Treatment of conditions related to CXCR2 loss-of-function

Abstract

The present invention relates to compounds and methods useful for treating a condition resulting from a deficiency or loss-of-function in CXCR2. The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders.

Description

TREATMENT OF CONDITIONS RELATED TO CXCR2 LOSS-OF-FUNCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Appl. No. 63 / 678,405, filed August 1, 2024, U.S. Provisional Appl. No. 63 / 729,198, filed December 6, 2024, and U.S. Provisional Appl. No. 63 / 842,455, filed July 11, 2025, the content of each of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to compounds and methods useful for treating a condition resulting from a deficiency or loss-of-function in CXCR2. The invention also provides pharmaceutically acceptable compositions comprising compounds of the present invention and methods of using said compositions in the treatment of various disorders.BACKGROUND OF THE INVENTION

[0003] The CXCR4 and CXCR2 chemokine receptor axes play pivotal but opposing roles in regulating neutrophil retention and release from the bone marrow (BM), respectively. Gain-of- function (GoF) variants in the gene encoding CXCR4 are more commonly associated with WHIM syndrome, a rare combined primary immunodeficiency disorder. Subjects with WHIM syndrome frequently exhibit neutropenia, recurrent and severe infections, accumulation of neutrophils in the bone marrow, and neutrophil myelokathexis. Additional clinical manifestations in WHIM subjects may include warts, hypogammaglobulinemia, and lymphocytopenia (see e.g, Geier, C.B., et al. J. Clin. Immunol. 2022, 42: 1748-65; and Heusinkveld, L.E., et al. J. Clin. Immunol. 2019, 39:532- 56). Interestingly, subjects with loss-of-function (LoF) variants in CXCR2 display similar phenotypic features, such as neutropenia, increased infection susceptibility, and neutrophil myelokathexis, as observed in subjects with WHIM syndrome (see e.g., Marin-Esteban, V., et al. Haematologica, 2022, 107:765-9; and Auer, P.L., etal. Nat. Genet. 2014, 46:629-34). Mavorixafor (Xolremdi™), an orally bioavailable CXCR4 antagonist, has demonstrated clinically meaningful increases in absolute neutrophil and lymphocyte counts, and concomitant reductions in infections in subjects with WHIM syndrome, leading to its recent approval by the U.S. Food and Drug Administration (see e.g., Badolato, R., et al. 2024, Blood, doi: 10.1182 / blood.2023022658).

[0004] Although CXCR4 antagonists have shown to be effective in treating subjects with symptoms resulting from gain-of-function mutations in CXCR4, there is a need for therapies that treat similar symptoms resulting from loss-of-function mutations in CXCR2. The present invention addresses this need and provides other related advantages.SUMMARY OF THE INVENTION

[0005] In some embodiments, the present disclosure provides methods of treating condition resulting from a deficiency or loss-of-function in CXCR2 in a subject in need thereof. Such methods include administering to the subject an effective amount of a CXCR4 antagonist or a pharmaceutically acceptable salt or composition thereof.

[0006] In some embodiments, the present disclosure provides methods for reducing the risk of an infection in a subject with deficiency or loss-of-function in CXCR2. Such methods may comprise administering to the subject an effective amount of a CXCR4 inhibitor or a pharmaceutically acceptable salt or composition thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 depicts a graphical representation of the study design for a pharmacologically- induced model of CXCR2 loss-of-function in mice.

[0008] FIG. 2 depicts the absolute neutrophil counts (ANC) in peripheral blood of CXCR2 loss-of-function and wild-type mice in a pharmacologically-induced mouse model of CXCR2 loss- of-function.

[0009] FIG. 3 depicts the mature neutrophil counts in bone marrow of murine subjects as determined by flow cytometry.

[0010] FIG. 4 depicts bone marrow myeloid / erythroid ratio in the bone marrow of WT and CXCR2 loss-of-function mice in the presence or absence of a CXCR4 antagonist.

[0011] FIG. 5 depicts the identification of MK-like cells in bone marrow of CXCR2 loss-of- function mice.

[0012] FIG. 6A depicts the experimental design for a pharmacological mouse model of CXCR2 LOF.

[0013] FIG. 6B depicts blood neutrophil counts in in the blood of CXCR2 and CXCR4 antagonist-treated mice 4 hours after the last dose on day 7. Data are represented as mean + SEMwith 6 mice per group. Statistics were calculated using the two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.00001. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of the mean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.

[0014] FIG. 6C depicts a summary of microscopic examination of blood smears.

[0015] FIG. 6D depicts blood lymphocyte counts in in the blood of CXCR2 and CXCR4 antagonist-treated mice 4 hours after the last dose on day 7.

[0016] FIG. 6E depicts blood monocyte counts in in the blood of CXCR2 and CXCR4 antagonist-treated mice 4 hours after the last dose on day 7.

[0017] FIG. 7A depicts the effect of CXCR4 antagonist on red blood cell (RBC) counts, determined in the blood of CXCR2 and CXCR4 antagonist-treated mice 4 hours after the last dose on day 7. Data are represented as mean + SEM with 6 mice per group. Statistics were calculated using the two-way ANOVA followed by Bonferroni post hoc test. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of the mean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.

[0018] FIG. 7B depicts the effect of CXCR4 antagonist on blood platelet counts, determined in the blood of CXCR2 and CXCR4 antagonist-treated mice 4 hours after the last dose on day 7.

[0019] FIG. 8A depicts absolute counts of mature neutrophils of CXCR2 and CXCR4 antagonist-treated mice determined 4 hours after the last dose on day 7. BM mature neutrophils were defined as CD49b-B220-CD3-Siglec-F’CDl 15-LY6CintLy6G+CDl lb+CD101+. Data are represented as mean + SEM with 6 mice per group. Statistics were calculated using the two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.0001. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of the mean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.

[0020] FIG. 8B depicts M / E ratio in bone marrow (BM) of CXCR2 and CXCR4 antagonist- treated mice determined 4 hours after the last dose on day 7.

[0021] FIG. 9A depicts the effect of CXCR4 antagonist on absolute counts of BM hematopoietic stem cells (HSC), determined 4 hours after the last dose on day 7. Data are represented as mean + SEM with 6 mice per group. Statistics were calculated using the two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.0001. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of themean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist. HSC defined as CD49b’B220'CD3"CD45+Sca-l+c-Kit+; CMP defined as CD49b"B220"CD3"CD45 Sca-l’c- Kit+CD34+CD 16 / 32"; GMP defined as CD49b"B220"CD3"CD45 Sca-l"c-Kit+CD34+CD16 / 32+; Immature neutrophils defined as CD49b"B220'CD3"Siglec-F"CDl 15 LY6CintLy6G+CDl lb+CD101".

[0022] FIG. 9B depicts the effect of CXCR4 antagonist on absolute counts of Common Myeloid Progenitor (CMP), determined 4 hours after the last dose on day 7.

[0023] FIG. 9C depicts the effect of CXCR4 antagonist on absolute counts of Granulocytemonocyte progenitor (GMP) , determined 4 hours after the last dose on day 7.

[0024] FIG. 9D depicts the effect of CXCR4 antagonist on absolute counts of immature neutrophils, determined 4 hours after the last dose on day 7.

[0025] FIG. 10A depicts BM smears from CXCR2 and CXCR4 antagonist-treated mice collected 4 hours after the last dose on day 7 and stained with May-Grunwald Giemsa. Arrows indicate neutrophils exhibiting myelokathexis-like morphologic features, characterized by nuclear hypersegmentation, an increased number of discernible nuclear lobes sometimes separated by thin chromatin strands. Scale bar: 7 pm.

[0026] FIG. 10B depicts a summary of the number of mice exhibiting neutrophil myelokethaxis in BM. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.

[0027] FIG. 11A depicts MK-like neutrophil in blood smears of control and CXCR2 LOF mice. Blood smears were collected 4 hours after the last dose on day 7 and stained with May- Grunwald Giemsa. Scale bar: 7 pm.

[0028] FIG. 11B depicts the frequency of MK-like neutrophil in peripheral blood of control and CXCR2 LOF mouse. Data are represented as mean + SEM with 6 mice per group. Statistics were calculated using the two-way ANOVA followed by Bonferroni post hoc test. CXCR4, C-X- C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of the mean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.

[0029] FIG. 12A depicts the experimental design for Streptococcus Pneumonia infection model. Mice were treated with CXCR2 and CXCR4 antagonist and then inoculated with Streptococcus pneumonia before analysis of bacterial burden, neutrophil count and mortality.

[0030] FIG. 12B depicts bacterial burden in lung tissue homogenates was quantitated at 24 hours postinfection. Data (mean + SEM) were from two independent experiments with 9-12 mice per group. Statistics were calculated using by two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of the mean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.

[0031] FIG. 12C depicts the mortality rate as determined at 48 hours postinfection. Data were from two independent experiments with 20 mice / group. Statistics were calculated using Fisher’s exact test. * p < 0.05, ** p < 0.01.

[0032] FIG. 12D depicts survival as monitored up to 15 days. A Kaplan-Meier plot is used to show survival of mice from each group. Summary of two independent experiment with N= 24 mice / group; * p < 0.05; log-rank test.

