disorders, in a patient, such as a human patient.
- a C-X-C chemokine receptor type 2 (CXCR2) agonist such as Gro-b or a variant thereof, such as a truncated form of Gro- b (e.g, Gro-b T), as described herein, optionally in combination with a C-X-C chemokine receptor type 4 (CXCR4) antagonist, such as l,r-[l,4-phenylenebis(methylene)]-bis- 1,4,8, 1 l-tetra-azacyclotetradecane or a variant thereof, may be administered to a subject in amounts sufficient to mobilize hematopoietic stem and progenitor cells.
- CXCR2 C-X-C chemokine receptor type 2
- CXCR4 C-X-C chemokine receptor type 4
- l,r-[l,4-phenylenebis(methylene)]-bis- 1,4,8, 1 l-tetra-azacyclotetradecane or a variant thereof may be administered to
- compositions and methods described herein may be used to mobilize hematopoietic stem and progenitor cells from a stem cell niche within a donor, such as a human donor, into the circulating peripheral blood of the donor while reducing the mobilization of other cells of the hematopoietic lineage, such as white blood cells, neutrophils, lymphocytes, and monocytes.
- a donor such as a human donor
- the compositions and methods described herein thus enable the selective mobilization of hematopoietic stem and progenitor cells in a donor, which may then be isolated from a donor for therapeutic use.
- the hematopoietic stem or progenitor cells may be mobilized from the bone marrow of the donor to the peripheral blood, from which the hematopoietic stem or progenitor cells may be collected and/or isolated. Upon collection of the mobilized cells, the withdrawn hematopoietic stem or progenitor cells may then be infused into a patient, which may be the donor or another subject, such as a subject that is HLA-matched to the donor, for the treatment of one or more pathologies of the hematopoietic system. In some embodiments, the withdrawn hematopoietic stem or progenitor cells are first expanded ex vivo prior to infusion of these cells, and/or progeny thereof, into the patient. The
- compositions and methods described herein provide the important clinical benefit of enabling the production of populations of cells that are enriched in hematopoietic stem cells relative to other cell types, such as leukocytes, neutrophils, and monocytes.
- the populations of mobilized hematopoietic stem and progenitor cells produced using the compositions and methods described herein are particularly suitable for hematopoietic stem cell transplantation therapy, optionally preceded by ex vivo expansion in order to increase the quantity of hematopoietic stem and progenitor cells available for infusion into a patient.
- Leukocytosis in the donor. Leukocytosis may lead to adverse effects such as splenic rupture, renal dysfunction, acute febrile noninfectious pneumonitis (i.e., pulmonary toxicity), cardiovascular toxicity ( e.g ., hypercoagulation, heart attack, dyspnea, angina, arrhythmia, atherosclerotic plaque rupture due to proinflammatory actions related o high neutrophil counts), neurological disturbances (e.g., blurred vision, headache, and retinal hemorrhage), and sickle cell crisis. See, e.g, D’ Souza et al. (2008) Transfusion Medicine Reviews 22(4):280-290.
- hematopoietic stem cells are capable of differentiating into a multitude of cell types in the hematopoietic lineage and can thus be administered to a patient in order to populate or repopulate a cell type that is defective or deficient in the patient.
- the patient may be one, for example, that is suffering from one or more blood disorders, such as an autoimmune disease, cancer, hemoglobinopathy, or other hematopoietic pathology, and is therefore in need of hematopoietic stem cell transplantation.
- the invention thus provides methods of treating a variety of hematopoietic conditions, such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency- severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among others.
- hematopoietic conditions such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency- severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among others.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + cells to leukocytes of from about 0.0008 to about 0.0021 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- the ratio of CD34 + cells to leukocytes in the sample may be about
- the ratio of CD34 + cells to leukocytes in the sample is from about 0.0010 to about 0.0018, such as a ratio of
- hematopoietic stem cells to leukocytes in the sample of about 0.00100, 0.00101, 0.00102,
- the ratio of CD34 + cells to leukocytes in the sample is about 0.0014.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + cells relative to leukocytes by a ratio of from about 3.40: 1 to about 6.90: 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the peripheral blood of the donor may be enriched with CD34 + cells relative to leukocytes by a ratio of about 3.40:1, 3.45:1, 3.50:1, 3.55:1, 3.60:1, 3.65:1, 3.70:1, 3.75:1, 3.80:1, 3.85:1, 3.90:1, 3.95:1, 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1, 4.30:1, 4.35:1, 4.40:1, 4.45:1,
- the peripheral blood of the donor is enriched with CD34+ cells relative to leukocytes by a ratio of from about 3.5:1 to about 6.8:1, about 3.6:1 to about 6.7:1, about 3.8:1 to about 6.6:1, about 3.9:1 to about 6.5:1, about 4:1 to about 6.4:1, about 4.1:1 to about 6.3:1, about 4.2:1 to about 6.2:1, about 4.3:1 to about 6.1:1, about 4.4:1 to about 6:1, about 4.5:1 to about 6:1, about 4.6:1 to about 5.9:1, about 4.7:1 to about 5.8:1, or about 4.8:1 to about 5.7:1.
- the peripheral blood of the donor is enriched with CD34 + cells relative to leukocytes by a ratio of about from about 4.0: 1 to about 6.0:1, such as a ratio of about 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1, 4.30:1, 4.35:1, 4.40:1, 4.45:1, 4.50:1, 4.55:1, 4.60:1, 4.65:1, 4.70:1, 4.75:1, 4.80:1, 4.85:1, 4.90:1, 4.95:1, 5.00: 1, 5.05: 1, 5.10: 1, 5.15: 1, 5.20: 1, 5.25: 1, 5.30: 1, 5.35: 1, 5.40: 1, 5.45: 1, 5.50: 1, 5.55: 1, 5.60: 1, 5.65: 1, 5.70: 1, 5.75: 1, 5.80: 1, 5.85: 1, 5.90: 1, 5.95: 1, or 6.00: 1.
- the peripheral blood of the donor is enriched with CD34 + cells relative to leukocytes by a ratio of about from about 4.5: 1 to about 5.5: 1, such as a ratio of about 4.50: 1, 4.55: 1, 4.60: 1, 4.65: 1, 4.70: 1, 4.75: 1, 4.80: 1, 4.85: 1, 4.90: 1, 4.95: 1, 5.00: 1, 5.05: 1, 5.10: 1, 5.15: 1, 5.20: 1, 5.25: 1, 5.30: 1, 5.35: 1, 5.40: 1, 5.45: 1, or 5.50.
- the peripheral blood of the donor is enriched with CD34 + cells relative to leukocytes by a ratio of about 5.1 : 1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of at least about 38,000 cells/ml, such as a density of CD34 + cells of from about 38,000 cells/ml to about 100,000 cells/ml, about 40,000 cells/ml to about 90,000 cells/ml, about 50,000 cells/ml to about 80,000 cells/ml, or about 60,000 cells/ml to about 70,000 cells/ml (e.g., about 38,00 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000 cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml,
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 38,000 cells/ml to about 100,000 cells/ml, and having a density of leukocytes of from about 2.3 x 10 7 cells/ml to about 5.3 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 40,000 cells/ml to about 80,000 cells/ml, and having a density of leukocytes of from about 2.5 x 10 7 cells/ml to about 5 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 50,000 cells/ml to about 90,000 cells/ml, and having a density of leukocytes of from about 3 x 10 7 cells/ml to about 4 x 10 7 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + cells to neutrophils of from about 0.0018 to about 0.0058 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- the ratio of CD34 + cells to neutrophils in the sample may be about
- the ratio of CD34 + cells to neutrophils in the sample is from about 0.002 to about 0.0056, about 0.0022 to about 0.0054, about 0.0024 to about 0.0052, about 0.0026 to about 0.005, about 0.0028 to about 0.0048, or about 0.003 to about 0.0046.
- the ratio of CD34 + cells to neutrophils in the sample is from about 0.0026 to about 0.0046, such as a ratio of CD34 + cells to neutrophils in the sample of about 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00277,
- the ratio of CD34 + cells to neutrophils in the sample is about 0.0036.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + cells relative to neutrophils by a ratio of from about 2.1 : 1 to about 8.1 : 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the peripheral blood of the donor may be enriched with CD34 + cells relative to neutrophils by a ratio of about 2.10:1, 2.15:1, 2.20:1, 2.25:1, 2.30:1, 2.35:1, 2.40:1, 2.45:1, 2.50:1, 2.55:1, 2.60:1, 2.65:1, 2.70:1, 2.75:1, 2.80:1, 2.85:1, 2.90:1, 2.95:1, 3.00:1, 3.05:1, 3.10:1, 3.15:1,
- the peripheral blood of the donor is enriched with CD34 + cells relative to neutrophils by a ratio of from about 2.5:1 to about 7:1, about 2.6: 1 to about 6.9:1, about 2.7:1 to about 6.8:1, about 2.8:1 to about 6.7:1, about 2.9:1 to about 6.6:1, about 3:1 to about 6.5:1, about 3.2:1 to about 6.4:1, about 3.3:1 to about 6.3:1, about 3.4:1 to about 6.2:1, or about 3.5 : 1 to about 6.1:1
- the peripheral blood of the donor is enriched with CD34 + cells relative to neutrophils by a ratio of from about from about 5.4: 1 to about 7.4:1, such as a ratio of about 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1,
- the peripheral blood of the donor is enriched with CD34 + cells relative to neutrophils by a ratio of about 6.4:1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + cells to lymphocytes of from about 0.0021 to about 0.0094 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the ratio of CD34 + cells to lymphocytes in the sample may be about 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216, 0.00217, 0.00218, 0.00219,
- the ratio of CD34 + cells to lymphocytes in the sample is from about 0.0022 to about 0.0093, about 0.0023 to about 0.0092, about 0.0024 to about 0.0091, about 0.003 to about 0.0085, about 0.0035 to about 0.0075, or about 0.0045 to about 0.0065.
- the ratio of CD34 + cells to lymphocytes in the sample is from about 0.0025 to about 0.0035, such as a ratio of CD34 + cells to lymphocytes in the sample of about 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 0.00257, 0.00258, 0.00259, 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267,
- the ratio of CD34 + cells to lymphocytes in the sample is about 0.0031.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + cells relative to lymphocytes by a ratio of from about 4.8: 1 to about 8.4: 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the peripheral blood of the donor may be enriched with CD34 + cells relative to lymphocytes by a ratio of about 4.80: 1, 4.85: 1, 4.90: 1, 4.95: 1, 5.00: 1, 5.05: 1, 5.10: 1, 5.15: 1, 5.20: 1, 5.25: 1, 5.30: 1, 5.35: 1, 5.40: 1, 5.45: 1, 5.50: 1, 5.55: 1, 5.60: 1, 5.65: 1, 5.70: 1, 5.75: 1, 5.80: 1, 5.85: 1,
- the peripheral blood of the donor is enriched with CD34 + cells relative to lymphocytes by a ratio of from about 5: 1 to about 7:1, about 5.5:1 to about 6.5:1, or about 5.2:1 to about 5.7:1.
- the peripheral blood of the donor is enriched with CD34 + cells relative to lymphocytes by a ratio of from about 5.0:1 to about 6.5:1, such as a ratio of about 5.00:1, 5.05:1, 5.10:1, 5.15:1, 5.20:1, 5.25:1, 5.30:1, 5.35:1, 5.40:1, 5.45:1, 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1,
- the peripheral blood of the donor is enriched with CD34 + cells relative to lymphocytes by a ratio of about 5.7:1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of at least about 38,000 cells/ml, such as a density of CD34 + cells of from about 38,000 cells/ml to about 100,000 cells/ml, about 40,000 cells/ml to about 90,000 cells/ml, about 50,000 cells/ml to about 80,000 cells/ml, or about 60,000 cells/ml to about 70,000 cells/ml (e.g., about 38,00 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000 cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml,
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 38,000 cells/ml to about 100,000 cells/ml, and having a density of lymphocytes of from about 1 x 10 7 cells/ml to about 2.3 x 10 7 cells/ml.
- the method includes
- a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 40,000 cells/ml to about 80,000 cells/ml, and having a density of lymphocytes of from about 1.3 x 10 7 cells/ml to about 2.3 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 50,000 cells/ml to about 90,000 cells/ml, and having a density of lymphocytes of from about 1.5 x 10 7 cells/ml to about 2 x 10 7 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + cells to monocytes of from about 0.0071 to about 0.0174 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist.
- the ratio of CD34 + cells to monocytes in the sample may be about 0.00710, 0.00711, 0.00712,
- the ratio of CD34 + cells to monocytes in the sample is from 0.008 to about 0.016, about 0.009 to about 0.015, about 0.01 to about 0.014, or about 0.011 to about 0.013.
- the ratio of CD34 + cells to monocytes in the sample is from about 0.0100 to about 0.0140, such as a ratio of CD34 + cells to monocytes in the sample of about 0.0100, 0.0101, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0120, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, 0.0130, 0.0131, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139, or 0.0140.
- the ratio of CD34 + cells to monocytes in the sample is about 0.0118.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of at least about 38,000 cells/ml, such as a density of CD34 + cells of from about 38,000 cells/ml to about 100,000 cells/ml, about 40,000 cells/ml to about 90,000 cells/ml, about 50,000 cells/ml to about 80,000 cells/ml, or about 60,000 cells/ml to about 70,000 cells/ml (e.g., about 38,00 cells/ml, 39,000 cells/ml, 40,000 cells/ml, 41,000 cells/ml, 42,000 cells/ml, 43,000 cells/ml, 44,000 cells/ml, 45,000 cells/ml (e.g., about 38,
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 38,000 cells/ml to about 100,000 cells/ml, and having a density of monocytes of from about 3.4 x 10 6 cells/ml to about 6 x 10 6 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 40,000 cells/ml to about 80,000 cells/ml, and having a density of monocytes of from about 4 x 10 6 cells/ml to about 5.5 x 10 6 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + cells of from about 50,000 cells/ml to about 90,000 cells/ml, and having a density of monocytes of from about 4 x 10 6 cells/ml to about 5 x 10 6 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + cells relative to monocytes by a ratio of from about 1.1 : 1 to about 2.3 : 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the peripheral blood of the donor may be enriched with CD34 + cells relative to monocytes by a ratio of about 1.10: 1, 1.15: 1, 1.20: 1, 1.25: 1, 1.30: 1, 1.35: 1, 1.40: 1, 1.45: 1, 1.50: 1, 1.55: 1, 1.60: 1, 1.65: 1, 1.70: 1, 1.75: 1, 1.80: 1, 1.85: 1, 1.90: 1, 1.95: 1, 2.00: 1, 2.05: 1, 2.10: 1, 2.15: 1, 2.20: 1, 2.25: 1, or 2.30: 1.
- the peripheral blood of the donor is enriched with CD34 + cells relative to monocytes by a ratio of about from about 1.3 : 1 to about 1.9: 1, such as a ratio of about 1.30: 1, 1.35: 1, 1.40: 1, 1.45: 1, 1.50: 1, 1.55: 1, 1.60: 1, 1.65: 1, 1.70: 1, 1.75: 1, 1.80: 1, 1.85: 1, or 1.90: 1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a frequency of CD34 + cells of from about 0.051% to about 0.140% in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g., a human donor
- the population of cells may have a frequency of CD34 + cells of about
- the population of cells has a frequency of CD34 + cells of from about 0.050% to about 0.120%, about 0.060% to about 0.110%, or about 0.080% to about 0.100%. In some embodiments, the population of cells has a frequency of CD34 + cells of from about 0.080% to about 0.120%, such as a frequency of hematopoietic stem cells of about 0.080%, 0.081%, 0.082%, 0.083%, 0.084%, 0.085%, 0.086%, 0.087%, 0.088%, 0.089%, 0.090%, 0.091%, 0.092%, 0.093%, 0.094%, 0.095%, 0.096%, 0.097%, 0.098%, 0.099%, 0.100%, 0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%, 0.108%, 0.109%, 0.110%, 0.111%, 0.112%, 0.113%, 0.114%, 0.115%, 0.116%, 0.117%, 0.118%, 0.109%, 0.
- the population of cells has a frequency of CD34 + cells of about 0.097%.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to induce an increase in the frequency of CD34 + cells in the peripheral blood of the donor by at least 3-fold as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist (e.g., by from about 3.4-fold to about 7.1 -fold, such as by about 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4.0-fold, 4.
- a mammalian donor e.g a human donor
- the frequency of CD34 + cells in the peripheral blood of the donor is increased by from about 4-fold to about 7-fold, about 4.5-fold to about 6.5-fold, or about 5-fold to about 6-fold following administration of the CXCR2 agonist and CXCR4 antagonist.
- the frequency of CD34 + cells in the peripheral blood of the donor is increased by from about 4.0-fold to about 6.0-fold following administration of the CXCR2 agonist and CXCR4 antagonist, such as by about 4.0-fold, 4.l-fold, 4.2-fold, 4.3-fold, 4.4- fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5.0-fold, 5.l-fold, 5.2-fold, 5.3-fold, 5.4- fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, or 6.0-fold.
- the frequency of CD34 + cells in the peripheral blood of the donor is increased by about 4.8-fold.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + CD90 + CD45RA cells to leukocytes of from about 0.0003 to about 0.0016 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- the ratio of CD34 + CD90 + CD45RA cells to leukocytes in the sample may be about 0.00030, 0.00031, 0.00032, 0.00033, 0.00034,
- the ratio of CD34 + CD90 + CD45RA cells to leukocytes in the sample is from about 0.0008 to about 0.0010. In some embodiments, the ratio of CD34 + CD90 + CD45RA cells to leukocytes in the sample is from about 0.0006 to about 0.0012, such as a ratio of hematopoietic stem cells to leukocytes in the sample of about 0.00060, 0.00061, 0.00062, 0.00063, 0.00064, 0.00065, 0.00066, 0.00067, 0.00068, 0.00069, 0.00070, 0.00071, 0.00072, 0.00073, 0.00074, 0.00075, 0.00076, 0.00077, 0.00078, 0.00079, 0.00080, 0.00081, 0.00082, 0.00083, 0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094, 0.00095, 0.00096, 0.00097, 0.00098
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + CD90 + CD45RA cells relative to leukocytes by a ratio of from about 5.5:1 to about 26.9: 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- the peripheral blood of the donor may be enriched with CD34 + CD90 +
- CD45RA cells relative to leukocytes by a ratio of about 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1,
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to leukocytes by a ratio of about from about 5.5:1 to about 6.5:1, such asaratio of about 5.50:1, 5.55:1, 5.60:1, 5.65:1, 5.70:1, 5.75:1, 5.80:1, 5.85:1, 5.90:1, 5.95:1, 6.00:1, 6.05:1, 6.10:1, 6.15:1, 6.20:1, 6.25:1, 6.30:1, 6.35:1, 6.40:1, 6.45:1, or 6.50:1.
