GPCR inhibitors and uses thereof

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 369,738, filed on July 8, 2022, and U.S. Provisional Patent Application No. 63 / 351,101, filed on June 10, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] The invention disclosed herein generally relates to the mobilization of stem cells and immune cells.

[0004] The bone marrow is highly innervated by the sympathetic nervous system. Traumatic stress in humans and rodent models has shown sustained elevated levels of norepinephrine (a ligand for the β-adrenergic receptor), which is 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 the β-adrenergic receptor inhibitor propranolol was shown to restore bone marrow function and increase erythroid progenitor colony growth in response to anemia (Alamo et al. 2017). In patients with multiple myeloma, 28-day cycles of propranolol administration shifted cell differentiation from a myeloid bias to a CD34 + Stem cells and upregulation of genes associated with this phenotype (Knight et al. 2020). Therefore, beta blockers appear to have the potential to improve hematopoietic stem cell mobilization by restoring bone marrow function.

[0005] CXC chemokine receptor 4 (CXCR4) belongs to the superfamily of G protein-coupled receptors (GPCRs). The binding of the chemokine CXCL12 (also known as SDF-1) to its receptor CXCR4 plays an important role in the homing and retention of hematopoietic stem cells (HSCs) in the bone marrow. Blocking the CXCL12 / CXCR4 axis can cause 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) have been used clinically 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's lymphoma and multiple myeloma. However, typically, the G-CSF regimen involves repeated multi-day injections and is associated with adverse side effects such as severe bone pain. However, successful ASCT in patients with lymphoma and MM is often hampered by poor mobilization, with at least 15% of patients failing to generate >2 × 10 6 CD34+ Target cell dose of cells / kg (Olivieri et al. 2012). BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1A-1B Shows self-cell (WBC) mobilization in C57 / BL6 and Balb / c mice after treatment with GPC100 ( Figure 1A ) and time-dependent WBC count ( Figure 1B ).

[0007] Figure 2A-2B Injection of vehicle and GPC100 or vehicle and AMD3100 ( Figure 2A ) and injection of G-CSF and GPC100 or G-CSF and AMD3100 ( Figure 2B ) after the circulating WBC count.

[0008] Figure 3 Data from 6 studies are presented, demonstrating that 7-day pretreatment with propranolol significantly increased GPC100-induced peripheral blood WBC counts compared with 7-day vehicle pretreatment.

[0009] Figure 4 Data are shown for mice administered GPC100 after propranolol pretreatment, showing leukocyte mobilization to an extent comparable to those receiving G-CSF.

[0010] Figure 5A-5B Cumulative data from several experiments are shown, showing leukocytes from combination treatment with G-CSF and AMD3100 ( Figure 5A ) and lymphocytes ( Figure 5B ) in the mobilization of the people.

[0011] Figure 6A-6B Shown in WBC ( Fig. 6A ) and progenitor cells ( Figure 6B ) enhanced G-CSF mobilization by GPC100 with or without propranolol pretreatment.

[0012] Figures 7A-7C Data pooled from four experiments are shown, indicating that when combined with G-CSF, GPC100 mobilized more WBCs than AMD3100, and the addition of propranolol did not alter total WBC counts in any group ( Fig. 7A ), lymphocyte counts showed that propranolol enhanced their mobilization when added to the combination of G-CSF and GPC100 ( Figure 7B ), the distribution of WBC differential counts indicated that the addition of propranolol increased the entry of lymphocytes into the peripheral blood ( Figure 7C ).

[0013] Figures 8A-8Dshowed that pretreatment with propranolol enhanced the leukocyte ( Fig. 8A ), lymphocytes ( Figure 8B ), neutrophils ( Figure 8C ) and monocytes ( Fig.8D )’s mobilization.

[0014] Fig. 9 Data from three studies are presented, showing a significant increase in GPC100-induced mobilization after propranolol pretreatment.

[0015] Figures 10A-10D The data presented show that, in contrast to the SOC regimen, which primarily mobilizes neutrophils, leukocytes mobilized by pretreatment with propranolol ( Fig. 10A ), lymphocytes ( Fig. 10B ), neutrophils ( Fig. 10C ) and monocytes ( Fig. 10D ) increased, mainly due to lymphocytes.

[0016] Fig.11 The data presented indicate that, in contrast to the SOC regimen, which primarily mobilizes neutrophils, the increase in WBC mobilization due to propranolol pretreatment was primarily attributable to lymphocytes.

[0017] Fig.12 Greater changes were shown in the SOC group in both studies, with SOC alone also resulting in a decrease in platelet count.

[0018] Fig.13 Greater changes were shown in the SOC group in both studies, with SOC alone also resulting in a decrease in platelet count.

[0019] Fig.14 Data showing hematopoietic stem cell mobilization as measured by flow cytometry with the dosing regimen, no significant differences were observed from the standard of care for LSK cells.

[0020] Fig.15 Data showing hematopoietic stem cell mobilization as measured by flow cytometry with dosing regimens, no significant differences were observed from the standard of care for Lin-CXCR4+ cells.

[0021] Fig.16 The data presented demonstrate that the addition of propranolol to the combination of G-CSF and GPC100 resulted in the greatest mobilization of WBCs, with mobilization significantly increased compared to SOC or G-CSF and GPC100 combination treatment.

[0022] Figures 17A-17C The data presented demonstrate that the addition of propranolol to the combination of G-CSF and GPC100 induces neutrophil ( Fig.17A ), lymphocytes ( Fig. 17B ) and monocytes ( Fig. 17C ) with a significant increase in mobilization compared with SOC or the combination of G-CSF and GPC100 treatment.

[0023] Fig.18 GPC100 in combination with propranolol was shown to increase lymphocytes, whereas GPC100 in combination with G-CSF increased neutrophils.

[0024] Figures 19A-19B showed that increased mobilized circulating WBC ( Fig.19A ) and colony forming units ( Fig.19B ).

[0025] Figures 20A-20B The triple combination of G-CSF, GPC100 and propranolol was shown to produce the highest number of colony forming units ( Fig. 20A ) and burst forming units ( Fig. 20B ).

[0026] Fig.21 is a schematic representation of studies focused on mobilization into peripheral blood.

[0027] Fig. 22 Data are shown for GPC100-induced leukocyte mobilization in mice.

[0028] Fig.23 Data showing time-dependent GPC100-induced leukocyte mobilization in mice.

[0029] Figures 24A-24C It was shown that 7-day propranolol administration enhanced the GPC100-induced leukocyte ( Fig.24A ), lymphocytes ( Fig. 24B ) and neutrophils ( Fig.24C ) but had no effect on blood cell counts when given alone.

[0030] Figures 25A-25C Nadolol was shown to enhance GPC100-induced leukocyte Fig.25A ), lymphocytes ( Fig.25B ) and neutrophils ( Fig.25C )’s mobilization.

[0031] Figures 26A-26C It was shown that administration of β-blockers together with single GPC100 for 7 days did not increase LSK and Lin-CXCR4+ cells.

[0032] Figures 27A-27C It was observed that propranolol enhanced the leukocyte ( Fig.27A ), lymphocytes ( Fig.27B) and neutrophils ( Fig.27C )’s mobilization.

[0033] Fig.28 Lymphocytes were shown to be increased with GPC100 and beta blockers, while neutrophils were increased with G-CSF+AMD3100.

[0034] Figures 29A-29B Shown are LSK cells ( Fig.29A ) and Lin-CXCR4+ cells ( Fig.29B ) multiples of the combined data.

[0035] Figures 30A-30B Shown are LSK cells ( Fig. 30A ) and Lin-CXCR4+ cells ( Fig. 30B ) multiples of the combined data.

[0036] Figures 31A-31C White blood cells ( Fig.31A ), lymphocytes ( Fig.31B ) and neutrophils ( Fig. 31C ) Mobilization data for the triple combination.

[0037] Fig.32 Data for triplicate combinations of leukocyte populations are shown.

[0038] Fig.33 It is a schematic diagram of the hematopoietic hierarchy.

[0039] Fig.34 is a schematic diagram of the colony forming unit assay.

[0040] Fig.35 The data shown indicate that the triple combination mobilized the highest number of progenitor cells.

[0041] Figures 36A-36B The triple combination that produced the highest number of colony forming units ( Fig.36A ) and burst forming units ( Fig.36B ) data.

[0042] Fig.37 Images of BFU-E colonies (left) and CFU-GM colonies (right) from G-CSF and GPC-100 combination treatment (top) and G-CSF+AMD3100 combination treatment (bottom) are shown.

[0043] Figures 38A-38BThe data presented showed that the triple combination was associated with a decrease in circulating WBC( Fig.38A ) and progenitor cells measured by total colony-forming units ( Fig.38B ) is related to the maximum increase in

[0044] Figures 39A-39B G-CSF combination therapy ( Fig.39A ) and GPC100 treatment with and without propranolol ( Fig.39B ) after .

[0045] Figures 40A-40B The results from the study on the effect of propranolol on the induction of GPC100 in leukocytes ( Fig.40A ) and lymphocytes ( Fig.40B ) Cumulative data from three studies on the effects of mobilization.

[0046] Figures 41A-41B Propranolol enhanced the leukocyte ( Fig.41A ) and lymphocytes ( Fig.41B )’s mobilization is comparable to that of the SOC.

[0047] Figures 42A-42C show data from studies using single agent GPC100, AMD3100 or G-CSF induced WBC mobilization of leukocytes (Figure 42A), lymphocytes (Figure 42B), and neutrophils (Figure 42C).

[0048] Figures 43A-43B showed similar results to Study 4 ( Fig.43A ) and Study 5( Fig.43B Data on the effect of propranolol on GPC100-induced mobilization in the absence or presence of G-CSF compared to standard of care (SOC) in WBCs of 54 cases.

[0049] Figures 44A-44B showed similar results to Study 4 ( Fig.44A ) and Study 5( Fig.44B Data on the effect of propranolol on GPC100-induced mobilization in the absence or presence of G-CSF compared to standard of care (SOC) in lymphocytes from patients with leukemia ().

[0050] Figures 45A-45B showed similar results to Study 4 ( Fig.45A ) and Study 5( Fig.45B Data on the effect of propranolol on GPC100-induced mobilization in the absence or presence of G-CSF compared to standard of care (SOC) in neutrophils from patients with leukemia ().

[0051] Figures 46A-46C The results show that the difference between GPC100 and AMD3100 in white blood cells ( Fig.46A ), lymphocytes ( Fig.46B ) and neutrophils ( Fig.46C )Data from a comparative study of mobilization.

[0052] Figures 47A-47C Propranolol showed the effect of GPC100 induced leukocytes ( Fig.47A ), lymphocytes ( Fig.47B ) and neutrophils ( Fig.47C ) data on the effects of mobilization and comparison with standard of care.

[0053] Figures 48A-48B Combined data from six studies are presented showing that propranolol treatment 7 days prior to GPC100 resulted in an increase in WBC ( Fig.48A ) and lymphocytes ( Fig.48B ) cell count increased significantly.

[0054] Figures 49A-49B The standard of care regimen was shown to mobilize more WBCs ( Fig.49A ), but standard of care did not mobilize more lymphocytes than propranolol and GPC100 ( Fig.49B ).

[0055] Figures 50A-50B The addition of propranolol to the G-CSF and GPC100 combination mobilized significantly more WBCs compared to standard care ( Fig.50A ) and lymphocytes ( Fig.50B ).

[0056] Figures 51A-51B Combined data from six studies are presented showing that propranolol treatment 7 days prior to GPC100 resulted in an increase in WBC ( Fig.51A ) and lymphocytes ( Fig.51B ) cell count increased significantly.

[0057] Figures 52A-52B The standard of care regimen was shown to mobilize more WBCs ( Fig.52A ), rather than lymphocytes ( Fig.52B ).

[0058] Figures 53A-53B The addition of propranolol to the G-CSF and GPC100 combination mobilized significantly more WBCs compared to the standard of care for stem cell mobilization ( Fig.53A ) and lymphocytes ( Fig.53B ).

[0059] Fig.54 Displays the distribution of WBC counts.

[0060] Figures 55A-55C The WBC ( Fig.55A ), lymphocytes ( Fig.55B ) and neutrophils ( Fig.55C ) mobilization data.

[0061] Figures 56A-56C The results show that the difference between GPC100 and AMD3100 in white blood cells ( Fig.56A ), lymphocytes ( Fig.56B ) and neutrophils ( Fig.56C )Data from a comparative study of mobilization.

[0062] Figures 57A-57C The data presented show that propranolol has an increased inhibitory effect on GPC100-induced leukocytes ( Fig.57A ), lymphocytes ( Fig.57B ) and neutrophils ( Fig.57C )The impact of mobilization.

[0063] Figures 58A-58B Combined data from three studies are presented showing that when combined with G-CSF, significantly more WBC mobilization was observed with GPC100 compared to AMD3100 ( Fig.58A ) and lymphocytes ( Fig.58B ).

[0064] Figures 59A-59F In vitro activity assays ( Fig.59A ), mobilization assay ( Fig.59B ), CXCR4 and B2AR in MDA-MB-231 cells expressing CXCR4 and B2AR ( Fig.59C ) and control cells expressing only CXCR4 ( Fig.59D ) and in the presence of GPC-100 ( Fig.59E ) or AMD3100( Fig.59F )-inhibited MDA-MB-231 cells, GPC100 inhibition of CXCR4 was modulated by propranolol.

[0065] Fig.60A -60E shows a single GPC100 ( Fig.60A ), propranolol followed by a dose of GPC100 or AMD3100 ( Fig.60B ) in vivo mobilization activity, and the triple combination for mobilization of ( Fig.60C ), progenitor cells ( Fig.60D) and mouse HSC ( FIG60E ).

[0066] Figures 61A-61C It was shown that in 3 studies ( Fig.61A , 61B and 61C) increase in WBC mobilization in AMD3100 compared to vehicle.

[0067] Fig.62 Mobilization of hematopoietic stem cells measured in LSK cells by flow cytometry is shown.

[0068] Figures 63A-63B It was shown that when propranolol was titrated to 5-40 mg / kg, IP ( Fig.63A ) and by pretreatment with propranolol (20 mg / kg, IP) for 7 days ( Fig.63B ) WBC mobilization during drug administration.

[0069] Figures 64A-64D It is shown that the vector ( Fig.64A )、GPC-100( Fig.64B ) and propranolol and GPC-100 ( Fig.64C ) flow cytometry analysis of LSK cells also showed that LSK cell mobilization by GPC-100 ( Fig.64D ) was enhanced by propranolol.

[0070] Fig.65 Mobilization induced by the combination of GPC-100 and propranolol compared to standard of care G-CSF is shown.

[0071] Fig.66 WBC mobilization was shown to be significantly greater compared to the increase in WBC counts by G-CSF alone (4.5-fold) or G-CSF plus AMD3100 (6.6-fold).

[0072] Figures 67A-67D The total CFU after triple combination (CFU-GM+BFU) is shown ( Fig.67A )、BFU( Fig.67C ) and CFU-GM (transparent frame) and BFU (solid frame) ( Fig.67B ) and WBC migration ( Fig.67D ).

[0073] Figures 68A-68F Display carrier ( Fig.68A ); G-CSF and GPC-100 ( Fig.68D ); G-CSF, propranolol and GPC-100 ( Fig.68E ); and G-CSF and AMD3100 ( Fig.68F ) after treatment, and MSC( Fig.68B) and WBC( Fig.68C )’s mobilization.

[0074] Abbreviations

[0075] Unless otherwise specified, the following terms including those disclosed herein are Abbreviations: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 cells (CSC), CC chemokine receptor type 2 (CCR2), chemokine chemotaxis C-type kinase receptor 1 (CMKLR1), cholinergic receptor muscarinic 1 (CHRM1), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic obstructive pulmonary disease (COPD), complement C5a receptor 1 (C5AR1), C-terminal fragment of Venus (VC), CXC 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 Basic and Clinical Pharmacology Receptor Nomenclature and Drug Classification Committee (NC-IUPHAR), μ-opioid receptor (MOR), Motilin receptor (MLNR), Multiple myeloma (MM), Multiple infection (MOI), Myelodysplastic syndrome (MDS), Neurotensin receptor 1 (NTSR1) 、Non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), N-terminal fragment of Venus (VN), patient-derived cells (PDC), patient-derived xenografts (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), reverse transcription-quantitative 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 cells (VSMC), WHIM syndrome (warts, hypogammaglobulinemia, infection, and ineffective chronic myeloid agranulocytosis), green fluorescent protein (GFP), and yellow fluorescent protein (YFP). DETAILED DESCRIPTION

[0076] Blood cells play a key role in maintaining the health and viability of animals (including humans). White blood cells are part of the body's immune system that helps the body fight infections and other diseases, including the immune system's granulocytes (neutrophils, eosinophils and basophils / mast cells), monocytes / macrophages, and lymphocytes (T and B cells). White blood cells are continuously replaced via the hematopoietic system through the effects of colony stimulating factors (CSFs) and various cytokines on stem cells and progenitor cells in hematopoietic tissues.