[0033] FIG. 12E depicts total neutrophil counts in lung tissue homogenates quantified at 72 hours post infection. Data (mean + SEM) were from two independent experiments with 9-12 mice per group. Statistics were calculated using by two-way ANOVA followed by Bonferroni post hoc test. * p < 0.05, ** p < 0.01. CXCR4, C-X-C chemokine receptor 4; CXCR2, C-X-C chemokine receptor 2; SEM, standard error of the mean; Veh., vehicle; CXCR4i, CXCR4 antagonist; CXCR2i, CXCR2 antagonist.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0034] CXCR4 and CXCR2 are chemokine receptors which are known to regulate bone marrow neutrophil release. Individuals possessing gain-of-function mutations in CXCR4 may exhibit the rare autosomal dominant WHIM syndrome, which is characterized by a number of debilitating symptoms including warts, hypogrammaglobulinemia, reccurent and severe infections, and myelokathexis. This results from an accumulation of senescent neutrophils in the bone marrow of such subjects. Certain CXCR4 antagonists, including FDA-approved mavorixafor, are known to treat WHIM syndrome and its accompanying symptoms.

[0035] It has now been found that CXCR4 antagonists are useful in correcting common pathogenic phenotypes associated with CXCR2 loss-of-function mutations.Methods

[0036] In some embodiments, provided herein are methods for treating a condition resulting from a deficiency or loss-of-function in CXCR2 in a subject in need thereof, comprising administering to the subject an effective amount of a CXCR4 antagonist or a pharmaceutically acceptable salt or composition thereof.

[0037] For instance, in some embodiments, the condition is neutropenia. In some embodiments, the condition is severe chronic neutropenia (SCN), chronic idiopathic neutropenia (CIN), congenital neutropenia, or cyclic neutropenia.

[0038] As used herein, the term “neutropenia” means that a subject has an absolute neutrophil count (ANC)thatis at or below about 1500 cells per pL. “Mild neutropenia” is generally described as a subject having an ANC between 1000 and 1500 cells / uL. “Moderate neutropenia” is generally understood to refer to subjects having an ANC between 500 and 1500 cells / uL. As used herein, “severe neutropenia” means that the subject has an ANC that is at or below 500 cells / pL. A beneficial treatment may comprise a treatment that significantly increases a subject’s neutrophil counts, even though the subj ect still has an ANC <1500 cells per uL, thus remaining ‘neutropenic’ . For example, a subject with severe neutropenia [ANC< 500 cells / uL] may be treated using the methods of the present invention, until the subject’s ANC is raised to 1000 cell / uL. In the expert judgement of the treating physician, such a result would likely be considered a successful treatment, though the subject would continue to be characterized as having mild-to-moderate neutropenia. In some embodiments, a subject with a CXCR2 mutation has mild neutropenia, moderate neutropenia, or sever neutropenia.

[0039] As used herein, the term “chronic neutropenia” is defined as neutropenia lasting for a period of at least three (3) months. In some embodiments, a subject with chronic neutropenia has a CXCR2 mutation.

[0040] The term “idiopathic” as applied herein to neutropenia means that all other causes have been excluded, for example, the subject’s neutropenia is not attributable to drugs, or to a specific identified genetic, infectious, inflammatory, autoimmune or malignant cause. In some embodiments, a subject with idiopathic neutropenia may also have a mutation in CXCR2.

[0041] The term “cyclic neutropenia” is a neutropenia characterized by recurrent episodes of abnormally low levels of neutrophils, where episodes often occur every few weeks and last for a few days at a time.

[0042] In some embodiments, the subject is exhibiting one or more of warts, infections, recurrent and severe infections, accumulation of neutrophils in the bone marrow, increased M / E ratio in the bone marrow, neutrophil myelokathexis, hypogammaglobulinemia, and lymphocytopenia.

[0043] In some embodiments, treatment of particular sub-populations of subjects with mavorixafor, or a pharmaceutically acceptable salt thereof, is particularly effective.

[0044] In some embodiments, the subject is male. In some embodiments, the subject is female.

[0045] In some embodiments, the subject is less than 50 years old. In some embodiments, the subject is at least 50 years old.

[0046] Also described herein are methods of correcting an imbalance of an immune cell population in a subject with a deficiency or loss-of-function mutation in CXCR2, comprising administering to the subject an effective amount of a CXCR4 antagonist or a pharmaceutically acceptable salt or composition thereof.

[0047] In some embodiments, the subject has one or more mutation in at least one copy of the CXCR2 gene.

[0048] In some embodiments, the subject has one or more mutations in the CXCR2 gene. In some embodiments, the one or more mutations are selected from Argl44Cys, Arg212Trp, Argl84Ter, Arg289Cys, and His323fs.

[0049] In certain embodiments of the methods described herein, the subject has a chronic immune cell imbalance. In some embodiments, the subject has an acute immune cell imbalance. In some embodiments, the immune cell imbalance is associated with a congenital primary immunodeficiency disease (PID). In some embodiments, the immune cell imbalance is associated with a disease state. In some embodiments, the disease state is cancer. In some embodiments, the cancer is renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cancer, melanoma, pancreatic cancer, ovarian cancer, non-small cell lung cancer, Waldenstrom’s macroglobulinemia (WM). In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the PID is WHIM syndrome, chronic neutropenia or severe chronic neutropenia (SCN). In some embodiments, the subject has been subjected to genetic testing and determined not to have a mutation in the subject’s CXCR4 gene. In some embodiments, the subject has been subjected to genetic testing and determined not to have a mutation in either of the subj ect’ s CXCR4 or MYD88 genes. In some embodiments, the subject has been subjected to genetic testing and determined tohave a loss-of-function mutation in the subject’s CXCR2 gene.

[0050] Variants of the human CXCR2 are known, including several identified as pathogenic or likely pathogenic, for example at R153, K158, R184, R208, R236, R248, and H323. Other variants have been identified as potentially having moderate impact or uncertain significance, for example, R251, V252, 1253, A255, C263, E284, C286, R288, R289, A295, E300, H306, P311, A315, K320, R322, G324, L329 (see e.g., https: / / www.uniprot.org / uniprotkb / P25025 / variant- viewer).

[0051] Without being bound by theory, it is believed that the methods described herein comprise administering a CXCR4 antagonist where the mechanism of action underlying observed pathogenic phenotypes in patients with loss-of-function mutations in CXCR2 is unrelated to the antagonist’s activity toward CXCR4. In some embodiments, the subject does not have a mutation in the subject’s CXCR4 gene. In some embodiments, the subject does not have abnormal expression of CXCR4. In some embodiments, the subject’s CXCR4 signaling is normal or within a normal range for a subject of the same sex and similar age and weight. In some embodiments, the subject’s disease does not involve a mutation or gain-of-function in a CXCR4 gene. In some embodiments, the subject does not have a MYD88 or CXCR4 mutation. In some embodiments, the subject does not have WHIM syndrome, chronic neutropenia or severe chronic neutropenia (SCN).

[0052] The cells of the immune system can be categorized as lymphocytes (T-cells, B-cells and NK cells), neutrophils, and monocytes / macrophages. These are all types of white blood cells. The major proteins of the immune system are predominantly signaling proteins (often called cytokines), antibodies, and complement proteins.

[0053] In some embodiments, a method provided by the present invention corrects an imbalance in B-cells in the subject. B-cells (sometimes called B-lymphocytes) are specialized cells of the immune system whose major function is to produce antibodies (also called immunoglobulins or gamma-globulins). B-cells develop in the bone marrow from hematopoietic stem cells. As part of their maturation in the bone marrow, B-cells are trained or educated so that they do not produce antibodies to healthy tissues. When mature, B-cells can be found in the bone marrow, lymph nodes, spleen, some areas of the intestine, and the bloodstream.

[0054] In some embodiments, a method provided by the present invention corrects an imbalance in T-cells in the subject. T-cells (sometimes called T-lymphocytes and often named inlab reports as CD3 cells) directly attack cells infected with viruses, and they also act as regulators of the immune system. T-cells develop from hematopoietic stem cells in the bone marrow but complete their development in the thymus. The thymus is a specialized organ of the immune system in the chest. Within the thymus, immature lymphocytes develop into mature T-cells and T-cells with the potential to attack normal tissues are eliminated. The thymus is essential for this process, and T-cells cannot develop if the fetus does not have a thymus. Mature T-cells leave the thymus and populate other organs of the immune system, such as the spleen, lymph nodes, bone marrow and blood. Each T-cell reacts with a specific antigen, just as each antibody molecule reacts with a specific antigen.

[0055] T-cells have different abilities to recognize antigen and are varied in their function. There are “killer” or cytotoxic T-cells (often denoted in lab reports as CD8 T-cells), helper T-cells (often denoted in lab reports as CD4 T-cells), and regulatory T-cells. Each has a different role to play in the immune system. Killer, or cytotoxic, T-cells perform the actual destruction of infected cells. Killer T-cells protect the body from certain bacteria and viruses that have the ability to survive and even reproduce within the body’s own cells. Killer T-cells also respond to foreign tissues in the body, such as a transplanted organ. The killer cell must migrate to the site of infection and directly bind to its target to ensure its destruction. Helper T-cells assist B-cells to produce antibodies and assist killer T-cells in their attack on foreign substances. Regulatory T-cells suppress or turn off other T-lymphocytes.