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to leukocytes by a ratio of about 6.0:1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of at least about 16,000 cells/ml, such as a density of from about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000 cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about 60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to about 50,000 cells/ml ( e.g ., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 1
- a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 20,000 cells/ml to about 75,000 cells/ml, and having a density of leukocytes of from about 2.3 x 10 7 cells/ml to about 5.3 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 30,000 cells/ml to about 60,000 cells/ml, and having a density of leukocytes of from about 2.5 x 10 7 cells/ml to about 5 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density of leukocytes of from about 3 x 10 7 cells/ml to about 4 x 10 7 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + CD90 + CD45RA cells to neutrophils of from about 0.0007 to about 0.0043 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- the ratio of CD34 + CD90 + CD45RA cells to neutrophils in the sample may be about 0.00070, 0.00071, 0.00072, 0.00073, 0.00074,
- the ratio of CD34 + CD90 + CD45RA cells to neutrophils in the sample is from about 0.002 to about 0.003. In some embodiments, the ratio of CD34 + CD90 + CD45RA cells to neutrophils in the sample is from about 0.0014 to about 0.0034, such as a ratio of CD34 + CD90 +
- CD45RA cells to neutrophils in the sample of about 0.00140, 0.00141, 0.00142, 0.00143,
- the ratio of CD34 + CD90 + CD45RA cells to neutrophils in the sample is about 0.0024.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + CD90 + CD45RA cells relative to neutrophils by a ratio of from about 3.5:1 to about 22.0: 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the peripheral blood of the donor may be enriched with CD34 + CD90 + CD45RA cells relative to neutrophils by a ratio of about 3.50:1, 3.55:1, 3.60:1, 3.65:1, 3.70:1, 3.75:1, 3.80:1, 3.85:1, 3.90:1, 3.95:1, 4.00:1, 4.05:1, 4.10:1, 4.15:1, 4.20:1, 4.25:1,
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to neutrophils by a ratio of about from about 7.0:1 to about 10:1.
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to neutrophils by a ratio of from about 7.00:1 to about 9.00:1, such as a ratio of about 7.00:1, 7.05:1, 7.10:1, 7.15:1, 7.20:1, 7.25:1, 7.30:1, 7.35:1, 7.40:1, 7.45:1, 7.50:1, 7.55:1, 7.60:1, 7.65:1, 7.70:1, 7.75:1,
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to neutrophils by a ratio of about 8.2:1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of at least about 16,000 cells/ml, such as a density of from about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000 cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about 60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to about 50,000 cells/ml ( e.g ., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 1
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 20,000 cells/ml to about 75,000 cells/ml, and having a density of neutrophils of from about 1 x 10 7 cells/ml to about 2.5 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 30,000 cells/ml to about 60,000 cells/ml, and having a density of neutrophils of from about 1.3 x 10 7 cells/ml to about 2.3 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density of neutrophils of from about 1.5 x 10 7 cells/ml to about 2 x 10 7 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + CD90 + CD45RA cells to lymphocytes of from about 0.0008 to about 0.0069 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- the ratio of CD34 + CD90 + CD45RA cells to lymphocytes in the sample may be about 0.00080, 0.00081, 0.00082, 0.00083, 0.00084,
- the ratio of CD34 + CD90 + CD45RA cells to lymphocytes in the sample is from about 0.0011 to about 0.0031, such as a ratio of CD34 + CD90 + CD45RA cells to lymphocytes in the sample of about 0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124, 0.00125,
- the ratio of CD34 + CD90 + CD45RA cells to lymphocytes in the sample is about 0.0021.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + CD90 + CD45RA cells relative to lymphocytes by a ratio of from about 5.6: 1 to about 37.0: 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- the peripheral blood of the donor may be enriched with CD34 + CD90 + CD45RA cells relative to lymphocytes by a ratio of about 5.60:1, 5.65:1, 5.70:1, 5.75:1,
- CD45RA cells relative to lymphocytes by a ratio of about from about 8.0: 1 to about 10.0:1, such as a ratio of about 8.00: 1, 8.05: 1, 8.10: 1, 8.15: 1, 8.20: 1, 8.25: 1, 8.30: 1, 8.35: 1, 8.40: 1,
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to lymphocytes by a ratio of about 9.3 : 1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of at least about 16,000 cells/ml, such as a density of from about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000 cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about 60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to about 50,000 cells/ml (e.g., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 19,000 cells
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 20,000 cells/ml to about 75,000 cells/ml, and having a density of lymphocytes of from about 1 x 10 7 cells/ml to about 2.3 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 30,000 cells/ml to about 60,000 cells/ml, and having a density of lymphocytes of from about 1.3 x 10 7 cells/ml to about 2.3 x 10 7 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density of lymphocytes of from about 1.5 x 10 7 cells/ml to about 2 x 10 7 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + CD90 + CD45R a cells to monocytes of from about 0.0028 to about 0.0130 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist.
- the ratio of CD34 + CD90 + CD45RA cells to monocytes in the sample may be about 0.00280. 0.00281. 0.00282. 0.00283. 0.00284. 0.00285. 0.00286. 0.00287. 0.00288
- the ratio of CD34 + CD90 + CD45RA cells to monocytes in the sample is from about 0.0063 to about 0.0083, such as a ratio of CD34 + CD90 + CD45RA cells to monocytes in the sample of about 0.00630, 0.00631, 0.00632, 0.00633, 0.00634, 0.00635, 0.00636, 0.00637, 0.00638, 0.00639, 0.00640, 0.00641, 0.00642, 0.00643, 0.00644, 0.00645, 0.00646, 0.00647, 0.00648, 0.00649, 0.00650, 0.00651, 0.00652, 0.00653, 0.00654, 0.00655, 0.00656, 0.00657, 0.00658, 0.00659, 0.00660, 0.00661, 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,
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + CD90 + CD45RA cells relative to monocytes by a ratio of from about 1.5: 1 to about 8.5: 1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- the peripheral blood of the donor may be enriched with CD34 + CD90 + CD45RA cells relative to monocytes by a ratio of about 1.50: 1, 1.55: 1, 1.60: 1, 1.65: 1,
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of at least about 16,000 cells/ml, such as a density of from about 20,000 cells/ml to about 75,000 cells/ml, about 25,000 cells/ml to about 70,000 cells/ml, about 30,000 cells/ml to about 65,000 cells/ml, about 35,000 cells/ml to about 60,000 cells/ml, about 40,000 cells/ml to about 55,000 cells/ml, or about 45,000 cells/ml to about 50,000 cells/ml (e.g., about 16,000 cells/ml, 17,000 cells/ml, 18,000 cells/ml, 19,000 cells
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 20,000 cells/ml to about 75,000 cells/ml, and having a density of monocytes of from about 3.4 x 10 6 cells/ml to about 6 x 10 6 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 30,000 cells/ml to about 60,000 cells/ml, and having a density of monocytes of from about 4 x 10 6 cells/ml to about 5.5 x 10 6 cells/ml.
- the method includes administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a density of CD34 + CD90 + CD45RA cells of from about 40,000 cells/ml to about 50,000 cells/ml, and having a density of monocytes of from about 4 x 10 6 cells/ml to about 5 x 10 6 cells/ml.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a ratio of CD34 + CD90 + CD45RA cells to CD34 + cells of from about 0.393 to about 0.745 in a sample of peripheral blood of the donor following administration of the CXCR2 agonist.
- a mammalian donor e.g ., a human donor
- the ratio of CD34 + CD90 + CD45RA cells to CD34 + cells in the sample may be about 0.393, 0.394, 0.395, 0.396, 0.397, 0.398, 0.399, 0.401, 0.402, 0.403, 0.404, 0.405, 0.406, 0.407, 0.408, 0.409, 0.410, 0.411, 0.412, 0.413, 0.414, 0.415, 0.416, 0.417, 0.418,
- the ratio of CD34 + CD90 + CD45RA cells to CD34 + cells in the sample is from about 0.625 to about 0.725, such as a ratio of CD34 + CD90 + CD45RA cells to CD34 + cells in the sample of about 0.625, 0.626, 0.627, 0.628, 0.629, 0.630, 0.631, 0.632, 0.633, 0.634, 0.635, 0.636, 0.637, 0.638, 0.639, 0.640, 0.641, 0.642, 0.643, 0.644, 0.645, 0.646, 0.647,
- the ratio of CD34 + CD90 + CD45RA cells to CD34 + cells in the sample is about 0.676.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to enrich the peripheral blood of the donor with CD34 + CD90 + CD45RA cells relative to CD34 + cells by a ratio of from about 1.1 : 1 to about 4.8:1 as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist.
- the peripheral blood of the donor may be enriched with CD34 + CD90 + CD45RA cells relative to monocytes by a ratio of about 1.10:1, 1.15:1, 1.20:1, 1.25:1,
- the peripheral blood of the donor is enriched with CD34 + CD90 + CD45RA cells relative to CD34 + cells by a ratio of about 1.2:1.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to produce a population of cells having a frequency of CD34 + CD90 + CD45RA cells of from about 0.020% to about 0.110% in a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist.
- a mammalian donor e.g ., a human donor
- the population of cells may have a frequency of CD34 + CD90 + CD45RA cells of about 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.040%, 0.041%, 0.042%, 0.043%, 0.044%, 0.045%, 0.046%, 0.047%, 0.048%, 0.049%, 0.050%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.071%, 0.072%, 0.073%, 0.074%, 0.075%, 0.076%,
- the population of cells has a frequency of CD34 + CD90 + CD45RA cells of from about 0.046% to about 0.086%, such as a frequency of hematopoietic stem cells of about 0.046%, 0.047%, 0.048%, 0.049%, 0.050%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%,
- the population of cells has a frequency of CD34 + CD90 + CD45RA cells of about 0.066%.
- the invention features a method of mobilizing a population of hematopoietic stem cells from the bone marrow of a mammalian donor (e.g ., a human donor) into peripheral blood, the method including administering to the donor a CXCR2 agonist and a CXCR4 antagonist in amounts sufficient to induce an increase in the frequency of CD34 + CD90 + CD45RA cells in the peripheral blood of the donor by at least 3-fold as assessed by comparing a sample of peripheral blood of the donor following administration of the CXCR2 agonist and CXCR4 antagonist to a sample of peripheral blood of the donor prior to administration of the CXCR2 agonist and CXCR4 antagonist (e.g., by from about 5.1 -fold to about 25.7-fold, such as by about 5.
- a mammalian donor e.g ., a human donor
- the frequency of CD34 + CD90 + CD45RA cells in the peripheral blood of the donor is increased by from about 5.1 -fold to about 7.1 -fold following administration of the CXCR2 agonist and CXCR4 antagonist, such as by about 5. l-fold, 5.2-fold, 5.3-fold, 5.4- fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6.0-fold, 6. l-fold, 6.2-fold, 6.3-fold, 6.4- fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7.0-fold, or 7. l-fold.
- the CXCR2 agonist and CXCR4 antagonist such as by about 5. l-fold, 5.2-fold, 5.3-fold, 5.4- fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6.0-fold, 6. l-fold, 6.2-fold, 6.3-fold,
- the frequency of CD34 + CD90 + CD45RA cells in the peripheral blood of the donor is increased by about 5.8-fold.
- the invention features a method of mobilizing a population of hematopoietic stem cells, from the bone marrow of a mammalian donor into peripheral blood, the method comprising administering to the donor mobilizing amounts of a CXCR2 agonist and a CXCR4 antagonist; acquiring an input value for each of one or more parameters in TABLE 2 characterizing a sample of peripheral blood of the donor, and releasing the sample for ex vivo expansion of the hematopoietic stem cells or for use in the treatment of one or more stem cell disorders in a mammalian patient if the input value for each of the one or more parameters meets the corresponding reference criterion for each of the one or more parameters.
- the one or more reference parameters are a set of parameters listed in any one of TABLES 3-6 herein.
- the sample is isolated from the donor at from about 3 hours to about 5 hours following administration of the CXCR2 agonist and CXCR4 antagonist (e.g ., at about 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, 4.0 hours, 4.1 hours,
- the sample is isolated from the donor at about 4 hours following administration of the CXCR2 agonist and CXCR4 antagonist.
- the CXCR2 agonist is Gro-b T or a variant thereof.
- the CXCR2 agonist may 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.
- the CXCR2 agonist is a peptide having from about 85% to 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, such as a peptide having from about 86% to about 100%, from about 87% to about 99%, about 88% to about 98%, about 89%, to about 97%, about 90% to about 96%, or about 91% to about 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.
- the CXCR2 agonist is a peptide having an amino acid sequence that differs from that of SEQ ID NO: 2 only by way of one or more conservative amino acid substitutions (e.g ., only by way of from 1 to 10 conservative amino acid substitutions, from 1 to 5 conservative amino acid substitutions, or from 1 to 3 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions).
- the CXCR2 agonist is Gro-b T.
- the Gro-b T is not covalently modified.
- the Gro-b T is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene glycol moiety.
- the CXCR2 agonist is Gro-b or a variant thereof.
- the CXCR2 agonist may be a peptide having at least about 85% (e.g., about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 1.
- the CXCR2 agonist is a peptide having from about 85% to 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, such as a peptide having from about 86% to about 100%, from about 87% to about 99%, about 88% to about 98%, about 89%, to about 97%, about 90% to about 96%, or about 91% to about 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.
- the CXCR2 agonist is a peptide having an amino acid sequence that differs from that of SEQ ID NO: 1 only by way of one or more conservative amino acid substitutions (e.g, only by way of from 1 to 10 conservative amino acid substitutions, from 1 to 5 conservative amino acid substitutions, or from 1 to 3 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions).
- the CXCR2 agonist is Gro-b.
- the Gro-b T is not covalently modified.
- the Gro-b is not covalently modified with a polyalkylene glycol moiety, such as a polyethylene glycol moiety.
- the CXCR2 agonist e.g, Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of from about 50 pg/kg to about 1 mg/kg, such as a dose of about 50 pg/kg, 55 pg/kg, 60 pg/kg, 65 pg/kg, 70 pg/kg, 75 pg/kg, 80 pg/kg, 85 pg/kg, 90 pg/kg, 95 pg/kg, 100 pg/kg, 105 pg/kg, 1 10 pg/kg, 115 gg/kg, 120 pg/kg, 125 pg/kg, 130 pg/kg, 135 pg/kg, 140 pg/kg, 145 pg/kg, 150 pg/kg, 155 gg/kg, 160 pg/kg, 165 pg/kg, 170 pg
- the CXCR2 agonist e.g, Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of from about 50 gg/kg to about 300 gg/kg, such as a dose of from about 100 gg/kg to about 250 gg/kg, or from about 125 gg/kg to about 225 gg/kg.
- the CXCR2 agonist e.g, Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- 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 selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 pg/kg to about 1 mg/kg (e.g., a dose of 50 pg/kg, 55 pg/kg, 60 pg/kg, 65 pg/kg, 70 pg/kg, 75 pg/kg, 80 pg/kg, 85 pg/kg, 90 pg/kg, 95 pg/kg, 100 pg/kg, 105 pg/kg, 110 pg/kg,
- a mammalian donor e.g, a human donor
- a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants
- the CXCR2 agonist e.g ., Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of from about 50 pg/kg to about 300 pg/kg, such as a dose of about 50 pg/kg, 55 pg/kg, 60 pg/kg, 65 pg/kg, 70 pg/kg, 75 pg/kg, 80 pg/kg, 85 pg/kg, 90 pg/kg, 95 pg/kg, 100 pg/kg, 105 pg/kg, 110 pg/kg, 115 pg/kg, 120 pg/kg, 125 pg/kg, 130 pg/kg, 135 pg/kg,
- the CXCR2 agonist e.g ., Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of from about 100 pg/kg to about 250 pg/kg, such as a dose of about 100 gg/kg, 105 pg/kg, 110 pg/kg, 115 pg/kg, 120 pg/kg, 125 pg/kg, 130 pg/kg, 135 pg/kg, 140 gg/kg, 145 pg/kg, 150 pg/kg, 155 pg/kg, 160 pg/kg, 165 pg/kg, 170 pg/kg, 175 pg/kg, 180 gg/kg, 185 pg/kg, 190 pg/kg, 195 gg/kg, 200 gg/kg, 205 gg/kg, 210 gg
- the CXCR2 agonist e.g., Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of about 150 pg/kg.
- the CXCR2 agonist e.g, Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of from about 50 pg/kg per day to about 1 mg/kg per day, such as a dose of about 50 pg/kg per day, 55 pg/kg per day, 60 pg/kg per day, 65 pg/kg per day, 70 pg/kg per day, 75 pg/kg per day, 80 pg/kg per day, 85 pg/kg per day, 90 pg/kg per day, 95 pg/kg per day, 100 pg/kg per day, 105 pg/kg per day, 110 pg/kg per day, 115 pg/kg per day, 120 pg/kg per day, 125 pg/kg per day, 130 pg/kg per day, 135 pg/kg per day, 140 pg/kg per day,
- the CXCR2 agonist e.g ., Gro-b or Gro-b T, such as unmodified Gro-b or Gro- b T
- the CXCR2 agonist is administered to the donor at a dose of from about 50 pg/kg per day to about 300 pg/kg per day, such as a dose of from about 100 pg/kg per day to about 250 pg/kg per day, or from about 125 pg/kg per day to about 225 pg/kg per day, or from about 125 pg/kg per day to about 175 pg/kg per day.
- the CXCR2 agonist e.g., Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered to the donor at a dose of about 150 pg/kg per day.
- the CXCR2 agonist may be administered as a single dose. In other embodiments, the CXCR2 agonist may be administered as two or more doses.
- a human equivalent dose may be derived from animal dosage data using a conversion factor.
- HED human equivalent dose
- Nair and Jacob, ./. Basic Clin. Pharma. (2016) 7:27-31 disclose methods extrapolation of dose between species.
- HED may be derived from rhesus monkey dose by multiplying the rhesus monkey dose by about 0.324.
- the CXCR2 agonist e.g ., Gro-b or Gro-b T, such as unmodified Gro-b or Gro-b T
- the CXCR2 agonist is administered intravenously to the donor.
- the CXCR4 antagonist is a compound represented by formula (I)
- A includes a monocyclic or bicyclic fused ring system including at least one nitrogen atom and B is H or a substituent of from 1 to 20 atoms;
- A’ includes a monocyclic or bicyclic fused ring system including at least one nitrogen atom and B’ is H or a substituent of from 1 to 20 atoms; or
- each R is independently H or Ci-C 6 alkyl, n is 1 or 2, and X is an aryl or heteroaryl group or a mercaptan;
- linker is a bond, optionally substituted Ci-C 6 alkyl ene, optionally substituted Ci-C 6 heteroalkylene, optionally substituted C 2 -C 6 alkenylene, optionally substituted C 2 -C 6 heteroalkenylene, optionally substituted C 2 -C 6 alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroaryl ene.
- Z and Z’ are each independently a cyclic polyamine containing from 9 to 32 ring members, of which from 2 to 8 are nitrogen atoms separated from one another by 2 or more carbon atoms. Z and Z’ may be identical substituents. In some embodiments, Z and/or Z’ is a cyclic polyamine including from 10 to 24 ring members, such as a cyclic polyamine including 14 ring members. In some embodiments, Z includes 4 nitrogen atoms. Z and/or Z’ may be, for example, 1,4,8, 1 l-tetraazocy cl otetradecane.
- the linker is represented by formula (ID)
- ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group;
- X and Y are each independently optionally substituted Ci-C 6 alkylene, optionally substituted Ci-C 6 heteroalkylene, optionally substituted C 2 -C 6 alkenylene, optionally substituted C 2 -C 6 heteroalkenylene, optionally substituted C 2 -C 6 alkynylene, or optionally substituted C 2 -C 6 heteroalkynylene.
- the linker is represented by formula (IE)
- ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group;
- X and Y are each independently optionally substituted Ci-C 6 alkylene, optionally substituted Ci-C 6 heteroalkylene, optionally substituted C 2 -C 6 alkenylene, optionally substituted C 2 -C 6 heteroalkenylene, optionally substituted C 2 -C 6 alkynylene, or optionally substituted C 2 -C 6 heteroalkynylene.
- X and Y are each independently optionally substituted Ci-C 6 alkylene.
- X and Y are identical substituents, such as identical alkylene substituents (e.g ., methylene, ethylene, propylene, or butylene substituents).
- the CXCR4 antagonist is plerixafor or a pharmaceutically acceptable salt thereof. In some embodiments, the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered subcutaneously to the donor.
