GPCR heteromeric inhibitors and uses thereof

Abstract

The present invention relates to inhibitors of cancer-associated CXC receptor 4 (CXCR4)-G protein-coupled receptor (GPCR) heteromers (CXCR4-GPCR heteromers), in which CXCR4 forms functional heteromers with other G protein-coupled receptors (GPCRx). More specifically, the present invention relates to ADRB2, which forms a heteromer with CXCR4 and results in enhanced signaling downstream of CXCR4 upon costimulation with a CXCR4 agonist and an ADRB2 agonist. The present invention also provides pharmaceutical compositions and kits comprising the CXCR4 inhibitor and the ADRB2 inhibitor, as well as methods for treating cancer and for use in the diagnosis and / or treatment of cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 022,845, filed May 11, 2020, and U.S. Provisional Patent Application No. 62 / 849,755, filed May 17, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application incorporates by reference the Sequence Listing entitled "14462-012-228_SEQ_LISTING.txt," which was filed herewith in ASCII text format, created on May 11, 2020, and is 1,516 bytes in size.

[0003] Field The present invention relates to the field of cancer therapy. In particular, provided herein are pharmaceutical compositions comprising a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, as well as methods of suppression, methods of treatment, pharmaceutical kits for use, and pharmaceutical compositions for use using the same. [Background technology]

[0004] G protein-coupled receptors (GPCRs) are seven-transmembrane domain cell surface receptors that are coupled to G proteins. GPCRs mediate diverse sensory and physiological responses by perceiving stimuli including light, odorants, hormones, neurotransmitters, chemokines, small lipid molecules, and nucleotides. Approximately 800 GPCR genes exist in the human genome, and more than half of these are predicted to encode sensory receptors, such as olfactory, visual, and taste receptors (Bjarnadottir, TK, et al. (2006) Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse, Genomics 88, 263-273). The remaining 350 GPCRs play important physiological roles in embryonic development, behavior, mood, cognition, blood pressure regulation, heart rate and digestive processes, immune system regulation and inflammation, homeostasis, and cancer growth and metastasis (Filmore, D. (2004) It's a GPCR world. Modern Drug Discovery American Chemical Society 2004 (November), 24-28; Overington, JP, et al. (2006) How many drug targets are there? Nat Rev Drug Discov 5, 993-996). GPCRs are involved in many diseases and are the targets of approximately 40% of all prescription drugs (Filmore, D. (2004)).

[0005] CXC receptor 4 (CXCR4) is a member of the chemokine receptor family, a GPCR. CXCR4 is expressed on most hematopoietic cell types, bone marrow stem cells, endothelial progenitor cells, vascular endothelial cells, neurons and neural stem cells, microglia, and astrocytes (Klein, RS, et al., (2004) Immune and nervous system CXCL12 and CXCR4: parallel roles in patterning and plasticity, Trends Immunol 25, 306-314; Griffith, JW, et al., (2014) Chemokines and chemokine receptors: positioning cells for host defense and immunity, Annu Rev Immunol 32, 659-702). CXCR4 responds to its ligand, the C-X-C motif chemokine ligand 12 (CXCL12), also known as stromal cell-derived factor 1 (SDF-1), and plays an essential role in the embryonic development of the hematopoietic, cardiovascular, and nervous systems (Griffith, JW, et al., (2014)). CXCR4 was discovered as a coreceptor for human immunodeficiency virus (HIV) and plays an important role in hematopoietic stem cell (HSC) homing to the bone marrow, inflammation, tissue immune surveillance, and tissue regeneration in adults (Chatterjee, S., et al., (2014) The intricate role of CXCR4 in cancer, Adv Cancer Res 124, 31-82).CXCR4 is involved in various immune and autoimmune diseases, such as HIV infection, ischemia, wound healing, rheumatoid arthritis, systemic lupus erythematosus (SLE), interstitial pneumonia, vascular disease, multiple sclerosis, pulmonary fibrosis, and allergic airway diseases (Chu, T. et al., (2017) CXCL12 / CXCR4 / CXCR7 Chemokine Axis in the Central Nervous System: Therapeutic Targets for Remyelination in Demyelinating Diseases, Neuroscientist 23, 627-648; Debnath, B., et al., (2013) Small molecule inhibitors of CXCR4, Theranostics 3, 47-75; and Domanska, U. M., et al., (2013) A review on CXCR4 / CXCL12 axis in oncology: no place to hide, Eur J Cancer 49, 219-230). CXCR4 has also been implicated in a wide variety of cancers and is thought to have multiple potential roles in malignancies. CXCR4 is overexpressed in more than 23 types of human cancers, including breast cancer, lung cancer, brain cancer, kidney cancer (or renal cell carcinoma), pancreatic cancer, ovarian cancer, prostate cancer, melanoma, leukemia, multiple myeloma, gastrointestinal cancer, and soft tissue sarcoma, and is considered a marker of poor prognosis (Domanska, U. M., et al., (2013); Chatterjee, S., et al., (2014); and Furusato, B., et al., (2010) CXCR4 and cancer, Pathol Int 60, 497-505).

[0006] The formation of CXCR4 and GPCR heteromers has been studied within a limited number of GPCR families, such as the chemokine, adrenergic, and opioid receptor families. Given the central role and increased expression of CXCR4 in various pathologies, it is likely that various CXCR4-GPCR heteromers exist that confer unique characteristics to specific diseases.

[0007] Adrenergic receptor beta2, sometimes referred to as beta-2 adrenergic receptor or β2 adrenoceptor ("ADRB2"), is a transmembrane beta-adrenergic receptor that interacts with epinephrine (a hormone and neurotransmitter (also known as the ligand, adrenaline)) whose signaling mediates physiological responses such as smooth muscle relaxation and bronchodilation via downstream L-type calcium channel interactions (Gregorio, GG, et al, (2017) Single-molecule analysis of ligand efficacy in beta2AR-G-protein activation, Nature 547, 68-73). The formation of heteromers of CXCR4 and ADRB2 (CXCR4-ADRB2 heteromers), co-expression of CXCR4 and ADRB2 (p2-AR) in cardiomyocytes, and the physical association of CXCR4 with ADRB2 using co-immunoprecipitation and bioluminescence resonance energy transfer (LaRocca, TJ, et al. (2010) beta2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4, J Cardiovasc Pharmacol 56, 548-559; Nakai, A., et al. (2014) Control of lymphocyte egress from lymph nodes through beta2-adrenergic receptors, J Exp Med 211, 2583-2598).

[0008] Given the key role and increased expression of CXCR4 in various disease states, it is likely that CXCR4-ADRB2 heteromers exist that confer unique characteristics to specific diseases. Thus, there is a need in the art to identify and develop inhibitors of CXCR4-ADRB2 heteromers for use as CXCR4-ADRB2 heteromer-targeted cancer therapeutics that have increased efficacy and reduced side effects compared to CXCR4 inhibitor monotherapy.

[0009] Provided herein are inhibitors of CXCR4-ADRB2 heteromers, or combinations of inhibitors of CXCR4-ADRB2 heteromers, and pharmaceutical compositions or kits containing same, as well as methods of treatment and use, wherein enhanced downstream signaling results from the formation of functional CXCR4-ADRB2 heteromers (e.g., enhanced downstream signaling for CXCR4 or enhanced downstream signaling for ADRB2); the presence of such CXCR4-ADRB2 heteromers in a subject, such as in the subject's cells, is associated with a disease, such as cancer. Summary of the Invention

[0010] In one aspect, provided herein is a method for suppressing enhanced downstream signaling resulting from CXCR4-ADRB2 heteromers in cells of a subject suffering from cancer, the method comprising administering to the subject (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor, wherein (i) the enhanced downstream signaling is due to CXCR4-ADRB2 heteromers; and (ii) administration of the combination of inhibitors suppresses the enhanced downstream signaling from the CXCR4-ADRB2 heteromers in the cancer subject.

[0011] In another aspect, provided herein is a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising administering to the subject (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor, wherein (i) enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers; and (ii) administration of the inhibitor combination suppresses enhanced downstream signaling from the CXCR4-ADRB2 heteromers in the cancer subject.

[0012] In another aspect, provided herein is a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, the method comprising: (a) determining whether the subject's cells contain CXCR4-ADRB2 heteromers, and if so, whether enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers; and (b) if the subject's cells contain the CXCR4-ADRB2 heteromers, administering to the cancer subject (i) a CXCR4 inhibitor that is blixafor; and (ii) an ADRB2 inhibitor.

[0013] In another aspect, a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers, comprises: (1) obtaining or obtaining a biological sample from the subject to determine whether the subject has cells containing CXCR4-ADRB2 heteromers; and (i) determining whether the subject's cells contain the CXCR4-ADRB2 heteromers; or (ii) determining whether a combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromers in cell(s) derived from the subject; or determining the heteromer-specific properties of the cell(s) derived from the subject that contain the CXCR4-ADRB2 heteromers. and (2) performing or having performed an assay on the biological sample to determine whether an inhibitor of CXCR4-ADRB2 alters or reduces the progression of cancer in a subject having cells containing the CXCR4-ADRB2 heteromer; and (3) if the subject does not have cells containing the CXCR4-ADRB2 heteromer, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor; and (4) if the subject does not have cells containing the CXCR4-ADRB2 heteromer, administering to the cancer subject a single inhibitor, either blixafor or an ADRB2 inhibitor.

[0014] In another aspect, a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers and where enhanced downstream signaling is due to CXCR4-ADRB2 heteromers comprises: (1) determining whether the subject has cells containing CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject; and (i) determining whether the subject's cells contain the CXCR4-ADRB2 heteromers; or (ii) performing or having performed an assay on the biological sample to determine whether a combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in cell(s) from the subject; alters the heteromer-specific properties of cell(s) from the subject that contain the CXCR4-ADRB2 heteromer; or reduces the progression of cancer in the subject having cells containing the CXCR4-ADRB2 heteromers; and (2) determining whether the subject has cells containing the CXCR4-ADRB2 heteromers by (3) if the subject has cells containing the CXCR4-ADRB2 heteromer, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor; and (4) if the subject does not have cells containing the CXCR4-ADRB2 heteromer, administering to the cancer subject a single inhibitor, either blixafor or an ADRB2 inhibitor, wherein (a) cancer progression in the subject having cells containing the CXCR4-ADRB2 heteromer is reduced by 5% to 100% more upon administration of the combination of blixafor and an ADRB2 inhibitor to the cancer subject compared to administration of either blixafor or an ADRB2 inhibitor alone; and (b) the efficacy of blixafor, when administered in combination with an ADRB2 inhibitor to the subject having cells containing CXCR4-ADRB2 heteromers, is increased by 5% to 2000% compared to the efficacy of blixafor when administered as a single inhibitor;and / or (c) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to a subject having such cells containing CXCR4-ADRB2 heteromers, is increased in the range of 5-2000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor.

[0015] In another aspect, a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is due to CXCR4-ADRB2 heteromers, comprises: (1) determining whether the subject's cells contain the CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromers are present in the subject's cells; wherein the assay performed on the biological sample is a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or an immunoassay. (i) being or including one or more of enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNA sequencing, RT-qPCR, microarray, or fluorescent animal assay; and (2) if the subject's cells have cells containing the CXCR4-ADRB2 heteromer, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor; and (3) if the subject's cells do not contain the CXCR4-ADRB2 heteromer, administering to the cancer subject a single inhibitor, either blixafor or an ADRB2 inhibitor.

[0016] In another aspect, a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is due to CXCR4-ADRB2 heteromers, comprises: (1) determining whether the subject's cells contain CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromers are present in the subject's cells; wherein the assay performed on the biological sample is or includes one or more of a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, proximity-based assay, co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNAseq, RT-qPCR, microarray, or fluorescent animal assay; and (2) determining whether the subject's cells contain CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromers are present in the subject's cells. (3) if the subject's cells do not contain the CXCR4-ADRB2 heteromer, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor; and (4) if the subject's cells do not contain the CXCR4-ADRB2 heteromer, administering to the cancer subject a single inhibitor, either blixafor or an ADRB2 inhibitor, wherein (a) cancer progression in the subject having the cells containing the CXCR4-ADRB2 heteromer is reduced by 5% to 100% or more upon administration of the combination of blixafor and an ADRB2 inhibitor to the cancer subject compared to administration of either blixafor or an ADRB2 inhibitor alone; and (b) the efficacy of blixafor, when administered in combination with an ADRB2 inhibitor to the subject having the cells containing CXCR4-ADRB2 heteromer, is increased by 5% to 2000% compared to the efficacy of blixafor when administered as a single inhibitor;and / or (c) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to a subject having such cells containing CXCR4-ADRB2 heteromers, is increased in the range of 5-2000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor.

[0017] In another aspect, provided herein is a pharmaceutical kit for use in treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising: (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor; wherein the enhanced downstream signaling is due to CXCR4-ADRB2 heteromers.

[0018] In another aspect, provided herein is a pharmaceutical composition for use in treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, the pharmaceutical composition comprising: (a) a CXCR4 inhibitor that is blixafor; (b) an ADRB2 inhibitor; and (c) a pharmaceutically acceptable carrier, wherein the enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromers.

[0019] In another aspect, provided herein is a pharmaceutical composition comprising: (a) a CXCR4 inhibitor that is blixafor; (b) an ADRB2 inhibitor; and (c) a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0020] [Figure 1] A schematic diagram of the bimolecular fluorescence complementation (BiFC) assay is shown. GPCR A is fused to the N-terminal fragment (VN) of the yellow fluorescent protein (YFP) Venus, and GPCR B is fused to the C-terminal fragment (VC) of Venus. When GPCRs A and B form a heteromer, the complementary VN and VC become close enough to form a functional Venus.

[0021] [Figure 2]Figure 1 shows the identification of CXCR4 interacting with ADRB2 using a BiFC assay. Representative images showing negative BiFC signals are CXCR4-VN and HA-VC (A), and CXCR4-VN and GCGR-VC (C). Representative images showing positive BiFC signals are CXCR4-VN and CXCR4-VC (B), and CXCR4-VN and ADRB2-VC (D).

[0022] [Figure 3] Figures A-B show the principle of GPCR co-internalization studies. Cells co-expressing CXCR4-GFP and GPCRx are treated with a GPCRx-specific agonist. (A) CXCR4 and GPCRx do not physically interact with each other. The GPCRx agonist induces internalization of GPCRx but not CXCR4-GFP. (B) CXCR4 and GPCRx physically interact and form a heteromer. The GPCRx agonist induces internalization of GPCRx, and CXCR4-GFP is co-internalized with GPCRx.

[0023] [Figure 4] Panels A-B show the co-internalization of CXCR4-EGFP upon stimulation of GPCRx with its corresponding agonist (control: CXCR4-GFP (A)). Adenoviruses encoding CXCR4-EGFP and GPCRx-VC were co-transduced into U-2 OS cells to examine whether the following GPCRx partners form heteromers with CXCR4-EGFP and induce co-internalization of CXCR4-EGFP: GPCRx represents ADRB2 (B).

[0024] [Figure 5]A–D show enhanced calcium responses in cells coexpressing CXCR4 and ADRB2 upon costimulation with their respective selective agonists. MDA-MB-231 human breast cancer cells were transduced with adenoviruses encoding CXCR4 and HA-VC (A), ADRB2 and HA-VC (B), or CXCR4 and ADRB2 (C). Adenovirus encoding HA-VC was used to adjust the total amount of transduced adenovirus. Cells were allowed to express GPCRs for 2 days, incubated with Cal-520 AM for 2 hours, and treated with 15 nM CXCL12, 100 nM salmeterol (an ADRB2-selective agonist), or CXCL12 and salmeterol. Calcium mobilization was measured using a FlexStation 3 Multi-Mode Microplate Reader. Results were normalized to baseline activity. Data represent three independent experiments (mean ± SEM). (D) Calcium mobilization was quantified by calculating the area under the curve (AUC) for each graph in A–C. Data were normalized to the CXCL12-stimulated calcium response in cells expressing CXCR4 alone. Total represents the sum of the responses obtained after individual stimulation with CXCL12 and salmeterol in cells coexpressing CXCR4 and ADRB2 to allow visualization of synergistic effects. ***P<0.001, Student's t-test.

[0025] [Figure 6]Figure 5 shows that co-administration of both antagonists effectively suppressed the enhanced calcium response observed when cells expressing CXCR4 and ADRB2 and containing CXCR4-ADRB2 heteromers were simultaneously stimulated with CXCL12 and the ADRB2 agonist salmeterol. MDA-MB-231 cells were co-transduced with adenoviruses encoding CXCR4 and ADRB2. Cells were incubated with Cal-520 AM for 2 hours, incubated with an ADRB2 antagonist or vehicle for 30 minutes, and stimulated with the indicated amounts of CXCL12, the ADRB2 agonist salmeterol, or both CXCL12 and the ADRB2 agonist salmeterol. Calcium mobilization was quantified as described in Figure 5D. Data represent three independent experiments (mean ± SEM). *P<0.05, **P<0.01, ***P<0.001, Student's t-test.

[0026] [Figure 7] The principle of internalization inhibition studies is shown below. Cells co-expressing CXCR4-GFP and GPCRx were treated with CXCL12 (SDF-1) and / or a GPCRx-specific antagonist. Scenario A: CXCR4 and GPCRx form heteromers. CXCR4 agonist CXCL12 (SDF-1) induced the internalization of CXCR4-GFP alone or CXCR4-GFP in combination with GPCRx. Scenario B: GPCRx antagonists did not induce CXCR4-GFP internalization. Scenario C: CXCL12 (SDF-1)-stimulated CXCR4-GFP internalization was inhibited by a GPCRx-specific antagonist.

[0027] [Figure 8]Figure 1 shows inhibition of internalization of CXCR4-ADRB2 heteromers by ADRB2 antagonists. CXCR4-GFP-expressing U-2 OS cells were transduced with adenovirus encoding ADRB2. Treatment with CXCR4 agonist CXCL12 induced CXCR4-ADRB2 internalization. However, treatment with ADRB2 antagonist carvedilol did not induce internalization. Cotreatment with CXCL12 and carvedilol partially inhibited CXCL12-induced CXCR4-ADRB2 internalization.

[0028] [Figure 9] Figure 1 shows the effect of an ADRB2 antagonist on the survival of patient-derived cells (PDCs) from cancer patients. Effect of an ADRB2 antagonist (carvedilol) on PDC survival. Carvedilol induced a significant decrease in cell viability (IC50 = 11.69 μM).

[0029] [Figure 10] Figures A-C show the detection of CXCR4-ADRB2 heterodimers in U-2 OS cells overexpressing CXCR4 and ADRB2 by PLA and RT-qPCR. CXCR4-GFP-expressing U-2 OS cells were transduced with adenovirus encoding ADRB2 at various MOIs for 2 days. PLA was then performed on CXCR4-ADRB2-coexpressing U-2 OS cells. A: Images of CXCR4-ADRB2 heterodimer detection by PLA. B: Increased PLA signal is proportional to the ADRB2 expression level in a dose-dependent manner. C: Data from RT-qPCR showing endogenous ADRB2 expression levels in U-2 OS cells.

[0030] [Figure 11]Panels A and B show the detection of CXCR4-ADRB2 heteromers in PDCs by PLA. PDCs originating from GBM (sample IDs: 986T, 948T, 783T, 777T, 352T1, 352T2, 578T, 559T, 464T, 448T, and 096T) were seeded onto chamber slides, and CXCR4-ADRB2 heteromers were detected by PLA using antibodies specific for CXCR4 and ADRB2. A: Images of CXCR4-ADRB2 heteromer detection. Nuclei were visualized with DAPI staining, and CXCR4-ADRB2 heteromers are indicated by small dots. B: Percentage of CXCR4-ADRB2 heteromers in PDCs.

[0031] [Figure 12] Panels A and B show results from the detection of CXCR4-GPCRx heteromers in PDX. CXCR4-ADRB2 heteromers were detected in PDXs originating from GBM (sample IDs: 777T, 783T, 948T, and 559T) by PLA using antibodies specific for CXCR4 and ADRB2. A: Images of CXCR4-ADRB2 heteromer detection. Nuclei were visualized by DAPI staining, and CXCR4-ADRB2 heteromers are indicated by small dots. B: Percentage of CXCR4-ADRB2 heteromers in PDX.

[0032] [Figure 13]Enhanced calcium responses in cells coexpressing CXCR4 and ADRB2 upon costimulation with an ADRB2 agonist are shown. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and ADRB2. Cells were cultured for 3 days, stained with Cal-520 AM, and treated with either CXCL12 (30 nM) alone, increasing doses of salmeterol alone, or increasing doses of salmeterol in combination with 30 nM CXCL12. Calcium mobilization was measured using a FlexStation 3. Totals represent the sum of the responses evoked by 30 nM CXCL12 alone (open squares) and the indicated doses of ADRB2 ligand alone (filled circles). The sum graph is delineated by inverted triangles and dashed lines. Statistical significance between the sum (inverted triangles) and cotreatment (filled squares) at each point was determined by Student's t-test. *P<0.05, **P<0.01, ***P<0.001; data represent the mean ± standard deviation (n=3).

[0033] [Figure 14] Figure 1 shows that cotreatment with an anti-CXCR4 antibody and an ADRB2 antagonist effectively suppressed the enhanced calcium response observed when cells expressing CXCR4-ADRB2 heteromers were simultaneously stimulated with CXCL12 and an ADRB2 agonist. MDA-MB-231 cells were cotransduced with adenoviruses encoding CXCR4 and ADRB2. Cells were treated with the indicated concentrations of an ADRB2 antagonist (carvedilol), an anti-CXCR4 antibody (12G5), or vehicle and incubated with Cal 6 for 2 hours. Cells were then stimulated with the indicated amounts of CXCL12, an ADRB2 agonist (salmeterol), or both CXCL12 and ADRB2 agonists.

[0034] [Figure 15](A) Images of three mice implanted with parental A549 cells, A549-CXCR4 stably overexpressing CXCR4, and A549-CXCR4-ADRB2 stably overexpressing the CXCR4-ADRB2 heteromer, at day 28 post-implantation. (B) A graph comparing tumor growth of implanted cells from (A). Tumor growth was monitored every 3 or 4 days by measuring tumor length (L) and width (W) and calculating tumor volume based on the following formula: volume = 0.5LW2. Results are expressed as the mean ± standard deviation of three mice.

[0035] [Figure 16] Figures AB show ERK activation upon stimulation with CXCL12 and / or salmeterol in CXCR4-ADRB2 heteromer-expressing MDA-MB-231 cells. Figure A shows Western blot analysis of phospho-ERK1 / 2 Thr202 / Tyr204 and total ERK1 / 2 in MDA-MB-231 cells, MDA-MB-231-CXCR4, and MDA-MB-231-CXCR4-ADRB2 treated with CXCL12 (10 nM) and / or salmeterol (10 nM) for 20 minutes. Figure B shows densitometry analysis (iBright Analysis Software) of phospho-ERK1 / 2 Thr202 / Tyr204 protein expression relative to total ERK protein levels.

[0036] [Figure 17] Figures AB show ERK activation upon stimulation with CXCL12 and / or salmeterol in CXCR4-ADRB2 heteromer-expressing A549 cells. Figure A shows Western blot analysis of phospho-ERK1 / 2 Thr202 / Tyr204 and total ERK1 / 2 in A549, A549-CXCR4, and A549-CXCR4-ADRB2 cells treated with CXCL12 (10 nM) and / or salmeterol (10 nM) for 10 minutes. Figure B shows densitometry analysis (iBright Analysis Software) of phospho-ERK1 / 2 Thr202 / Tyr204 protein expression relative to total ERK protein levels.