[0056] Natural killer (NK) cells are so named because they easily kill cells infected with viruses. They are said to be “natural killer” cells as they do not require the same thymic education that T-cells require. NK cells are derived from the bone marrow and are present in relatively low numbers in the bloodstream and in tissues. They are important in defending against viruses and possibly preventing cancer as well.

[0057] In some embodiments, a method provided by the present invention corrects an imbalance in neutrophils in the subject. Neutrophils or polymorphonuclear leukocytes (polys or PMN’s) are the most numerous of all the types of white blood cells, making up about half or more of the total. They are also called granulocytes and appear on lab reports as part of a complete blood count (CBC with differential). They are found in the bloodstream and can migrate into sites of infection within a matter of minutes. These cells, like the other cells in the immune system, develop from hematopoietic stem cells in the bone marrow. Neutrophils increase in number in thebloodstream during infection and are in large part responsible for the elevated white blood cell count seen with some infections. They are capable of leaving the bloodstream and accumulating in tissues during the first few hours of an infection. Their major role is to ingest bacteria or fungi and kill them.

[0058] In some embodiments, a method provided by the present invention corrects an imbalance in monocytes (monocytopenia) in the subject. Monocytes are closely related to neutrophils and are found circulating in the bloodstream. They make up 5-10 percent of the white blood cells. They also line the walls of blood vessels in organs like the liver and spleen. Here they capture microorganisms in the blood as the microorganisms pass by. Monocytopenia is a reduction in blood monocyte count (ANC) to < 500 / mcL (< 0.5 * 109 / L). Risk of certain infections is increased. It is diagnosed by complete blood count with differential. Typical treatment includes hematopoietic stem cell transplantation.

[0059] Macrophages are essential for killing fungi and certain bacteria. Macrophages live longer than neutrophils and are especially important for slow growing or chronic infections. Macrophages can be influenced by T-cells and often collaborate with T-cells in killing microorganisms.

[0060] In some embodiments, the subject has an imbalance of an immune cell population selected from T-cells, B-cells, NK cells, neutrophils, and monocytes. In some embodiments, the subject has leukopenia, neutropenia, or monocytopenia. In some embodiments, the subject exhibits a low total white blood cell (WBC) count.

[0061] Accordingly, in some embodiments, provided herein are methods for treating infections or reducing the risk of an infection in a subject with deficiency or loss-of-function in CXCR2, comprising administering to the subject an effective amount of a CXCR4 inhibitor or a pharmaceutically acceptable salt or composition thereof.

[0062] In some embodiments, the patient has an elevated risk of an infection selected from respiratory tract infections, periodontitis, otitis media, stomatitis, urinary tract infections, pyelonephritis, skin abscesses, cellulitis, and sepsis. In some embodiments, the patient has increased susceptibility to infection by Streptococcus pneumoniae, Klebsiella Pneumoniae, Escherichia coli, Staphylococcus aureus, or Toxoplasma gondii.

[0063] In some embodiments, the infection is pneumonia.

[0064] The methods described herein may increase or decrease absolute neutrophil count(ANC) and / or increase absolute lymphocyte count (ALC) in the subject, for example in the subject’s blood. In some embodiments, the ANC and / or ALC is increased or decreased in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50% of that of the pretreatment baseline counts.

[0065] In some embodiments, the methods described herein increase absolute neutrophil count (ANC) to a level greater than or equal to 500 / pL and / or increase absolute lymphocyte count (ALC) to a level greater than or equal to 1000 / pL.

[0066] In some embodiments, said subject originally exhibits ANC less than 600 / pL and / or ALC less than 1000 / pL before treatment. In some embodiments, said subject originally exhibits ANC less than 500 / pL and / or ALC less than 650 / pL before treatment. In some embodiments, the subject originally exhibits an ANC less than 400 / pL and / or ALC less than 650 / pL before treatment with the CXCR4 antagonist or a pharmaceutically acceptable salt thereof

[0067] In some embodiments, a method described herein results in an increase or decrease in ANC levels to at least about 500 / pL, at least about 600 / pL, at least about 700 / pL, at least about 800 / pL, at least about 900 / pL, at least about 1000 / pL, at least about 1,100 / pL, at least about 1,200 / pL, at least about 1,300 / pL, at least about 1, 400 / pL, at least about 1, 500 / pL, or to about that of a human with a normally-functioning immune system, on at least 85% of assessments.

[0068] In some embodiments, a method described herein results in and increase or decrease in ALC to at least about 1000 / pL, about 1,200 / pL, or about 1, 500 / pL, or to about that of a human with a normally-functioning immune system, on at least 85% of assessments.

[0069] In some embodiments, a method described herein results in a lowered frequency of infections in the subject, such as at least 10%; at least 25%; or at least 50% less infections. In some embodiments, the method reduces the frequency of a respiratory tract infection. In some embodiments, a method described herein results in lowered severity and / or duration of infections.

[0070] In some embodiments, the respiratory tract infection is pneumonia.

[0071] In some embodiments, a method described herein results in increased levels of total circulating WBC, neutrophils, and / or lymphocytes. In some embodiments, cell counts of WBC, neutrophils, and / or lymphocytes increase to approximately 1.4x baseline. In some embodiments, cell counts of WBC, neutrophils, and / or lymphocytes increase to approximately 1 ,6x baseline, 1.8x baseline, or 2. Ox baseline. In some embodiments, cell counts of WBC, neutrophils, and / or lymphocytes increase to approximately 2.9x baseline. In some embodiments, cell counts oflymphocytes increase to approximately 2.9x baseline. In some embodiments, cell counts of neutrophils increase to approximately 2.7x baseline and lymphocytes to approximately 1.9x baseline.

[0072] In some embodiments, the methods described herein achieve an ANC of at least 500 cells / pL and / or an ALC of at least 1000 / pL.

[0073] In some embodiments of the methods described herein, the method achieves an ANC of between about 1,000 cells / pL and 10,000 cells / pL. In some embodiments, the method achievse an ANC of between about 1,000 cells / pL and 10,000 cells / pL; between about 1,000 cells / pL and 9,000 cells / pL; between about 1000 cells / pL and 8,000 cells / pL; between about 1,000 cells / pL and 7,000 cells / pL; between about 1,000 cells / pL and 6,000 cells / pL; between about 1,000 cells / pL and 5,000 cells / pL; between about 1,000 cells / pL and 4,000 cells / pL; between about 1,000 cells / pL and 3,000 cells / pL; between about 1,000 cells / pL and 2,000 cells / pL; between about 2,000 cells / pL and 10,000 cells / pL; between about 2,000 cells / pL and 9,000 cells / pL; between about 2,000 cells / pL and 8,000 cells / pL; between about 2,000 cells / pL and 7,000 cells / pL; between about 2,000 cells / pL and 6,000 cells / pL; between about 2,000 cells / pL and 5,000 cells / pL; between about 2,000 cells / pL and 4,000 cells / pL; between about 2,000 cells / pL and 3,000 cells / pL; between about 3,000 cells / pL and 10,000 cells / pL; between about 3,000 cells / pL and 9,000 cells / pL; between about 3,000 cells / pL and 8,000 cells / pL; between about 3,000 cells / pL and 7,000 cells / pL; between about 3,000 cells / pL and 6,000 cells / pL; between about 3,000 cells / pL and 5,000 cells / pL; between about 3,000 cells / pL and 4,000 cells / pL; between about 4,000 cells / pL and 10,000 cells / pL; between about 4,000 cells / pL and 9,000 cells / pL; between about 4,000 cells / pL and 8,000 cells / pL; between about 4,000 cells / pL and 7,000 cells / pL; between about 4,000 cells / pL and 6,000 cells / pL; between about 4,000 cells / pL and 5,000 cells / pL; between about 5,000 cells / pL and 10,000 cells / pL; between about 5,000 cells / pL and 9,000 cells / pL; between about 5,000 cells / pL and 8,000 cells / pL; between about 5,000 cells / pL and 7,000 cells / pL; between about 5,000 cells / pL and 6,000 cells / pL; between about 6,000 cells / pL and 10,000 cells / pL; between about 6,000 cells / pL and 9,000 cells / pL; between about 6,000 cells / pL and 8,000 cells / pL; between about 6,000 cells / pL and 7,000 cells / pL; between about 7,000 cells / pL and 10,000 cells / pL; between about 7,000 cells / pL and 9,000 cells / pL; between about 7,000 cells / pL and 8,000 cells / pL; between about 8,000 cells / pL and 10,000 cells / pL; between about 8,000 cells / pL and 9,000 cells / pL; or between about 9,000cell s / pl. and 10,000 cells / pL. Tn some embodiments, the method achieves an ANC of about 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; or 10,000 cells / pL.