- the CXCR4 antagonist (e.g, plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 50 gg/kg to about 500 m /1 ⁇ , such as a dose of about 50 mm/1 ⁇ m, 55 mm/1 ⁇ m, 60 mm/1 ⁇ m, 65 mm/1 ⁇ m, 70 mm/1 ⁇ m, 75 mm/1 ⁇ m, 80 gg/kg, 85 mm/kg, 90 mm/kg, 95 gg/kg, 100 gg/kg, 105 gg/kg, 1 10 gg/kg, 1 15 gg/kg, 120 gg/kg, 125 gg/kg, 130 gg/kg, 135 gg/kg, 140 gg/kg, 145 gg/kg, 150 gg/kg, 155 gg/kg, 160 gg/kg, 165 gg/kg, 170 gg/kg, 175 gg/kg, 180 gg/kg, 185 gg/kg, 190 gg/kg, 100 gg
- the CXCR4 antagonist e.g ., plerixafor or a pharmaceutically acceptable salt thereof
- the CXCR4 antagonist is administered to the donor at a dose of from about 200 gg/kg to about 300 gg/kg, such as a dose of about 240 hg/kg.
- the CXCR4 antagonist (e.g., plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 50 gg/kg per day to about 500 gg/kg per day, such as a dose of about 50 gg/kg per day, 55 gg/kg per day, 60 gg/kg per day, 65 gg/kg per day, 70 gg/kg per day, 75 gg/kg per day, 80 gg/kg per day, 85 gg/kg per day, 90 gg/kg per day, 95 gg/kg per day, 100 gg/kg per day, 105 gg/kg per day, 110 gg/kg per day, 115 gg/kg per day, 120 gg/kg per day, 125 gg/kg per day, 130 gg/kg per day, 135 gg/kg per day, 140 gg/kg per day, 145 gg/kg per day, 150 gg/kg per day, 155 gg/kg per day,
- the CXCR4 antagonist (e.g, plerixafor or a pharmaceutically acceptable salt thereof) is administered to the donor at a dose of from about 200 gg/kg per day to about 300 gg/kg per day, such as a dose of about 240 gg/kg per day.
- the CXCR4 antagonist may be administered as a single dose. In other embodiments, the CXCR4 antagonist may be administered as two or more doses.
- the CXCR2 agonist and the CXCR4 antagonist are administered to the donor concurrently.
- the CXCR4 antagonist is administered to the donor prior to administration of the CXCR2 agonist.
- the CXCR4 antagonist may be administered to the donor from about 1 minute to about 180 minutes prior to administration of the CXCR2 agonist, such as from about 15 minutes to about 180 minutes, about 30 minutes to about 180 minutes, about 40 minutes to about 160 minutes, about 50 minutes to about 150 minutes, about 60 minutes to about 140 minutes, about 70 minutes to about 130 minutes, about 60 minutes to about 120 minutes, about 70 minutes to about 110 minutes, or about 80 minutes to about 100 minutes ( e.g ., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes, about 170 minutes, about 175 minutes, or about 180 minutes prior to
- the CXCR4 antagonist is administered to the donor from about 30 minutes to about 60 minutes prior to administration of the CXCR2 agonist (e.g., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes prior to administration of the CXCR2 agonist). In some embodiments, the CXCR4 antagonist may be administered to the donor about 45 minutes prior to administration of the CXCR2 agonist.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g, a human donor), wherein the ratio of CD34 + cells to leukocytes in the population is from about 0.0008 to about 0.0021.
- the ratio of CD34 + cells to leukocytes may be about 0.00080, 0.00081, 0.00082, 0.00083, 0.00084, 0.00085, 0.00086, 0.00087, 0.00088, 0.00089, 0.00090, 0.00091, 0.00092, 0.00093, 0.00094, 0.00095, 0.00096,
- the ratio of CD34 + cells to leukocytes is from about 0.0010 to about 0.0018, such as a ratio of CD34 + cells to leukocytes of about 0.00100, 0.00101, 0.00102, 0.00103, 0.00104, 0.00105, 0.00106, 0.00107, 0.00108, 0.00109, 0.00110, 0.00111, 0.00112, 0.00113,
- the ratio of CD34 + cells to leukocytes is about 0.0014.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g ., a human donor), wherein the ratio of CD34 + cells to neutrophils in the population is from about 0.0018 to about 0.0058.
- the ratio of CD34 + cells to neutrophils may be about 0.00180, 0.00181, 0.00182, 0.00183, 0.00184, 0.00185, 0.00186, 0.00187, 0.00188, 0.00189, 0.00190, 0.00191, 0.00192, 0.00193, 0.00194, 0.00195, 0.00196,
- the ratio of CD34 cells to neutrophils is from about 0.0026 to about 0.0046, such as a ratio of CD34 + cells to neutrophils of about 0.00260, 0.00261, 0.00262, 0.00263, 0.00264, 0.00265, 0.00266, 0.00267, 0.00268, 0.00269, 0.00270, 0.00271, 0.00272, 0.00273, 0.00274, 0.00275, 0.00276, 0.00277, 0.00278, 0.00279, 0.00280, 0.00281, 0.00282, 0.00283, 0.00284, 0.00285, 0.00286, 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
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g, a human donor), wherein the ratio of CD34 + cells to lymphocytes in the population is from about 0.0021 to about 0.0094. In some embodiments, the ratio of CD34 + cells to lymphocytes may be about 0.00210, 0.00211, 0.00212, 0.00213, 0.00214, 0.00215, 0.00216,
- the ratio of CD34 cells to lymphocytes is from about 0.0025 to about 0.0035, such as a ratio of CD34 + cells to lymphocytes of about 0.00250, 0.00251, 0.00252, 0.00253, 0.00254, 0.00255, 0.00256, 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, 0.00290, 0.00
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor e.g ., a human donor), wherein the ratio of CD34 + cells to monocytes in the population is from about 0.0071 to about 0.0174.
- the ratio of CD34 + cells to monocytes may be about 0.00710, 0.00711, 0.00712, 0.00713, 0.00714, 0.00715, 0.00716,
- the ratio of CD34 + cells to monocytes is from about 0.0100 to about 0.0140, such as a ratio of CD34 + cells to monocytes of about 0.0100, 0.0101, 0.0103, 0.0104, 0.0105, 0.0106, 0.0107, 0.0108, 0.0109, 0.0110, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115,
- the ratio of CD34 + cells to monocytes is about 0.0118.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor e.g ., a human donor), wherein the frequency of CD34 + cells in the population is from about 0.051% to about 0.140%.
- the population of cells may have a frequency of CD34 + cells of about 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%,
- the population of cells has a frequency of CD34 + cells of from about 0.080% to about 0.120%, such as a frequency of CD34 + cells of about 0.080%, 0.081%, 0.082%, 0.083%, 0.084%, 0.085%, 0.086%, 0.087%, 0.088%, 0.089%, 0.090%, 0.091%, 0.092%, 0.093%, 0.094%, 0.095%, 0.096%, 0.097%, 0.098%, 0.099%, 0.100%, 0.101%, 0.102%, 0.103%, 0.104%, 0.105%, 0.106%, 0.107%, 0.108%, 0.109%, 0.110%, 0.111%, 0.112%, 0.113%, 0.114%, 0.115%, 0.116%, 0.117%,
- the population of cells has a frequency of CD34 + cells of about 0.097%.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor e.g ., a human donor), wherein the ratio of CD34 + CD90 + CD45RA cells to leukocytes in the population is from about 0.0003 to about 0.0016.
- the ratio of CD34 + CD90 + CD45RA cells to leukocytes may be about 0.00030, 0.00031, 0.00032, 0.00033, 0.00034, 0.00035, 0.00036, 0.00037, 0.00038, 0.00039, 0.00040, 0.00041, 0.00042, 0.00043,
- the ratio of CD34 + CD90 + CD45RA cells to leukocytes is from about 0.0006 to about 0.0012, such as a ratio of CD34 + CD90 + CD45RA cells to leukocytes of about 0.00060, 0.00061, 0.00062, 0.00063, 0.00064, 0.00065, 0.00066, 0.00067, 0.00068, 0.00069, 0.00070,
- the ratio of CD34 + CD90 + CD45RA cells to leukocytes is about 0.0009.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g ., a human donor), wherein the ratio of CD34 + CD90 + CD45RA cells to neutrophils in the population is from about 0.0007 to about 0.0043. In some embodiments, the ratio of CD34 CD90 + CD45RA cells to neutrophils may be about 0.00070, 0.00071,
- the ratio of CD34 + CD90 + CD45RA cells to neutrophils is from about 0.0014 to about 0.0034, such as a ratio of CD34 + CD90 + CD45RA cells to neutrophils of about 0.00140, 0.00141, 0.00142, 0.00143, 0.00144, 0.00145, 0.00146,
- the ratio of CD34 + CD90 + CD45RA cells to neutrophils is about 0.0024.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor (e.g, a human donor), wherein the ratio of CD34 + CD90 + CD45RA cells to lymphocytes in the population is from about 0.0008 to about 0.0069. In some embodiments, the ratio of CD34 + CD90 + CD45RA cells to lymphocytes may be about 0.00080, 0.00081, 0.00082,
- the ratio of CD34 + CD90 + CD45RA cells to lymphocytes is from about 0.0011 to about 0.0031, such as a ratio of CD34 + CD90 + CD45RA cells to lymphocytes of about 0.00110, 0.00111, 0.00112, 0.00113, 0.00114, 0.00115, 0.00116, 0.00117, 0.00118, 0.00119, 0.00120, 0.00121, 0.00122, 0.00123, 0.00124, 0.00125, 0.00126,
- the ratio of CD34 + CD90 + CD45RA - cells to lymphocytes is about 0.0021.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor e.g ., a human donor), wherein the ratio of CD34 + CD90 + CD45RA cells to monocytes in the population is from about 0.0028 to about 0.0130. In some embodiments, the ratio of CD34 +
- CD90 + CD45RA cells to monocytes may be about 0.00280, 0.00281, 0.00282, 0.00283,
- the ratio of CD34 + CD90 + CD45RA cells to monocytes is from about 0.0063 to about 0.0083, such as a ratio of CD34 + CD90 + CD45RA cells to monocytes of about
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor e.g ., a human donor), wherein the ratio of CD34 + CD90 + CD45RA cells to CD34 + cells in the population is from about 0.393 to about 0.745. In some embodiments, the ratio of CD34 +
- CD90 + CD45RA cells to CD34 + cells may be about 0.393, 0.394, 0.395, 0.396, 0.397, 0.398,
- the ratio of CD34 + CD90 + CD45RA cells to CD34 + cells is from about 0.625 to about 0.725, such as a ratio of CD34 + CD90 + CD45RA cells to CD34 + cells of about 0.625, 0.626, 0.627, 0.628, 0.629, 0.630, 0.631, 0.632, 0.633, 0.634, 0.635, 0.636, 0.637, 0.638, 0.639, 0.640, 0.641, 0.642, 0.643,
- the ratio of CD34 + CD90 + CD45RA cells to CD34 + cells is about 0.676.
- the invention features a pharmaceutical composition including a population of hematopoietic stem cells or progeny thereof isolated from a mammalian donor e.g ., a human donor), wherein the frequency of CD34 + CD90 + CD45RA cells in the population is from about 0.020% to about 0.110%.
- the population of cells may have a frequency of CD34 + CD90 + CD45RA cells of about 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.040%, 0.041%, 0.042%, 0.043%, 0.044%, 0.045%, 0.046%, 0.047%, 0.048%, 0.049%, 0.050%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.071%, 0.072%, 0.073%, 0.074%, 0.075%, 0.076%,
- the population of cells has a frequency of CD34 + CD90 + CD45RA - cells of from about 0.046% to about 0.086%, such as a frequency of hematopoietic stem cells of about 0.046%, 0.047%, 0.048%, 0.049%, 0.050%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.060%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.070%, 0.071%, 0.072%, 0.073%, 0.074%, 0.075%, 0.076%, 0.077%, 0.078%, 0.079%, 0.080%, 0.081%, 0.082%, 0.083%, 0.084%, 0.085%, or 0.086%.
- the population of cells has a frequency of CD34 + CD90 + CD45RA cells of about 0.066%.
- the invention features a method of treating a stem cell disorder in a mammalian patient (e.g ., a human patient), the method including mobilizing a population of hematopoietic stem cells in a mammalian donor (e.g., a human donor) in accordance with any of the above-described methods, and infusing a therapeutically effective amount of the hematopoietic stem cells, or progeny thereof, into the patient.
- a mammalian donor e.g., a human donor
- the invention features a method of treating a stem cell disorder in a mammalian patient (e.g, a human patient), the method including infusing into the patient a therapeutically effective amount of the hematopoietic stem cells mobilized by any of the above-described methods, or progeny thereof.
- the invention features a method of treating a stem cell disorder in a mammalian patient (e.g, a human patient), the method including administering to the patient any one or more of the above-described pharmaceutical compositions.
- the stem cell disorder is a hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
- the stem cell disorder is a myelodysplastic disorder.
- the stem cell disorder may be an immunodeficiency disorder, such as a congenital immunodeficiency or an acquired immunodeficiency, for example, human immunodeficiency virus or acquired immune deficiency syndrome.
- the stem cell disorder is a metabolic disorder, such as a metabolic disorder selected from glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.
- a metabolic disorder selected from glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.
- the stem cell disorder is cancer.
- the cancer may be, for example, leukemia, lymphoma, multiple myeloma, and neuroblastoma. In some embodiments, tumor necrosis, tumor necrosis, tumor necrosis, tumor necrosis, tumor necrosis, and tumor necrosis.
- the cancer is a hematological cancer.
- 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.
- the stem cell disorder is a disorder selected from the group consisting of adenosine deaminase deficiency and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.
- the stem cell disorder is an autoimmune disorder, such as an autoimmune disorder selected from multiple sclerosis, human systemic lupus, rheumatoid arthritis, inflammatory bowel disease, treating psoriasis, Type 1 diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune
- an autoimmune disorder selected from multiple sclerosis, human systemic lupus, rheumatoid arthritis, inflammatory bowel disease, treating psoriasis, Type 1 diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepati
- lymphoproliferative syndrome autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, coeliac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture' s syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto' s thyroiditis, Hidradenitis suppurativa, idiopathic and/or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis
- the hematopoietic stem cells are autologous with respect to the patient. In some embodiments, the hematopoietic stem cells are allogeneic with respect to the patient, and may be, for example, HLA-matched with respect to the patient.
- the hematopoietic stem cells have been genetically modified to disrupt an endogenous gene, such as a gene encoding a major histocompatibility complex protein.
- the hematopoietic stem cells may be genetically modified to disrupt an endogenous by way of, for example, a CRISPR-associated protein, such as caspase 9, or another nuclease described herein, such as a transcription activator-like effector nuclease, a meganuclease, or a zinc finger nuclease.
- 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.
- the hematopoietic stem cells, or progeny thereof localize to hematopoietic tissue and/or reestablish hematopoiesis following infusion of the hematopoietic stem cells, or progeny thereof, into the patient.
- the hematopoietic stem cells, or progeny thereof upon infusion into the patient, give rise to recovery of a population of cells selected from the group consisting of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells.
- the disclosure relates to a method of mobilizing CD34 dim cells from the bone marrow of a human donor into peripheral blood, the method comprising
- a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 pg/kg to about 1,000 pg/kg and (ii) a CXCR4 antagonist.
- the disclosure relates to a method of performing an allogeneic hematopoietic stem cell transplant in a patient in need thereof, the method comprising infusing into the patient a therapeutically effective amount of allogeneic hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a human donor into peripheral blood of the human donor by a method comprising administering to the donor (i) a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 pg/kg to about 1,000 pg/kg and (ii) a CXCR4 antagonist.
- the disclosure relates to a method of preventing, reducing the risk of developing, or reducing the severity of a post-transplant infection in a patient in need thereof, the method comprising infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a human donor into peripheral blood of the human donor by a method comprising administering to the human donor (i) a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 m /1 ⁇ to about 1,000 mm/1 ⁇ and (ii) a CXCR4 antagonist.
- the disclosure relates to a method of preventing, reducing the risk of developing, or reducing the severity of graft versus host disease (GVHD) in a patient in need thereof, the method comprising infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a human donor into peripheral blood of the human donor by a method comprising administering to the human donor (i) a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 pg/kg to about 1,000 pg/kg and (ii) a CXCR4 antagonist.
- a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof
- the CD34 dim cells are present in a higher amount in the peripheral blood than if the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone. In certain embodiments, the CD34 dim cells are capable of suppressing alloreactive T lymphocyte proliferation when administered to a recipient.
- the CXCR2 agonist is Gro-b T. In certain embodiments, the CXCR2 agonist is administered to the donor at a dose of from about 100 pg/kg to about 250 pg/kg. In certain embodiments, the CXCR2 agonist is administered to the donor at a dose of from about 125 pg/kg to about 225 pg/kg. In certain embodiments, the CXCR2 agonist is administered to the donor at a dose of about 150 pg/kg. In certain embodiments, the CXCR2 agonist is administered intravenously to the donor.
- the CXCR4 antagonist is administered subcutaneously to the donor.
- the CXCR4 antagonist is plerixafor or a pharmaceutically acceptable salt thereof.
- the plerixafor or pharmaceutically acceptable salt thereof is administered to the donor at a dose of from about 50 pg/kg to about 500 pg/kg.
- the plerixafor or pharmaceutically acceptable salt thereof is administered to the donor at a dose of from about 200 pg/kg to about 300 pg/kg.
- the plerixafor or pharmaceutically acceptable salt thereof is
- the method further includes testing a sample of peripheral blood for the presence of CD34 dim cells and releasing the sample for ex vivo expansion of the CD34 dim cells.
- the disclosure relates to the population of CD34 dim cells derived from any of the above methods, or a composition comprising the same.
- FIG. IB provides a graph showing that grafts containing cells mobilized by Gro-b T and plerixaflor led to higher relative numbers of competitive repopulating units (CRU) at week 16 than did grafts containing cells mobilized by G-CSF alone.
- CRU competitive repopulating units
- FIG. 2A is a graph demonstrating the pharmacokinetic profile of various dosages of Gro-b T when administered intravenously to Rhesus monkeys.
- FIG. 2B is a graph demonstrating the pharmacokinetic profile of various dosages of Gro-b T when administered subcutaneously to Rhesus monkeys. In all experiments, Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 3A shows a series of graphs demonstrating the mobilization response of leukocytes (white blood cells,“WBCs”) to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- Leukocyte response is shown both in terms of the quantity of cells mobilized (top) and the fold change in leukocyte density relative to baseline leukocyte density prior to administration (bottom).
- FIG. 3B shows a series of graphs demonstrating the mobilization response of leukocytes (white blood cells,“WBCs”) to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- Leukocyte response is shown both in terms of the quantity of cells mobilized (top) and the fold change in leukocyte density relative to baseline leukocyte density prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 4A shows a series of graphs demonstrating the mobilization response of neutrophils to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys. Neutrophil response is shown both in terms of the quantity of cells mobilized (top) and the fold change in neutrophil density relative to baseline neutrophil density prior to administration (bottom).
- FIG. 4B shows a series of graphs demonstrating the mobilization response of neutrophils to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys. Neutrophil response is shown both in terms of the quantity of cells mobilized (top) and the fold change in neutrophil density relative to baseline neutrophil density prior to administration (bottom). In all experiments, Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 5A shows a series of graphs demonstrating the mobilization response of lymphocytes to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys. Lymphocyte response is shown both in terms of the quantity of cells mobilized (top) and the fold change in lymphocyte density relative to baseline lymphocyte density prior to administration (bottom).
