GPCR inhibitor and its use

Abstract

The present invention relates to methods and compositions for mobilizing cells in a subject by blocking CXCR4, β-adrenergic receptors, GPCRs, or any combination thereof. In some embodiments, the cells are stem cells. In some embodiments, the cells are immune cells.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 369,738, filed Jul. 8, 2022; and U.S. Provisional Patent Application No. 63 / 351,101, filed Jun. 10, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] (Background of the Invention) The invention disclosed herein generally relates to the mobilization of stem cells and immune cells.

Background Art

[0003] The bone marrow is highly innervated by the sympathetic nervous system. Traumatic stress in human and rodent models has shown persistently elevated levels of norepinephrine (a ligand for β - adrenergic receptors) associated with bone marrow dysfunction (Bible et al., 2014, Bible et al., 2015a, Bible et al., 2015b). In a rat model of traumatic stress, daily administration of propranolol, a β - adrenergic receptor inhibitor, has been shown to restore bone marrow function and increase erythroid progenitor cell colony growth in response to anemia (Alamo et al., 2017). In patients with multiple myeloma, a 28 - day cycle of propranolol administration changed cell differentiation away from a myeloid bias to up - regulation of CD34+ stem cells and genes associated with this phenotype (Knight et al., 2020). Thus, β - blockers may have the potential to improve hematopoietic stem cell mobilization by restoring bone marrow function.

[0004] CXC chemokine receptor 4 (CXCR4) belongs to the superfamily of G protein-coupled receptors (GPCRs). The binding of chemokine CXCL12 (also known as SDF-1) to its receptor CXCR4 plays an important role in the homing and engraftment of hematopoietic stem cells (HSCs) in the bone marrow. Blocking the CXCL12 / CXCR4 axis can induce the rapid mobilization of HSCs from the bone marrow to the peripheral blood (Domingues et al., 2017). CXCR4 antagonists such as burixafor (also known as GPC-100 or TG-0054) and plerixafor (also known as AMD3100 or Mozobil) are clinically used in combination with granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell mobilization and subsequent autologous stem cell transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. However, usually, the G-CSF regimen requires repeated injections for several days and is associated with adverse side effects such as severe bone pain. However, the success of ASCT in lymphoma and MM patients is often hampered by insufficient mobilization, and at least 15% of patients are unable to produce the target cell dose of >2×10 6 CD34+ cells / kg (Olivieri et al., 2012).

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0073] (Abbreviations) Unless otherwise indicated, the following are abbreviations for the terms disclosed in this specification: acute myeloid leukemia (AML), adenosine A3 receptor (ADORA3), adenosine receptor A2b (ADORA2B), adenovirus high-throughput system (AdHTS), adenylate cyclase-activating polypeptide 1 (pituitary) receptor type I (ADCYAP1R1), adrenergic receptor alpha-1A (ADRA1A), adrenergic receptor beta-2 (ADRB2), apelin receptor (APLNR), atypical chemokine receptor 3 (ACKR3), bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer (BRET), bovine serum albumin (BSA), calcitonin receptor (CALCR), cancer stem cell (CSC), C-C chemokine receptor type 2 (CCR2), chemerin chemokine-like receptor 1 (CMKLR1), muscarinic acetylcholine receptor 1 (CHRM1), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic obstructive pulmonary disease (COPD), complement C5a receptor 1 (C5AR1), C-terminal fragment of Venus (VC), C-X-C motif chemokine ligand 12 (CXCL12), CXC receptor 4 (CXCR4), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), delta-opioid receptor (OPRD), endothelin receptor type B (EDNRB), enzyme-linked immunosorbent assay (ELISA), formalin-fixed paraffin-embedded (FFPE), fluorescence resonance energy transfer (FRET), G protein-coupled receptor (GPCR), galanin receptor 1 (GALR1), glioblastoma multiforme (GBM), glucagon receptor (GCGR), GPCR heteromer identification technology (GPCR-HIT), granulocyte colony-stimulating factor (G-CSF), hematopoietic stem cell (HSC), hepatocellular carcinoma (HCC), histamine receptor H1 (HRH1), human immunodeficiency virus (HIV), International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), mu-opioid receptor (MOR), motilin receptor (MLNR), multiple myeloma (MM), multiplicity of infection (MOI), myelodysplastic syndrome (MDS), neurotensin receptor 1 (NTSR1), non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), N-terminal fragment of Venus (VN), patient-derived cell (PDC), patient-derived xenograft (PDX), positron emission tomography (PET),Computed tomography (CT), programmed cell death ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), prostaglandin E receptor 2 (PTGER2), prostaglandin E receptor 3 (PTGER3), proximity ligation assay (PLA), quantitative reverse transcription polymerase chain reaction (RT-qPCR), single photon emission computed tomography (SPECT), small lymphocytic lymphoma (SLL), small cell lung cancer (SCLC), somatostatin receptor 2 (SSTR2), stromal cell-derived factor 1 (SDF-1), systemic lupus erythematosus (SLE), tachykinin receptor 3 (TACR3), threshold cycle (Ct), time-resolved FRET (TR-FRET), tumor microenvironment (TME), vascular endothelial growth factor (VEGF), vascular smooth muscle cell (VSMC), WHIM syndrome (warts, hypogammaglobulinemia, infections, and myeloid cell retention), green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are included.

[0074] (Detailed Description of the Invention) Blood cells play a crucial role in maintaining the health and viability of animals, including humans. White blood cells, which are part of the body's immune system and help the body fight infections and other diseases, include the immune system's granulocytes (neutrophils, eosinophils, and basophils / mast cells), monocytes / macrophages, and lymphocytes (T and B cells). White blood cells are constantly replaced through the hematopoietic system by the action of colony-stimulating factors (CSFs) and various cytokines on stem cells and progenitor cells in hematopoietic tissue.

[0075] One of the best-known of these is granulocyte colony-stimulating factor (G-CSF), which is approved for use in counteracting the negative effects of chemotherapy by stimulating the production of white blood cells and progenitor cells (peripheral blood stem cell mobilization). For the hematopoietic effects of G-CSF, see, for example, U.S. Patent No. 5,582,823, which is incorporated herein by reference.

[0076] The generation and maturation of blood cells is a complex process. Mature blood cells are derived from hematopoietic progenitor (precursor) cells and stem cells that exist within specific hematopoietic tissues, including the bone marrow. Within the scope of these environments, hematopoietic cells proliferate and differentiate before entering the circulation.

[0077] The chemokine receptor CXCR4 and its natural ligand, stromal cell-derived factor 1 (SDF-1), appear to be important in this process (for reviews, see Maekawa, T. et al., Internal Med. (2000) 39:90-100; Nagasawa, T. et al., Int. J. Hematol. (2000) 72:408-411). This has been demonstrated by reports that CXCR4- or SDF-1-knockout mice exhibit embryonic lethality and hematopoietic defects (Ma, Q. et al., Proc. Natl. Acad. Sci USA (1998) 95:9448-9453; Tachibana, K. et al., Nature (1998) 393:591-594; Zou, Y-R. et al., Nature (1998) 393:595-599). CD34+ progenitor cells are known to express CXCR4 and to require SDF-1 produced by bone marrow stromal cells for chemotaxis and engraftment (Peled, A. et al., Science (1999) 283:845-848). In addition, in vitro, SDF-1 is also known to have chemotactic effects on both CD34+ cells (Aiuti, A. et al., J. Exp. Med. (1997) 185:111-120; Viardot, A. et al., Ann. Hematol. (1998) 77:194-197) and progenitor / stem cells (Jo, D-Y. et al., J. Clin. Invest. (2000) 105: 101-111).In addition, SDF-1 is an important chemoattractant for several other more committed precursors and mature blood cells, including T-lymphocytes and monocytes (Bleul, C. et al., J. Exp. Med. (1996) 184:1101-1109), pro- and pre-B lymphocytes (Fedyk, E. R. et al., J. Leukoc. Biol. (1999) 66:667-673; Ma, Q. et al., Immunity (1999) 10:463-471), and megakaryocytes (Hodohara, K. et al., Blood (2000) 95:769-775; Riviere, C. et al., Blood (1999) 95:1511-1523; Majka, M. et al., Blood (2000) 96:4142-4151; Gear, A. et al., Blood (2001) 97:937-945; Abi-Younes, S. et al., Circ. Res. (2000) 86:131-138), which signal through the CXCR4 receptor.

[0078] Thus, SDF-1 appears to be able to control the positioning and differentiation of cells bearing the CXCR4 receptor, whether those cells be stem cells (i.e., cells that are CD34+), and / or progenitor cells (either CD34+ or CD34− that are capable of giving rise to the formation of specific types of colonies in response to specific stimuli), or more differentiated cells.

[0079] In recent years, there has been considerable attention focused on the number of CD34+ cells mobilized into the pool of peripheral blood progenitor cells used for autologous stem cell transplantation. The CD34+ population is the component thought to be mainly responsible for improving the recovery time after chemotherapy, and these cells are most likely to be responsible for long-term engraftment and hematopoietic recovery (Croop, J. M. et al., Bone Marrow Transplantation (2000) 26:1271-1279). The mechanism by which CD34+ cells re-engraft may be due to the chemotactic effect of SDF-1 on CXCR4-expressing cells (Voermans, C., Blood, 2001, 97, 799-804; Ponomaryov, T. et al., J. Clin. Invest. (2000) 106:1331-1339). For example, it has been shown that adult hematopoietic stem cells have the ability to restore damaged heart tissue in mice (Jackson, K. et al., J. Clin. Invest. (2001) 107:1395-1402; Kocher, A. et al., Nature Med. (2001) 7:430-436). Thus, the role of the CXCR4 receptor in regulating cell positioning and differentiation has taken on considerable importance.

[0080] As used herein, the term "progenitor cell" means a cell that can form differentiated hematopoietic or myeloid cells in response to a stimulus. The presence of progenitor cells can be evaluated by the ability of the cells in a sample to form various types of colony-forming units, including, for example, CFU-GM (colony-forming unit, granulocyte-macrophage); CFU-GEMM (colony-forming unit, multipotent); BFU-E (burst-forming unit, erythroid); HPP-CFC (high-proliferative potential colony-forming cell); or other types of differentiated colonies that can be obtained in culture using known protocols.

[0081] As used herein, a "stem" cell is a progenitor cell in a less differentiated form. Usually, such cells are often CD34 positive. However, some stem cells do not contain this marker. These CD34+ cells can be assayed using fluorescence-activated cell sorting (FACS), and thus their presence can be evaluated in a sample using this technique. Generally, CD34+ cells are present only at low concentrations in the blood, but are present in large numbers in the bone marrow. Although other types of cells, such as endothelial cells and mast cells, may also exhibit this marker, CD34 is considered an indicator of the presence of stem cells.

[0082] As used herein, the term "CXCR4" refers to C-X-C motif chemokine receptor 4, which is also identified by a unique database identifier (ID) and aliases as shown in Table 1 (Chatterjee et al., 2014; Debnath et al., 2013; Domanska et al., 2013; Guo et al., 2016; Peled et al., 2012; Roccaro et al., 2014; Walenkamp et al., 2017). Table 1 also provides the names of CXCR4, and GPCRx, which forms heteromers with CXCR4 and synergistically enhances the Ca2+ response upon co-stimulation with agonists of both. (Table 1)

Table 1

[0083] As used herein, the term "GPCRx" refers to a GPCR used in this study to investigate whether the GPCR interacts with CXCR4 and whether it exhibits properties distinct from those of the individual protomers including ADCYAP1R1 (ADCYAP receptor type I), ADORA2B (adenosine A2b receptor), ADORA3 (adenosine A3 receptor), ADRB2 (adrenergic receptor beta 2), APLNR (apelin receptor), C5AR1 (complement C5a receptor 1), CALCR (calcitonin receptor), CCR5 (chemokine (C-C motif) receptor 5), CHRM1 (muscarinic acetylcholine receptor 1), GALR1 (galanin receptor 1), EDNRB (endothelin receptor type B), HRH1 (histamine receptor H1), MLNR (motilin receptor), NTSR1 (neurotensin receptor 1), PTGER2 (prostaglandin E receptor 2), PTGER3 (prostaglandin E receptor 3), SSTR2 (somatostatin receptor 2), and TACR3 (tachykinin receptor 3), which are also identified by unique database identifiers (IDs) and aliases as shown in Table 1.

[0084] As used herein, the term "inhibitor" means a molecule that inhibits or suppresses the enhanced function of CXCR4, β - adrenergic receptor, GPCR, the heteromer of CXCR4 and β - adrenergic receptor, and / or the CXCR4 - GPCRx heteromer. Non - limiting examples of the inhibitors of the present invention that can be used for cell mobilization include GPCRx antagonists, GPCRx inverse agonists, GPCRx positive and negative allosteric modulators, CXCR4 - GPCRx heteromer - specific antibodies or antigen - binding portions thereof including single - domain antibody - like scaffolds, bivalent ligands having a pharmacophore selective for CXCR4 linked to a pharmacophore selective for GPCRx by a spacer arm, bispecific antibodies against CXCR4 and GPCRx, radiolabeled CXCR4 ligands linked to GPCRx ligands, and small - molecule ligands that inhibit heteromer - selective signal transduction. Specific examples of inhibitors for GPCRx that form a heteromer with CXCR4 and enhance the Ca2+ response upon co - stimulation with agonists of both are listed in Table 2.

[0085] As used herein, the term "antagonist" means a type of receptor ligand or drug that is also called a blocker, binds to a receptor and blocks or attenuates a biological response by blocking it. An antagonist has affinity but no efficacy for its cognate receptor, and its binding interferes with interactions and inhibits the function of agonists or inverse agonists at the cognate receptor. Specific examples of antagonists for GPCRx that form a heteromer with CXCR4 and enhance the Ca2+ response upon co - stimulation with agonists of both are listed in Table 2. (Table 2. Examples of inhibitors against CXCR4 and ADRB2)

Table 2

[0086] As used herein, the term "heteromer" refers to a macromolecular complex composed of at least two GPCR units [protomers] having biochemical properties that are significantly different from the biochemical properties of their individual components. Heteromerization can be evaluated by in situ hybridization, immunohistochemistry, RNAseq, reverse transcription quantitative PCR (RT-qPCR, real-time PCR), microarray, proximity ligation assay (PLA), time-resolved FRET (TR-FRET), whole-body single photon emission computed tomography (SPECT), or positron emission tomography / computed tomography (PET / CT).

[0087] As used herein, the phrase "effective amount" means an amount sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery is determined by several variables including the period over which individual dosage units are to be used, the bioavailability of the agent, the route of administration, and the like.

[0088] As used herein, the phrase "therapeutically effective amount" means an amount of a therapeutic agent (e.g., an inhibitor, antagonist, or any other therapeutic agent as defined herein) sufficient to reduce, ameliorate, and / or arrest the severity and / or duration of cancer and / or symptoms associated therewith. The therapeutically effective amount of a therapeutic agent can be an amount necessary to reduce, ameliorate, or arrest the progression or exacerbation of cancer, reduce, ameliorate, or arrest the recurrence, progression, or onset of cancer, and / or an amount necessary to enhance or augment the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an inhibitor, antagonist, or any other therapeutic agent as defined herein).

[0089] The term "therapeutic agent" means any agent that can be used in the treatment, amelioration, prevention, or management of cancer and / or symptoms associated therewith. In certain embodiments, the therapeutic agent means an inhibitor of the CXCR4-GPCRx heteromer of the present invention. The therapeutic agent can be a drug that is well-known to be useful in the treatment, amelioration, prevention, or management of cancer and / or symptoms associated therewith, or that has been used or is currently being used for them.

[0090] As used herein, the terms "intracellular Ca2+ assay", "calcium mobilization assay", or variations thereof mean cell-based assays that measure calcium flux associated with GPCR activation or inhibition. This method utilizes calcium-sensitive fluorescent dyes that are taken up into the cytoplasm of most cells. The dye binds to calcium released from intracellular stores, increasing its fluorescence. Changes in fluorescence intensity are directly correlated with the amount of intracellular calcium released into the cytoplasm in response to ligand activation of the receptor of interest.

[0091] As used herein, the term "proximity-based assay" means biophysical and biochemical techniques that can monitor the proximity and / or binding of two protein molecules in vitro (in cell lysates) and in live cells, including bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), proximity ligation assay (PLA), cysteine crosslinking, and co-immunoprecipitation (Ferre et al., 2009; Gomes et al., 2016).