[0074] In some embodiments of the methods described herein, subjects are treated with an effective amount of a CXCR4 antagonist, or a pharmaceutically acceptable salt or composition thereof, either as a single agent (monotherapy), or in combination (e.g., a combination therapy) with other treatments (e.g., for neutropenia). In some embodiments, the combination therapy comprises treatment with an effective amount of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), a variant of G-CSF or GM-CSF (e.g., a pegylated version), bone marrow transplantation, treatment with cord blood stem cells, or a combination thereof.

[0075] In some embodiments, G-CSF is co-administered to the patient at a starting dosage of about 6 mcg / kg as a twice daily subcutaneous injection (for a patient having congenital neutropenia); or about 5 mcg / kg as a single daily subcutaneous injection (for a patient having idiopathic or cyclic neutropenia). In some embodiments, the patient is already receiving G-CSF and continues chronic dosing at a dosage sufficient to maintain clinical benefits, such as daily administration in the amount of about 6 mcg / kg (for patients having congenital neutropenia); about 2.1 mcg / kg (for patients having cyclic neutropenia); or about 1.2 mcg / kg (for patients having idiopathic neutropenia).

[0076] In another aspect, the present invention provides a method for treating neutropenia, comprising administering to a patient in need thereof an effective amount of a CXCR4 antagonist (e.g., mavorixafor), or a pharmaceutically acceptable salt or composition thereof, in combination with an effective amount of G-CSF or GM-CSF, or a variant thereof, wherein the effective amount of G-CSF or GM-CSF, or a variant thereof is less than the approved dosage as a monotherapy for a similar patient being treated with the G-CSF or GM-CSF, or a variant thereof.CXCR4 Inhibitors

[0077] As described herein, a variety of CXCR4 inhibitors may be used in accordance with the present disclosure.

[0078] In some embodiments, the CXCR4 inhibitor is mavorixafor (X4P-001; AMD11070), or a pharmaceutically acceptable salt thereof. Mavorixafor is currently in clinical development in subjects with cancer (renal cell carcinoma), Waldenstrom’s Macroglobulinemia, and with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome. The chemicalformula is: C21H27N5; and molecular weight is 349.48 amu. The chemical structure of mavorixafor is as follows according to Formula I:

[0079] In some embodiments, the CXCR4 inhibitor is one of those described in the following documents, or a pharmaceutically acceptable salt thereof: WO2017223229, WO2017223239, WO2017223243, W02019126106, WO2020 / 264292, W02003 / 022785, W02003 / 055876, W02004 / 106493, W02004 / 091518, W02004 / 093817, W02006 / 049764, W02005 / 090308, WO2021 / 263203, W02023059903, or W02006 / 039250. Each of the foregoing documents is hereby incorporated by reference in its entirety.

[0080] In some embodiments, the CXCR4 inhibitor is selected from mavorixafor,or a pharmaceutically acceptable salt or composition thereof.

[0081] In some embodiments, the CXCR4 inhibitor is mavorixafor or a pharmaceutically acceptable salt or composition thereof.

[0082] In some embodiments, the CXCR4 inhibitor is Compound A:Compound A or a pharmaceutically acceptable salt thereof.

[0083] In some embodiments, the CXCR4 inhibitor is selected from the following:or a pharmaceutically acceptable salt thereof.

[0084] In some embodiments, the CXCR4 inhibitor is not plerixafor or a pharmaceutically acceptable salt thereof.

[0085] In some embodiments, the CXCR4 inhibitor is one of those described in Table 1, below.Each document listed in Table 1 is hereby incorporated by reference in its entirety.Table 1: Exemplary CXCR4 InhibitorsDosage and Formulation

[0086] In some embodiments, the dosage of CXCR4 inhibitor is a well -tolerated dose that achieves a satisfactory therapeutic result, without causing any severe or treatment-limiting toxicities.

[0087] As used herein, the term “well -tolerated” in reference to a dose of CXCR4 inhibitor (e.g., mavorixafor) means a dose that can be given to a subject without the subject experiencing any treatment-limiting toxicities. As used herein, “treatment-limiting toxicities” (TLTs) means that the subject experiences one or more of the toxicities in Table 2:Table 2: Treatment-Limiting ToxicitiesGrading: As defined by the National Cancer Institute [NCI] Common Terminology Criteria for Adverse Events, version 4.03).Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase; TLT = treatment-limiting toxicity.

[0088] In some embodiments, a CXCR4 inhibitor (e.g., mavorixafor), or a pharmaceutically acceptable salt thereof, can be administered orally (PO) once daily (QD). In some embodiments, the CXCR4 inhibitor is administered orally (PO) once daily (QD). In some embodiments, the CXCR4 inhibitor is administered orally (PO) twice daily (BD).

[0089] In some embodiments, the CXCR4 inhibitor described herein is mavorixafor, or a pharmaceutically acceptable salt thereof.

[0090] In certain embodiments, the mavorixafor, pharmaceutically acceptable salt thereof, or composition comprising mavorixafor or a pharmaceutically acceptable salt thereof is administered orally (PO) once daily (QD) or twice daily (BID), in an amount from about 25 mg to about 800 mg daily. In certain embodiments, the dosage composition may be provided twice a day in divideddosage, approximately 12 hours apart. In other embodiments, the dosage composition may be provided once daily. The terminal half-life of mavorixafor has been generally determined to be between about 12 to about 24 hours, or approximately 14.5 hrs. In certain embodiments, the dosage of mavorixafor useful in the invention is from about 25 mg to about 1200 mg daily. In other embodiments, the dosage of mavorixafor useful in the invention may range from about 25 mg to about 1000 mg daily, from about 50 mg to about 800 mg daily, from about 50 mg to about 600 mg daily, from about 50 mg to about 500 mg daily, from about 50 mg to about 400 mg daily, from about 100 mg to about 800 mg daily, from about 100 mg to about 600 mg daily, from about 100 mg to about 500 mg daily, from about 100 mg to about 400 mg daily; from about 200 mg to about 800 mg daily, from about 200 mg to about 600 mg daily, from about 300 mg to about 600 mg daily, from about 200 mg to about 500 mg daily from about 200 mg to about 400 mg daily.

[0091] In other embodiments, the dosage of mavorixafor or a pharmaceutically acceptable salt thereof is administered in a dosage range from about 100 mg to about 800 mg daily, from about 200 mg to about 600 mg daily, from about 300 mg to about 500 mg daily, or from about 350 mg to about 450 mg daily; or in a daily dosage of about 100 mg / day; 125 mg / day; 150 mg / day; 175 mg / day; 200 mg / day; 225 mg / day; 250 mg / day; 275 mg / day; 300 mg / day; 325 mg / day; 350 mg / day; 400 mg / day; 425 mg / day; 450 mg / day; 475 mg / day; 500 mg / day; 525 mg / day; 550 mg / day; 575 mg / day; 600 mg / day; 625 mg / day; 650 mg / day; 675 mg / day; 700 mg / day; 725 mg / day; 750 mg / day; 775 mg / day or 800 mg / day. In unusual cases, the dosage of mavorixafor or a pharmaceutically acceptable salt thereof may be administered in an amount in excess of 800 mg / day, while taking care to minimize or avoid any adverse effects of such administration.

[0092] In certain embodiments, a method disclosed herein may comprise administering a composition comprising mavorixafor, or a pharmaceutically acceptable salt thereof, one or more diluents, a disintegrant, a lubricant, a flow aid, and a wetting agent. In some embodiments, a disclosed method comprises administering a composition comprising 25 mg to 1200 mg mavorixafor, or a pharmaceutically acceptable salt thereof, microcrystalline cellulose, dibasic calcium phosphate dihydrate, croscarmellose sodium, sodium stearyl fumarate, colloidal silicon dioxide, and sodium lauryl sulfate. In some embodiments, a disclosed method comprises administering a unit dosage form wherein said unit dosage form comprises a composition comprising 25 mg to 200 mg mavorixafor, or a pharmaceutically acceptable salt thereof, microcrystalline cellulose, dibasic calcium phosphate dihydrate, croscarmellose sodium, sodiumstearyl fumarate, colloidal silicon dioxide, and sodium lauryl sulfate. In certain embodiments, a disclosed method comprises administering a unit dosage form comprising a composition comprising mavorixafor, or a pharmaceutically acceptable salt thereof, present in an amount of about 25 mg, about 40 mg, about 50 mg, about 80 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, or about 1200 mg. In some embodiments, a provided composition (or unit dosage form) is administered to the subject once per day, twice per day, three times per day, or four times per day. In some embodiments, a provided composition (or unit dosage form) is administered to the subject once per day or twice per day.

[0093] In some embodiments, a disclosed method comprises administering a unit dosage form comprising a composition comprising:(a) mavorixafor, or a pharmaceutically acceptable salt thereof, as about 10-30% by weight of the composition;(b) microcrystalline cellulose as about 60-80% by weight of the composition;(c) croscarmellose sodium as about 5-10% by weight of the composition;(d) sodium stearyl fumarate as about 0.5-2% by weight of the composition; and(e) colloidal silicon dioxide as about 0.1-1.0 % by weight of the composition.

[0094] In some embodiments, a disclosed method comprises administering a unit dosage form comprising a composition comprising:(a) mavorixafor, or a pharmaceutically acceptable salt thereof, as about 15% by weight of the composition;(b) microcrystalline cellulose as about 78% by weight of the composition;(c) croscarmellose sodium as about 6% by weight of the composition;(d) sodium stearyl fumarate as about 1% by weight of the composition; and(e) colloidal silicon dioxide as about 0.2% by weight of the composition.