- FIG. 5B shows a series of graphs demonstrating the mobilization response of lymphocytes to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys. Lymphocyte response is shown both in terms of the quantity of cells mobilized (top) and the fold change in lymphocyte density relative to baseline lymphocyte density prior to administration (bottom). In all experiments, Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 6A shows a series of graphs demonstrating the mobilization response of monocytes to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys. Monocyte response is shown both in terms of the quantity of cells mobilized (top) and the fold change in monocyte density relative to baseline monocyte density prior to administration (bottom).
- FIG. 6B shows a series of graphs demonstrating the mobilization response of monocytes to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys. Monocyte response is shown both in terms of the quantity of cells mobilized (top) and the fold change in monocyte density relative to baseline monocyte density prior to administration (bottom). In all experiments, Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 7A shows a series of graphs demonstrating the mobilization response of CD34 + cells to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- CD34 + cell response is shown both in terms of the frequency of CD34 + cells in the sample obtained from peripheral blood of the subjects (top) and the fold change in CD34 + cell frequency relative to baseline CD34 + cell frequency prior to administration (bottom).
- FIG. 7B shows a series of graphs demonstrating the mobilization response of CD34 + cells to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- CD34 + cell response is shown both in terms of the frequency of CD34 + cells in the sample obtained from peripheral blood of the subjects (top) and the fold change in CD34 + cell frequency relative to baseline CD34 + cell frequency prior to administration (bottom).
- FIG. 8A shows a series of graphs demonstrating the mobilization response of CD34 + cells to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- CD34 + cell response is shown both in terms of the quantity of cells mobilized (top) and the fold change in CD34 + cell density relative to baseline CD34 + cell density prior to
- FIG. 8B shows a series of graphs demonstrating the mobilization response of CD34 + cells to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- CD34 + cell response is shown both in terms of the quantity of cells mobilized (top) and the fold change in CD34 + cell density relative to baseline CD34 + cell density prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 9A shows a series of graphs demonstrating the mobilization response of hematopoietic stem cells (CD34 + CD90 + CD45RA cells) to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- CD34 + CD90 + CD45RA cell response is shown both in terms of the frequency of CD34 + CD90 + CD45RA cells in the sample obtained from peripheral blood of the subjects (top) and the fold change in CD34 + CD90 + CD45RA cell frequency relative to baseline CD34 + CD90 + CD45RA cell frequency prior to administration (bottom).
- FIG. 9A shows a series of graphs demonstrating the mobilization response of hematopoietic stem cells (CD34 + CD90 + CD45RA cells) to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- CD34 + CD90 + CD45RA cell response is shown both in terms of the frequency of CD34 + CD90 + CD45RA cells in the sample obtained from peripheral blood of the subjects (top) and the fold change
- CD34 + CD90 + CD45RA cells hematopoietic stem cells
- FIG. 9B shows a series of graphs demonstrating the mobilization response of hematopoietic stem cells (CD34 + CD90 + CD45RA cells) to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- CD34 + CD90 + CD45RA cell response is shown both in terms of the frequency of CD34 + CD90 + CD45RA cells in the sample obtained from peripheral blood of the subjects (top) and the fold change in CD34 + CD90 + CD45RA cell frequency relative to baseline CD34 + CD90 + CD45RA cell frequency prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 10A shows a series of graphs demonstrating the mobilization response of hematopoietic stem cells (CD34 + CD90 + CD45RA cells) to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- CD34 + CD90 + CD45RA cell response is shown both in terms of the quantity of cells mobilized (top) and the fold change in CD34 + CD90 + CD45RA cell density relative to baseline CD34 + CD90 + CD45RA cell density prior to administration (bottom).
- FIG. 10B shows a series of graphs demonstrating the mobilization response of hematopoietic stem cells (CD34 + CD90 + CD45RA cells) to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- CD34 + cell response is shown both in terms of the quantity of cells mobilized (top) and the fold change in CD34 + CD90 + CD45RA cell density relative to baseline CD34 + CD90 + CD45RA cell density prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 11 shows a series of graphs demonstrating the increase in the quantity of colony-forming units (CFU) of hematopoietic stem cells achieved by the intravenous administration of various dosages of Gro-b T to Rhesus monkeys.
- CFR response is shown both in terms of the concentration of CFUs (top) and the fold change in CFU concentration relative to baseline CFU concentration prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 12A shows a series of graphs demonstrating the response of plasma matrix metalloproteinase 9 (MMP9) to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys. Plasma MMP9 response is shown both in terms of absolute
- FIG. 12B shows a series of graphs demonstrating the response of plasma MMP9 to various dosages of Gro-b T upon
- Plasma MMP9 response is shown both in terms of absolute concentration (top) and the fold change in plasma MMP9 concentration relative to baseline MMP9 concentration prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 13A shows a series of graphs demonstrating the response of plasma tissue inhibitor of matrix metalloproteinase 1 (TIMP-l) to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- Plasma TIMP-l response is shown both in terms of absolute concentration (top) and the fold change in plasma TIMP-l concentration relative to baseline TIMP-l concentration prior to administration (bottom).
- FIG. 13B shows a series of graphs demonstrating the response of plasma TIMP-l to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- Plasma TIMP-l response is shown both in terms of absolute concentration (top) and the fold change in plasma TIMP-l concentration relative to baseline TIMP-l concentration prior to administration (bottom).
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 14A is a graph showing the response of the molar ratio of plasma MMP9 to plasma TIMP-l to various dosages of Gro-b T upon intravenous administration to Rhesus monkeys.
- FIG. 14B is a graph showing the response of the molar ratio of plasma MMP9 to plasma TIMP-l to various dosages of Gro-b T upon subcutaneous administration to Rhesus monkeys.
- Gro-b T was administered to subjects concurrently with plerixafor.
- FIG. 15 provides representative flow plots from blood samples taken four hours post mobilization from Rhesus monkeys. Intravenous administration of 450 pg/kg Gro-b T and subcutaneous administration of 1 mg/kg plerixafor (AMD3100) leads to the mobilization of a population of CD34 dim cells.
- AMD3100 1 mg/kg plerixafor
- FIG. 16 provides representative flow plots from blood samples taken at baseline versus four hours post-mobilization from Rhesus monkeys. Mobilization was induced by (1) intravenous administration of 450 pg/kg Gro-b T and subcutaneous administration of 1 mg/kg plerixafor (AMD3100) or (2) subcutaneous administration of 1 mg/kg plerixafor (AMD3100). The combination of Gro-b T and plerixafor (as compared to plerixafor alone) leads to enhanced mobilization of O ⁇ 34 ⁇ 111 cells.
- FIG. 17 is a graph quantifying the concentration of CD34 dim cells in peripheral blood from untreated Rhesus monkeys (“Unmobilized”), Rhesus monkeys that have been treated with intravenous administration of 450 pg/kg Gro-b T and subcutaneous administration of 1 mg/kg plerixafor (“Gro-b T + plerixafor”), Rhesus monkeys that have been treated with subcutaneous administration of 1 mg/kg plerixafor (“plerixafor”) and Rhesus monkeys that have been treated with subcutaneous administration of 50 pg/kg (q.d. x 5) G-CSF (“G-CSF”).
- CD34 dim cells were present at a significantly higher concentration in blood mobilized using Gro-b T plus plerixafor.
- FIG. 18 is a graph depicting the composition of unmobilized cells and grafts mobilized by G-CSF, Gro-b T and AMD3100 and AMD3100 alone. As shown, grafts mobilized using Gro-b T and AMD3100 show a 3 fold increase in CD34 dim cells and a 3 fold increase in T-cells as compared to grafts mobilized using G-CSF.
- FIG. 19 provides graphs showing that Gro-b T and AMD3100 mobilized CD34 dim cells suppressed T-cell proliferation as measured by carboxyfluorescein succinimidyl ester (CFSE) staining after four days in culture.“Beads” indicates stimulation of T-cells using anti-CD2/CD3/CD28 coated microbeads.
- CFSE carboxyfluorescein succinimidyl ester
- FIG. 20 provides a survival curve showing that by day 24, all mice (13/13) transplanted with unmobilized PBMCs had died of aGVHD compared to 5/16 mice transplanted with AMD3100 mobilized peripheral blood, 3/16 mice transplanted with G-CSF mobilized PBMCs and none of the mice transplanted with Gro-b T and AMD3100 mobilized PBMCs.
- 15/16 mice transplanted with Gro-b T and AMD3100 remained alive, whereas only 10/16 mice transplanted with AMD3100 mobilized PBMCs and 11/16 mice transplanted with G-CSF mobilized PBMCs remained alive.
- FIG. 21A shows rhesus CD45+CD3+ T-cell numbers in mice at day 14 post- transplant with unmobilized PBMCs, PBMCs mobilized with Gro-b T and plerixafor (i.e., AMD3100), PBMCs mobilized with plerixafor alone, and PBMCs mobilized with G-CSF alone.
- FIG. 21B shows T-cell numbers in mice at day 14 post-transplant with unmobilized PBMCs, Gro-b T and plerixafor (i.e., AMD3100) mobilized PBMCs, and Gro-b T and plerixafor mobilized PBMCs that have been depleted of CD34dim cells.
- FIG. 21A shows rhesus CD45+CD3+ T-cell numbers in mice at day 14 post- transplant with unmobilized PBMCs, PBMCs mobilized with Gro-b T and plerixafor (i.e., AMD3100), PBMCs mobilized with plerix
- 21C provides a survival curve of mice transplanted with unmobilzied PBMCs, Gro-b T and AMD3100 mobilized PBMCs, and Gro-b T and AMD3100 mobilized PBMCs that have been depleted of CD34dim cells.
- the present invention provides compositions and methods for mobilizing
- the subject may be a hematopoietic stem and progenitor cell donor (i.e ., a donor), such as a mammalian donor ( e.g ., a human donor).
- a hematopoietic stem and progenitor cell donor i.e ., a donor
- a mammalian donor e.g ., a human donor
- the compositions and methods described herein can additionally be used for the treatment of one or more stem cell disorders in a patient, such as a human patient.
- a C-X-C chemokine receptor type 2 (CXCR2) agonist such as Gro-b or a variant thereof, such as a truncated form of Gro- b (e.g., Gro-b T), as described herein, optionally in combination with a C-X-C chemokine receptor type 4 (CXCR4) antagonist, such as l,r-[l,4-phenylenebis(methylene)]-bis- 1,4,8, 1 l-tetra-azacyclotetradecane or a variant thereof, may be administered to a donor, as described herein, in amounts sufficient to mobilize hematopoietic stem and progenitor cells.
- CXCR2 C-X-C chemokine receptor type 2
- CXCR4 C-X-C chemokine receptor type 4
- compositions and methods described herein are capable of mobilizing hematopoietic stem and progenitor cells from a stem cell niche within a donor into circulating peripheral blood while reducing the mobilization of other cells of the hematopoietic lineage, such as leukocytes, neutrophils, lymphocytes, and monocytes.
- the compositions and methods described herein thus enable the selective mobilization of hematopoietic stem and progenitor cells in a donor, which may then be isolated from a donor for therapeutic use.
- the invention is based, in part, on the discovery that administration of a CXCR2 agonist, such as Gro-b, Gro-b T, or a variant thereof, optionally in combination with a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, at particular doses can provide the important clinical benefit of mobilizing populations of cells that are enriched in hematopoietic stem cells relative to other cell types, such as leukocytes, neutrophils, and monocytes. This ability is advantageous, as these other cell types may be undesirable for administration to a human patient undergoing hematopoietic stem cell transplant therapy.
- the populations of mobilized hematopoietic stem and progenitor cells produced using the compositions and methods described herein are particularly suitable for hematopoietic stem cell transplantation therapy.
- the hematopoietic stem or progenitor cells may be isolated for ex vivo expansion and/or for therapeutic use.
- the withdrawn cells may be infused into a patient, such as the donor or another subject (e.g ., a subject that is HLA-matched to the donor) for the treatment of one or more pathologies of the hematopoietic system.
- the mobilized cells may be withdrawn and then expanded ex vivo , such as by contacting the cells with an aryl hydrocarbon receptor antagonist, so as to produce a population of hematopoietic stem cells having a sufficient quantity of cells for transplantation.
- hematopoietic stem cells are capable of differentiating into a multitude of cell types in the hematopoietic lineage, and can thus be administered to a patient in order to populate or repopulate a cell type that is defective or deficient in the patient.
- the patient may be one, for example, that is suffering from one or more blood disorders, such as an autoimmune disease, cancer, hemoglobinopathy, or other hematopoietic pathology, and is therefore in need of hematopoietic stem cell transplantation.
- the invention thus provides methods of treating a variety of hematopoietic conditions, such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency- severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among others.
- hematopoietic conditions such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency- severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immune deficiency syndrome, as well as cancers and autoimmune diseases, among others.
- the sections that follow provide a description of CXCR4 antagonists and CXCR2 agonists that can be administered to a donor so as to induce mobilization of a population of hematopoietic stem or progenitor cells from a stem cell niche into peripheral blood, from which the cells may subsequently be isolated and infused into a patient for the treatment, for example, of one or more stem cell disorders, such as a cancer, autoimmune disease, of metabolic disorder described herein.
- the following sections additionally describe methods of determining whether populations of cells mobilized with a CXCR2 agonist and/or a CXCR antagonist are suitable for release for ex vivo expansion and/or for therapeutic applications.
- the term“about” refers to a value that is within 10% above or below the value being described.
- the term“about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.
- the terms "acquire” and “acquiring” means obtaining possession of a physical entity, or a value, such as a numerical value, directly acquiring or indirectly acquiring the physical entity or value.
- Directly acquiring means performing a process (e.g ., performing an assay or test on a sample or analyzing a sample) to obtain the physical entity or value.
- Indirectly acquiring refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value).
- Directly acquiring a physical entity includes performing a process, e.g, analyzing a sample, such as a sample of hematopoietic cells isolated from a donor that has undergone or is undergoing a hematopoietic stem cell mobilization regimen described herein.
- Directly acquiring a value includes performing a process, such as an assay, on a sample or another substance, e.g, performing an analytical process which includes determining the quantity of hematopoietic stem cells in a sample, the ratio of hematopoietic stem cells to cells of another type within the hematopoietic lineage, or the frequency of hematopoietic stem cells among the total quantity of cells in a sample.
- affinity refers to the strength of the non-covalent interaction between two or more molecules, such as two or more proteins (e.g ., a
- Affinity can be expressed quantitatively, for example, as an equilibrium dissociation constant (K d ) or, in cases in which one of the binding partners is an enzyme, as an inhibition constant (3 ⁇ 4).
- Binding affinity can be determined using standard techniques, such as enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance assays, and isothermal titration calorimetry assays, among others.
- ELISA enzyme-linked immunosorbent assays
- surface plasmon resonance assays surface plasmon resonance assays
- isothermal titration calorimetry assays among others.
- antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab', F(ab') 2 , Fab, Fv, rlgG, and scFv fragments.
- the term“monoclonal antibody” is meant to include both intact molecules, as well as antibody fragments (including, for example, Fab and F(ab') 2 fragments) that are capable of specifically binding to a target protein.
- the Fab and F(ab') 2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.
- antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen.
- the antigen binding function of an antibody can be performed by fragments of a full-length antibody.
- the antibody fragments can be, for example, a Fab, F(ab’) 2 , scFv, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody.
- binding fragments encompassed of the term“antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH!
- a dAb which consists of a V H or a V L domain
- CDR complementarity determining region
- the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston el al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
- scFv single chain Fv
- These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.
- Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.
- bispecific antibody refers to, for example, a monoclonal, often a human or humanized antibody that is capable of binding at least two different antigens.
- CD34 dim cells refers to a population of cells, of which at least a portion of the population expresses the markers CD34, CD1 lb, and CD45 and does not substantially express the markers CD3, CD8, or CD20, wherein CD34 and CD45 are expressed at a relatively low level.
- This cell population exhibits characteristics of monocytes, for example, the ability to suppress alloreactive T lymphocyte proliferation. (D’Aveni et al. (2015) Science Translational Medicine 7(281): 1-12.
- a population of CD34 dim cells may be CD14+.
- CD34 dim cell population when viewing a flow cytometry plot as a population of cells that is CD34 + but has a brightness between the base level of fluorescence (e.g, autofluorescence) in the cell population being examined and the brightness of a CD34 + bright cell (e.g., a hematopoietic stem cell and/or a CD34 + CD90 + cell) population.
- a CD34 dim cell population exhibits between 5% and 95% of the brightness of a CD34 + bright cell (e.g., a hematopoietic stem cell) population, but is brighter than a CD34 cell population.
- CD34 dim cells exhibit 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%
- the CD34 dim cells are at least 5% brighter, at least 10% brighter, at least 20% brighter or at least 30% brighter than CD34 cells, but is less bright than CD34 + bright cells (e.g., a hematopoietic stem cells).
- CD34 dim cells are identified in a cell sample by using flow cytometry with magnetic beads instead of fluorescence.
- the magnetic beads will selectively pull down CD34 + bright cells, leaving CD34 dim cells in the cell sample.
- CD34 dim cells are identified by measuring the number of copies of CD34 expressed by the cells.
- CD34 dim cells can exhibit between 5% and 95% of the number of copies of CD34 as compared to CD34 + bright cells (e.g., hematopoietic stem cells), or between 10% and 90%, 10% and 80%, 10% and 70%, 10% and 60%, 10% and 50%, 10% and 40%, 10% and 30%, 10% and 20%, 20% and 90%, 20% and
- CD34 + bright cells e.g., hematopoietic stem cells
- CDR complementarity determining region
- FRs framework regions
- the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions.
- variable domains of native heavy and light chains each contain four framework regions that primarily adopt a b-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the b-sheet structure.
- the CDRs in each chain are held together in close proximity by the framework regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Rabat et al ., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda,
- an agent as described herein such as a CXCR4 antagonist (e.g ., plerixafor or a variant thereof) and/or a CXCR2 agonist (e.g., Gro-b or a variant or truncation thereof, such as Gro-b T) can be administered to a subject over a period of time, for example, by intravenous or subcutaneous injection.
- An agent is considered to have“completed administration” once the prescribed dosage of the agent has been administered to the subject in its entirety.
- agents are considered to have“completed administration” once the prescribed dosages of all agents in a particular regimen have been administered to the subject in their entirety.
- the terms“conservative mutation,”“conservative substitution,” or “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in TABLE 1 below.
- conservative amino acid families include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W.
- a conservative mutation or substitution is therefore one that substitutes one amino acid for a member of the same amino acid family (e.g ., a substitution of Ser for Thr or Lys for Arg).
- CRU competitive repopulating unit
- the term“donor” refers to a subject, such as a mammalian subject (e.g., a human subject) from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient.
- the one or more cells may be, for example, a population of hematopoietic stem or progenitor cells.
- the term“diabody” refers to a bivalent antibody containing two polypeptide chains, in which each polypeptide chain includes V H and V L domains joined by a linker that is too short (e.g, a linker composed of five amino acids) to allow for
- the term“triabody” refers to trivalent antibodies containing three peptide chains, each of which contains one V H domain and one V L domain joined by a linker that is exceedingly short (e.g, a linker composed of 1-2 amino acids) to permit intramolecular association of V H and V L domains within the same peptide chain.
- peptides configured in this way typically trimerize so as to position the V H and V L domains of neighboring peptide chains spatially proximal to one another (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-48).
- the term“disrupt” with respect to a gene refers to preventing the formation of a functional gene product.
- a gene product is functional only if it fulfills its normal (wild-type) functions.
- Disruption of the gene prevents expression of a functional factor encoded by the gene and comprises an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and/or a promoter and/or an operator that is necessary for expression of the gene in the animal.