[0037] [Figure 18A] (Figure 1 shows the effect of CXCR4 antagonists on CXCR4-CXCL12-mediated proliferation.) A549 double-negative cells (RLuc-Luc2P) and A549-CXCR4-ADRB2 cells overexpressing CXCR4 and ADRB2 were seeded in 96-well plates and stimulated with CXCL12 and / or the indicated drugs (AMD3100 (10 μM)) in serum-free conditions for 72 hours. Fluorescence from triplicate wells was measured, and data are presented as the mean fluorescence ratio (drug-treated / vehicle) ± SEM. [Figure 18B] Figure 1 shows the effect of CXCR4 antagonists on CXCR4-CXCL12-mediated proliferation. A549 double-negative cells (RLuc-Luc2P) and A549-CXCR4-ADRB2 cells overexpressing CXCR4 and ADRB2 were seeded in 96-well plates and stimulated with CXCL12 and / or the indicated drugs (LY2510924 (10 μM)) in serum-free conditions for 72 hours. Fluorescence from triplicate wells was measured, and data are presented as the mean fluorescence ratio (drug-treated / vehicle) ± SEM. [Figure 18C] Figure 1 shows the effect of CXCR4 antagonists on CXCR4-CXCL12-mediated proliferation. A549 double-negative cells (RLuc-Luc2P) and A549-CXCR4-ADRB2 cells overexpressing CXCR4 and ADRB2 were seeded in 96-well plates and stimulated with CXCL12 and / or the indicated drug (AMD070 (1 μM)) in serum-free conditions for 72 hours. Fluorescence from triplicate wells was measured, and data are presented as the mean fluorescence ratio (drug-treated / vehicle) ± SEM. [Figure 18D](Figure 1 shows the effect of CXCR4 antagonists on CXCR4-CXCL12-mediated proliferation. A549 double-negative cells (RLuc-Luc2P) and A549-CXCR4-ADRB2 cells overexpressing CXCR4 and ADRB2 were seeded in 96-well plates and stimulated with CXCL12 and / or the indicated drug (TG-0054 (10 μM)) in serum-free conditions for 72 hours. Fluorescence from triplicate wells was measured, and data are presented as the mean fluorescence ratio (drug-treated / vehicle) ± SEM. [Figure 18E] Figure 1 shows the effect of CXCR4 antagonists on CXCR4-CXCL12-mediated proliferation. A549 double-negative cells (RLuc-Luc2P) and A549-CXCR4-ADRB2 cells overexpressing CXCR4 and ADRB2 were seeded in 96-well plates and stimulated with CXCL12 and / or the indicated drugs (BKT-140 (10 μM)) in serum-free conditions for 72 hours. Fluorescence from triplicate wells was measured, and data are presented as the mean fluorescence ratio (drug-treated / vehicle) ± SEM.

[0038] [Figure 19A] 1 shows the correlation between CXCR4-ADRB2 heteromer expression and tumor growth. 1 shows the detection of CXCR4-ADRB2 heteromers by PLA in A549 parental cells, the CXCR4 homomer-expressing A549-CXCR4 cell line, and the CXCR4-ADRB2 heteromer-expressing A549-CXCR4-ADRB2 cell line. [Figure 19B] 1 shows the correlation between CXCR4-ADRB2 heteromer expression and tumor growth. Quantification of CXCR4-ADRB2 heteromers by PLA in A549, A549-CXCR4, and A549-CXCR4-ADRB2. [Figure 19C] 1 shows the correlation between CXCR4-ADRB2 heteromeric expression and tumor growth. 2 shows a comparison of tumor growth between mice bearing A549 parental cells and mice bearing A549-CXCR4-ADRB2 cells.

[0039] [Figure 20A]1 shows the correlation between CXCR4-ADRB2 heteromer expression and tumor growth. 1 shows the detection of CXCR4-ADRB2 heteromers by PLA in MDA-MB-231 parental cells, the CXCR4 homomer-expressing MDA-MB-231-CXCR4 cell line, and the CXCR4-ADRB2 heteromer-expressing MDA-MB-231-CXCR4-ADRB2 cell line. [Figure 20B] 1 shows the correlation between CXCR4-ADRB2 heteromer expression and tumor growth. Quantification of CXCR4-ADRB2 heteromers by PLA in MDA-MB-231, MDA-MB-231-CXCR4, and MDA-MB-231-CXCR4-ADRB2. [Figure 20C] 1 shows the correlation between CXCR4-ADRB2 heteromeric expression and tumor growth. 2 shows a comparison of tumor growth among mice bearing MDA-MB-231 parental cells, MDA-MB-231-CXCR4 cells, or MDA-MB-231-CXCR4-ADRB2 cells.

[0040] [Figure 21A] Figure 1 shows the effect of a CXCR4 inhibitor alone on tumor growth in CXCR4-ADRB2 heteromer-expressing A549 xenograft mice. Mice implanted with A549 cell lines expressing CXCR4-ADRB2 heteromers were dose-dependently treated with AMD3100, one of various CXCR4 inhibitors, and the tumor size was compared. [Figure 21B] Figure 1 shows the effect of a single CXCR4 inhibitor on tumor growth in CXCR4-ADRB2 heteromer-expressing A549 xenograft mice. Mice implanted with A549 cell lines expressing CXCR4-ADRB2 heteromers were dose-dependently treated with various CXCR4 inhibitors, including LY2510924, and tumor size was compared. [Figure 21C]Figure 1 shows the effect of a single CXCR4 inhibitor on tumor growth in CXCR4-ADRB2 heteromer-expressing A549 xenograft mice. Mice implanted with A549 cell lines expressing CXCR4-ADRB2 heteromers were given AMD070, one of various CXCR4 inhibitors, in a dose-dependent manner, and tumor size was compared. [Figure 21D] Figure 1 shows the effect of a single CXCR4 inhibitor on tumor growth in CXCR4-ADRB2 heteromer-expressing A549 xenograft mice. Mice implanted with A549 cell lines expressing CXCR4-ADRB2 heteromers were given TG-0054, one of various CXCR4 inhibitors, in a dose-dependent manner, and tumor size was compared.

[0041] [Figure 22A] Figure 1 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing cells were implanted into mice and treated with a CXCR4 inhibitor and the ADRB2 inhibitor carvedilol, either alone or in combination. Figure 2 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing MDA-MB-231 cells were orthotopically implanted into mice and treated with a CXCR4 inhibitor (AMD3100) and the ADRB2 inhibitor carvedilol, either alone or in combination. Results are expressed as the mean ± standard deviation of five animals. [Figure 22B] Figure 1 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing cells were implanted into mice and treated with a CXCR4 inhibitor and the ADRB2 inhibitor carvedilol, either alone or in combination. Figure 2 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing MDA-MB-231 cells were orthotopically implanted into mice and treated with a CXCR4 inhibitor (LY2510924) and the ADRB2 inhibitor carvedilol, either alone or in combination. Results are expressed as the mean ± standard deviation of five animals. [Figure 22C]Figure 1 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing cells were implanted into mice and treated with a CXCR4 inhibitor and the ADRB2 inhibitor carvedilol, either alone or in combination. Figure 2 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing MDA-MB-231 cells were orthotopically implanted into mice and treated with a CXCR4 inhibitor (AMD070) and the ADRB2 inhibitor carvedilol, either alone or in combination. Results are expressed as the mean ± standard deviation of five animals. [Figure 22D] Figure 1 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing cells were implanted into mice and treated with a CXCR4 inhibitor and the ADRB2 inhibitor carvedilol, either alone or in combination. Figure 2 shows the relative tumor growth when CXCR4-ADRB2 heteromer-expressing A549 cells were implanted into mice (A549 xenograft model) and treated with AMD3100 (a CXCR4 inhibitor) and carvedilol (an ADRB2 inhibitor), either alone or in combination. Tumor growth was monitored every 3 or 4 days by measuring the length (L) and width (W) of the tumor and calculating the tumor volume based on the following formula: volume = 0.5LW2. Results are expressed as the mean ± standard deviation of 10 animals.

[0042] [Figure 23A] Ligand-assisted TR-FRET signals observed in A549 cells transiently infected with Ad-CXCR4 and Ad-ADRB2 at the indicated MOIs and labeled with TZ14011-tb and propranolol-g2 in the presence or absence of propranolol (1 μM) as a competitor are shown.

[0043] [Figure 23B]Antibody-mediated TR-FRET signals observed in U2OS cells transiently infected with Ad-CXCR4 and Ad-ADRB2 at the indicated MOIs are shown, followed by incubation with rabbit anti-CXCR4 and mouse anti-ADRB2 antibodies. Terbium cryptate-labeled goat anti-rabbit IgG and Alexa Fluor 647-labeled goat anti-mouse IgG were used for TR-FRET.

[0044] [Figure 24] Figure 1 shows CXCR4-ADRB2 heteromer detection by PLA and quantification of CXCR4 or ADRB2 through RNA expression levels by RT-qPCR in solid tumor cancer cell lines: A549 (lung cancer), U2OS (osteosarcoma), and MDA-MB-231 (breast cancer). A shows the RNA expression levels of CXCR4 and ADRB2 by RT-qPCR. B shows images of CXCR4 and ADRB2 heteromers detected by PLA. C shows quantification of CXCR4 and ADRB2 heteromers by PLA.

[0045] [Figure 25] Figures A-C show CXCR4-ADRB2 heteromer detection by PLA and quantification of CXCR4 or ADRB2 through RNA expression levels by RT-qPCR in hematological cancer cell lines: HL60 (leukemia), U937 (leukemia), and RPMI 8226 (myeloma). Figure A shows the RNA expression levels of CXCR4 and ADRB2 by RT-qPCR. Figure B shows images of CXCR4 and ADRB2 heteromers detected by PLA. Figure C shows quantification of CXCR4 and ADRB2 heteromers by PLA.

[0046] [Figure 26]Figures A-B show enhanced calcium responses in cells expressing CXCR4-ADRB2 heteromers upon costimulation with CXCL12 and the ADRB2 agonist formoterol. Figure A shows enhanced calcium responses in U937 cells expressing CXCR4-ADRB2 heteromers upon costimulation with CXCL12 and the ADRB2 agonist formoterol. Figure B shows enhanced calcium responses in HL-60 cells expressing CXCR4-ADRB2 heteromers upon costimulation with CXCL12 and the ADRB2 agonist formoterol. DETAILED DESCRIPTION OF THE INVENTION

[0047] Abbreviation Unless otherwise indicated, the following include abbreviations for terms disclosed herein: acute myeloid leukemia (AML), adenoviral high-throughput system (AdHTS), adrenergic receptor beta 2 (ADRB2), bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer (BRET), bovine serum albumin (BSA), cancer stem cell (CSC), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic obstructive pulmonary disease (COPD), Venus C-terminal fragment (VC), C-X-C motif chemokine CXCL12, CXC receptor 4 (CXCR4), enzyme-linked immunosorbent assay (ELISA), formalin-fixed paraffin-embedded (FFPE), fluorescence resonance energy transfer (FRET), G protein-coupled receptor (GPCR), glioblastoma multiforme (GBM), glucagon receptor (GCGR), GPCR heteromer identification technology (GPCR-HIT), granulocyte colony-stimulating factor (G-CSF), hematopoietic stem cells (HSC), hepatocellular carcinoma (HCC), human immunodeficiency virus (HIV), International Society of Pharmacology (ISSP) National Committee on Immunotherapy and Immunotherapy for Cancer (NC-IUPHAR), multiple myeloma (MM), multiplicity of infection (MOI), myelodysplastic syndrome (MDS), 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), proximity ligation assay (PLA), quantitative reverse transcription polymerase chain reaction (RT-qPCR, or qRT-PCR, or qPCR, or reverse transcription). (sometimes referred to as real-time PCR), single-photon emission computed tomography (SPECT), small lymphocytic lymphoma (SLL), small cell lung cancer (SCLC), stromal cell-derived factor 1 (SDF-1), systemic lupus erythematosus (SLE), threshold cycle (Ct), time-resolved FRET (TR-FRET), tumor microenvironment (TME), vascular smooth muscle cells (VSMC), WHIM syndrome (warts, hypogammaglobulinemia, immunocompromise, myelocatechus), green fluorescent protein (GFP), and yellow fluorescent protein (YFP).

[0048] Detailed Description of the Invention As used herein, the articles "a," "an," and "the" refer to one or to more than one of the grammatical objects of the article. By way of example, "a sample" refers to one sample or to more than one sample.

[0049] As used herein, the term "subject" refers to a mammal. A subject can be a human or non-human mammal, such as a dog, cat, cow, horse, mouse, rat, rabbit, or transgenic species thereof. A subject can be a patient or a cancer patient.

[0050] As used herein, the term "sample" refers to a substance or mixture of substances containing one or more components of interest. A sample from a subject refers to a sample obtained from a subject, including samples of biological tissue or fluid origin obtained, delivered, or collected in vivo or in situ. A sample can be obtained from a site of a subject containing precancerous or cancerous cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions, and cells isolated from a mammal. In certain embodiments, a sample can be a biological sample, such as a biological fluid sample or a biological tissue sample. A sample can also be a tissue biopsy. In certain embodiments, a biological sample includes, but is not limited to, a lymph node, a blood sample, a plasma sample, whole blood, partially purified blood, serum, bone marrow, a cell lysate, a cell culture, a cell line, a tissue, an oral tissue, a gastrointestinal tissue, an organ, an organelle, a biological fluid, a urine sample, a skin sample, a saliva sample, a cerebrospinal fluid sample, or an ocular fluid sample.

[0051] As used herein, the terms "treat," "treating," or "treatment," when used in relation to a cancer patient, refer to actions that reduce the severity of cancer or delay or slow the progression of cancer, including (a) inhibiting the growth of cancer or halting the development of cancer, and (b) causing regression of cancer or delaying or minimizing one or more symptoms associated with the presence of cancer.

[0052] As used herein, the terms "administer," "administering," or "administration" refer to the act of delivering or causing to be delivered a compound, a combination of compounds, or a pharmaceutical composition comprising a compound to the body of a subject by methods described herein or otherwise known in the art. Administering a compound, a combination of compounds, or a pharmaceutical composition comprising a compound includes formulating the compound, a combination of compounds, or a pharmaceutical composition comprising a compound for delivery to the patient's body. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, suspensions, etc.; intravenous (IV), intramuscular (IM), or intraperitoneal (IP); injectable dosage forms such as subcutaneous (SC), intracranial (IC); transdermal dosage forms including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0053] As used herein, the phrase "therapeutic agent" refers to any agent that can be used in the treatment, amelioration, prevention, or management of cancer and / or cancer-related symptoms. In certain embodiments, a therapeutic agent refers to an inhibitor of a CXCR4-ADRB2 heteromer of the present invention. A therapeutic agent can be an agent that is known to be useful, or is being used, or is currently being used, for the treatment, amelioration, prevention, or management of cancer and / or cancer-related symptoms.

[0054] As used herein, the phrase "effective amount" refers to an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, doses, or dosages. Such delivery depends on several variables, including the period for which the individual dosage unit is to be used, the bioavailability of the agent, the route of administration, etc.

[0055] As used herein, the term "therapeutically effective amount" of a compound (e.g., a therapeutic agent such as an inhibitor, antagonist, or any other therapeutic agent provided herein) or combination of compounds when used in connection with a disease or disorder refers to an amount sufficient to achieve a therapeutic effect in the treatment or management of the disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound refers to an amount of a compound that, when used alone or in combination with other therapies, would provide a therapeutic effect in the treatment or management of a disease or disorder. The term encompasses an amount that improves overall treatment, alleviates or avoids symptoms, or enhances the therapeutic efficacy of another therapeutic agent. The term also refers to an amount of a compound sufficient to elicit the biological or medical response in a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human that a researcher, veterinarian, physician, or clinician is seeking.

[0056] As used herein, the term "heteromer" refers to a macromolecular complex composed of a CXCR4 unit (or sometimes referred to as a CXCR4 protomer when identified in the context of a CXCR4-containing heteromer) and an ADRB2 unit (or sometimes referred to as an ADRB2 protomer when identified in the context of an ADRB2-containing heteromer) that has biochemical properties that are demonstrably different from the biochemical properties of a CXCR4 monomer or an ADRB2 monomer, respectively, or that are demonstrably different from the biochemical properties of both a CXCR4 monomer and an ADRB2 monomer, respectively. Heteromerization can be assessed by in situ hybridization, immunohistochemistry, RNAseq, reverse transcription-quantitative PCR (sometimes referred to as RT-qPCR, or qRT-PCR, or qPCR, or real-time PCR), expression levels of each monomer or protomer of an identified heteromer (e.g., expression levels of CXCR4 and ADRB2 as associated with CXCR4-ADRB2 heteromers), microarrays, proximity ligation assays (PLA), time-resolved FRET (TR-FRET), whole-body single-photon emission computed tomography (SPECT), or positron emission tomography / computed tomography (PET / CT).

[0057] As used herein, the phrases "intracellular Ca2+ assay," "calcium mobilization assay," or variations thereof, refer to a cell-based assay for measuring calcium flux associated with GPCR activation or inhibition, such as CXCR4 activation or inhibition and / or ADRB2 activation or inhibition. This method utilizes a calcium-sensitive fluorescent dye that is incorporated into the cytoplasm of most cells. The dye binds to calcium released from intracellular stores, increasing its fluorescence. The change in fluorescence intensity directly correlates with the amount of intracellular calcium released into the cytoplasm in response to ligand activation of the receptor of interest. In certain embodiments, the GPCR is CXCR4. In certain embodiments, the GPCR is ADRB2. In certain embodiments, the cell-based assay measures calcium flux associated with CXCR4-ADRB2 heteromeric activation or inhibition.

[0058] As used herein, the phrase "proximity-based assay" refers to biophysical and biochemical techniques that can monitor the proximity and / or binding of two protein molecules in vitro (in cell lysates) and in living cells, such as the proximity and / or binding of CXCR4 protein (monomer or unit) and ADRB2 protein (monomer or unit), and includes 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, S., et al., (2009) Building a new conceptual framework for receptor heteromers, Nat Chem Biol 5, 131-134; Gomes, I., et al., (2016) G Protein-Coupled Receptor Heteromers, Annu Rev Pharmacol Toxicol 56, 403-425).

[0059] Alternative methods for detecting CXCR4-ADRB2 heteromer formation include, but are not limited to, immunostaining (Bushlin, T., et al., (2012) Dimerization with cannabinoid receptors allosterically modulates delta opioid receptor activity during neuropathic pain, PLoS One 7, e49789; Decaillot, FM, et al., (2008) Cell surface targeting of mu-delta opioid receptor heterodimers by RTP4, Proc Natl Acad Sci USA 105, 16045-16050); immunoelectron microscopy (Fernandez-Duenas, V., et al., (2015) Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats, Dis Model Mech 8, 57-63); BRET (Pfleger, KD, et al., (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET), Nat Methods 3, 165-174); time-resolved FRET assay (Fernandez-Duenas, V., et al., 2015); in situ hybridization (He, SQ, et al., (2011) Facilitation of mu-opioid receptor activity by preventing delta-opioid receptor-mediated coding, Neuron 69, 120-131); FRET (Lohse, MJ, et al.,(2012) Fluorescence / bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling, Pharmacol Rev 64, 299-336; GPCR heteromer identification technology (GPCR-HIT, Dimerix Bioscience) (Mustafa, S., et al.,(2011) G protein-coupled receptor heteromer identification technology: identification and profiling of GPCR heteromers, J Lab Autom 16, 285-291); BRET, FRET, BiFC, bimolecular luminescence complementation, enzyme cleavage assay, and β-arrestin recruitment assay using the Tango Tango GPCR Assay System (Thermo Fisher Scientific) (Mustafa, S., et al.,(2010) Uncovering GPCR heteromer-biased ligands, Drug Discovery Today Technol 7, e1-e94); PRESTO-Tango system (Kroeze, WK, et al. al., (2015) PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome, Nat Struct Mol Biol 22, 362-369; Secretion Modulation / Aggregation Technology (ARIAD Pharmaceuticals) (Hansen, JL, et al., (2009) Lack of evidence for AT1R / B2R heterodimerization in COS-7, HEK293, and NIH3T3 cells: how common is the AT1R / B2R heterodimer? J Biol Chem 284, 1831-1839); Receptor Selection and Amplification Technology (ACADIA Pharmaceuticals) (Hansen, JL, et al., 2009); DimerScreen (Cara Therapeutics) (Mustafa, S., 2010); Dimer / Interacting Protein Translocation Assay (Patobios) (Mustafa, S., 2010); co-immunoprecipitation (Abd Alla, J., et al., (2009) Calreticulin enhances B2 bradykinin receptor maturation and heterodimerization, Biochem Biophys Res Commun 387, 186-190); GPCR internalization assay using surface enzyme-linked immunosorbent assay (ELISA) (Decaillot, FM, et al., 2008) or flow cytometry (Law, PY, et al., (2005) Heterodimerization of mu- and delta-opioid receptors occurs at the cell surface only and requires receptor-G protein interactions, J Biol Chem 280, 11152-11164); whole-cell phosphorylation assay (Pfeiffer, M., et al., (2002) Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization, J Biol Chem 277, 19762-19772); and proximity ligation assay (PLA) (Frederick, AL, et al., (2015) Evidence against dopamine D1 / D2 receptor heteromers, Mol Psychiatry 20, 1373-1385).

[0060] Alternative methods for detecting changes in pharmacological, signaling, and / or trafficking properties in cells expressing both CXCR4 and ADRB2 include, but are not limited to, radioligand binding assays (Bushlin, T., et al., 2012; Pfeiffer, M., et al., 2002); cell surface biotinylation and immunoblotting (He, SQ, et al., 2011); immunostaining (Bushlin, T., et al., 2012; Decaillot, FM, et al., 2008); immunoelectron microscopy (Fernandez-Duenas, V., et al., 2015); [35S]GTP-γS binding assay (Bushlin, T., et al., 2012); Fura 2-acetomethoxyester (Molecular Probes), Fluo-4 NW calcium dye (Thermo Fisher Scientific), or FLIPR5 dye (Molecular Probes). calcium (Calcium) imaging or assay using dyes such as Calcium Imaging Devices; cAMP assay using radioimmunoassay kits (Amersham Biosciences); AlphaScreen (PerkinElmer Life Sciences); parameter cyclic AMP assay (R&D Systems); femto cAMP kit (Cisbio); cAMP Direct Immunoassay Kit (Calbiochem) or GloSensor cAMP assay (Promega); GTPase assay (Pello, O.M., et al., (2008) Ligand stabilization of CXCR4 / delta-opioid receptor heterodimers reveals a mechanism for immune response regulation, Eur J Immunol 38, 537-549); PKA activation (Stefan, E., et al.,(2007) Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo, Proc Natl Acad Sci USA 104, 16916-16921); ERK1 / 2 and / or Akt / PKB phosphorylation assays (Callen, L., et al.,(2012) Cannabinoid receptors CB1 and CB2 form functional heteromers in the brain, J Biol Chem 287, 20851-20865); reporter assays such as cAMP response element (CRE); serum response element (SRE); serum response factor response element (SRF-RE); and secreted alkaline phosphatase assays (Decaillot, FM, et al., 2011); measurement of inositol 1-phosphate production using TR-FRET or [3H]myo-inositol (Mustafa, S., et al.,(2012) Identification and profiling of Novel alpha1A-adrenoceptor-CXC chemokine receptor 2 heteromer, J Biol Chem 287, 12952-12965); RT-qPCR to measure downstream target gene expression (Mustafa, S., et al., 2012); and adenylyl cyclase activity (George, SR, et al., (2000) Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. J Biol Chem 275, 26128-26135); next-generation sequencing (NGS); and any other assay that can detect changes in receptor function as a result of receptor heterodimerization.