[0077] One of the most widely known is granulocyte colony stimulating factor (G-CSF), which has been approved for use in counteracting the negative effects of chemotherapy by stimulating the production of leukocytes and progenitor cells (peripheral blood stem cell mobilization). Regarding the hematopoietic effect of G-CSF, see, e.g., U.S. Patent No. 5,582,823, which is incorporated herein by reference.

[0078] The development and maturation of blood cells is a complex process. Mature blood cells are derived from hematopoietic precursor (progenitor) cells and stem cells present in specific hematopoietic tissues, including the bone marrow. In these environments, hematopoietic cells proliferate and differentiate before entering the circulation.

[0079] The chemokine receptor CXCR4 and its natural ligand stromal cell derived factor-1 (SDF-1) appear to be important in this process (for review, see Maekawa, T., et al., Internal Med. (2000) 39:90-100; Nagasawa, T., et al., Int. J. Hematol. (2000) 72:408-411). This is confirmed 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, YR., et al., Nature (1998) 393:595-599). It is known that CD34+ progenitor cells express CXCR4 and require SDF-1 produced by bone marrow stromal cells for chemoattraction and engraftment (Peled, A., et al., Science (1999) 283: 845-848). It is also known that, in vitro, SDF-1 has a chemotactic effect 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, DY., et al., J. Clin. Invest. (2000) 105: 101-111). SDF-1 is also an important chemoattractant, signaling through the CXCR4 receptor for several other more committed progenitor and mature blood cells, including T-lymphocytes and monocytes (Bleul, C., et al., J. Exp. Med. (1996) 184: 1101-1109), pro-B lymphocytes, and pre-B lymphocytes. 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).

[0080] Thus, it appears that SDF-1 is able to control the localization and differentiation of cells bearing the CXCR4 receptor, whether these cells are stem cells (i.e., are CD34+ cells) and / or progenitor cells (which are either CD34+ or CD34- and can lead to the formation of a specific type of colony in response to a specific stimulus) or slightly more differentiated cells.

[0081] Recently, considerable attention has been focused on the number of CD34+ cells mobilized in the peripheral blood progenitor cell population for autologous stem cell transplantation. The CD34+ population is considered to be the component that is primarily responsible for improving recovery time after chemotherapy, and the cells are most likely responsible for long-term engraftment and hematopoietic recovery (Croop, JM, et al., Bone Marrow Transplantation (2000) 26: 1271-1279). The mechanism of CD34+ cell re-engraftment 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, adult hematopoietic stem cells have been shown to be able to restore damaged cardiac tissue in mice (Jackson, K., et al., J. Clin. Invest. (2001) 107: 1395-1402; Kocher, A., et al., Nature Med. (2001) 7: 430-436). Therefore, the role of the CXCR4 receptor in controlling cell localization and differentiation is considered to be quite important.

[0082] As used herein, the term "progenitor cell" refers to a cell that can form differentiated hematopoietic or myeloid cells in response to certain stimuli. The presence of progenitor cells can be assessed by the ability of the cells in the sample to form various types of colony-forming units, including, for example, CFU-GM (colony-forming units, granulocyte-macrophages); CFU-GEMM (colony-forming units, multipotent); BFU-E (burst-forming units, erythroid); HPP-CFC (high proliferation potential colony-forming cells); or other types of differentiated colonies that can be obtained in culture using known protocols.

[0083] "Stem" cells used herein are less differentiated forms of progenitor cells. Typically, such cells are usually CD34 positive. However, some stem cells do not contain this marker. These CD34+ cells can be measured using fluorescence activated cell sorting (FACS), so their presence in the sample can be assessed using this technique. Typically, CD34+ cells are only present at low levels in the blood, but are present in large quantities in the bone marrow. Although other types of cells such as endothelial cells and mast cells can also show this marker, CD34 is considered to be an indicator of the presence of stem cells.

[0084] As used herein, the term "CXCR4" refers to CXC motif chemokine receptor 4, also identified by a unique database identifier (ID) and alternative names 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 nomenclature of CXCR4 and GPCRx, which forms heteromers with CXCR4 and synergistically enhances Ca2+ responses when co-stimulated with two agonists.

[0085] Table 1

[0086]

[0087]

[0088]

[0089]

[0090] *GCID: Genecards ID

[0091] HGNC: HUGO Gene Nomenclature Committee

[0092] As used herein, the term "GPCRx" refers to GPCRs that were used in this study to investigate whether these GPCRs interact with CXCR4 and show properties different from those of the individual protomers, including ADCYAP1R1 (ADCYAP receptor type I), ADORA2B (adenosine A2b receptor), ADORA3 (adenosine A3 receptor), ADRB2 (adrenergic receptor β2), APLNR (apelin receptor), C5AR1 (complement C5a receptor 1), CALCR (calcitonin receptor), CCR 5 (chemokine (CC motif) receptor 5), CHRM1 (cholinergic muscarinic 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), also identified by unique database identifiers (IDs) and alternative names as shown in Table 1.

[0093] The term "inhibitor" as used herein refers to a molecule that inhibits or suppresses the enhanced function of CXCR4, β-adrenergic receptors, GPCRs, heteromers of CXCR4 and β-adrenergic receptors, and / or CXCR4-GPCRx heteromers. Non-limiting examples of 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 connected to a pharmacophore selective for GPCRx via a spacer arm, bispecific antibodies for CXCR4 and GPCRx, radiolabeled CXCR4 ligands connected to GPCRx ligands, and small molecule ligands that inhibit heteromer selective signaling. Table 2 lists some examples of inhibitors for GPCRx that form heteromers with CXCR4 and enhance Ca2+ responses after costimulation with two agonists.

[0094] The term "antagonist" as used herein refers to a type of receptor ligand or drug that blocks or attenuates a biological response by binding to and blocking a receptor, also referred to as a blocker. Antagonists have affinity for their cognate receptors but are ineffective, and their binding disrupts the interaction and inhibits the function of agonists or inverse agonists on cognate receptors. Table 2 lists some examples of antagonists for GPCRx that form heteromers with CXCR4 and enhance Ca2+ responses after costimulation with two agonists.

[0095] Table 2. Examples of inhibitors targeting CXCR4 and ADRB2

[0096]

[0097] As used herein, the term "heteromer" refers to a macromolecular complex composed of at least two GPCR units [protomers] that has biochemical properties that are distinct from those of its individual components. Heteromerization can be assessed 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).

[0098] As used herein, the phrase "effective amount" refers to an amount sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on many variables, including the time period over which a single dosage unit is used, the bioavailability of the agent, the route of administration, etc.

[0099] As used herein, the phrase "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., an inhibitor, antagonist, or any other therapeutic agent provided herein) sufficient to reduce, improve, and / or prevent the severity and / or duration of cancer and / or symptoms associated therewith. A therapeutically effective amount of a therapeutic agent can be an amount required to reduce, improve, or prevent the development or progression of cancer, slow down, improve, or prevent the recurrence, development, or onset of cancer, and / or improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than administering an inhibitor, antagonist, or any other therapeutic agent provided herein).

[0100] The phrase "therapeutic agent" refers to any agent that can be used to treat, improve, prevent or control cancer and / or symptoms associated therewith. In certain embodiments, the therapeutic agent refers to an inhibitor of the CXCR4-GPCRx heteromer of the present invention. The therapeutic agent can be a well-known agent that can be used or has been used or is currently used to treat, improve, prevent or control cancer and / or symptoms associated therewith.

[0101] As used herein, the phrases "intracellular Ca2+ assay," "calcium mobilization assay," or variations thereof, refer to a cell-based assay that measures calcium flux associated with GPCR activation or inhibition. The method utilizes a calcium-sensitive fluorescent dye that is taken up into the cytoplasm of most cells. The dye binds calcium released from intracellular stores and its fluorescence increases. The change in fluorescence intensity is directly related to the amount of intracellular calcium released into the cytoplasm in response to ligand activation of the target receptor.

[0102] The phrase "proximity-based assays" as used herein refers to biophysical and biochemical techniques capable of monitoring the proximity and / or binding of two protein molecules in vitro (in cell lysates) and in living cells, including bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), proximity ligation assay (PLA), cysteine ​​cross-linking and co-immunoprecipitation (Ferre et al., 2009; Gomes et al., 2016).

[0103] Disclosed herein are 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. In some embodiments, mobilizing cells in a subject includes blocking CXCR4. In some embodiments, mobilizing cells in a subject includes blocking β-adrenergic receptors. In some embodiments, mobilizing cells in a subject includes blocking GPCRs. In some embodiments, mobilizing cells in a subject includes blocking CXCR4 and β-adrenergic receptors. In some embodiments, mobilizing cells in a subject includes blocking CXCR4 and GPCRs. In some embodiments, mobilizing cells in a subject includes blocking CXCR4-GPCR heteropolymers.

[0104] 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. Also 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. In embodiments, blocking β-adrenergic receptor signaling is performed before blocking CXCR4 signaling. In some embodiments, blocking β-adrenergic receptor signaling is performed at a first specific time interval before blocking CXCR4 signaling. In some embodiments, the first specific time interval is between 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 embodiments, blocking beta-adrenergic receptor signaling continues after blocking CXCR4 signaling is terminated. In some embodiments, after blocking CXCR4 signaling is terminated, blocking β-adrenergic receptor signaling continues for a second specific time interval. In some embodiments, the second specific time interval is between 5 minutes and 10 minutes, 10 minutes and 20 minutes, 20 minutes and 30 minutes, 30 minutes and 40 minutes, 40 minutes and 50 minutes, 50 minutes and 1 hour, 1 hour and 2 hours, 2 hours and 3 hours, 3 hours and 4 hours, 4 hours and 5 hours, 5 hours and 6 hours, 6 hours and 12 hours, 12 hours and 24 hours, 1 day and 2 days, 2 days and 3 days, 3 days and 4 days, 4 days and 5 days, 5 days and 6 days, 6 days and 7 days, 7 days and 8 days, 8 days and 9 days, 9 days and 10 days, 10 days and 11 days, 11 days and 12 days, 12 days and 13 days, 13 days and 14 days, or 14 days or more days.

[0105] In embodiments, blocking CXCR4 signaling comprises administering to the subject a CXCR4 inhibitor.

[0106] In embodiments, blocking beta-adrenergic receptor signaling comprises administering a beta-adrenergic receptor inhibitor to the subject. In embodiments, blocking CXCR4 signaling comprises administering a CXCR4 inhibitor to the subject, and blocking beta-adrenergic receptor signaling comprises administering a beta-adrenergic receptor inhibitor to the subject. In embodiments, the cell is a stem cell. In some embodiments, the cell is an immune cell.

[0107] Disclosed herein is a method of 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 of 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 between 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 embodiments, administration of the beta-adrenergic receptor inhibitor continues after administration of the CXCR4 inhibitor is terminated. In some embodiments, the administration of the beta-adrenergic receptor inhibitor continues for a second specific time interval after the administration of the CXCR4 inhibitor is terminated. In some embodiments, the second specific time interval is between 5 minutes and 10 minutes, 10 minutes and 20 minutes, 20 minutes and 30 minutes, 30 minutes and 40 minutes, 40 minutes and 50 minutes, 50 minutes and 1 hour, 1 hour and 2 hours, 2 hours and 3 hours, 3 hours and 4 hours, 4 hours and 5 hours, 5 hours and 6 hours, 6 hours and 12 hours, 12 hours and 24 hours, 1 day and 2 days, 2 days and 3 days, 3 days and 4 days, 4 days and 5 days, 5 days and 6 days, 6 days and 7 days, 7 days and 8 days, 8 days and 9 days, 9 days and 10 days, 10 days and 11 days, 11 days and 12 days, 12 days and 13 days, 13 days and 14 days, or 14 days or more days.

[0108] In the embodiment, the β-adrenergic receptor inhibitor is an ADRB2 inhibitor. In the embodiment, the β-adrenergic receptor inhibitor is selected from alpranolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloclorolol, ICI 118551, ICYP, labetalol, levobetataxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A and timolol. In the embodiment, the β-adrenergic receptor inhibitor is selected from propranolol, nadolol and ICI118551. In the embodiment, the β-adrenergic receptor inhibitor is propranolol.

[0109] In an embodiment, the CXCR4 inhibitor is selected from 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-11-9[P2G] dimer, SDF1P2G, T134, T140, T22, TC 14012, TG-0054 (brixafor), 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). Brixafor is also known as GPC-100 or TG-0054. Plerixafor is also known as AMD3100 or Mozobil. In embodiments, the CXCR4 inhibitor is selected from AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (brixafor), PF-06747143, POL6326, and ulolumab (MDX1338 / BMS-936564). In embodiments, the CXCR4 inhibitor is TG-0054 (brixafor). In embodiments, the CXCR4 inhibitor is AMD3100 (plerixafor). In embodiments, the CXCR4 inhibitor is ulolumab (MDX1338 / BMS-936564).

[0110] In embodiments, administering a CXCR4 inhibitor to a subject comprises administering TG-0054 (brixafor) and propranolol. In embodiments, administering a CXCR4 inhibitor to a subject comprises administering AMD3100 (plerixafor) and propranolol. In embodiments, administering a CXCR4 inhibitor to a subject comprises administering ulolumab (MDX1338 / BMS-936564) and propranolol.

[0111] In embodiments, the method further comprises administering G-CSF to the subject. In embodiments, administering a β-adrenergic receptor inhibitor and a CXCR4 inhibitor to a 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 a CXCR4 inhibitor and G-CSF to a subject 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 a CXCR4 inhibitor and G-CSF to a subject in the absence of a β-adrenergic receptor inhibitor. In some embodiments, administering a CXCR4 inhibitor to a subject comprises administering TG-0054 (brixafor) and propranolol. In embodiments, administering a CXCR4 inhibitor to a subject comprises administering AMD3100 (plerixafor) and propranolol. In embodiments, administering a CXCR4 inhibitor to a subject comprises administering ululumab (MDX1338 / BMS-936564) and propranolol.

[0112] In embodiments, relative to the amount of cell mobilization induced only by CXCR4 inhibitors, the combination of CXCR4 inhibitors and G-CSF is used to induce the increased amount of cell mobilization. In embodiments, relative to the amount of cell mobilization induced only by CXCR4 inhibitors, the combination of CXCR4 inhibitors and G-CSF is used to mobilize the increased amount of cells. In some embodiments, relative to the amount of cell mobilization induced only by CXCR4 inhibitors, the increased amount of cell mobilization is 1.1 times to 1.2 times, 1.2 times to 1.3 times, 1.3 times to 1.4 times, 1.4 times to 1.5 times, 1.5 times to 1.6 times, 1.6 times to 1.7 times, 1.7 times to 1.8 times, 1.8 times to 1.9 times, 1.9 times to 2 times, 2 times to 2.5 times, 2.5 times to 3 times, 3 times to 4 times, 4 times to 5 times, 5 times to 10 times, or 10 times or more times. In some embodiments, the amount of increased cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 5%-10% more, 10%-20% more, 20%-30% more, 30%-40% more, 40%-50% more, 50%-60% more, 60%-70% more, 70%-80% more, 80%-90% more, 90%-100% more, 100%-120% more, 120%-140% more, 140%-160% more, 160%-180% more, 180%-200% more, 200%-250% more, 250%-300% more, 300%-400% more, 400%-500% more, 500%-750% more, 750%-1000% more, or 1000% more or more.

[0113] In embodiments, relative to the amount of cell mobilization induced only by CXCR4 inhibitors, the cell mobilization of the amount of increase induced by the combination of CXCR4 inhibitors and beta-adrenergic receptor inhibitors is applied. In embodiments, relative to the amount of cell mobilization induced only by CXCR4 inhibitors, the cell of the amount of increase is mobilized by the combination of CXCR4 inhibitors and beta-adrenergic receptor inhibitors. In some embodiments, relative to the amount of cell mobilization induced only by CXCR4 inhibitors, the amount of increase of cell mobilization is 1.1 times to 1.2 times, 1.2 times to 1.3 times, 1.3 times to 1.4 times, 1.4 times to 1.5 times, 1.5 times to 1.6 times, 1.6 times to 1.7 times, 1.7 times to 1.8 times, 1.8 times to 1.9 times, 1.9 times to 2 times, 2 times to 2.5 times, 2.5 times to 3 times, 3 times to 4 times, 4 times to 5 times, 5 times to 10 times, or 10 times or more times. In some embodiments, the amount of increased cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 5%-10% more, 10%-20% more, 20%-30% more, 30%-40% more, 40%-50% more, 50%-60% more, 60%-70% more, 70%-80% more, 80%-90% more, 90%-100% more, 100%-120% more, 120%-140% more, 140%-160% more, 160%-180% more, 180%-200% more, 200%-250% more, 250%-300% more, 300%-400% more, 400%-500% more, 500%-750% more, 750%-1000% more, or 1000% more or more.