[0092] Disclosed herein are methods and compositions directed to mobilizing cells in a subject by blocking CXCR4, β - adrenergic receptor, GPCR, or any combination thereof. In some embodiments, the cells are stem cells. In some embodiments, the cells are immune cells. In some embodiments, mobilizing cells in the subject comprises blocking CXCR4. In some embodiments, mobilizing cells in the subject comprises blocking the β - adrenergic receptor. In some embodiments, mobilizing cells in the subject comprises blocking GPCR. In some embodiments, mobilizing cells in the subject comprises blocking CXCR4 and the β - adrenergic receptor. In some embodiments, mobilizing cells in the subject comprises blocking CXCR4 and GPCR. In some embodiments, mobilizing cells in the subject comprises blocking the CXCR4 - GPCR heteromer.

[0093] Disclosed herein is a method of mobilizing cells in a subject, said method comprising blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject. Also disclosed herein is a method of inducing cell mobilization in a subject, said method comprising blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject. In an embodiment, blocking the β-adrenergic receptor signaling is performed prior to blocking the CXCR4 signaling. In some embodiments, blocking the β-adrenergic receptor signaling is performed at a first specific time interval prior to blocking the CXCR4 signaling. In some embodiments, the first specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more. In an embodiment, blocking the β-adrenergic receptor signaling is continued after blocking the CXCR4 signaling has ended. In some embodiments, blocking the β-adrenergic receptor signaling is continued for a second specific time interval after blocking the CXCR4 signaling has ended. In some embodiments, the second specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more.

[0094] In an embodiment, blocking the CXCR4 signaling includes administering a CXCR4 inhibitor to the subject.

[0095] In an embodiment, blocking the β - adrenergic receptor signaling includes administering a β - adrenergic receptor inhibitor to the subject. In an embodiment, blocking the CXCR4 signaling includes administering a CXCR4 inhibitor to the subject, and blocking the β - adrenergic receptor signaling includes administering a β - adrenergic receptor inhibitor to the subject. In an embodiment, the cell is a stem cell. In some embodiments, the cell is an immune cell.

[0096] Disclosed herein is a method for mobilizing stem cells in a subject, the method comprising administering a β - adrenergic receptor inhibitor and a CXCR4 inhibitor to the subject. Also disclosed herein is a method for inducing stem cell mobilization in a subject, the method comprising administering a β - adrenergic receptor inhibitor and a CXCR4 inhibitor to the subject. In some embodiments, administering the β - adrenergic receptor inhibitor is performed before administering the CXCR4 inhibitor. In some embodiments, administering the β - adrenergic receptor inhibitor is performed at a first specific time interval before administering the CXCR4 inhibitor. In some embodiments, the first specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more. In an embodiment, administering the β - adrenergic receptor inhibitor is continued even after administering the CXCR4 inhibitor is completed. In some embodiments, administering the β - adrenergic receptor inhibitor is continued for a second specific time interval after administering the CXCR4 inhibitor is completed. In some embodiments, the second specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more.

[0097] In an embodiment, the β - adrenergic receptor inhibitor is an ADRB2 inhibitor. In an embodiment, the β - adrenergic receptor inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204 - 545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol. In an embodiment, the β - adrenergic receptor inhibitor is selected from the group consisting of propranolol, nadolol, and ICI 118551. In an embodiment, the β - adrenergic receptor inhibitor is propranolol.

[0098] In an embodiment, the CXCR4 inhibitor is selected from the group consisting of ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (Plerixafor), AMD3465, ATI 2341, BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, CX549, D-[Lys3] GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, Isothiourea-1a, Isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012, TG-0054 (Brixafort), USL311, viral macrophage inflammatory protein-II (vMIP-II), WZ811, [64Cu]-AMD3100, [64Cu]-AMD3465, [68Ga]pentixafor, [90Y]pentixather, [99mTc]O2-AMD3100, [177Lu]pentixather, and 508MCl (Compound 26). Brixafort is also referred to as GPC-100 or TG-0054. Plerixafor is also referred to as AMD3100 or Mozobil. In an embodiment, the CXCR4 inhibitor is selected from the group consisting of AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (Plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (Brixafort), PF-06747143, POL6326, and Ulocuplumab (MDX1338 / BMS-936564). In an embodiment, the CXCR4 inhibitor is TG-0054 (Brixafort). In an embodiment, the CXCR4 inhibitor is AMD3100 (Plerixafor). In an embodiment, the CXCR4 inhibitor is Ulocuplumab (MDX1338 / BMS-936564).

[0099] In an embodiment, administering the CXCR4 inhibitor to the subject includes administering TG-0054 (bryxafol) and propranolol. In an embodiment, administering the CXCR4 inhibitor to the subject includes administering AMD3100 (plerixafor) and propranolol. In an embodiment, administering the CXCR4 inhibitor to the subject includes administering ulocuplumab (MDX1338 / BMS-936564) and propranolol.

[0100] In an embodiment, the method further includes administering G-CSF to the subject. In an embodiment, administering the β-adrenergic receptor inhibitor and the CXCR4 inhibitor to the subject is performed in the absence of G-CSF. Disclosed herein is a method of mobilizing stem cells in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor. Also disclosed herein is a method of inducing stem cell mobilization in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor. In some embodiments, administering the CXCR4 inhibitor to the subject includes administering TG-0054 (bryxafol) and propranolol. In an embodiment, administering the CXCR4 inhibitor to the subject includes administering AMD3100 (plerixafor) and propranolol. In an embodiment, administering the CXCR4 inhibitor to the subject includes administering ulocuplumab (MDX1338 / BMS-936564) and propranolol.

[0101] In an embodiment, administering the combination of the CXCR4 inhibitor and the G-CSF induces an improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone. In an embodiment, administering the combination of the CXCR4 inhibitor and the G-CSF mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone. In some embodiments, the improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 1.1-fold to 1.2-fold, 1.2-fold to 1.3-fold, 1.3-fold to 1.4-fold, 1.4-fold to 1.5-fold, 1.5-fold to 1.6-fold, 1.6-fold to 1.7-fold, 1.7-fold to 1.8-fold, 1.8-fold to 1.9-fold, 1.9-fold to 2-fold, 2-fold to 2.5-fold, 2.5-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold, or 10-fold or more. In some embodiments, the improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is a 5% to 10% increase, 10% to 20% increase, 20% to 30% increase, 30% to 40% increase, 40% to 50% increase, 50% to 60% increase, 60% to 70% increase, 70% to 80% increase, 80% to 90% increase, 90% to 100% increase, 100% to 120% increase, 120% to 140% increase, 140% to 160% increase, 160% to 180% increase, 180% to 200% increase, 200% to 250% increase, 250% to 300% increase, 300% to 400% increase, 400% to 500% increase, 500% to 750% increase, 750% to 1000% increase, or 1000% or more.

[0102] In an embodiment, administering the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor induces an improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone. In an embodiment, administering the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone. In some embodiments, the improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 1.1-fold to 1.2-fold, 1.2-fold to 1.3-fold, 1.3-fold to 1.4-fold, 1.4-fold to 1.5-fold, 1.5-fold to 1.6-fold, 1.6-fold to 1.7-fold, 1.7-fold to 1.8-fold, 1.8-fold to 1.9-fold, 1.9-fold to 2-fold, 2-fold to 2.5-fold, 2.5-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold, or 10-fold or more. In some embodiments, the improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is a 5% to 10% increase, 10% to 20% increase, 20% to 30% increase, 30% to 40% increase, 40% to 50% increase, 50% to 60% increase, 60% to 70% increase, 70% to 80% increase, 80% to 90% increase, 90% to 100% increase, 100% to 120% increase, 120% to 140% increase, 140% to 160% increase, 160% to 180% increase, 180% to 200% increase, 200% to 250% increase, 250% to 300% increase, 300% to 400% increase, 400% to 500% increase, 500% to 750% increase, 750% to 1000% increase, or 1000% or more.

[0103] In an embodiment, administering a combination of the CXCR4 inhibitor, the β-adrenergic receptor inhibitor, and the G-CSF induces an improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone. In an embodiment, administering a combination of the CXCR4 inhibitor, the β-adrenergic receptor inhibitor, and the G-CSF mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone. In an embodiment, administering a combination of TG-0054 (bryxalfor) and the G-CSF induces an improved amount of cell mobilization as compared to the amount of cell mobilization induced by AMD3100 (plerixafor) and the G-CSF. In an embodiment, administering a combination of the TG-0054 (bryxalfor) and the G-CSF mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the AMD3100 (plerixafor) and the G-CSF. In some embodiments, the improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 1.1-fold to 1.2-fold, 1.2-fold to 1.3-fold, 1.3-fold to 1.4-fold, 1.4-fold to 1.5-fold, 1.5-fold to 1.6-fold, 1.6-fold to 1.7-fold, 1.7-fold to 1.8-fold, 1.8-fold to 1.9-fold, 1.9-fold to 2-fold, 2-fold to 2.5-fold, 2.5-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold, or 10-fold or more. In some embodiments, the improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is a 5% to 10% increase, 10% to 20% increase, 20% to 30% increase, 30% to 40% increase, 40% to 50% increase, 50% to 60% increase, 60% to 70% increase, 70% to 80% increase, 80% to 90% increase, 90% to 100% increase, 100% to 120% increase, 120% to 140% increase, 140% to 160% increase, 160% to 180% increase, 180% to 200% increase, 200% to 250% increase, 250% to 300% increase, 300% to 400% increase, 400% to 500% increase, 500% to 750% increase, 750% to 1000% increase, or 1000% or more.In an embodiment, the improved amount of cell mobilization or apheresis is measured by a method selected from the group consisting of a complete blood count (CBC) analysis, flow cytometry, and a colony forming unit (CFU) assay. In an embodiment, the improved amount of the cell mobilization or apheresis is measured by flow cytometry. In an embodiment, the flow cytometry is performed on (Lin-Sca1+c-Kit+) LSK cells. In an embodiment, the improved amount of the cell mobilization or apheresis is measured by a colony forming unit (CFU) assay.

[0104] In an embodiment, the subject has a CXCR4 protomer in the cell. In an embodiment, the subject has an ADRB2 protomer in the cell. In an embodiment, the subject has a CXCR4 protomer and an ADRB2 protomer in the cell. In an embodiment, the subject has a CXCR4-ADRB2 heteromer in the cell. In an embodiment, i) the CXCR4-ADRB2 heteromer has an improved amount of downstream calcium mobilization compared to the downstream calcium mobilization from the CXCR4 protomer or the ADRB2 protomer; and ii) the administered combination of inhibitors suppresses the enhanced downstream calcium mobilization from the CXCR4-ADRB2 heteromer in the stem cells.

[0105] In an embodiment, the cell is a stem cell. In an embodiment, the stem cell is selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, endothelial progenitor cells, nervous system stem cells, epithelial stem cells, skin stem cells, and cancer stem cells. In an embodiment, the stem cell is a hematopoietic stem cell or a hematopoietic progenitor cell. In an embodiment, the hematopoietic stem cell or the hematopoietic progenitor cell is mobilized from bone marrow to peripheral blood. In an embodiment, the mobilized hematopoietic stem cell or hematopoietic progenitor cell is collected for transplantation into a cancer patient. In an embodiment, the cancer is selected from the group consisting of lymphoma, leukemia, and myeloma. In an embodiment, the cancer is non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or multiple myeloma (MM). In an embodiment, the stem cell is a mesenchymal stem cell. In an embodiment, the mesenchymal stem cell is mobilized from bone marrow to peripheral blood. In an embodiment, the mesenchymal stem cell is mobilized for the treatment of a condition selected from the group consisting of neuropathy, myocardial ischemia, myocardial infarction, diabetes, tissue repair, bone and cartilage diseases, autoimmune diseases, graft-versus-host disease, Crohn's disease, multiple sclerosis, systemic lupus erythematosus, and systemic sclerosis. In an embodiment, the stem cell is a cancer stem cell. In an embodiment, the cancer stem cell is mobilized into the blood. In an embodiment, the cancer stem cell is mobilized for the treatment of cancer.

[0106] In an embodiment, the cell is an immune cell. In an embodiment, the immune cell is a leukocyte. In an embodiment, the leukocyte is a lymphocyte. In an embodiment, the lymphocyte is selected from the group consisting of T cells, B cells, and natural killer (NK) cells. In an embodiment, the lymphocyte is a T cell. In an embodiment, the lymphocyte is a natural killer (NK) cell. In an embodiment, the leukocyte is a granulocyte. In an embodiment, the granulocyte is selected from the group consisting of neutrophils, eosinophils, and basophils. In an embodiment, the granulocyte is a neutrophil. In an embodiment, the leukocyte is a monocyte. In an embodiment, the immune cell is mobilized from bone marrow to peripheral blood. In an embodiment, the immune cell is mobilized from lymph nodes to peripheral blood. In an embodiment, the mobilized immune cell is used in adoptive cell therapy (ACT). In an embodiment, the adoptive cell therapy (ACT) is chimeric antigen receptor (CAR) T cell therapy. In an embodiment, the adoptive cell therapy (ACT) is natural killer (NK) cell therapy. In an embodiment, the adoptive cell therapy (ACT) is modified T cell receptor (TCR) therapy. In an embodiment, the adoptive cell therapy (ACT) is tumor-infiltrating lymphocyte (TIL) therapy.

[0107] In some embodiments of the present invention, mobilizing cells in the subject includes blocking CXCR4. Many antiviral agents that inhibit HIV replication by inhibiting CXCR4, a co-receptor required for the fusion and entry of T-tropic HIV strains, also inhibit the binding and signaling induced by the natural ligand chemokine SDF-1 (also known as CXCL12). Without wishing to be bound by any theory, agents that inhibit the binding of SDF-1 to CXCR4 can, due to such inhibition, result in an increase in the mobilization of stem cells and / or progenitor cells to the periphery. Enhancing the mobilization of stem cells and / or progenitor cells to the peripheral blood can assist in the treatment of protocols that have a detrimental effect on the bone marrow, such as those that cause neutropenia, a known side effect of chemotherapy and radiotherapy. Also, agents that inhibit the binding of SDF-1 to CXCR4 can lead to better success in bone marrow transplantation, improve wound healing and burn treatment, and assist in the recovery of damaged organ tissue. Also, this fights the bacterial infections commonly seen in leukemia. This is used to mobilize and collect CD34+ cells via apheresis with and without the use of other mobilizing factors. The cells collected are used in treatments that require stem cell transplantation.

[0108] In some embodiments of the present invention, mobilizing stem cells in the subject includes blocking the CXCR4-GPCR heteromer. Various CXCR4-GPCR heteromers with distinct physiological and pharmacological properties have been reported, but their role in stem cell mobilization or the potential to develop stem cell mobilization therapeutics that target CXCR4-GPCR heteromers has not been clearly understood or recognized.

[0109] In this technical field, GPCRs are thought to function as monomers that interact with heterotrimeric G proteins upon ligand binding, and drugs have been developed based on monomeric or homomeric GPCRs (Milligan, 2008). In recent years, this view has changed significantly based on the discovery that GPCRs can form heteromers and that heteromerization is essential for some GPCRs. GPCR heteromerization is known to alter GPCR maturation and cell surface delivery, ligand binding affinity, signaling strength and pathways, and receptor desensitization and recycling (Terrillon and Bouvier, 2004; Ferre et al., 2010; Rozenfeld and Devi, 2010; Gomes et al., 2016; Farran, 2017). Different GPCR heteromers exhibit distinct functional and pharmacological properties, and GPCR heteromerization can vary depending on cell type, tissue, and disease or pathological state (Terrillon and Bouvier, 2004; Ferre et al., 2010; Rozenfeld and Devi, 2010; Gomes et al., 2016; Farran, 2017). Currently, GPCR heteromerization is regarded as a common phenomenon, and elucidating GPCR heteromerization opens up new avenues for understanding the functions of receptors, their roles in physiology, disease, and pathological states. Therefore, the identification of GPCR heteromers and their functional properties provides new opportunities for developing new pharmaceuticals with fewer side effects, higher efficacy, and increased tissue selectivity, or for discovering new uses for existing drugs (Ferre et al., 2010; Rozenfeld and Devi, 2010; Farran, 2017).

[0110] Apheresis is a standard technique for obtaining a larger number of immune cells as a starting material for adoptive cell therapy (ACT), a treatment based on transferring cells into a patient (1-3). Apheresis may involve passing a patient's blood through a device that separates one specific component and returns the rest to the patient's blood circulation. Thus, apheresis is an extracorporeal therapy. Depending on the substance to be removed, various processes are employed in apheresis. When separation by density is required, centrifugation is the most commonly used method. Other methods include absorption onto beads coated with absorbent material and filtration. Centrifugation methods can be classified into two basic categories: continuous flow centrifugation (CFC) and intermittent flow centrifugation.