- the disrupted gene may be disrupted by, e.g, removal of at least a portion of the gene from a genome of the animal, alteration of the gene to prevent expression of a functional factor encoded by the gene, an interfering RNA, or expression of a dominant negative factor by an exogenous gene.
- Materials and methods of genetically modifying hematopoietic stem/progenitor cells are detailed in US 8,518,701; US 2010/0251395; and US 2012/0222143, the disclosures of each of which are incorporated herein by reference in their entirety (in case of conflict, the instant specification is controlling).
- Various techniques known in the art can be used to inactivate genes to make knock out animals and/or to introduce nucleic acid constructs into animals to produce founder animals and to make animal lines, in which the knockout or nucleic acid construct is integrated into the genome.
- Such techniques include, without limitation, pronuclear microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA, 82:6148-6152), gene targeting into embryonic stem cells (Thompson et al. (1989) Cell, 56:313-321), electroporation of embryos (Lo (1983 )Mol. Cell.
- An animal that is genomically modified is an animal wherein all of its cells have the genetic modification, including its germ line cells.
- the animals may be inbred and progeny that are genomically modified may be selected.
- Cloning for example, may be used to make a mosaic animal if its cells are modified at the blastocyst state, or genomic modification can take place when a single-cell is modified. Animals that are modified so they do not sexually mature can be homozygous or heterozygous for the modification, depending on the specific approach that is used. If a particular gene is inactivated by a knock out modification, homozygosity would normally be required. If a particular gene is inactivated by an RNA interference or dominant negative strategy, then heterozygosity is often adequate.
- a“dual variable domain immunoglobulin” refers to an antibody that combines the target-binding variable domains of two monoclonal antibodies via linkers to create a tetravalent, dual-targeting single agent (see, for example, Gu et al. (2012) Meth. Enzymol., 502:25-41).
- the term“endogenous” describes a substance, such as a molecule, cell, tissue, or organ (e.g ., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is found naturally in a particular organism, such as a human patient.
- a hematopoietic stem cell or a cell of hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte
- the term“engraftment potential” is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate a tissue, whether such cells are naturally circulating or are provided by transplantation.
- the term encompasses all events surrounding or leading up to engraftment, such as tissue homing of cells and colonization of cells within the tissue of interest.
- the engraftment efficiency or rate of engraftment can be evaluated or quantified using any clinically acceptable parameter as known to those of skill in the art and can include, for example, assessment of competitive repopulating units (CRU); incorporation or expression of a marker in tissue(s) into which stem cells have homed, colonized, or become engrafted; or by evaluation of the progress of a subject through disease progression, survival of hematopoietic stem and progenitor cells, or survival of a recipient.
- Engraftment can also be determined by measuring white blood cell counts in peripheral blood during a post-transplant period. Engraftment can also be assessed by measuring recovery of marrow cells by donor cells in a bone marrow aspirate sample.
- exogenous describes a substance, such as a molecule, cell, tissue, or organ (e.g ., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is not found naturally in a particular organism, such as a human patient.
- Exogenous substances include those that are provided from an external source to an organism or to cultured matter extracted therefrom.
- framework region or“FW region” includes amino acid residues that are adjacent to the CDRs of an antibody or antigen -binding fragment thereof.
- FW region residues may be present in, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies, among others.
- hematopoietic progenitor cells includes pluripotent cells capable of differentiating into several cell types of the hematopoietic system, including, without limitation, granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T- cells, among others. Hematopoietic progenitor cells are committed to the hematopoietic cell lineage and generally do not self-renew. Hematopoietic progenitor cells can be identified, for example, by expression patterns of cell surface antigens, and include cells having the following immunophenotype: Lin KLS + Flk2 CD34 + .
- Hematopoietic progenitor cells include short-term hematopoietic stem cells, multi-potent progenitor cells, common myeloid progenitor cells, granulocyte-monocyte progenitor cells, and megakaryocyte-erythrocyte progenitor cells.
- the presence of hematopoietic progenitor cells can be determined functionally, for example, by detecting colony -forming unit cells, e.g ., in complete methylcellulose assays, or phenotypically through the detection of cell surface markers using flow cytometry and cell sorting assays described herein and known in the art.
- HSCs hematopoietic stem cells
- granulocytes e.g, promyelocytes, neutrophils, eosinophils, basophils
- erythrocytes e.g, reticulocytes, erythrocytes
- thrombocytes e.g, megakaryoblasts, platelet producing megakaryocytes, platelets
- monocytes e.g, monocytes, macrophages
- dendritic cells e.g, NK cells, B-cells and T-cells
- lymphocytes e.g, NK cells, B-cells and T-cells.
- CD34 + cells are immature cells that express the CD34 cell surface marker. In humans,
- CD34 + cells are believed to include a subpopulation of cells with the stem cell properties defined above, whereas in mice, HSCs are CD34-.
- HSCs also refer to long term repopulating HSCs (LT-HSC) and short term repopulating HSCs (ST-HSC).
- LT-HSCs and ST-HSCs are differentiated, based on functional potential and on cell surface marker expression.
- human HSCs are CD34 + , CD38 , CD45RA , CD90 + , CD49F + , and lin (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD 10, CD11B, CD 19, CD20, CD56, CD235A).
- bone marrow LT-HSCs are CD34-, SCA- 1+, C-kit+, CD135-, Slamfl/CDl50+, CD48-, and lin- (negative for mature lineage markers including Terl l9, CDl lb, Grl, CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs are CD34 + , SCA-l + , C-kit + , CD135 , Slamfl/CDl50 + , and lin (negative for mature lineage markers including Terl l9, CDl lb, Grl, CD3, CD4, CD8, B220, IL7ra).
- ST- HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions.
- LT-HSC have greater self-renewal potential (i.e., they survive throughout adulthood, and can be serially transplanted through successive recipients), whereas ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time, and do not possess serial transplantation potential). Any of these HSCs can be used in the methods described herein.
- ST-HSCs are particularly useful because they are highly proliferative and thus, can more quickly give rise to differentiated progeny.
- hematopoietic stem cell functional potential refers to the functional properties of hematopoietic stem cells which include 1) multi -potency (which refers to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g ., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g, megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g, monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g, NK cells, B-cells and T-cells), 2) self- renewal (which refers to the ability of hematopoietic stem cells to give rise to daughter cells that have equivalent potential as the mother cell, and further that this
- multi -potency which refers to the
- MHC Major histocompatibility complex antigens
- HLA human leukocyte antigens
- MHC/HLA antigens are target molecules that are recognized by T cells and NK cells as being derived from the same source of hematopoietic stem cells as the immune effector cells ("self) or as being derived from another source of hematopoietic reconstituting cells ("non- self).
- HLA class I and HLA class II Two main classes of HLA antigens are recognized: HLA class I and HLA class II.
- HLA class I antigens (A, B, and C in humans) render each cell recognizable as "self”
- HLA class II antigens DR, DP, and DQ in humans
- MHC class I A, B and C
- MHC class II DP, DQ and DR
- HLA-B genes B5, and Bw4l
- Allelic gene products differ in one or more amino acids in the a and/or b domain(s).
- Large panels of specific antibodies or nucleic acid reagents are used to type HLA haplotypes of individuals, using leukocytes that express class I and class II molecules.
- the genes commonly used for HLA typing are the six MHC Class I and Class II proteins, two alleles for each of HLA- A; HLA-B and HLA-DR.
- the HLA genes are clustered in a "super-locus" present on chromosome position 6p2l, which encodes the six classical transplantation HLA genes and at least 132 protein coding genes that have important roles in the regulation of the immune system as well as some other fundamental molecular and cellular processes.
- the complete locus measures roughly 3.6 Mb, with at least 224 gene loci.
- haplotypes i.e. the set of alleles present on a single chromosome, which is inherited from one parent, tend to be inherited as a group.
- the set of alleles inherited from each parent forms a haplotype, in which some alleles tend to be associated together. Identifying a patient's haplotypes can help predict the probability of finding matching donors and assist in developing a search strategy, because some alleles and haplotypes are more common than others and they are distributed at different frequencies in different racial and ethnic groups.
- HLA-matched refers to a donor-recipient pair in which none of the HLA antigens are mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplant therapy.
- HLA-matched ⁇ i.e., where all of the 6 alleles are matched) donor- recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells are less likely to recognize the incoming graft as foreign, and are thus less likely to mount an immune response against the transplant.
- HLA-mismatched refers to a donor-recipient pair in which at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplant therapy.
- HLA-A, HLA-B and HLA-DR HLA-mismatched
- HLA-mismatched donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched donor-recipient pairs, as endogenous T cells and NK cells are more likely to recognize the incoming graft as foreign in the case of an HLA-mismatched donor-recipient pair, and such T cells and NK cells are thus more likely to mount an immune response against the transplant.
- human antibody refers to an antibody in which
- a human antibody can be produced in a human cell (for example, by recombinant expression) or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (such as heavy chain and/or light chain) genes.
- a human antibody is a single chain antibody, it can include a linker peptide that is not found in native human antibodies.
- an Fv can contain a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
- linker peptides are considered to be of human origin.
- Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, for example, PCT Publication Nos. WO
- the term“humanized” antibody refers to a non-human antibody that contains minimal sequences derived from non-human immunoglobulin.
- a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non human immunoglobulin. All or substantially all of the FW regions may also be those of a human immunoglobulin sequence.
- the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
- Fc immunoglobulin constant region
- patients that are“in need of’ a hematopoietic stem cell transplant include patients that exhibit a defect or deficiency in one or more blood cell types, as well as patients having a stem cell disorder, autoimmune disease, cancer, or other pathology described herein.
- Hematopoietic stem cells generally exhibit 1) multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g ., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g, reticulocytes, erythrocytes), thrombocytes (e.g, megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g ., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal, and can thus give rise to daughter cells that have equivalent potential as the mother cell, and 3) the ability to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re-establish productive and sustained hema
- Hematopoietic stem cells can thus be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re-constitute the defective or deficient population of cells in vivo.
- the patient may be suffering from cancer, and the deficiency may be caused by administration of a chemotherapeutic agent or other medicament that depletes, either selectively or non-specifically, the cancerous cell population.
- the patient may be suffering from a hemoglobinopathy (e.g, a non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
- a hemoglobinopathy e.g, a non-malignant hemoglobinopathy
- the subject may be one that is suffering from adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
- ADA SCID adenosine deaminase severe combined immunodeficiency
- the subject may have or be affected by an inherited blood disorder (e.g, sickle cell anemia) or an autoimmune disorder. Additionally or alternatively, the subject may have or be affected by a malignancy, such as neuroblastoma or a hematologic cancer.
- the subject may have a leukemia, lymphoma, or myeloma.
- 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
- 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.
- the subject is in need of chimeric antigen receptor T-cell (CART) therapy.
- the subject has or is otherwise affected by a metabolic storage disorder.
- the subject may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease,
- sphingolipidoses metachromatic leukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wi scott- Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1 :319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it pertains to pathologies that may be treated by administration of hematopoietic stem cell transplant therapy.
- IgM hyper immunoglobulin M
- a patient“in need of’ a hematopoietic stem cell transplant may one that is or is not suffering from one of the foregoing pathologies, but nonetheless exhibits a reduced level (e.g ., as compared to that of an otherwise healthy subject) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B -lymphocytes.
- endogenous cell types within the hematopoietic lineage such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, e
- FACS fluorescence activated cell sorting
- leukocyte refers to a heterogeneous group of nucleated blood cell types, and excludes erythrocytes and platelets. Leukocytes can be divided into two general groups: polymorphonucleocytes, which include neutrophils, eosinophils, and basophils, and mononucleocytes, which include lymphocytes and monocytes.
- Polymorphonucleocytes contain many cytoplasmic granules and a multilobed nucleus and include the following: neutrophils, which are generally amoeboid in shape, phagocytic, and stain with both basic and acidic dyes, and eosinophils and basophils, which contain cytoplasmic granules that stain with acidic dyes and with basic dyes, respectively.
- neutrophils which are generally amoeboid in shape, phagocytic, and stain with both basic and acidic dyes
- eosinophils and basophils which contain cytoplasmic granules that stain with acidic dyes and with basic dyes, respectively.
- lymphocyte refers to a mononuclear leukocyte that is involved in the mounting of an immune response.
- lymphocytes include B lymphocytes, T lymphocytes, and NK cells.
- the terms“mobilize” and“mobilization” refer to processes by which a population of hematopoietic stem or progenitor cells is released from a stem cell niche, such as the bone marrow of a subject, into circulation in the peripheral blood. Mobilization of hematopoietic stem and progenitor cells can be monitored, for example, by assessing the quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from a subject.
- the peripheral blood sample may be withdrawn from the subject, and the quantity or concentration of hematopoietic stem or progenitor cells in the peripheral blood sample may subsequently be assessed, following the administration of a hematopoietic stem or progenitor cell mobilization regimen to the subject.
- the mobilization regimen may include, for example, a CXCR4 antagonist, such as a CXCR4 antagonist described herein ( e.g ., plerixafor or a variant thereof), and a CXCR2 agonist, such as a CXCR2 agonist described herein (e.g., Gro-b or a variant thereof, such as a truncation of Gro-b, for example, Gro-b T).
- a CXCR4 antagonist such as a CXCR4 antagonist described herein (e.g ., plerixafor or a variant thereof)
- a CXCR2 agonist such as a CXCR2 agonist described herein (e.g., Gro-b or
- administration of the mobilization regimen may be compared to the quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from the subject prior to administration of the mobilization regimen.
- An observation that the quantity or concentration of hematopoietic stem or progenitor cells has increased in the peripheral blood of the subject following administration of the mobilization regimen is an indication that the subject is responding to the mobilization regimen, and that hematopoietic stem and progenitor cells have been released from one or more stem cell niches, such as the bone marrow, into peripheral blood circulation.
- HSCs are CD34 + , CD38 , CD45RA , CD90 + , CD49F + , and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A).
- Additional methods for determining the quantity or concentration of hematopoietic stem or progenitor cells in a peripheral blood sample isolated from a subject include assays that quantify the number of colony-forming units (CFUs) in the sample, which is a measure of the quantity of viable hematopoietic stem or progenitor cells that, upon incubation with an appropriate culture medium, give rise to an individual population of hematopoietic stem or progenitor cells.
- CFUs colony-forming units
- the term“mobilizing amount” refers to a quantity of one or more agents, such as a quantity of a CXCR4 antagonist and/or a CXCR2 agonist described herein (In some embodiments, a quantity of plerixafor, or a variant thereof, and/or Gro-b, or a variant thereof, such as a truncation of Gro-b, for example, Gro-b T) that mobilizes a population of hematopoietic stem or progenitor cells upon administration to a subject, such as a mammalian subject ( e.g ., a human subject).
- a mammalian subject e.g ., a human subject
- Exemplary mobilizing amounts of these agents include amounts sufficient to effectuate the release of a population of, for example, from about 20 to about 40 CD34 + cells/pL of peripheral blood, such as from about 21 to about 39 CD34 + cells/pL of peripheral blood, about 22 to about 38 CD34 + cells/pL of peripheral blood, about 23 to about 37 CD34 + cells/pL of peripheral blood, about 24 to about 36 CD34 + cells/pL of peripheral blood, about 25 to about 35 CD34 + cells/pL of peripheral blood, about 26 to about 34 CD34 + cells/pL of peripheral blood, about 27 to about 33 CD34 + cells/pL of peripheral blood, about 28 to about 32 CD34 + cells/pL of peripheral blood, or about 29 to about 31 CD34 + cells/pL of peripheral blood (e.g., about 20 CD34 + cells/pL of peripheral blood, 21 CD34 + cells/pL of peripheral blood, 22 CD34 + cells/pL of peripheral blood, 23 CD34 + cells/pL of peripheral blood, 24, CD34 + cells/pL of
- mobilizing amounts of a CXCR2 agonist include from about 50 pg/kg of recipient to about 1 mg/kg of recipient, such as from about 50 pg/kg to about 300 pg/kg, 100 pg/kg to about 250 pg/kg, or about 150 pg/kg.
- Mobilizing amounts of a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof include from about 50 pg/kg of recipient to about 500 pg/kg of recipient, such as from about 200 pg/kg to about 300 pg/kg, or about 240 pg/kg.
- the term“monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
- a monocyte refers to a CDl4 + and CD34- peripheral blood mononuclear cell (PBMC), which is generally capable of differentiating into a macrophage and/or dendritic cell upon activation by one or more foreign substances, such as, a microbial product.
- a monocyte may express elevated levels of the CD 14 surface antigen marker, and may express at least one biomarker selected from CD64, CD93, CD 180, CD328 (also known as sialic acid-binding Ig-like lectin 7 or Siglec7), and CD329 (sialic acid-binding Ig-like lectin 9 or Siglec9), as well as the peanut agglutinin protein (PNA).
- PBMC peripheral blood mononuclear cell
- a“peptide” refers to a single-chain polyamide containing a plurality of amino acid residues, such as naturally-occurring and/or non-natural amino acid residues, that are consecutively bound by amide bonds.
- Examples of peptides include shorter fragments of full-length proteins, such as full-length naturally-occurring proteins.
- the term“recipient” refers to a patient that receives a transplant, such as a transplant containing a population of hematopoietic stem cells.
- the transplanted cells administered to a recipient may be, e.g ., autologous, syngeneic, or allogeneic cells.
- sample refers to a specimen (e.g, blood, blood component (e.g, serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g, placental or dermal), pancreatic fluid, chorionic villus sample, and cells) taken from a subject.
- a sample may be, for example, withdrawn peripheral blood from a donor that is undergoing or has undergone a hematopoietic stem or progenitor cell mobilization regimen described herein.
- scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain.
- scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (V L ) (e.g, CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (V H ) (e.g, CDR-H1, CDR-H2, and/or CDR-H3) separated by a linker.
- V L variable region of an antibody light chain
- V H variable region of an antibody heavy chain
- the linker that joins the V L and V H regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids.
- linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (for example, linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (for example, a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (for example, linkers containing glycosylation sites).
- linkers containing D-amino acids for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
- hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
- variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived.
- nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g ., in CDR and/or framework residues) so as to preserve or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.
- stem cell disorder broadly refers to any disease, disorder, or condition that may be treated or cured by engrafting or transplanting a population of hematopoietic stem or progenitor cells in a target tissue within a patient.
- Type I diabetes has been shown to be cured by hematopoietic stem cell transplant, along with various other disorders.
- Exemplary diseases that can be treated by infusion of hematopoietic stem or progenitor cells into a patient are sickle cell anemia, thalassemias, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
- Additional diseases that may be treated by transplantation of hematopoietic stem and progenitor cells as described herein include blood disorders (e.g., sickle cell anemia) and autoimmune disorders, such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn’s disease.
- Additional diseases that may be treated using hematopoietic stem and progenitor cell transplant therapy include cancer, such as a cancer described herein.
- Exemplary stem cell disorders are malignancies, such as a neuroblastoma or a hematologic cancers, such as leukemia, lymphoma, and myeloma.
- the cancer may be acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin’s lymphoma.
- Additional diseases treatable using hematopoietic stem or progenitor cell transplant therapy include myelodysplastic syndrome.
- the patient has or is otherwise affected by a metabolic storage disorder.
- the patient may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses,
- metachromatic leukodystrophy or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wi scott- Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in“Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education Book, 1 :319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it pertains to pathologies that may be treated by administration of hematopoietic stem or progenitor cell transplant therapy.
- IgM hyper immunoglobulin M
- stem cell niche refers to a microenvironment within a donor, such as a mammalian donor (e.g ., a human donor) in which endogenous hematopoietic stem or progenitor cells reside.
- a mammalian donor e.g ., a human donor
- An exemplary stem cell niche is bone marrow tissue.
- 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.