[0061] As used herein, the term "ADRB2" refers to the beta-2 adrenergic receptor (also called beta-2 adrenergic receptor or β2 adrenoceptor), a transmembrane beta adrenergic receptor that interacts with epinephrine (a hormone and neurotransmitter (another name for its ligand, adrenaline)), whose signaling mediates physiological responses such as smooth muscle relaxation and bronchodilation via downstream L-type calcium channel interactions (Gregorio, GG, et al., 2017). ADRB2 is also identified by its unique exemplary database identifier (ID) and alternative names as shown in Table 1. ADRB2 functions in the muscular system, such as smooth muscle relaxation, motor nerve terminals, and glycogenolysis, and in the circulatory system, such as myocardial contraction and increased cardiac output. In the normal eye, beta-2 stimulation by salbutamol increases intraocular pressure via the retina. In the digestive system, ADRB2 induces glycogenolysis and gluconeogenesis in the liver and insulin secretion from the pancreas (Fitzpatrick, D., et al., (2004) "Table 20:2" (Mass: Sunderland)). Activation of ADRB2 can stimulate signaling pathways that promote tumor growth and metastasis, including the Ras-mediated Raf proto-oncogene serine / threonine-protein kinase (Raf) / dual specificity mitogen-activated protein kinase kinase (MEK) / extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K) / RAC serine / threonine-protein kinase (Akt), and cAMP / protein kinase A / mitogen-activated protein kinase pathways (Quoc Lu'o'ng, KV, et al., (2012) Cancer Manag Res 4: 431-445), which promote cell proliferation and invasion and suppress apoptosis of cancer cells, which can enhance tumor growth and promote metastasis (Antoni, MH, et al., (2006) Nat Rev Cancer 6: 240-248; Thaker, PH, et al., (2006) Nat Med 12: 939-944).

[0062] As used herein, the term "CXCR4" refers to the C-X-C motif chemokine receptor 4, which is also identified by its unique exemplary database identifier (ID) and alternative names as shown in Table 1 (Chatterjee, S., et al., 2014; Debnath, B., et al., 2013; Domanska, U.M., et al., 2013; Guo, F., et al., (2016) CXCL12 / CXCR4: a symbiotic bridge linking cancer cells and their stromal neighbors in oncogenic communication networks, Oncogene 35, 816-826; Peled, A., et al., (2012) Development of novel CXCR4-based therapeutics, Expert Opin Investig Drugs 21, 341-353; Roccaro, A.M., et al., (2014) SDF-1 inhibition targets the bone marrow niche for cancer therapy, Cell Rep 9, 118-128; Walenkamp, ​​AME, et al., (2017) CXCR4 Ligands: The Next Big Hit? J Nucl Med 58, 77S-82S). CXCL12 binding to CXCR4 initiates a wide variety of signaling pathways downstream of ligand binding, resulting in a variety of responses, including chemotaxis, cell survival and / or proliferation, increased intracellular calcium, and gene transcription. Chemotaxis is mediated by either PKC or Gα iIt has been shown that MAPK signaling is mediated through Erk / 1 / 2, which can signal through Erk / 1 / 2 (Mellado, M., et al., (2001) Annu Rev Immunol; 19:397-421; Bendall, LJ, et al., (2005) Cancer Res; 65:3290-8). The pair of chemokine CXCL12 and chemokine receptor CXCR4 plays an important role in many stages of tumorigenesis. CXCR4 is overexpressed in various human cancers, and this overexpression correlates with an increased risk of recurrence and poor overall survival in multiple cancers, including breast, lung, kidney, colon, ovarian, and brain cancers, as well as lymphoma and leukemia (Balkwill, F., (2004) Nat Rev Cancer; 4: 540-50. 1-5; Orimo, A., et al., (2005) Cell; 121: 335-48; Domanska, UM, et al., 2013). The important role of CXCR4 in cancer and other diseases has led to the development of selective CXCR4 inhibitors for clinical use. [Table 1]

[0063] As used herein, the term "CXCL12" (or stromal cell-derived factor 1 ("SDF-1")) refers to a potent chemotactic factor for lymphocytes. During embryogenesis, CXCL12 directs the migration of hematopoietic cells from the fetal liver to bone, and in adults, CXCL12 plays an important role in angiogenesis by recruiting endothelial progenitor cells via a CXCR4-dependent mechanism. CXCL12 is also expressed in the splenic red pulp and lymph node medullary cords (Pitt, et al., (2015) Cancer Cell, 27:755-768; and Zhao, et al., (2011) Proc. Natl. Acad. Sci. USA 108:337-342). Exemplary amino acid sequences and corresponding coding nucleic acid sequences for human CXCL12 can be found in GENBANK Accession Nos. NP_954637.1 and NM_199168.3, respectively.

[0064] Salmeterol is a long-acting beta-2 adrenergic receptor agonist (LABA) with an arylalkyl group with a chain length of 11 atoms from the amine. This bulkiness is thought to make the compound more lipophilic and selective for beta-2 adrenergic receptors. First marketed and manufactured by Glaxo (now GlaxoSmithKline, GSK) in the 1980s, salmeterol was launched in 1990 as Serevent®. This product is sold by GSK under the trademark Allen & Hanburys in the UK. Salmeterol is used for the maintenance and prevention of asthma symptoms and chronic obstructive pulmonary disease (COPD) symptoms (2010 Global initiative for chronic obstructive disease). Symptoms of bronchospasm include shortness of breath, wheezing, cough, and chest tightness. Salmeterol is also used to prevent dyspnea during exercise (exercise-induced bronchoconstriction).

[0065] As used herein, the term "ADRB2 inhibitor" refers to a molecule that inhibits or suppresses the function of an ADRB2 monomer or an ADRB2 unit or protomer of a CXCR4-ADRB2 heteromer. Non-limiting examples of ADRB2 inhibitors that can be used in the treatment methods, inhibition methods, pharmaceutical compositions, and / or pharmaceutical kits provided herein, and their methods and uses, include, but are not limited to, ADRB2 antagonists, ADRB2 inverse agonists, ADRB2-positive allosteric modulators, ADRB2-negative allosteric modulators, ADRB2-specific antibodies or antigen-binding portions thereof comprising single-domain antibody-like scaffolds, bivalent ligands having a pharmacophore selective for ADRB2 connected by a spacer arm to a pharmacophore selective for CXCR4, bispecific antibodies against ADRB2 and CXCR4, radiolabeled ADRB2 ligands linked to a CXCR4 ligand, and small molecule ligands that inhibit CXCR4-ADRB2 heteromer-selective signaling. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the function of an ADRB2 monomer. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the function of the ADRB2 unit or promoter of a CXCR4-ADRB2 heteromer. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the function of a CXCR4-ADRB2 heteromer. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the enhanced response of an ADRB2 monomer upon stimulation with an ADRB2 agonist. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the enhanced response of an ADRB2 unit of a CXCR4-ADRB2 heteromer upon stimulation with an ADRB2 agonist. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the enhanced response of an ADRB2 unit of a CXCR4-ADRB2 heteromer upon costimulation with an ADRB2 agonist and a CXCR4 agonist. In certain embodiments, the ADRB2 inhibitor inhibits or suppresses the enhanced response of a CXCR4-ADRB2 heteromer upon costimulation with an ADRB2 agonist and a CXCR4 agonist. In certain embodiments, the enhanced response is an enhanced Ca2+ response.In certain embodiments, the enhanced response is enhanced cancer progression in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell proliferation in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell migration in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced metastasis in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced tumor growth in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced angiogenesis in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the cell(s) are cancer cell(s). In certain embodiments, the cell(s) are derived from a subject. In certain embodiments, the cell(s) are derived from a biological sample obtained from a subject. Certain examples of ADRB2 inhibitors are listed in Table 2.

[0066] As used herein, the term "CXCR4 inhibitor" refers to a molecule that inhibits or suppresses the function of a CXCR4 monomer or a CXCR4 unit or protomer of a CXCR4-ADRB2 heteromer. Non-limiting examples of CXCR4 inhibitors that can be used in the treatment methods, inhibition methods, pharmaceutical compositions, and / or pharmaceutical kits provided herein, and their methods and uses, include, but are not limited to, CXCR4 antagonists, CXCR4 inverse agonists, CXCR4-positive allosteric modulators, CXCR4-negative allosteric modulators, CXCR4-specific antibodies or antigen-binding portions thereof comprising single-domain antibody-like scaffolds, bivalent ligands having a pharmacophore selective for CXCR4 connected to a pharmacophore selective for ADRB2 by a spacer arm, bispecific antibodies against CXCR4 and ADRB2, radiolabeled CXCR4 ligands linked to ADRB2 ligands, and small molecule ligands that inhibit CXCR4-ADRB2 heteromer-selective signaling. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the function of a CXCR4 monomer. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the function of the CXCR4 unit or protomer of a CXCR4-ADRB2 heteromer. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the function of the CXCR4-ADRB2 heteromer. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the enhanced response of a CXCR4 monomer upon stimulation with a CXCR4 agonist. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the enhanced response of a CXCR4 unit of a CXCR4-ADRB2 heteromer upon stimulation with a CXCR4 agonist. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the enhanced response of a CXCR4 unit of a CXCR4-ADRB2 heteromer upon costimulation with a CXCR4 agonist and an ADRB2 agonist. In certain embodiments, the CXCR4 inhibitor inhibits or suppresses the enhanced response of a CXCR4-ADRB2 heteromer upon costimulation with a CXCR4 agonist and an ADRB2 agonist. In certain embodiments, the enhanced response is an enhanced Ca 2+ response.In certain embodiments, the enhanced response is enhanced cancer progression in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell proliferation in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell migration in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced metastasis in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced tumor growth in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced angiogenesis in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the cell(s) are cancer cell(s). In certain embodiments, the cell(s) are derived from a subject. In certain embodiments, the cell(s) are derived from a biological sample obtained from a subject. Certain examples of CXCR4 inhibitors are listed in Table 2.

[0067] As used herein, the term "antagonist" refers to a class of receptor ligands or drugs that block or attenuate a biological response by binding to and blocking the receptor, also called blockers. Antagonists have affinity for their cognate receptors, and their binding disrupts the interaction of an agonist or inverse agonist at the cognate receptor, inhibiting its function. For example, an ADRB2 antagonist binds to the ADRB2 receptor. to and / or thatThe ADRB2 antagonist may be an ADRB2 ligand or ADRB2 drug that blocks or attenuates a biological response by blocking it. In certain embodiments, the ADRB2 antagonist may disrupt the interaction of an ADRB2 agonist or ADRB2 inverse agonist with ADRB2 (e.g., an ADRB2 monomer, or an ADRB2 protomer or unit of a CXCR4-ADRB2 heteromer) and / or inhibit the function of an ADRB2 agonist or ADRB2 inverse agonist with ADRB2 (e.g., an ADRB2 monomer, or an ADRB2 protomer or unit of a CXCR4-ADRB2 heteromer). In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the function of an ADRB2 monomer. In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the function of an ADRB2 unit or protomer of a CXCR4-ADRB2 heteromer. In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the function of a CXCR4-ADRB2 heteromer. In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the enhanced response upon stimulation of the ADRB2 monomer with an ADRB2 agonist. In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the enhanced response upon stimulation of the ADRB2 unit of a CXCR4-ADRB2 heteromer with an ADRB2 agonist. In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the enhanced response upon costimulation of the ADRB2 unit of a CXCR4-ADRB2 heteromer with an ADRB2 agonist and a CXCR4 agonist. In certain embodiments, the ADRB2 antagonist blocks, inhibits, or suppresses the enhanced response upon costimulation of the CXCR4-ADRB2 heteromer with an ADRB2 agonist and a CXCR4 agonist. For example, the CXCR4 antagonist can be a CXCR4 ligand or a CXCR4 drug that blocks or attenuates a biological response by binding to and / or blocking the CXCR4 receptor. In certain embodiments, a CXCR4 antagonist may disrupt the interaction of a CXCR4 agonist or CXCR4 inverse agonist with CXCR4 (e.g., a CXCR4 monomer, or a CXCR4 protomer or unit of a CXCR4-ADRB2 heteromer), and / or may inhibit the CXCR4 agonist or CXCR4Inverse agonists can inhibit the function of CXCR4 (e.g., a CXCR4 monomer, or a CXCR4 protomer or unit of a CXCR4-ADRB2 heteromer). In certain embodiments, a CXCR4 antagonist blocks, inhibits, or suppresses the function of a CXCR4 monomer. In certain embodiments, a CXCR4 antagonist blocks, inhibits, or suppresses the function of a CXCR4 unit or protomer of a CXCR4-ADRB2 heteromer. In certain embodiments, a CXCR4 antagonist blocks, inhibits, or suppresses the function of a CXCR4-ADRB2 heteromer. In certain embodiments, a CXCR4 antagonist blocks, inhibits, or suppresses the response enhanced upon stimulation of a CXCR4 monomer with a CXCR4 agonist. In certain embodiments, a CXCR4 antagonist blocks, inhibits, or suppresses the response enhanced upon stimulation of a CXCR4 unit of a CXCR4-ADRB2 heteromer with a CXCR4 agonist. In certain embodiments, the CXCR4 antagonist blocks, inhibits, or suppresses the enhanced response upon costimulation of the CXCR4 unit of a CXCR4-ADRB2 heteromer with an ADRB2 agonist and a CXCR4 agonist. In certain embodiments, the CXCR4 antagonist blocks, inhibits, or suppresses the enhanced response upon costimulation of the CXCR4-ADRB2 heteromer with an ADRB2 agonist and a CXCR4 agonist. In certain embodiments, the enhanced response is an enhanced Ca2+ response. In certain embodiments, the enhanced response is enhanced cancer progression in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell proliferation in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell migration in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced metastasis in subjects with cell(s) containing the CXCR4-ADRB2 heteromer, hi certain embodiments, the enhanced cancer progression is enhanced tumor growth in subjects with cell(s) containing the CXCR4-ADRB2 heteromer.In certain embodiments, the enhanced cancer progression is enhanced angiogenesis in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the cell(s) are cancer cell(s). In certain embodiments, the cell(s) are derived from a subject. In certain embodiments, the cell(s) are from a biological sample obtained from the subject. Certain examples of CXCR4 antagonists and ADRB2 antagonists are listed in Table 2. [Table 2]

[0068] As used herein, the phrases "protein-protein interaction inhibitor," "PPI inhibitor," or variations thereof, refer to any molecule capable of interfering with protein-protein interactions. Unlike enzyme-substrate interactions, which involve distinct binding pockets, protein-protein interactions are transient interactions or associations between proteins over relatively large regions and are often driven by electrostatic interactions, hydrophobic interactions, hydrogen bonds, and / or van der Waals forces. PPI inhibitors can include, but are not limited to, membrane-permeable peptides or lipids fused to peptide sequences that interfere with GPCR heteromeric interfaces, such as the transmembrane helices, intracellular loops, or C-terminal tails of CXCR4 and / or ADRB2 units. PPI inhibitors of CXCR4-ADRB2 heteromers can be, for example, membrane-permeable or cell-penetrating peptides (CPPs) conjugated to peptides that target the CXCR4-ADRB2 heteromeric interface(s), or cell-permeable lipidated peptides that target the CXCR4-ADRB2 heteromeric interface(s).

[0069] For example, membrane-permeable or cell-permeable peptides include HIV-1 TAT peptides, such as TAT48-60 and TAT49-57; penetratins, such as pAntp(43-58); polyarginines (Rn, such as R5 to R12); Diatos Peptide Vector 1047 (DPV1047, Vectocell®); MPG (HIV fusion with the nuclear localization signal (NLS) of SV40 large T antigen) gp41; Pep-1 (tryptophan-rich cluster fused to the NLS of SV40 large T antigen); pVEC peptide (vascular endothelial cadherin); ARF(1-22) based on the p14 alternative reading frame (ARF) protein; N-terminal BPrPr(1-28) of unprocessed bovine prion protein; model amphipathic peptides (MAP); transportan; azurin-derived p28 peptide; amphipathic β-sheet peptides, such as VT5; proline-rich CPPs, such as Bac7 (Bac1-24); hydrophobic CPPs, such as C105Y derived from α1-antitrypsin; PFVYLI derived from synthetic C105Y; Pep-7 peptide (CHL8 peptide phage clone); and modified hydrophobic CPPs, such as stapled peptides and prenylated peptides (Guidotti, G., et al., (2017) Cell-Penetrating Peptides: From Basic Research to Clinics, Trends Pharmacol Sci 38, 406-424; Kristensen, M., et al., (2016) Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos, Int J Mol Sci 17). Membrane-penetrating or cell-penetrating peptides may further include, for example, TAT-derived cell-penetrating peptides, signal sequence-based (e.g., NLS) cell-penetrating peptides, hydrophobic membrane translocation sequence (MTS) peptides, and arginine-rich molecular transporters.Cell-permeable lipidated peptides include, for example, pepducins, such as ICL1 / 2 / 3, and C-tail short-chain palmitoylated peptides (Covic, L., et al., (2002) Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides, Proc Natl Acad Sci USA, 99, 643-648; and O'Callaghan, K., et al., (2012) Turning receptors on and off with intracellular pepducins: new insights into G protein-coupled receptor drug development. J Biol Chem 287, 12787-12796).

[0070] The peptide(s) targeting the CXCR4-ADRB2 heteromeric interface can be, for example, the transmembrane domain of CXCR4, the transmembrane domain of ADRB2, the intracellular loop of CXCR4, the intracellular loop of ADRB2, the C-terminal domain of CXCR4 or the C-terminal domain of ADRB2, the extracellular loop of CXCR4, the extracellular loop of ADRB2, the N-terminal region of CXCR4 or the N-terminal region of ADRB2.

[0071] As used herein, terms such as "express," "expression," or "expressing," when used in reference to a gene, refer to the process by which the information carried by a gene is manifested as a phenotype, including transcription of the gene into messenger RNA (mRNA), the subsequent translation of the mRNA molecule into a polypeptide chain, and its assembly into the final protein. In certain embodiments, diseases such as cancer may be characterized with respect to the expression of particular genes, such as with respect to the expression of the final protein from a particular gene. For example, cancer may be characterized as a CXCR4-expressing cancer, an ADRB2-expressing cancer, or a CXCR4-expressing and ADRB2-expressing cancer.

[0072] As used herein, the phrase "expression level" when used in reference to a gene refers to the amount or accumulation of an expression product of the gene, such as, for example, the amount of the gene's RNA product (the gene's RNA level) or the amount of the gene's protein product (the gene's protein level). When a gene has more than one allele, the expression level of the gene refers to the total accumulation of the expression products of all alleles present for that gene, unless otherwise specified. For example, cancer may be associated with the expression level (amount) of a particular product of the gene, such as a particular RNA product of the gene or a particular protein product of the gene. For example, cancer may be associated with the expression level (amount) of a particular product of the gene, such as a particular RNA product of the gene or a particular protein product of the gene. For example, cancer may have a particular expression level of the CXCR4 gene. For example, cancer may have a particular expression level of the ADRB2 gene. For example, cancer may have a particular expression level of the CXCR4 gene and a particular expression level of the ADRB2 gene. For example, cancer may have a particular expression level of the CXCR4 protein. For example, cancer may have a particular expression level of the ADRB2 protein. For example, a cancer may have a particular expression level of CXCR4 protein and a particular expression level of ADRB2 protein.

[0073] As used herein, the term "reference" when used in reference to a quantifiable value refers to a predetermined value that can be used to determine the significance of the value as measured in a sample.

[0074] As used herein, the phrase "reference expression level" refers to a predetermined expression level of a gene that can be used to determine the significance of the expression level of the gene in a cell or sample. The reference expression level of a gene can be the expression level of the gene in a reference cell determined by a person of ordinary skill in the art. For example, the reference expression level of the CXCR4 gene can be its average expression level in cells such as T cells or cancer cells. Therefore, if the expression level of the CXCR4 gene in a cell (or a sample of cells) is higher than its average expression level in cells such as T cells or cancer cells, it can be determined that the cell (or a sample of cells) is indicative of a CXCR4-expressing cell (or a CXCR4-expressing sample of cells). For example, the reference expression level of the ADRB2 gene can be its average expression level in cells such as T cells or cancer cells. Therefore, if the expression level of the ADRB2 gene in a cell (or a sample of cells) is higher than its average expression level in cells such as T cells or cancer cells, it can be determined that the cell is indicative of an ADRB2-expressing cell (or an ADRB2-expressing sample of cells). The reference expression level of a gene may also be a cutoff value determined by a person of ordinary skill in the art through statistical analysis of the expression levels of the gene in various sample cell populations. For example, a cancer may have a CXCR4 gene expression level in a sample that is higher than the reference level of the CXCR4 gene. For example, a cancer may have a CXCR4 protein expression level in a sample that is higher than the reference level of the CXCR4 protein. For example, a cancer may have an ADRB2 gene expression level in a sample that is higher than the reference level of the ADRB2 gene. For example, a cancer may have an ADRB2 protein expression level in a sample that is higher than the reference level of the ADRB2 protein. For example, a cancer may have a CXCR4 gene expression level and an ADRB2 gene expression level in a sample that are higher than the reference level of the CXCR4 gene and the reference level of the ADRB2 gene, respectively.For example, a cancer may have a CXCR4 protein expression level and an ADRB2 protein expression level in a sample that are higher than the reference CXCR4 protein level and the reference ADRB2 protein level, respectively. In certain embodiments, for example, by analyzing the expression level of a gene in a sample cell population having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of cells known to express the gene, one of ordinary skill in the art can determine a cutoff value as the reference expression level of the gene that can be used to indicate the percentage of cells expressing the gene in a cell population of unknown composition.

[0075] As used herein, the terms "selecting" and "selected" in reference to a subject (e.g., a cancer subject, such as a cancer patient) refer to the fact that a particular subject has a predetermined criterion or set of predetermined criteria, e.g., the subject has a CX3 level above a criterion level. CR The term "selectively treating a subject" is used to mean that a particular subject is specifically selected from a larger group of subjects based on the subject having a predetermined criterion or set of predetermined criteria, e.g., the subject having a CXCR4 expression level above a reference level, the subject having an ADRB2 expression level above a reference level, or the subject having CXCR4 expression and ADRB2 expression levels above the respective reference levels. CR Similarly, "selectively administering" refers to administering treatment to a subject (e.g., a cancer subject, such as a cancer patient) who has been specifically selected from a larger group of subjects based on (and thereby) having a predetermined criterion or set of predetermined criteria, e.g., the subject has an ADRB2 expression level above a reference level, the subject has an ADRB2 expression level above a reference level, or the subject has CXCR4 expression and ADRB2 expression levels above the respective reference levels. CRSelecting, selectively treating, and selectively administering refer to administering a drug to a subject (e.g., a cancer subject, such as a cancer patient) who has been specifically selected from a larger group of subjects based on (and thereby) having an ADRB2 expression level above a baseline level, the subject having an ADRB2 expression level above a baseline level, or the subject having CXCR4 expression and ADRB2 expression levels above the respective baseline levels. Selecting, selectively treating, and selectively administering refer to delivering an individualized therapy for a disease or disorder, e.g., cancer, based on the subject's biology, rather than delivering a standard treatment regimen to a subject based solely on the subject having the disease or disorder (e.g., cancer).

[0076] Traditionally, GPCRs were thought to function as monomers that interact with heterotrimeric G proteins upon ligand binding, and drugs were developed based on either monomeric or homomeric GPCRs (Milligan, G. (2008) A day in the life of a G protein-coupled receptor: the contribution to function of G protein-coupled receptor dimerization, Br J Pharmacol 153 Suppl 1, S216-229). This view has changed dramatically with 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, signal transduction strength and pathways, and receptor desensitization and regeneration (Terrillon, S., et al., (2004) Roles of G-protein-coupled receptor dimerization, EMBO Rep 5, 30-34; Ferre, S., et al., (2009); Rozenfeld, R., et al., (2010) Receptor heteromerization and drug discovery, Trends Pharmacol Sci 31, 124-130; Gomes, I., et al., (2016); Farran, B. (2017) An update on the physiological and therapeutic relevance of GPCR oligomers, Pharmacol Res 117, 303-327). Different GPCR heteromers exhibit distinct functional and pharmacological properties, and GPCR heteromerization can vary depending on cell type, tissue, and disease or pathology (Terrillon, S., et al., 2004; Ferre, S., et al., 2009; Rozenfeld, R., et al., 2010; Gomes, I., et al., 2016; Farran, B., 2017).GPCR heteromerization is now considered a common phenomenon, and elucidating it opens new avenues for understanding receptor function, physiology, and its role in diseases and pathologies. Therefore, identifying GPCR heteromers and their functional properties offers new opportunities for developing new drugs with fewer side effects, higher efficacy, and improved tissue selectivity, or for finding new uses for existing drugs (Ferre, S., et al., 2009; Rozenfeld, R., et al., 2010; Farran, B., 2017).