[0095] In some embodiments, a disclosed method comprises administering a unit dosage form comprising a composition comprising:(a) mavorixafor, or a pharmaceutically acceptable salt thereof, as about 10-20% by weight of the composition;(b) microcrystalline cellulose as about 25-40% by weight of the composition;(c) dibasic calcium phosphate dihydrate as about 35-55% by weight of the composition;(d) croscarmellose sodium as about 4-15% by weight of the composition;(e) sodium stearyl fumarate as about 0.3-2% by weight of the composition;(f) colloidal silicon dioxide as about 0.1-1.5% by weight of the composition; and(g) sodium lauryl sulfate as about 0.1-1.5% by weight of the composition.

[0096] In some embodiments, a disclosed method comprises administering a unit dosage form comprising a composition comprising:(a) mavorixafor, or a pharmaceutically acceptable salt thereof, as about 13% by weight of the composition;(b) microcrystalline cellulose as about 32% by weight of the composition;(c) dibasic calcium phosphate dihydrate as about 44% by weight of the composition;(d) croscarmellose sodium as about 8% by weight of the composition;(e) sodium stearyl fumarate as about 1.4% by weight of the composition;(f) colloidal silicon dioxide as about 0.4% by weight of the composition; and(g) sodium lauryl sulfate as about 0.7% by weight of the composition.

[0097] In some embodiments, a disclosed method comprises administering a unit dosage form comprising a composition comprising:(a) mavorixafor, or a pharmaceutically acceptable salt thereof, as about 35-75% by weight of the composition;(b) microcrystalline cellulose as about 5-28% by weight of the composition;(c) dibasic calcium phosphate dihydrate as about 7-30% by weight of the composition;(d) croscarmellose sodium as about 2-10% by weight of the composition;(e) sodium stearyl fumarate as about 0.3-2.5% by weight of the composition;(f) colloidal silicon dioxide as about 0.05-1.2% by weight of the composition; and(g) sodium lauryl sulfate as about 0.2-1.2% by weight of the composition.

[0098] Inasmuch as it may be desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound in accordance with the invention, may conveniently be combined in the form of a kit suitable for co-administration of the compositions. Thus the kit of the invention includes two ormore separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

[0099] The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically includes directions for administration and may be provided with a memory aid.

[0100] The examples below explain the invention in more detail. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0101] The contents of each document cited in the specification are herein incorporated by reference in their entireties.EXEMPLIFICATIONExample 1: CXCR4 Antagonism Corrects Peripheral Neutropenia and Mature Neutrophil Accumulation in Bone Marrow in a Pharmacological Mouse Model of CXCR2 Loss-of- function

[0102] A pharmacological mouse model of CXCR2 loss-of-function phenotypes was developed using the CXCR2 antagonist navarixin to recapitulate the common phenotypes seen in WHIM and CXCR2 deficiency subjects, and to explore the impact of CXCR4 antagonism (e.g., with mavorixafor) to correct these defects in this mouse model of CXCR2 loss-of-function. Previously, it was shown that the compound AMD3100 (plerixafor) failed to mobilize neutrophil release in a murine mixed chimera bone marrow cell model of CXCR2 loss-of-function (see Eash, et al. J. Clin. Invest. 2010, 120:2423-2431).

[0103] In this study, we investigated the effects of the CXCR4 inhibitor Compound A, whichhas the following structure:

[0104] Female BALB / c mice were orally administered with the CXCR2 antagonist (3 mg / kg) and subsequently with the CXCR4 antagonist Compound A (10 mg / kg, 2 hours after the navarixin dose) or vehicle control daily for 7 days. Blood and BM were collected 4 hours after Compound A dose for enumeration and flow cytometry analysis. At day 7, blood and BM smears were prepared and stained with May-Grunwald Giemsa for morphological analysis.

[0105] The CXCR2 loss-of-function mice, induced by navarixin, recapitulated many of the phenotypes observed in subjects with WHIM syndrome and CXCR2 deficiency including peripheral blood neutropenia, accumulation of segmented neutrophils and an elevated myeloid / erythroid (M / E) ratio in BM. Furthermore, a large proportion of neutrophils in BM and blood of CXCR2 loss-of-function mice displayed a myelokathexis-like morphology characteristic of that seen in WHIM, including nuclear hypersegmentation, an increased number of discernible nuclear lobes, and thinner intranuclear chromatin strands. Treatment with the CXCR4 antagonist Compound A resulted in the correction of these pathogenic phenotypes including normalization of ANC in peripheral blood, reversal of mature neutrophil accumulation in BM, and normalization of the M / E ratio in BM. Moreover, chronic dosing with the CXCR4 antagonist daily for 7 days appeared to reduce the frequency of MK neutrophils in BM as well as the number of CXCR2 LoF mice with this BM defect.

[0106] These data demonstrate that loss-of-function in the CXCR2 pathway induced by navarixin results in similar phenotypes as observed in subjects with WHIM syndrome, including peripheral blood neutropenia, accumulation of segmented neutrophils, many of which display myelokathexis-like morphology, and an elevated M / E ratio in BM. Furthermore, the data implicate the potential of CXCR4 antagonist therapy to correct or normalize these pathogenic phenotypes in subjects where CXCR2 is dysfunctional.Example 2: Results from an experimental mouse model of CXCR2 loss-of-function phenotypes

[0107] As described in Example 1, a pharmacologically-induced model of CXCR2 loss-of- function was developed to characterize pathogenic phenotypes and response to CXCR4 antagonist. FIG. 1 depicts a graphical representation of the study.

[0108] Absolute neutrophil counts (ANC) in the peripheral blood of WT and loss-of-function mouse subjects was measured daily over seven days of treatment. The results, which are shown in FIG. 2, indicate that CXCR2 loss-of-function mice develop peripheral neutropenia, and CXCR4 antagonism elevates ANC and corrects neutropenia in those mice. Microscopic examination of blood smears confirms neutropenia and recovery with CXCR4 Antagonist Compound A in CXCR2 loss-of-function mice. The data show that CXCR2 blockade with Navarixin impairs CXCR2-dependent mobilization of neutrophils to blood of mice resulting in peripheral neutropenia. Treatment with CXCR4 antagonist overcomes this CXCR2-dependent mobilization defect resulting in ANC elevation and correction of peripheral neutropenia.

[0109] Mature neutrophil counts in bone marrow of murine subjects was analyzed by flow cytometry. FIG. 3 shows the results of this flow cytometric analysis, which indicate that mature (segmented) neutrophils accumulate in bone marrow CXCR2 loss-of-function mice and that CXCR4 antagonism with Compound A corrects the accumulation of neutrophils in bone marrow of those same mice. FIG. 4 shows the bone marrow myeloid / erythroid ratio in the bone marrow of WT and CXCR2 loss-of-function mice in the presence or absence of a CXCR4 antagonist. Increased bone marrow myeloid / erythroid (M / E) ratio is observed in CXCR2 loss-of-function mice induced by CXCR2 antagonist navarixin. CXCR4 antagonist Compound A normalizes this increased bone marrow M / E ratio.

[0110] As described above, myelokathexis (retention of neutrophils in bone marrow) is observed in some human patients with CXCR2 loss-of-function mutations, as well as being a diagnostic feature in bone marrow and blood of patients with CXCR4 gain-of-function. In this study, neutrophil myelokathexis is seen in the bone marrow (5 / 5 mice) and blood (2 / 5 mice) of navarixin-induced CXCR2 loss-of-function mice. CXCR4 antagonist treatment (QD, 7 days) appears to reduce the frequency and number of CXCR2 LoF mice with myelokathexis-like neutrophils in bone marrow. No significant change in MK frequency was observed in blood of CXCR2 LoF mice after 7-day QD treatment with CXCR4 antagonist. A higher percent of MK-like cells were present in the navarixin group than in the navarixin + Compound A group. These results are shown in the cellular data depicted in FIG. 5.[001111 In summary, CXCR2 loss-of-function mice exhibit many similar pathogenic phenotypes to patients with CXCR2 loss-of-function, including peripheral blood neutropenia, accumulation of mature (segmented) neutrophils in bone marrow, increased myeloid-to-erythroid (MZE) ratio in bone marrow, and myelokathexis-like neutrophils in bone marrow and peripheral blood. Treatment of these mice with CXCR4 antagonist Compound A for 7 days resulted in Correction of neutropenia correction of segmented neutrophil accumulation in bone marrow, normalization of M / E ratio in bone marrow, and apparent reduction in frequency and number of mice with myelokathexis-like neutrophils in bone marrow.Example 3: Protocol for measuring pneumonia severity in a pharmacological mouse model of CXCR2 loss-of-function

[0112] Mice were orally gavaged with the CXCR2 antagonist navarixin at 3 mg / kg / day and subsequently with the CXCR4 antagonist Compound A at 10 mg / kg / day daily for 7 days, 2 hours after navarixin dose. Vehicle only was given as control in all experiments (10% DMSO+ 40% PEG300+ 5% Tween80 + 45% saline and 50 mM citrate Buffer, pH4.0). Blood samples were collected from the mice every 7 days, 4 hours post-dosing, for complete blood cell count analysis using a Sysmex XN-350 hematology analyzer. Peripheral blood was obtained via submandibular puncture. BM samples were collected 4 hours after the final Compound A dose on day 7 for numeration and flow cytometry analysis. Euthanasia of the mice was conducted using a gradual increase in CO2 concentration.