[0111] CFC historically required two venipunctures because "continuous" meant that blood was simultaneously drawn, spun, and returned. Newer systems can use a single venipuncture. The main advantage of CFC is that the extracorporeal volume used in this procedure (calculated by the volume of the apheresis chamber, the donor's hematocrit, and the donor's total blood volume) is small, which can be advantageous in the elderly and for children. Intermittent flow centrifugation works in cycles where blood is drawn, spun / processed, and then the unused portion is returned to the donor in a bolus. The main advantage is a single venipuncture site. An anticoagulant is automatically mixed with the blood as it is pumped from the body into the apheresis machine to prevent the blood from clotting.

[0112] When the component to be removed causes severe symptoms of the disease in the patient, various apheresis techniques can always be used. Generally, apheresis needs to be performed rather frequently, and this is an invasive procedure. Therefore, it is generally employed when other means of controlling a particular disease have failed, or when the symptoms are of such a nature that waiting for drug therapy to become effective could cause the risk or occurrence of complications. Apheresis techniques include: (1) Plasma exchange - removal of the liquid portion of the blood to remove harmful substances, where plasma is replaced with a replacement solution; (2) LDL apheresis - removal of low-density lipoprotein in patients with familial hypercholesterolemia; (3) Photopheresis - used to treat graft-versus-host disease, cutaneous T-cell lymphoma, and rejection in heart transplantation; (4) Immunoadsorption using a staphylococcal protein A-agarose column - removal of alloantibodies and autoantibodies (those in autoimmune diseases, transplant rejection, hemophilia) by guiding plasma through a protein A-agarose column (protein A is a cell wall component produced by some strains of Staphylococcus aureus and binds to the Fc region of IgG); (5) Leukapheresis - removal of malignant white blood cells in those with leukemia and a very high white blood cell count causing symptoms; (6) Erythrocytapheresis - removal of erythrocytes (red blood cells) in those with iron overload as a result of hereditary hemochromatosis or transfusion hemosiderosis; (7) Plateletpheresis - removal of platelets in those with symptoms due to extreme elevation of platelet count, such as those associated with essential thrombocythemia or polycythemia vera; and (8) Leukocytapheresis - separation of excess white blood cells from leukemia patients while reusing the rest of the patient's blood.

[0113] Apheresis is a difficult procedure, inconvenient and expensive. With the rapid increase in adoptive cell transfer (ACT) including chimeric antigen receptor (CAR)-T, CAR-natural killer (NK), tumor-infiltrating lymphocytes (TIL), and engineered T cell receptors (TCR), there is an increasing need for apheresis technology for the routine production of pure immune cells (2). The industry supplying starting materials for good manufacturing practice (GMP)-grade ACT is also growing rapidly (4-5). Therefore, stem cell mobilization techniques that can control the types of immune cells and improve the yield of apheresis are important.

[0114] Enhanced stem cell mobilization (SCM) or cell mobilization methods as disclosed herein can further supplement or facilitate conventional apheresis procedures. In specific embodiments, enhanced SCM or cell mobilization is particularly beneficial for apheresis techniques for leukapheresis. In some embodiments, administering a CXCR4 antagonist to a subject further enhances apheresis by enhancing SCM or cell mobilization. In some embodiments, administering a β-adrenergic receptor antagonist in combination with a CXCR4 antagonist to a subject enhances SCM or cell mobilization and / or further enhances apheresis by replacing the granulocyte colony-stimulating factor (G-CSF) component of the treatment regimen with a non-selective β-blocker such as propranolol. In some embodiments, the enhancement of SCM then serves for the production of CAR-T cells for hematopoietic stem cell transplantation (HSCT) or cancer immunotherapy. Currently, CXCR4 inhibitors such as plerixafor (Mozobil), which are approved as stem cell mobilizing agents, are used with G-CSF as a standard treatment to provide concentrated hematopoietic stem and progenitor cells from healthy donors sold as the product "mobilized leukopak".

[0115] Disclosed herein is a method for enhancing apheresis in a subject, the method comprising blocking CXCR4 signaling and β - adrenergic receptor signaling in the subject. Also disclosed herein is a method for enhancing apheresis by inducing cell mobilization in a subject, the method comprising blocking CXCR4 signaling and β - adrenergic receptor signaling in the subject. Further disclosed herein is a method for enhancing apheresis by mobilizing cells in a subject, the method comprising blocking CXCR4 signaling and β - adrenergic receptor signaling in the subject. In an embodiment, blocking the β - adrenergic receptor signaling is performed before blocking the CXCR4 signaling. In some embodiments, blocking the β - adrenergic receptor signaling is performed at a first specific time interval before blocking the CXCR4 signaling. In some embodiments, the first specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more. In an embodiment, blocking the β - adrenergic receptor signaling continues after blocking the CXCR4 signaling has ended. In some embodiments, blocking the β - adrenergic receptor signaling continues for a second specific time interval after blocking the CXCR4 signaling has ended.In some embodiments, the second specific time interval is 5 to 10 minutes, 10 to 20 minutes, 20 to 30 minutes, 30 to 40 minutes, 40 to 50 minutes, 50 minutes to 1 hour, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, 5 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 3 days, 3 to 4 days, 4 to 5 days, 5 to 6 days, 6 to 7 days, 7 to 8 days, 8 to 9 days, 9 to 10 days, 10 to 11 days, 11 to 12 days, 12 to 13 days, 13 to 14 days, or 14 days or more.

[0116] In an embodiment, blocking the CXCR4 signaling includes administering a CXCR4 inhibitor to the subject.

[0117] Disclosed herein is a method for enhancing apheresis in a subject, the method comprising administering a β - adrenergic receptor inhibitor and a CXCR4 inhibitor to the subject. Also disclosed herein is a method for enhancing apheresis by inducing cell mobilization in a subject, the method comprising administering a β - adrenergic receptor inhibitor and a CXCR4 inhibitor to the subject. Further disclosed herein is a method for enhancing apheresis by mobilizing cells in a subject, the method comprising administering a β - adrenergic receptor inhibitor and a CXCR4 inhibitor to the subject. In some embodiments, administering the β - adrenergic receptor inhibitor is performed at a first specific time interval prior to administering the CXCR4 inhibitor. In some embodiments, the first specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more. In an embodiment, administering the β - adrenergic receptor inhibitor continues even after administering the CXCR4 inhibitor has ended. In some embodiments, administering the β - adrenergic receptor inhibitor continues for a second specific time interval after terminating administering the CXCR4 inhibitor. In some embodiments, the second specific time interval is 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 11 days, 11 days to 12 days, 12 days to 13 days, 13 days to 14 days, or 14 days or more.

[0118] In an embodiment, the β-adrenergic receptor inhibitor is an ADRB2 inhibitor. In an embodiment, the β-adrenergic receptor inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol. In an embodiment, the β-adrenergic receptor inhibitor is selected from the group consisting of propranolol, nadolol, and ICI 118551. In an embodiment, the β-adrenergic receptor inhibitor is propranolol.

[0119] In an embodiment, the CXCR4 inhibitor is selected from the group consisting of ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI 2341, BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, CX549, D-[Lys3] GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012, TG-0054 (plerixafor), USL311, viral macrophage inflammatory protein-II (vMIP-II), WZ811, [64Cu]-AMD3100, [64Cu]-AMD3465, [68Ga]pentixafor, [90Y]pentixather, [99mTc]O2-AMD3100, [177Lu]pentixather, and 508MCl (Compound 26). In an embodiment, the CXCR4 inhibitor is selected from the group consisting of AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (plerixafor), PF-06747143, POL6326, and urocuplumab (MDX1338 / BMS-936564). In an embodiment, the CXCR4 inhibitor is TG-0054 (plerixafor). In an embodiment, the CXCR4 inhibitor is AMD3100 (plerixafor). In an embodiment, the CXCR4 inhibitor is urocuplumab (MDX1338 / BMS-936564).

[0120] In an embodiment, administering the CXCR4 inhibitor to the subject comprises administering TG-0054 (bryxalofol) and propranolol. In an embodiment, administering the CXCR4 inhibitor to the subject comprises administering AMD3100 (plerixafor) and propranolol. In an embodiment, administering the CXCR4 inhibitor to the subject comprises administering ulocuplumab (MDX1338 / BMS-936564) and propranolol.

[0121] In an embodiment, the method further comprises administering G-CSF to the subject. In an embodiment, administering the β-adrenergic receptor inhibitor and the CXCR4 inhibitor to the subject is performed in the absence of G-CSF. Disclosed herein is a method of enhancing apheresis in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor. Further disclosed herein is a method of enhancing apheresis by inducing cell mobilization in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor. Also disclosed herein is a method of enhancing apheresis by mobilizing cells in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor. In an embodiment, administering a combination of the CXCR4 inhibitor and G-CSF induces an improved amount of apheresis compared to the amount of apheresis induced by the CXCR4 inhibitor alone. In an embodiment, administering a combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor induces an improved amount of apheresis compared to the amount of apheresis induced by the CXCR4 inhibitor alone. In an embodiment, administering a combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor and G-CSF induces an improved amount of apheresis compared to the amount of apheresis induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone. In an embodiment, administering a combination of the TG-0054 (bryxalfor) and G-CSF induces an improved amount of apheresis compared to the amount of apheresis induced by the AMD3100 (plerixafor) and G-CSF.In some embodiments, the amount of cell mobilization improved as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 1.1-fold to 1.2-fold, 1.2-fold to 1.3-fold, 1.3-fold to 1.4-fold, 1.4-fold to 1.5-fold, 1.5-fold to 1.6-fold, 1.6-fold to 1.7-fold, 1.7-fold to 1.8-fold, 1.8-fold to 1.9-fold, 1.9-fold to 2-fold, 2-fold to 2.5-fold, 2.5-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 10-fold, or 10-fold or more. In some embodiments, the amount of cell mobilization improved as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone is a 5% to 10% increase, 10% to 20% increase, 20% to 30% increase, 30% to 40% increase, 40% to 50% increase, 50% to 60% increase, 60% to 70% increase, 70% to 80% increase, 80% to 90% increase, 90% to 100% increase, 100% to 120% increase, 120% to 140% increase, 140% to 160% increase, 160% to 180% increase, 180% to 200% increase, 200% to 250% increase, 250% to 300% increase, 300% to 400% increase, 400% to 500% increase, 500% to 750% increase, 750% to 1000% increase, or 1000% or more. In an embodiment, the improved amount of cell mobilization or apheresis is measured by a method selected from the group consisting of a complete blood count (CBC) analysis, flow cytometry, and a colony-forming unit (CFU) assay. In an embodiment, the improved amount of cell mobilization or apheresis is measured by flow cytometry. In an embodiment, the flow cytometry is performed on (Lin-Sca1+c-Kit+) LSK cells. In an embodiment, the improved amount of cell mobilization or apheresis is measured by a colony-forming unit (CFU) assay.

[0122] Details of further information regarding ADRB2, which is evaluated herein as forming a heteromer with CXCR4, are shown below.

[0123] The beta-2 adrenergic receptor (β2 adrenergic receptor), also known as ADRB2, is a transmembrane β-adrenergic receptor that interacts with the hormone and neurotransmitter epinephrine (ligand synonym, adrenaline) whose signaling through downstream L-type calcium channel interactions mediates physiological responses such as smooth muscle relaxation and bronchodilation (Gregorio et al., 2017). ADRB2 functions in the muscular system such as smooth muscle relaxation, motor nerve endings, glycogenolysis, etc., and in the cardiovascular system such as myocardial contraction and increased cardiac output. In a normal eye, β2 stimulation by salbutamol increases intraocular pressure via the net. In the digestive system, ADRB2 induces glycogenolysis and gluconeogenesis in the liver and insulin secretion from the pancreas (Fitzpatrick, 2004).

[0124] ADRB2 signaling in cardiomyocytes is regulated by its interaction with CXCR4 (LaRocca et al., 2010). Norepinephrine weakens the invasion of MDA-MB-231 breast cancer cells via CXCR4 expression and the corresponding ADRB2 (Wang et al., 2015a). ADRB2 is expressed in several cancers such as pancreatic cancer, prostate cancer (Braadland et al., 2014; Xu et al., 2017), kidney cancer, and breast cancer (Choy et al., 2016).

[0125] Another method for detecting heteromer formation includes: immunostaining (Bushlin et al., 2012; Decaillot et al., 2008); immunoelectron microscopy (Fernandez-Duenas et al., 2015); BRET (Pfleger and Eidne, 2006); time-resolved FRET assay (Fernandez-Duenas et al., 2015); in situ hybridization (He et al., 2011); FRET (Lohse et al., 2012); GPCR heteromer identification techniques using BRET, FRET, BiFC, bimolecular fluorescence complementation, enzyme fragmentation assay, and Tango Tango GPCR assay system (Thermo Fisher Scientific) (Mustafa, 2010); β-arrestin mobilization assay (GPCR-HIT, Dimerix Bioscience) (Mustafa and Pfleger, 2011); PRESTO-Tango system (Kroeze et al., 2015); regulated secretion / aggregation technology (ARIAD Pharmaceuticals) (Hansen et al., 2009); receptor selection and amplification technology (ACADIA Pharmaceuticals) (Hansen et al., 2009); DimerScreen (Cara Therapeutics) (Mustafa, 2010); dimer / interacting protein translocation assay (Patobios) (Mustafa, 2010); co-immunoprecipitation (Abd Alla et al., 2009); GPCR internalization assay using surface enzyme-linked immunosorbent assay (ELISA) (Decaillot et al., 2008) or flow cytometry (Law et al., 2005); whole cell phosphorylation assay (Pfeiffer et al., 2002); and proximity ligation assay (PLA) (Frederick et al., 2015), but not limited to these.

[0126] As another method for detecting changes in pharmacological properties, signal transduction properties, and / or transport properties in cells expressing both CXCR4 and GPCRx: radioligand binding assays (Bushlin et al., 2012; Pfeiffer et al., 2002); cell surface biotinylation and immunoblotting (He et al., 2011); immunostaining (Bushlin et al., 2012; Decaillot et al., 2008); immunoelectron microscopy (Fernandez-Duenas et al., 2015); [35S]GTPγS binding assays (Bushlin et al., 2012); calcium imaging or assays using dyes such as Fura 2-acetomethoxyester (Molecular Probes), Fluo-4 NW calcium dye (Thermo Fisher Scientific), or FLIPR5 dye (Molecular Devices); cAMP assays using radioimmunoassay kits (Amersham Biosciences); AlphaScreen (PerkinElmer Life Sciences); parameter cyclic AMP assays (R&D Systems); femto cAMP kits (Cisbio); cAMP direct immunoassay kits (Calbiochem) or GloSensor cAMP assays (Promega); GTPase assays (Pello et al., 2008); PKA activation (Stefan et al., 2007); ERK1 / 2 and / or Akt / PKB phosphorylation assays (Callen et al., 2012); Src and STAT3 phosphorylation assays (Rios et al., 2006); reporter assays such as cAMP response element (CRE); nuclear factor of activated T cells response element (NFAT-RE); serum response element (SRE); serum response factor response element (SRF-RE); and NF-κB-response element luciferase reporter assays; secreted alkaline phosphatase assays (Decaillot et al., 2011); measurement of inositol 1-phosphate production using TR-FRET or [3H]myo-inositol (Mustafa et al., 2012);RT-qPCR for measuring downstream target gene expression (Mustafa et al., 2012); and adenylyl cyclase activity (George et al., 2000); next-generation sequencing (NGS); and any other assay capable of detecting changes in receptor function as a result of receptor heterodimerization, including but not limited to these.

[0127] As used herein, the phrase "protein-protein interaction inhibitor", "PPI inhibitor", or variations thereof means any molecule capable of interfering with protein-protein interactions. Unlike enzyme-substrate interactions involving defined binding pockets that are specifically disclosed, protein-protein interactions are transient interactions or associations between proteins over a relatively large area and are often driven by electrostatic interactions, hydrophobic interactions, hydrogen bonds, and / or van der Waals forces. PPI inhibitors can include, but are not limited to, membrane-permeable peptides or lipids fused to peptide sequences that disrupt GPCR heteromer interfaces, such as transmembrane helices, intracellular loops, or C-terminal tails of GPCRx. A PPI inhibitor of the CXCR4-GPCRx heteromer can be, for example, a membrane-permeable peptide or cell-permeable peptide (CPP) conjugated to a peptide targeting the CXCR4-GPCRx heteromer interface(s), or a cell-permeable lipidated peptide targeting the CXCR4-GPCRx heteromer interface(s).