- a patient such as a human patient, that is in need of hematopoietic stem cell transplantation may receive treatment that includes a population of hematopoietic stem cells so as to treat a stem cell disorder, such as a cancer, autoimmune disease, or metabolic disorder described herein.
- the hematopoietic stem cells that are transplanted into the patient may be, for example, a population of hematopoietic stem cells that has been mobilized and withdrawn from a donor in accordance with the compositions and methods described herein.
- the hematopoietic stem cells that are transplanted into the patient may be mobilized within a donor by administration of a CXCR4 antagonist and/or a CXCR2 agonist to the donor.
- the term“transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection and the like.
- the terms“treat” or“treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder or to promote a beneficial phenotype in the patient being treated.
- Beneficial or desired clinical results include, but are not limited to, promoting the engraftment of exogenous hematopoietic cells in a patient following hematopoietic stem or progenitor cell transplant therapy.
- the benefits include a more rapid engraftment of transplanted cells, e.g., neutrophils and platelets.
- neutrophil recovery occurs within about 5-20 days post- transplant, about 5-15 days post-transplant, about 5-10 days post-transplant, about 7-12 days post-transplant, about 8-12 days post transplant, about 9-15 days post-transplant, about 10-15 days post-transplant, or about 10 days post-transplant.
- platelet recovery occurs within about 10-20 days post-transplant, about 10-15 days post-transplant, about 15-20 days post-transplant, about 12-18 days post transplant, about 12-17 days post transplant, about 13-18 days post-transplant, about 12-17 days post-transplant, or about 15 days post-transplant.
- Additional beneficial results include an increase in the cell count or relative concentration of hematopoietic stem cells in a patient in need of a hematopoietic stem or progenitor cell transplant following administration of an exogenous hematopoietic stem or progenitor cell graft to the patient.
- Beneficial results of therapy described herein may also include an increase in the cell count or relative
- hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte, following and subsequent hematopoietic stem cell transplant therapy.
- Additional beneficial results may include the reduction in quantity of a disease-causing cell population, such as a population of cancer cells or autoimmune cells.
- variants and“derivative” are used interchangeably and refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein.
- a variant or derivative of a compound, peptide, protein, or other substance described herein may retain or improve upon the biological activity of the original material.
- vector includes a nucleic acid vector, such as a plasmid, a
- DMA vector a plasmid, a RNA vector, virus, or other suitable replicon.
- Expression vectors described herein may contain a polynucleotide sequence as well as, for example, additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
- Certain vectors that can be used for the expression of peptides and proteins, such as those described herein, include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
- Suitable vectors for expression of peptides and proteins described herein contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements may include, for example, 5’ and 3’ untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
- the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.
- alkyl refers to a straight- or branched-chain alkyl group having, for example, from 1 to 20 carbon atoms in the chain.
- alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.
- alkylene refers to a straight- or branched-chain divalent alkyl group. The divalent positions may be on the same or different atoms within the alkyl chain. Examples of alkylene include methylene, ethylene, propylene, isopropylene, and the like.
- heteroalkyl refers to a straight or branched-chain alkyl group having, for example, from 1 to 20 carbon atoms in the chain, and further containing one or more heteroatoms (e.g ., oxygen, nitrogen, or sulfur, among others) in the chain.
- heteroatoms e.g ., oxygen, nitrogen, or sulfur, among others
- heteroalkyl ene refers to a straight- or branched-chain divalent heteroalkyl group.
- the divalent positions may be on the same or different atoms within the heteroalkyl chain.
- the divalent positions may be one or more heteroatoms.
- alkenyl refers to a straight- or branched-chain alkenyl group having, for example, from 2 to 20 carbon atoms in the chain.
- alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, tert-butyl enyl, hexenyl, and the like.
- alkenylene refers to a straight- or branched-chain divalent alkenyl group. The divalent positions may be on the same or different atoms within the alkenyl chain. Examples of alkenylene include ethenylene, propenylene, isopropenylene, butenylene, and the like.
- heteroalkenyl refers to a straight- or branched-chain alkenyl group having, for example, from 2 to 20 carbon atoms in the chain, and further containing one or more heteroatoms (e.g ., oxygen, nitrogen, or sulfur, among others) in the chain.
- heteroalkenylene refers to a straight- or branched-chain divalent heteroalkenyl group.
- the divalent positions may be on the same or different atoms within the heteroalkenyl chain.
- the divalent positions may be one or more heteroatoms.
- alkynyl refers to a straight- or branched-chain alkynyl group having, for example, from 2 to 20 carbon atoms in the chain.
- alkynyl groups include propargyl, butynyl, pentynyl, hexynyl, and the like.
- 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.
- heteroalkynyl refers to a straight- or branched-chain alkynyl group having, for example, from 2 to 20 carbon atoms in the chain, and further containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur, among others) in the chain.
- heteroalkynylene refers to a straight- or branched-chain divalent heteroalkynyl group.
- the divalent positions may be on the same or different atoms within the heteroalkynyl chain.
- the divalent positions may be one or more heteroatoms.
- cycloalkyl refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 carbon ring atoms.
- cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3. l.0]hexane, and the like.
- 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.
- heterocycloalkyl refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure that is saturated and has, for example, from 3 to 12 ring atoms per ring structure selected from carbon atoms and heteroatoms selected from, e.g ., nitrogen, oxygen, and sulfur, among others.
- the ring structure may contain, for example, one or more oxo groups on carbon, nitrogen, or sulfur ring members.
- heterocycloalkylene refers to a divalent heterocyclolalkyl group.
- the divalent positions may be on the same or different atoms within the ring structure.
- aryl refers to a monocyclic or multicyclic aromatic ring system containing, for example, from 6 to 19 carbon atoms.
- Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, and the like. The divalent positions may be one or more heteroatoms.
- arylene refers to a divalent aryl group.
- the divalent positions may be on the same or different atoms.
- heteroaryl refers to a monocyclic heteroaromatic, or a bicyclic or a tricyclic fused-ring heteroaromatic group.
- Heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, l,2,3-triazolyl, l,2,4-triazolyl, l,2,3-oxadiazolyl, l,2,4-oxadia-zolyl, l,2,5-oxadiazolyl, 1,3,4- oxadiazolyl, l,3,4-triazinyl, l,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, [2,3-dihydro]benzofuryl, [2,3-dihydro]benzofuryl,
- the term“heteroarylene” refers to a divalent heteroaryl group.
- the divalent positions may be on the same or different atoms.
- the divalent positions may be one or more heteroatoms.
- the foregoing chemical moieties such as“alkyl”,“alkylene”,“heteroalkyl”,“heteroalkylene”, “alkenyl”,“alkenylene”,“heteroalkenyl”,“heteroalkenylene”,“alkynyl”,“alkynylene”, “heteroalkynylene”,“cycloalkyl”,“cycloalkylene”,“heterocyclolalkyl”, heterocycloalkylene”,“aryl,”“arylene”,“heteroaryl”, and“heteroarylene” groups can optionally be substituted.
- the term“optionally substituted” refers to a compound or moiety containing one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituents, as permitted by the valence of the compound or moiety or a site thereof, such as a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkyl aryl, alkyl heteroaryl, alkyl cycloalkyl, alkyl heterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfmyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like.
- substitution may include situations in which neighboring substituents have undergone ring closure, such as ring closure of vicinal functional substituents, to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals, formed by ring closure, for example, to furnish a protecting group.
- ring closure such as ring closure of vicinal functional substituents, to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals, formed by ring closure, for example, to furnish a protecting group.
- the present invention is based, in part, on the discovery that hematopoietic stem and progenitor cells can be mobilized by administering particular doses of a CXCR2 agonist, such as Gro-b, Gro-b T, or a variant thereof, optionally in combination with a CXCR4 antagonist to a mammalian donor (e.g ., a human donor) while reducing the mobilization of other cell types, such as leukocytes, neutrophils, lymphocytes, and monocytes.
- a CXCR2 agonist such as Gro-b, Gro-b T, or a variant thereof
- This property is particularly beneficial in the context of hematopoietic stem cell transplant therapy, as hematopoietic stem cells that are mobilized and isolated from a donor using the compositions and method described herein have reduced quantities of cell types that are undesirable for administration to a human patient suffering from a stem cell disorder.
- CXCR2 agonists such as Gro-b, Gro-b T, or a variant thereof, when administered intravenously at a dose of from about 50 pg/kg to about 1 mg/kg, preferably from about 100 pg/kg to about 250 pg/kg, and even more preferably at a dose of about 150 pg/kg, exhibit the ability to rapidly mobilize hematopoietic stem and progenitor cells in a donor (e.g ., a mammalian donor, such as a human donor) while reducing the mobilization of other cells of the hematopoietic lineage that may be undesirable for infusion into a patient (e.g., a mammalian patient, such as a human patient) that is undergoing hematopoietic stem cell transplant therapy.
- hematopoietic stem cells mobilized in a donor by administration of a CXCR2 agonist, such as Gro-b, Gro-b T, or a variant thereof, and optionally, a CXCR4 antagonist, such as plerixafor or a pharmaceutically acceptable salt thereof, are suitable for release for ex vivo expansion and/or for therapeutic use one may acquire an input value for each of one or more parameters set forth in TABLE 2 that characterize a sample of peripheral blood of the donor.
- the one or more parameters may be compared to the corresponding reference criterion for each parameter, and if the reference criterion is satisfied by the ample of hematopoietic stem cells, the cells isolated from the donor may be released for expansion ex vivo and/or for infusion into a patient for therapeutic use (e.g, for the treatment of one or more stem cell disorders described herein).
- a donor e.g ., a mammalian donor, such as a human donor
- parameters for determining whether a population of hematopoietic stem cells obtained from a donor one may select one or more input parameters listed in TABLE 2.
- a donor e.g ., a mammalian donor, such as a human donor
- the parameters used for determining whether a population of hematopoietic stem cells obtained from a donor are a combination of parameters as set forth in any one of TABLES 3-6, below.
- Exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described herein are Gro-b and variants thereof.
- Gro-b also referred to as growth-regulated protein b, chemokine (C-X-C motif) ligand 2 (CXCL2), and macrophage inflammatory protein 2-a (MIP2-a)
- CXCL2 chemokine ligand 2
- MIP2-a macrophage inflammatory protein 2-a
- MMP9 may induce mobilization of hematopoietic stem and progenitor cells from stem cell niches, such as the bone marrow, to circulating peripheral blood by stimulating the degradation of proteins such as stem cell factor, its corresponding receptor, CD117, and CXCL12, all of which generally maintain hematopoietic stem and progenitor cells immobilized in bone marrow.
- exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described herein are truncated forms of Gro-b, such as those that feature a deletion at the N-terminus of Gro-b of from 1 to 8 amino acids (e.g, peptides that feature an N-terminal deletion of 1 amino acids, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, or 8 amino acids).
- CXCR2 agonists that may be used in conjunction with the compositions and methods described herein include Gro-b T, which is characterized by a deletion of the first four amino acids from the N-terminus of Gro-b.
- Gro-b T exhibits particularly advantageous biological properties, such as the ability to induce hematopoietic stem and progenitor cell mobilization with a potency superior to that of Gro-b by multiple orders of magnitude.
- Gro-b and Gro-b T are described, for example, in US Patent No. 6,080,398, the disclosure of which is incorporated herein by reference in its entirety.
- exemplary CXCR2 agonists that may be used in conjunction with the compositions and methods described herein are variants of Gro-b containing an aspartic acid residue in place of the asparagine residue at position 69 of SEQ ID NO: 1.
- This peptide referred to herein as Gro-b N69D, retains the hematopoietic stem and progenitor cell- mobilizing functionality of Gro-b, yet induces this effect with a superior potency.
- CXCR2 agonists that may be used with the compositions and methods described herein include variants of Gro-b T containing an aspartic acid residue in place of the asparagine residue at position 65 of SEQ ID NO: 2.
- This peptide referred to herein as Gro-b T N65D, not only retains hematopoietic stem and progenitor cell-mobilizing capacity, but exhibits a potency that is substantially greater than that of Gro-b T.
- Gro-b N69D and Gro-b T N65D are described, for example, in US Patent No. 6,447,766, the disclosure of which is incorporated herein by reference in its entirety.
- CXCR2 agonists that may be used in conjunction with the compositions and methods described herein include other variants of Gro-b, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-b.
- CXCR2 agonists that may be used in conjunction with the compositions and methods described herein include peptides having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 ( e.g ., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1).
- the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 1 only by way of one or more conservative amino acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 1 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
- CXCR2 agonists useful in conjunction with the compositions and methods described herein are variants of Gro-b T, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-b T.
- the CXCR2 agonist may be a peptide having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 2 ( e.g ., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2).
- the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 2 only by way of one or more conservative amino acid
- the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 2 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
- CXCR2 agonists useful in conjunction with the compositions and methods described herein are variants of Gro-b N69D, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-b N69D.
- the CXCR2 agonist may be a peptide having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 3 (e.g., a peptide having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3).
- the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 3 only by way of one or more conservative amino acid
- the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 3 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid substitutions.
- CXCR2 agonists useful in conjunction with the compositions and methods described herein are variants of Gro-b T N65D, such as peptides that have one or more amino acid substitutions, insertions, and/or deletions relative to Gro-b T N65D.
- the CXCR2 agonist may 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%, 99.%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4).
- the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 4 only by way of one or more conservative amino acid substitutions. In some embodiments, in some embodiments, the amino acid sequence of the CXCR2 agonist differs from that of SEQ ID NO: 4 by no more than 20, no more than 15, no more than 10, no more than 5, or no more than 1 nonconservative amino acid
- the CXCR2 agonist is an antibody or antigen-binding fragment thereof that binds CXCR2 and activates CXCR2 signal transduction.
- the CXCR2 agonist may be an antibody or antigen-binding fragment thereof that binds the same epitope on CXCR2 as Gro-b or a variant or truncation thereof, such as Gro-b T, as assessed, for example, by way of a competitive CXCR2 binding assay.
- the CXCR2 agonist is an antibody or an antigen-binding fragment thereof that competes with Gro-b or a variant or truncation thereof, such as Gro-b T, for binding to CXCR2.
- the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen binding fragment thereof, a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab’) 2 molecule, and a tandem di-scFv.
- the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.
- the peptidic CXCR2 agonists described herein, such as Gro-b, Gro-b T, and variants thereof, may be prepared synthetically, for instance, using solid phase peptide synthesis techniques.
- Systems and processes for performing solid phase peptide synthesis include those that are known in the art and have been described, for instance, in US Patent Nos. 9,169,287; 9,388,212; 9,206,222; 6,028,172; and 5,233,044, among others, the disclosures of each of which are incorporated herein by reference as they pertain to protocols and techniques for the synthesis of peptides on solid support.
- Solid phase peptide synthesis is a process in which amino acid residues are added to peptides that have been immobilized on a solid support, such as a polymeric resin (e.g ., a hydrophilic resin, such as a polyethylene-glycol-containing resin, or hydrophobic resin, such as a polystyrene-based resin).
- a polymeric resin e.g ., a hydrophilic resin, such as a polyethylene-glycol-containing resin, or hydrophobic resin, such as a polystyrene-based resin.
- Peptides such as those containing protecting groups at amino, hydroxy, thiol, and carboxy substituents, among others, may be bound to a solid support such that the peptide is effectively immobilized on the solid support.
- the peptides may be bound to the solid support via their C termini, thereby immobilizing the peptides for subsequent reaction in at a resin-liquid interface.
- the process of adding amino acid residues to immobilized peptides can include exposing a deprotection reagent to the immobilized peptides to remove at least a portion of the protection groups from at least a portion of the immobilized peptides.
- the deprotection reagent exposure step can be configured, for instance, such that side-chain protection groups are preserved, while N-terminal protection groups are removed.
- an exemplary amino protecting contains a fluorenylmethyloxycarbonyl (Fmoc) substituent.
- a deprotection reagent containing a strongly basic substance, such as piperidine e.g., a piperidine solution in an appropriate organic solvent, such as dimethyl formamide (DMF)
- a strongly basic substance such as piperidine
- DMF dimethyl formamide
- Other protecting groups suitable for the protection of amino substituents include, for instance, the tert-butyloxy carbonyl (Boc) moiety.
- a deprotection reagent comprising a strong acid, such as trifluoroacetic acid (TFA) may be exposed to immobilized peptides containing a Boc-protected amino substituent so as to remove the Boc protecting group by an ionization process.
- peptides can be protected and deprotected at specific sites, such as at one or more side-chains or at the N- or C -terminus of an immobilized peptide so as to append chemical functionality regioselectively at one or more of these positions.
- This can be used, for instance, to derivatize a side-chain of an immobilized peptide, or to synthesize a peptide, e.g, from the C-terminus to the N- terminus.
- the process of adding amino acid residues to immobilized peptides can include, for instance, exposing protected, activated amino acids to the immobilized peptides such that at least a portion of the activated amino acids are bonded to the immobilized peptides to form newly-bonded amino acid residues.
- the peptides may be exposed to activated amino acids that react with the deprotected N-termini of the peptides so as to elongate the peptide chain by one amino acid.
- Amino acids can be activated for reaction with the deprotected peptides by reaction of the amino acid with an agent that enhances the electrophilicity of the backbone carbonyl carbon of the amino acid.
- phosphonium and uronium salts can, in the presence of a tertiary base ( e.g .,
- DIPEA diisopropylethylamine
- TEA triethylamine
- Other reagents can be used to help prevent racemization that may be induced in the presence of a base.
- These reagents include carbodiimides (for example, DCC or WSCDI) with an added auxiliary nucleophile (for example, l-hy dr oxy-benzotri azole (HOBt), l-hydroxy-azabenzotriazole (HO At), or HOSu) or derivatives thereof.
- TBTU Another reagent that can be utilized to prevent racemization is TBTU.
- These types of compounds can also increase the rate of carbodiimide-mediated couplings, as well as prevent dehydration of Asn and Gln residues.
- Typical additional reagents include also bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N- methylmorpholine (NMM).
- DIPEA N,N-diisopropylethylamine
- TEA triethylamine
- NMM N- methylmorpholine
- the solid phase peptide synthesis techniques described above, synthetic Gro-b, Gro-b T, and variants thereof that may be used in conjunction with the compositions and methods described herein may have a purity of, e.g., at least about 95% relative to the deamidated versions of these peptides (i.e., contain less than 5% of the corresponding deamidated peptide).
- synthetic Gro- b, Gro-b T, and variants thereof that may be used in conjunction with the compositions and methods described herein may have a purity of about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or more, relative to the deamidated versions of these peptides (e.g ., the Asn69 deamidated version of SEQ ID NO: 1 or the Asn65 deamidated version of SEQ ID NO: 2).
- synthetic Gro-b, Gro-b T, and variants thereof may have, for instance, a purity of from about 95% to about 99.99%, such as a purity of from about 95% to about 99.99%, about 96% to about 99.99%, about 97% to about 99.99%, about 98% to about 99.99%, about 99% to about 99.99%, about 99.9% to about 99.99%, about 95% to about 99.5%, about 96% to about 99.5%, about 95% to about 99%, or about 97% to about 99% relative to the deamidated versions of these peptides (e.g., the Asn69 deamidated version of SEQ ID NO: 1 or the Asn65 deamidated version of SEQ ID NO: 2).
- the deamidated versions of these peptides e.g., the Asn69 deamidated version of SEQ ID NO: 1 or the Asn65 deamidated version of SEQ ID NO: 2.