[0114] In embodiments, relative to the amount of cell mobilization induced by only CXCR4 inhibitors and beta-adrenergic receptor inhibitors, the combination of CXCR4 inhibitors, beta-adrenergic receptor inhibitors and G-CSF induces an increased amount of cell mobilization. In embodiments, relative to the amount of cell mobilization induced by only CXCR4 inhibitors and beta-adrenergic receptor inhibitors, the combination of CXCR4 inhibitors, beta-adrenergic receptor inhibitors and G-CSF mobilizes an increased amount of cells. In embodiments, relative to the amount of cell mobilization induced by AMD3100 (plerixafor) and G-CSF, the combination of TG-0054 (brixafor) and G-CSF induces an increased amount of cell mobilization. In embodiments, relative to the amount of cell mobilization induced by AMD3100 (plerixafor) and G-CSF, the combination of TG-0054 (brixafor) and G-CSF mobilizes an increased amount of cells. In some embodiments, the amount of increase in cell mobilization is between 1.1-fold and 1.2-fold, 1.2-fold and 1.3-fold, 1.3-fold and 1.4-fold, 1.4-fold and 1.5-fold, 1.5-fold and 1.6-fold, 1.6-fold and 1.7-fold, 1.7-fold and 1.8-fold, 1.8-fold and 1.9-fold, 1.9-fold and 2-fold, 2-fold and 2.5-fold, 2.5-fold and 3-fold, 3-fold and 4-fold, 4-fold and 5-fold, 5-fold and 10-fold, or 10-fold or more relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone. In some embodiments, the amount of increase in cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 5%-10% more, 10%-20% more, 20%-30% more, 30%-40% more, 40%-50% more, 50%-60% more, 60%-70% more, 70%-80% more, 80%-90% more, 90%-100% more, or more. 100%-120%, more than 120%-140%, more than 140%-160%, more than 160%-180%, more than 180%-200%, more than 200%-250%, more than 250%-300%, more than 300%-400%, more than 400%-500%, more than 500%-750%, more than 750%-1000%, or more than 1000% or more. In embodiments, the increased amount of cell mobilization or apheresis is measured by a method selected from complete blood count (CBC) analysis, flow cytometry, and colony forming unit (CFU) assay. In embodiments, the increased amount of cell mobilization or apheresis is measured by flow cytometry. In embodiments, flow cytometry is performed on (Lin-Sca1+c-Kit+)LSK cells. In embodiments, the increased amount of cell mobilization or apheresis is measured by a colony forming unit (CFU) assay.

[0115] In embodiments, the subject has CXCR4 protomers in cells. In embodiments, the subject has ADRB2 protomers in cells. In embodiments, the subject has CXCR4 protomers and ADRB2 protomers in cells. In embodiments, the subject has CXCR4-ADRB2 heteromers in cells. In embodiments, i) the CXCR4-ADRB2 heteromers have an increased amount of downstream calcium mobilization relative to downstream calcium mobilization from CXCR4 protomers or ADRB2 protomers; and ii) the combination of inhibitors administered inhibits the enhanced downstream calcium mobilization of the CXCR4-ADRB2 heteromers in stem cells.

[0116] In embodiments, the cell is a stem cell. In embodiments, stem cells are selected from hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, endothelial progenitor cells, neural stem cells, epithelial stem cells, skin stem cells and cancer stem cells. In embodiments, stem cells are hematopoietic stem cells or hematopoietic progenitor cells. In embodiments, hematopoietic stem cells or hematopoietic progenitor cells are mobilized from the bone marrow to peripheral blood. In embodiments, mobilized hematopoietic stem cells or hematopoietic progenitor cells are collected for transplantation to patients with cancer. In embodiments, cancer is selected from lymphoma, leukemia and myeloma. In embodiments, the cancer is non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) or multiple myeloma (MM). In embodiments, stem cells are mesenchymal stem cells. In embodiments, mesenchymal stem cells are mobilized from the bone marrow to peripheral blood. In embodiments, mesenchymal stem cells are mobilized for the treatment of a condition selected from neurological disorders, 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 embodiments, the stem cells are cancer stem cells. In embodiments, cancer stem cells are mobilized into the blood. In embodiments, cancer stem cells are mobilized for the treatment of cancer.

[0117] In embodiments, the cell is an immune cell. In embodiments, the immune cell is a leukocyte. In embodiments, the leukocyte is a lymphocyte. In embodiments, the lymphocyte is selected from T cells, B cells and natural killer (NK) cells. In embodiments, the lymphocyte is a T cell. In embodiments, the lymphocyte is a natural killer (NK) cell. In embodiments, the leukocyte is a granulocyte. In embodiments, the granulocyte is selected from neutrophils, eosinophils and basophils. In embodiments, the granulocyte is a neutrophil. In embodiments, the leukocyte is a monocyte. In embodiments, immune cells are mobilized from the bone marrow to the peripheral blood. In embodiments, immune cells are mobilized from lymph nodes to the peripheral blood. In embodiments, mobilized immune cells are used for adoptive cell therapy (ACT). In embodiments, the adoptive cell therapy (ACT) is a chimeric antigen receptor (CAR) T cell therapy. In embodiments, adoptive cell therapy (ACT) is a natural killer (NK) cell therapy. In embodiments, adoptive cell therapy (ACT) is an engineered T cell receptor (TCR) therapy. In embodiments, the adoptive cell therapy (ACT) is tumor infiltrating lymphocyte (TIL) therapy.

[0118] 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 (co-receptor required for fusion and entry into T-tropic HIV strains) also inhibit binding and signaling induced by natural ligands, chemokine SDF-1 (also known as CXCL12). Although it is not desired to be bound by any theory, the medicament that inhibits SDF-1 from binding to CXCR4 can achieve an increase in the mobilization of stem cells and / or progenitor cells to the periphery through such inhibition. Enhancing the mobilization of stem cells and / or progenitor cells to peripheral blood helps to alleviate the impact of treatment regimens that adversely affect the bone marrow, such as those that cause leukopenia, a known side effect of chemotherapy and radiotherapy. The medicament that inhibits SDF-1 from binding to CXCR4 also enhances the success of bone marrow transplantation, enhances wound healing and burn treatment, and contributes to the recovery of damaged organ tissue. They also combat bacterial infections that are prevalent in leukemia. They are used to mobilize and harvest CD34+ cells with and without other mobilization factors by apheresis. The harvested cells are used for treatment requiring stem cell transplantation.

[0119] In some embodiments of the invention, mobilizing stem cells in a subject comprises blocking CXCR4-GPCR heteromers. Various CXCR4-GPCR heteromers with different physiological and pharmacological properties have been reported, but their roles in stem cell mobilization or the possibility of developing stem cell mobilization therapeutics targeting CXCR4-GPCR heteromers have not been clearly understood or recognized.

[0120] In the art, GPCRs are thought to act as monomers that interact with heterotrimeric G proteins upon ligand binding, and drugs are developed based on monomeric or homomeric GPCRs (Milligan 2008). Recently, this view has changed dramatically 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, as well as 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 display different functional and pharmacological properties, and GPCR heteromerization can vary depending on cell type, tissue, and disease or pathological conditions (Terrillon and Bouvier 2004; Ferre et al., 2010; Rozenfeld and Devi 2010; Gomes et al., 2016; Farran 2017). GPCR heteromerization is now considered a universal phenomenon, and deciphering GPCR heteromerization opens new avenues for understanding receptor function, physiology, and roles in disease and pathological conditions. Therefore, the identification of GPCR heteromers and their functional properties provides new opportunities to develop new drugs or discover new uses for old drugs with fewer side effects, higher efficacy, and increased tissue selectivity ((Ferre et al., 2010; Rozenfeld and Devi 2010; Farran 2017).

[0121] Apheresis is a standard practice for obtaining large numbers of immune cells as starting material for adoptive cell therapy (ACT) based on the transfer of cells into a patient for treatment (1-3). Apheresis may involve passing a patient's blood through a device that separates out a specific component and returns the remainder to the patient's blood circulation. Apheresis is therefore an extracorporeal therapy. Depending on the substance being removed, different methods are used in apheresis. If separation by density is required, centrifugation is the most common method. Other methods include absorption on beads coated with absorbent material and filtration. Centrifugation can be divided into two basic categories: continuous flow centrifugation (CFC) and intermittent flow centrifugation.

[0122] CFC historically required two venipunctures because "continuous" meant that the blood was collected, spun, and returned simultaneously. Newer systems can use a single venipuncture. The main advantage of CFC is the low extracorporeal volume used in the procedure (calculated by the volume of the apheresis chamber, the donor's hematocrit, and the donor's total blood volume), which is advantageous in the elderly and children. The intermittent flow centrifuge cycle works by drawing blood, spinning / processing the blood, and then returning the unused portion to the donor in the form of a bolus. The main advantage is the single venipuncture site. To stop the blood from clotting, an anticoagulant is automatically mixed with the blood as it is pumped from the body into the apheresis machine.

[0123] A variety of apheresis techniques can be used whenever the component being removed causes serious symptoms of disease in the patient. Generally, apheresis must be performed fairly frequently and is an invasive procedure. Therefore, it is often used if other methods of controlling a particular disease have failed, or if the symptoms are of a nature that will cause pain or risk of complications if medications are effective. Apheresis techniques include: (1) plasmapheresis - the removal of the liquid portion of the blood to remove harmful substances, in which the plasma is replaced with a replacement solution; (2) LDL apheresis - the removal of low-density lipoprotein in patients with familial hypercholesterolemia; (3) photochemotherapy - used to treat graft-versus-host disease, cutaneous T-cell lymphoma, and cardiac transplant rejection; (4) immunoadsorption with staphylococcal protein A-agarose columns - the removal of alloantibodies and autoantibodies by directing plasma through a protein A-agarose column (protein A is a cell wall component produced by several strains of Staphylococcus aureus that binds to the Fc region of IgG) (5) leukapheresis - removal of malignant white blood cells from people with leukemia and those with high white blood cell counts that cause symptoms; (6) erythrapheresis - removal of red blood cells (erythrocytes) from patients with iron overload due to hereditary hemochromatosis or transfusional iron overload; (7) plateletpheresis - removal of platelets from patients with symptoms of extremely elevated platelet counts, such as essential thrombocythaemia or polycythaemia vera; and (8) leukapheresis - separation of excess white blood cells from leukemia patients while recycling the rest of the blood.

[0124] Apheresis is a difficult procedure that is inconvenient and expensive. With the rapid growth of ACTs, including CAR-T, CAR-NK, tumor-infiltrating lymphocytes (TIL), and engineered T cell receptors (TCR), the demand for apheresis technology for routine production of pure immune cells is increasing (2). The industry that provides GMP-grade starting materials for ACTs is also growing rapidly (4-5). Therefore, stem cell mobilization technologies that can control immune cell types and increase the yield of apheresis have become important.

[0125] Enhanced stem cell mobilization (SCM) or cell mobilization methods disclosed herein can further enhance or promote conventional single blood component procedures. In a specific embodiment, enhanced stem cell mobilization (SCM) or cell mobilization is particularly beneficial for single blood component technology for leukocyte single collection. In some embodiments, CXCR4 antagonists are administered to subjects to further increase single blood components by increasing SCM or cell mobilization. In some embodiments, beta-adrenergic receptor antagonists are administered to subjects in combination with CXCR4 antagonists, by increasing SCM or cell mobilization, and / or replacing the G-CSF component of the treatment regimen with non-selective beta-blockers such as propranolol, further increasing single blood components. In some embodiments, the enhancement of SCM is beneficial to HSCT (hematopoietic stem cell transplantation) or the manufacture of CAR-T cells for cancer immunotherapy. Currently, CXCR4 inhibitors, such as plerixafor (Mozobil), which has been approved as a stem cell mobilizer, are used with G-CSF as the standard of care to provide enriched hematopoietic stem and progenitor cells from healthy donors, marketed as the product "mobilized leukopaks."

[0126] 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. Also 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 embodiments, blocking β-adrenergic receptor signaling is performed prior to blocking CXCR4 signaling. In some embodiments, blocking β-adrenergic receptor signaling is performed at a first specific time interval prior to blocking CXCR4 signaling. In some embodiments, the first specific time interval is between 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 embodiments, blocking beta-adrenergic receptor signaling continues after blocking CXCR4 signaling is terminated. In some embodiments, after blocking CXCR4 signaling, blocking β-adrenergic receptor signaling continues for a second specific time interval. In some embodiments, the second specific time interval is between 5 minutes and 10 minutes, 10 minutes and 20 minutes, 20 minutes and 30 minutes, 30 minutes and 40 minutes, 40 minutes and 50 minutes, 50 minutes and 1 hour, 1 hour and 2 hours, 2 hours and 3 hours, 3 hours and 4 hours, 4 hours and 5 hours, 5 hours and 6 hours, 6 hours and 12 hours, 12 hours and 24 hours, 1 day and 2 days, 2 days and 3 days, 3 days and 4 days, 4 days and 5 days, 5 days and 6 days, 6 days and 7 days, 7 days and 8 days, 8 days and 9 days, 9 days and 10 days, 10 days and 11 days, 11 days and 12 days, 12 days and 13 days, 13 days and 14 days, or 14 days or more days.

[0127] In embodiments, blocking CXCR4 signaling comprises administering to the subject a CXCR4 inhibitor.

[0128] 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. Also 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, administration of the β-adrenergic receptor inhibitor is performed at a first specific time interval prior to administration of the CXCR4 inhibitor. In some embodiments, the first specific time interval is between 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 embodiments, administration of the beta-adrenergic receptor inhibitor continues after administration of the CXCR4 inhibitor is terminated. In some embodiments, the administration of the beta-adrenergic receptor inhibitor continues for a second specific time interval after the administration of the CXCR4 inhibitor is terminated. In some embodiments, the second specific time interval is between 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 days.

[0129] In the embodiment, the β-adrenergic receptor inhibitor is an ADRB2 inhibitor. In the embodiment, the β-adrenergic receptor inhibitor is selected from alpranolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloclorolol, ICI 118551, ICYP, labetalol, levobetataxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A and timolol. In the embodiment, the β-adrenergic receptor inhibitor is selected from propranolol, nadolol and ICI118551. In the embodiment, the β-adrenergic receptor inhibitor is propranolol.

[0130] In an embodiment, the CXCR4 inhibitor is selected from 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 (ITlt), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-11-9[P2G] dimer, SDF1P2G, T134, T140, T22, TC 14012, TG-0054 (brixafor), 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 embodiments, the CXCR4 inhibitor is selected from AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (brixafor), PF-06747143, POL6326, and ulolumab (MDX1338 / BMS-936564). In embodiments, the CXCR4 inhibitor is TG-0054 (brixafor). In embodiments, the CXCR4 inhibitor is AMD3100 (plerixafor). In embodiments, the CXCR4 inhibitor is ulolumab (MDX1338 / BMS-936564).

[0131] In embodiments, administering a CXCR4 inhibitor to a subject comprises administering TG-0054 (brixafor) and propranolol. In embodiments, administering a CXCR4 inhibitor to a subject comprises administering AMD3100 (plerixafor) and propranolol. In embodiments, administering a CXCR4 inhibitor to a subject comprises administering ulolumab (MDX1338 / BMS-936564) and propranolol.