[0128] For example, as the membrane-permeable peptide or cell-permeable peptide: TAT 48-60 and TAT 49-57HIV-1 TAT peptides such as; penetratin such as pAntp(43-58); polyarginine (Rn such as R5-R12); Diatos peptide vector 1047 (DPV1047, Vectocell (registered trademark)); MPG (HIV gp41 fused to the nuclear localization signal (NLS) of SV40 large T antigen); Pep-1 (tryptophan-rich cluster fused to the NLS of SV40 large T antigen); pVEC peptide (vascular endothelial cadherin); p14 alternative reading frame (ARF) protein-based ARF(1-22); N-terminus of unprocessed bovine prion protein BPrPr (1-28); model amphipathic peptide (MAP); transporteran; azurin-derived p28 peptide; amphipathic □-sheet peptides such as VT5; proline-rich CPPs such as Bac 7 (Bac1-24); hydrophobic CPPs such as C105Y derived from □1-antitrypsin; PFVYLI derived from synthetic C105Y; Pep-7 peptide (CHL8 peptide phage clone); and modified hydrophobic CPPs such as stapled peptides and prenylated peptides (Guidotti et al., 2017; Kristensen et al., 2016). Membrane-permeable peptides or cell-permeable peptides can further include, for example, TAT-derived cell-permeable peptides, signal sequence-based (e.g., NLS) cell-permeable peptides, hydrophobic membrane translocating sequence (MTS) peptides, and arginine-rich molecular transporters. Examples of cell-permeable lapidated peptides include pepducins such as ICL1 / 2 / 3, C-tail short-chain palmitoylated peptides (Covic et al., 2002; O'Callaghan et al., 2012).

[0129] Peptides (plural possible) that target the CXCR4-GPCRx heteromeric interface can be, for example, the transmembrane domain of CXCR4, the transmembrane domain of GPCRx, the intracellular loop of CXCR4, the intracellular loop of GPCRx, the C-terminal domain of CXCR4, or the C-terminal domain of GPCRx, the extracellular loop of CXCR4, the extracellular loop of GPCRx, the N-terminal region of CXCR4, or the N-terminal region of GPCRx.

[0130] Modifications that do not substantially affect the activities of the various embodiments of the present invention are also provided within the scope of the definitions of the present invention provided herein. Accordingly, the following examples are intended to illustrate, but not limit, the invention disclosed herein.

Example

[0131] (Example) (Example 1. Combinatorial blockade of β2-adrenergic receptor and CXCR4 signaling in stem cell mobilization: Preclinical evidence) To identify novel CXCR4-GPCRx heteromers, recombinant adenoviruses encoding 143 GPCRs fused to the N-terminal fragment (VN) of the yellow fluorescent protein Venus and 147 GPCRs fused to the C-terminal fragment (VC) of Venu were generated as described by Song et al. (Song et al., 2014; SNU patent; Song's dissertation). CXCR4-GPCR heteromers in which the two complementary VN and VC fragments of Venus reconstitute a fluorescent signal only when the two fragments are in close proximity via an interaction between the two different proteins to which they are fused were identified using a bimolecular fluorescence complementation (BiFC) assay (Figure 1) (Hu et al., 2002).

[0132] In preclinical studies, the ability of propranolol, a non-selective β-adrenergic receptor blocker, to improve GPC100-induced stem cell mobilization was evaluated after 7 days of treatment in a mouse model. These effects were further evaluated by the addition of G-CSF to GPC100 and in comparison to current standard therapeutic treatments for stem cell mobilization such as G-CSF alone or in combination with AMD3100.

[0133] (Materials and Methods) (Compounds)

[0134] Propranolol (MedChem Express, Princeton, NJ) was administered intraperitoneally (IP) at 20 mg / kg once daily for 7 days. Recombinant murine G-CSF (Peprotech, Cranbury, NJ) was administered subcutaneously (SC) at 0.1 mg / kg / dose twice daily for 5 days. AMD3100 (MedChem Express, Princeton, NJ) was administered subcutaneously once at 5 mg / kg on day 7. GPC100 was administered intravenously (IV) once at 30 mg / kg on day 7. GPC100 was obtained from TaiGen Biotechnology (Taiwan) by GPCR Therapeutics. All compounds were reconstituted in PBS. Vehicle controls were given PBS intravenously, intraperitoneally, or subcutaneously depending on the drug combinations used in the study.

[0135] (Mice)

[0136] C57BL / 6 and BALB / c mice (female, 6 - 9 weeks old) were purchased from Jackson Laboratory and housed on a 12 - hour light - dark cycle with free access to food and water. All mice were housed in an animal facility accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) and the IACUC (Institutional Animal Care and Use Committee) of Crown Bioscience (San Diego, CA) or Explora Biolabs (San Carlos, CA).

[0137] (Experimental design)

[0138] For the pilot study, C57BL / 6 and BALB / c mice were administered GPC100 (30 mg / kg, IV) or vehicle (IV) as a single dose, and blood was collected 1 hour later. Another group of C57 / BL6 mice received a single dose of GPC100 (30 mg / kg, IV), and blood was collected at 30 minutes, 1 hour, and 2 hours after injection. The time point for sample collection after GPC100 was set at 2 hours based on the maximal WBC mobilization (Figure 1B). All subsequent tests were performed in C57 / BL6 female mice. This is because this mouse strain is more rigorously evaluated in stem cell mobilization tests.

[0139] To determine the effect of propranolol on GPC100-induced mobilization, mice were given vehicle (IP) or propranolol (20 mg / kg, IP) for 7 days. On day 7, GPC100 (30 mg / kg, IV) was co-administered (Table 3). To determine whether propranolol alone changes blood cell counts, it was administered for 7 days, followed by an intravenous vehicle injection on day 7. Mice received a 7-day treatment with propranolol or vehicle; GPC100 or vehicle was co-administered on day 7, and the effect of propranolol alone, GPC100 alone, or their combination on total blood cell counts in peripheral blood was determined (Table 3). (Table 3 Dosage regimen for treatment with the combination of propranolol and GPC100) [Table 3] (Table 4 Dosage regimen for standard treatment) [Table 4] (Table 5 Dosage regimen for the combination of propranolol and G-CSF + AMD3100) [Table 5]

[0140] In another experiment, mice received G-CSF (0.1 mg / kg, SC, BID) for 5 days (days 2 - 6) with or without propranolol. GPC100 (30 mg / kg, IV) was co-administered with propranolol or alone on day 7 (Table 5).

[0141] The effect of propranolol on GPC100-induced mobilization was compared to the current standard treatment for stem cell mobilization, i.e., G-CSF with or without AMD3100. In this study, G-CSF (0.1 mg / kg, SC, BID) was administered for 5 days, followed by a single injection of vehicle (SC) or AMD3100 (5 mg / kg, SC) the next day (Table 4).

[0142] Blood was collected by terminal cardiac puncture 2 hours after GPC100 administration and 1 hour after AMD3100 administration. Total blood cell counts were obtained using an Abaxis hematology analyzer (Abaxis, Union City, CA). Circulating white blood cell counts were used as an indicator of stem cell mobilization in all studies.

[0143] The effect of propranolol on GPC100-induced mobilization was evaluated in all six studies. In four studies, this effect was compared to the standard treatment regimen of the combination of G-CSF and AMD3100. In the last three studies, G-CSF was added to GPC100 with (3 doses) or without (2 doses) propranolol. The effects of GPC100, AMD3100, and G-CSF in combination with vehicle were evaluated in one study. Data points were not removed unless the sample showed clotting prior to CBC analysis.

[0144] Mice treated with vehicle had an average of 3.4 + / - 1.8 x 10 3 white blood cells per microliter of peripheral blood and 2.6 + / - 1.2 x 10 3 lymphocytes per cell. Although not shown in the graphs of the data, vehicle-treated mice were included as controls in all studies.

[0145] (Colony-Forming Unit (CFU) Assay)

[0146] In one of the six studies, mobilization of hematopoietic progenitor cells was evaluated by CFU assay in addition to white blood cell counts. Mice were dosed by Crown Bioscience (San Diego, CA) and blood in heparinized tubes was sent to Reach Bio Research (Seattle, WA) at room temperature overnight. Approximately 8 x 10 4Individual cells were incubated in methylcellulose-based medium supplemented with cytokines known to support erythroid and myeloid precursors. Cultures were incubated in a humidified incubator for approximately 7 days, and then colonies were scored by trained personnel.

[0147] (Statistical analysis)

[0148] Data analysis was performed using Prism (GraphPad), and all data were presented as mean values (mean ± SD). Data presented in one figure were obtained during the same experiment. Comparison of data across various dosing conditions was performed using repeated measures one-way ANOVA followed by Tukey's multiple comparison test. Differences between two groups were determined using the Mann-Whitney test. For all tests, P < 0.05 was considered statistically significant.

[0149] (Results)

[0150] GPC100 increased the circulating white blood cell count in mice.

[0151] Single intravenous administration of GPC100 (30 mg / kg) resulted in a rapid increase in circulating WBC, which reflects stem cell mobilization, in C57 / BL6 and Balb / c mice (Figure 1A). To determine the time course of GPC-100-induced mobilization, GPC-100 (30 mg / kg) was intravenously administered to naive C57 / Bl6 mice, and peripheral blood was collected at 0.5 hours, 1 hour, and 2 hours post-injection in various groups. A time-dependent increase in white blood cell count was observed, and sample collection at 2 hours post-injection was selected for subsequent tests (Figure 1B). Furthermore, C57 / BL6 mice were preferentially selected over Balb / c mice for future tests because hematopoietic stem cell mobilization has been rigorously evaluated in this mouse strain (Broxmeyer et al., 2005).

[0152] Administration of GPC100 resulted in significantly more peripheral blood WBCs when administered with or without G-CSF compared to AMD3100.

[0153] Single injection of GPC100 resulted in a greater increase in circulating WBCs compared to a single injection of AMD3100 after 7 days of vehicle treatment (Figure 2A). When both GPC100 and AMD3100 were combined with 5 days of G-CSF treatment, a further increase in white blood cell count was observed in all three tests. However, this increase was more pronounced with GPC100 compared to AMD3100. This supports further clinical evaluation of GPC100 as a potent stem cell mobilizer (Figure 2B).

[0154] Propranolol enhanced the GPC100-induced mobilization of WBCs into the peripheral blood.

[0155] Data from six tests showed that pretreatment with propranolol for 7 days significantly increased the GPC100-induced white blood cell count in the peripheral blood compared to 7 days of vehicle pretreatment (Figure 3). When propranolol was administered alone for 7 days without GPC100 on day 7, no change in blood cell count was observed (Table 6). These data indicate that propranolol may improve bone marrow cell engorgement, thereby enabling GPC100 to mobilize more cells. (Table 6: Seven-day treatment with propranolol alone does not change blood cell count)

Table 6

[0156] The increase in white blood cell count resulting from the combined treatment of propranolol and GPC100 is comparable to that by G-CSF.

[0157] Mice administered GPC100 after propranolol pretreatment mobilized white blood cells to a similar extent as mice administered G-CSF in 3 out of 6 mice (Figure 4). Since propranolol can be safely administered orally in patients, its administration will not cause the inconveniences and side effects associated with G-CSF. This justifies further preclinical studies comparing the two groups.

[0158] The standard treatment regimen of the combination of G-CSF and AMD3100 mobilizes significantly more WBCs than the combination of propranolol and GPC100, but not for lymphocytes.

[0159] Data collected across four trials showed significant variability in mobilization by the combination treatment of G-CSF and AMD3100 (Figures 5A and 5B). Mobilization induced by the standard treatment regimen was significantly greater for total white blood cell count than the combination treatment of GPC100 and propranolol (Figure 5A). However, comparison of lymphocyte counts showed that the standard treatment regimen was comparable to the combination treatment of GPC100 and propranolol (Figure 5B).

[0160] The addition of propranolol to GPC100 and G-CSF resulted in mobilization of more WBCs and hematopoietic progenitor cells compared to the standard treatment regimen.

[0161] The addition of G-CSF enhanced mobilization by GPC100 with or without propranolol pretreatment. In this experiment, the numbers of WBCs and colony-forming units (progenitor cells) in peripheral blood were compared in the same mice. The three-drug combination containing propranolol, G-CSF, and GPC100 was shown to cause the greatest mobilization for both WBCs (Figure 6A) and progenitor cells (Figure 6B) compared to the G-CSF combination treatment in the absence of propranolol. When similar tests were repeated, the three-drug combination was significantly better than the combination treatment of AMD3100 and G-CSF (Figure 7A).

[0162] Propranolol enhances the GPC100-induced mobilization of lymphocytes into the peripheral blood.

[0163] Data pooled from all four experiments showed that when combined with G-CSF, GPC100 mobilized more WBCs compared to AMD3100. The addition of propranolol to the combination treatment of GPC100 and G-CSF mobilized more WBCs, including lymphocytes, compared to the combination of G-CSF and AMD3100 (Figures 7A and 7B). The lymphocyte count was not affected by the addition of propranolol to the combination of G-CSF and GPC100 (Figure 7B). The distribution of the WBC fraction numbers showed that the addition of propranolol could increase lymphocyte transport into the peripheral blood (Figure 7C). The lymphocyte data in Figure 7C are shown as a percentage of the total white blood cell count (Figure 7C), where the lymphocyte count in the vehicle / vehicle group was 2.6+ / -1.2×103 cells / μL).

[0164] This provides compelling preclinical evidence for further investigation of the effect of propranolol on GPC100-induced lymphocyte mobilization.

[0165] (Explanation) In the tests described herein, propranolol was shown to increase GPC100-induced mobilization in the absence of G-CSF. However, the addition of G-CSF further enhanced the mobilizing effect of GPC100, with or without propranolol. Whether propranolol enhances mobilization by the combined treatment of GPC100 and G-CSF remains unclear. In patients with multiple myeloma, propranolol has been shown to inhibit molecular risk markers in hematopoietic stem cell transplantation, a phenomenon that is currently being investigated by evaluating changes in inflammatory cytokines after propranolol treatment in mice. The tests disclosed herein were performed in naive or non-tumor-bearing mice that may not have the stress response present in tumor-bearing mice. Future tests will investigate the combined blockade of β-adrenergic and CXCR4 signaling in tumor-bearing C57 / BL6 mice to measure stem cell mobilization.

[0166] An increase in lymphocyte mobilization has been observed in all experiments in the propranolol treatment group, which may have clinical relevance as described below. In clinical trials, a high T cell content was shown to be associated with rapid hematopoietic reconstitution, a decrease in relapse, and an increase in disease-free survival in patients receiving peripheral blood stem cell transplantation compared to patients receiving bone marrow transplantation (reference by Stem Cell Trialists’ Collaborative Group, J Clin Onc 2005). Similarly, in both non-human primates and cancer patients, a single injection of AMD3100 resulted in an improvement in the number of lymphocytes in peripheral blood, including effector T cells and regulatory T cells, which is associated with GVHD protective properties (reference by Kean et al., Blood 2014, reference by Greef et al., Blood 2014). A sufficient amount of T lymphocytes is important in the production process of CAR-T cells. Some CAR-T products that are currently under clinical investigation or commercially available rely on patient-derived T cells. Patient-derived T cells may be insufficient in number or may be affected by several pretreatment lines and / or actual disease-related treatments (e.g., advanced AML) (reference by Fesnak et al., Transfus Med Rev 2016). This suggests that lymphocyte mobilization is important for both allogeneic hematopoietic stem cell transplantation designed to reduce the risk of GVHD and strategies designed to mobilize both effector and regulatory lymphocyte populations for adoptive cell therapy.

[0167] Previous studies have documented CXCL12 / CXCR4-mediated lymphocyte homing in bone marrow, lymph nodes, high endothelial venules, small blood vessels, thymus, and gastrointestinal tract (Bunting et al., Immunol Cell Biol 2011). It has also been reported that β2-adrenergic receptor interacts with CXCR4 to promote lymphocyte engraftment in lymph nodes (Nakai et al., JEM 2014). Therefore, an increase in lymphocyte transport into the peripheral blood is expected after blocking both CXCR4 receptor signaling and β-adrenergic receptor signaling. Further investigation of the phenotypic profile of immune cells, including lymphocytes mobilized by treatment with the combination of propranolol and GPC100, is underway. Results from the studies disclosed herein will provide additional information regarding the types of lymphocyte subsets that can be recovered by treatment with the combination of GPC100 and propranolol and their importance in the development of therapies.

[0168] (Example 2. Pretreatment with propranolol, a β-adrenergic receptor antagonist, enhances stem cell mobilization by blxafol (GPC100), a CXCR4 antagonist) (Materials and methods and study design) The study subjects were C57 / BL6 female mice. Peripheral blood was collected by terminal cardiac puncture 2 hours after vehicle or GPC100 and 1 hour after AMD3100. Total blood cell counts were determined by a hematology analyzer. (Table 7) [Table 7]

[0169] (Results) It was observed that propranolol caused an increase in GPC100-induced mobilization. See Table 3. Propranolol alone did not change the blood cell count. This is the first study showing enhanced mobilization by propranolol pretreatment (Figures 8A - 8D). Furthermore, a significant increase in GPC100-induced mobilization after propranolol pretreatment was also observed in a total of three studies (Figure 9).