- CXCR4 antagonists for use in conjunction with the compositions and methods described herein are compounds represented by formula (I)
- A includes a monocyclic or bicyclic fused ring system including at least one nitrogen atom and B is H or a substituent of from 1 to 20 atoms; and wherein Z’ is:
- linker is a bond, optionally substituted alkylene (e.g ., optionally substituted Ci- C 6 alkylene), optionally substituted heteroalkylene (e.g., optionally substituted Ci-C 6 heteroalkyl ene), optionally substituted alkenyl ene (e.g, optionally substituted C 2 -C 6 alkenyl ene), optionally substituted heteroalkenylene (e.g, optionally substituted C 2 -C 6 heteroalkenyl ene), optionally substituted alkynylene (e.g, optionally substituted C 2 -C 6 alkynylene), optionally substituted heteroalkynylene (e.g, optionally substituted C 2 -C 6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted alkylene (e.g ., optionally substituted Ci- C 6 alkylene), optionally substituted heteroalkylene (e.g., optionally substituted Ci-C 6 heteroalky
- heterocycloalkyl ene optionally substituted arylene, or optionally substituted heteroaryl ene.
- Z and Z’ may each independently a cyclic polyamine containing from 9 to 32 ring members, of which from 2 to 8 are nitrogen atoms separated from one another by 2 or more carbon atoms. In some embodiments, Z and Z’ are identical substituents. As an example, Z may be a cyclic polyamine including from 10 to 24 ring members. In some embodiments, Z may be a cyclic polyamine that contains 14 ring members. In some embodiments, Z includes 4 nitrogen atoms. In some embodiments, Z is 1,4,8,11 -tetraazocyclotetradecane.
- the linker is represented by formula (ID)
- ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted
- X and Y are each independently optionally substituted alkylene (e.g, optionally substituted Ci-C 6 alkylene), optionally substituted heteroalkylene (e.g, optionally substituted Ci-C 6 heteroalkylene), optionally substituted alkenyl ene (e.g, optionally substituted C 2 -C 6 alkenyl ene), optionally substituted heteroalkenylene (e.g, optionally substituted C 2 -C 6 heteroalkenylene), optionally substituted alkynylene (e.g, optionally substituted C 2 -C 6 alkynylene), or optionally substituted heteroalkynylene (e.g, optionally substituted C 2 -C 6 heteroalkynylene).
- optionally substituted alkylene e.g, optionally substituted Ci-C 6 alkylene
- optionally substituted heteroalkylene e.g, optionally substituted Ci-C 6 heteroalkylene
- optionally substituted alkenyl ene e.g
- the linker may be represented by formula (IE) wherein ring D is an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted
- X and Y are each independently optionally substituted alkyl ene (e.g ., optionally substituted Ci-C 6 alkyl ene), optionally substituted heteroalkylene (e.g., optionally substituted Ci-C 6 heteroalkyl ene), optionally substituted C 2 -C 6 alkenyl ene (e.g, optionally substituted C 2 -C 6 alkenyl ene), optionally substituted heteroalkenylene (e.g, optionally substituted C 2 -C 6 heteroalkenyl ene), optionally substituted alkynylene (e.g, optionally substituted C 2 -C 6 alkynylene), or optionally substituted heteroalkynylene (e.g, optionally substituted C 2 -C 6 heteroalkynylene).
- optionally substituted alkyl ene e.g ., optionally substituted Ci-C 6 alkyl ene
- optionally substituted heteroalkylene e.g.
- X and Y are each independently optionally substituted Ci-C 6 alkylene. In some embodiments, X and Y are identical substituents. In some embodiments, X and Y may be each be methylene, ethylene, n-propylene, n-butylene, n-pentylene, or n-hexylene groups. In some embodiments, X and Y are each methylene groups.
- the linker may be, for example, l,3-phenylene, 2,6-pyridine, 3,5-pyridine, 2,5- thiophene, 4,4'-(2,2'-bipyrimidine), 2,9-(l,l0-phenanthroline), or the like.
- the linker is 1, 4-phenyl ene-bis-(m ethyl ene).
- CXCR4 antagonists useful in conjunction with the compositions and methods described herein include plerixafor (also referred to herein as“AMD3100” and“Mozibil”), or a pharmaceutically acceptable salt thereof, represented by formula (II), 1,1 '-[1,4- phenylenebis(m ethylene)] -bis- 1 ,4,8, 11 -tetra-azacyclotetradecane.
- CXCR4 antagonists that may be used in conjunction with the compositions and methods described herein include variants of plerixafor, such as a compound described in US Patent No. 5,583, 131, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: l,r-[l,3-phenylenebis(methylene)]-bis- 1,4,8, 1 l-tetra-azacyclotetradecane; l,r-[l,4-phenylene-bis-(methylene)]-bis-l,4,8,l 1- tetraazacyclotetradecane; bis-zinc or bis-copper complex of l,l'-[l,4-phenylene-bis- (methylene)]-bis-l,4,8, 1 l-tetraazacyclotetradecane; 1,1 '-[3,3 '-biphenylene-bis-(methylene)]- bis-l,4,8,l l-tetraazacyclotetradecane; 11,11 '-[l,4-phenylene-bis-(methylene)]-bis-l,4,7,l 1- tetraazacyclotetradecane
- the CXCR4 antagonist is a compound described in US 2006/0035829, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: 3,7,l l,l7-tetraazabicyclo(l3.3.l)heptadeca- l(l7),l3,l5-triene;
- the CXCR4 antagonist may be a compound described in WO 2001/044229, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: N-[4-(l l-fluoro-l, 4, 7-triazacyclotetradecanyl)-l, 4-phenyl enebis(methylene)]- 2-(aminomethyl)pyridine; N-[4-(l 1,1 l-difluoro-l,4,7-triazacyclotetradecanyl)-l,4- phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[4-(l,4,7-triazacyclotetradecan-2- onyl)-l,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine; N-[l2-(5-oxa-l,9- di
- CXCR4 antagonists useful in conjunction with the compositions and methods described herein include compounds described in WO 2000/002870, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: N-[l,4,8, 1 l-tetraazacycl otetra-decanyl-l, 4-phenyl enebis-(m ethyl ene)]-2- (aminomethyl)pyridine; N-[l,4,8, 1 l-tetraazacycl otetra-decanyl-l, 4- phenylenebis(methylene)]-N-methyl-2-(aminomethyl)pyridine; N-[l,4,8,l 1- tetraazacyclotetra-decanyl-l,4-phenylenebis(methylene)]-4-(aminomethyl)pyridine; N- [l,4,8,l
- the CXCR4 antagonist is a compound selected from the group consisting of: l-[2,6-dimethoxypyrid-4-yl(methylene)]-l,4,8,l l-tetraazacyclotetradecane; 1- [2-chloropyrid-4-yl(methylene)]-l,4,8,l l-tetraazacyclotetradecane; l-[2,6-dimethylpyrid-4- yl(methylene)]-l,4,8,l l-tetraazacyclotetradecane; l-[2-methylpyrid-4-yl(methylene)]- 1,4, 8,1 l-tetraazacyclotetradecane; l-[2,6-dichloropyrid-4-yl(methylene)]-l,4,8,l 1- tetraazacyclotetradecane; l-[2-chloropyrid-5-yl(methylene)]
- the CXCR4 antagonist is a compound described in US Patent No. 5,698,546, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: 7,7'-[l,4-phenylene-bis(methylene)]bis-3,7, 11,17- tetraazabicyclo[ 13.3.1 Jheptadeca- 1 (17), 13 , 15-triene; 7,7 '-[ 1 , 4-phenyl ene- bis(methylene)]bis[l5-chloro-3,7,l l,l7-tetraazabicyclo [l3.3.
- the CXCR4 antagonist is a compound described in US Patent No. 5,021,409, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: 2,2'-bicyclam, 6,6'-bicyclam; 3,3 '-(bis-l,5,9, 13- tetraaza cyclohexadecane); 3,3'-(bis-l,5,8,l l,l4-pentaazacyclohexadecane); methylene (or polymethylene) di-l-N-l,4,8,l l-tetraaza cyclotetradecane; 3,3 '-bis-l,5,9, 13- tetraazacyclohexadecane; 3,3'-bis-l,5,8,l l,l4-pentaazacyclohexadecane; 5,5'-bis-bis-
- the CXCR4 antagonist is a compound described in WO 2000/056729, the disclosure of which is incorporated herein by reference as it pertains to CXCR4 antagonists.
- the CXCR4 antagonist may be a compound selected from the group consisting of: N-(2-pyridinylmethyl)-N'-(6,7,8,9-tetrahydro-5H- cyclohepta[b]pyridin-9-yl)-l,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(5, 6,7,8- tetrahydro-8-quinolinyl)-l,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'-(6,7-dihydro- 5H-cyclopenta[b]pyridin-7-yl)-l,4-benzenedimethanamine; N-(2-pyridinylmethyl)-N'- (l,2,3,4
- CXCR4 antagonists that may be used to in conjunction with the compositions and methods described herein include those described in WO 2001/085196, WO 1999/050461, WO 2001/094420, and WO 2003/090512, the disclosures of each of which are incorporated herein by reference as they pertain to compounds that inhibit CXCR4 activity or expression.
- hematopoietic and progenitor cells Prior to infusion into a patient, hematopoietic and progenitor cells may be expanded ex vivo , for example, by contacting the cells with an aryl hydrocarbon receptor antagonist.
- Aryl hydrocarbon receptor antagonists useful in conjunction with the compositions and methods described herein include those described in US Patent No. 9,580,426, the disclosure of which is incorporated herein by reference in its entirety.
- aryl hydrocarbon receptor antagonists include those represented by formula (III)
- L is selected from— NR 5a (CH 2 ) 2-3 ,— NR 5a (CH 2 ) 2 NR 5b— — NR 5a (CH 2 ) 2 S— ,—
- R 5a and R 5b are independently selected from hydrogen and Ci -4 alkyl;
- Ri is selected from thiophenyl, lH-benzoimidazolyl, isoquinolinyl, lH-imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, and thiazolyl; In some embodiments, wherein the thiophenyl, lH-benzoimidazolyl, isoquinolinyl, 1H- imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, or thiazolyl of Ri can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, C l-4 alkyl, Ci -4 alkoxy, halo, halo- sub stituted-C l- alkyl, halo- sub stituted-C
- R 8a and R 8b are independently selected from hydrogen and Ci -4 alkyl;
- R 2 is selected from— S(0) 2 NR 6a R6 b ,— NR a C(0)R 6b— ,— NR 6a C(0)NR 6b Rr,c, phenyl, 1H- pyrrol opyri din-3 -yl, lH-pyrrolopyridin-5-yl, lH-indolyl thiophenyl, pyridinyl, lH-l,2,4- triazolyl, 2-oxoimidazolidinyl, lH-pyrazolyl, 2-oxo-2,3-dihydro-lH-benzoimidazolyl and lH-indazolyl; wherein R 6a , R f , b and R 6c are independently selected from hydrogen and Ci.
- Rv a and R 7b are independently selected from hydrogen and Ci -4 alkyl;
- R 3 is selected from hydrogen, Ci- alkyl and biphenyl
- Rr is selected from C M O alkyl, prop-l-en-2-yl, cyclohexyl, cyclopropyl, 2-(2-oxopyrrolidin-l- yl)ethyl, oxetan-2-yl, oxetan-3-yl, benzhydryl, tetrahydro-2H-pyran-2-yl, tetrahydro-2H- pyran-3-yl, phenyl, tetrahydrofuran-3-yl, and benzyl, (4-pentylphenyl)(phenyl)methyl and 1- (l-(2-oxo-6,9, l2-trioxa-3-azatetradecan-l4-yl)-lH-l,2,3-triazol-4-yl)ethyl wherein said alkyl, cyclopropyl, cyclohexyl, 2-(2-oxopyrrolidin-l-yl)
- aryl hydrocarbon receptor antagonists useful in conjunction with the compositions and methods described herein include SR-l, represented by formula (1), below.
- Peptides and proteins described herein can be expressed in host cells, for example, by delivering to the host cell a nucleic acid encoding the corresponding peptide or protein.
- CXCR2 agonists such as Gro-b, Gro-b T, Gro-b N69D, Gro-b T N65D, and variants thereof
- Peptides and proteins described herein can be expressed in host cells, for example, by delivering to the host cell a nucleic acid encoding the corresponding peptide or protein.
- the sections that follow describe a variety of techniques that can be used for the purposes of introducing nucleic acids encoding peptides and proteins described herein to a host cell for the purposes of recombinant expression.
- Techniques that can be used to introduce a polynucleotide, such as nucleic acid encoding a CXCR2 agonist, such as Gro-b, Gro-b T, Gro-b N69D, Gro-b T N65D, or a variant thereof, into a cell e.g ., a mammalian cell, such as a human cell
- a cell e.g ., a mammalian cell, such as a human cell
- electroporation can be used to permeabilize mammalian cells (e.g., human cells) by the application of an electrostatic potential to the cell of interest.
- Mammalian cells such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al. ( 1987) Nucleic Acids Research 15: 1311, the disclosure of which is incorporated herein by reference. A similar technique,
- NucleofectionTM utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell. NucleofectionTM and protocols useful for performing this technique are described in detail, e.g, in Distler el al. (2005) Experimental Dermatology 14:315, as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.
- Additional techniques useful for the transfection of host cells for the purposes of recombinant peptide and protein expression include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human cell. Squeeze-poration is described in detail, e.g ., in Sharei et al. (2013) Journal of Visualized Experiments 8l :e50980, the disclosure of which is incorporated herein by reference.
- Lipofection represents another technique useful for transfection of cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in US Patent No. 7,442,386, the disclosure of which is incorporated herein by reference.
- Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex.
- exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane are activated dendrimers (described, e.g. , in Dennig (2003) Topics in Current Chemistry 228:227, the disclosure of which is incorporated herein by reference) and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for example, in Gulick et al. (1997) Current Protocols in
- Magnetic beads are another tool that can be used to transfect cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010/0227406, the disclosure of which is incorporated herein by reference.
- Another useful tool for inducing the uptake of exogenous nucleic acids by cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g. , in Rhodes el al. (2007) Methods in Cell Biology 82:309, the disclosure of which is incorporated herein by reference.
- Microvesicles represent another potential vehicle that can be used to introduce a nucleic acid encoding a peptide or protein described herein into a host cell for the purpose of recombinant expression.
- microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g ., a genome-modifying protein, such as a nuclease can be used to efficiently deliver proteins into a cell that subsequently catalyze the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence.
- a genome-modifying protein such as a nuclease
- Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous nucleic acids encoding peptides and proteins described herein, such as CXCR2 agonists, including Gro-b, Gro-b T, Gro-b N69D, Gro-b T N65D, and variants thereof, into host cells for the purpose of recombinant expression.
- Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes may be incorporated into the genome of a cell, for example, by way of generalized or specialized transduction. These processes may occur as part of the natural replication cycle of a viral vector, and may not require added proteins or reagents in order to induce gene integration.
- viral vectors that may be used to introduce a nucleic acid molecule encoding a peptide or protein described herein into a host cell for recombinant expression include parvovirus, such as adeno-associated virus (AAV), retrovirus, adenovirus (e.g, Ad5, Ad26, Ad34, Ad35, and Ad48), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g, influenza virus), rhabdovirus (e.g, rabies and vesicular stomatitis virus), paramyxovirus (e.g.
- AAV adeno-associated virus
- retrovirus e.g, Ad5, Ad26, Ad34, Ad35, and Ad48
- coronavirus e.g, Ad5
- negative strand RNA viruses such as orthomyxovirus (e.g, influenza virus), rhabdovirus (e.g, rabies and vesicular stomatitis virus), paramyxo
- RNA viruses such as picomavirus and alphavirus
- double stranded DNA viruses including adenovirus, herpesvirus (e.g, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g, vaccinia, modified vaccinia Ankara (MV A), fowlpox and canarypox).
- herpesvirus e.g, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
- poxvirus e.g, vaccinia, modified vaccinia Ankara (MV A), fowlpox and canarypox.
- viruses useful for delivering polynucleotides encoding peptides and proteins described herein to host cells for recombinant expression purposes include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis vims, for example.
- retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al.
- murine leukemia viruses include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor vims, bovine leukemia vims, feline leukemia vims, feline sarcoma vims, avian leukemia vims, human T-cell leukemia vims, baboon endogenous vims, Gibbon ape leukemia vims, Mason Pfizer monkey vims, simian immunodeficiency vims, simian sarcoma vims, Rous sarcoma vims and lentivimses.
- vectors are described, for example, in ETS Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene delivery and recombinant protein and peptide expression.
- hematopoietic stem cell transplant therapy can be administered to a subject in need of treatment so as to populate or repopulate one or more blood cell types, such as a blood cell lineage that is deficient or defective in a patient suffering from a stem cell disorder.
- Hematopoietic stem and progenitor cells exhibit multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g ., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g, reticulocytes, erythrocytes), thrombocytes (e.g, megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g, monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g, NK cells, B-cells and T-cells).
- granulocytes e.g ., promyelocytes, neutrophils, eosinophils, basophils
- erythrocytes e.g, reticulocytes, erythrocytes
- thrombocytes e.g
- Hematopoietic stem cells are additionally capable of self-renewal, and can thus give rise to daughter cells that have equivalent potential as the mother cell, and also feature the capacity to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re- establish productive and sustained hematopoiesis.
- hematopoietic stem and progenitor cells represent a useful therapeutic modality for the treatment of a wide array of disorders in which a patient has a deficiency or defect in a cell type of the hematopoietic lineage.
- the deficiency or defect may be caused, for example, by depletion of a population of endogenous cells of the hematopoietic system due to administration of a chemotherapeutic agent (e.g, in the case of a patient suffering from a cancer, such as a hematologic cancer described herein).
- the deficiency or defect may be caused, for example, by depletion of a population of endogenous hematopoietic cells due to the activity of self-reactive immune cells, such as T lymphocytes or B lymphocytes that cross-react with self antigens ( e.g ., in the case of a patient suffering from an autoimmune disorder, such as an autoimmune disorder described herein).
- the deficiency or defect in cellular activity may be caused by aberrant expression of an enzyme (e.g., in the case of a patient suffering from various metabolic disorders, such as a metabolic disorder described herein).
- hematopoietic stem cells can be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re-constitute the defective or deficient population of cells in vivo , thereby treating the pathology associated with the defect or depletion in the endogenous blood cell population.
- Hematopoietic stem and progenitor cells can be used to treat, e.g, a non-malignant hemoglobinopathy (e.g, a hemoglobinopathy selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome).
- a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment.
- the hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of cells that are damaged or deficient in the patient.
- hematopoietic stem and progenitor cells can be used to treat an immunodeficiency, such as a congenital immunodeficiency.
- an immunodeficiency such as a congenital immunodeficiency.
- the compositions and methods described herein can be used to treat an acquired immunodeficiency (e.g, an acquired immunodeficiency selected from the group consisting of HIV and AIDS).
- an acquired immunodeficiency e.g, an acquired immunodeficiency selected from the group consisting of HIV and AIDS.
- a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment.
- Hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of immune cells (e.g, T lymphocytes, B lymphocytes, NK cells, or other immune cells) that are damaged or deficient in the patient.
- a metabolic disorder e.g, a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, and metachromatic leukodystrophy.
- a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment.
- the hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of hematopoietic cells that are damaged or deficient in the patient.
- hematopoietic stem or progenitor cells can be used to treat a malignancy or proliferative disorder, such as a hematologic cancer or
- a CXCR4 antagonist and/or a CXCR2 agonist may be administered to a donor, such as a donor identified as likely to exhibit release of a population of hematopoietic stem and progenitor cells from a stem cell niche, such as the bone marrow, into circulating peripheral blood in response to such treatment.
- hematopoietic stem and progenitor cells thus mobilized may then be withdrawn from the donor and administered to a patient, where the cells may home to a hematopoietic stem cell niche and re-constitute a population of cells that are damaged or deficient in the patient, such as a population of hematopoietic cells that is damaged or deficient due to the administration of one or more chemotherapeutic agents to the patient.