[0132] In embodiments, the method further comprises administering G-CSF to the subject. In embodiments, administering a β-adrenergic receptor inhibitor and a CXCR4 inhibitor to a subject is performed in the absence of G-CSF. Disclosed herein is a method for enhancing apheresis in a subject, the method comprising: administering a CXCR4 inhibitor and G-CSF to a subject in the absence of a β-adrenergic receptor inhibitor. Also disclosed herein is a method for enhancing apheresis by inducing cell mobilization in a subject, the method comprising: administering a CXCR4 inhibitor and G-CSF to a subject in the absence of a β-adrenergic receptor inhibitor. Also disclosed herein is a method for enhancing apheresis by mobilizing cells in a subject, the method comprising: administering a CXCR4 inhibitor and G-CSF to a subject in the absence of a β-adrenergic receptor inhibitor. In embodiments, administration of a combination of a CXCR4 inhibitor and G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by a CXCR4 inhibitor alone. In embodiments, administration of a combination of a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor induces an increased amount of apheresis relative to the amount of apheresis induced by a CXCR4 inhibitor alone. In embodiments, administration of a combination of a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor and G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor alone. In embodiments, administration of a combination of TG-0054 (brixafor) and G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by AMD3100 (plerixafor) and G-CSF. In some embodiments, the amount of increase in cell mobilization is between 1.1-fold and 1.2-fold, 1.2-fold and 1.3-fold, 1.3-fold and 1.4-fold, 1.4-fold and 1.5-fold, 1.5-fold and 1.6-fold, 1.6-fold and 1.7-fold, 1.7-fold and 1.8-fold, 1.8-fold and 1.9-fold, 1.9-fold and 2-fold, 2-fold and 2.5-fold, 2.5-fold and 3-fold, 3-fold and 4-fold, 4-fold and 5-fold, 5-fold and 10-fold, or 10-fold or more relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.In some embodiments, the amount of increase in cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone is 5%-10% more, 10%-20% more, 20%-30% more, 30%-40% more, 40%-50% more, 50%-60% more, 60%-70% more, 70%-80% more, 80%-90% more, 90%-100% more, or more. 100%-120%, more than 120%-140%, more than 140%-160%, more than 160%-180%, more than 180%-200%, more than 200%-250%, more than 250%-300%, more than 300%-400%, more than 400%-500%, more than 500%-750%, more than 750%-1000%, or more than 1000% or more. In embodiments, the increased amount of cell mobilization or single blood components is measured by a method selected from complete blood count (CBC) analysis, flow cytometry, and colony forming unit (CFU) assay. In embodiments, the increased amount of cell mobilization or single blood components is measured by flow cytometry. In embodiments, flow cytometry is performed on (Lin-Scal+c-Kit+)LSK cells. In embodiments, the increased amount of cell mobilization or apheresis is measured by a colony forming unit (CFU) assay.

[0133] Further information regarding ADRB2 (assessed herein as forming a heteromer with CXCR4) is detailed below:

[0134] ADRB2--beta-2 adrenergic receptor (β2 adrenergic receptor), also known as ADRB2, is a transcellular beta-adrenergic receptor that interacts with epinephrine, a hormone and neurotransmitter (ligand synonym, adrenal hormone), whose signaling mediates physiological responses such as smooth muscle relaxation and bronchial dilation through L-type calcium channel interactions (Gregorio et al., 2017). ADRB2 plays a role in the muscular system (such as smooth muscle relaxation, motor nerve endings, glycogenolysis) and the circulatory system (such as myocardial contraction, increased cardiac output). In the normal eye, stimulation of beta-2 by salbutamol increases intraocular pressure through the network. In the digestive system, ADRB2 induces glycogenolysis and gluconeogenesis in the liver and insulin secretion in the pancreas (Fitzpatrick, 2004).

[0135] ADRB2 signaling in cardiomyocytes is regulated by interaction with CXCR4 (LaRocca et al., 2010). Norepinephrine attenuates CXCR4 expression and the corresponding invasion of MDA-MB-231 breast cancer cells through 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), renal cancer, and breast cancer (Choy et al., 2016).

[0136] Alternative methods for detecting heteromer formation include, but are not limited to: 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); β-arrestin recruitment assay, using GPCR Heteromer Identification Technology (GPCR-HIT, Dimerix Bioscience) (Mustafa and Pfleger, 2011), using BRET, FRET, BiFC, bimolecular luminescent complementation, enzyme fragmentation assay and Tango Tango GPCR detection system (Thermo Fisher Scientific) Scientific)(Mustafa, 2010); 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).

[0137] Alternative methods for detecting changes in pharmacological, signaling, and / or trafficking properties in cells expressing both CXCR4 and GPCRx include, but are not limited to: 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]GTPrS binding assays (Bushlin et al., 2012); calcium imaging or assays using dyes such as Fura2-acetylmethoxy ester (Molecular Probes), Fluo-4NW calcium dye (Thermo Fisher Scientific), or FLIPR5 dye (Molecular Devices); cAMP assays using radioimmunoassay kits (Amersham Biosciences); AlphaScreen (PerkinElmer Life Sciences); parametric cyclic AMP assays (R&D Biotechnology, Inc., New York, NY); cAMP direct immunoassay kit (Calbiochem) or GloSensor cAMP assay (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 assay; secreted alkaline phosphatase assay (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 that can detect alterations in receptor function due to receptor heterodimerization.

[0138] As used herein, the phrase "protein-protein interaction inhibitor", "PPI inhibitor" or variants thereof refers to any molecule that can interfere with protein-protein interactions. Unlike enzyme-substrate interactions, which involve well-defined binding pockets, protein-protein interactions are transient interactions or associations between proteins over relatively large areas and are typically driven by electrostatic interactions, hydrophobic interactions, hydrogen bonds, and / or van der Waals forces. PPI inhibitors may include, but are not limited to, membrane permeable peptides or lipids fused to peptide sequences that disrupt GPCR heteromeric interfaces (e.g., transmembrane helices, intracellular loops, or the C-terminal tail of GPCRx). The PPI inhibitor of CXCR4-GPCRx heteromers, for example, can be a membrane permeable peptide or cell penetrating peptide (CPP) conjugated to a peptide targeting the CXCR4-GPCRx heteromeric interface, or can be a cell penetrating lipidated peptide targeting the CXCR4-GPCRx heteromeric interface.

[0139] For example, the membrane permeable peptide or cell penetrating peptide includes: HIV-1 TAT peptide, such as TAT 48-60 and TAT 49-57 ; membrane-penetrating peptides, such as pAntp (43-58); polyarginine (Rn such as R5 to R12); Diatos peptide vector 1047 (DPV1047, ); MPG (HIV gp41 fused to the nuclear localization signal (NLS) of the SV40 large T antigen); Pep-1 (tryptophan-rich cluster fused to the NLS of the SV40 large T antigen); pVEC peptide (vascular endothelial cadherin); ARF (1-22) based on the p14 alternative reading frame (ARF) protein; the N-terminus of the unprocessed bovine prion protein BPrPr (1-28); model amphipathic peptide (MAP); transport peptide (Transportan); azurin-derived p28 peptide; amphipathic β-fold peptides, such as VT5; Proline-rich CPPs, such as Bac7 (Bacl-24); hydrophobic CPPs, such as α1-antitrypsin-derived C105Y; 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 penetrating peptides may further include, for example, TAT-derived cell penetrating peptides, cell penetrating peptides based on signal sequences (e.g., NLS), hydrophobic membrane transport sequence (MTS) peptides, and arginine-rich molecular transporters. Cell penetrating lipidated peptides include, for example, pepducins, such as ICL1 / 2 / 3, C-tail-short palmitoylated peptides (Covic et al., 2002; O'Callaghan et al., 2012).

[0140] The peptide targeting 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.

[0141] It should be understood that modifications that do not substantially affect the activity of the various embodiments of the invention are also provided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but not to limit the invention disclosed herein.

[0142] Example

[0143] Example 1. Combined blockade of β2 adrenergic receptor and CXCR4 signaling in stem cell mobilization: preclinical evidence.

[0144] To identify novel CXCR4-GPCRx heteromers, recombinant adenovirus 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 Venus were prepared as described by Song et al. (Song et al., 2014; SNU patent; Song, paper). CXCR4-GPCR heteromers were identified using a bimolecular fluorescence complementation (BiFC) assay (Figure 1), in which the two complementary VN and VC fragments of Venus reconstruct a fluorescent signal only when the two fragments are close enough through the interaction between the two different proteins fused to them (Hu et al., 2002).

[0145] Preclinical studies evaluated the ability of propranolol, a non-selective beta-adrenergic receptor blocker, to improve GPC100-induced stem cell mobilization in a mouse model after seven days of treatment. These effects were further evaluated by adding G-CSF to GPC100 and compared to current standard of care treatments for stem cell mobilization, such as G-CSF alone or in combination with AMD3100.

[0146] Materials and methods

[0147] Compound

[0148] Propranolol (MedChem Express, Princeton, NJ) was administered intraperitoneally (IP) at 20 mg / kg once a day for seven days. Recombinant mouse G-CSF (Peprotech, Cranbury, NJ) was administered subcutaneously (SC) at 0.1 mg / kg / dose twice a day for 5 days. AMD3100 (MedChem Express, Princeton, NJ) was administered once subcutaneously on day 7 at 5 mg / kg. GPC100 was administered once intravenously (IV) at 30 mg / kg on day 7. GPC10 was obtained from TaiGen Biotechnology, Taiwan, China by GPCR Therapeutics. All compounds were reconstituted in PBS. Vehicle controls received PBS intravenously, intraperitoneally, or subcutaneously, depending on the drug combination used in the study.

[0149] Mouse

[0150] C57BL / 6 and BALB / c mice (female, 6-9 weeks old) were purchased from Jackson Laboratory and maintained in a 12-hour light / dark cycle with free access to food and water. All mice were housed in the laboratory animal facilities of Crown Bioscience (San Diego, CA) or Explora Biolabs (San Carlos, CA), which have been accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) and IACUC (Institutional Animal Care and Use Committee).

[0151] Experimental design

[0152] For preliminary studies, C57BL / 6 and BALB / c mice were administered a single dose of GPC100 (30 mg / kg, IV) or vehicle (IV) and blood was collected one hour later. Another group of C57 / BL6 mice received a single dose of GPC100 (30 mg / kg, IV) and blood was collected 30 minutes, 1 hour, and 2 hours after injection. Based on maximum WBC mobilization, the time point for sample collection after GPC100 was established at 2 hours ( Figure 1B ). All subsequent studies were performed in C57 / BL6 female mice because this mouse strain has been more rigorously evaluated in stem cell mobilization studies.

[0153] To determine the effect of propranolol on GPC100-induced mobilization, mice received 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 alters blood cell counts, it was administered for 7 days followed by an intravenous vehicle injection on day 7. Mice were treated with propranolol or vehicle for 7 days; GPC100 or vehicle was co-administered on day 7 to determine the effect of propranolol alone, GPC100 alone, or a combination thereof on total blood cell counts in peripheral blood (Table 3).

[0154] Table 3 Dosing regimen for propranolol and GPC100 combination therapy.

[0155]

[0156] Table 4 Dosing regimen for standard of care treatment

[0157]

[0158] Table 5 Dosing regimen of the combination of propranolol and G-CSF+AMD3100

[0159]

[0160]

[0161] In another study, mice received G-CSF (0.1 mg / kg, SC, BID) for five consecutive days (days 2 to 6) with or without propranolol. On day 7, GPC100 (30 mg / kg, IV) was co-administered with propranolol or alone (Table 5).

[0162] The effect of propranolol on GPC100-induced mobilization was compared with the current standard of care for stem cell mobilization, 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) on the following day (Table 4).

[0163] Blood was collected by terminal cardiac puncture 2 hours after GPC100 administration and 1 hour after AMD3100 administration. Complete blood cell counts were obtained by Abaxis hematology analyzer (Abaxis, Union City, CA). In all studies, circulating WBC counts were used as an indicator of stem cell mobilization.

[0164] The effect of propranolol on GPC100-induced mobilization was evaluated in all six studies. In four studies, the effect was compared with the standard of care treatment of G-CSF and AMD3100 combination. In the last three studies, G-CSF was added to GPC100 with (triple) or without (double) propranolol. The effect of GPC100, AMD3100 and G-CSF in combination with vehicle was evaluated in one study. No data point was deleted unless the sample showed clotting before CBC analysis.

[0165] Mice treated with vehicle showed 3.4+ / -1.8×10 3 The mean WBC count was 2.6 + / - 1.2 × 10 cells / μL. 3 Lymphocyte counts were calculated as 10 cells / μL. Vehicle-treated mice were included as controls in all studies, although not shown in the data graphs.

[0166] Colony forming unit (CFU) assay

[0167] In one of the six studies, mobilization of hematopoietic progenitor cells was evaluated by CFU assay in addition to WBC counts. Mice were dosed at Crown Bioscience (Sandiego, CA), and blood was shipped overnight at room temperature in heparinized tubes to Reach Bio Research (Seattle, CA). Approximately 8 × 10 4 The cells were incubated in a methylcellulose-based medium supplemented with cytokines known to support erythroid and myeloid progenitor cells. The cultures were incubated in a humidified incubator for approximately 7 days, after which colonies were scored by trained personnel.

[0168] Statistical analysis

[0169] Data analysis was performed using Prism (GraphPad), and all data are expressed as mean values ​​(mean ± SD). The data shown in one figure were generated during the same experiment. Data comparisons under different dosing conditions were performed using repeated measures one-way analysis of variance followed by Turkey'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.

[0170] result

[0171] GPC100 increases circulating WBC counts in mice

[0172] A single intravenous administration of GPC100 (30 mg / kg) resulted in a rapid increase in circulating WBCs in C57 / BL6 and Balb / c mice, reflecting stem cell mobilization ( Figure 1A To determine the time course of GPC-100-induced mobilization, GPC-100 (30 mg / kg) was administered intravenously to naive C57 / B16 mice were used, and peripheral blood was collected from different groups at 0.5, 1, and 2 hours after injection. A time-dependent increase in WBC counts was observed, and the sample collection at 2 hours after injection was selected for subsequent studies ( Figure 1B ). In addition, C57 / BL6 mice were chosen rather than Balb / c mice for further studies because hematopoietic stem cell mobilization has been rigorously evaluated in this mouse strain (Broxmeyer et al. 2005).

[0173] GPC100 administration resulted in significantly more WBCs in peripheral blood compared to AMD3100 when administered with or without G-CSF.

[0174] A 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 GPC100 and AMD3100 were combined with 5 days of G-CSF treatment, further increases in WBC counts were observed in all three studies. However, this increase was more significant for GPC100 compared to AMD3100. This supports further evaluation of GPC100 as an effective stem cell mobilizer in the clinic ( Figure 2B ).

[0175] Propranolol enhances GPC100-induced WBC mobilization into peripheral blood.

[0176] Data from 6 studies showed that 7-day pretreatment with propranolol significantly increased GPC100-induced peripheral blood WBC counts compared with 7-day vehicle pretreatment ( Figure 3 When propranolol was administered alone for seven days without GPC100 on day 7, no changes in blood cell counts were observed (Table 6). These data suggest that propranolol can improve bone marrow cellularity, thereby enabling GPC100 to mobilize more cells.

[0177] Table 6 Treatment with propranolol alone for 7 days does not change blood cell counts

[0178]

[0179] The combination treatment of propranolol and GPC100 increased WBC counts comparable to that achieved by G-CSF.

[0180] In 3 of 6 mice, mice that were pretreated with propranolol and then administered GPC100 mobilized leukocytes to an extent comparable to that of mice that received G-CSF ( Figure 4 ). Since propranolol can be safely administered orally to patients, its administration does not cause the inconvenience and side effects associated with G-CSF. More preclinical studies comparing these two groups are needed.

[0181] The standard of care treatment of G-CSF and AMD3100 combination mobilized significantly more WBCs, but not lymphocytes, than the propranolol and GPC100 combination.

[0182] Data collected from 4 studies showed that the combination of G-CSF and AMD3100 resulted in significant changes in mobilization ( Figure 5A -B). Mobilization induced by standard of care treatment was significantly greater than total WBC counts for the GPC100 and propranolol combination treatment. ( Figure 5A ). However, comparison of lymphocyte counts showed that the standard of care regimen was comparable to the combination of GPC100 and propranolol ( Figure 5B ).

[0183] The addition of propranolol to GPC100 and G-CSF resulted in greater mobilization of WBCs and hematopoietic progenitor cells compared to standard of care treatment.

[0184] The addition of G-CSF enhanced the mobilization of GPC100 with or without pretreatment with propranolol. This experiment compared the number of WBC and colony forming units (progenitor cells) in the peripheral blood of the same mice. Fig. 6A ) and progenitor cells ( Figure 6B ), the triple combination including propranolol, G-CSF and GPC100 was shown to induce the greatest mobilization compared to the G-CSF combination treatment without propranolol. When a similar study was repeated, the triple combination was significantly superior to the combination treatment of AMD3100 and G-CSF ( Fig. 7A ).

[0185] Propranolol enhances GPC100-induced lymphocyte mobilization into peripheral blood

[0186] The data pooled from all four experiments indicated that GPC100 mobilized more WBCs compared to AMD3100 when combined with G-CSF. The addition of propranolol to the GPC100 and G-CSF combination treatment mobilized more WBCs, including lymphocytes ( Fig. 7A -B). Adding propranolol to the combination of G-CSF and GPC100 did not affect lymphocyte counts ( Figure 7BThe distribution of WBC differential counts indicated that the addition of propranolol increased the influx of lymphocytes into the peripheral blood ( Figure 7C ). Figure 7C The lymphocyte data are expressed as % of the total leukocyte count ( Figure 7C ), of which the lymphocyte count in the vehicle / vehicle group was 2.6+ / -1.2×10 3 cells / μL).