[0077] Identifying true GPCR heteromers requires thorough and critical evaluation. To distinguish GPCR heteromers from simple associations of GPCRs, researchers in the field and the International Union of Pharmacology Committee on Receptor Nomenclature (NC-IUPHAR) have declared that GPCR heteromers are "macromolecular complexes consisting of at least two (functional) receptor units [protomers] with biochemical properties distinct from those of their individual components" and that these heteromers exist in natural tissues (Ferre, S., et al., 2009; Gomes, I., et al., 2016; Pin, J.P., et al., (2007) International Union of Basic and Clinical Pharmacology. LXVII. Recommendations for the recognition and nomenclature of G protein-coupled receptor heteromultimers, Pharmacol Rev 59, 5-13). They proposed three criteria for demonstrating GPCR heteromers: (1) using proximity-based techniques, including co-immunoprecipitation, in situ hybridization, or proximity ligation assays, heteromers must exhibit appropriate colocalization and interactions that enable allosterism in cells / tissues expressing both receptors but not in cells / tissues lacking one receptor; (2) heteromers must exhibit distinct properties, such as altered signaling, ligand binding, and / or trafficking, only in cells / tissues expressing both receptors but not in cells / tissues lacking one receptor; and (3) heteromer-selective reagents must alter heteromer-specific properties. Heteromer-selective reagents include heteromer-selective antibodies, membrane-permeable peptides, and bivalent / bifunctional ligands (Gomes, I., et al., 2016; Pin, J.P., et al., 2007).Although many GPCR heteromers have been identified in vitro using recombinant receptors expressed in heterologous cells, only a few have demonstrated novel properties, and few have demonstrated evidence of GPCR heteromerization in native tissues due to technical challenges (Gomes, I., et al., 2016). The NC-IUPHAR announced that to be recognized as a novel GPCR heteromer, evidence must be provided that meets at least two of the three criteria (Pin, JP, et al., 2007).

[0078] As disclosed herein, one or more of the following methods may be utilized to establish whether criterion 1 of three is met in determining the presence / existence of CXCR4-ADRB2 heteromers (regarding whether the heteromeric components are colocalized and physically interact directly or through an intermediate protein that acts as a vehicle for allosterism), including, but not limited to, co-internalization assays; co-localization assays (which determine co-localization of receptor protomers within a cellular compartment), such as in situ hybridization, immunohistochemistry, or immunoelectron microscopy; proximity-based assays, such as proximity-based biophysical techniques, such as resonance energy transfer (RET), bioluminescence RET (BRET), fluorescence RET (FRET), time-resolved fluorescence RET (TR-FRET), antibody-mediated FRET, ligand-mediated FRET, bimolecular fluorescence complementation (BiFC), expression levels of CXCR4 and ADRB2, and proximity ligation assays (PLA); co-immunoprecipitation assays; or fluorescent animals. For example, BiFC, co-internalization assays, CXCR4 and ADRB2 expression levels, or PLA were used to assess whether CXCR4-ADRB2 heteromers fulfilled criterion 1 of three.

[0079] As disclosed herein, to establish whether criterion 2 of the three (with respect to whether a CXCR4-ADRB2 heteromer exhibits properties distinct from those of the individual protomers), e.g., whether a CXCR4-ADRB2 heteromer results in enhanced downstream signaling, e.g., enhanced calcium mobilization (e.g., as determined by a calcium mobilization assay), a two-step approach was utilized for CXCR4-ADRB2 heteromers that met criterion 1 of the three described above: (1) determining the presence / absence of enhanced downstream signaling, e.g., enhanced calcium mobilization (synergism), in the context of the individual protomers—HA-VC and In cells co-expressing either protomer (either CXCR4 or ADRB2), calcium mobilization is compared (a) upon costimulation with CXCL12 and an ADRB2 agonist and (b) upon stimulation with either CXCL12 alone or an ADRB2 agonist alone; and (2) to determine the presence or absence of enhanced downstream signaling, e.g., enhanced calcium mobilization (synergy), in the context of CXCR4-ADRB2 heteromers - calcium mobilization is compared (a) upon costimulation with CXCL12 and an ADRB2 agonist and (b) upon the sum of stimulation with either CXCL12 alone or an ADRB2 agonist alone in cells co-expressing CXCR4 and ADRB2. As disclosed herein, to meet criterion 2 of the three and be considered a CXCR4-ADRB2 heteromer that results in enhanced downstream signaling, e.g., enhanced calcium mobilization, there must be (1) the absence of enhanced downstream signaling, e.g., enhanced calcium mobilization, in the context of either protomer (i.e., in the context of CXCR4 and HA-VC or in the context of ADRB2 and HA-VC), and (2) the presence of enhanced downstream signaling, e.g., enhanced calcium mobilization, in the context of the CXCR4-ADRB2 heteromer.In both the protomer and CXCR4-ADRB2 heteromer contexts, the concentration of CXCL12 (as a single agent or in combination with an ADRB2 agonist) used to stimulate the cells and the concentration of the ADRB2 agonist (either an endogenous agonist or a known selective agonist for ADRB2) used to stimulate the cells (as a single agent or in combination with CXCL12) were independently 100-fold or less than the EC concentration. For example, the concentration of CXCL12 (as a single agent or in combination with an ADRB2 agonist) used to stimulate the cells was 15 nM (approximately the EC concentration for CXCR4).

[0080] As disclosed herein, to establish whether criterion 3 of 3 is met in determining the presence / existence of CXCR4-ADRB2 heteromers (regarding whether a heteromer-selective reagent alters heteromer-specific properties), cells from a subject that meet criterion 1 of 3 and criterion 2 of 3 are placed in the presence of an antagonist (a CXCR4 antagonist, an ADRB2 antagonist, or a CXCR4-ADRB2 heteromer antagonist), e.g., subject-derived cells containing CXCR4-ADRB2 heteromers are subjected to cell proliferation.

[0081] In some embodiments, a method of treatment, suppression, pharmaceutical composition, or pharmaceutical kit as disclosed herein comprises or relies on establishing that an association of CXCR4 and ADRB2 in a cell meets at least two of the following criteria or characteristics to be considered a CXCR4-ADRB2 heteromer, including: 1) the CXCR4-ADRB2 heteromeric components within the cell are colocalized and physically interact, either directly or through an intermediate protein that acts as a vehicle for allostery, as determined by one or more of the following: co-internalization assay, co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, CXCR4 and ADRB2 expression levels, proximity-based assay, co-immunoprecipitation assay, or fluorescent animal assay; 2) the calcium mobilization assay determines whether either CXCR4 or ADRB2, in the context of their individual protomers within the cell, upon costimulation with CXCL12 and an ADRB2 agonist, results from single agonist stimulation with either CXCL12 or an ADRB2 agonist; a) an amount of calcium mobilization that is equal to or less than the sum of the amounts of calcium mobilization resulting from CXCR4-ADRB2 stimulation with either CXCL12 or an ADRB2 agonist; b) an amount of enhanced calcium mobilization, such that upon costimulation with CXCL12 and an ADRB2 agonist, the CXCR4-ADRB2 heteromer exhibits enhanced calcium mobilization compared to the sum of the amounts of calcium mobilization resulting from single-agonist stimulation with either CXCL12 or an ADRB2 agonist; or 3) a CXCR4-ADRB2 heteromer-selective reagent i) alters the heteromer-specific properties of CXCR4-ADRB2 heteromers in cells derived from the subject, ii) alters the heteromer-specific function of CXCR4-ADRB2 heteromers in cells derived from the subject, iii) alters the heteromer-specific properties of cells derived from the subject containing CXCR4-ADRB2 heteromers, or iv) reduces cellular progression of cell(s) derived from the subject containing CXCR4-ADRB2 heteromers (e.g., reduced cancer progression of cell(s) derived from the subject containing the CXCR4-ADRB2 heteromers, reduced cellular proliferation of cell(s) derived from the subject containing the CXCR4-ADRB2 heteromers,reduced cell migration of subject-derived cell(s) containing the CXCR4-ADRB2 heteromer, reduced metastasis of subject-derived cell(s) containing the CXCR4-ADRB2 heteromer, and / or reduced angiogenesis of subject-derived cell(s) containing the CXCR4-ADRB2 heteromer. In some embodiments, the CXCR4-ADRB2 heteromer-selective reagent alters the heteromer-specific properties of CXCR4-ADRB2 heteromers in cells derived from a subject as determined by at least one of the following methods: PLA, radioligand binding assay, [35S]GTP-γS binding assay, calcium assay, cAMP assay, GTPase assay, PKA activation, ERK1 / 2 and / or Akt / PKB phosphorylation assay, Src and STAT3 phosphorylation assay, CRE reporter assay, NFAT-RE reporter assay, SRE reporter assay, SRF-RE reporter assay, secreted alkaline phosphatase assay, inositol 1-phosphate production assay, adenylyl cyclase activity assay, analysis of target gene expression by RT-PCR, by RT-qPCR, by RNAseq, by next-generation sequencing (NGS), or by microarray. In some embodiments, the CXCR4-ADRB2 heteromer-selective reagent alters a heteromer-specific function of CXCR4-ADRB2 heteromers in cells from a subject, as determined by at least one of the following methods: PLA, radioligand binding assay, [35S]GTP-γS binding assay, calcium assay, cAMP assay, GTPase assay, PKA activation, ERK1 / 2 and / or Akt / PKB phosphorylation assay, Src and STAT3 phosphorylation assay, CRE reporter assay, NFAT-RE reporter assay, SRE reporter assay, SRF-RE reporter assay, NF-kB-RE reporter assay, secreted alkaline phosphatase assay, inositol 1-phosphate production assay, adenylyl cyclase activity assay, analysis of target gene expression by RT-PCR, by RT-qPCR, by RNAseq, or by microarray.CXCR4-ADRB2 heteromer-selective reagents alter heteromer-specific properties of subject-derived cells containing CXCR4-ADRB2 heteromers, including assays for cancer cell proliferation, migration, invasion, and drug resistance (survival), modulation of immune cell function, angiogenesis, vasculogenesis, metastasis, drug resistance, tissue microarray (TMA), and cancer cell-tumor microenvironment (TME) interactions. For example, in some embodiments, a method of treatment, inhibition method, pharmaceutical composition, or pharmaceutical kit as disclosed herein comprises or relies on establishing that the association of CXCR4 and ADRB2 in a cell meets at least two of the following criteria or characteristics to be considered a CXCR4-ADRB2 heteromer, including: 1) the CXCR4-ADRB2 heteromeric components within the cell are co-localized and physically interact, either directly or through an intermediate protein that acts as a vehicle for allostery, as determined by one or more of the following: co-internalization assay, bimolecular fluorescence complementation (BiFC), CXCR4 and ADRB2 expression levels, or proximity ligation assay (PLA); 2) the CXCR4-ADRB2 heteromeric components within the cell are co-localized and physically interact, either directly or through an intermediate protein that acts as a vehicle for allostery, as determined by a calcium mobilization assay; a) an enhanced calcium mobilization such that upon costimulation with CXCL12 and an ADRB2 agonist, the CXCR4-ADRB2 heteromer exhibits enhanced calcium mobilization compared to the sum of the calcium mobilization resulting from single agonist stimulation with either CXCL12 or an ADRB2 agonist; or 3) a CXCR4-ADRB2 heteromer-selective reagent i) alters the heteromer-specific properties of CXCR4-ADRB2 heteromers in cells derived from the subject, ii) alters the heteromer-specific function of CXCR4-ADRB2 heteromers in cells derived from the subject, or iii) alters the heteromer-specific properties of cells derived from the subject containing CXCR4-ADRB2 heteromers.or iv) reducing cancer progression in a subject harboring cells containing the CXCR4-ADRB2 heteromer (e.g., reducing cancer progression in cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer, reducing cell proliferation in cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer, reducing cell migration in cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer, reducing metastasis in cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer, and / or reducing angiogenesis in cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer). In some embodiments, a method of treatment, suppression method, pharmaceutical composition, or pharmaceutical kit as disclosed herein includes or relies on establishing that the association of CXCR4 and ADRB2 in a cell satisfies criteria 1 and 2 to be considered a CXCR4-ADRB2 heteromer. In some embodiments, a method of treatment, a method of inhibition, a pharmaceutical composition, or a pharmaceutical kit as disclosed herein comprises or relies on establishing that the association of CXCR4 and ADRB2 in a cell satisfies criteria 1 and 3 to be considered a CXCR4-ADRB2 heteromer. In some embodiments, a method of treatment, a method of inhibition, a pharmaceutical composition, or a pharmaceutical kit as disclosed herein comprises or relies on establishing that the association of CXCR4 and ADRB2 in a cell satisfies criteria 2 and 3 to be considered a CXCR4-ADRB2 heteromer. In some embodiments, a method of treatment, a method of inhibition, a pharmaceutical composition, or a pharmaceutical kit as disclosed herein comprises or relies on establishing that the association of CXCR4 and ADRB2 in a cell satisfies criteria 1, 2, and 3 to be considered a CXCR4-ADRB2 heteromer.

[0082] As disclosed herein, CXCR4-ADRB2 heteromers that meet at least two of the three criteria can have, cause, or result in enhanced downstream signaling. Enhanced downstream signaling can be due to the CXCR4-ADRB2 heteromer, e.g., agonism of the CXCR4-ADRB2 heteromer, CXCR4 agonism of the CXCR4-ADRB2 heteromer, ADRB2 agonism of the CXCR4-ADRB2 heteromer, and / or CXCR4 agonism and ADRB2 agonism of the CXCR4-ADRB2 heteromer. In some embodiments, enhanced downstream signaling can be downstream of CXCR4, ADRB2, or CXCR4-ADRB2 heteromers. In some embodiments, enhanced downstream signaling can be from CXCR4-ADRB2 heteromers compared to downstream signaling from CXCR4 protomers or ADRB2 protomers in the context of each individual protomer. In some embodiments, enhanced downstream signaling can be from CXCR4-ADRB2 heteromers compared to downstream signaling from CXCR4 protomers in the context of each individual protomer. In some embodiments, enhanced downstream signaling can be from CXCR4-ADRB2 heteromers compared to downstream signaling from ADRB2 protomers in the context of each individual protomer. In some embodiments, enhanced downstream signaling can be from CXCR4-ADRB2 heteromers compared to downstream signaling from CXCR4 protomers and ADRB2 protomers in the context of each individual protomer. Enhanced downstream signaling from CXCR4-ADRB2 heteromers can, in some embodiments, be inhibited in a cancer subject, e.g., in cancer cells of a subject. In some embodiments, enhanced downstream signaling from CXCR4-ADRB2 heteromers can be an amount of enhanced calcium mobilization (or an amount of synergistic calcium mobilization), which can be determined by an intracellular Ca2+ assay, e.g., a calcium mobilization assay.

[0083] As disclosed herein, CXCR4-ADRB2 heteromers that meet at least two of the three criteria can have, cause, or result in enhanced downstream signaling, where the enhanced downstream signaling is enhanced calcium mobilization. The enhanced calcium mobilization from CXCR4-ADRB2 heteromers can be at least 10% greater than the sum of the calcium mobilization resulting from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist, as determined by calcium mobilization assays upon costimulation with CXCL12 and an ADRB2 agonist. In some embodiments, the enhanced calcium mobilization from CXCR4-ADRB2 heteromers can be at least 10% greater than the sum of the calcium mobilization resulting from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist, as determined by calcium mobilization assays upon costimulation with CXCL12 and an ADRB2 agonist. For example, the amount of enhanced calcium mobilization (or synergistic amount of calcium mobilization) from a CXCR4-ADRB2 heteromer, as determined by a calcium mobilization assay upon costimulation with CXCL12 and a selective ADRB2 agonist, can be at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 90% greater, at least 100% greater, at least 150% greater, or at least 200% greater than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist.In some embodiments, the amount of enhanced calcium mobilization (or synergistic amount of calcium mobilization) from CXCR4-ADRB2 heteromers, as determined by calcium mobilization assays upon costimulation with CXCL12 and an ADRB2 agonist, can be 10-100% greater than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either a CXCL12 or an ADRB2 agonist, for example, 25-100% greater, 50-100% greater, 75-100% greater, or 100-200% greater than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either a CXCL12 or an ADRB2 agonist, as determined by calcium mobilization assays upon costimulation with CXCL12 and an ADRB2 agonist.

[0084] In some embodiments, according to the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein, the CXCR4-ADRB2 heteromers satisfy at least two of three criteria, thereby having, causing, or resulting in enhanced downstream signaling, where the enhanced downstream signaling is an amount of calcium mobilization that is enhanced, as determined by a calcium mobilization assay, such that: a) upon costimulation with CXCL12 and an ADRB2 agonist, either CXCR4 or ADRB2 in the context of the individual protomers in the cell results in an amount of calcium mobilization that is equal to or less than the sum of the amounts of calcium mobilization that result from single agonist stimulation with either CXCL12 or an ADRB2 agonist; and b) upon costimulation with CXCL12 and an ADRB2 agonist, the CXCR4-ADRB2 heteromers exhibit enhanced calcium mobilization compared to the sum of the amounts of calcium mobilization that result from single agonist stimulation with either CXCL12 or an ADRB2 agonist. For example, in some embodiments, enhanced downstream signaling from CXCR4-ADRB2 heteromers is an amount of enhanced calcium mobilization such that i) calcium mobilization from protomer CXCR4 or ADRB2 in the context of the individual protomers in cells is non-synergistic as determined by calcium mobilization assays, and ii) calcium mobilization from CXCR4-ADRB2 heteromers in cells is synergistic as determined by calcium mobilization assays. In some embodiments, the context of the individual protomers can be such that upon costimulation with CXCL12 and an ADRB2 agonist, a) the individual protomer CXCR4 in the cell in the absence of the individual protomer ADRB2, or b) the individual protomer ADRB2 in the cell in the absence of the individual protomer CXCR4, results in an amount of calcium mobilization equal to or less than the sum of the amounts of calcium mobilization resulting from single agonist stimulation with either CXCL12 or an ADRB2 agonist, as determined by calcium mobilization assays.In some embodiments, the context of each individual protomer can independently be such that a) each individual protomer CXCR4 in a cell in the absence of each individual protomer ADRB2, and b) each individual protomer ADRB2 in a cell in the absence of each individual protomer CXCR4, upon costimulation with CXCL12 and an ADRB2 agonist, results in calcium mobilization that is equal to or less than the sum of the calcium mobilization resulting from single-agonist stimulation of the cell with either a CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay. For example, in some embodiments, upon costimulation with CXCL12 and an ADRB2 agonist, a CXCR4-ADRB2 heteromer can result in calcium mobilization that is greater than the sum of the calcium mobilization resulting from single-agonist stimulation of the cell with either a CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay.

[0085] Provided herein is a method for suppressing enhanced downstream signaling resulting from CXCR4-ADRB2 heteromers in cells of a subject suffering from cancer, comprising administering to the subject: (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor, wherein (i) the enhanced downstream signaling is due to CXCR4-ADRB2 heteromers; and (ii) the combination of the administered inhibitors suppresses the enhanced downstream signaling from the CXCR4-ADRB2 heteromers in the cancer subject. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0086] Provided herein is a method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising administering to the subject: (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor, wherein (i) enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers; and (ii) the combination of the administered inhibitors suppresses enhanced downstream signaling from the CXCR4-ADRB2 heteromers in the cancer subject. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0087] Provided herein are methods for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, the method comprising: (a) determining whether the subject's cells contain CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers; and (b) if the subject's cells contain the CXCR4-ADRB2 heteromers, administering to the cancer subject: (i) a CXCR4 inhibitor that is blixafor; and (ii) an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0088] A method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers, comprising: (1) obtaining or obtaining a biological sample from the subject to determine whether the subject has cells containing CXCR4-ADRB2 heteromers; and (i) determining whether the subject's cells contain the CXCR4-ADRB2 heteromers; or (ii) determining whether a combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromers in cell(s) derived from the subject; or altering the heteromer-specific properties of cell(s) derived from the subject that contain the CXCR4-ADRB2 heteromers. (2) performing or having performed an assay on a biological sample to determine whether a CXCR4-ADRB2 heteromer reduces or decreases the progression of cancer in a subject having cells containing the CXCR4-ADRB2 heteromer; and (3) administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor, if the subject has cells containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0089] A method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is due to CXCR4-ADRB2 heteromers, comprising: (1) determining whether the subject has cells containing CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject; and (i) determining whether the subject's cells contain the CXCR4-ADRB2 heteromers; or (ii) performing or having performed an assay on the biological sample to determine whether a combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in cell(s) derived from the subject; alters the heteromer-specific properties of cell(s) derived from the subject that contain the CXCR4-ADRB2 heteromer; or reduces the progression of cancer in the subject having cells containing the CXCR4-ADRB2 heteromers; and (2) determining whether the subject has cells containing the CXCR4-ADRB2 heteromers by performing or having performed an assay on the biological sample to determine whether the combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in cell(s) derived from the subject; alters the heteromer-specific properties of cell(s) derived from the subject that contain the CXCR4-ADRB2 heteromer; or reduces the progression of cancer in the subject having cells containing the CXCR4-ADRB2 heteromers. (3) if the subject has cells containing CXCR4-ADRB2 heteromers, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor; and (4) if the subject does not have cells containing CXCR4-ADRB2 heteromers, administering to the cancer subject a single inhibitor, either blixafor or an ADRB2 inhibitor, wherein (a) cancer progression in the subject having cells containing CXCR4-ADRB2 heteromers is reduced by 5% to 100% more upon administration of the combination of blixafor and an ADRB2 inhibitor to the cancer subject compared to administration of either blixafor or an ADRB2 inhibitor alone; and (b) the efficacy of blixafor, when administered in combination with an ADRB2 inhibitor to the subject having cells containing CXCR4-ADRB2 heteromers, is increased by 5% to 2000% compared to the efficacy of blixafor when administered as a single inhibitor;and / or (c) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to a subject having such cells containing CXCR4-ADRB2 heteromers, is increased in the range of 5-2000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol;