[0170] The propranolol-induced increase in mobilization was comparable to the current standard treatment in the preclinical model. See Tables 3, 5, and 8. It was observed that the increase in WBC mobilized by propranolol pretreatment was mainly due to lymphocytes, while the SOC regimen mainly mobilized neutrophils (Figures 10A - 10D and Figure 11). In both studies, large variations were observed in the SOC group, and SOC alone also resulted in a decrease in platelet count (Figures 12 and 13). (Table 8. It was shown that no changes in platelet count, RBC, or hemoglobin levels were observed in mice treated with GPC100 and / or propranolol compared to mice treated with standard treatment or vehicle by complete blood count analysis)

Table 8

[0171] In the determination of hematopoietic stem cell mobilization by flow cytometry in the dosing regimen, no significant difference from the standard treatment was observed. In mice, hematopoietic stem cells lack the lineage marker (Lin-) and express the Sca1 and cKit markers (LSK cell profile). Also, CXCR4 is expressed in hematopoietic stem cells. Data for LSK cells (Figure 14) and Lin-CXCR4+ cells (Figure 15) are shown.

[0172] In summary, it was observed that single intravenous administration of GPC100 / Blissafol caused an increase in rapidly circulating WBC, an indicator of stem cell mobilization. Furthermore, CBC analysis of three mobilization trials showed that pretreatment with propranolol for 7 days enhanced GPC100-induced mobilization. Mobilization with the combined pretreatment of propranolol and GPC100 was at a level comparable to that of the combined treatment of G-CSF and AMD3100. While the combination of G-CSF+AMD3100 was observed to mobilize more neutrophils, the combination of β-blocker+GPC100 was observed to mobilize more lymphocytes. Further determination of hematopoietic stem cell mobilization by flow analysis will provide additional findings.

[0173] (Example 3. Combined blockade of the CXCR4 and β-adrenergic receptor signaling pathways induces stem cell mobilization comparable to current standard therapy) The combined blockade of two signaling pathways was investigated with respect to their ability to drive the CXCR4 receptor and the β-adrenergic receptor. CXCR4 blockade will be determined by the administration of both Blissafol and Prelissafol. The effects of the combination of propranolol+Prelissafol and propranolol+G-CSF+Prelissafol were also tested.

[0174] (Example 4. Addition of propranolol / β-blocker improves stem cell mobilization by combined treatment with Blissafol (GPC100) and G-CSF - three-drug combination test design) A new group was added to this study to determine whether propranolol improves the response to the combination of G-CSF and GPC100. The dosing regimens are shown in Tables 9 and 10. GPC-100 and G-CSF resulted in a greater number of mobilized circulating WBCs and progenitor cells compared to AMD3100 and G-CSF. The three-drug combination resulted in the greatest number of mobilized WBCs and progenitor cells. G-CSF was administered at 0.1 mg / kg, SC, twice daily for 5 days; 12 hours after the final G-CSF injection, GPC100 was administered alone, at 30 mg / kg, IV. Samples were taken 2 hours after GPC100 administration (Table 9). Propranolol was administered once daily for 7 days; G-CSF was administered twice daily for 5 days starting on the second day; 12 hours after the final G-CSF injection, GPC100 was co-administered with propranolol; samples were taken 2 hours after GPC100 administration (Table 10). (Table 9. Title: Dosing Regimen for Combination Treatment of G-CSF and GPC100)

Table 9

Table 10

[0175] WBC Mobilization in the G-CSF Combination Test

[0176] The addition of propranolol to the combination of G-CSF and GPC100 caused the greatest mobilization of WBCs, and a significant increase in mobilization was observed compared to the SOC and the combination treatment of G-CSF and GPC100 (Figures 16 and 17A - 17C). Subsequently, changes in white blood cell subsets during treatment were tested. It was observed that GPC100 increased lymphocytes when combined with propranolol, while GPC100 increased neutrophils when combined with G-CSF (Figure 18). An increase in the colony-forming unit assay was observed only in the group using G-CSF (Figure 19B). Compared with AMD3100 and G-CSF, GPC-100 and G-CSF resulted in a greater number of mobilized circulating WBCs and progenitor cells (Figure 19A). The three-drug combination resulted in the greatest number of mobilized WBCs and progenitor cells (Figures 19A and 19B and Figures 20A and 20B).

[0177] In summary, the combination treatment of G-CSF with blinatumomab mobilized more WBCs and hematopoietic progenitor cells in the peripheral blood compared to the combination treatment with AMD3100. The addition of propranolol for 7 days to the combination treatment of G-CSF and GPC100 resulted in the greatest number of mobilized WBCs and mobilized hematopoietic progenitor cells in the colony formation assay.

[0178] (Example 5. Addition of propranolol / β-blocker improved stem cell mobilization by combination treatment with CXCR4 antagonists (e.g., blinatumomab, plerixafor) and G-CSF) The combined blockade of two signaling pathways was tested in combination with G-CSF for stem cell mobilization with respect to its ability to drive the CXCR4 receptor and the β-adrenergic receptor. CXCR4 blockade was determined by the administration of both blinatumomab and plerixafor. The combinations of propranolol + plerixafor and propranolol + G-CSF + plerixafor were tested.

[0179] (Example 6. In vivo pharmacology) A mouse study design was conducted to investigate the effects of CXCR4 and B2AR blockade on HSC mobilization. Bone marrow replenishes itself in response to cells leaving, so the number of cells in the bone marrow may not be counted as decreased at the time of sampling. In the study, attention was paid to mobilization into the peripheral blood (Figures 21 and Table 11). (Table 11. Combination tests using β-blockers and GPC100 for stem cell mobilization in mice at GPCR Therapeutics)

Table 11

[0180] The role of CXCR4 antagonists in stem cell mobilization. Binding of the chemokine CXCL12 to its receptor CXCR4 plays an important role in the homing and engraftment of HSCs in the bone marrow. In preclinical studies, single intravenous administration of the CXCR4 antagonist GPC100 has been shown to result in a rapid increase in circulating WBCs, an indicator of stem cell mobilization, in C57 / Bl6 and Balb / c mice. CXCR4 antagonists such as plerixafor (AMD3100) and blinatumomab (GPC100) are clinically approved in the United States and Europe for use in combination with G-CSF for hematopoietic stem cell mobilization and subsequent autologous stem cell transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. G-CSF regimens require repeated injections over several days and are associated with adverse side effects such as severe bone pain. Inadequate mobilization has also been reported in up to 40% of patients. Therefore, an alternative approach to improving hematopoietic stem cell mobilization with CXCR4 antagonists is needed.

[0181] There is a need to improve stem cell mobilization for several reasons, including the following. ASCT is increasingly being used to treat hematological malignancies. However, the success of ASCT in lymphoma and MM patients is often hampered by inadequate mobilization, and at least 15% of patients require >2×106 Unable to produce the target cell dose of CD34+ cells / kg (Olivieri et al., 2012). Also, newer therapies for MM patients approved in recent years may have an adverse effect on mobilization. For example, recent studies have shown that MM patients who received induction with daratumumab prior to ASCT showed more inadequate mobilization (Hulin et al., 2021). Also, the use of daratumumab is associated with an increased rate of febrile neutropenia, which leads to increased antibiotic use and prolonged hospitalization (Papaiakovou et al., 2021). This further increases the patient burden, another factor to be considered in the treatment of MM patients. MM patients have been found to have a higher symptom burden and worse health-related quality of life (HRQoL) compared to patients with other hematological malignancies (Johnsen et al., 2009).

[0182] GPC100-induced mobilization in mice (Figure 22). Single intravenous (IV) administration of GPC-100 (30 mg / kg), a potent and selective CXCR4 antagonist, resulted in a rapid increase in the circulating white blood cell count (WBC), which reflects stem cell mobilization, in C57 / Bl6 and Balb / c mice. To determine the time course of GPC-100-induced mobilization, GPC-100 (30 mg / kg) was administered intravenously to naive C57 / Bl6 mice, and peripheral blood was collected at 0.5 hours, 1 hour, and 2 hours after injection in various groups. A time-dependent increase in the white blood cell count was observed, and sample collection at 2 hours after injection was selected for subsequent tests. C57 / Bl6 mice were selected over Balb / c mice for future tests because HSC mobilization has been well-evaluated in this mouse strain (Broxmeyer et al., 2005).

[0183] GPC-100 (30 mg / kg, IV) alone was observed to induce time-dependent WBC mobilization (Figure 23). In future studies, blood sampling will be taken 2 hours after GPC administration. The planned time-course study is as follows: 0.5, 1, 2, 3, 4 hours after GPC100 administration. A dose-response for IV GPC100 administration will be conducted (dose to be determined (TBD)).

[0184] The rationale for enhancing CXCR4-induced mobilization using β-blockers. The bone marrow is highly innervated by the sympathetic nervous system. Traumatic stress in human and rodent models has been shown to be associated with persistently elevated levels of norepinephrine (a ligand for β-adrenergic receptors), which are associated with bone marrow dysfunction (Bible et al., 2014; Bible et al., 2015a; Bible et al., 2015b). Therefore, in future studies, the potential of β-blockers to improve GPC100-induced mobilization by restoring bone marrow function will be evaluated. In other studies shown elsewhere, 7-day intraperitoneal administration of the non-selective β-blocker propranolol (20 mg / kg) and nadolol (5 mg / kg) or the selective β2-receptor antagonist ICI-118,551 (5 mg / kg) alone did not affect total blood cell counts. This dose of propranolol will be selected for future studies where β-adrenergic blockade is involved.

[0185] (Example 7. Leukocyte mobilization induced by GPC-100 + / - β-blocker) The study was conducted with the dosing schedules shown in Tables 12 and 13. Propranolol, nadolol, or ICI-118,551 was administered once daily for 7 days. GPC100 was co-administered with propranolol on day 7 (Table 12). Propranolol, nadolol, or ICI-118,551 was administered once daily for 7 days; vehicle was co-administered intravenously with propranolol on day 7 (Table 13). (Table 12. Dosing schedule for combination treatment of β-blocker and GPC100)

Table 12

[0186] Leukocyte mobilization induced by GPC-100+ / -β-blocker. Administration of propranolol for 7 days enhanced GPC100-induced mobilization, but had no effect on blood cell counts when administered alone (Figs. 24A - 24C). It was observed that β-blockers administered for 3 days or simultaneously with GPC100 did not change blood cell counts (data not shown). Future studies will: (1) confirm that hematopoietic stem cells are mobilized; (2) mouse hematopoietic stem cells lack lineage markers (lin-) and express SCA1 and cKit. Therefore, the LSK cell profile determined by flow cytometry (Reach Bio); (3) be conducted to repeat the CBC. Studies were performed to determine stem cell mobilization. It was observed that nadolol enhanced GPC100-induced mobilization (Figs. 25A - 25C). Furthermore, 7-day administration of β-blocker with a single dose of GPC100 did not appear to increase LSK and Lin-CXCR4+ cells (Figs. 26A - 26C). In future studies, the experiments will be repeated and a standard treatment group will be added.

[0187] Tests were conducted and compared with G-CSF+AMD3100 (Table 14). Propranolol (20 mg / kg IP) was administered once daily for 7 days. On day 7, GPC100 (30 mg / kg IV) was co-administered with propranolol. Peripheral blood was collected by cardiac puncture 2 hours after injection. These results were compared with the current standard treatment for mobilization, i.e., combination treatment with G-CSF and AMD3100 (plerixafor). G-CSF (0.1 mg / kg SC) was administered twice daily for 5 days, followed by a single injection of AMD3100 (5 mg / kg SC) 12 hours later on day 6. Based on the literature report, peripheral blood was collected 1 hour after AMD3100 (Hoggatt et al., 2018). (Table 14. Dosage regimen based on the literature)

Table 14

[0188] Propranolol was observed to enhance GPC100-induced mobilization (Figures 27A - 27C). This effect was comparable to the standard treatment (G-CSF+AMD3100 / plerixafor). The combination of propranolol+GPC100 was observed to mobilize more lymphocytes. Also, the SOC was observed to mobilize more neutrophils (driven by G-CSF).

[0189] Tests were conducted using the observation that lymphocytes increased with GPC100 and β-blocker, while neutrophils increased with G-CSF+AMD3100 (Figure 28). The fold changes for combined tests 2 and 3 for LSK (Figure 30A) and Lin-CXCR4+ (Figure 30B) are shown in Figures 29A and 29B. For LSK flow, the combination of propranolol+GPC100 was comparable to the SOC. In future tests, this experiment will be repeated using a larger blood volume and additional G-CSF.

[0190] Experiments were conducted to test the addition of a three-drug combination using G-CSF (Table 15). The three-drug combination yielded the best results (Figures 31A - 31C and 32), and propranolol + GPC100 was comparable to SOC. (Table 15. Three-drug combination dosing schedule)

Table 15

[0191] The colony-forming unit assay was performed (Figures 33 and 34). The CFU assay is based on the ability of hematopoietic precursors to proliferate and differentiate into colonies in a semi-solid medium in response to cytokine stimulation. The number and type of colonies counted in the CFU assay provide information about the frequency and type of progenitor cells present in the original cell population and their ability to proliferate and differentiate. The three-drug combination mobilized the highest number of progenitor cells (Figures 35, 36A and 36B, and 37). Furthermore, the three-drug combination was associated with the greatest increase in circulating WBC and progenitor cells compared to other drug groups (Figures 38A and 38B). Additionally, GPC100 + G-CSF mobilized more WBC and progenitors compared to AMD3100 + G-CSF. No difference was observed between the vehicle group and GPC100 + / - propranolol. The CFU assay was designed for myeloid progenitors only and not for lymphoid progenitors (Figures 39A and 39B). G-CSF was found to mobilize myeloid progenitors, and the assay was found to be G-CSF-dependent. Data on the effect of propranolol on GPC100-induced mobilization from three tests (Tests 1, 3, 4) are shown in Figures 40A and 40B. Propranolol was found to enhance GPC100-induced mobilization in the three tests.

[0192] The effect of propranolol on GPC100-induced mobilization was tested compared to standard therapy. Propranolol enhanced GPC100-induced mobilization to a similar extent as SOC (Figures 41A and 41B). It was observed that propranolol + GPC100 mobilized more lymphocytes compared to SOC. The effect of propranolol on GPC100- and AMD3100-induced mobilization was tested with and without combination with G-CSF (Tables 16, 17). (Table 16)

Table 16

Table 17

[0193] GPC100-, AMD3100-, or G-CSF-induced WBC mobilization (single agent) was tested (Figures 42A - 42C). Maximum mobilization was observed with G-CSF, while GPC100 mobilized more lymphocytes than AMD3100 or G-CSF. Furthermore, GPC100 mobilized more WBC than AMD3100, and G-CSF mobilized more neutrophils than GPC100 or AMD3100.

[0194] The effect of propranolol on GPC100-induced mobilization was tested in WBC compared to standard therapy in the absence or presence of G-CSF. Data from Test 4 are shown in Figure 43A, and data from Test 5 are shown in Figure 43B. The effect of the triple combination was not observed in Test 5, and the effect of SOC was considerably higher than previously observed.

[0195] The effect of propranolol on GPC100-induced mobilization was tested in lymphocytes compared to standard therapy in the absence or presence of G-CSF. Data from Test 4 are shown in Figure 44A, and data from Test 5 are shown in Figure 44B. The effect of the triple combination was not observed in Test 5, and the effect of SOC was considerably higher than previously observed.

[0196] The effect of propranolol on GPC100-induced mobilization was tested in neutrophils compared to standard therapy in the absence or presence of G-CSF. Data from Study 4 are shown in Figure 45A, and data from Study 5 are shown in Figure 45B. The effect of the triple combination was not observed in Study 5, and the effect of SOC was considerably higher than previously observed.

[0197] A comparative study was conducted between GPC100 and AMD3100 (Figures 46A - 46C). This was the first study to show the effect of the triple combination of propranolol and AMD3100. It was observed that propranolol slightly increased AMD3100-induced mobilization of lymphocytes.

[0198] The effect of propranolol on GPC100-induced mobilization with or without G-CSF was tested and compared to standard therapy (Figures 47A - 47C). Propranolol significantly enhanced GPC100-induced mobilization. When combined with G-CSF, GPC100 mobilized more WBCs compared to AMD3100. The combination of propranolol and GPC100 resulted in an increase in circulating lymphocytes at a level similar to standard therapy (G-CSF + AMD3100).

[0199] Composite data from all 6 studies are shown in Figures 48A and 48B. Propranolol treatment for 7 days prior to GPC100 resulted in significantly improved WBC and lymphocyte cell counts in peripheral blood compared to GPC100 alone.