[0187] This provides compelling preclinical evidence for further investigation of the effect of propranolol on GPC100-induced lymphocyte mobilization.

[0188] discuss

[0189] The studies described herein show that propranolol increases GPC100-induced mobilization in the absence of G-CSF. However, the addition of G-CSF further enhanced the mobilization effect of GPC100, regardless of whether propranolol was used. Whether propranolol enhances mobilization through combination therapy of GPC100 and G-CSF remains unclear. In patients with multiple myeloma, propranolol has been shown to suppress molecular risk markers in hematopoietic stem cell transplantation, a phenomenon currently being studied in mice by evaluating changes in inflammatory cytokines after propranolol treatment. The studies disclosed herein were conducted in naive or non-tumor-bearing mice, which may not have the stress response present in tumor-bearing mice. Future studies will investigate combined blockade of beta-adrenergic and CXCR4 signaling in tumor-bearing C57 / BL6 mice to measure stem cell mobilization.

[0190] Increased lymphocyte mobilization was observed in the propranolol treatment group in all experiments, which may have clinical relevance as described below. Clinical studies have shown that high T cell content in patients receiving peripheral blood stem cell transplantation is associated with rapid hematopoietic reconstitution, reduced relapse, and increased disease-free survival compared with patients receiving bone marrow transplantation (Stem Cell Trialists' Collaborative Group J Clin Onc 2005). Similarly, in non-human primates and cancer patients, a single injection of AMD3100 resulted in an increase in lymphocyte counts in peripheral blood including effector T cells and regulatory T cells, which was associated with GVHD-protective properties (Kean et al. Blood 2014, Greef et al. Blood 2014). A sufficient amount of T lymphocytes is critical in the manufacturing process of CAR-T cells. Some CAR-T products that are undergoing clinical research or are commercially available rely on autologous patient-derived T cells. T cells from patients may be insufficient in number or affected by various pre-treatments and / or actual disease-related treatments (e.g., progressive AML) (Fesnak et al. Transfus Med Rev 2016). This suggests that lymphocyte mobilization is significant for allogeneic hematopoietic stem cell transplantation to reduce the risk of GVHD and for strategies designed to mobilize both effector and regulatory lymphocyte populations for adoptive cell therapy.

[0191] Previous studies have demonstrated CXCL12 / CXCR4-mediated lymphocyte homing in the 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 receptors interact with CXCR4 to promote the retention of lymphocytes in lymph nodes (Nakai et al. JEM 2014). Therefore, it is expected that the transport of lymphocytes into the peripheral blood will increase after blocking CXCR4 and β-adrenergic receptor signaling. The phenotypic characteristics of immune cells, including lymphocytes, mobilized by the combination treatment of propranolol and GPC100 are being further studied. The research results disclosed herein will provide more information about the types of lymphocyte subsets that can be collected by the combination treatment of GPC100 and propranolol and their importance in treatment development.

[0192] Example 2. Pretreatment with the β-adrenergic receptor antagonist propranolol enhances stem cell mobilization by the CXCR4 antagonist brixafor (GPC100)

[0193] Materials and methods and study design

[0194] The subjects of this study were C57 / BL6 female mice. Peripheral blood was collected by terminal cardiac puncture 2 hours after vehicle or GPC100 and 1 hour after AMD3100. Complete blood counts were determined using a hematology analyzer.

[0195] Table 7

[0196]

[0197]

[0198] result

[0199] It was observed that propranolol induced and augmented GPC100-induced mobilization. See Table 3. Propranolol alone did not alter blood cell counts. This is the first study to show that propranolol pretreatment enhances mobilization ( Fig. 8A -D). In addition, a significant increase in GPC100-induced mobilization after propranolol pretreatment was also observed in a total of 3 studies ( Fig. 9 ).

[0200] The propranolol-induced increase in mobilization was comparable to the current standard of care in preclinical models. See Tables 3, 5, and 8. The increase in mobilized WBCs from propranolol pretreatment was observed to be primarily due to lymphocytes, whereas the SOC regimen was primarily due to neutrophils ( Fig. 10A -D and Fig.11 In both studies, large changes were observed in the SOC group, with SOC alone also resulting in a decrease in platelet count ( Fig.12 and Fig.13 ).

[0201] Table 8. Complete blood count analysis showed that in mice treated with GPC100 and / or propranolol, no changes in the number of platelets, RBCs or hemoglobin levels were observed compared to those treated with standard of care treatment or vehicle.

[0202]

[0203] No significant differences from standard of care were observed in hematopoietic stem cell mobilization measured by flow cytometry with the dosing regimen. In mice, hematopoietic stem cells lack lineage markers (Lin-) and express Scal and cKit markers (LSK cell lineage). CXCR4 is also expressed on hematopoietic stem cells. Data are for LSK cells ( Fig.14 ) and Lin-CXCR4+ cells display ( Fig.15 ).

[0204] In summary, it was observed that a single intravenous administration of GPC100 / brixafor caused a rapid increase in circulating WBCs - an indication of stem cell mobilization. In addition, CBC analysis from 3 mobilization studies showed that pretreatment with propranolol for 7 days enhanced the mobilization induced by GPC100. The mobilization level of propranolol and GPC100 combination pretreatment was comparable to G-CSF and AMD3100 combination treatment. It was observed that G-CSF+AMD3100 combined mobilization more neutrophils, while beta blocker+GPC100 combined mobilization more lymphocytes were observed. Further determination of hematopoietic stem cell mobilization by flow analysis will provide more insights.

[0205] Example 3. Combined blockade of CXCR4 and β-adrenergic receptor signaling pathways induces stem cell mobilization comparable to current standard of care

[0206] The ability of the combination of two signaling pathways to block the drive of CXCR4 and beta-adrenergic receptors is being investigated. CXCR4 blockade will be determined by administering brixafor and plerixafor. The effect of the combination of propranolol + plerixafor and propranolol + G-CSF + plerixafor will also be investigated.

[0207] Example 4. Addition of propranolol / beta blocker improves stem cell mobilization via combination therapy with brixafor (GPC100) and G-CSF-triple combination

[0208] Study Design

[0209] New groups were added to this study to determine whether propranolol improves the response to the combination of G-CSF and GPC100. The dosing regimen is given in Tables 9 and 10. GPC-100 & G-CSF resulted in a greater number of mobilized circulating WBCs and progenitor cells compared to AMD3100 & G-CSF. The triple combination produced the highest number of mobilized WBCs and progenitor cells. G-CSF was administered twice a day at 0.1 mg / kg, SC for 5 days; GPC100 was administered alone at 30 mg / kg IV 12 hours after the last injection of G-CSF. Samples were collected 2 hours after GPC100 administration (Table 9). Propranolol was administered once a day for 7 days; G-CSF was administered twice a day starting the next day for a total of 5 days; GPC100 was co-administered with propranolol 12 hours after the last injection of G-CSF; samples were collected 2 hours after GPC100 administration (Table 10).

[0210] Table 9. Title: Dosing regimen for G-CSF and GPC100 combination therapy

[0211]

[0212]

[0213] Table 10. Title: Dosing regimen for combination therapy of propranolol, G-CSF and GPC100

[0214]

[0215] WBC Mobilization in G-CSF Combination Studies

[0216] The addition of propranolol to the combination of G-CSF and GPC100 resulted in the greatest mobilization of WBCs, with a significant increase in mobilization observed compared to SOC or the combined treatment of G-CSF and GPC100 ( Fig.16 and Fig.17A -C). Subsequently, changes in leukocyte subsets after treatment were investigated. It was observed that the combination of GPC100 and propranolol increased lymphocytes, while the combination of GPC100 and G-CSF increased neutrophils ( Fig.18 Increased colony forming unit assays were observed only in the group with G-CSF ( Fig.19B GPC-100&G-CSF resulted in a greater number of mobilized circulating WBCs and progenitor cells compared to AMD3100&G-CSF ( Fig.19A The triple combination produced the highest number of mobilized WBCs and progenitor cells ( Fig.19A -B and Fig. 20A -B).

[0217] In conclusion, G-CSF combination treatment with brixafor mobilized more WBCs and hematopoietic progenitor cells in peripheral blood compared with combination treatment with AMD3100. In colony formation assays, the addition of 7 days of propranolol to combination treatment with G-CSF and GPC100 resulted in the greatest number of mobilized WBCs as well as mobilized hematopoietic progenitor cells.

[0218] Example 5. Addition of propranolol / beta blocker improves stem cell mobilization by combination therapy with CXCR4 antagonists (eg, brixafor, plerixafor) and G-CSF.

[0219] Combination blockade of both signaling pathways will be investigated for their ability to drive CXCR4 and β-adrenergic receptors in combination with G-CSF for stem cell mobilization. CXCR4 blockade will be determined by administration of brixafor and plerixafor. Combinations with propranolol + plerixafor and propranolol + G-CSF + plerixafor will be investigated.

[0220] Example 6. In vivo pharmacology

[0221] A mouse study design was performed to investigate the effects of CXCR4 and B2AR blockade on HSC mobilization. The bone marrow replenishes itself in response to the departure of cells, so the number of cells in the bone marrow may not be accounted for by the reduction at the time of sample collection. The study focused on mobilization into peripheral blood. Fig.21 and Table 11).

[0222] Table 11. Combination studies of beta blockers and GPC100 targeting stem cell mobilization in mice at GPCR Therapeutics

[0223]

[0224] 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 retention of HSCs in the bone marrow. Preclinical studies have shown that a single intravenous administration of the CXCR4 antagonist GPC100 resulted in a rapid increase in circulating WBCs in C57 / B16 and Balb / c mice, an indication of stem cell mobilization. CXCR4 antagonists such as plerixafor (AMD3100) and brixafor (GPC100) are clinically approved in the United States and Europe in combination with G-CSF for hematopoietic stem cell mobilization and subsequent autologous stem cell transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma. The G-CSF regimen involves repeated multi-day injections and is associated with adverse side effects such as severe bone pain. It has been reported that up to 40% of patients have poor mobilization. Therefore, an alternative method to improve hematopoietic stem cell mobilization by CXCR4 antagonists is needed.

[0225] Improved stem cell mobilization is needed for several reasons, including the following. ASCT is increasingly used to treat hematological malignancies. However, successful ASCT in patients with lymphoma and MM is often hampered by poor mobilization, with at least 15% of patients unable to generate the >2 × 10 required for ASCT. 6 CD34 +cells / kg (Olivieri et al. 2012). Newer therapies approved in recent years for MM patients may also have a negative impact on mobilization. For example, recent studies have shown that MM patients who received daratumumab induction prior to ASCT had poor mobilization (Hulin et al. 2021). The use of daratumumab has also been associated with an increased rate of neutropenic fever, leading to increased antibiotic use and prolonged hospital stay (Papaiakovou et al. 2021). This further increases the patient burden, which is another factor to consider in the treatment of MM patients. MM patients have been found to have a higher symptom burden and poorer health-related quality of life (HRQoL) compared to patients with other hematological malignancies (Johnsen et al. 2009).

[0226] GPC100-induced mobilization in mice. Fig. 22 ). A single intravenous (IV) administration of GPC-100 (30 mg / kg), a potent and selective antagonist of CXCR4, resulted in a rapid increase in circulating white blood cell counts (WBC) in C57 / B16 and Balb / c mice, reflecting stem cell mobilization. To determine the time course of GPC-100-induced mobilization, GPC-100 (30 mg / kg) was administered intravenously to naive C57 / B16 mice, and peripheral blood was collected in different groups at time points of 0.5, 1, and 2 hours after injection. A time-dependent increase in WBC counts was observed, and a sample collection at 2 hours after injection was selected for subsequent studies. C57 / B16 mice were selected rather than Balb / c for future studies because HSC mobilization has been rigorously evaluated in this mouse strain (Broxmeyer et al. 2005).

[0227] GPC-100 alone (30 mg / kg, IV) was observed to induce a time-dependent WBC mobilization ( Fig.23 ). Future studies will utilize sample collection 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 of IV GPC100 administration will be performed (dose TBD).

[0228] Rationale for using beta-blockers to enhance CXCR4-induced mobilization. The bone marrow is highly innervated by the sympathetic nervous system. Traumatic stress in humans and rodent models has shown sustained elevated levels of norepinephrine (a ligand for beta-adrenergic receptors), which is associated with bone marrow dysfunction (Bible et al. 2014, Bible et al. 2015a, Bible et al. 2015b). Therefore, future studies will evaluate the potential of beta-blockers to improve GPC100-induced mobilization by restoring bone marrow function. In studies presented elsewhere, 7 days of intraperitoneal administration of the non-selective beta-blockers propranolol (20 mg / kg) and nadolol (5 mg / kg) or the selective beta-2 receptor antagonist ICI-118,551 (5 mg / kg) alone did not affect total blood cell counts. This dose of propranolol was selected for future studies involving beta-adrenergic blockade.

[0229] Example 7. Leukocyte mobilization induced by GPC-100 + / - beta blocker

[0230] The study was conducted using the dosing regimens 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 with propranolol intravenously on day 7 (Table 13).

[0231] Table 12. Dosing regimen for β-blocker and GPC100 combination therapy

[0232]

[0233] Table 13. Dosing regimen for beta-blocker therapy

[0234]

[0235]

[0236] Leukocyte mobilization induced by GPC-100 + / - beta blocker. 7-day administration of propranolol enhanced GPC100-induced mobilization but had no effect on blood cell counts when given alone ( Fig.24A-C). It was observed that administration of beta blockers for 3 days or concurrently with GPC100 did not alter blood cell counts (data not shown). Future studies will be conducted to: (1) confirm that hematopoietic stem cells are being mobilized; (2) mouse hematopoietic stem cells lack lineage markers (1in-) and express SCA1 and cKit. Therefore, the LSK cell profile was determined by flow cytometry (Reach Bio); (3) repeat CBC. Studies were conducted to determine stem cell mobilization. It was observed that nadolol enhanced GPC100-induced mobilization ( Fig.25A -C). In addition, 7 days of β-blocker administration together with single GPC100 did not seem to increase LSK and lin-CXCR4+ cells ( Figures 26A-26C ). Future studies will repeat the experiment and add a standard care group.

[0237] Study was conducted to study the comparison with G-CSF+AMD3100 (Table 14). Propranolol (20mg / kg IP) was administered once a day for 7 days. On the 7th day, GPC100 (30mg / 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 of care for mobilization, i.e., the combined treatment with G-CSF and AMD3100 (plerixafor). G-CSF (0.1mg / kg SC) was administered for 5 days, twice a day, followed by a single injection of AMD3100 (5mg / kg SC) 12 hours later on the 6th day. Based on literature reports (Hoggatt et al. 2018), peripheral blood was collected 1 hour after AMD3100.

[0238] Table 14. Dosage regimen based on literature

[0239]

[0240] It was observed that propranolol enhanced GPC100-induced mobilization ( Fig.27A -C). This effect was comparable to the standard of care (G-CSF + AMD3100 / plerixafor). The propranolol + GPC100 combination was observed to mobilize more lymphocytes. SOC was also observed to mobilize more neutrophils (G-CSF driven).

[0241] A study was conducted and observed that lymphocytes increased with GPC100 and beta blockers, while neutrophils increased with G-CSF+AMD3100 ( Fig.28 ). For LSK( Fig. 30A ) and Lin-CXCR4+( Fig. 30B ), the fold changes for combination studies 2 and 3 are shown in Fig.29A-B. For LSK flow, the propranolol + GPC100 combination was comparable to SOC. Future studies will repeat this experiment with a larger blood volume and added G-CSF.

[0242] Experiments were performed to investigate the triple combination of adding G-CSF (Table 15). The triple combination produced the best results ( Fig.31A -C and Fig.32 ), propranolol+GPC100 was equivalent to SOC.

[0243] Table 15. Triple combination dosing regimen.

[0244]

[0245] Colony forming unit assay ( Fig.33 and Fig.34 The CFU assay is based on the ability of hematopoietic progenitor cells to proliferate and differentiate into colonies in semisolid culture media in response to cytokine stimulation. The number and type of colonies counted in the CFU assay provides information about the frequency and type of progenitor cells present in the original cell population and their ability to proliferate and differentiate. The triple combination mobilized the highest number of progenitor cells ( Fig.35 , Figures 36A-36B and Fig.37 In addition, the triple combination was associated with the greatest increase in circulating WBCs as well as progenitor cells compared with the other drug groups ( Figures 38A-38B ). In addition, GPC100+G-CSF mobilized more WBC and progenitor cells compared to AMD3100+G-CSF. No differences were observed between the vehicle group and GPC100+ / - propranolol. The CFU assay is designed only for myeloid progenitor cells, not lymphoid progenitor cells ( Figures 39A-39B ). G-CSF was observed to mobilize myeloid progenitor cells and the assay was observed to be G-CSF dependent. Figures 40A-40B Data from 3 studies (Studies 1, 3, 4) are shown regarding the effect of propranolol on GPC100-induced mobilization. Propranolol enhancement of GPC100-induced mobilization was observed in 3 studies.