[0090] A method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers, comprising: (1) determining whether the subject's cells contain the CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromers are present in the subject's cells; wherein the assay performed on the biological sample is a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, or an immunoassay. (2) if the subject's cells contain the CXCR4-ADRB2 heteromer, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is blixafor; and (3) if the subject's cells do not contain the CXCR4-ADRB2 heteromer, administering to the cancer subject a single inhibitor, either blixafor or an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0091] A method for treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers and in which enhanced downstream signaling is due to CXCR4-ADRB2 heteromers, comprising: (1) determining whether the subject's cells contain CXCR4-ADRB2 heteromers by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromers are present in the subject's cells; wherein the assay performed on the biological sample is or includes one or more of a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNA sequencing, RT-qPCR, a microarray, or a fluorescent animal assay; and (2) if the subject's cells contain the CXCR4-ADRB2 heteromers, administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the CXCR4 inhibitor is bridging the heteromers. and (3) administering to the cancer subject a single inhibitor of either blixafor or an ADRB2 inhibitor if the subject's cells do not contain the CXCR4-ADRB2 heteromer, wherein (a) the progression of cancer in the subject having the cells containing the CXCR4-ADRB2 heteromer is reduced by 5% to 100% or more upon administration of the combination of blixafor and an ADRB2 inhibitor to the cancer subject compared to administration of either blixafor or an ADRB2 inhibitor alone; and (b) the effectiveness of blixafor. and / or (c) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to a subject having cells containing CXCR4-ADRB2 heteromers, is increased by 5 to 2000% compared to the efficacy of blixafor when administered as a single inhibitor; and / or (d) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to a subject having cells containing CXCR4-ADRB2 heteromers, is increased by 5 to 2000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor.In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0092] Provided herein is a pharmaceutical kit for use in treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, the pharmaceutical kit comprising: (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor; wherein the enhanced downstream signaling results from the CXCR4-ADRB2 heteromers. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0093] Provided herein is a pharmaceutical composition for use in treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising: (a) a CXCR4 inhibitor that is blixafor; (b) an ADRB2 inhibitor; and (c) a pharmaceutically acceptable carrier, wherein the enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromers. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0094] Provided herein are pharmaceutical compositions comprising: (a) a CXCR4 inhibitor that is blixafor; (b) an ADRB2 inhibitor; and (c) a pharmaceutically acceptable carrier. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0095] Provided herein is a method for suppressing enhanced downstream signaling resulting from CXCR4-ADRB2 heteromers in a cell, the method comprising administering to the cell: (a) a CXCR4 inhibitor, which is blixafor; and (b) an ADRB2 inhibitor; wherein (i) the enhanced downstream signaling is due to CXCR4-ADRB2 heteromers; and (ii) contacting the cell with blixafor and the ADRB2 inhibitor suppresses the enhanced downstream signaling from the CXCR4-ADRB2 heteromers in the cell. In certain embodiments, the method further comprises determining whether the cell contains CXCR4-ADRB2 heteromers. In a specific embodiment, the cell is a subject's cell. In a specific embodiment, the subject's cell is a cancer cell. In a specific embodiment, the cell is a cancer cell. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0096] Provided herein is a pharmaceutical kit for use in suppressing enhanced downstream signaling in a cell due to CXCR4-ADRB2 heteromers, the pharmaceutical kit comprising: (a) a CXCR4 inhibitor that is blixafor; and (b) an ADRB2 inhibitor; wherein (i) the enhanced downstream signaling is due to CXCR4-ADRB2 heteromers. In a specific embodiment, the cell is a subject's cell. In a specific embodiment, the subject's cell is a cancer cell. In a specific embodiment, the cell is a cancer cell. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0097] Provided herein is a pharmaceutical composition for use in suppressing enhanced downstream signaling in a cell due to CXCR4-ADRB2 heteromers, the pharmaceutical composition comprising: (a) a CXCR4 inhibitor that is blixafor; (b) an ADRB2 inhibitor; and (c) a pharmaceutically acceptable carrier; wherein the enhanced downstream signaling is due to CXCR4-ADRB2 heteromers. In a specific embodiment, the cell is a subject's cell. In a specific embodiment, the subject's cell is a cancer cell. In a specific embodiment, the cell is a cancer cell. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0098] In some embodiments, the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein further comprise detecting the presence of CXCR4-ADRB2 heteromers in a subject with cancer. In some embodiments, the methods and uses provided herein further comprise identifying CXCR4-ADRB2 heteromers in a subject with cancer. In some embodiments, the methods and uses provided herein further comprise obtaining a biological sample from a subject. In some embodiments, the methods and uses provided herein further comprise performing an assay on the biological sample obtained from the subject. In some embodiments, the methods and uses provided herein further comprise (i) obtaining or having obtained a biological sample from a subject with cancer; (ii) performing or having performed a diagnostic assay to determine the presence, identity, or presence and identity of CXCR4-ADRB2 heteromers in the biological sample obtained from the subject with cancer; and (iii) selecting an ADRB2 inhibitor for administration in combination with blixafor to suppress enhanced downstream signaling due to CXCR4-ADRB2 heteromers. In some embodiments, the methods and uses provided herein further comprise determining whether cells of the subject contain CXCR4-ADRB2 heteromers and performing an assay on a biological sample obtained from the subject. In some embodiments, the methods and uses provided herein further comprise determining whether cells of the subject contain CXCR4-ADRB2 heteromers, obtaining a biological sample from the subject, and performing an assay on the biological sample obtained from the subject. In some embodiments, the methods and uses provided herein further comprise the biological sample obtained from the subject containing CXCR4-ADRB2 heteromers. In some embodiments, the subject's biological sample is a biological fluid sample. In some embodiments, the biological fluid sample is a blood sample, plasma sample, saliva sample, cerebrospinal fluid sample, ocular fluid sample, or urine sample. In some embodiments, the methods and uses provided herein further comprise performing a liquid biopsy on the biological fluid sample.In some embodiments, the subject's biological sample is a biological tissue sample. In some embodiments, the biological tissue sample is an organ tissue sample, a bone tissue sample, or a tumor tissue sample. In some embodiments, the methods and uses provided herein further comprise performing a tissue sample assay on the biological tissue sample. In some embodiments, the methods and uses provided herein comprise the subject's cells containing CXCR4-ADRB2 heteromers. In some embodiments, the cells are cancer cells. In some embodiments, the subject's cells are cancer cells. In some embodiments, the subject is a patient.

[0099] In some embodiments, the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein further comprise a method in which the CXCR4-ADRB2 heteromer exhibits any of the following characteristics: (1) that the components of the CXCR4-ADRB2 heteromer colocalize and physically interact within cells, either directly or through an intermediate protein that acts as a vehicle for allostery; (2) that enhanced downstream signaling results from the CXCR4-ADRB2 heteromer; and / or (3) that the combination of blixafor and an ADRB2 inhibitor: (i) alters the heteromer-specific properties of the CXCR4-ADRB2 heteromer in a cell(s) derived from a subject; (ii) alters the heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from a subject; (iii) alters the heteromer-specific properties of a cell(s) derived from a subject that contain the CXCR4-ADRB2 heteromer; and / or (iv) reduces cancer progression in a subject having cells that contain the CXCR4-ADRB2 heteromer. This includes having two or more of the following:

[0100] In some embodiments, the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein include characterizing CXCR4-ADRB2 heteromers as having the following characteristic: the CXCR4-ADRB2 heteromeric components within a cell are colocalized and physically interact, either directly or through an intermediate protein that acts as a vehicle for allosterism. In specific embodiments, the methods and uses provided herein further include performing an assay to identify or determine the colocalization of the CXCR4 and ADRB2 components of CXCR4-ADRB2 heteromers in a cell and their physical interaction, either directly or through an intermediate protein that acts as a vehicle for allosterism. In specific embodiments, assays for determining colocalization and physical interaction of CXCR4-ADRB2 heteromeric components in cells include one or more of a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, a proximity-based assay, a co-immunoprecipitation assay, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNA sequencing, RT-PCR, RT-qPCR, CXCR4 expression levels, ADRB2 expression levels, CXCR4 and ADRB2 expression levels, a microarray, or a fluorescent animal assay. In specific embodiments, the assay is a co-internalization assay. In specific embodiments, the assay is a co-localization assay. In specific embodiments, the assay is an in situ hybridization assay. In specific embodiments, the assay is a co-immunoprecipitation assay. In specific embodiments, the assay is a proximity-based assay. In specific embodiments, the proximity-based assay is or includes resonance energy transfer (RET), bioluminescence RET (BRET), fluorescence RET (FRET), time-resolved fluorescence RET (TR-FRET), antibody-mediated FRET, ligand-mediated FRET, bimolecular fluorescence complementation (BiFC), or proximity ligation assay (PLA). In specific embodiments, the TR-FRET is ligand-mediated TR-FRET. In specific embodiments, the TR-FRET is antibody-mediated TR-FRET.In a specific embodiment, the assay is bimolecular fluorescence complementation (BiFC). In a specific embodiment, the assay is proximity ligation assay (PLA). In a specific embodiment, the assay is enzyme-linked immunosorbent assay (ELISA). In a specific embodiment, the assay is a flow cytometry assay. In a specific embodiment, the assay determines the expression level of CXCR4, the expression level of ADRB2, and / or the expression levels of CXCR4 and ADRB2. In a specific embodiment, the assay determines the expression level of CXCR4, the expression level of ADRB2, and / or the expression levels of CXCR4 and ADRB2 via RNA-seq. In a specific embodiment, the assay determines the expression level of CXCR4, the expression level of ADRB2, and / or the expression levels of CXCR4 and ADRB2 via RT-PCR. In a specific embodiment, the assay determines the expression level of CXCR4, the expression level of ADRB2, and / or the expression levels of CXCR4 and ADRB2 via RT-qPCR. In a specific embodiment, the assay is a microarray assay. In a specific embodiment, the assay is a fluorescent animal assay. In a specific embodiment, the assay determines the presence of CXCR4-ADRB2 heteromers in a biological sample obtained from the subject. In a specific embodiment, the assay determines the presence of CXCR4-ADRB2 heteromers in the subject. In a specific embodiment, the assay determines the presence of CXCR4-ADRB2 heteromers in the subject's cells.

[0101] In some embodiments, the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein include those characterized as having the following characteristic: enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromer. In a specific embodiment, the CXCR4-ADRB2 heteromer results in enhanced downstream signaling. In a specific embodiment, the enhanced downstream signaling is attributable to the presence of the CXCR4-ADRB2 heteromer in cells of the subject. In a specific embodiment, the CXCR4-ADRB2 heteromer results in enhanced downstream signaling in cells of the subject. In a specific embodiment, the enhanced downstream signaling is attributable to agonism of the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced downstream signaling is attributable to CXCR4 agonism of the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced downstream signaling is attributable to ADRB2 agonism of the CXCR4-ADRB2 heteromer. In specific embodiments, the enhanced downstream signaling results from CXCR4 agonism and ADRB2 agonism of CXCR4-ADRB2 heteromers. In specific embodiments, the enhanced downstream signaling is downstream of CXCR4, ADRB2, or CXCR4-ADRB2 heteromers. In specific embodiments, the enhanced downstream signaling is downstream of CXCR4. In specific embodiments, the enhanced downstream signaling is downstream of ADRB2. In specific embodiments, the enhanced downstream signaling is downstream of CXCR4-ADRB2 heteromers. In specific embodiments, the enhanced downstream signaling from CXCR4-ADRB2 heteromers is compared to downstream signaling from CXCR4 protomers or ADRB2 protomers, respectively, in the context of the individual protomers. In specific embodiments, the enhanced downstream signaling from CXCR4-ADRB2 heteromers is compared to downstream signaling from CXCR4 protomers in the context of the individual protomers.In a specific embodiment, the enhanced downstream signaling from a CXCR4-ADRB2 heteromer is compared to downstream signaling from an ADRB2 protomer in the context of each individual protomer. In a specific embodiment, the enhanced downstream signaling from a CXCR4-ADRB2 heteromer is compared to downstream signaling from a CXCR4 protomer and an ADRB2 protomer in the context of each individual protomer. In a specific embodiment, the enhanced downstream signaling is enhanced calcium mobilization. In a specific embodiment, the enhanced response is enhanced cancer progression in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced cancer progression is enhanced cell proliferation in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced cancer progression is enhanced cell migration in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced cancer progression is enhanced metastasis in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced cancer progression is enhanced tumor growth in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the enhanced cancer progression is enhanced angiogenesis in a subject having cell(s) containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the cell(s) are cancer cell(s). In a specific embodiment, the cell(s) are subject-derived cell(s). In a specific embodiment, the cell(s) are derived from a biological sample obtained from the subject.

[0102] In some embodiments, the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein include characterizing CXCR4-ADRB2 heteromers as having the following characteristics: enhanced downstream signaling is attributable to CXCR4-ADRB2 heteromers; and the amount of enhanced calcium mobilization is determined by an intracellular Ca2+ assay. In a specific embodiment, the intracellular Ca2+ assay is a calcium mobilization assay. In a specific embodiment, the calcium mobilization assay determines that CXCR4-ADRB2 heteromers result in enhanced downstream signaling. In a specific embodiment, the calcium mobilization assay determines that enhanced downstream signaling is attributable to the presence of CXCR4-ADRB2 heteromers. In a specific embodiment, CXCR4-ADRB2 heteromers exhibit enhanced amounts of calcium mobilization, as determined by calcium mobilization assays, such that: a) upon costimulation with CXCL12 and an ADRB2 agonist, either CXCR4 or ADRB2, in the context of the individual protomers within a cell, results in an amount of calcium mobilization that is equal to or less than the sum of the amounts of calcium mobilization that result from single-agonist stimulation with either a CXCL12 or an ADRB2 agonist; and b) upon costimulation with CXCL12 and an ADRB2 agonist, CXCR4-ADRB2 heteromers exhibit enhanced calcium mobilization compared to the sum of the amounts of calcium mobilization that result from single-agonist stimulation with either a CXCL12 or an ADRB2 agonist. In a specific embodiment, calcium mobilization from protomer CXCR4 or ADRB2, in the context of the individual protomers within a cell, is non-synergistic, as determined by calcium mobilization assays; and calcium mobilization from CXCR4-ADRB2 heteromers within a cell is synergistic, as determined by calcium mobilization assays.In a specific embodiment, as determined by calcium mobilization assays, in the context of the individual protomers: a) individual protomer CXCR4 in cells in the absence of individual protomer ADRB2, or b) each individual protomer ADRB2 in cells in the absence of individual protomer CXCR4, upon costimulation with CXCL12 and an ADRB2 agonist, results in an amount of calcium mobilization that is equal to or less than the sum of the amounts of calcium mobilization that result from single agonist stimulation with either CXCL12 or an ADRB2 agonist. In a specific embodiment, as determined by calcium mobilization assays, in the context of the individual protomers: a) individual protomer CXCR4 in cells in the absence of individual protomer ADRB2, and b) each individual protomer ADRB2 in cells in the absence of individual protomer CXCR4, independently, upon costimulation with CXCL12 and an ADRB2 agonist, results in an amount of calcium mobilization that is equal to or less than the sum of the amounts of calcium mobilization that result from single agonist stimulation with either CXCL12 or an ADRB2 agonist. In a specific embodiment, costimulation of a CXCR4-ADRB2 heteromer with CXCL12 and an ADRB2 agonist results in an amount of calcium mobilization that is greater than the sum of the amounts of calcium mobilization that result from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay. In a specific embodiment, the amount of calcium mobilization that results from costimulation of a CXCR4-ADRB2 heteromer is an amount of calcium mobilization that is enhanced relative to the sum of the amounts of calcium mobilization that result from single-agonist stimulation of the CXCR4-ADRB2 heteromer, as determined by a calcium mobilization assay. In a specific embodiment, the amount of calcium mobilization that is enhanced due to a CXCR4-ADRB2 heteromer is an amount of calcium mobilization upon costimulation with CXCL12 and an ADRB2 agonist that is greater than the sum of the amounts of calcium mobilization that result from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay.In a specific embodiment, the amount of calcium mobilization enhanced by CXCR4-ADRB2 heteromers is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 90% greater upon costimulation with CXCL12 and an ADRB2 agonist than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay. In a specific embodiment, the amount of calcium mobilization enhanced by CXCR4-ADRB2 heteromers is at least 100% greater upon costimulation with CXCL12 and an ADRB2 agonist than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay. In a specific embodiment, the amount of calcium mobilization enhanced is the amount of synergistic calcium mobilization. In specific embodiments, the amount of synergistic calcium mobilization from cells containing CXCR4-ADRB2 heteromers is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, or at least 90% greater upon costimulation with CXCL12 and an ADRB2 agonist than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either a CXCL12 or an ADRB2 agonist, as determined by a calcium mobilization assay.

[0103] In some embodiments, the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein include those in which a CXCR4-ADRB2 heteromer is characterized as having the following characteristic: an enhanced response (or enhanced downstream signaling) resulting from stimulation of the CXCR4-ADRB2 heteromer, such as costimulation of the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced response (or enhanced downstream signaling) is an enhanced amount of calcium mobilization. In certain embodiments, calcium mobilization is determined by an intracellular Ca2+ assay. In a specific embodiment, the intracellular Ca2+ assay is a calcium mobilization assay. In certain embodiments, the enhanced response is enhanced cancer progression in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell proliferation in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced cell migration in a subject having a cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced metastasis in subjects having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced tumor growth in subjects having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the enhanced cancer progression is enhanced angiogenesis in subjects having cell(s) containing the CXCR4-ADRB2 heteromer. In certain embodiments, the cell(s) are cancer cell(s). In certain embodiments, the cell(s) are derived from a subject. In certain embodiments, the cell(s) are derived from a biological sample obtained from a subject.

[0104] In some embodiments, the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein include wherein the CXCR4-ADRB2 heteromer is characterized as having one or more characteristics selected from the group consisting of: (1) the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific properties of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; (2) the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; (3) the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific properties of a cell(s) derived from the subject containing a CXCR4-ADRB2 heteromer; and (4) the combination of blixafor and an ADRB2 inhibitor reduces cancer progression in a cell(s) derived from the subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the CXCR4-ADRB2 heteromer is characterized such that the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific properties of the CXCR4-ADRB2 heteromer in a cell(s) derived from a subject. In a specific embodiment, the CXCR4-ADRB2 heteromer is characterized such that the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from a subject. In a specific embodiment, the CXCR4-ADRB2 heteromer is characterized such that the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific properties of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the CXCR4-ADRB2 heteromer is characterized such that the combination of blixafor and an ADRB2 inhibitor reduces cancer progression in a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the decreased cancer progression comprises a decrease in cancer progression of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer, hi a specific embodiment, the decreased cancer progression comprises a decrease in cell proliferation of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer.In a specific embodiment, the decreased cancer progression comprises decreased cell migration of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the decreased cancer progression comprises decreased metastasis of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the decreased cancer progression comprises decreased angiogenesis of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0105] In some embodiments, the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein include those characterized as having the following characteristic: a greater amount of downstream ERK signaling resulting from costimulation of the CXCR4-ADRB2 heteromer with a CXCR4 agonist and an ADRB2 agonist than the amount of downstream ERK signaling resulting from stimulation of the CXCR4-ADRB2 heteromer with either a CXCR4 agonist or an ADRB2 agonist alone. In some embodiments, the amount of downstream ERK signaling resulting from costimulation with a CXCR4 agonist and an ADRB2 agonist is greater than the amount resulting from stimulation with a CXCR4 agonist alone. In some embodiments, the amount of downstream ERK signaling resulting from costimulation with a CXCR4 agonist and an ADRB2 agonist is at least 5% greater than the amount resulting from stimulation with a CXCR4 agonist alone. In some embodiments, the amount of downstream ERK signaling resulting from costimulation with a CXCR4 agonist and an ADRB2 agonist is at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% greater than the amount resulting from stimulation with a CXCR4 agonist alone. In some embodiments, the amount of downstream ERK signaling resulting from costimulation with a CXCR4 agonist and an ADRB2 agonist is 5-15%, 10-25%, 20-50%, 40-75%, or 60-100% greater than the amount resulting from stimulation with a CXCR4 agonist alone. In some embodiments, the amount of downstream ERK signaling resulting from costimulation with a CXCR4 agonist and an ADRB2 agonist is at least 5% greater than the amount resulting from stimulation with an ADRB2 agonist alone. In some embodiments, the amount of downstream ERK signaling resulting from co-stimulation with a CXCR4 agonist and an ADRB2 agonist is at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% greater than the amount resulting from stimulation with the ADRB2 agonist alone.In some embodiments, the amount of downstream ERK signaling resulting from costimulation with a CXCR4 agonist and an ADRB2 agonist is 5-15%, 10-25%, 20-50%, 40-75%, or 60-100% greater than the amount resulting from stimulation with the ADRB2 agonist alone. In specific embodiments, the CXCR4 agonist is CXCL12 and the ADRB2 agonist is salmeterol.

[0106] In some embodiments, the methods of suppression, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein may be used to demonstrate that the administered combination of blixafor and an ADRB2 inhibitor (i) alters the heteromer-specific properties of CXCR4-ADRB2 heteromers in a cell(s) derived from the subject; (ii) alters the heteromer-specific function of CXCR4-ADRB2 heteromers in a cell(s) derived from the subject; (iii) )Cor (iv) altering the heteromer-specific characteristics of a cell(s) derived from a subject that contain CXCR4-ADRB2 heteromers; or (iv) reducing cancer progression in a subject having cells containing the CXCR4-ADRB2 heteromers. In a specific embodiment, the administered combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific characteristics of a CXCR4-ADRB2 heteromer in a cell(s) derived from a subject. In a specific embodiment, the administered combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific function of a CXCR4-ADRB2 heteromer in a cell(s) derived from a subject. In a specific embodiment, the administered combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific characteristics of a cell(s) derived from a subject that contain CXCR4-ADRB2 heteromers. In a specific embodiment, the administered combination of blixafor and an ADRB2 inhibitor reduces cancer progression in a subject having cells containing the CXCR4-ADRB2 heteromers. In a specific embodiment, the reduced cancer progression comprises a reduction in cancer progression of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the reduced cancer progression comprises a reduction in cell proliferation of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the reduced cancer progression comprises a reduction in cell migration of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the reduced cancer progression comprises a reduction in metastasis of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the reduced cancer progression comprises a reduction in angiogenesis of a cell(s) derived from a subject containing the CXCR4-ADRB2 heteromer. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0107] In some embodiments, the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein comprise administering a combination of blixafor and an ADRB2 inhibitor to suppress enhanced downstream signaling from the CXCR4-ADRB2 heteromer in a cancer subject, e.g., from the CXCR4-ADRB2 heteromer in a cell or from the CXCR4-ADRB2 heteromer in a cell of a cancer subject. In specific embodiments, the CXCR4-ADRB2 heteromeric component comprises individual protomers of CXCR4 and ADRB2. In some embodiments, cells containing either CXCR4 or ADRB2 in the context of individual protomers comprise (i) individual protomers CXCR4 in the presence or absence of individual protomers ADRB2; or (ii) individual protomers ADRB2 in the presence or absence of individual protomers CXCR4, respectively. In some embodiments, cells containing CXCR4 in the context of individual protomers comprise individual protomers CXCR4 in the presence or absence of individual protomers ADRB2. In a specific embodiment, cells containing CXCR4 in the context of individual protomers contain the individual protomer CXCR4 in the absence of the individual protomer ADRB2. In a specific embodiment, cells containing CXCR4 in the context of individual protomers contain the individual protomer CXCR4 in the presence of the individual protomer ADRB2. In some embodiments, cells containing ADRB2 in the context of individual protomers contain the individual protomer ADRB2 in the presence or absence of the individual protomer CXCR4. In a specific embodiment, cells containing ADRB2 in the context of individual protomers contain the individual protomer ADRB2 in the absence of the individual protomer CXCR4. In a specific embodiment, cells containing ADRB2 in the context of individual protomers contain the individual protomer ADRB2 in the presence of the individual protomer CXCR4. In a specific embodiment, the CXCR4 inhibitor is blixafor and the ADRB2 inhibitor is carvedilol.