- hematopoietic stem or progenitor cells may be infused into a patient in order to repopulate a population of cells depleted during cancer cell eradication, such as during systemic chemotherapy.
- Exemplary hematological cancers that can be treated by way of administration of hematopoietic stem and progenitor cells in accordance with the compositions and methods described herein are acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin’s lymphoma, as well as other cancerous conditions, including neuroblastoma.
- Hematopoietic stem or progenitor cells mobilized to the peripheral blood of a subject may be withdrawn (e.g, harvested or collected) from the subject by any suitable technique.
- the hematopoietic stem or progenitor cells may be withdrawn by a blood draw.
- hematopoietic stem or progenitor cells mobilized to a subject’s peripheral blood as contemplated herein may be harvested (i.e., collected) using apheresis.
- apheresis may be used to enrich a donor’s blood with mobilized hematopoietic stem or progenitor cells.
- Additional diseases that can be treated by the administration of hematopoietic stem and progenitor cells to a patient include, without limitation, adenosine deaminase deficiency and severe combined immunodeficiency, hyper immunoglobulin M syndrome, Chediak- Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.
- hematopoietic stem and progenitor cells can be used to treat autoimmune disorders.
- transplanted hematopoietic stem and progenitor cells may home to a stem cell niche, such as the bone marrow, and establish productive hematopoiesis. This, in turn, can re-constitute a population of cells depleted during autoimmune cell eradication, which may occur due to the activity of self-reactive lymphocytes (e.g ., self-reactive T lymphocytes and/or self-reactive B lymphocytes).
- self-reactive lymphocytes e.g ., self-reactive T lymphocytes and/or self-reactive B lymphocytes.
- autoimmune diseases that can be treated by way of administering
- hematopoietic stem and progenitor cells to a patient include, without limitation, psoriasis, psoriatic arthritis, Type 1 diabetes mellitus (Type 1 diabetes), rheumatoid arthritis (RA), human systemic lupus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn'
- Kawasaki's disease lichen planus, Lyme disease, Meniere disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia,
- MCTD mixed connective tissue disease
- OMS opsoclonus myoclonus syndrome
- optic neuritis Ord's thyroiditis
- pemphigus vulgaris pernicious anemia
- polychondritis polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter' s syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff person syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, collagenous colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis”), and Wegener' s granulomatosis.
- a method of harvesting hematopoietic stem cells from a human subject comprises administering a CXCR2 agonist and a CXCR4 antagonist to the human subject and harvesting the hematopoietic stem cells from peripheral blood of the human subject.
- a method of transplanting hematopoietic stem cells into a human patient in need thereof comprises administering a CXCR2 agonist and a CXCR4 antagonist to a hematopoietic stem cell donor, harvesting the hematopoietic stem cells from peripheral blood of the donor, and transplanting the harvested hematopoietic stem cells into the patient.
- the patient is the donor.
- withdrawn hematopoietic stem or progenitor cells may be re-infused into the patient, such that the cells may subsequently home hematopoietic tissue and establish productive hematopoiesis, thereby populating or repopulating a line of cells that is defective or deficient in the patient (e.g ., a population of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen- presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B- lymphocytes).
- the transplanted hematopoietic stem or progenitor cells are least likely to undergo graft rejection, as the infused cells are derived from the patient and
- the patient and the donor may be distinct.
- the patient and the donor are related, and may, for example, be HLA-matched.
- HLA-matched donor-recipient pairs have a decreased risk of graft rejection, as endogenous T cells and NK cells within the transplant recipient are less likely to recognize the incoming hematopoietic stem or progenitor cell graft as foreign, and are thus less likely to mount an immune response against the transplant.
- Exemplary HLA-matched donor-recipient pairs are donors and recipients that are genetically related, such as familial donor-recipient pairs ( e.g ., sibling donor-recipient pairs).
- the patient and the donor are HLA-mismatched, which occurs when at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient.
- HLA-mismatched occurs when at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient.
- one haplotype may be matched between the donor and recipient, and the other may be mismatched.
- CD34 dim cells are particularly useful, in part because CD34 dim cells are capable of suppressing alloreactive T lymphocyte proliferation when administered to a recipient, thereby reducing the risk of graft vs. host disease (GVHD).
- GVHD graft vs. host disease
- administration of a CXCR2 agonist and a CXCR4 antagonist according to the methods disclosed herein mobilizes hematopoietic stem cells comprising CD34 dim cells from the bone marrow of a donor into peripheral blood.
- the CD34 dim cells are present in a higher amount in the peripheral blood as compared to peripheral blood from an unmobilized mammal.
- the CD34 dim cells are present in a higher amount in the peripheral blood than if the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
- the methods disclosed herein are useful in performing an allogeneic hematopoietic stem cell transplant in a patient in need thereof.
- the method can include infusing into the patient a therapeutically effective amount of allogeneic
- the method includes administering to the donor (i) a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 pg/kg to about 1,000 pg/kg and (ii) a CXCR4 antagonist.
- CD34 dim cells have been shown to increase overall survival (OS), decrease non-relapse mortality (NRM, i.e., the time to death without relapse/recurrence), and lower the risk of infection (e.g ., cytomegalovirus (CMV) infection) in a patient having a standard risk disease receiving an allogenic hematopoietic cell transplant.
- OS overall survival
- NAM non-relapse mortality
- CMV cytomegalovirus
- the methods of treating a stem cell disorder in a human patient disclosed herein can include infusing into the patient a therapeutically effective amount of the hematopoietic stem cells mobilized by any one of the methods disclosed herein, wherein the mobilized hematopoietic stem cells comprise CD34 dim cells, and wherein the treatment results in increased OS, decreased NRM, and/or lowered risk of infection (e.g., CMV infection).
- a therapeutically effective amount of the hematopoietic stem cells mobilized by any one of the methods disclosed herein wherein the mobilized hematopoietic stem cells comprise CD34 dim cells
- the treatment results in increased OS, decreased NRM, and/or lowered risk of infection (e.g., CMV infection).
- the methods described herein can be used in preventing, reducing the risk of developing, or reducing the severity of a post-transplant infection in a patient in need thereof.
- the method can include infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a human donor into peripheral blood of the human donor according to the methods described herein, for example, administering to the human donor (i) a CXCR2 agonist selected from the group consisting of Gro-b, Gro-b T, and variants thereof at a dose of from about 50 pg/kg to about 1,000 pg/kg and (ii) a CXCR4 antagonist.
- the infection is a CMV infection.
- the disclosure relates to a method of preventing, reducing the risk of developing, or reducing the severity of graft versus host disease (GVHD) in a patient in need thereof, wherein the method includes infusing into the patient a therapeutically effective amount of hematopoietic stem cells, wherein the hematopoietic stem cells were mobilized from bone marrow of a mammalian donor into peripheral blood by the methods described herein, e.g, including administering to the mammalian donor a CXCR2 agonist and a CXCR4 antagonist.
- GVHD graft versus host disease
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise at least 1%, at least 2% at least 5% at least 10%, at least 15%, at least 20% or at least 20% or more CD34 dim cells as compared to peripheral blood from an unmobilized mammal.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise at from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 15%, from about 1% to about 20%, from about 2% to about 25%, 2% to about 5%, from about 2% to about 10%, from about 2% to about 15%, from about 2% to about 20%, from about 2% to about 25%, from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 15% to about 20%, from about 15% to about 20%, from about 15% to about 25% CD34 dim cells as compared to peripheral blood from an unmobilized mammal.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least lO-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 dim cells as compared to peripheral blood from an unmobilized mammal.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise between about 1.5-fold and 30-fold, between about 5-fold and about 25-fold, between about lO-fold and about 20-fold, or between about l2-fold and about l7-fold more CD34 dim cells as compared to peripheral blood from an unmobilized mammal.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise at least 1%, at least 2% at least 5% at least 10%, at least 15%, at least 20% or at least 20% or more CD34 dim cells than if the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise at from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 15%, from about 1% to about 20%, from about 2% to about 25%, 2% to about 5%, from about 2% to about 10%, from about 2% to about 15%, from about 2% to about 20%, from about 2% to about 25%, from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 15% to about 20%, from about 15% to about 20%, from about 15% to about 25% CD34 dim cells than if the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least lO-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 dim cells than if the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
- the hematopoietic stem and progenitor cells mobilized from the bone marrow of a donor into peripheral blood comprise between about 1.5-fold and 30-fold, between about 5-fold and about 25-fold, between about lO-fold and about 20-fold, or between about l2-fold and about 17-fold more CD34 dim cells than if the hematopoietic stem cells were mobilized using the CXCR4 antagonist alone.
- hematopoietic stem cells obtained from a donor may be genetically modified, for example, by disrupting an endogenous gene. This strategy can be used, for example, to silence the expression of one or more major
- CRISPR clustered regularly interspaced short palindromic repeats
- Cas9 Cas9 nuclease
- Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site.
- highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization.
- RNA:DNA hybridization As a result, one can theoretically design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al. (2013) Nature
- Biotechnology 31 :227 the disclosure of which is incorporated herein by reference
- CRISPR/Cas to modulate gene expression has been described in, e.g. , US 8,697,359, the disclosure of which is incorporated herein by reference.
- Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a gene of interest in a hematopoietic stem cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).
- ZFNs zinc finger nucleases
- TALENs transcription activator-like effector nucleases
- these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes.
- ZFNs and TALENs in genome editing applications is described, e.g. , in Urnov et al. (2010) Nature Reviews Genetics 11 :636; and in Joung et al. (2013) Nature Reviews Molecular Cell Biology 14:49, the disclosure of both of which are incorporated herein by reference.
- Additional genome editing techniques that can be used to incorporate polynucleotides encoding target genes into the genome of a hematopoietic stem cell include the use of ARCUSTM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA.
- ARCUSTM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA.
- the use of these enzymes for the incorporation of genes encoding target genes into the genome of a mammalian cell is advantageous in view of the defined structure- activity relationships that have been established for such enzymes.
- meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target gene into the nuclear DNA of a hematopoietic stem cell.
- These single-chain nucleases have been described extensively in, e.g. , US 8,021,867 and US 8,445,251, the disclosures of each of which are incorporated herein by reference.
- the two agents may be administered to the donor concurrently.
- the two agents may be administered to the donor concurrently.
- the CXCR4 antagonist and the CXCR2 agonist may be co-formulated with one another and administered in the same pharmaceutical composition.
- the CXCR4 antagonist and the CXCR2 agonist may be formulated in distinct pharmaceutical compositions and administered separately but simultaneously to the donor.
- the CXCR4 antagonist is administered to the donor prior to administration of the CXCR2 agonist.
- the CXCR4 antagonist may be administered to the donor from about 30 minutes to about 180 minutes prior to administration of the CXCR2 agonist, such as from about 40 minutes to about 160 minutes, about 50 minutes to about 150 minutes, about 60 minutes to about 140 minutes, about 70 minutes to about 130 minutes, about 60 minutes to about 120 minutes, about 70 minutes to about 110 minutes, or about 80 minutes to about 100 minutes ( e.g ., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 125 minutes, about 130 minutes, about 135 minutes, about 140 minutes, about 145 minutes, about 150 minutes, about 155 minutes, about 160 minutes, about 165 minutes,
- the CXCR4 antagonist is administered to the donor from about 30 minutes to about 60 minutes prior to administration of the CXCR2 agonist (e.g., about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes prior to administration of the CXCR2 agonist). In some embodiments, the CXCR4 antagonist may be administered to the donor about 45 minutes prior to administration of the CXCR2 agonist.
- Isolation of the population of hematopoietic stem or progenitor cells may commence from about 10 minutes to about 60 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist (e.g, about 10 minutes to about 1.9 hours, about 20 minutes to about 1.8 hours, about 25 minutes to about 1.7 hours, about 30 minutes to about 1.6 hours, about 40 minutes to about 1.5 hours (e.g, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or about 120 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist).
- the CXCR4 antagonist and the CXCR2 agonist e.g, about 10 minutes to about 1.9 hours, about 20 minutes to about 1.8 hours, about 25 minutes to about 1.7 hours, about 30 minutes to about 1.6 hours, about 40 minutes to about 1.5 hours (e.g, about 10 minutes, about 15 minutes, about 20 minutes,
- isolation of the population of hematopoietic stem or progenitor cells may commence from about 10 minutes to about 20 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist (e.g, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist). In some embodiments, isolation of the population of hematopoietic stem or progenitor cells commences about 15 minutes following completion of the administration of the CXCR4 antagonist and the CXCR2 agonist.
- the population of hematopoietic stem or progenitor cells is isolated from the donor over a period of from about 15 minutes to about 6 hours, such as from about 20 minutes to about 4.5 hours, about 30 minutes to about 4 hours, about 40 minutes to about 3.5 hours, about 50 minutes to about 3 hours, or about 1 hour to about 2 hours ( e.g ., over a period of about 15 minutes, about 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, or about 360 minutes).
- the population of hematopoietic stem and progenitor cells may be isolated from the donor over a period of from about 30 minutes to about 1 hour (e.g., over a period of about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes).
- the hematopoietic stem or progenitor cells may be harvested by apheresis. In some embodiments, the hematopoietic stem or progenitor cells may be harvested by drawing peripheral blood from the donor (i.e., subject).
- the CXCR4 antagonists and CXCR2 agonists described herein may be administered to a patient by a variety of routes, such as intravenously, subcutaneously, intramuscularly, or parenterally.
- routes such as intravenously, subcutaneously, intramuscularly, or parenterally.
- the most suitable route for administration in any given case will depend on the particular agent administered, the patient, pharmaceutical formulation methods,
- the CXCR2 agonist e.g, Gro-b, Gro-b T, or a variant thereof
- the CXCR2 agonist may be administered to a donor intravenously.
- CXCR2 agonists such as those described herein, rapidly give rise to populations of cells that are enriched in CD34 + CD90 + CD45RA cells (hematopoietic stem cells), and reduce the mobilization of other cell types, such as leukocytes, neutrophils, lymphocytes, and monocytes. This property is described in further detail in Example 1, below.
- the CXCR2 agonists and CXCR4 antagonists contemplated herein may each be formulated into a pharmaceutical composition for administration to a subject, such as a mammalian subject (e.g ., a human subject).
- a subject such as a mammalian subject (e.g ., a human subject).
- contemplated herein are pharmaceutical compositions comprising a CXCR2 agonist and/or a CXCR4 antagonist, in admixture with one or more suitable diluents, carriers, and/or excipients.
- Pharmaceutical compositions may include sterile aqueous suspensions.
- a pharmaceutical composition may be administered to a subject, such as a human subject, alone or in combination with pharmaceutically acceptable carriers, the proportion of which may be determined by the quantity of active pharmaceutical ingredient (i.e., CXCR2 agonist and/or a CXCR4 antagonist), chosen route of administration, and standard pharmaceutical practice.
- active pharmaceutical ingredient i.e., CXCR2 agonist and/or a CXCR4 antagonist
- Contemplated CXCR2 agonists and CXCR4 antagonists may be administered to a subject, such as a mammalian subject (e.g., a human subject), by one or more routes of administration.
- a subject such as a mammalian subject (e.g., a human subject)
- contemplated CXCR2 agonists and CXCR4 antagonists may be administered to a subject by intravenous, intraperitoneal, intramuscular, intraarterial, or subcutaneous infusion, among others.
- Contemplated CXCR2 agonists and CXCR4 antagonists may be administered to a subject in one or more doses.
- a CXCR2 agonist and/or CXCR4 antagonist may be administered as a single dose or in two, three, four, five, or more doses.
- subsequent doses may be provided during the same day or one or more days, weeks, months, or years following the initial dose.
- contemplated CXCR2 agonists and CXCR4 antagonists described herein may be administered to a subject, such as a human subject one or more times daily, weekly, monthly, or yearly, depending on such factors as, for instance, the subject's age, body weight, sex, the subject’s diet, and the subject’s excretion rate.
- the contemplated CXCR2 agonists and CXCR4 antagonists are each administered in a single dose once per day.
- Hematopoietic stem or progenitor cells and pharmaceutical compositions described herein may be administered to a subject in one or more doses. When multiple doses are administered, subsequent doses may be provided one or more days, weeks, months, or years following the initial dose.
- the hematopoietic stem cells and pharmaceutical compositions described herein may be administered to a subject, such as a human subject suffering from one or more diseases, conditions, or disorders described herein, one or more times daily, weekly, monthly, or yearly, depending on such factors as, for instance, the subject's age, body weight, sex, severity of the diseases being treated, the subject’s diet, and the subject’s excretion rate.
- Example 1 The effects of Gro-b T on the mobilization of hematopoietic stem cells in mice and Rhesus monkeys
- hematopoietic stem and progenitor cells for both autologous and allogeneic transplantation.
- the most common clinical hematopoietic stem cell mobilization protocol is five days of Filgrastim (G-CSF). This regimen requires daily injections, has been associated with bone pain and often results in unpredictably low yields. A rapid mobilization method that ideally only required a single treatment and had robust and predictable kinetics would be a significant improvement over the current standard of care.
- a unique CXCR2 agonist, Gro-b T induces rapid mobilization of stem and progenitor cells 15 minutes after a single injection.
- CRU competitive repopulating units
- FIG. 2A shows the pharmacokinetic profile of various dosages of Gro-b T when administered intravenously to Rhesus monkeys.
- FIG. 2B shows the pharmacokinetic profile of various dosages of Gro-b T when administered subcutaneously to Rhesus monkeys. In all experiments, Gro-b T was administered to subjects concurrently with plerixafor.
- CD34 + cells mobilized in response to Gro- b T + AMD3100 are enriched for primitive CD34 + CD90+ CD45RA- stem and progenitor cells.
- the data shown in FIGS. 9 and 10 include the percentage of whole blood accounted for by CD34 + CD90 + CD45RA hematopoietic stem and progenitor cells for each treatment group. Absolute numbers and fold change in CD34 + CD90 + CD45RA cells per pL of peripheral blood are shown.
- Data shown in FIGS. 9 and 10 are expressed as mean ⁇ SEM and represent 5 animals per group. Statistical significance was determined based on 2-way ANOVA with post-hoc Dunnett’s multiple comparisons test (* p ⁇ 0.05. **p ⁇ 0.01, *** p ⁇
- Gro-b T and AMD3100 mobilizes hematopoietic stem and progenitor cells with colony forming potential.
- the number of CFET per mL of peripheral blood was enumerated after seven days of culture in methylcellulose.
- Data shown in FIG. 11 are expressed as mean ⁇ SEM and represent 3-5 animals per group. Statistical significance was determined based on 2-way ANOVA with post-hoc Dunnett’s multiple comparisons test (* p ⁇ 0.05).
- the ratio of MMP-9 to TIMP-l is additionally elevated following treatment with Gro-b T and AMD3100 (FIGS. 12-14).
Abstract
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WO2021087406A1 (en) * | 2019-11-01 | 2021-05-06 | Magenta Therapeutics, Inc. | Dosing regimens for the mobilization of hematopoietic stem and progentor cells |
WO2021222313A1 (en) * | 2020-04-27 | 2021-11-04 | Magenta Therapeutics, Inc. | Methods and compositions for transducing hematopoietic stem and progenitor cells in vivo |
US11260079B2 (en) | 2017-12-06 | 2022-03-01 | Magenta Therapeutics, Inc. | Dosing regimens for the mobilization of hematopoietic stem and progenitor cells |
WO2022197776A1 (en) * | 2021-03-16 | 2022-09-22 | Magenta Therapeutics, Inc. | Dosing regimens for hematopoietic stem cell mobilization for stem cell transplants in multiple myeloma patients |
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