[0113] For BM single-cell suspensions, femora and tibiae were flushed with DPBS. Cell collection was carried out in DPBS supplemented with 5% fetal bovine serum and filtered through a 70-pm nylon strainer to eliminate debris and fat. Red blood cell lysis was performed using ACK Lysis Buffer. The total number of BM suspension cells was quantified using a Countstar® Rigel S2 cell counter.

[0114] For lung single-cell suspensions, lung tissue was rinsed in ice-cold DPBS and chopped with scissors. The minced tissue was then incubated with a dissociation mix containing 20 pg / mL DNase I and 1 mg / mL Collagenase, diluted in DMEM, at 37°C for 30 minutes, with shaking every 10 minutes. Following enzymatic digestion, the tissue was further dissociated using a gentleMACS Octo Dissociator. The resulting cell suspension was filtered through a 70 pm strainer to obtain asingle-cell suspension. Red blood cells were lysed using ACK Lysis Buffer, and the suspended cells were washed twice with cold DPBS. Finally, the cells were counted using Countstar® Rigel S2, and appropriate aliquots were collected for flow cytometry analysis.

[0115] Flow Cytometry: Single-cell suspensions were initially stained with Live / Dead (R3) dye to differentiate between live and dead cells, followed by incubation with an Fc blocker for 5 minutes at 4°C in the dark. Subsequently, cells were stained with specific antibodies in a cell staining buffer for 30 min at 4°C in the dark. Table 3 lists the antibodies used in the study. In some experiments, stained samples were fixed with a Fix buffer before analysis. Analyses were performed using either the Attune NxT V6 or the FACSCanto II Plus, with data analyzed using FlowJo software.Table 3: Flow Cytometry Antibodies

[0116] Bone marrow and blood smear analysis: Peripheral blood and BM aspirate were collected 4h after last dosing on day 7. Blood and BM smears were stained with May-Grunwald Giemsa , and evaluated under light microscopy using a BX 43 Olympus microscope with images captured using a xlOO objective. Blood smears were assessed at low magnification (xlO) for a qualitative analysis of total neutrophil counts. Subsequently, high magnification (x50 and xlOO, oil immersion) was used to evaluate the monolayer area of the blood smear and the morphology of the neutrophils. For BM smears, two BM smears per animal were examined microscopically. The smears were evaluated at low magnification (xlO) for their overall quality (cellularity, smearing artifacts, cell lysis, blood contamination, etc). Then, for smears of adequate quality, approximately 15 to 20 high-power fields were evaluated at high magnification (x50 and xlOO, oil immersion). The hematopoietic cells were evaluated for their maturation sequences and morphology, the number of myeloid and erythroid cells was counted and a myeloid to erythroid ratio was calculated.

[0117] Infection of mice with Streptococcus pneumoniae: Streptococcus pneumoniae 6301 was grown overnight at 37°C and 5% CO2 in Tryptic Soy Agar supplemented with 5% sterile defibrillated sheep blood. The bacteria were then transferred to nutrient broth liquid medium supplemented with 10% horse serum and incubated overnight under the same conditions. Following this, the bacteria were subcultured for approximately 4 hours in NB liquid medium to reach the mid-logarithmic phase for inoculum preparation.

[0118] Mice were treated with CXCR2 and CXCR4 antagonists as outlined above. On the day of infection, the mice were anesthetized using isoflurane and subsequently inoculated intratracheally with Streptococcus pneumoniae at a dose of 3 * 104CFU per mouse to establish a lung infection model. Following the inoculation, lung tissue samples were collected at 24 and 72 hours post-infection to assess bacterial counts and the recruitment of neutrophils.

[0119] Determination of bacterial loads in lung tissue homogenate: Bacterial loads in the lungs of mice infected with Streptococcus pneumoniae were assessed across various experimental groups by analyzing lung tissue homogenates. Mice were euthanized using CO2, after which their lungs were excised, weighed, and homogenized in DPBS. To quantify the bacterial burden, tenfold serial dilutions of the lung tissue homogenates were prepared and plated on TSA supplemented with 5% sheep blood. The plates were then incubated at 35 ± 2 °C in a CO2 incubatorovernight to facilitate bacterial growth. The actual bacterial load of each organ was calculated according to the corresponding dilution times.[001201 Statistical analysis: The data were presented as mean + standard error of the mean (SEM) and the number of mice per experiments was indicated in the figure legends. All statistical analyses were conducted using Prism software. The significance of differences between two independent groups was calculated using Fisher’s exact test. The significance of differences between multiple groups was determined by two-way ANOVA followed by Bonferroni post hoc test. The log-rank test was used to evaluate survival differences between groups of mice. P values <0.05 were considered statistically significant.Example 4: Results of the Experiments of Example 3

[0121] CXCR4 antagonism corrects peripheral blood neutropenia in a pharmacological mouse model of CXCR2 LOF: As described in detail in Example 3 above, mice were treated with the CXCR2 antagonist navarixin (3 mg / kg) and subsequently with the orally bioavailable CXCR4 antagonist Compound A (10 mg / kg) by daily gavage for 7 days. Blood cell counts were measured 4 hours after the last dose (FIG. 6A). Treatment with the CXCR2 antagonist resulted in profound peripheral blood neutropenia, evidenced by a ~65% reduction in circulating neutrophil levels compared to control mice (FIG. 6B). The effect of the CXCR2 antagonist did not impact circulating lymphocyte, monocyte, red blood cell, or platelet counts (FIG 6D, FIG. 6E, FIG. 7A, FIG. 7B). This observation aligns with the hematological phenotypes observed in patients with CXCR2 LOF variants, suggesting that pharmacologically induced CXCR2 LOF replicates the neutropenia phenotype seen in patients. Hence, mice treated with the CXCR2 antagonist are referred to as CXCR2 LOF mice hereafter.

[0122] CXCR4 antagonist treatment led to a significant increase in absolute neutrophil count and corrected circulating neutropenia in CXCR2 LOF mice (FIG. 6B). Blood smear analysis corroborated these results, confirming that the CXCR2 LOF mice exhibited circulating neutropenia, which was reversed by CXCR4 antagonism (FIG. 6C). Consistent with previous preclinical models, treatment with the CXCR4 antagonists also led to increases in the absolute numbers of neutrophils, lymphocytes, and monocytes in control mice (FIG. 6D, FIG. 6E). While CXCR4 antagonism corrected peripheral blood neutropenia in CXCR2 LOF mice (FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E), it did not appear to affect the count of red blood cells or platelets in both CXCR2 LOF and control mice (FIG. 7A, FIG. 7B), consistent with a previous reports. Altogether,these results indicate that CXCR4 antagonism corrected peripheral blood neutropenia in CXCR2 LOF mice.[001231 CXCR4 antagonism normalizes neutrophil accumulation in bone marrow in a pharmacological mouse model of CXCR2 LOF: Impaired CXCR2 signaling has been demonstrated to lead to increased retention of neutrophils in BM and an elevated myeloid-to- erythroid (M / E) ratio, which are characteristic features observed in patients with CXCR2 LOF variants. Given these significant associations, it was investigated whether the pharmacologically induced CXCR2 LOF mouse model exhibited similar BM phenotypes and evaluated the effects of CXCR4 antagonism on these abnormalities.

[0124] The CXCR2 LOF mice exhibited a markedly higher count of mature neutrophils in BM and an increased M / E ratio compared to control mice (FIG. 8A, FIG. 8B). Furthermore, no significant differences were observed in the counts of immature neutrophils, as well as hematopoietic stem and progenitor cell subset counts between CXCR2 LOF and control mice (FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E). This finding suggests that the observed accumulation of mature neutrophils is not due to enhanced expansion but is likely a consequence of their impaired egress from BM resulting from CXCR2 blockade. Treatment with the CXCR4 antagonist significantly normalized the accumulation of mature neutrophils in BM and led to a correction of the M / E ratio in CXCR2 LOF mice (FIG. 8A, FIG. 8B). Consistent with previous findings, treatment with the CXCR4 antagonist led to a decrease in BM mature neutrophil counts (FIG. 8A) while simultaneously increasing their numbers in peripheral blood (FIG. 6B) in control mice. This supports the notion that BM serves as an important reservoir for neutrophils mobilized by CXCR4 antagonism. In summary, these findings indicate that CXCR4 antagonism effectively reverses the accumulation of mature neutrophils in BM and normalizes the M / E ratio in CXCR2 LOF mice.