[0200] Data from 4 studies with the addition of a standard treatment group are shown in Figures 49A and 49B. It was observed that the standard treatment regimen mobilized more WBCs compared to the combination of propranolol and GPC100. However, there was no difference in lymphocyte mobilization. Also, the standard treatment group showed a large variability reflecting the response of mobilized patients in the clinic.

[0201] Data from three trials with the addition of the G-CSF combination group are shown in FIGS. 50A and 50B. It was observed that the addition of propranolol to the combination of G-CSF and GPC100 mobilized significantly more WBCs and lymphocytes compared to standard treatment. When combined with G-CSF, GPC100 was observed to mobilize significantly more WBCs compared to AMD3100. However, when propranolol was added, there was significantly more lymphocyte mobilization.

[0202] (Example 8. Effect of Propranolol on GPC100-Induced Mobilization) (Role of CXCR4 Antagonists in Stem Cell Mobilization)

[0203] The binding of the chemokine CXCL12 to its receptor CXCR4 plays an important role in the homing and engraftment of HSCs in the bone marrow. In preclinical studies, it has been shown that single intravenous administration of the CXCR4 antagonist GPC100 resulted in a rapid increase in circulating WBCs, an indicator of stem cell mobilization, in C57 / Bl6 and Balb / c mice. CXCR4 antagonists such as plerixafor (AMD3100) and blinatumomab (GPC100) are clinically approved in the United States and Europe for use in combination with G-CSF for hematopoietic stem cell mobilization and subsequent autologous stem cell transplantation in patients with non-Hodgkin lymphoma and multiple myeloma. G-CSF regimens require repeated injections over several days and are associated with adverse side effects such as severe bone pain. Inadequate mobilization has also been reported in up to 40% of patients.

[0204] (Lymphocyte Mobilization)

[0205] In patients receiving peripheral blood stem cell transplantation as compared to those receiving bone marrow transplantation, high T cell content has been associated with rapid hematopoietic reconstitution, decreased relapse, and increased disease-free survival, highlighting the importance of lymphocyte mobilization (Stem Cell Trialists’ Collaborative Group, J Clin Onc 2005). In studies in non-human primates, single injection of the CXCR4 antagonist AMD3100 has been shown to result in improved lymphocyte numbers in peripheral blood, including effector T cells and Tregs and Tem associated with GVHD protective properties (Kean et al., Blood 2014). Similarly, allogeneic stem cell grafts recovered from healthy donors after single administration of AMD3100 contained higher numbers of both effector and regulatory T cells compared to grafts recovered after G-CSF (Greef et al., Blood 2014). This is important for both alloHSCT and adoptive cell therapy strategies designed to mobilize both effector and regulatory lymphocyte populations. Previous studies have documented CXCL12 / CXCR4-mediated lymphocyte homing in bone marrow, lymph nodes, high endothelial venules, small blood vessels, thymus, and gastrointestinal tract (Bunting et al., Immunol Cell Biol 2011).

[0206] (Lymphocyte Mobilization for CAT-T Therapy)

[0207] Efficient leukapheresis to provide sufficient amounts of T lymphocytes is an important step in the production process of CAR-T cells. Some currently investigated CAR-T cell products are based on allogeneic T cells from healthy donors, and some clinically investigated or commercially available CAR-T products rely on the patient's own T cells. Patient-derived T cells may be few in number or may be hampered by several lines of pretreatment and actual disease-related treatments (e.g., advanced AML) (Fesnak et al., Transfus Med Rev 2016).

[0208] (Lymphocyte mobilization and β-blockade)

[0209] Stem cells in leukapheresis products pose a risk of malignant transformation during the process of gene modification by viral transduction, which indicates the risk that can be caused by stem cell mobilization with G-CSF. It has been reported that the β2-adrenergic receptor interacts with CXCR4 to promote the homing of lymphocytes in lymph nodes (Nakai et al., JEM, 2014). Therefore, in this study, the effect of combined blockade of β-adrenergic receptor signaling and CXCR4 signaling, which increases the transport of lymphocytes to peripheral blood, was determined.

[0210] Study design. Effect of propranolol on GPC100-induced mobilization (Table 18). C57 / BL6 mice were given the non-selective β-blocker propranolol (20 mg / kg, IP) once a day for 7 days. On the 7th day, GPC100 (30 mg / kg, IV) was co-administered. Based on preliminary data showing that the maximum mobilization occurs 2 hours after a single intravenous administration of GPC100, blood was collected 2 hours after drug administration. (Table 18. Dosage schedule) [Table 18]

[0211] Composite data from all 6 tests are shown in Figures 51A and 51B. It was observed that pretreatment with propranolol for 7 days before GPC100 resulted in significantly improved WBC and lymphocyte cell counts in peripheral blood compared to GPC100 alone.

[0212] Test design. The effect of propranolol on GPC100-induced mobilization was tested compared to the standard treatment (G-CSF + AMD3100) for stem cell mobilization (Table 19). Propranolol (20 mg / kg IP) was administered once a day for 7 days. On the 7th day, GPC100 (30 mg / kg IV) was co-administered with propranolol. Peripheral blood was collected by cardiac puncture 2 hours after injection. The results were compared with the current standard treatment for mobilization, i.e., the combined treatment with G-CSF and AMD3100 (plerixafor). G-CSF (0.1 mg / kg SC) was administered twice a day for 5 days, followed by a single injection of AMD3100 (5 mg / kg SC) on the 6th day 12 hours later. Based on the literature report (Hoggatt et al., 2018), peripheral blood was collected 1 hour after AMD3100. (Table 19. Dosage regimen based on the literature)

Table 19

[0213] Data from four additional tests with a standard treatment group are shown in Figures 52A and 52B. It was observed that the standard treatment regimen mobilized more WBCs compared to the combination of propranolol and GPC100. However, there was no difference in lymphocyte mobilization. Lymphocyte mobilization by treatment with the combination of propranolol and GPC100 was comparable to the combined treatment with G-CSF and AMD3100, suggesting the possibility of omitting G-CSF to obtain lymphocytes in peripheral blood.

[0214] Test design. The effect of propranolol on GPC100-induced mobilization with or without G-CSF was tested (Table 20). (Table 20. Three-drug combination dosing schedule)

Table 20

[0215] Data from three trials with the addition of the G-CSF combination group are shown in FIGS. 53A and 53B. It was observed that the addition of propranolol to the combination of G-CSF and GPC100 mobilized significantly more WBCs and lymphocytes compared to the standard treatment for stem cell mobilization. When combined with G-CSF, GPC100 was observed to mobilize significantly more WBCs compared to AMD3100. However, when propranolol was added, there was significantly more lymphocyte mobilization.

[0216] The distribution of the WBC fraction is shown in FIG. 54. It was observed that G-CSF mainly mobilizes neutrophils. Furthermore, the addition of propranolol to G-CSF slightly decreased neutrophil mobilization, while the addition of propranolol to GPC100 slightly increased the number of lymphocytes in circulation.

[0217] (Example 9. Comparison of mobilization between GPC100 and AMD3100) GPC100, AMD3100, or G-CSF-induced WBC mobilization was tested (FIGS. 55A - 55C). The greatest mobilization was observed with G-CSF. While GPC100 mobilized more lymphocytes than AMD3100 or G-CSF, GPC100 mobilized more WBCs than AMD3100, and G-CSF mobilized more neutrophils than GPC100 or AMD3100.

[0218] In previous experiments, a comparison was made between GPC100 and AMD3100 (FIGS. 56A - 56C). It was the first test showing the effect of a three-drug combination using propranolol and AMD3100. It was observed that propranolol slightly increased the AMD3100-induced mobilization of lymphocytes.

[0219] The effect of propranolol on GPC100-induced mobilization, with or without G-CSF, was tested and compared to standard therapy (Figures 57A - 57C). When combined with G-CSF, GPC100 was observed to mobilize more WBCs compared to AMD3100. The combination of propranolol and GPC100 resulted in an increase in circulating lymphocytes at levels similar to standard therapy (G-CSF + AMD3100).

[0220] Data from three additional studies with a G-CSF combination group are shown in Figures 58A and 58B. When combined with G-CSF, GPC100 was observed to mobilize significantly more WBCs compared to AMD3100. However, when propranolol was added, there was significantly more lymphocyte mobilization.

[0221] (Example 10. GPC100 and Propranolol as a Cell Mobilization Therapy for Autologous Stem Cell Transplantation (ASCT)) The success of autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM) is often hampered by inadequate mobilization, and approximately 1 in 7 patients are unable to reach an adequate number of CD34+ cells / kg. Small molecule inhibitors of CXCR4, such as GPC100 and plerixafor, interfere with the CXCL12 / CXCR4 axis, which is important for the migration and engraftment of hematopoietic stem cells (HSCs) in the bone marrow. Here, the inventors provide evidence that propranolol (Pro), a β2-adrenergic receptor (B2AR) blocker (BB), and GPC100 in combination with G-CSF have the potential to be a top-class mobilization therapy for ASCT.

[0222] The in vitro activity of GPC100 was investigated in cell-based assays (Figures 59A and 59B). In a FRET ligand binding assay in HEK cells, GPC100 potently inhibited the binding of CXCL12 to CXCR4 with a binding affinity approximately 30-fold better than AMD3100 (Ki of 1.6 vs 40 nM, respectively). In a cell migration assay using the multiple myeloma cell line MM1.S, in which GPC100 inhibited CXCL12-mediated migration with an IC50 of 30 nM, potent inhibition of CXCR4 was reproduced as compared to an IC50 of 80 nM for AMD3100.

[0223] Previous studies have shown that stress hormones such as epinephrine and norepinephrine exert a stimulatory effect on cancer progression by regulating tumor formation, growth, and metastasis via B2AR signaling. In a recent study of 208 MM patients, the overall survival was significantly longer in 37% of patients who reported using BB for ≥ 3 months after diagnosis compared to patients who did not use BB (107 months vs 86 months, Hwa et al., Eur J Haematol 2021). Furthermore, it has been demonstrated that BBs such as Pro can change bone marrow-derived cells to differentiate into a phenotype consistent with CD34+ stem cells and stem cell-related genes away from the bias towards myeloid lineage (Knight et al., Blood Adv 2020).

[0224] To investigate the interaction between CXCR4 and B2AR blockade in vitro, the inventors conducted interaction tests and functional tests (Figs. 59C to 59F). Using proximity ligation assay (PLA) in breast cancer cell line MDA-MB-231 that endogenously expresses CXCR4 and B2AR, the inventors detected CXCR4 and B2AR heteromers, while knockout of B2AR expression led to a reduction in PLA signals, confirming the proximity of CXCR4 and B2AR. In addition, the inventors demonstrated the functional significance of CXCR4 and B2AR using a Ca2+ flux assay in MDA-MB-231 cells in which synergistic effects were shown when co-stimulated with CXCL12 and salmeterol, a B2AR agonist. Inhibition of Ca2+ flux by GPC100 but not by AMD3100 was enhanced approximately 30-fold by co-treatment with Pro (1.3 vs. 30 nM). Collectively, the inventors' in vitro results suggested that GPC100 inhibition of CXCR4 can be regulated by Pro.

[0225] To obtain preclinical proof-of-concept, the inventors determined the mobilization of white blood cells (WBC) by complete blood count (CBC) analysis, progenitor cells by colony-forming unit (CFU) assay, and hematopoietic stem cells (HSC) by flow cytometry in C57 / BL6 mice after GPC100 combination treatment. First, administration of GPC100 alone resulted in more WBC mobilization into peripheral blood compared to AMD3100 alone (Figure 60A). Next, mice were treated with Pro for 7 days, followed by a single administration of GPC100 or AMD3100 on day 7 (Figure 60B). The inventors' data demonstrated that combination treatment of GPC100 and Pro mobilized more WBC compared to the combination of AMD3100 and Pro. Finally, the inventors determined whether the three-drug combination of G-CSF+GPC100+Pro was more beneficial than the current standard treatment for autologous stem cell transplantation (ASCT): G-CSF alone or G-CSF combined with AMD3100 (Figure 60C). The inventors demonstrated that the three-drug combination resulted in the highest WBC mobilization. In addition, the inventors confirmed the correlation between WBC count and CFU as an indicator of progenitor cells (Figure 60D), and the mobilized Lin- / sca-1+ / c-Kit+ (LSK) population as an indicator of mouse HSC (Figure 60E), indicating that WBC mobilization is a predictor of progenitor cell and stem cell mobilization.

[0226] The inventors' findings support the use of GPC100 and Pro with or without G-CSF for stem cell mobilization. This treatment strategy makes it possible to eliminate daily repeated injections of G-CSF, improve the quality of life of patients, and provide treatment options for patients who experience adverse effects with G-CSF. Furthermore, treatment with G-CSF, GPC100, and Pro will prove to be the best-in-class mobilization therapy for ASCT in MM patients, particularly those MM patients who have failed to mobilize with standard treatment.

[0227] All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0228] Preferred embodiments have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The following claims are intended to define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are intended to be included therein.

[0229] (Example 11. GPC100 induces WBC and stem cell mobilization in mice) Multiple myeloma (MM) is a major hematologic malignancy with an estimated 34,920 cases per year in the United States and approximately 588,161 cases worldwide (Cowan et al., 2022). Autologous stem cell transplantation (ASCT) is associated with the comprehensive management of eligible MM patients and has improved anti-cancer response and survival compared to conventional chemotherapy (Devarakonda et al., 2021; Holsteain and McCarthy, 2016; Li and Zhu, 2019; Kumar et al., 2008). Part of the success of ASCT depends on harvesting a sufficient number of hematopoietic stem cells (HSCs), which is mainly achieved by mobilizing HSCs from the bone marrow (BM) into the peripheral blood (PB) (Arora, Majhail, and Liu, 2019; Balassa, Danby, and dRocha, 2019). HSCs are phenotypically characterized by the expression of CD34. A minimum of about 2×10 6 CD34+ cells / kg are used for HSC collection, but numbers suitable for improved engraftment and survival are >5 - 6×10 6It is CD34+ cells / kg (Toor et al., 2004; Tricot et al., 1995). Granulocyte colony-stimulating factor (G-CSF) is the standard treatment in clinical practice for HSC mobilization (DiPersio et al., 2009). However, G-CSF cannot mobilize an optimal number of HSCs in at least 40-50% of MM patients (DiPersio et al., 2009; Demirer et al., 196). Some patients have received treatment with G-CSF in combination with plerixafor (AMD3100), a small molecule CXCR4 antagonist (DiPersio et al., 2009). Even with this combination treatment, 15-35% of MM patients do not mobilize a sufficient number of cells (DiPersio et al., 2009). In a recent phase 3 clinical trial, the combination of G-CSF and motixafortide, a peptide inhibitor of CXCR4, mobilized significantly more CD34+ cells compared to G-CSF + placebo (Crees et al., 2023). Although this is promising, as data has accumulated, it has been suggested that MM therapies such as daratumumab or lenalidomide may have a negative impact on HSC mobilization (Hulin et al., 2021; Popat et al., 2021). Furthermore, G-CSF is contraindicated for stem cell collection in conditions such as sickle cell disease (Fitzhugh et al., 2009). These factors highlight the unresolved issues of optimal HSC mobilization in MM patients and the unresolved issue of expanding ASCT to the entire spectrum of other eligible diseases (Pusic et al., 2008; Giralt et al., 2014).

[0230] CXCR4 is a member of the chemokine G protein-coupled receptor (GPCR) family and is expressed on HSCs (Wu et al., 2010; Mezzapelle et al., 2022; Guo et al., 2016). CXCR4 signaling mediated by its natural ligand CXCL12 plays a role in the cell chemotaxis, engraftment, and survival of HSCs in the BM (Guo et al., 2016). GPC-100, also known as Blinatumomab or TG-0054, is a novel small molecule antagonist of CXCR4 with high binding affinity for CXCR4. GPC-100 has been clinically tested in MM patients as an HSC mobilizer in combination with G-CSF (NCT02104427) (Schuster et al., 2021), and it has been shown to induce an increase in HSCs of >5.0×10 6 CD34+ cells / kg in 1-2 leukapheresis sessions (Setia et al., 2015). This result was comparable to the background results with G-CSF + AMD3100 treatment.