[0246] The effect of propranolol on GPC100-induced mobilization was investigated compared with standard of care. Propranolol enhanced GPC100-induced mobilization comparable to SOC ( Fig.41A -B). Compared with SOC, propranolol + GPC100 was observed to mobilize more lymphocytes. The effect of propranolol on GPC100 and AMD3100-induced mobilization was studied with and without combination with G-CSF (Table 16, Table 17).

[0247] Table 16

[0248]

[0249] Table 17

[0250]

[0251] The WBC mobilization (single agent) of GPC100, AMD3100 or G-CSF induction was studied (Figure 42 A-42C). Maximum mobilization was observed with G-CSF, and GPC100 mobilized more lymphocytes than AMD3100 or G-CSF. In addition, GPC100 mobilized more WBC than AMD3100, and G-CSF mobilized more neutrophils than GPC100 or AMD3100.

[0252] The effect of propranolol on GPC100-induced mobilization was investigated in the absence or presence of G-CSF compared with standard of care in WBC. Data from Study 4 were Fig.43A , while data from Study 5 are shown in Fig.43B No effect of the triple combination effect was observed in Study 5, and the SOC effect was much higher than previously observed.

[0253] The effect of propranolol on GPC100-induced mobilization was investigated in the absence or presence of G-CSF compared with standard of care in lymphocytes. Data from Study 4 were Fig.44A , while data from Study 5 are shown in Fig.44B No effect of the triple combination effect was observed in Study 5, and the SOC effect was much higher than previously observed.

[0254] The effect of propranolol on GPC100-induced mobilization was investigated in the absence or presence of G-CSF compared with standard of care in neutrophils. Data from Study 4 were Fig.45A , while data from Study 5 are shown in Fig.45B No effect of the triple combination effect was observed in Study 5, and the SOC effect was much higher than previously observed.

[0255] A comparative study between GPC100 and AMD3100 was conducted ( Fig.46A -C). This is the first study to show the effect of propranolol and a triple combination with AMD3100. Propranolol was observed to slightly increase AMD3100-induced lymphocyte mobilization.

[0256] The effects of propranolol on GPC100-induced mobilization in the presence or absence of G-CSF were investigated and compared with standard of care ( Fig.47A-C). 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 similar level to the standard of care (G-CSF+AMD3100).

[0257] The combined data from all six studies are as follows Fig.48A -B. Propranolol treatment 7 days before GPC100 resulted in a significant increase in WBC and lymphocyte counts in peripheral blood compared to GPC100 alone.

[0258] The data from the four studies that added standard care to the Fig.49A -B. It was observed that the standard care regimen mobilized more WBCs compared to the propranolol and GPC100 combination. However, there was no difference in lymphocyte mobilization. The standard care group also showed high variability, which reflects the response of mobilized patients in the clinic.

[0259] The data from the three studies that included the G-CSF combination group are as follows Fig.50A -B. 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 of care. When combined with G-CSF, GPC100 was observed to mobilize significantly more WBCs compared to AMD3100. However, with the addition of propranolol, the mobilization of lymphocytes was significantly increased.

[0260] Example 8. Effect of propranolol on GPC100-induced mobilization

[0261] Role of CXCR4 antagonists in stem cell mobilization

[0262] The binding of the chemokine CXCL12 to its receptor CXCR4 in the bone marrow plays an important role in the homing and retention of HSCs. Preclinical studies have shown that a single intravenous administration of the CXCR4 antagonist GPC100 resulted in a rapid increase in circulating WBCs in C57 / B16 and Balb / c mice, an indication of stem cell mobilization. CXCR4 antagonists such as plerixafor (AMD3100) and brixafor (GPC100) are clinically approved in the United States and Europe in combination with G-CSF for hematopoietic stem cell mobilization and subsequent autologous stem cell transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma. The G-CSF regimen involves repeated multi-day injections and is associated with adverse side effects such as severe bone pain. Poor mobilization has been reported in up to 40% of patients.

[0263] Lymphocyte mobilization

[0264] Compared with patients receiving bone marrow transplantation, high T cell content is associated with rapid hematopoietic reconstitution, reduced relapse, and increased disease-free survival in patients receiving peripheral blood stem cell transplantation, which highlights the importance of lymphocyte mobilization (StemCell Trialists'Collaborative Group J Clin Onc 2005). Studies in non-human primates have shown that a single injection of the CXCR4 antagonist AMD3100 resulted in an increase in lymphocyte counts in the peripheral blood, including effector T cells, as well as Treg and Tem, which was associated with GVHD-protective properties (Kean et al. Blood 2014). Similarly, allogeneic stem cell transplants harvested from healthy donors after a single dose of AMD3100 contained a greater number of effector T cells and regulatory T cells compared to transplants harvested after G-CSF. (Greef et al. Blood 2014). This is important for allo-HSCT and strategies designed to mobilize effector lymphocyte populations and regulatory lymphocyte populations for adoptive cell therapy. Previous studies have demonstrated CXCL12 / CXCR4-mediated homing of lymphocytes to the bone marrow, lymph nodes, high endothelial venules, small blood vessels, thymus, and gastrointestinal tract (Bunting et al. Immunol Cell Biol 2011).

[0265] Lymphocyte mobilization for CAT-T therapy

[0266] Efficient leukapheresis to provide sufficient T lymphocytes is a key step in the CAR-T cell production process. Some CAR-T cell products currently under investigation are based on allogeneic T cells from healthy donors, while some clinical studies or commercially available CAR-T products rely on T cells from the patient's own body. T cells from patients may be reduced in number or hampered by several series of pretreatments and actual disease-related treatments (e.g., progressive AML) (Fesnak et al. TransfusMed Rev 2016).

[0267] Lymphocyte mobilization and beta blockade

[0268] Stem cells in leukapheresis products are at risk of malignant transformation during genetic modification by viral transduction, suggesting a possible risk associated with stem cell mobilization by G-CSF. It has been reported that the β2-adrenergic receptor interacts with CXCR4 to promote lymphocyte retention in lymph nodes (Nakai et al. JEM 2014). Therefore, this study determined the effect of combined blockade of β2-adrenergic receptor and CXCR4 signaling on increasing lymphocyte trafficking into peripheral blood.

[0269] Study design. Effect of propranolol on GPC100-induced mobilization (Table 18). C57 / BL6 mice received the non-selective beta blocker propranolol (20 mg / kg, IP) once a day for 7 days. On the 7th day, GPC100 (30 mg / kg, IV) was co-administered. Blood was collected 2 hours after dosing based on preliminary data showing that maximum mobilization occurred 2 hours after a single intravenous dose of GPC100.

[0270] Table 18. Dosage regimen

[0271]

[0272] The combined data from all six studies are as follows Fig.51A It was observed that 7 days of propranolol treatment prior to GPC100 resulted in a significant increase in WBC and lymphocyte counts in peripheral blood compared to GPC100 alone.

[0273] Study design. The effect of propranolol on GPC100-induced mobilization compared to the standard of care for stem cell mobilization (G-CSF+AMD3100) was studied (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 of care for mobilization, i.e., combined treatment with G-CSF and AMD3100 (plerixafor). G-CSF (0.1 mg / kg SC) was administered for 5 days, twice a day, followed by a single injection of AMD3100 (5 mg / kg SC) 12 hours after the 6th day. Based on literature reports (Hoggatt et al. 2018), peripheral blood was collected 1 hour after AMD3100.

[0274] Table 19. Dosage regimen based on literature

[0275]

[0276] The data from the four studies that added standard care to the Fig.52A -B. It was observed that the standard of care regimen mobilized more WBCs compared to the propranolol and GPC100 combination. However, there was no difference in lymphocyte mobilization. Lymphocyte mobilization with the propranolol and GPC100 combination treatment was comparable to that with the G-CSF and AMD3100 combination treatment, suggesting the possibility of eliminating G-CSF to gain lymphocytes in the peripheral blood.

[0277] Study Design. The effect of propranolol on GPC100-induced mobilization with or without G-CSF was investigated (Table 20).

[0278] Table 20. Triple Combination Dosing Regimen

[0279]

[0280] The data from the three studies that included the G-CSF combination group are as follows Fig.53A -B. 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 of care for stem cell mobilization. When combined with G-CSF, GPC100 was observed to mobilize significantly more WBCs compared to AMD3100. However, with the addition of propranolol, the mobilization of lymphocytes was significantly increased.

[0281] Distribution of WBC counts Fig.54 As shown. It was observed that G-CSF mainly mobilized neutrophils. In addition, the addition of propranolol to G-CSF slightly reduced neutrophil mobilization, while the addition of propranolol to GPC100 slightly increased the lymphocyte count in the circulation.

[0282] Example 9. Comparison of mobilization between GPC100 and AMD3100

[0283] The WBC mobilization induced by GPC100, AMD3100 or G-CSF was studied. Fig.55A -C). The greatest mobilization was observed with G-CSF. GPC100 mobilized more lymphocytes than AMD3100 or G-CSF, while GPC100 mobilized more WBCs than AMD3100, and G-CSF mobilized more neutrophils than GPC100 or AMD3100.

[0284] In previous experiments, a comparison between GPC100 and AMD3100 was performed ( Fig.56A -C). The first study showed the effect of propranolol and triple combination with AMD3100. It was observed that propranolol slightly increased AMD3100-induced lymphocyte mobilization.

[0285] The effects of propranolol on GPC100-induced mobilization with or without G-CSF were investigated compared with standard of care ( Fig.57A -C). When combined with G-CSF, GPC100 was observed to mobilize more WBCs than AMD3100. The combination of propranolol and GPC100 resulted in an increase in circulating lymphocytes at a similar level to the standard of care (G-CSF + AMD3100).

[0286] The data from the three studies that included the G-CSF combination group are as follows Fig.58A-B. When combined with G-CSF, GPC100 was observed to mobilize significantly more WBCs compared to AMD3100. However, with the addition of propranolol, the mobilization of lymphocytes was significantly increased.

[0287] Example 10. GPC100 and propranolol as cell mobilization therapy for autologous stem cell transplantation (ASCT)

[0288] Successful autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM) is often hampered by poor mobilization, with approximately 1 in 7 patients failing to achieve adequate numbers of CD34+ cells / kg. Small molecule inhibitors of CXCR4 such as GPC100 and plerixafor are used to disrupt the CXCL12 / CXCR4 axis, which is critical for migration and retention of hematopoietic stem cells (HSCs) in the bone marrow. Here, we provide evidence that GPC100 in combination with propranolol (Pro), a β2 adrenergic receptor (B2AR) blocker (BB), and G-CSF has the potential to be an optimal in-class mobilization therapy for ASCT.

[0289] In vitro activity of GPC100 was investigated in a cell-based assay ( Figures 59A-59B ). In a FRET ligand binding assay in HEK cells, GPC100 inhibited CXCL12 binding to CXCR4 more effectively than AMD3100, with approximately 30-fold better binding affinity (Ki of 1.6 vs 40 nM, respectively). Potent inhibition of CXCR4 was reproduced in a cell migration assay using the multiple myeloma cell line MM1.S, where GPC100 inhibited CXCL12-mediated migration with an IC50 of 30 nM compared to an IC50 of 80 nM for AMD3100.

[0290] Previous studies have shown that stress hormones such as adrenaline and norepinephrine play a stimulatory role in cancer progression by regulating tumorigenesis, proliferation, and metastasis via B2AR signaling. In a recent study of 208 MM patients, the 37% of patients who reported BB use for ≥3 months after diagnosis had significantly longer overall survival compared with those who did not use BB (107 vs 86 months, Hwa et al., Eur J Haematol 2021). In addition, BBs such as Pro have been shown to enable bone marrow-derived cells to differentiate from myeloid bias to a phenotype consistent with CD34+ stem cells and stem cell-related genes (Knight et al., Biod Adv 2020).

[0291] To investigate the interaction between CXCR4 and B2AR blockade in vitro, we performed interaction and functional studies ( Figures 59C-59F). Using the proximity ligation assay (PLA) in the breast cancer cell line MDA-MB-231, which endogenously expresses CXCR4 and B2AR, we detected CXCR4 and B2AR heteromers, and knockdown of B2AR expression resulted in a decrease in PLA signal, confirming the proximity of CXCR4 and B2AR. We also demonstrated the functional consequences of CXCR4 and B2AR using a Ca2+ flux assay in MDA-MB-231 cells, demonstrating synergy when co-stimulated with CXCL12 and salmeterol, a B2AR agonist. Inhibition of Ca2+ flux by GPC100, but not AMD3100, was enhanced by co-treatment with Pro by approximately 30-fold (1.3 vs 30 nm). In summary, our in vitro results suggest that GPC100 inhibition of CXCR4 can be regulated by Pro.

[0292] To obtain preclinical proof of concept, we determined white blood cell (WBC) mobilization by complete blood count (CBC) analysis, progenitor cell mobilization by colony forming unit (CFU) assay, and HSC mobilization by flow cytometry in C57 / BL6 mice after GPC100 combination treatment. First, administration of GPC100 alone resulted in greater WBC mobilization into the peripheral blood compared to administration of AMD3100 alone ( Fig.60A Next, mice were treated with Pro for 7 days and then given a single dose of GPC100 or AMD3100 on day 7 ( Fig.60B ). Our data suggest that combination therapy with GPC100 and Pro mobilizes more WBCs compared to the combination of AMD3100 and Pro. Finally, we determined whether the triple combination of G-CSF+GPC100+Pro is superior to the current ASCT standard of care: G-CSF alone or the combination of G-CSF and AMD3100 ( Fig.60C ). We demonstrate that the triple combination results in the highest mobilization of WBCs. Furthermore, we show that WBC mobilization is a predictor of progenitor and stem cell mobilization, as we see a significant difference between WBC counts and CFUs indicative of progenitor cells ( Fig.60D ) and the Lin- / sca-1+ / c-kit+ (LSK) population ( FIG. 60E ), which indicates the mobilization of mouse HSCs.

[0293] Our findings support the use of GPC100 and Pro (with or without G-CSF) for stem cell mobilization. This treatment strategy allows the elimination of repeated daily injections of G-CSF, improving the quality of life of patients, as well as providing a treatment option for patients who experience side effects of G-CSF. Furthermore, treatment with G-CSF, GPC100, and Pro may prove to be the optimal in-class mobilization therapy for ASCT in MM patients, especially those who cannot be mobilized with standard of care.

[0294] All publications and patent applications mentioned in this specification are herein 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.

[0295] Although preferred embodiments have been shown and described herein, it will be apparent to those skilled in the art that these 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 covered thereby.

[0296] Example 11. GPC100 induces WBC and stem cell mobilization in mice

[0297] Multiple myeloma (MM) is a major hematological 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) involves the overall management of eligible MM patients and has improved anticancer response and survival compared with conventional chemotherapy (Devarakonda et al., 2021; Holsteain and McCarthy, 2016; Li and Zhu, 2019; Kumar et al., 2008). The success of ASCT depends in part on harvesting a sufficient number of hematopoietic stem cells (HSCs), which are mainly obtained by mobilizing HSCs from the bone marrow (BM) into the peripheral blood (PB) (Arora, Majhail, and Liu, 2019; Balassa, Danby, and Rocha, 2019). The phenotypic characteristic of HSCs is the expression of CD34. Approximately 2×10 6 CD34 + A minimum number of cells / kg is used for HSC harvest, while the preferred number for improved engraftment and survival is >5-6×10 6 CD34 +Cells / kg (Toor et al., 2004; Tricot et al., 1995). Granulocyte colony stimulating factor (G-CSF) is the clinical standard of care for HSC mobilization (DiPersio et al., 2009). However, G-CSF fails to mobilize the optimal number of HSCs in at least 40-50% of MM patients (DiPersio et al., 2009; Demirer et al., 196). Some patients are treated with G-CSF combined with the small molecule CXCR4 antagonist plerixafor (AMD3100) (DiPersio et al., 2009). Even with this combination treatment, 15-35% of MM patients fail to mobilize a sufficient number of cells (DiPersio et al., 2009). In a recent phase 3 clinical study, the combination of G-CSF and motixafortide (a peptide inhibitor of CXCR4) mobilized significantly more CD34+ cells compared to G-CSF plus placebo (Crees et al., 2023). Although this is promising, accumulating data suggest that MM therapies such as daratumumab or lenalidomide may negatively affect HSC mobilization (Hulin et al., 2021; Popat et al., 2021). In addition, G-CSF is contraindicated for stem cell collection in conditions such as sickle cell disease (Fitzhugh et al., 2009). These factors highlight the unmet need for optimal HSC mobilization in MM patients and also emphasize the expansion of ASCT to other disease indications (Pusic et al., 2008; Giralt et al., 2014).