[0125] CXCR4 antagonism reduces incidence of myelokathexis phenotype in a pharmacological mouse model of CXCR2 LOF: Myelokathexis is characterized by the abnormal retention of neutrophils in BM with distinct neutrophil morphology, including condensed chromatin with long, thin, often redundant chromatin strands separating nuclear lobes. This condition is a diagnostic feature in BM and blood of patients with CXCR4 GOF variants and has also been observed in a subset of patients exhibiting LOF variants in CXCR2. Consistent with observations in patients with CXCR4 GOF or CXCR2 LOF, myelokathexis-like (MK-like)neutrophils, characterized by nuclear hypersegmentation, an increased number of discernible nuclear lobes, and thinner intranuclear chromatin strands, were identified at a significantly elevated frequency in the BM of pharmacologically induced CXCR2 LOF mice, in contrast to control mice (FIG. 10A). Notably, MK-like neutrophils were present in BM of all CXCR2 LOF mice (N=5 / 5, 100%) (FIG. 10B). Administration of the CXCR4 antagonist over seven days appeared to diminish both the frequency of MK neutrophils in the BM, and the overall number of mice exhibiting neutrophil myelokathexis (N=3 / 6, 50%) (FIG. 10A, FIG. 10B). MK-like neutrophils were also detected in the peripheral blood of pharmacologically induced CXCR2 LOF mice, but not in control mice (FIG. 11 A, FIG. 11B). These MK-like neutrophils constituted approximately 7% of the total blood neutrophils in CXCR2 LOF mice (FIG. 11 A, FIG. 11B). Additionally, the morphology and frequency of MK-like neutrophils in the blood of CXCR2 LOF mice remained unchanged from baseline levels following one week of treatment with the CXCR4 antagonist, consistent with previous findings in CXCR4 GOF mouse model. Taken together, these data indicate that CXCR4 antagonism appears to reduce the frequency of MK neutrophils in the BM and the incidence of myelokathexis phenotype in a pharmacological mouse model of CXCR2 LOF.

[0126] Patients with CXCR2 LOF variants often exhibit increased susceptibility to upper respiratory infections. Previous preclinical studies have demonstrated that Cxcr2" mice, as well as mice subjected to pharmacological blockade of the CXCR2 receptor using CXCR2 antagonists orthose deficient in the CXCR2 chemokine CXCL1, show heightened vulnerability to challenges with Streptococcus pneumoniae . Therefore, experiments were conducted to investigate whether pharmacologically induced CXCR2 LOF mice also display increased susceptibility to Streptococcus pneumoniae infection and whether CXCR4 antagonism could mitigate this susceptibility.

[0127] To this end, mice were treated with the CXCR2 antagonist navarixin (3 mg / kg) and the CXCR4 antagonist Compound A (10 mg / kg) by daily oral gavage for 4 or up to 14 days. Mice were subsequently challenged with Streptococcus pneumoniae. Bacterial load and total neutrophil counts were analyzed, and mortality as well as overall survival were monitored (FIG. 12A). In line with previous reports, pharmacologically induced CXCR2 LOF mice exhibited a significantly increased bacterial load in lung tissue homogenates compared to control mice at day 1 post- pneumococcal challenge (FIG. 12B). Additionally, CXCR2 LOF mice displayed progressivemortality, with a 46% mortality rate by day 2 post-infection, compared to 23% in control mice. Treatment with the CXCR4 antagonist effectively reduced both bacterial load in lung tissues and the mortality rate in CXCR2 LOF mice, while having no significant effect on these parameters in control mice (FIG. 12B, FIG. 12C). When mice were monitored up to 14 days post-infection, CXCR2 LOF mice also exhibited significantly reduced overall survival compared to control mice, and this reduction appeared to improve with treatment with the CXCR4 antagonist (FIG. 12D).

[0128] Moreover, previous studies have indicated that the increased susceptibility to infection observed in Cxcr2' ' and Cxcll' ' mice is linked to impaired neutrophil recruitment into infected tissues. These findings corroborate this, as total neutrophil counts in lung tissue homogenates were diminished in CXCR2 LOF mice compared to controls. Treatment with the CXCR4 antagonist restored neutrophil counts in lung tissues (FIG. 12E), suggesting that CXCR4 antagonism may normalize neutrophil infiltration into infected tissues, thereby facilitating bacterial clearance. In conclusion, these data indicate that CXCR4 antagonism reduces pneumonia severity in a pharmacological mouse model of CXCR2 LOF, and this effect is likely associated with the normalization of neutrophil numbers in infected tissues.

[0129] Discussion: In these experiments, the impact of a CXCR4 antagonist on the pathogenic phenotypes seen in patients with chronic neutropenia, including abnormalities in blood and BM neutrophil levels, as well as infection susceptibility, in a pharmacologically induced CXCR2 LOF mouse model, were investigated. The findings demonstrate that CXCR4 antagonism effectively corrected peripheral blood neutropenia, normalized mature neutrophil accumulation in BM, and M / E ratio and MK-like phenotype in BM of CXCR2 LOF mice. Additionally, CXCR4 antagonism mitigated the severity of pneumonia in CXCR2 LOF mice and facilitated neutrophil emigration into bacterial infected tissues. These findings suggest the potential therapeutic benefits of CXCR4 antagonist therapy in reversing peripheral blood neutropenia and other pathogenic phenotypes associated with CXCR2 LOF variants in patients.

[0130] To investigate the relationship between CXCR2 LOF variants and pathogenic features, genetic inactivation of CXCR2 in mice would normally be the preferred model. However, the complete genetic ablation of CXCR2 in mice does not replicate the picture seen in patients, resulting in phenotypic discrepancies such as peripheral blood neutrophilia, extramedullary hematopoiesis and altered retinal glial cell distribution. These phenotypes underscores the compensatory mechanisms that may arise from gene deletion, limiting the utility of this model forstudying patient phenotypes. In contrast, Cxcr2 mixed BM chimeras exhibit patient-like features, including circulating neutropenia and BM myelokathaxis, but present challenges like immune interactions, donor cell variability, and complications from conditioning regimens and immunosuppression. Alternatively, a pharmacological approach using small molecule inhibitors may offer a more precise and reversible method to study CXCR2-dependent neutrophil trafficking. In this study, pharmacologically induced CXCR2 LOF in mice displayed pathogenic phenotypes similar to those described in patients with germline CXCR2 LOF variants, such as peripheral blood neutropenia, accumulation of mature neutrophils in BM, increased M / E ratio, neutrophils exhibiting myelokathexis-like morphology and heightened susceptibility to infections. Previous research has demonstrated that genetic inactivation of Cxcr2 in mice activates G-CSF production, thereby promoting BM granulopoiesis and increased BM neutrophil counts. The observed abnormalities in BM neutrophil counts and M / E ratio in the CXCR2 LOF mouse model are likely due to impaired neutrophil release from the BM rather than enhanced granulopoiesis, as pharmacological inhibition of CXCR2 for a duration of 7 days did not affect BM hematopoietic stem and progenitor cell subsets or immature neutrophil counts.

[0131] Neutrophil release from BM to peripheral blood is tightly regulated through the interplay of CXCR4 and CXCR2 signaling. Disruptions in either pathway can lead to increased neutrophil retention in the BM and reduced peripheral blood neutrophil levels, a common feature in patients with CXCR4 GOF and CXCR2 LOF variants. Preclinical studies have shown that CXCR2 signaling is not essential for neutrophil mobilization in the absence of CXCR4, and that neutrophil mobilization via the CXCR2 chemokine CXCL1 is significantly enhanced by transient CXCR4 inhibition, highlighting CXCR4's dominant role in neutrophil trafficking from the BM. These results further support this finding, demonstrating that chronic treatment with a CXCR4 antagonist increased blood neutrophil counts and corrected peripheral blood neutropenia in CXCR2 LOF mice. Additionally, these data indicated that the CXCR4 antagonist effectively reversed neutrophil accumulation in the BM and normalized the M / E ratio in these mice. These results suggest that selectively blocking the CXCR4 receptor disrupts the retention signals that normally sequester neutrophils within the BM of CXCR2 LOF mice, thereby facilitating their release into circulation. The observation that CXCR4 treatment reduced BM mature neutrophil counts while increasing their number in blood of both control and CXCR2 LOF mice suggests that the BM is a key source of CXCR4 antagonist-mobilized neutrophils, corroborating previousfindings. Additionally, continuous dosing with an oral CXCR4 antagonist did not affect the counts of hematopoietic stem and progenitor cells or immature neutrophils in the BM of either control or CXCR2 LOF mice. This indicates that CXCR4 antagonism effectively mobilizes neutrophils into peripheral blood without disrupting BM reserve.