[0231] Previous studies have suggested that CXCR4 physically interacts with the β2 - adrenergic receptor or β2AR (gene ADRB2) in cells that ectopically overexpress both the β2 - adrenergic receptor and β2AR (gene ADRB2) (Nakai et al., 2014; LaRocca et al., 2010; Nakai, Leach, and Suzuki, 2021). In lymph nodes, the CXCR4 - β2AR complex was thought to enhance lymphocyte homing by CXCR4 and inhibit their mobilization (Nakai et al., 2014). Also, β2AR is expressed on HSCs, and adrenergic signaling plays a role in the regulation of the HSC niche in the BM (Spiegel et al., 2008; Saba et al., 2015; Maestroni, 2020; Katayama et al., 2006). Epinephrine and norepinephrine, the natural ligands of β2AR, affect turnover and transport and have been shown to reduce the proliferation and differentiation capacity of HSCs (Hanoun et al., 2015; Schraml et al., 2009). When human HSCs were co - stimulated with G - CSF and a β2AR agonist, the expression of CXCR4 on HSCs increased, suggesting that the interaction between the β2AR agonist and G - CSF in the BM niche promotes HSC homing by CXCR4 and impairs mobilization by G - CSF (Saba et al., 2015).

[0232] Studies have shown an association between the use of β - adrenergic inhibitors (β - blockers) and positive survival outcomes in several cancer types, including MM (Hwa et al., 2017; Hwa et al., 2021). The MM niche is known to cause dysregulation of HSC function leading to changes in gene expression and blood cell differentiation (Bruns et al., 2012; Knight et al., 2020). In a phase II biomarker - driven randomized trial, in MM patients, propranolol, an FDA - approved non - selective β - blocker, was shown to change cell differentiation from a bias towards myeloid lineage to up - regulation of CD34+ cells and enhance engraftment (Knight et al., 2020). Furthermore, propranolol demonstrated the ability to inhibit the BM sympathetic nervous system - induced shift to a pattern of high inflammatory gene expression, named Conserved Transcriptional Response to Adversity (CTRA), which is associated with poor outcomes in ASCT from the basal gene expression profile (Knight et al., 2020). In another study, BM samples from MM patients showed that propranolol could enhance the differentiation of HSCs into megakaryocyte - erythroid precursors and decrease the number of granulocyte - monocyte progenitor cells, which are known to contribute to the tumor - promoting niche (Nair et al., 2022). Therefore, considering the positive effects of propranolol on HSC proliferation and differentiation and the potential crosstalk between β2AR and CXCR4 in the BM, co - inhibition of these two pathways may improve HSC mobilization.

[0233] In this study, the efficacy of in vivo mobilization of GPC - 100 compared to AMD3100 is reported. Furthermore, this report demonstrates the enhancement of in vivo mobilization by GPC - 100 in combination with propranolol, proposing a new direction for clinical application in stem cell mobilization.

[0234] (Method) In vivo mobilization: C57BL / 6J or Balb / c mice (female, 6 - 9 weeks old) were randomized to each study such that all treatment groups had equivalent age and body weight distributions. The studies were conducted in facilities accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care and the Institutional Animal Care and Use Committee. PB was collected by cardiac puncture 2 hours after GPC - 100 administration on day 7 and 1 hour after AMD3100 administration. Blood samples were processed for complete blood count (CBC) analysis using an Abaxis VetScan HM5 hematology analyzer. (Table 21: Dosage for in vivo mobilization)

Table 21

[0235] Colony - forming unit (CFU) assay: 8×10 5 individual mononuclear cells isolated from PB after CBC analysis were added to tubes of semi - solid methylcellulose medium (StemCell Technologies) known to support erythroid and myeloid precursors (Kronstein - Wiedemann, 2019). After 7 days, colonies showing the appearance of granulocyte - macrophage precursors (CFU - GM) and burst - forming erythroid units (BFU - E) formed and were counted by blinded experimenters. Total CFU was calculated as the sum of CFU - GM and BFU - U colonies.

[0236] Flow cytometry: To determine the mobilization of mouse HSCs characterized as Lineage - Sca - 1+c - Kit+ (LSK cells) (Challen et al., 2009), mononuclear cells isolated from PB after CBC analysis were stained with anti - lineage cocktail, c - Kit, and Sca - 1 antibodies. Samples were acquired on a Cytek Aurora spectral flow cytometer (Fremont, CA) and data were analyzed using CellEngine software. Gating was determined using FMO controls. c - Kit + Sca - 1 +The total number of μL of blood LSK cells was determined using the percentage of cells. (Table 22: Antibodies used in flow cytometry)

Table 22

[0237] Statistical analysis: Data analysis was performed using GraphPad Prism, and all data were presented as mean ± SEM. Comparison of data across dosing conditions was performed using the Mann–Whitney test or one-way ANOVA. For all tests, P < 0.05 was considered statistically significant.

[0238] Experimental design: First, mobilization of WBC and LSK stem cells by GPC-100 was determined after a single IV administration. In subsequent studies, WBC mobilization was used as a marker of stem cell mobilization. To identify the dose of propranolol to be used in combination with GPC-100, propranolol was administered IP at 5, 10, 20, and 40 mg / kg for 7 days, followed by coadministration of GPC-100 on day 7. Propranolol was administered at 20 mg / kg because this dose significantly improved GPC-100-induced mobilization. Mobilization of LSK stem cells was determined by flow for the combination of propranolol and GPC-100. Next, for WBC mobilization, the combination of GPC-100 and propranolol was compared with G-CSF alone. In this study, G-CSF was administered twice daily for 5 days. Finally, the three-drug combination using G-CSF, GPC-100, and propranolol was investigated for WBC and stem cell mobilization in phenotypic analysis and colony-forming unit assays compared with G-CSF + AMD3100. In all studies, blood was collected 2 h after GPC-100, 1 h after AMD3100, and 12 h after G-CSF.

[0239] (Results) Single administration of GPC-100 (30 mg / kg, IV) induced WBC mobilization in PB, which peaked at 2 hours. In a number of tests in mice, peak mobilization by AMD3100 (5 mg / kg, SC) at the 1-hour time point has been reported (e.g., Broxmeyer et al., 2005). Therefore, PB white blood cell counts after GPC-100 and after AMD3100 were determined at the time point and dose at which maximum mobilization was observed for each antagonist. GPC-100 induced an increase in PB white blood cell counts in both C57 / BL6 and balb / c mouse strains (Figure 1A). When compared with AMD3100 in three separate tests (Figure 61A, Figure 61B, Figure 61C), GPC-100 resulted in a 2- to 3-fold increase in WBC compared to vehicle, while AMD3100 resulted in a <2-fold increase. The increase in WBC by both antagonists included an increase in lymphocytes and neutrophils. No changes in platelet count, hemoglobin, or other red blood cell parameters were observed. Determination of LSK cells by flow cytometry also showed that GPC-100 also mobilized hematopoietic stem cells (Figure 62).

[0240] To evaluate the effect of β2AR blockade in vivo, mice were administered propranolol. The dose of propranolol was selected based on a dose escalation (5 - 40 mg / kg, IP) when combined with GPC-100 (Figure 63A). Pretreatment with propranolol (20 mg / kg, IP) for 7 days significantly improved GPC-100-induced mobilization (Figure 63B). Also, phenotypic analysis of LSK cells by flow cytometry also showed that GPC-100-induced LSK cell mobilization was enhanced by propranolol (Figure 64A - Figure 64D).

[0241] Next, mobilization by the combination of GPC-100 and propranolol was compared with G-CSF, a standard treatment. In the mobilization of WBC, propranolol induced a 4.1-fold increase, while G-CSF induced a 4.5-fold increase of the same degree (Figure 65).

[0242] A combination of three agents, G-CSF, GPC-100, and propranolol, was compared with the current standard ASCT treatment, namely G-CSF alone or in combination with AMD3100. The three-agent combination and the combination of G-CSF and GPC-100 each induced an 8.2-fold and 8.4-fold increase in WBC mobilization, respectively, which was significantly higher compared to the white blood cell counts increased by G-CSF alone (4.5-fold) or G-CSF + AMD3100 (6.6-fold) (Figure 66).

[0243] Further experiments were conducted to determine whether the increased white blood cell counts in circulation reflected hematopoietic stem and progenitor cell (HSPC) mobilization. A CFU assay was performed to measure the mobilized HSPCs based on their ability to form CFU-GM and BFU-E colonies. The three-agent combination resulted in a 47-fold increase in CFU compared to the vehicle control, as compared to a 35-fold and 27-fold increase in CFU by G-CSF + GPC-100 treatment and G-CSF + AMD3100 treatment, respectively (Figures 67A - 67D).

[0244] Phenotypic analysis showed that the G-CSF + AMD3100 treatment resulted in a 13-fold increase in LSK cells in the PB compared to the vehicle. As a comparison, the combinations of G-CSF and GPC-100 with and without propranolol resulted in a 20-fold and 24-fold increase in LSK cells, respectively (Figures 68A - 68F). The patterns of LSK and CFU numbers across the various drug combinations were consistent with the white blood cell counts from matched samples (Figures 67A - 67D and 68A - 68F), which supported the use of white blood cell counts as a surrogate marker for stem cell mobilization.

[0245] This study demonstrates that GPC-100 is a potent hematopoietic mobilizer and its mobilizing effect is enhanced by propranolol. This study also shows that the increase in GPC-100-induced mobilization by G-CSF is superior to that of the combination of G-CSF and AMD3100. The addition of propranolol to G-CSF and GPC-100 significantly mobilized more hematopoietic stem cells capable of differentiating into multipotent precursors. This data and previous reports have shown that HSPC mobilization is associated with a concomitant increase in circulating WBCs (Vater et al., 2013; Almeida-Neto et al., 2020; Abraham et al., 2007; Lee et al., 2014). The effect of propranolol observed in this study may be explained by an independent effect of propranolol on HSPCs or an interaction between β2AR and CXCR4.

[0246] This study also shows that the combination of propranolol and GPC-100 showed a 4-fold increase compared to the vehicle control WBCs, while G-CSF induced a 4.5-fold increase. This observation is important to suggest the possibility of similar HSC mobilization without the use of G-CSF. Excluding G-CSF from the treatment can reduce the risk of severe bone pain and moderate to severe side effects of G-CSF such as rare splenic rupture.

[0247] The addition of propranolol to G-CSF and GPC-100 (triple combination) increased the number of PB CFUs, which was significantly greater than that of G-CSF+AMD3100. This indicates that the triple combination mobilized a greater number of viable cells capable of differentiating into functionally myeloid and erythroid multipotent precursors. Furthermore, phenotypic analysis revealed more LSK cells in the PB with G-CSF and GPC-100 treatment with or without propranolol compared to G-CSF+AMD3100. This study was performed in naive mice, and in models where the BM niche and HSC differentiation are impaired, the effect of propranolol will likely be amplified (Giles et al., 2016).

[0248] In summary, the preclinical findings of the present inventors support the addition of propranolol to GPC-100-induced stem cell mobilization for ASCT in MM patients. The three-drug combination of GPC-100, propranolol, and G-CSF can, in some cases, be class-leading and can target patient populations in which other mobilization regimens have failed. Propranolol complements mobilization therapy for a greater stem cell yield in fewer apheresis sessions and will prove to be a safe, accessible, and inexpensive option that reduces the economic burden on patients and the healthcare system. A related clinical trial has been registered as a two-arm phase 2 clinical trial (NCT05561751) with GPC-100 + propranolol arm and GPC-100, propranolol, and G-CSF arm.

[0249] (Exemplary Embodiments) In an embodiment, disclosed herein is a method of mobilizing cells in a subject, the method comprising blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject.

[0250] In an embodiment, disclosed herein is a method of inducing cell mobilization in a subject, the method comprising blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject.

[0251] In an embodiment, disclosed herein is a method of enhancing apheresis in a subject, the method comprising blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject.

[0252] In an embodiment, disclosed herein is a method of enhancing apheresis by inducing cell mobilization in a subject, the method comprising blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject.

[0253] In an embodiment, disclosed herein is a method for enhancing apheresis by mobilizing cells in a subject, the method comprising blocking CXCR4 signaling and β - adrenergic receptor signaling in the subject.

[0254] In an embodiment, blocking the β - adrenergic receptor signaling is performed before blocking the CXCR4 signaling.

[0255] In an embodiment, blocking the β - adrenergic receptor signaling continues even after blocking the CXCR4 signaling has ended.

[0256] In an embodiment, blocking the CXCR4 signaling comprises administering a CXCR4 inhibitor to the subject.

[0257] In an embodiment, blocking the β - adrenergic receptor signaling comprises administering a β - adrenergic receptor inhibitor to the subject.

[0258] In an embodiment, the cells are stem cells.

[0259] In an embodiment, blocking the CXCR4 signaling comprises administering a CXCR4 inhibitor to the subject, and blocking the β - adrenergic receptor signaling comprises administering a β - adrenergic receptor inhibitor to the subject.

[0260] In an embodiment, the cells are stem cells.

[0261] In an embodiment, disclosed herein is a method for mobilizing stem cells in a subject, the method comprising administering a β - adrenergic receptor inhibitor and a CXCR4 inhibitor to the subject.

[0262] In an embodiment, disclosed herein is a method of inducing stem cell mobilization in a subject, the method comprising administering to the subject a β-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0263] In an embodiment, disclosed herein is a method of enhancing apheresis in a subject, the method comprising administering to the subject a β-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0264] In an embodiment, disclosed herein is a method of enhancing apheresis by inducing cell mobilization in a subject, the method comprising administering to the subject a β-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0265] In an embodiment, disclosed herein is a method of enhancing apheresis by mobilizing cells in a subject, the method comprising administering to the subject a β-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0266] In an embodiment, administering the β-adrenergic receptor inhibitor is performed before administering the CXCR4 inhibitor.

[0267] In an embodiment, administering the β-adrenergic receptor inhibitor is continued even after administering the CXCR4 inhibitor has been completed.

[0268] In an embodiment, the method further comprises administering G-CSF to the subject.

[0269] In an embodiment, administering the β-adrenergic receptor inhibitor and the CXCR4 inhibitor to the subject is performed in the absence of G-CSF.

[0270] In an embodiment, disclosed herein is a method of mobilizing stem cells in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

[0271] In an embodiment, disclosed herein is a method of inducing stem cell mobilization in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

[0272] In an embodiment, disclosed herein is a method of enhancing apheresis in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

[0273] In an embodiment, disclosed herein is a method of enhancing apheresis by inducing cell mobilization in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

[0274] In an embodiment, disclosed herein is a method of enhancing apheresis by mobilizing cells in a subject, the method comprising administering to the subject a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

[0275] In an embodiment, the β-adrenergic receptor inhibitor is an ADRB2 inhibitor.

[0276] In an embodiment, the β-adrenergic receptor inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

[0277] In an embodiment, the β-adrenergic receptor inhibitor is selected from the group consisting of propranolol, nadolol, and ICI 118551.

[0278] In an embodiment, the β-adrenergic receptor inhibitor is propranolol.

[0279] In an embodiment, the CXCR4 inhibitor is selected from the group consisting of ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI 2341, BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, CX549, D-[Lys3] GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012, TG-0054 (brixtafol), USL311, viral macrophage inflammatory protein-II (vMIP-II), WZ811, [64Cu]-AMD3100, [64Cu]-AMD3465, [68Ga]pentixafor, [90Y]pentixather, [99mTc]O2-AMD3100, [177Lu]pentixather, and 508MCl (Compound 26).

[0280] In an embodiment, the CXCR4 inhibitor is selected from the group consisting of AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (bryxalfor), PF-06747143, POL6326, and urocuplumab (MDX1338 / BMS-936564).

[0281] In an embodiment, the CXCR4 inhibitor is TG-0054 (bryxalfor).

[0282] In an embodiment, the CXCR4 inhibitor is AMD3100 (plerixafor).

[0283] In an embodiment, the CXCR4 inhibitor is urocuplumab (MDX1338 / BMS-936564).

[0284] In an embodiment, administering the CXCR4 inhibitor to the subject includes administering TG-0054 (bryxalfor) and propranolol.

[0285] In an embodiment, administering the CXCR4 inhibitor to the subject includes administering AMD3100 (plerixafor) and propranolol.

[0286] In an embodiment, administering the CXCR4 inhibitor to the subject includes administering urocuplumab (MDX1338 / BMS-936564) and propranolol.

[0287] In an embodiment, administering a combination of the CXCR4 inhibitor and the G-CSF induces an improved amount of cell mobilization compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0288] In an embodiment, administering the combination of the CXCR4 inhibitor and the G-CSF mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0289] In an embodiment, administering the combination of the CXCR4 inhibitor and the G-CSF induces an improved amount of apheresis as compared to the amount of apheresis induced by the CXCR4 inhibitor alone.

[0290] In an embodiment, administering the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor induces an improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0291] In an embodiment, administering the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0292] In an embodiment, administering the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor induces an improved amount of apheresis as compared to the amount of apheresis induced by the CXCR4 inhibitor alone.