[0108] In some embodiments, cancer progression is synergistically reduced upon administration of the combination of inhibitors compared to administration of a CXCR4 inhibitor or an ADRB2 inhibitor as a single inhibitor to the subject, hi some embodiments, the reduction in cancer progression upon administration of the combination of inhibitors is greater than the sum of the reductions in the level of progression achieved by administering a CXCR4 inhibitor or an ADRB2 inhibitor as a single inhibitor to the subject. In some embodiments, in the methods of inhibition, methods of treatment, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein, the progression of cancer in a subject having cancer cells containing a CXCR4-ADRB2 heteromer is reduced by 5-100% or more upon administration of the combination of inhibitors compared to administration of a CXCR4 inhibitor or an ADRB2 inhibitor as a single inhibitor to the subject, e.g., by 5-100% or more, 10-100% or more, 20-100% or more, 30-100% or more, 40-100% or more, 50-100% or more, 60-100% or more, 75-100% or more, 5-75% or more, 5-50% or more, or 5-25% or more upon administration of the combination of inhibitors compared to administration of a CXCR4 inhibitor or an ADRB2 inhibitor as a single inhibitor to the subject. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol. In specific embodiments, cancer progression is determined by the percentage change in tumor size. In specific embodiments, cancer progression is determined by the percentage change in tumor size over a period of 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0109] In some embodiments, in the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein, the efficacy of a CXCR4 inhibitor, when administered in combination with an ADRB2 inhibitor to a subject having cancer cells containing CXCR4-ADRB2 heteromers, is increased by 5-5000% compared to the efficacy of the CXCR4 inhibitor when administered as a single inhibitor, e.g., 5-4500%, 5-4000%, 5-3500%, 5-3000%, 5-2500%, 5-2000%, 5-1750%, 5-1500%, 5-1250%, 5-6000%, 5-7000%, 5-8000%, 5-8500%, 5-9000%, 5-9500%, 5-1000%, 5-1150%, 5-1250%, 5-1300%, 5-1400%, 5-1500%, 5-1650%, 5-1750%, 5-1850%, 5-2000%, 5-2150%, 5-2250%, 5-2350%, 5-2450%, 5-2500%, 5-3000%, 5-3500%, 5-3000%, 5-2500%, 5-3000%, 5-4000%, 5-4500%, 5-4000%, 5-3500%, 5-3000%, 5-2500%, 5-2000%, 5-1750%, 5-1500%, 5-1250%, 5-1500%, 5-2350%, 5-2450%, 5-3000%, 5-3500%, 5-3000%, 5- ~1000%, 5~900%, 5~800%, 5~700%, 5~500%, 5~400%, 5~250%, 5~200%, 5~100%, 5~75%, 5~50%, 5~40%, 5~30%, 5~25%, 1000~3000%, 2000~4000%, 3000~5000%, 3500~4500%, 4000~5000%, 100~2000%, 200~2000%, 300~20 In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol. In a specific embodiment, the increased efficacy of a CXCR4 inhibitor against CXCR4-ADRB2 heteromers in a subject's cells (e.g., in suppressing downstream signaling enhanced from CXCR4-ADRB2 heteromers) is determined by a change in the IC50 value when the CXCR4 inhibitor is administered in combination with an ADRB2 inhibitor compared to the IC50 value when the CXCR4 inhibitor is administered as a single inhibitor.In specific embodiments, determining the IC50 value of a CXCR4 inhibitor using the assays disclosed herein can utilize a concentration of the ADRB2 inhibitor in the range of 1 to 10 μM, e.g., a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In specific embodiments, determining the IC50 value of a CXCR4 inhibitor using the assays disclosed herein can utilize an ADRB2 inhibitor at its IC90 concentration relative to ADRB2, e.g., a concentration in the range of 1 to 1,000 nM, e.g., 1 to 500 nM, 100 to 750 nM, or 600 to 1,000 nM, e.g., a concentration of 10, 25, 50, 100, 250, 500, 600, 700, 800, 900, or 1,000 nM.

[0110] In some embodiments, in the methods of treatment, methods of suppression, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein, the efficacy of an ADRB2 inhibitor, when administered in combination with a CXCR4 inhibitor to a subject having cancer cells containing CXCR4-ADRB2 heteromers, is increased by 5 to 5000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor, e.g., 5 to 4500%, 5 to 4000%, 5 to 3500%, 5 to 3000%, 5 to 2500%, 5 to 2000%, 5 to 1750%, 5 to 1500%, 5 to 1250%, 5 to 1500%, 5 to 2000%, 5 to 3000%, 5 to 4500%, 5 to 4000%, 5 to 3500%, 5 to 3000%, 5 to 2500%, 5 to 2000%, 5 to 1750%, 5 to 1500%, 5 to 1250%, 5 to 1500%, 5 to 25 ...3000%, 5 to 4500%, 5 to 4000%, 5 to 3500%, 5 to 3000%, 5 to 2500%, 5 to 2000%, 5 to 1750%, 5 to 1500%, 5 to 1000%, 5-900%, 5-800%, 5-700%, 5-500%, 5-400%, 5-250%, 5-200%, 5-100%, 5-75%, 5-50%, 5-40%, 5-30%, 5-25%, 1000-3000%, 2000-4000%, 3000-5000%, 3500-4500%, 4000-5000%, 100-2000%, 200-2000%, 300-200 The increase in serum vasoconstriction is in the range of 0%, 500-2000%, 750-2000%, 1000-2000%, 1250-2000%, 1500-2000%, 5-1500%, 25-1500%, 50-1500%, 75-1500%, 100-1500%, 200-1500%, 300-1500%, 500-1500%, 750-1500%, 1000-1500%, or 1250-1500%. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol. In a specific embodiment, the increased efficacy of an ADRB2 inhibitor against CXCR4-ADRB2 heteromers in a subject's cells (e.g., in suppressing downstream signaling enhanced from CXCR4-ADRB2 heteromers) is determined by a change in the IC50 value when the ADRB2 inhibitor is administered in combination with a CXCR4 inhibitor compared to the IC50 value when the ADRB2 inhibitor is administered as a single inhibitor.In specific embodiments, determining IC50 values ​​of ADRB2 inhibitors using the assays disclosed herein can utilize a CXCR4 inhibitor concentration in the range of 1-10 μM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. In specific embodiments, determining IC50 values ​​of ADRB2 inhibitors using the assays disclosed herein can utilize a CXCR4 inhibitor at its IC90 concentration relative to CXCR4, e.g., a concentration in the range of 1-1,000 nM, e.g., 1-500 nM, 100-750 nM, or 600-1,000 nM, e.g., 10, 25, 50, 100, 250, 500, 600, 700, 800, 900, or 1,000 nM.

[0111] In some embodiments, the methods of treatment, suppression methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the method or use suppresses enhanced downstream signaling resulting from CXCR4-ADRB2 heteromers in a cancer subject by 5-2000 fold compared to administration of a single inhibitor, e.g., suppresses enhanced downstream signaling resulting from the CXCR4-ADRB2 heteromers in a cancer subject by 5-1750 fold, 5-1500 fold, 5-1250 fold, 5-1000 fold, 5-900 fold, 5-800 fold, or 5-900 fold compared to administration of a single inhibitor. 00x, 5~700x, 5~500x, 5~400x, 5~250x, 5~200x, 5~100x, 5~75x, 5~50x, 5~40x, 5~30x , 5~25x, 100~2000x, 200~2000x, 300~2000x, 500~2000x, 750~2000x, 1000~2000x, 1 In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0112] In some embodiments, the methods of treatment, suppression methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor, wherein the method or use suppresses enhanced downstream signaling due to CXCR4-ADRB2 heteromers in a cancer subject by 5 to 2000 times compared to suppression of downstream signaling from either CXCR4 protomers or ADRB2 protomers in the context of each individual protomer, e.g., suppresses enhanced downstream signaling due to CXCR4-ADRB2 heteromers in a cancer subject by 5 to 2000 times compared to suppression of downstream signaling from either CXCR4 protomers or ADRB2 protomers in the context of each individual protomer. 5-1750-fold, 5-1500-fold, 5-1250-fold, 5-1000-fold, 5-900-fold, 5-800-fold, 5-700-fold, 5-500-fold, 5-400-fold, 5-250-fold, 5-200-fold, 5-100-fold, 5-75-fold, 5-50-fold, 5-40-fold, 5-30-fold, 5-25-fold, 100-2000-fold, 200-2000-fold, 300-2000-fold, 500-2000-fold, 600-650-fold, 700-750-fold, 800-850-fold, 900-950-fold, 1000-1200-fold, 1300-1400-fold, 1400-1500-fold, 1500-1600-fold, 1600-1750-fold, 1700-1800-fold, 1800-1900-fold, 1900-2000-fold, 2100-2200-fold, 2200-2300-fold, 2300-2400-fold, 2400-2500-fold, 2500-2600-fold, 2600-2700-fold, 2700-2800-fold, 2800-2900-fold, 2900-3000-fold, 3000-3100-fold, 3100-3200-fold, 3200-3300-fold, 3300-3 In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0113] Initial testing and evaluation of an inhibitor or combination of inhibitors for efficacy or therapeutic effectiveness in suppressing downstream signaling enhanced from CXCR4-ADRB2 heteromers according to the methods disclosed herein and / or in determining IC50 values ​​according to the assays disclosed herein can utilize a concentration of the inhibitor (or each inhibitor in a combination at a concentration in the range of 1-10 μM), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. If the inhibition of signal by the inhibitor is not found to be sufficient to provide a determinable measurement, a higher concentration of the inhibitor can be used to better assess a determinable measurement, such as an IC50 value. If the inhibition of signal by the inhibitor is very strong, a lower concentration of the inhibitor can be used to better assess a determinable measurement, such as an IC50 value.

[0114] In some embodiments, the methods provided herein are methods for treating, ameliorating, or preventing a disease in a subject in need thereof. In some embodiments, the methods provided herein may comprise administering to a subject a therapeutically effective amount of a pharmaceutical composition provided herein. For example, the pharmaceutical compositions provided herein may comprise a CXCR4 inhibitor, an ADRB2 inhibitor, or a combination of a CXCR4 inhibitor and an ADRB2 inhibitor; and a pharmaceutically acceptable carrier. Diseases that can be treated or prevented using the methods of the present invention include, but are not limited to, cancer, tumor, metastasis, and / or angiogenesis. For example, in some embodiments, the methods provided herein are useful for treating cancer or related conditions in which cells of the cancer, tumor, and / or microenvironment express CXCR4-ADRB2 heteromers. In some embodiments, the cancer is a hematological cancer or solid tumor. In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is selected from the group consisting of lymphoma, leukemia, myeloma, and multiple myeloma. In some embodiments, the hematological cancer is selected from the group consisting of multiple myeloma, acute myeloid leukemia, acute monocytic leukemia, diffuse large B-cell lymphoma, B-cell acute lymphoblastic leukemia, Hodgkin's lymphoma, acute promyelocytic leukemia, chronic eosinophilic leukemia, and Burkitt's lymphoma. In some embodiments, non-limiting examples of cancers or tumors that can be treated, ameliorated, or prevented using the methods of the present invention include tumors of the gastrointestinal tract, such as breast cancer, lung cancer, small cell carcinoma of the lung, hepatocellular carcinoma, brain cancer, kidney cancer, pancreatic cancer or pancreatic adenocarcinoma, ovarian cancer, prostate cancer, MalignantThese include melanoma, lymphoma, leukemia, multiple myeloma, renal cell carcinoma, soft tissue sarcoma, gastrointestinal cancer, stomach cancer, colon cancer, colorectal cancer, colorectal adenocarcinoma, bladder adenocarcinoma, esophageal cancer, and adenocarcinoma of the stomach, esophagus, throat, and genitourinary tract. In some embodiments, the lymphoma is a B-cell lymphoma, a T-cell lymphoma, or an NK-cell lymphoma. In some embodiments, the lymphoma is a relapsed or refractory lymphoma. In some embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), follicular center lymphoma, transformed lymphoma, moderately differentiated lymphocytic lymphoma, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small cleaved cell lymphoma (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL), ... (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL), follicular center lymphoma (FCL layerIn some embodiments, the leukemia is selected from the group consisting of: (a) acute lymphocytic leukemia (ALL), T-cell acute lymphocytic leukemia (T-ALL), B-cell acute lymphoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia (AML), acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia; (b) chronic myeloid (granulomatous) leukemia ... or (c) a chronic leukemia selected from the group consisting of chronic myelogenous leukemia (chronic myeloid leukemia; CML), chronic myelogenous leukemia (chronic myeloid leukemia; CML), and chronic lymphocytic leukemia (CLL); or (c) chronic myelogenous monocytic leukemia (CMML), chronic eosinophilic leukemia, juvenile myelogenous monocytic leukemia (JMML), polycythemia vera, natural killer cell leukemia (NK leukemia), or hairy cell leukemia. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a benign tumor or cancer. In some embodiments, the solid tumor is a carcinoma or sarcoma. In some embodiments, the solid tumor is selected from the group consisting of pancreatic adenocarcinoma, pancreatic tubular adenocarcinoma, renal cell carcinoma, breast adenocarcinoma, breast carcinoma, breast tubular adenocarcinoma, ovarian serous adenocarcinoma, ovarian clear cell adenocarcinoma, ovarian mucinous cystadenocarcinoma, uterine carcinosarcoma, endometrial adenocarcinoma, endometrial stromal sarcoma, endometrial carcinoma, gastric tubular adenocarcinoma, gastric adenosquamous carcinoma, multiple endocrine neoplasia, Malignant Melanoma, thyroid cancer, prostate cancer, hepatocellular carcinoma, intrahepatic cholangiocarcinoma, lung adenocarcinoma, small cell lung carcinoma, adenosquamous lung carcinoma, malignant epithelioid mesothelioma, glioblastoma, medulloblastoma, astrocytoma, Alveolar In some embodiments, the solid tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, synovium, mesothelioma, malignant epithelioid mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, colorectal cancer, esophageal cancer, colon cancer, lymphoid malignancies, pancreatic cancer, pancreatic adenocarcinoma, pancreatic tubular adenocarcinoma, breast cancer, breast adenocarcinoma, breast tubular adenocarcinoma, lung cancer, small cell lung carcinoma, lung adenocarcinoma, adenosquamous lung carcinoma, AlveolarRhabdomyosarcoma, ovarian cancer, ovarian clear cell adenocarcinoma, ovarian mucinous cystadenocarcinoma, ovarian serous adenocarcinoma, prostate cancer, hepatocellular carcinoma, soft tissue sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma origin Cancer, gastrointestinal cancer, gastric tubular adenocarcinoma, gastric adenosquamous carcinoma, kidney cancer, intrahepatic cholangiocarcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, chorioepithelioma, Wilms' tumor, uterine carcinosarcoma, endometrial adenocarcinoma, endometrial stromal sarcoma, endometrial cancer, cervical cancer, testicular tumor, seminoma, bladder cancer, MalignantThe solid tumor is selected from the group consisting of melanoma, multiple myeloma, multiple endocrine neoplasia, CNS tumor, glioblastoma, astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis. In some embodiments, the solid tumor is selected from the group consisting of breast cancer, lung cancer, and hepatocellular carcinoma. In some embodiments, the solid tumor is breast cancer. In some embodiments, the solid tumor is lung cancer. In some embodiments, the solid tumor is hepatocellular carcinoma. In some embodiments, the method for treating cancer is a method for inhibiting downstream signaling enhanced by CXCR4-ADRB2 heteromers. In some embodiments, the method for inhibiting downstream signaling enhanced by CXCR4-ADRB2 heteromers is a method for treating cancer. In some embodiments, the cancer is a CXCR4-expressing cancer. In some embodiments, the cancer is an ADRB2-expressing cancer. In some embodiments, the cancer is a CXCR4-expressing cancer and an ADRB2-expressing cancer. In some embodiments, the CXCR4 expression level in the subject is higher than a baseline level. In some embodiments, the ADRB2 expression level in the subject is higher than a baseline level. In some embodiments, the CXCR4 expression level and the ADRB2 expression level in the subject are higher than their respective baseline levels. In some embodiments, the CXCR4 expression level in the cells is higher than a baseline level. In some embodiments, the ADRB2 expression level in the cells is higher than a baseline level. In some embodiments, the CXCR4 expression level and the ADRB2 expression level in the cells are higher than their respective baseline levels.

[0115] In some embodiments, the suppression method, treatment method, pharmaceutical kit for use, or pharmaceutical composition for use provided herein further comprises determining the expression level of the CXCR4 gene in a cell, in a subject, or in a sample obtained from the subject, wherein if the expression level is higher than a reference level of the CXCR4 gene, the cell, subject, or sample obtained from the subject is determined to have a CXCR4-expressing cancer. In a specific embodiment, the method or use provided herein determines the expression level of the CXCR4 gene in a cell. In a specific embodiment, the method or use provided herein determines the expression level of the CXCR4 gene in a subject. In a specific embodiment, the method or use provided herein determines the expression level of the CXCR4 gene in a sample obtained from the subject. In a specific embodiment, the cancer is a CXCR4-expressing cancer. In a specific embodiment, the CXCR4 gene expression level in the cell is higher than the reference level. In a specific embodiment, the CXCR4 gene expression level in the subject is higher than the reference level. In a specific embodiment, the CXCR4 gene expression level in the sample obtained from the subject is higher than the reference level. In a specific embodiment, in which the CXCR4 gene expression level is higher than the reference level, the methods or uses provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0116] In some embodiments, the suppression method, treatment method, pharmaceutical kit for use, or pharmaceutical composition for use provided herein further comprises determining the expression level of the ADRB2 gene in a cell, in a subject, or in a sample obtained from the subject, wherein if the expression level is higher than a reference level of the ADRB2 gene, the cell, subject, or sample obtained from the subject is determined to have an ADRB2-expressing cancer. In a specific embodiment, the method or use provided herein determines the expression level of the ADRB2 gene in a cell. In a specific embodiment, the method or use provided herein determines the expression level of the ADRB2 gene in a subject. In a specific embodiment, the method or use provided herein determines the expression level of the ADRB2 gene in a sample obtained from the subject. In a specific embodiment, the cancer is an ADRB2-expressing cancer. In a specific embodiment, the ADRB2 gene expression level in the cell is higher than the reference level. In a specific embodiment, the ADRB2 gene expression level in the subject is higher than the reference level. In a specific embodiment, the ADRB2 gene expression level in the sample obtained from the subject is higher than the reference level. In a specific embodiment, in which the ADRB2 gene expression level is higher than the reference level, the methods or uses provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0117] In some embodiments, the suppression method, treatment method, pharmaceutical kit for use, or pharmaceutical composition for use provided herein further comprises determining the expression levels of the CXCR4 gene and the ADRB2 gene in a cell, in a subject, or in a sample obtained from the subject, wherein if the expression levels of the CXCR4 gene and the ADRB2 gene are higher than the respective reference levels of the CXCR4 gene and the ADRB2 gene, the cell, subject, or sample obtained from the subject is determined to have a CXCR4-expressing and ADRB2-expressing cancer. In a specific embodiment, the method or use provided herein determines the expression levels of the CXCR4 gene and the ADRB2 gene in a cell. In a specific embodiment, the method or use provided herein determines the expression levels of the CXCR4 gene and the ADRB2 gene in a subject. In a specific embodiment, the method or use provided herein determines the expression levels of the CXCR4 gene and the ADRB2 gene in a sample obtained from the subject. In a specific embodiment, the cancer is a CXCR4-expressing and ADRB2-expressing cancer. In a specific embodiment, the expression levels of the CXCR4 gene and the ADRB2 gene in the cell are higher than the respective reference levels. In a specific embodiment, the expression levels of the CXCR4 gene and the ADRB2 gene in the subject are higher than their respective reference levels. In a specific embodiment, the expression levels of the CXCR4 gene and the ADRB2 gene in a sample obtained from the subject are higher than their respective reference levels. In a specific embodiment in which the expression levels of the CXCR4 gene and the ADRB2 gene are higher than their respective reference levels, the methods or uses provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0118] In some embodiments, the suppression method, treatment method, pharmaceutical kit for use, or pharmaceutical composition for use provided herein further comprises determining the expression level of CXCR4 protein in a cell, in a subject, or in a sample obtained from the subject, wherein if the expression level is higher than a reference level of CXCR4 protein, the cell, subject, or sample obtained from the subject is determined to have a CXCR4-expressing cancer. In a specific embodiment, the method or use provided herein determines the expression level of CXCR4 protein in a cell. In a specific embodiment, the method or use provided herein determines the expression level of CXCR4 protein in a subject. In a specific embodiment, the method or use provided herein determines the expression level of CXCR4 protein in a sample obtained from the subject. In a specific embodiment, the cancer is a CXCR4-expressing cancer. In a specific embodiment, the expression level of CXCR4 protein in the cell is higher than the reference level. In a specific embodiment, the expression level of CXCR4 protein in the subject is higher than the reference level. In a specific embodiment, the expression level of CXCR4 protein in the sample obtained from the subject is higher than the reference level. In a specific embodiment, in which the CXCR4 protein expression level is higher than the baseline level, the methods or uses provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0119] In some embodiments, the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein further comprise determining the expression level of ADRB2 protein in a cell, in a subject, or in a sample obtained from the subject, wherein if the expression level is higher than a reference level of ADRB2 protein, the cell, subject, or sample obtained from the subject is determined to have an ADRB2-expressing cancer. In a specific embodiment, the methods or uses provided herein determine the expression level of ADRB2 protein in a cell. In a specific embodiment, the methods or uses provided herein determine the expression level of ADRB2 protein in a subject. In a specific embodiment, the methods or uses provided herein determine the expression level of ADRB2 protein in a sample obtained from the subject. In a specific embodiment, the cancer is an ADRB2-expressing cancer. In a specific embodiment, the expression level of ADRB2 protein in the cell is higher than the reference level. In a specific embodiment, the expression level of ADRB2 protein in the subject is higher than the reference level. In a specific embodiment, the expression level of ADRB2 protein in the sample obtained from the subject is higher than the reference level. In a specific embodiment, in which the ADRB2 protein expression level is higher than the baseline level, the methods or uses provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0120] In some embodiments, the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein further comprise determining the expression levels of CXCR4 protein and ADRB2 protein in a cell, in a subject, or in a sample obtained from the subject, wherein if the expression levels of CXCR4 protein and ADRB2 protein are higher than the respective reference levels of CXCR4 protein and ADRB2 protein, the cell, subject, or sample obtained from the subject is determined to have a CXCR4-expressing and ADRB2-expressing cancer. In a specific embodiment, the method or use provided herein determines the expression levels of CXCR4 protein and ADRB2 protein in a cell. In a specific embodiment, the method or use provided herein determines the expression levels of CXCR4 protein and ADRB2 protein in a subject. In a specific embodiment, the method or use provided herein determines the expression levels of CXCR4 protein and ADRB2 protein in a sample obtained from the subject. In a specific embodiment, the cancer is a CXCR4-expressing and ADRB2-expressing cancer. In a specific embodiment, the expression levels of CXCR4 protein and ADRB2 protein in the cell are higher than the respective reference levels. In a specific embodiment, the CXCR4 protein and ADRB2 protein expression levels in the subject are higher than their respective reference levels. In a specific embodiment, the CXCR4 protein and ADRB2 protein expression levels in a sample obtained from the subject are higher than their respective reference levels. In a specific embodiment in which the CXCR4 protein and ADRB2 protein expression levels are higher than their respective reference levels, the methods or uses provided herein comprise administering a CXCR4 inhibitor and an ADRB2 inhibitor. In a specific embodiment, the CXCR4 inhibitor is blixafor. In a specific embodiment, the ADRB2 inhibitor is carvedilol.

[0121] In some embodiments, pharmaceutical compositions provided herein (sometimes referred to herein as "pharmaceutical formulations") comprise a combination of a CXCR4 inhibitor and an ADRB2 inhibitor and a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions provided herein comprise a CXCR4 inhibitor and a pharmaceutically acceptable carrier. In some embodiments, pharmaceutical compositions provided herein comprise an ADRB2 inhibitor and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include standard pharmaceutical carriers, additives, or stabilizers known in the art, such as physiologically acceptable carriers, excipients, or stabilizers, e.g., phosphate-buffered saline solution, water, and emulsions, e.g., oil-based and aqueous emulsions, and any of various types of wetting agents. These pharmaceutical compositions can be prepared in a liquid unit dosage form sufficient to deliver a CXCR4 inhibitor, an ADRB2 inhibitor, or a combination of a CXCR4 inhibitor and an ADRB2 inhibitor to a target area of ​​a subject requiring treatment, or in any other dosage form. For example, pharmaceutical compositions can be prepared in any manner appropriate for the selected mode of administration, e.g., intravascular, intramuscular, subcutaneous, intradermal, intrathecal, etc. Other optional ingredients, such as pharmaceutical grade stabilizers, buffers, preservatives, additives, etc., can be readily selected by one of ordinary skill in the art. The preparation of pharmaceutical compositions having due regard for pH, isotonicity, stability, etc. is within the level of skill in the art.