[0132] G-CSF was approved ~30 years ago to treat multiple forms of neutropenia including SCN by elevating circulating absolute neutrophil counts and lowering rates of infection. Previous studies indicate that G-CSF-mediated neutrophil mobilization from BM into peripheral blood is dependent on CXCR2 signaling as G-CSF was ineffective in mobilizing neutrophils in Cxcr2~ ~ mice. Additionally, the ability of G-CSF to mobilize neutrophils and correct blood neutropenia in a Cxcr2 mixed BM chimeras mouse model was significantly compromised, suggesting the need for alternative therapies for treatment of peripheral blood neutropenia in CXCR2 deficient patients. Notably, the correction of peripheral neutropenia observed in the CXCR2 LOF mice following CXCR4 antagonist treatment suggests a promising alternative for patients with CXCR2 LOF who do not adequately respond to G-CSF. Future studies will be required to address whether CXCR4 antagonist can correct peripheral blood neutropenia in a Cxcr2 / ' mixed BM chimeras and patients with CXCR2 LOF

[0133] Myelokathexis is characterized by the abnormal accumulation of neutrophils in BM, where these neutrophils exhibit a distinct morphology. These clinical features are commonly observed in patients with GOF variants of CXCR4, and in a subset of patients with LOF variants in CXCR2. A MK-like phenotype has been previously reported in BM of a Cxcr2' ~ mixed BM chimeras. The pharmacologically induced CXCR2 LOF mouse model showed a high frequency of MK-like neutrophils in the BM, along with a notable increased incidence of mice exhibiting myelokathexis phenotype. MK-like neutrophils were also detected in the peripheral blood of CXCR2 LOF mice, mirroring observations in a mouse model with CXCR4 GOF. Furthermore, the data indicate that treatment with an oral CXCR4 antagonist appeared to reduce the incidence of myelokathexis phenotype and the frequency of MK-like neutrophils in the BM of CXCR2 LOF mice. While chronic treatment with the CXCR4 antagonist appeared to decrease the frequency of MK-like neutrophils in the BM of CXCR2 LOF mice, their frequency in the blood remained unchanged at the end of 7 days of treatment with CXCR4 antagonist, aligning with prior observations in a mouse model with CXCR4 GOF as well as in patients with CXCR4 GOF variants. This suggests that once released from the BM by the CXCR4 antagonist, MK-likeneutrophils may redistribute to organs other than peripheral blood, or that newly released MK-like neutrophils have a relatively short half-life, making them difficult to detect in the peripheral blood. Nevertheless, further studies are needed to address these questions comprehensively.

[0134] Previous preclinical studies have showed that deficiency in Cxcr2 or its chemokine Cxcll or pharmacological inhibition of these molecules resulted in impaired neutrophil recruitment into infected tissues. This impairment increased infection susceptibility to various pathogens, such as Streptococcus pneumoniae, Klebsiella Pneumoniae, Escherichia coh, Staphylococcus aureus, and Toxoplasma gondii. Consistent with previous findings, this study revealed that pharmacologically induced CXCR2 LOF with navarixin exhibited increased susceptibility to Streptococcus pneumoniae infection, evidenced by increased bacterial load in the lungs, elevated mortality, and decreased overall survival. This increased susceptibility to pneumonia appears to be associated with impaired neutrophil recruitment to the lungs. It was reported that CXCR2 deficiency impairs neutrophil migration toward CXCR2 ligands, while their ability to migrate towards another neutrophil chemoattractant, fMLP, remains unaffected. These findings imply that CXC7?2-deficient neutrophils may still be effectively recruited by alternative chemoattractant signals, such as fMLP, and possibly other chemoattractive factors that are typically generated at infection sites during bacterial challenges. This observation raises the possibility that treatments aimed at promoting neutrophil release into peripheral blood may enhance their emigration into bacterially infected tissues, thereby aiding in bacterial eradication. Supporting this hypothesis, these data indicate that treatment with a CXCR4 antagonist, which normalized the abnormal accumulation of mature neutrophils in BM, promoting their release into peripheral blood, alleviated pneumonia severity, lowered bacterial counts in the lungs, reduced mortality, and restored lung neutrophil levels. These findings in CXCR2 LOF mice are consistent with previous reports in patients with WHIM syndrome indicating that CXCR4 antagonist treatment results in clinically significant improvements in peripheral blood neutrophil levels and reductions in infection rates. Furthermore, it was observed that the CXCR4 antagonist also enhanced neutrophil recruitment to the lungs without significantly impacting infection susceptibility in control mice. This raises questions regarding the extent of neutrophil recruitment necessary for effective bacterial clearance and whether a threshold exists below which diminished recruitment compromises lung immunity against S. pneumoniae. Future studies are warranted to explore these questions further.

[0135] These experiments provide evidence that CXCR4 antagonism effectively mitigates pathogenic phenotypes in a pharmacologically induced CXCR2 LOF mouse model. The observed corrections include the reversal of peripheral blood neutropenia, normalization of mature neutrophil accumulation within BM, restoration of the M / E ratio as well as a reduction in the frequency of MK-like neutrophils in BM and the incidence of mice exhibiting neutrophil myelokathexis phenotype. Furthermore, the results demonstrate that CXCR4 antagonism reduces the severity of pneumonia in the CXCR2 LOF mice and facilitates the emigration of neutrophils into bacterially infected lung. These findings highlight the potential of CXCR4 antagonist therapy as a promising strategy for improving peripheral blood neutropenia and other clinical manifestations in individuals with CXCR2 deficiencies, as well as in various subsets of SCN.

Claims

CLAIMSWe claim:

1. A method for treating a condition resulting from a deficiency or loss-of-function in CXCR2 in a subject in need thereof, comprising administering to the subject an effective amount of a CXCR4 antagonist or a pharmaceutically acceptable salt or composition thereof.

2. The method of claim 1, wherein the condition is neutropenia.

3. The method of claim 1 or 2, wherein the condition is severe chronic neutropenia (SCN), chronic idiopathic neutropenia (CIN), congenital neutropenia, or cyclic neutropenia.

4. The method of claims 2 or 3, wherein the subject originally exhibited absolute neutrophil count (ANC) less than 600 / pL and / or absolute leukocyte count (ALC) less than 1000 / pL before treatment with the CXCR4 antagonist or a pharmaceutically acceptable salt thereof.

5. The method of any one of claims 2-4, wherein the subject originally exhibited ANC less than 400 / pL and / or ALC less than 650 / pL before treatment with the CXCR4 antagonist or a pharmaceutically acceptable salt thereof.

6. The method of any one of claims 1-5, wherein the subject is exhibiting one or more of infections, recurrent and severe infections, accumulation of neutrophils in the bone marrow, increased M / E ratio in the bone marrow, neutrophil myelokathexis, hypogammaglobulinemia, and lymphocytopenia.

7. The method of any one of claims 1-6, wherein the patient is infected with Streptococcus pneumoniae, Klebsiella Pneumoniae, Escherichia coli, Staphylococcus aureus, or Toxoplasma gondii.

8. A method for reducing the risk of an infection in a subject with deficiency or loss-of- function in CXCR2, comprising administering to the subject an effective amount of a CXCR4inhibitor or a pharmaceutically acceptable salt or composition thereof.

9. The method of claim 8, wherein the subject has an absolute neutrophil count less than about 500 cells / pL.

10. The method of claim 8 or 9, wherein the subject has an elevated risk of an infection selected from respiratory tract infections, periodontitis, otitis media, stomatitis, urinary tract infections, pyelonephritis, skin abscesses, cellulitis, and sepsis.

11. The method of claim 10, wherein the patient has an elevated risk of infection by Streptococcus pneumoniae, Klebsiella Pneumoniae, Escherichia coli, Staphylococcus aureus, or Toxoplasma gondii.

12. The method of claim 10 or 11, wherein the infection is pneumonia.

13. The method of any one of claims 1-12, wherein the subject has one or more mutations in at least one copy of the CXCR2 gene.

14. The method of claim 13, wherein the one or more mutations are selected from Argl44Cys, Arg212Trp, Argl84Ter, Arg289Cys, and His323fs.

15. The method of any one of claims 1-14, wherein the CXCR4 antagonist is selected fromor a pharmaceutically acceptable salt or composition of any of the foregoing.

16. The method of any one of claims 1-14, wherein the CXCR4 inhibitor is mavorixafor or a pharmaceutically acceptable salt or composition thereof.

17. The method of any one of claims 1-15, wherein the CXCR4 antagonist isor a pharmaceutically acceptable salt thereof.

18. The method of any one of claims 1-15, wherein the CXCR4 antagonist is not plerixafor.

19. The method of any one of claims 1-18, wherein the method achieves an absolute neutrophil count (ANC) of at least 500 cells / pL and / or an absolute leukocyte count (ALC) of at least 1000 cells / pL.

20. The method of any one of claims 1-19, wherein the method achieves an absolute neutrophil count (ANC) of about 500 cells / pL to 3,000 cells / pL.

21. The method of any one of claims 1-20, wherein the method achieves an absolute neutrophil count (ANC) of about 500 cells / pL to 3,000 cells / pL and an absolute leukocyte count (ALC) of about 1,000 to 3,000 cells / pL.

22. The method of any one of claims 1-21, wherein the method achieves an absolute neutrophil count (ANC) that is at least 2.0 times the ANC prior to administration of the CXCR4 inhibitor and / or an absolute leukocyte count (ALC) that is at least 2.0 times the ALC prior to administration of the CXCR4 inhibitor.

23. The method of any one of claims 1-21, wherein the subject does not have a genetic abnormality associated with a gain-of-function mutation in the CXCR4 gene.

24. The method of any one of claims 1-23, wherein the subject is administered an effective amount of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), a variant of G-CSF or GM-CSF (e.g., a pegylated version), bone marrow transplantation, treatment with cord blood stem cells, or a combination thereof.

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