[0298] 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 cell chemotaxis and the retention and survival of HSCs in the BM (Guo et al., 2016). GPC-100, also known as brixafor or TG-0054, is a novel small molecule antagonist of CXCR4 with high binding affinity to CXCR4. The combination of GPC-100 and G-CSF has been tested clinically as an HSC activator in MM patients (NCT02104427) (Schuster, 2021) and has been shown to induce a decrease in HSC activity with >5.0x10 in 1-2 leukocyte apheresis courses. 6 CD34 + The number of HSC cells / kg was increased (Setia et al., 2015). This result is comparable to historical results of G-CSF plus AMD3100 treatment.

[0299] Previous studies have shown that CXCR4 physically interacts with the β-2-adrenergic receptor or β2AR (gene ADRB2) in cells that ectopically overexpress both receptors (Nakai et al., 2014; LaRocca et al., 2010; Nakai, Leach, and Suzuki, 2021). In lymph nodes, the CXCR4-β2AR complex is thought to enhance lymphocyte retention and inhibit their mobilization through CXCR4 (Nakai et al., 2014). β2AR is also expressed on HSCs and adrenergic signaling plays a role in regulating the HSC niche in the BM (Spiegel et al., 2008; Saba et al., 2015; Maestroni, 2020; Katayama et al., 2006). The natural ligands of β2AR, epinephrine and norepinephrine, affect turnover, trafficking, 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 β2AR agonists, CXCR4 expression on HSCs increased, suggesting that the interaction between β2AR agonists and G-CSF in the BM niche promotes CXCR4 retention on HSCs and impairs G-CSF mobilization (Saba et al., 2015).

[0300] Studies have noted an association between beta-adrenergic inhibitor (beta-blocker) use and positive survival outcomes in several cancer types, including MM (Hwa et al., 2017; Hwa et al., 2021). The MM microenvironment is known to cause HSC dysfunction, resulting in altered gene expression and changes in hematopoietic differentiation (Bruns et al., 2012; Knight et al., 2020). A phase 1I biomarker-driven randomized study showed that in MM patients, the FDA-approved non-selective beta-blocker propranolol shifted cell differentiation from myeloid bias to upregulation of CD34+ cells and enhanced engraftment (Knight et al., 2020). In addition, propranolol demonstrated the ability to inhibit the BM sympathetic nervous system-induced shift from a basal gene expression profile to a more inflammatory gene expression pattern, called the conservative transcriptional response to adversity (CTRA), which is associated with poorer outcomes in ASCT (Knight et al., 2020). In another study, BM samples from MM patients showed that propranolol increased HSC differentiation into megakaryocyte-erythroid progenitors and reduced the number of granulocyte-monocyte progenitors, which are known to contribute to the pro-tumorigenic niche (Nair et al., 2022). Therefore, considering the positive effects of propranolol on HSC proliferation and differentiation, and the possible crosstalk between β2AR and CXCR4 in BM, co-inhibition of these two pathways may improve HSC mobilization.

[0301] In this study, we report the in vivo mobilization efficacy of GPC-100 compared to AMD3100. Furthermore, this report demonstrates that GPC-100 in combination with propranolol enhances in vivo mobilization and suggests a new strategy for clinical application in stem cell mobilization.

[0302] method:

[0303] In vivo mobilization: C57BL / 6J or Balb / c mice (female, 6-9 weeks old) are randomized for each study so that all treatment groups contain similar age and weight distribution. The study was conducted in an institution approved by the Association for Assessment and Accreditation of Laboratory Animal Care International and Institutional Animal Care and Use Committee. PB was collected by cardiac puncture 2 hours after GPC-100 and 1 hour after AMD3100 on the 7th day. Blood samples were processed using Abaxis VetScan HM5 hematology analyzer to perform complete blood cell count (CBC) analysis.

[0304] Table 21: Dosing for in vivo mobilization

[0305]

[0306] Colony forming unit (CFU) assay: 8×10 5 Monocytes were added to tubes of semisolid methylcellulose medium (StemCell Technologies) known to support erythroid and myeloid progenitors (Kronstein-Wiedemann, 2019). After seven days, colonies of granulocyte-monocyte progenitors (CFU-GM) and burst-forming erythroid units (BFU-E) were shown to emerge and counted by a blinded experimenter. Total CFU was calculated as the sum of CFU-GM and BFU-U colonies.

[0307] Flow cytometry: To determine the mobilization of mouse HSCs characterized as LSK cells (Lineage-Sca-1+c-Kit+) (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 using a Cytek Aurora Spectrum flow cytometer (Fremont, CA) and data were analyzed using CellEngine software. Gating was determined using FMO controls. C-Kit + Sca-1 + The percentage of cells that are a subset of parental Lin- cells is used to determine the total number of LSK cells per μL of blood.

[0308] Table 22: Antibodies used in flow cytometry

[0309]

[0310] Statistical analysis: Data analysis was performed using GraphPad Prism, and all data are presented as mean ± SEM. Data comparisons for each dosing condition were performed using the Mann-Whitney test or one-way ANOVA. For all tests, P < 0.05 was considered statistically significant.

[0311] Experimental design: First, the mobilization of WBC and LSK stem cells by GPC-100 was determined after a single IV administration. For subsequent studies, WBC mobilization was used as a marker of stem cell mobilization. To determine the dose of propranolol used in combination with GPC-100, propranolol was administered at 5, 10, 20, and 40 mg / kg IP for 7 days, followed by co-administration 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 in combination with propranolol and GPC-100. Next, the combination of GPC-100 and propranolol was compared with G-CSF alone for WBC mobilization. For this study, G-CSF was administered for 5 days, twice a day. Finally, the triple combination of G-CSF, GPC-100, and propranolol was studied in phenotypic analysis and colony forming unit assays for WBC and stem cell mobilization compared with G-CSF plus AMD3100. For all studies, blood was collected 2 hours after GPC-100, 1 hour after AMD3100, and 12 hours after G-CSF.

[0312] result

[0313] A single administration of GPC-100 (30 mg / kg, IV) induced WBC mobilization in the PB, which peaked at 2 hours. Numerous studies in mice reported peak mobilization of AMD3100 (5 mg / kg, SC) at 1 hour (e.g., Broxmeyer et al. 2005). Therefore, PB WBC counts after GPC-100 and after AMD3100 were determined at the time point and dose at which the maximum mobilization of each antagonist was observed. GPC-100 caused an increase in PB WBC counts in C57 / BL6 and balb / c mouse strains ( Figure 1A When compared to AMD3100 in 3 separate studies ( Fig.61A , 61B , 61C), GPC-100 produced a 2-3 fold increase, while AMD3100 produced a <2 fold increase in WBC compared to vehicle. The increase in WBC caused by both antagonists included an increase in lymphocytes and neutrophils. No changes in platelet count, hemoglobin, or other red blood cell parameters were observed. GPC-100 also mobilized hematopoietic stem cells ( Fig.62 ).

[0314] To evaluate the effects of β2AR blockade in vivo, mice were administered propranolol. When combined with GPC-100, propranolol doses were selected based on dose titration (5-40 mg / kg, IP) Fig.63A Pretreatment with propranolol (20 mg / kg, IP) for 7 days significantly improved GPC-100-induced mobilization ( Fig.63B ). Phenotypic analysis of LSK cells by flow cytometry also showed that LSK cell mobilization by GPC-100 was enhanced by propranolol ( Figures 64A-64D ).

[0315] Next, mobilization of the GPC-100 and propranolol combination was compared to the standard of care, G-CSF. In mobilizing WBCs, propranolol induced a 4.1-fold increase, while G-CSF induced a comparable 4.5-fold increase ( Fig.65 ).

[0316] The triple combination of G-CSF, GPC-100, and propranolol was compared to the current ASCT standard of care, G-CSF alone or in combination with AMD3100. The triple combination and the combination of G-CSF and GPC-100 induced an 8.2-fold and 8.4-fold increase in WBC mobilization, respectively, which was significantly greater than the increase in WBC counts with G-CSF alone (4.5-fold) or G-CSF plus AMD3100 (6.6-fold) ( Fig.66 ).

[0317] Further experiments were performed to determine whether the increased WBC counts in the circulation reflect hematopoietic stem and progenitor cell (HSPC) mobilization. Based on the ability of mobilized HSPC to form CFU-GM and BFU-E colonies, CFU determination was performed to measure mobilized HSPC. Compared to the 35-fold and 27-fold growth generated by the vehicle from G-CSF plus GPC-100 and G-CSF plus AMD3100 treatment, the triple combination produced a 47-fold CFU growth ( Figures 67A-67D ).

[0318] Phenotypic analysis showed that G-CSF plus AMD3100 treatment resulted in a 13-fold increase in LSK cells in PB compared to vehicle. In contrast, the combination of G-CSF and GPC-100 resulted in a 20-fold and 24-fold increase in LSK cells in the presence and absence of propranolol, respectively ( Figures 68A-68F ). The LSK and CFU count patterns for different drug combinations were consistent with the WBC counts from matched samples ( Figures 67A-67D and 68A-68F), supporting the use of WBC count as a surrogate marker of stem cell mobilization.

[0319] The current studies show that GPC-100 is a potent hematopoietic mobilizer, and its mobilization effect is enhanced by propranolol. These studies also show that the increase in G-CSF mobilization induced by GPC-100 is superior to the combination of G-CSF and AMD3100. The addition of propranolol to G-CSF and GPC-100 mobilizes more hematopoietic stem cells that can differentiate into multipotent progenitor cells. The data and previous reports show that HSPC mobilization is associated with a concomitant increase in circulating WBCs (Vater et al., 2013; A1meida-Neto et al., 2020; Abraham et al., 2007; Lee et al., 2014). The effects of propranolol observed in this study may be explained by an independent effect of propranolol on HSPCs or by an interaction between β2AR and CXCR4.

[0320] This study also showed that the combination of propranolol and GPC-100 showed a 4-fold increase, while G-CSF induced a 4.5-fold increase in WBCs over vehicle control. This observation is important as it suggests the potential for comparable HSC mobilization without the use of G-CSF. Eliminating G-CSF from treatment may reduce the risk of moderate to severe side effects of G-CSF such as severe bone pain and rare spleen rupture.

[0321] The addition of propranolol to G-CSF and GPC-100 (triple combination) increased PB CFU counts, which were significantly greater than G-CSF plus AMD3100. This suggests that the triple combination mobilized a greater number of viable cells that were functionally capable of differentiating into myeloid and erythroid multipotent progenitors. Furthermore, phenotypic analysis showed that there were more LSK cells in PB treated with G-CSF and GPC-100 (with or without propranolol) compared to G-CSF plus AMD3100. This study was performed in naive mice, and the effects of propranolol may be amplified in models with impaired BM microenvironment and HSC differentiation (Giles et al., 2016).

[0322] In summary, our preclinical findings support the addition of propranolol to GPC-100-induced stem cell mobilization for ASCT in MM patients. The triple combination of GPC-100, propranolol, and G-CSF may be best in class and target patient populations where other mobilization regimens have failed. Propranolol has been shown to be a safe, accessible, and inexpensive option to supplement mobilization therapy for higher stem cell yields in fewer apheresis courses and reduce the economic burden on patients and the healthcare system. The relevant clinical study is registered as a two-arm Phase 2 clinical trial (NCT05561751) with a GPC-100 plus propranolol arm and a GPC-100, propranolol, and G-CSF arm.

[0323] Exemplary embodiments

[0324] In one 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.

[0325] In one 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.

[0326] In one 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.

[0327] In one 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.

[0328] In one embodiment, disclosed herein is a method of enhancing apheresis by mobilizing cells in a subject, the method comprising: blocking CXCR4 signaling and β-adrenergic receptor signaling in the subject.

[0329] In one embodiment, blocking beta-adrenergic receptor signaling is performed prior to blocking CXCR4 signaling.

[0330] In one embodiment, blocking of β-adrenergic receptor signaling continues after blocking of CXCR4 signaling has ceased.

[0331] In one embodiment, blocking CXCR4 signaling comprises administering to the subject a CXCR4 inhibitor.

[0332] In one embodiment, blocking beta-adrenergic receptor signaling comprises administering a beta-adrenergic receptor inhibitor to the subject.

[0333] In one embodiment, the cell is a stem cell.

[0334] In one embodiment, blocking CXCR4 signaling comprises administering to the subject a CXCR4 inhibitor, and blocking β-adrenergic receptor signaling comprises administering to the subject a β-adrenergic receptor inhibitor.

[0335] In one embodiment, the cell is a stem cell.

[0336] In one embodiment, disclosed herein is a method of mobilizing stem cells in a subject, the method comprising: administering to the subject a beta-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0337] In one 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.

[0338] In one embodiment, disclosed herein is a method of enhancing apheresis in a subject, the method comprising: administering to the subject a beta-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0339] In one embodiment, disclosed herein is a method of enhancing apheresis by inducing cell mobilization in a subject, the method comprising: administering to the subject a beta-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0340] In one embodiment, disclosed herein is a method of enhancing apheresis by mobilizing cells in a subject, the method comprising: administering to the subject a beta-adrenergic receptor inhibitor and a CXCR4 inhibitor.

[0341] In one embodiment, administration of the beta-adrenergic receptor inhibitor occurs prior to administration of the CXCR4 inhibitor.

[0342] In one embodiment, administration of the beta-adrenergic receptor inhibitor continues after administration of the CXCR4 inhibitor has ceased.

[0343] In one embodiment, the method further comprises administering G-CSF to the subject.

[0344] In one embodiment, administering the beta-adrenergic receptor inhibitor and the CXCR4 inhibitor to the subject is performed in the absence of G-CSF.

[0345] In one 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.

[0346] In one 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.

[0347] In one 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.

[0348] In one 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.

[0349] In one 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 beta-adrenergic receptor inhibitor.

[0350] In one embodiment, the beta-adrenergic receptor inhibitor is an ADRB2 inhibitor.

[0351] In one embodiment, the beta-adrenergic receptor inhibitor is selected from alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloclorolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

[0352] In one embodiment, the beta-adrenergic receptor inhibitor is selected from propranolol, nadolol, and ICI118551.

[0353] In one embodiment, the beta-adrenergic receptor inhibitor is propranolol.

[0354] In one embodiment, the CXCR4 inhibitor is selected from 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 (ITlt), KRH-1636, KRH-3955, LY2510924, MSX-122, N-[11C]methyl-AMD3465, POL6326, SDF-11-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012, TG-0054 (brixafor), 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).

[0355] In one embodiment, the CXCR4 inhibitor is selected from AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (brixafor), PF-06747143, POL6326 and ululumab (MDX1338 / BMS-936564).

[0356] In one embodiment, the CXCR4 inhibitor is TG-0054 (brixafor).

[0357] In one embodiment, the CXCR4 inhibitor is AMD3100 (plerixafor).

[0358] In one embodiment, the CXCR4 inhibitor is ululumab (MDX1338 / BMS-936564).

[0359] In one embodiment, administering a CXCR4 inhibitor to a subject comprises administering TG-0054 (brixafor) and propranolol.

[0360] In one embodiment, administering a CXCR4 inhibitor to a subject comprises administering AMD3100 (plerixafor) and propranolol.

[0361] In one embodiment, administering a CXCR4 inhibitor to a subject comprises administering ululumab (MDX1338 / BMS-936564) and propranolol.

[0362] In one embodiment, administration of a CXCR4 inhibitor in combination with G-CSF induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0363] In one embodiment, administration of a CXCR4 inhibitor in combination with G-CSF mobilizes an increased amount of cells relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0364] In one embodiment, administration of a combination of a CXCR4 inhibitor and G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by the CXCR4 inhibitor alone.

[0365] In one embodiment, administration of a combination of a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0366] In one embodiment, administration of a combination of a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor mobilizes an increased amount of cells relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

[0367] In one embodiment, administration of a combination of a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor induces an increased amount of apheresis relative to the amount of apheresis induced by the CXCR4 inhibitor alone.

[0368] In one embodiment, administration of a combination of a CXCR4 inhibitor, a β-adrenergic receptor inhibitor, and G-CSF induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

[0369] In one embodiment, administration of a combination of a CXCR4 inhibitor, a β-adrenergic receptor inhibitor, and G-CSF mobilizes an increased amount of cells relative to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

[0370] In one embodiment, administration of a combination of a CXCR4 inhibitor and a beta-adrenergic receptor inhibitor and G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by the CXCR4 inhibitor and the beta-adrenergic receptor inhibitor alone.