[0122] In some embodiments, a pharmaceutical composition provided herein that can be utilized in the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein comprises (a) a CXCR4 inhibitor that is blixafor; (b) an ADRB2 inhibitor; and (c) a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition provided herein, or a method of use provided herein utilizing the same, comprises the ADRB2 inhibitor being an ADRB2 antagonist, an ADRB2 inverse agonist, an ADRB2 partial antagonist, an ADRB2 allosteric modulator, an ADRB2 antibody, an ADRB2 antibody fragment, an ADRB2 ligand, or an antibody-drug conjugate. In a specific embodiment, a pharmaceutical composition provided herein, or a method of use provided herein utilizing the same, comprises the ADRB2 inhibitor being an antagonist ADRB2. In a specific embodiment, a pharmaceutical composition provided herein, or a method of use provided herein utilizing the same, comprises the ADRB2 inhibitor being an inverse agonist ADRB2. In specific embodiments, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is a partial antagonist of ADRB2. In specific embodiments, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is an allosteric modulator of ADRB2. In specific embodiments, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is an antibody to ADRB2. In specific embodiments, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is an antibody fragment of ADRB2. In specific embodiments, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is a ligand of ADRB2.In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is an antibody-drug conjugate. In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP 12177, cycloprolol, ICI 118551, ICYP, labetalol, levobetaxolol, levobunolol, LK 204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A, and timolol. In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use utilizing same, include where the ADRB2 inhibitor is carvedilol. In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use provided herein utilizing the same, comprise administering a therapeutically effective amount of blixafor to a subject. In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use provided herein utilizing the same, comprise administering a sub-therapeutically effective amount of blixafor to a subject. In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use provided herein utilizing the same, comprise administering a therapeutically effective amount of an ADRB2 inhibitor to a subject. In a specific embodiment, the pharmaceutical compositions provided herein, or methods of use provided herein utilizing the same, comprise administering a sub-therapeutically effective amount of an ADRB2 inhibitor to a subject.

[0123] In some embodiments, the suppression methods, treatment methods, pharmaceutical kits for use, or pharmaceutical compositions for use provided herein comprise administering blixafor as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the methods or uses provided herein comprise administering an ADRB2 inhibitor as a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the methods or uses provided herein comprise administering a combination of blixafor and an ADRB2 inhibitor sequentially, concomitantly, or simultaneously. In some embodiments, the methods or uses provided herein comprise administering a combination of blixafor and an ADRB2 inhibitor as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the methods or uses provided herein comprise administering blixafor and an ADRB2 inhibitor as a combined pharmaceutical composition. In some embodiments, the methods or uses provided herein comprise the combined pharmaceutical compositions comprising (a) a pharmaceutical composition comprising blixafor and a pharmaceutically acceptable carrier; and (b) a pharmaceutical composition comprising an ADRB2 inhibitor and a pharmaceutically acceptable carrier. In some embodiments, the methods or uses provided herein comprise administering a combination of pharmaceutical compositions sequentially, concomitantly, or simultaneously.

[0124] Pharmaceutical formulations containing CXCR4 inhibitors and ADRB2 inhibitors provided herein, pharmaceutical formulations containing CXCR4 inhibitors, and pharmaceutical formulations containing ADRB2 inhibitors can be prepared in the form of lyophilized formulations or aqueous solutions by mixing inhibitors having the desired purity with optional physiologically acceptable carriers, additives, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA) for storage. Acceptable carriers, additives, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight polypeptides of less than about 10 residues; proteins, such as serum These include albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0125] It is understood that modifications that do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate, but not limit, the invention disclosed herein. [Example]

[0126] Example 1. Assessment of CXCR4-GPCRx heteromer formation by BiFC assay To identify novel CXCR4-GPCRx heteromers, we prepared recombinant adenoviruses encoding 143 GPCRs fused to the N-terminal fragment (VN) of the yellow fluorescent protein Venus and 147 GPCRs fused to the C-terminal fragment (VC) of Venus (see, e.g., Song, YB, et al., (2014) Monitoring G protein-coupled receptor activation using an adenovirus-based beta-arrestin bimolecular fluorescence complementation assay, Anal Biochem 449, 32-41; Korean Patent No. 101029972B1, issued April 12, 2011; YB, (2012) “Global analysis of GPCR dimerization using AdBiFC assay,” A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, School of Biological Sciences, Seoul National University, 126 pages). We identified CXCR4-GPCRx heteromers using a bimolecular fluorescence complementation (BiFC) assay (Figure 1), in which the two complementary VN and VC fragments of Venus reconstitute a fluorescent signal only when they are sufficiently close together due to interactions between the two different proteins fused to them (Hu, CD, et al., (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation, Mol Cell 9, 789-798).

[0127] U-2 OS cells were seeded in 96-well plates and co-transduced with adenoviruses encoding CXCR4-VN and GPCRx-VC, or CXCR4-VC and GPCRx-VN, at an MOI of 30, to express GPCRs for 2 days. After staining with Hoechst 33342, BiFC and nuclear images were acquired from three fields per well using an IN Cell Analyzer 1000. Images of approximately 200 cells from each well were analyzed using multitarget analysis software within the IN Cell Developer ToolBox (GE Healthcare, Waukesha, WI). Cell boundaries were highlighted based on the Hoechst signal, and the fluorescence intensity per cell was measured. Cells with fluorescence intensity higher than the background level were considered BiFC-positive. Dead cells with extremely high fluorescence intensity were excluded from the cell count. Positive cells were scored, and the positive cell count ratio ("BiFC score") was calculated as (positive cells / total cells) × 100.

[0128] When CXCR4-VN was coexpressed with HA-VC (Figure 2A) or GCGR-VC (Figure 2C), a GPCR encoding the glucagon receptor, no yellow fluorescent protein (YFP) signal (BiFC signal) was observed. In contrast, when CXCR4-VN was coexpressed with CXCR4-VC (Figure 2B), BiFC signals were observed at the plasma membrane and cytoplasm. When CXCR4-VN was cotransfected with ADRB2-VC (Figure 2D), strong BiFC signals were observed at the plasma membrane and cytoplasm. Cells that showed BiFC fluorescence signals higher than the background level were counted as BiFC-positive cells, and a BiFC score was calculated.

[0129] Protein-protein interactions can be affected by fusion tags, such as fluorescent protein fragments in BiFC or Renilla luciferase in BRET, through interference with the expression, folding, or localization of the partner protein. The partner protein can also impair the expression or folding of the fusion tag, affecting the results of proximity-based assays. Therefore, the absence of a signal between two proteins in a particular combination does not necessarily indicate that the proteins do not interact; it simply indicates that the bound donor and acceptor molecules are in a specific conformation that prevents them from interacting (Eidne, KA, et al., (2002) Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells, Trends Endocrinol Metab 13, 415-421; Kerppola, TK, (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells, Nat Protoc 1, 1278-1286).

[0130] Therefore, CXCR4 and GPCRx that showed BiFC signals in either the combination of CXCR4-VN and GPCRx-VC, or CXCR4-VC and GPCRx-VN, were considered to be interacting proteins. The BiFC score of the known homomeric pair CXCR4-VN and CXCR4-VC was 9.9. Therefore, CXCR4-GPCRx pairs that showed BiFC scores equal to or higher than 10 were selected as potential CXCR-GPCRx heteromers. The CXCR4-GPCRx pair of CXCR4-ADRB2 showed a BiFC score of 24 and was therefore considered a potential CXCR-GPCRx heteromer. Evaluation of additional GPCRs using BiFC analysis was reported in International Application PCT / KR2018 / 016166, filed December 18, 2018.

[0131] Example 2. Assessment of CXCR4-GPCRx heteromer formation by co-internalization assay Some GPCR heteromers exhibit changes in trafficking properties as a result of heteromerization, such as maturation of the partner GPCR (GABA(B) receptor) (White, 1998) and agonist-mediated internalization of partner GPCRs from the cell surface (DOR-GRPR, A2A-D2R) (Hillion, J., et al., (2002) Coaggregation, cointernalization, and cosensitization of adenosine A2A receptors and dopamine D2 receptors, J Biol Chem 277, 18091-18097; Liu, XY, et al., (2011) Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids, Cell 147, 447-458; Torvinen, M., et al., (2005) Trafficking of adenosine A2A and dopamine D2 receptors, J Mol Neurosci 25, 191-200), and a change in the localization of the partner GPCR (DOR-CB1) from the intracellular compartment to the cell surface (Rozenfeld, R., et al., (2012) Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons, PLoS One 7, e29239).

[0132] GPCR heteromerization was confirmed using the co-internalization of co-expressed GPCR pairs in response to agonists selective for only one of the pair (Milligan, 2008). To investigate whether GPCRx regulates CXCR4 trafficking when co-expressed and whether CXCR4-GPCRx heteromers form, and to confirm the physical interaction between CXCR4 and GPCRx confirmed using BiFC assays, cells were co-transfected with adenoviruses encoding CXCR4-GFP and GPCRx, and GFP images were acquired before and 30 min after stimulation with GPCRx agonists. A decrease in GFP expression on the cell surface or the appearance of GFP granules inside the cells was considered to indicate CXCR4-GFP co-internalization with GPCRx (Figure 3A-B).

[0133] In Figures 4A-B, cells were stimulated with GPCRx agonists specific for CXCR4 (Figure 4A) and ADRB2 (Figure 4B) (10 nM CXCL12 (Figure 4A) and 100 nM formoterol (Figure 4B), respectively). Images were acquired before and 30 min after agonist stimulation and analyzed using an IN Cell Analyzer 2000. The concentrations of these GPCRx agonists were initially selected to be the EC50 concentration (or alternatively, the Ki or Kd concentration) for each specific GPCRx. If the signal generated was too strong, the concentration was reduced to below the EC50; if the signal generated was too weak, the concentration was increased to 10,000-fold or less above the EC50 concentration.

[0134] Stimulation of cells expressing CXCR4-GFP with CXCL12 resulted in relocation of GFP from the plasma membrane to distinct intracellular granules, indicating internalization of surface CXCR4-GFP into the cytoplasm (Figure 4A). Regarding CXCR4-ADRB2 heteromers identified by BiFC assay, ADRB2 induced CXCR4 internalization, as revealed by an increase in intracellular GFP granules and a decrease in GFP signal at the plasma membrane in cells coexpressing CXCR4-GFP and ADRB2 (Figure 4B), thus confirming heteromeric co-internalization. Evaluation of additional GPCRs related to induced CXCR4 internalization was reported in International Application PCT / KR2018 / 016166, filed December 18, 2018.

[0135] Example 3. Evaluation of enhanced CXCR4 downstream signaling and inhibition of enhanced signaling upon CXCR4-GPCRx heteromer formation using Ca 2+ mobilization assay To further investigate whether CXCR4-GPCRx heteromers exhibit properties distinct from those of the individual protomers (in cells lacking one receptor), calcium signaling was studied in cells expressing either or both GPCRs in the presence of either or both agonists. In this example, as described in Example 2, concentrations of these GPCRx agonists were initially selected at the EC50 concentration (or alternatively, the Ki or Kd concentration) for each specific GPCRx; if the Ca2+ signal generated was too strong, the concentration was reduced below the EC50; if the Ca2+ signal generated was too weak, the concentration was increased to 100-fold or less above the EC50 concentration.

[0136] When MDA-MB-231 human breast cancer cells were transduced with an adenovirus encoding CXCR4, stimulation with CXCL12 induced intracellular calcium mobilization (Figure 5A). Stimulation of cells with salmeterol, an ADRB2-selective agonist, did not induce a calcium response, demonstrating that the calcium response evoked by CXCL12 is mediated by CXCR4. Costimulation of cells with CXCL12 and salmeterol induced a calcium response comparable to that induced by CXCL12 alone. In cells overexpressing only ADRB2, CXCL12 alone did not induce a calcium response, whereas salmeterol alone induced a calcium response, indicating that salmeterol induces calcium responses via ADRB2 (Figure 5B). Costimulation with both agonists induced a calcium response comparable to that stimulated by salmeterol alone.

[0137] In cells overexpressing both CXCR4 and ADRB2, stimulation with each agonist induced calcium responses comparable to those seen in cells expressing CXCR4 or ADRB2 alone (Fig. 5, A and B versus C). In contrast, co-stimulation with both agonists simultaneously significantly increased calcium responses compared with those elicited by the individual agonists (Fig. 5, C and D). Enhanced calcium signaling was observed only in cells expressing both CXCR4 and ADRB2, but not in cells expressing either CXCR4 or ADRB2 alone. These results demonstrate that CXCR4-ADRB2 heteromers exhibit properties distinct from those of their respective individual GPCR protomers.

[0138] We further investigated whether GPCRx antagonists inhibited the enhanced calcium response in cells co-expressing CXCR4 and GPCRx upon costimulation with CXCL12 and a GPCRx ligand. Specifically, as shown in Figure 6, in cells co-expressing CXCR4 and ADRB2, administration of the ADRB2 antagonist carvedilol (10 μM) significantly suppressed the enhanced calcium signaling, and co-administration of both the CXCR4 antagonist (AMD3100; 10 μM) and the ADRB2 antagonist carvedilol (10 μM) resulted in even greater suppression of calcium signaling. These results demonstrate that CXCR4 antagonists, ADRB2 antagonists, or a combination of CXCR4 and ADRB2 antagonists can be used as therapeutic agents against CXCR4-ADRB2 heteromers. Evaluation of additional GPCRs with respect to calcium signaling in the presence of one or both of their respective agonists and one or both of their respective antagonists was reported in International Application PCT / KR2018 / 016166, filed December 18, 2018.

[0139] The coadministration results also suggest that small doses of antagonists targeting each protomer of the heteromer may provide a novel therapeutic tool to efficiently suppress CXCR4-GPCRx heteromeric responses while avoiding the side effects associated with high doses of each individual antagonist.

[0140] Example 4. Inhibition of Internalization by GPCRx Antagonists To further investigate whether co-internalization of CXCR4 heterodimers can be blocked by partner GPCRx antagonists, we performed an internalization inhibition assay. As shown in Figure 4B (GPCRx is ADRB2), when cells were co-transfected with CXCR4 and GPCRx, CXCR4-GFP-expressing U-2 OS cells were co-internalized by partner GPCRx-specific agonists (control: CXCR4-GFP (Figure 4A)). If CXCR4 forms a heterodimer with GPCRx and is co-internalized with partner GPCRx, it can be blocked by GPCRx-specific antagonists.

[0141] Adenovirus encoding a GPCRx (ADRB2) was transduced into U-2 OS cells stably expressing CXCR4-GFP. Two days later, images were acquired before and 20 minutes after stimulation of the cells with the CXCR4-specific agonist CXCL12 (20 nM) and / or a GPCRx-specific antagonist (10 μM). Using an IN Cell Analyzer 2500, CXCR4-GFP internalization was observed as GFP granules. A decrease in GFP expression on the cell surface or the appearance of GFP granules inside the cell was considered CXCR4-GFP co-internalization. In this case, the GPCRx assessed was ADRB2. Stimulation with the CXCR4 agonist CXCL12 induced CXCR4-GFP co-internalization with ADRB2 (Figure 8, left panel). Administration of the ADRB2 antagonist carvedilol had no effect on heteromeric internalization (Figure 8, center panel). CXCL12-stimulated CXCR4-GFP internalization with ADRB2 was inhibited by an ADRB2 antagonist (Figure 8, right panel). Evaluation of additional GPCRs for inhibition of internalization by GPCRx antagonists was reported in International Application PCT / KR2018 / 016166, filed December 18, 2018.

[0142] These data suggest that inhibition of CXCR4 heteromer internalization could therapeutically block aberrant downstream signals in cells overexpressing CXCR4-ADRB2 heteromers, such as cancer cells.

[0143] Example 5. Evaluation of phenotype-related effects of inhibitors of CXCR4-GPCRx heteromeric signaling on tumor growth by cell proliferation assay To develop therapeutics based on CXCR4-GPCRx heteromers, the effect of co-administration of a CXCR4 antagonist and a GPCRx antagonist on cell proliferation was evaluated, particularly with respect to ADRB2.

[0144] Single-cell suspensions of dissociated glioblastoma tissue were prepared from patients (provided by Samsung Seoul Hospital, Seoul, Korea). These cells were cultured under conditions optimal for the proliferation and non-differentiation of normal neural stem cells. The culture medium consisted of serum-free Neurobasal medium supplemented with basic FGF and EGF.

[0145] The effect of GPCRx antagonists on patient-derived cell (PDC) viability was assessed using ATPlite (PerkinElmer, Cat. No. 6016739 Reagent). ATPlite is a firefly luciferase-based adenosine triphosphate monitoring system. This luminescent assay is an alternative to colorimetric, fluorometric, and radioisotopic assays for quantitative assessment of proliferation and cytotoxicity in cultured mammalian cells. Cells were seeded at 500 cells / well in 40 μL of culture medium in 384-well plates. After overnight growth, cells were cultured for 7 days in the presence of several doses of GPCRx antagonists or DMSO alone. After 7 days of incubation, 15 μL of ATPlite was added to each well, and the plate was shaken at 700 rpm on an orbital shaker for 5 minutes. Luminescent signals were detected within 30 minutes using a PerkinElmer TopCount detector. Cell viability was calculated using the equation: Cell viability (%) = (OD of antagonist treatment / OD of DMSO alone treatment) x 100%.

[0146] When the ADRB2-specific antagonist carvedilol was administered to PDCs expressing CXCR4 and ADRB2, cell growth was significantly inhibited (IC50 = 11.69 μM, Figure 9). Evaluation of additional GPCRs on cell proliferation was reported in International Application PCT / KR2018 / 016166, filed December 18, 2018.

[0147] These results suggest that abnormal cell proliferation induced by CXCR4 heteromers can be blocked by ADRB2 antagonists in CXCR4-ADRB2 heteromer-expressing cells, and indicate that inhibition of cancer cell growth using ADRB2 antagonists in patients with CXCR4-ADRB2 heteromers can overcome the limitations of monotherapy using only CXCR4 inhibitors as cancer therapeutic agents.

[0148] Example 6. Assessment of CXCR4-GPCRx heteromer formation in patient-derived cells (PDCs) using proximity ligation assay (PLA) Various techniques have been used to investigate the presence of GPCR complexes in native tissues, including atomic force microscopy (Fotiadis, D., et al., (2006) Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors, Curr Opin Struct Biol 16, 252-259), co-immunoprecipitation (Gomes, I., et al., (2004) A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia, Proc Natl Acad Sci USA 101(14):5135-5139), and binding or functional assays (Wreggett, K.A., et al., (1995) Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors, J Biol Chem 270, 22488-22499). The most common method for monitoring interactions is based on resonance energy transfer, which is performed using labeled proteins. Labeling can be performed with selective probes such as antibodies or fluorescent ligands (Roess, DA, et al., (2000) Luteinizing hormone receptors are self-associated in the plasma membrane, Endocrinology 141, 4518-4523; Patel, RC, et al., (2002) Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells, Proc Natl Acad Sci USA 99, 3294-3299).

[0149] Bazin et al. employed a method based on time-resolved fluorescence resonance energy transfer (TR-FRET), which exhibits a much higher signal-to-noise ratio (Bazin, H., et al., (2002) Time-resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions, J Biotechnol 82, 233-250). FRET is based on the transfer of energy between two fluorophores, a donor and an acceptor, when they are in close proximity. Intermolecular interactions between biomolecules can be assessed by attaching fluorescent labels to each partner and detecting the level of energy transfer. Introducing a time delay of approximately 50-150 μs between the excitation of the system and the fluorescence measurement can eliminate nonspecific, short-lived emissions from the signal.

[0150] Proximity ligation assay (PLA) is a technique that extends the capabilities of traditional immunoassays to include the direct detection of proteins, protein interactions, and modifications with high specificity and sensitivity (Gullberg, M., et al., (2004) Cytokine detection by antibody-based proximity ligation, Proc Natl Acad Sci USA 101, 8420-8424). Two primary antibodies, raised in different species, recognize target antigens on a protein of interest. Secondary antibodies, called PLA probes, directed against the constant regions of the different primary antibodies bind to the primary antibodies. Each PLA probe carries a unique short DNA strand. When the PLA probes are in close proximity (i.e., when the original two proteins of interest are in close proximity or part of a protein complex, as shown in the figure), the DNA strands can be incorporated into rolling-circle DNA synthesis upon the addition of appropriate substrates and enzymes. The DNA synthesis reaction results in hundreds-fold amplification of the DNA circle. Fluorescently labeled complementary oligonucleotide probes are then added, which bind to the amplified DNA. The resulting high intensity of fluorescence can be easily visualized as a distinct bright spot when viewed under a fluorescence microscope (Gustafsdottir, SM, et al., (2005) Proximity ligation assays for sensitive and specific protein analyses, Anal Biochem 345, 2-9).

[0151] The CXCR4-overexpressing cell line U2OS-CXCR4 was infected with the ADRB2-expressing adenovirus Ad-ADRB2 at MOIs of 0, 2.5, 10, or 40 for 2 days. PLA was performed as previously described (Brueggemann, LI, et al., (2014) Differential protein kinase C-dependent modulation of Kv7.4 and Kv7.5 subunits of vascular Kv7 channels, J Biol Chem 289, 2099-2111; Tripathi, A., et al., (2014) CXC chemokine receptor 4 signaling upon coactivation with stromal cell-derived factor-1alpha and ubiquitin, Cytokine 65, 121-125). To perform PLA, infected cells were fixed in 4% paraformaldehyde (PFA) on 16-well tissue culture slides. Slides were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (1:200, Santacruz, Sc-53534), rabbit anti-ADRB2 (1:200, Thermoscientific, PA5-33333), and rabbit anti-CHRM1 (1:200, Ls bio, Ls-C313301) for 1 hour at 37°C in a humidified chamber. Slides were then washed and incubated with secondary anti-rabbit and anti-mouse antibodies conjugated to plus and minus Duolink II PLA probes (1 hour at 37°C). Slides were washed again and then incubated with ligation-ligase solution (37°C for 30 minutes), followed by amplification polymerase solution (37°C for 2 hours).The slides were then mounted with a minimal volume of Duolink II mounting medium with 4',6-diamidino-2-phenylindole (DAPI) for 15–30 min, and PLA signals [Duolink In Situ Detection Reagent green (λ excitation / emission 495 / 527 nm) or red (λ excitation / emission 575 / 623 nm) were identified as fluorescent puncta under an IN Cell analyzer 2500.

[0152] As shown in Figures 10A-10B, the PLA signal increased dose-dependently as the ADRB2 expression level increased. Figure 10A: Images of PLA signals from U2OS cells expressing CXCR4-ADRB2 heteromers at a range of MOIs (multiplicities of infection). Figure 10B: Red signal points were counted and calculated by normalization to the negative control. The PLA signal increased dose-dependently and proportionally to the ADRB2 expression level. To examine endogenous ADRB2 expression, qRT-PCR analysis was performed using ADRB2-specific primers. As shown in Figure 10C, the endogenous ADRB2 expression level in U2OS cells was extremely high, indicating that PLA signals were detected even in areas without virus infection (ADRB2 MOI 0).

[0153] Traditionally, glioblastoma (GBM) is the most common and deadly primary brain tumor. Preclinical cancer biology has largely relied on the use of in vitro human cancer cell lines and xenograft processes of these established cell lines. However, the process of establishing traditional cell lines results in the irreversible loss of important biological properties, and as a result, xenograft tumor models do not maintain the genomic and phenotypic characteristics present in the original tumor.

[0154] Patient-derived cells (PDCs) derived directly from glioblastoma harbor extensive similarities to normal neural stem cells and recapitulate the genotype, gene expression pattern, and in vivo biology of human glioblastoma.

[0155] To perform PLA on PDC samples, patient-derived cells were seeded onto 16-well tissue culture slides and fixed with 4% PFA. Slides were blocked with blocking solution provided by Duolink and incubated with mouse anti-CXCR4 (1:200, Santa Cruz, Sc-53534), rabbit anti-ADRB2 (1:200, Thermo Scientific, PA5-33333), and rabbit anti-CHRM1 (1:200, Lsbio, Ls-C313301) for 1 hour at 37°C in a humidified chamber. Slides were then washed and incubated with secondary anti-rabbit and anti-mouse antibodies conjugated to plus and minus Duolink II PLA probes (1 hour at 37°C). Slides were washed again and then incubated with ligation-ligase solution (30 minutes at 37°C) followed by amplification polymerase solution (2 hours at 37°C). Slides were then mounted with a minimal volume of Duolink II mounting medium with 4',6-diamidino-2-phenylindole (DAPI) for 15–30 min, and PLA signals [Duolink In Situ Detection Reagent green (λ excitation / emission 495 / 527 nm) or red (λ excitation / emission 575 / 623 nm) were identified as fluorescent puncta under an IN Cell analyzer 2500.