[0293] In an embodiment, administering the combination of the CXCR4 inhibitor, the β-adrenergic receptor inhibitor, and the G-CSF induces an improved amount of cell mobilization as compared to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

[0294] In an embodiment, administering the combination of the CXCR4 inhibitor, the β-adrenergic receptor inhibitor, and the G-CSF mobilizes cells in an improved amount as compared to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

[0295] In an embodiment, administering the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor with G-CSF induces an improved amount of apheresis compared to the amount of apheresis induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

[0296] In an embodiment, administering the combination of TG-0054 (bryxalfor) with G-CSF induces an improved amount of cell mobilization compared to the amount of cell mobilization induced by AMD3100 (plerixafor) and G-CSF.

[0297] In an embodiment, administering the combination of the TG-0054 (bryxalfor) with the G-CSF mobilizes cells in an improved amount compared to the amount of cell mobilization induced by the AMD3100 (plerixafor) and the G-CSF.

[0298] In an embodiment, administering the combination of the TG-0054 (bryxalfor) with the G-CSF induces an improved amount of apheresis compared to the amount of apheresis induced by the AMD3100 (plerixafor) and the G-CSF.

[0299] In an embodiment, the improved amount of cell mobilization or apheresis is measured by a method selected from the group consisting of complete blood count (CBC) analysis, flow cytometry, and colony-forming unit (CFU) assay.

[0300] In an embodiment, the improved amount of cell mobilization or apheresis is measured by flow cytometry.

[0301] In an embodiment, the flow cytometry is performed on (Lin-Sca1+c-Kit+) LSK cells.

[0302] In an embodiment, the improved amount of cell mobilization or apheresis is measured by a colony-forming unit (CFU) assay.

[0303] In an embodiment, the subject has a CXCR4 protomer within the cell.

[0304] In an embodiment, the subject has an ADRB2 protomer within the cell.

[0305] In an embodiment, the subject has a CXCR4 protomer and an ADRB2 protomer within the cell.

[0306] In an embodiment, the subject has a CXCR4-ADRB2 heteromer within the cell.

[0307] In an embodiment, i) the CXCR4-ADRB2 heteromer has an improved amount of downstream calcium mobilization compared to downstream calcium mobilization from the CXCR4 protomer or the ADRB2 protomer; and ii) the administered combination of inhibitors suppresses the enhanced downstream calcium mobilization from the CXCR4-ADRB2 heteromer within the stem cell.

[0308] In an embodiment, the cell is a stem cell.

[0309] In an embodiment, the stem cell is selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, endothelial progenitor cells, nervous system stem cells, epithelial stem cells, skin stem cells, and cancer stem cells.

[0310] In an embodiment, the stem cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

[0311] In an embodiment, the hematopoietic stem cell or the hematopoietic progenitor cell is mobilized from bone marrow to peripheral blood.

[0312] In an embodiment, the mobilized hematopoietic stem cell or hematopoietic progenitor cell is collected for transplantation into a cancer patient.

[0313] In an embodiment, the cancer is selected from the group consisting of lymphoma, leukemia, and myeloma.

[0314] In an embodiment, the cancer is non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or multiple myeloma (MM).

[0315] In an embodiment, the stem cells are mesenchymal stem cells.

[0316] In an embodiment, the mesenchymal stem cells are mobilized from bone marrow to peripheral blood.

[0317] In an embodiment, the mesenchymal stem cells are mobilized for the treatment of a condition selected from the group consisting of neuropathy, myocardial ischemia, myocardial infarction, diabetes, tissue repair, bone and cartilage diseases, autoimmune diseases, graft-versus-host disease, Crohn's disease, multiple sclerosis, systemic lupus erythematosus, and systemic sclerosis.

[0318] In an embodiment, the stem cells are cancer stem cells.

[0319] In an embodiment, the cancer stem cells are mobilized into the blood.

[0320] In an embodiment, the cancer stem cells are mobilized for the treatment of cancer.

[0321] In an embodiment, the cells are immune cells.

[0322] In an embodiment, the immune cells are white blood cells.

[0323] In an embodiment, the white blood cells are lymphocytes.

[0324] In an embodiment, the lymphocytes are selected from the group consisting of T cells, B cells, and natural killer (NK) cells.

[0325] In an embodiment, the lymphocyte is a T cell.

[0326] In an embodiment, the lymphocyte is a natural killer (NK) cell.

[0327] In an embodiment, the leukocyte is a granulocyte.

[0328] In an embodiment, the granulocyte is selected from the group consisting of neutrophils, eosinophils, and basophils.

[0329] In an embodiment, the granulocyte is a neutrophil.

[0330] In an embodiment, the leukocyte is a monocyte.

[0331] In an embodiment, the immune cell is mobilized from the bone marrow to the peripheral blood.

[0332] In an embodiment, the immune cell is mobilized from the lymph node to the peripheral blood.

[0333] In an embodiment, the mobilized immune cell is used in adoptive cell therapy (ACT).

[0334] In an embodiment, the adoptive cell therapy (ACT) is chimeric antigen receptor (CAR) T cell therapy.

[0335] In an embodiment, the adoptive cell therapy (ACT) is natural killer (NK) cell therapy.

[0336] In an embodiment, the adoptive cell therapy (ACT) is modified T cell receptor (TCR) therapy.

[0337] In an embodiment, the adoptive cell therapy (ACT) is tumor-infiltrating lymphocyte (TIL) therapy.

Claims

1. A pharmaceutical composition for use in a method for treating cancer, comprising administering to a subject a CXCR4 inhibitor and a beta-2-adrenergic receptor (ADRB2) inhibitor, either alone or in combination, wherein the treatment comprises stem cell transplantation.

2. 10. The pharmaceutical composition of claim 1, wherein said administering comprises administering said ADRB2 inhibitor and said CXCR4 inhibitor to said subject simultaneously, concurrently, or sequentially.

3. 10. The pharmaceutical composition of claim 1, wherein said administering comprises administering said ADRB2 inhibitor to said subject before administering said CXCR4 inhibitor to said subject.

4. 2. The pharmaceutical composition of claim 1, wherein administering the ADRB2 inhibitor begins a first specified time interval prior to administering the CXCR4 inhibitor, and the first specified time interval is 6 to 7 days.

5. 2. The pharmaceutical composition of claim 1, wherein the treatment comprises hematopoietic stem cell transplantation or hematopoietic progenitor cell transplantation.

6. 2. The pharmaceutical composition of claim 1, wherein the stem cell transplant comprises an autologous stem cell transplant.

7. 2. The pharmaceutical composition of claim 1, wherein the stem cell transplant comprises an allogeneic stem cell transplant.

8. 2. The pharmaceutical composition of claim 1, wherein the cancer is selected from lymphoma, leukemia, and myeloma.

9. 2. The pharmaceutical composition of claim 1, wherein the cancer is selected from non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma.

10. 2. The pharmaceutical composition of claim 1, wherein the cancer is multiple myeloma.

11. The CXCR4 inhibitor may be ALX40-4C, AMD070, plerixafor, AMD3465, ATI 2341, BKT140, CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, or TC. 14012, Blixafor, USL311, viral macrophage inflammatory protein-II, WZ811, [ 64 Cu]-AMD3100,[ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]Pentixatel,[ 99m Tc]O2-AMD3100,[ 177 10. The pharmaceutical composition of claim 1, wherein the compound is selected from the group consisting of [Lu]pentixatel, and 508MCl (Compound 26).

12. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is selected from blixafor, plerixafor, and urocuplumab.

13. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor.

14. 2. The pharmaceutical composition of claim 1, wherein the ADRB2 inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

15. 2. The pharmaceutical composition of claim 1, wherein the ADRB2 inhibitor is propranolol.

16. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is selected from the group consisting of blixafor, plerixafor, and urocuprumab, and the ADRB2 inhibitor is propranolol.

17. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is propranolol.

18. 10. The pharmaceutical composition of claim 1, wherein the method further comprises administering G-CSF to the subject.

19. The pharmaceutical composition of claim 1, wherein the method does not include administering G-CSF to the subject.

20. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the treatment further comprises administration of G-CSF.

21. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the treatment does not include administration of G-CSF.

22. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the treatment comprises administration of G-CSF.

23. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the treatment does not comprise administration of G-CSF.

24. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises allogeneic stem cell transplant, and the treatment comprises administration of G-CSF.

25. 2. The pharmaceutical composition of claim 1, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises allogeneic stem cell transplant, and the treatment does not comprise administration of G-CSF.

26. A composition for treating cancer comprising, alone or in combination, a CXCR4 inhibitor and a beta-2-adrenergic receptor (ADRB2) inhibitor, wherein the treatment comprises stem cell transplantation.

27. 27. The composition of claim 26, wherein the treatment comprises hematopoietic stem cell transplantation or hematopoietic progenitor cell transplantation.

28. 27. The composition of claim 26, wherein the stem cell transplant comprises an autologous stem cell transplant.

29. 27. The composition of claim 26, wherein the stem cell transplant comprises an allogeneic stem cell transplant.

30. 27. The composition of claim 26, wherein the cancer is selected from lymphoma, leukemia, or myeloma.

31. 27. The composition of claim 26, wherein the cancer is selected from non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, or multiple myeloma.

32. 27. The composition of claim 26, wherein the cancer is multiple myeloma.

33. The CXCR4 inhibitor may be ALX40-4C, AMD070, plerixafor, AMD3465, ATI 2341, BKT140, CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, or TC. 14012, Blixafor, USL311, viral macrophage inflammatory protein-II, WZ811, [ 64 Cu]-AMD3100,[ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]Pentixatel,[ 99m Tc]O2-AMD3100,[ 177 27. The composition of claim 26, wherein the compound is selected from the group consisting of: pentixatel, 508MCl (Compound 26), and 508MCl (Compound 26).

34. 27. The composition of claim 26, wherein the CXCR4 inhibitor is selected from blixafor, plerixafor, and urocuplumab.

35. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor.

36. 27. The composition of claim 26, wherein the ADRB2 inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

37. 27. The composition of claim 26, wherein the ADRB2 inhibitor is propranolol.

38. 27. The composition of claim 26, wherein the CXCR4 inhibitor is selected from the group consisting of blixafor, plerixafor, and urocuplumab, and the ADRB2 inhibitor is propranolol.

39. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is propranolol.

40. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the composition further comprises G-CSF.

41. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the composition does not include G-CSF.

42. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the composition further comprises G-CSF.

43. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the composition does not comprise G-CSF.

44. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises an allogeneic stem cell transplant, and the composition further comprises G-CSF.

45. 27. The composition of claim 26, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises an allogeneic stem cell transplant, and the composition does not comprise G-CSF.

46. A pharmaceutical composition for treating cancer, comprising, alone or in combination, a CXCR4 inhibitor and a beta-2-adrenergic receptor (ADRB2) inhibitor, and a pharmaceutically acceptable excipient, wherein the treatment comprises stem cell transplantation.

47. 47. The pharmaceutical composition of claim 46, wherein the treatment comprises hematopoietic stem cell transplantation or hematopoietic progenitor cell transplantation.

48. 47. The pharmaceutical composition of claim 46, wherein the stem cell transplant comprises an autologous stem cell transplant.

49. 47. The pharmaceutical composition of claim 46, wherein the stem cell transplant comprises an allogeneic stem cell transplant.

50. 47. The pharmaceutical composition of claim 46, wherein the cancer is selected from lymphoma, leukemia, or myeloma.

51. 47. The pharmaceutical composition of claim 46, wherein the cancer is selected from non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, or multiple myeloma.

52. 47. The pharmaceutical composition of claim 46, wherein the cancer is multiple myeloma.

53. The CXCR4 inhibitor may be ALX40-4C, AMD070, plerixafor, AMD3465, ATI 2341, BKT140, CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, or TC. 14012, Blixafor, USL311, viral macrophage inflammatory protein-II, WZ811, [ 64 Cu]-AMD3100,[ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]Pentixatel,[ 99m Tc]O2-AMD3100,[ 177 47. The pharmaceutical composition of claim 46, wherein the compound is selected from the group consisting of [Lu]pentixatel, and 508MCl (Compound 26).

54. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is selected from blixafor, plerixafor, and urocuplumab.

55. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor.

56. 47. The pharmaceutical composition of claim 46, wherein the ADRB2 inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

57. 47. The pharmaceutical composition of claim 46, wherein the ADRB2 inhibitor is propranolol.

58. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is selected from the group consisting of blixafor, plerixafor, and urocuplumab, and the ADRB2 inhibitor is propranolol.

59. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is propranolol.

60. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the pharmaceutical composition further comprises G-CSF.

61. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the pharmaceutical composition does not contain G-CSF.

62. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the pharmaceutical composition further comprises G-CSF.

63. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the pharmaceutical composition does not comprise G-CSF.

64. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises allogeneic stem cell transplant, and the pharmaceutical composition further comprises G-CSF.

65. 47. The pharmaceutical composition of claim 46, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises an allogeneic stem cell transplant, and the pharmaceutical composition does not comprise G-CSF.

66. A pharmaceutical composition for use in a method for mobilizing cells in a subject, the method comprising administering to the subject a CXCR4 inhibitor and a beta-2-adrenergic receptor (ADRB2) inhibitor, either alone or in combination.

67. 67. The pharmaceutical composition of claim 66, wherein said administering comprises administering the ADRB2 inhibitor and the CXCR4 inhibitor to the subject simultaneously, concurrently, or sequentially.

68. 67. The pharmaceutical composition of claim 66, wherein said administering comprises administering said ADRB2 inhibitor to said subject before administering said CXCR4 inhibitor to said subject.

69. 67. The pharmaceutical composition of claim 66, wherein administering the ADRB2 inhibitor begins a first specified time interval prior to administering the CXCR4 inhibitor, and the first specified time interval is 6 to 7 days.

70. 67. The pharmaceutical composition of claim 66, wherein the cells are stem cells.

71. 67. The pharmaceutical composition of claim 66, wherein the cells are stem cells and the mobilizing is used for cancer treatment, and the treatment comprises stem cell transplantation.

72. 72. The pharmaceutical composition of claim 71, wherein the treatment comprises hematopoietic stem cell transplantation or hematopoietic progenitor cell transplantation.

73. 72. The pharmaceutical composition of claim 71, wherein the stem cell transplant comprises an autologous stem cell transplant.

74. 72. The pharmaceutical composition of claim 71, wherein the stem cell transplant comprises an allogeneic stem cell transplant.

75. 72. The pharmaceutical composition of claim 71, wherein the cancer is selected from lymphoma, leukemia, or myeloma.

76. 72. The pharmaceutical composition of claim 71, wherein the cancer is selected from non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, or multiple myeloma.

77. 72. The pharmaceutical composition of claim 71, wherein the cancer is multiple myeloma.

78. The CXCR4 inhibitor may be ALX40-4C, AMD070, plerixafor, AMD3465, ATI 2341, BKT140, CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, or TC. 14012, Blixafor, USL311, viral macrophage inflammatory protein-II, WZ811, [ 64 Cu]-AMD3100,[ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]Pentixatel,[ 99m Tc]O2-AMD3100,[ 177 67. The pharmaceutical composition of claim 66, wherein the compound is selected from the group consisting of [Lu]pentixatel, and 508MCl (Compound 26).

79. 67. The pharmaceutical composition of claim 66, wherein the CXCR4 inhibitor is selected from blixafor, plerixafor, and urocuplumab.

80. 67. The pharmaceutical composition of claim 66, wherein the CXCR4 inhibitor is blixafor.

81. 67. The pharmaceutical composition of claim 66, wherein the ADRB2 inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

82. 67. The pharmaceutical composition of claim 66, wherein the ADRB2 inhibitor is propranolol.

83. 67. The pharmaceutical composition of claim 66, wherein the CXCR4 inhibitor is selected from the group consisting of blixafor, plerixafor, and urocuprumab, and the ADRB2 inhibitor is propranolol.

84. 67. The pharmaceutical composition of claim 66, wherein the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is propranolol.

85. 67. The pharmaceutical composition of claim 66, wherein the method further comprises administering G-CSF to the subject.

86. 67. The pharmaceutical composition of claim 66, wherein the method does not include administering G-CSF to the subject.

87. 72. The pharmaceutical composition of claim 71, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the treatment further comprises administration of G-CSF.

88. 72. The pharmaceutical composition of claim 71, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, and the treatment does not include administration of G-CSF.

89. 72. The pharmaceutical composition of claim 71, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the treatment comprises administration of G-CSF.

90. 72. The pharmaceutical composition of claim 71, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises autologous stem cell transplant, and the treatment does not comprise administration of G-CSF.

91. 72. The pharmaceutical composition of claim 71, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises an allogeneic stem cell transplant, and the treatment comprises administration of G-CSF.

92. 72. The pharmaceutical composition of claim 71, wherein the CXCR4 inhibitor is blixafor, the ADRB2 inhibitor is propranolol, the cancer is multiple myeloma, the stem cell transplant comprises an allogeneic stem cell transplant, and the treatment does not comprise administration of G-CSF.

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