[0371] In one embodiment, administration of a combination of TG-0054 (brixafor) and G-CSF induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by AMD3100 (plerixafor) and G-CSF.

[0372] In one embodiment, administration of a combination of TG-0054 (brixafor) and G-CSF mobilizes an increased amount of cells relative to the amount of cell mobilization induced by AMD3100 (plerixafor) and G-CSF.

[0373] In one embodiment, administration of a combination of TG-0054 (brixafor) and G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by AMD3100 (plerixafor) and G-CSF.

[0374] In one embodiment, the increased 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.

[0375] In one embodiment, the increased amount of cell mobilization or apheresis is measured by flow cytometry.

[0376] In one embodiment, flow cytometry is performed on (Lin-Sca1+c-Kit+) LSK cells.

[0377] In one embodiment, the increased amount of cell mobilization or apheresis is measured by a colony forming unit (CFU) assay.

[0378] In one embodiment, the subject has CXCR4 protomers in cells.

[0379] In one embodiment, the subject has ADRB2 protomers in cells.

[0380] In one embodiment, the subject has CXCR4 protomers and ADRB2 protomers in cells.

[0381] In one embodiment, the subject has CXCR4-ADRB2 heteromers in cells.

[0382] In one embodiment, i) the CXCR4-ADRB2 heteromer has an increased amount of downstream calcium mobilization relative to downstream calcium mobilization from CXCR4 protomers or ADRB2 protomers; and ii) the administered inhibitor combination inhibits enhanced downstream calcium mobilization from said CXCR4-ADRB2 heteromer in stem cells.

[0383] In one embodiment, the cell is a stem cell.

[0384] In one embodiment, the stem cell is selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, endothelial progenitor cells, neural stem cells, epithelial stem cells, skin stem cells, and cancer stem cells.

[0385] In one embodiment, the stem cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

[0386] In one embodiment, the hematopoietic stem cells or hematopoietic progenitor cells are mobilized from the bone marrow into the peripheral blood.

[0387] In one embodiment, mobilized hematopoietic stem cells or hematopoietic progenitor cells are collected for transplantation into a patient suffering from cancer.

[0388] In one embodiment, the cancer is selected from lymphoma, leukemia, and myeloma.

[0389] In one embodiment, the cancer is non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or multiple myeloma (MM).

[0390] In one embodiment, the stem cells are mesenchymal stem cells.

[0391] In one embodiment, the mesenchymal stem cells are mobilized from the bone marrow into the peripheral blood.

[0392] In one embodiment, the mesenchymal stem cells are mobilized for treating a condition selected from the group consisting of neurological disorders, 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.

[0393] In one embodiment, the stem cell is a cancer stem cell.

[0394] In one embodiment, the cancer stem cells are mobilized into the blood.

[0395] In one embodiment, the cancer stem cells are mobilized for use in treating cancer.

[0396] In one embodiment, the cell is an immune cell.

[0397] In one embodiment, the immune cells are leukocytes.

[0398] In one embodiment, the leukocytes are lymphocytes.

[0399] In one embodiment, the lymphocytes are selected from the group consisting of T cells, B cells, and Natural Killer (NK) cells.

[0400] In one embodiment, the lymphocytes are T cells.

[0401] In one embodiment, the lymphocytes are natural killer (NK) cells.

[0402] In one embodiment, the leukocytes are granulocytes.

[0403] In one embodiment, the granulocytes are selected from the group consisting of neutrophils, eosinophils, and basophils.

[0404] In one embodiment, the granulocytes are neutrophils.

[0405] In one embodiment, the leukocytes are monocytes.

[0406] In one embodiment, the immune cells are mobilized from the bone marrow into the peripheral blood.

[0407] In one embodiment, the immune cells are mobilized from lymph nodes into peripheral blood.

[0408] In one embodiment, the mobilized immune cells are used for adoptive cell therapy (ACT).

[0409] In one embodiment, the adoptive cell therapy (ACT) is chimeric antigen receptor (CAR) T cell therapy.

[0410] In one embodiment, the adoptive cell therapy (ACT) is natural killer (NK) cell therapy.

[0411] In one embodiment, the adoptive cell therapy (ACT) is an engineered T cell receptor (TCR) therapy.

[0412] In one embodiment, the adoptive cell therapy (ACT) is tumor infiltrating lymphocyte (TIL) therapy.

Claims

1. A method of mobilizing cells in a subject, the method comprising: Blocking CXCR4 signaling and beta-adrenergic receptor signaling in the subject.

2. A method of inducing cell mobilization in a subject, the method comprising: Blocking CXCR4 signaling and beta-adrenergic receptor signaling in the subject.

3. A method of enhancing apheresis in a subject, the method comprising: Blocking CXCR4 signaling and beta-adrenergic receptor signaling in the subject.

4. A method for enhancing apheresis by inducing cell mobilization in a subject, the method comprising: Blocking CXCR4 signaling and beta-adrenergic receptor signaling in the subject.

5. A method for enhancing apheresis by mobilizing cells in a subject, the method comprising: Blocking CXCR4 signaling and beta-adrenergic receptor signaling in the subject.

6. The method of any one of claims 1-5, wherein said blocking of β-adrenergic receptor signaling is performed prior to said blocking of CXCR4 signaling.

7. The method of any one of claims 1-5, wherein said blocking of β-adrenergic receptor signaling continues after said blocking of CXCR4 signaling is terminated.

8. The method of any one of claims 1-7, wherein said blocking CXCR4 signaling comprises administering a CXCR4 inhibitor to the subject.

9. The method of any one of claims 1-7, wherein said blocking β-adrenergic receptor signaling comprises administering to said subject a β-adrenergic receptor inhibitor.

10. The method of any one of claims 1-2 and 4-9, wherein the cells are stem cells.

11. The method of any one of claims 1-7, wherein said blocking CXCR4 signaling comprises administering to said subject a CXCR4 inhibitor, and said blocking β-adrenergic receptor signaling comprises administering to said subject a β-adrenergic receptor inhibitor.

12. The method of claim 11, wherein the cells are stem cells.

13. A method of mobilizing stem cells in a subject, the method comprising: A beta-adrenergic receptor inhibitor and a CXCR4 inhibitor are administered to the subject.

14. A method of inducing stem cell mobilization in a subject, the method comprising: A beta-adrenergic receptor inhibitor and a CXCR4 inhibitor are administered to the subject.

15. A method of enhancing apheresis in a subject, the method comprising: A beta-adrenergic receptor inhibitor and a CXCR4 inhibitor are administered to the subject.

16. A method for enhancing apheresis by inducing cell mobilization in a subject, the method comprising: A beta-adrenergic receptor inhibitor and a CXCR4 inhibitor are administered to the subject.

17. A method for enhancing apheresis by mobilizing cells in a subject, the method comprising: A beta-adrenergic receptor inhibitor and a CXCR4 inhibitor are administered to the subject.

18. The method of any one of claims 11-17, wherein said administering a beta-adrenergic receptor inhibitor is performed prior to said administering a CXCR4 inhibitor.

19. The method of any one of claims 11-17, wherein the administration of the beta-adrenergic receptor inhibitor continues after the administration of the CXCR4 inhibitor is terminated.

20. The method according to any one of claims 11 to 19, further comprising: G-CSF is administered to the subject.

21. The method of any one of claims 11-19, wherein administering the β-adrenergic receptor inhibitor and the CXCR4 inhibitor to the subject is performed in the absence of G-CSF.

22. A method of mobilizing stem cells in a subject, the method comprising: The subject is administered a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

23. A method of inducing stem cell mobilization in a subject, the method comprising: The subject is administered a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

24. A method of enhancing apheresis in a subject, the method comprising: The subject is administered a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

25. A method for enhancing apheresis by inducing cell mobilization in a subject, the method comprising: The subject is administered a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

26. A method for enhancing apheresis by mobilizing cells in a subject, the method comprising: The subject is administered a CXCR4 inhibitor and G-CSF in the absence of a β-adrenergic receptor inhibitor.

27. The method of any one of claims 9, 11-21, wherein the beta-adrenergic receptor inhibitor is an ADRB2 inhibitor.

28. The method of any one of claims 9, 11-21, wherein the beta-adrenergic receptor inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloclorolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol.

29. The method of claim 28, wherein the beta-adrenergic receptor inhibitor is selected from the group consisting of propranolol, nadolol, and ICI 118551.

30. The method of claim 29, wherein the beta-adrenergic receptor inhibitor is propranolol.

31. The method of any one of claims 8, 11-30, wherein the CXCR4 inhibitor is selected from 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-11-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC 14012, TG-0054 (brixafor), 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).

32. The method of claim 31, wherein the CXCR4 inhibitor is selected from AD-214, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), BKT140 (BL-8040; TF14016; 4F-benzoyl-TN14003), CTCE-9908, LY2510924, LY2624587, T140, TG-0054 (brixafor), PF-06747143, POL6326, and ululumab (MDX1338 / BMS-936564).

33. The method of claim 32, wherein the CXCR4 inhibitor is TG-0054 (brixafor).

34. The method of claim 32, wherein the CXCR4 inhibitor is AMD3100 (plerixafor).

35. The method of claim 32, wherein the CXCR4 inhibitor is ululumab (MDX1338 / BMS-936564).

36. The method of any one of claims 8, 11-21, and 27-30, wherein said administering to the subject said CXCR4 inhibitor and a beta-adrenergic receptor inhibitor comprises administering TG-0054 (brixafor) and propranolol.

37. The method of any one of claims 8, 11-21, and 27-30, wherein said administering to the subject said CXCR4 inhibitor and a beta-adrenergic receptor inhibitor comprises administering AMD3100 (plerixafor) and propranolol.

38. The method of any one of claims 8, 11-21, and 27-30, wherein the administering the CXCR4 inhibitor and the beta-adrenergic receptor inhibitor to the subject comprises administering ululumab (MDX1338 / BMS-936564) and propranolol.

39. The method of any one of claims 20-38, wherein administration of the combination of the CXCR4 inhibitor and the G-CSF induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

40. The method of any one of claims 20-38, wherein administration of the CXCR4 inhibitor in combination with the G-CSF mobilizes an increased amount of cells relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

41. The method of any one of claims 20-38, wherein administration of the combination of the CXCR4 inhibitor and the G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by the CXCR4 inhibitor alone.

42. The method of any one of claims 11-21 and 27-42, wherein administration of the combination of the CXCR4 inhibitor and the beta-adrenergic receptor inhibitor induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

43. The method of any one of claims 11-21 and 27-42, wherein administration of the CXCR4 inhibitor and the beta-adrenergic receptor inhibitor in combination mobilizes an increased amount of cells relative to the amount of cell mobilization induced by the CXCR4 inhibitor alone.

44. The method of any one of claims 11-21 and 27-42, wherein administration of the combination of the CXCR4 inhibitor and the beta-adrenergic receptor inhibitor induces an increased amount of apheresis relative to the amount of apheresis induced by the CXCR4 inhibitor alone.

45. The method of claim 20, wherein administering the combination of the CXCR4 inhibitor, the β-adrenergic receptor inhibitor, and the G-CSF induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

46. ​​The method of claim 20, wherein administration of the combination of the CXCR4 inhibitor, the beta-adrenergic receptor inhibitor, and the G-CSF mobilizes an increased amount of cells relative to the amount of cell mobilization induced by the CXCR4 inhibitor and the beta-adrenergic receptor inhibitor alone.

47. The method of claim 20, wherein administration of the combination of the CXCR4 inhibitor and the β-adrenergic receptor inhibitor and the G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by the CXCR4 inhibitor and the β-adrenergic receptor inhibitor alone.

48. The method of any one of claims 20-38, wherein administration of a combination of TG-0054 (brixafor) and the G-CSF induces an increased amount of cell mobilization relative to the amount of cell mobilization induced by AMD3100 (plerixafor) and the G-CSF.

49. The method of any one of claims 20-38, wherein administration of a combination of TG-0054 (brixafor) and the G-CSF mobilizes an increased amount of cells relative to the amount of cell mobilization induced by AMD3100 (plerixafor) and the G-CSF.

50. The method of any one of claims 20-38, wherein administration of a combination of TG-0054 (brixafor) and the G-CSF induces an increased amount of apheresis relative to the amount of apheresis induced by AMD3100 (plerixafor) and the G-CSF.

51. The method of any one of claims 1-50, wherein the increased amount of cell mobilization or apheresis is measured by a method selected from complete blood count (CBC) analysis, flow cytometry, and colony forming unit (CFU) assay.

52. The method of claim 51, wherein the increased amount of cell mobilization or apheresis is measured by flow cytometry.

53. The method of claim 51, wherein the flow cytometry is performed on (Lin-Scal+c-Kit+)LSK cells.

54. The method of claim 51, wherein the increased amount of cell mobilization or apheresis is measured by a colony forming unit (CFU) assay.

55. The method of any one of claims 1-54, wherein the subject has CXCR4 protomers in the cells.

56. The method of any one of claims 1-54, wherein the subject has ADRB2 protomers in the cells.

57. The method of any one of claims 1-54, wherein the subject has CXCR4 protomers and ADRB2 protomers in the cells.

58. The method of claim 57, wherein the subject has CXCR4-ADRB2 heteromers in the cells.

59. The method of claim 58, wherein: i) the CXCR4-ADRB2 heteromer has an increased amount of downstream calcium mobilization relative to downstream calcium mobilization from a CXCR4 protomer or an ADRB2 protomer; and ii) the administered inhibitor combination inhibits enhanced downstream calcium mobilization from said CXCR4-ADRB2 heteromers in said stem cells.

60. The method of any one of claims 1, 2, 4-11, 16, 17, 25, and 26, wherein the cell is a stem cell.

61. The method of claim 60, wherein the stem cell is selected from the group consisting of hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, endothelial progenitor cells, neural stem cells, epithelial stem cells, skin stem cells, and cancer stem cells.

62. The method of claim 61, wherein the stem cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

63. The method of claim 62, wherein the hematopoietic stem cells or the hematopoietic progenitor cells are mobilized from the bone marrow into peripheral blood.

64. The method of claim 63, wherein the mobilized hematopoietic stem cells or hematopoietic progenitor cells are collected for transplantation into a patient suffering from cancer.

65. The method of claim 64, wherein the cancer is selected from the group consisting of lymphoma, leukemia, and myeloma.

66. The method of claim 65, wherein the cancer is non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or multiple myeloma (MM).

67. The method of claim 61, wherein the stem cells are mesenchymal stem cells.

68. The method of claim 67, wherein the mesenchymal stem cells are mobilized from the bone marrow into the peripheral blood.

69. The method of claim 68, wherein the mesenchymal stem cells are mobilized for treating a condition selected from the group consisting of neurological disorders, 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.

70. The method of claim 61, wherein the stem cell is a cancer stem cell.

71. The method of claim 70, wherein the cancer stem cells are mobilized into the blood.

72. The method of claim 71, wherein the cancer stem cells are mobilized for treating cancer.

73. The method of any one of claims 1, 2, 4-11, 16, 17, 25, and 26, wherein the cell is an immune cell.

74. The method of claim 73, wherein the immune cells are leukocytes.

75. The method of claim 74, wherein the leukocytes are lymphocytes.

76. The method of claim 75, wherein the lymphocytes are selected from the group consisting of T cells, B cells, and natural killer (NK) cells.

77. The method of claim 76, wherein the lymphocytes are T cells.

78. The method of claim 76, wherein the lymphocytes are natural killer (NK) cells.

79. The method of claim 74, wherein the leukocytes are granulocytes.

80. The method of claim 79, wherein the granulocytes are selected from the group consisting of neutrophils, eosinophils, and basophils.

81. The method of claim 80, wherein the granulocytes are neutrophils.

82. The method of claim 74, wherein the leukocytes are monocytes.

83. The method of any one of claims 73-82, wherein the immune cells are mobilized from the bone marrow to the peripheral blood.

84. The method of any one of claims 73-82, wherein the immune cells are mobilized from lymph nodes to peripheral blood.

85. The method of any one of claims 73-82, wherein the mobilized immune cells are used for adoptive cell therapy (ACT).

86. The method of claim 85, wherein the adoptive cell therapy (ACT) is chimeric antigen receptor (CAR) T cell therapy.

87. The method of claim 85, wherein the adoptive cell therapy (ACT) is natural killer (NK) cell therapy.

88. The method of claim 85, wherein the adoptive cell therapy (ACT) is an engineered T cell receptor (TCR) therapy.

89. The method of claim 85, wherein the adoptive cell therapy (ACT) is tumor infiltrating lymphocyte (TIL) therapy.

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