[0156] As shown in Figure 11A-B, the PLA ratio correlates with CXCR4-ADRB2 heterodimerization, and the frequency of heteromerization varies among patients. The PLA ratio was calculated as the number of fluorescent puncta in the PDC sample divided by the number of fluorescent puncta in the negative control. The negative control (NC) represents the background fluorescent signal, as indicated by the number of puncta in the PLA treatment without primary antibody treatment (mouse anti-CXCR4, rabbit anti-ADRB2) and with secondary antibody conjugated to the plus and minus Duolink II PLA probes. These data demonstrate the quantitative analysis of CXCR4-ADRB2 heterodimerization in cancer patient samples. The evaluation of additional GPCRs involved in heteromerization in PDC was reported in International Application PCT / KR2018 / 016166, filed December 18, 2018.

[0157] Example 7. Evaluation of CXCR4-GPCRx heteromer formation in vivo using a PDX model To perform PLA on patient-derived xenografts (PDXs), FFPE samples from glioblastoma patients (provided by Samsung Seoul Hospital, Seoul, Korea) were used. After deparaffinization of the FFPE samples, heat-induced antigen retrieval was performed at 100°C for 15 minutes. The slides were blocked with blocking solution provided by Duolink and incubated with rabbit anti-CXCR4 (1:200, Thermoscientific, PA3305) and mouse anti-ADRB2 (1:200, Santacruz, Sc-271322) for 1 hour at 37°C in a humidified chamber. The remaining process was the same as described above (PLA on PDXs).

[0158] Nuclei were visualized by DAPI staining, and CXCR4-ADRB4 heteromers were stained as small dots by PLA in Figure 12A. As shown in Figure 12B, the PLA ratio varied among patients, demonstrating the potential for personalized medicine through companion diagnostics.

[0159] Example 8. Evaluation of CXCR4 downstream signaling enhanced upon CXCR4-GPCRx heteromer formation using Ca 2+ mobilization assay MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and ADRB2 (Figure 13). The cells were cultured for 3 days, stained with Cal-520 AM, and treated with either CXCL12 (30 nM) alone, increasing doses of salmeterol alone, or salmeterol in combination with 30 nM CXCL12. Calcium mobilization was measured using a FlexStation 3. In MDA-MB-231 cells overexpressing both CXCR4 and ADRB2, stimulation with salmeterol alone did not induce calcium mobilization in a dose-dependent manner (Figure 13). However, when the cells were costimulated with salmeterol in the presence of CXCL12, calcium signaling was significantly enhanced over a wide range of salmeterol concentrations, e.g., from 10 nM to 300 nM.

[0160] Example 9. Evaluation of enhanced CXCR4 downstream signaling and inhibition of enhanced signaling upon CXCR4-GPCRx heteromer formation using Ca 2+ mobilization assay In MDA-MB-231 cells overexpressing both CXCR4 and ADRB2, costimulation with CXCL12, a CXCR4 agonist, and salmeterol (an ADRB2-selective agonist) significantly increased calcium responses compared with those induced by monostimulation with each individual agonist (Figure 14). These results demonstrate that CXCR4-ADRB2 heteromers exhibit properties distinct from those of the individual GPCRs, CXCR4 and ADRB2.

[0161] We also investigated whether the enhanced calcium response in cells co-expressing CXCR4 and ADRB2 upon costimulation with CXCL12 and an ADRB2 ligand could be inhibited by an anti-CXCR4 antibody as a CXCR4 antagonist. As shown in Figure 14, administration of 2 μg of the anti-CXCR4 antibody 12G5 suppressed the enhanced calcium signaling in cells co-expressing CXCR4 and ADRB2. Furthermore, co-administration of both antagonists (carvedilol, an ADRB2 antagonist, and 12G5, a CXCR4 antagonist) resulted in a more significant suppression of calcium signaling. These results demonstrate that anti-CXCR4 antibodies and ADRB2 antagonists can be used as effective therapeutic agents (or combination therapies) for CXCR4-ADRB2 heteromer-mediated diseases.

[0162] Specifically, using a calcium mobilization assay, MDA-MB-231 human breast cancer cells were seeded at 20,000 cells per well in black, clear-bottom 96-well plates (Corning Costar, #3340) in 100 μL of RPMI 1640 supplemented with 10% FBS. The following day, cells were co-transduced with CXCR4 at 10 MOI and GPCRx at 30 MOI. Two days later, cells were treated with the indicated amounts of the ADRB2 antagonist carvedilol (Tocris), the anti-CXCR4 antibody 12G5 (Thermo Scientific, 35-8800), and incubated with Cal 6 (Molecular Devices FLIPR® Calcium 6 Assay Kit, catalog R8191) for 2 hours. Cells were then stimulated with the indicated amounts of CXCL12, an ADRB2 agonist, or both CXCL12 and ADRB2 agonists. Calcium mobilization was measured using a FlexStation 3 Multi-Mode Microplate Reader. Results were normalized to baseline activity. Calcium mobilization was quantified by calculating the area under the curve (AUC) for each graph. Data were normalized to the CXCL12-stimulated calcium response in cells expressing CXCR4 alone. Data represent three independent experiments (mean ± SEM). * P<0.05, Student's t test.

[0163] Example 10. Effect of CXCR4-ADRB2 heteromers on tumor growth To investigate the effect of CXCR4-ADRB2 heteromers on tumor growth, cell lines stably overexpressing CXCR4 alone ("A549-CXCR4 cells") or both CXCR4 and ADRB2 ("A549-CXCR4-ADRB2 cells") such that the cells contained CXCR4-ADRB2 heteromers were generated from A549 lung cancer cells and incubated with the same amount of cells (1 x 10 7 The tumor growth rates were compared by subcutaneous injection of 1000 cells / mouse into nude mice. As shown in Figure 15A-B, the tumor size on day 28 after implantation was 351.4 ± 214.7 mm for A549. 3 , and 726.9 ± 259.6 mm for A549-CXCR4 cells. 3 , and 1012.2 ± 556.1 mm for A549-CXCR4-ADRB2 cells. 3 The tumor growth rate in mice implanted with A549-CXCR4 cells (overexpressing CXCR4) was faster than that in mice bearing only parental A549, and the fastest tumor growth was observed in mice implanted with A549-CXCR4-ADRB2 cells (overexpressing both CXCR4 and ADRB2). These results suggest that the formation of CXCR4-ADRB2 heteromers is a Ca2+ signaling pathway. 2+ This suggests that the combination of α- and β-glucan synergistically enhances the response and thus promotes tumor growth.

[0164] Figure 15A shows images of three mice implanted with either parental A549 cells, A549-CXCR4 cells (stable overexpressing CXCR4), or A549-CXCR4-ADRB2 cells (stable overexpressing both CXCR4 and ADRB2) at 28 days post-implantation. These images demonstrate that the tumor size was largest (most accelerated) in the mice bearing A549-CXCR4-ADRB2 cells (stable overexpressing both CXCR4 and ADRB2). The tumor growth rate over time for these three different mice is graphically shown in Figure 15B. Tumor growth was monitored every 3 or 4 days by measuring the length (L) and width (W) of the tumor and calculating tumor volume based on the following formula: volume = 0.5LW 2 Compared with mice bearing parental A549 cells, mice bearing A549-CXCR4 cells showed relatively rapid tumor growth, with tumor growth being the most rapid in mice implanted with A549-CXCR4-ADRB2 cells (stably overexpressing CXCR4 and ADRB2).

[0165] Example 11. Ca2+ Mobilization Inhibition of CXCR4 Downstream Signaling Enhanced upon CXCR4-ADRB2 Heteromer Formation - Comparison of Single and Combined Inhibitor Treatments The degree of inhibition (Ca) of blixafor (a CXCR4 inhibitor, also referred to as TG-0054) was evaluated as a single treatment in MDA-MB-231 cells expressing only CXCR4 (in the context of monomers or individual protomers; "MDA-MB-231-CXCR4 cells"), as a single treatment in an MDA-MB-231 cell line overexpressing both CXCR4 and ADRB2 and containing CXCR4-ADRB2 heteromers ("MDA-MB-231-CXCR4-ADRB2 cells"), and as a co-treatment with an ADRB2 inhibitor (carvedilol; 10 μM) in MDA-MB-231-CXCR4-ADRB2 cells. 2+ The concentrations of agonists (measured as IC50 values ​​for the response) were compared.

[0166] MDA-MB-231 cells were transduced with adenovirus encoding either CXCR4 alone or both CXCR4 and ADRB2. The cells were cultured for 2 days and treated with blixafor (a CXCR4 inhibitor) alone or co-treated with blixafor and an ADRB2 inhibitor (carvedilol; 10 μM). The cells were then stained with Cal-6 for 2 hours and stimulated with a CXCR4 agonist (CXCL12, 20 nM) and an ADRB2 agonist (salmeterol, 1 μM). Calcium mobilization was measured using FlexStation 3, and the results are shown in Table 3, which shows the Ca2+ mobilization in (1) MDA-MB-231-CXCR4 cells treated with blixafor alone (column 2); (2) MDA-MB-231-CXCR4-ADRB2 cells treated simultaneously with blixafor and the ADRB2 inhibitor carvedilol (column 3); and (3) MDA-MB-231-CXCR4-ADRB2 cells treated with blixafor alone (column 4). + The IC50 of the response is shown. [Table 3] As shown in Table 3, Ca in MDA-MB-231-CXCR4-ADRB2 cells containing CXCR4-ADRB2 heteromers 2+ The IC50 value of the response was more than 4500-fold lower when treated in combination with the ADRB2 inhibitor carvedilol (column 3) compared to single treatment with the CXCR4 inhibitor TG-0054 alone (column 4). This result indicates that in MDA-MB-231-CXCR4-ADRB2 cells containing the CXCR4-ADRB2 heteromer, co-treatment with blixafor and an ADRB2 inhibitor significantly reduced Ca2+ levels compared to single treatment with blixafor alone. 2+ This suggests that the increased response is more effectively inhibited.

[0167] To determine whether CXCR4-ADRB2 heteromerization induces conformational changes and / or alters binding affinity for CXCR4 inhibitors, Ca 2+The IC50 values ​​of the response were compared between single treatment with blixafor (TG-0054) in MDA-MB-231-CXCR4 cells, which express only CXCR4, and MDA-MB-231-CXCR4-ADRB2 cells, which contain CXCR4-ADRB2 heteromers. The results showed that single treatment with the CXCR4 inhibitor altered the IC50 values ​​by approximately 1.4-fold in MDA-MB-231-CXCR4-ADRB2 cells (column 4) compared to MDA-MB-231-CXCR4 cells (column 2). This result suggests that cotreatment with a CXCR4 inhibitor, e.g., blixafor (TG-0054), and an ADRB2 inhibitor can dramatically increase the therapeutic efficacy of certain CXCR4 inhibitors against CXCR4-ADRB2 heteromer-containing subjects and / or subject cells / tissues compared to single treatment with the CXCR4 inhibitor.

[0168] As shown in Table 3, co-treatment with blixafor and carvedilol significantly reduced Ca in cells containing CXCR4-ADRB2 heteromers. 2+ This resulted in a decrease in the IC50 value of the response. Because excessive doses of ADRB2 antagonists can affect the activity of their counterpart CXCR4, changes in the IC50 values ​​of CXCR4 antagonists were measured in combination with a series of CXCR4 antagonists, including carvedilol at its IC90 concentration of 650 nM. The data are shown in Table 4. [Table 4] As shown in Table 4, the IC50 values ​​of CXCR4 inhibitors were determined based on the Ca2+ / Ca2+ binding in the context of the CXCR4-ADRB2 heteromer. 2+ The response was reduced when administered in combination with an ADRB2 inhibitor (column 3) compared to the single administration of a CXCR4 inhibitor alone (column 4). For example, Ca in the context of CXCR4-ADRB2 heteromers 2+The IC50 values ​​of the responses were reduced by approximately 10.5-fold (22.45 nM to 2.12 nM), approximately 29.3-fold (0.88 nM to 0.03 nM), approximately 45.7-fold (88.66 nM to 1.94 nM), and approximately 60.7-fold (90.54 nM to 1.49 nM) when carvedilol (600 nM, its IC90 concentration value against ADRB2 in the monomeric context) was co-administered with AMD3100, urocupulumab, BKT140, and TG-0054, respectively. These results suggest that co-administration of low doses of a CXCR4 inhibitor and an ADRB2 inhibitor may increase therapeutic efficacy against CXCR4-ADRB2 heteromer-containing subjects compared with single administration of a CXCR4 inhibitor.

[0169] Example 12. Stimulation with CXCL12 and / or salmeterol induces activation of ERK signaling in cell lines containing CXCR4-ADRB2 heteromers ERK1 / 2 regulates chemotaxis or survival in various cell types (Riol-Blanco, L., et al., (2005) The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed, J. Immunol. 174(7), 4070-4080; Klemke, R.L., et al., (1997) Regulation of cell motility by mitogen-activated protein kinase, J. Cell Biol. 137(2), 481-492; Copp, J., et al., (2009) ORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2, Cancer Res. 69(5), 1821-1827). ERK1 / 2 activity was assessed by stimulating CXCR4 or CXCR4-ADRB2 heteromers in cells with CXCL12 (a CXCR4 agonist) and / or salmeterol (an ADRB2 agonist). Specifically, CXCL12 stimulation of MDA-MB-231 cells expressing Rluc-luc2P ("MDA-MB-231 parental cells"), MDA-MB-231 cells expressing CXCR4 ("MDA-MB-231-CXCR4 cells"), and MDA-MB-231 cells containing CXCR4-ADRB2 heteromers ("MDA-MB-231-CXCR4-ADRB2 cells") induced ERK1 / 2 activation after 10 minutes, reaching a maximum level after 60 minutes. Salmeterol stimulation of parental MDA-MB-231 cells, MDA-MB-231-CXCR4 cells, and MDA-MB-231-CXCR4-ADRB2 cells also induced ERK activation after 20 minutes, which decayed to basal levels after 60 minutes (data not shown).

[0170] To determine the synergistic effect of CXCR4 and ADRB2 signaling, the level of ERK1 / 2 activation was measured after CXCL12 and salmeterol stimulation. MDA-MB-231 and A549 cells expressing Rluc-luc2P or CXCR4, or containing CXCR4-ADRB2 heteromers, were plated in 6-well plates at 5 × 10 cells per well. 5 Cells were seeded at a density of 1000 μg / ml. After 16 hours of serum starvation, cells were treated with 10 nM CXCL12 and / or 10 nM salmeterol for 20 minutes in MDA-MB-231 parental, MDA-MB-231-CXCR4, and MDA-MB-231-CXCR4-ADRB2 cells, and for 10 minutes in A549 cells expressing Rluc-luc2P ("A549 parental cells"), A549 cells expressing CXCR4 alone (either in the monomeric or individual protomer context; "A549-CXCR4 cells"), and an A549 cell line stably overexpressing both CXCR4 and ADRB2 to contain the CXCR4-ADRB2 heteromer ("A549-CXCR4-ADRB2 cells"). Each cell set was then harvested and subjected to Western blot analysis.

[0171] Stimulation with CXCL12 or salmeterol alone induced higher levels of ERK1 / 2 activation in MDA-MB-231-CXCR4 cells than in parental MDA-MB-231 cells. ERK1 / 2 phosphorylation was significantly increased in MDA-MB-231-CXCR4-ADRB2 cells compared with MDA-MB-231-CXCR4 cells. Simultaneous stimulation of MDA-MB-231 parental cells with CXCL12 and salmeterol induced ERK1 / 2 phosphorylation at levels similar to those stimulated with the single agonists. In MDA-MB-231-CXCR4 cells, ERK1 / 2 phosphorylation increased by the sum of the effects of each agonist alone, whereas in MDA-MB-231-CXCR4-ADRB2 cells, ERK1 / 2 activation was synergistically increased (Figure 16A-B).

[0172] ERK1 / 2 activation was also examined in A549 parental cells, A549-CXCR4 cells, and A549-CXCR4-ADRB2 cells. ERK1 / 2 phosphorylation was detected in A549-CXCR4 cells or A549-CXCR4-ADRB2 cells upon stimulation with CXCL12 or salmeterol alone. In A549 parental cells, ERK1 / 2 phosphorylation was not induced by stimulation with CXCL12 or salmeterol alone, but was significantly increased by costimulation with CXCL12 and salmeterol. In A549-CXCR4 cells, ERK1 / 2 phosphorylation was induced by each agonist alone, and costimulation with CXCL12 and salmeterol increased it to the sum of the two agonists. In A549-CXCR4-ADRB2 cells, ERK1 / 2 phosphorylation was significantly more induced by stimulation with salmeterol alone than by CXCL12 alone, and was significantly activated by co-stimulation with CXCL12 and salmeterol (Figures 17A-17B).

[0173] These results suggest that expression of CXCR4 and ADRB2 in cancer cells, especially when the cancer cells contain CXCR4-ADRB2 heteromers, can result in significant induction of downstream ERK activation and have profound effects on cancer cell proliferation and chemotaxis.

[0174] Example 13. Effect of CXCR4 antagonists on CXCR4 / CXCL12-mediated increased proliferation in A549 cell lines containing CXCR4-ADRB2 heteromers To investigate the effect of the presence of CXCR4-ADRB2 heteromers in cancer cells on cancer cell proliferation, the growth of A549 cells containing CXCR4-ADRB2 heteromers stably overexpressing both CXCR4 and ADRB2 ("A549-CXCR4-ADRB2 cells") was compared with that of A549 cells expressing Rluc-luc2P as a control (A549 double-negative cells; "A549 parental cells") and in response to the presence of a CXCL12 agonist.

[0175] 1 × 10 parental A549 cells or A549-CXCR4-ADRB2 cells 4Cells were plated into 96-well plates at a density of 100 cells / well and incubated in culture medium for 24 hours to allow attachment. After washing, serum-free medium ± CXCL12 was added under serum-free conditions with or without the indicated CXCR4 inhibitors (Figure 18A: AMD3100 (10 μM), Figure 18B: LY2510924 (10 μM), Figure 18C: AMD070 (1 μM), Figure 18D: TG-0054 (10 μM), and Figure 18E: BKT-140 (10 μM)). Cells were incubated for 72 hours (96 hours after seeding). At the end of the incubation, cell proliferation was assessed using Prestoblue Cell Viability Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0176] Figure 18A-E shows that the cell proliferation rate was observed to increase by approximately 20% in the presence of the CXCL12 agonist, and this increase was blocked by each of the CXCR4 antagonists tested. Under normal cell growth conditions (10% serum), no difference in cell proliferation rate was observed between A549 parental cells and A549-CXCR4-ADRB2 cells. However, under serum-free conditions, A549-CXCR4-ADRB2 cells (but not A549 parental cells) showed a dose-dependent increase in cell proliferation in the presence of CXCL12 (data not shown). A maximum increase of approximately 20% was achieved over 72 hours in the presence of 100 nM CXCL12. The CXCL12-mediated increase in cell proliferation attenuated as serum concentrations increased (data not shown). To confirm that the increase in cell proliferation rate was mediated by CXCR4 / CXCL12-specific signaling, several CXCR4-specific antagonists, AMD3100, LY210924, AMD070, TG-0054, and BKT-140, were evaluated. Each of the tested CXCR4 antagonists blocked the CXCL12 effect on cell proliferation. The CXCR4 / CXCL12 signaling axis increased cell proliferation rate by approximately 20% under serum-free conditions, consistent with previous reports that CXCR4 / CXCL12 induces increased proliferation only under suboptimal conditions (Balkwill, Fran (2004) Nature Reviews Cancer, Cancer and the Chemokine Network, 4:540-550).

[0177] Inhibitors targeting CXCR4 can only inhibit tumor growth to the same extent as increased by the presence of, for example, CXCL12, and the use of higher concentrations of CXCR4 inhibitors can result in cytotoxicity (data not shown). These results may explain the low efficacy of tumor growth inhibition and adverse effects observed in cancer treatment clinics associated with the use of CXCR4 inhibitors as the sole active agent. In subjects containing CXCR4-ADRB2 heteromers, treatment with a CXCR4 inhibitor alone cannot effectively inhibit tumor growth, whereas co-administration of a CXCR4 inhibitor and an ADRB2 inhibitor can effectively inhibit tumor growth.

[0178] Example 14. Correlation between CXCR4-ADRB2 heteromer abundance and tumor growth To investigate the correlation between the amount of CXCR4-ADRB2 heteromers and tumor growth, A549 cell lines stably overexpressing CXCR4 homomers ("A549-CXCR4 cells") and A549 cell lines stably overexpressing both CXCR4 and ADRB2 to contain CXCR4-ADRB2 heteromers ("A549-CXCR4-ADRB2 cells") were prepared. The amounts of CXCR4-ADRB2 heteromers were compared via PLA in the engineered cell lines. Figure 19A-B shows that there were approximately 30, approximately 80, and more than 150 CXCR4-ADRB2 heteromers detected in A549 parental cells, A549-CXCR4 cells, and A549-CXCR4-ADRB2 cells, respectively, as determined by PLA.

[0179] To determine whether the inclusion of CXCR4-ADRB2 heteromers would increase tumor growth in a dose-dependent manner, as determined by PLA at the cellular level, A549 parental cells and A549-CXCR4-ADRB2 cells were prepared and incubated with equal amounts of cells (1 × 10 7 The tumor growth rates were compared. Tumor growth was monitored every 3 or 4 days by measuring the length (L) and width (W) of the tumor and calculating the tumor volume based on the following formula: volume = 0.5LW 2 As shown in Figure 19C, the tumor size at day 44 after implantation was 562.8 ± 245.9 mm in A549 xenograft mice (with A549 parental cells). 3 , and 967.2 ± 493.0 mm in A549-CXCR4-CXCR4-ADRB2 xenograft mice (bearing A549-CXCR4-ADRB2 cells). 3 The tumor growth rate in mice implanted with A549-CXCR4-ADRB2 cells was faster than that in mice implanted with parental A549 cells. These results suggest that the formation of CXCR4-ADRB2 heteromers is related to Ca 2+This suggests that the two compounds synergistically enhance the response and therefore promote tumor growth.

[0180] The correlation between the presence and amount of CXCR4-ADRB2 heteromers and tumor growth was confirmed in the A549 lung cancer cell line and the MDA-MB-231 breast cancer cell line. Using the same method as in the A549 cell line, the MDA-MB-231-CXCR4 cell line overexpressing CXCR4 homomers ("MDA-MB-231-CXCR4 cells") and the MDA-MB-231 cell line containing CXCR4-ADRB2 heteromers by overexpressing both CXCR4 and ADRB2 ("MDA-MB-231-CXCR4-ADRB2 cells") were prepared, and the amounts of CXCR4-ADRB2 heteromer expression were then compared via PLA. As shown in Figure 20A-B, there are approximately 65, approximately 80, and more than 180 CXCR4-ADRB2 heteromers detected in MDA-MB-231 parental cells, MDA-MB-231-CXCR4 cells, and MDA-MB-231-CXCR4-ADRB2 cells, respectively. To determine whether the presence and amount of CXCR4-ADRB2 heteromers increases tumor growth in a dose-dependent manner as examined by PLA at the cellular level, MDA-MB-231 parental cells, MDA-MB-231-CXCR4 cells, and MDA-MB-231-CXCR4-ADRB2 cells were prepared and incubated in equal amounts (5 x 10 6 The tumor growth rates were compared by orthotopically injecting 1000 cells / mouse into the mammary fat pad of nude mice. As shown in Figure 20C, the tumor size at 67 days after implantation was 265.8 ± 161.8 mm in MDA-MB-231 xenograft mice (with MDA-MB-231 parental cells). 3 , and 459.2 ± 399.6 mm for MDA-MB-231-CXCR4 xenografts (containing MDA-MB-231-CXCR4 cells). 3 , and 1107.0 ± 184.8 mm in MDA-MB-231-CXCR4-ADRB2 xenograft mice (bearing MDA-MB-231-CXCR4-ADRB2 cells). 3Tumor size was observed in the following order: MDA-MB-231-CXCR4-ADRB2 xenograft mice, MDA-MB-231-CXCR4 xenograft mice, and MDA-MB-231 xenograft mice, indicating that tumor size increased according to the amount of CXCR4-ADRB2 heteromers present. These results suggest that as the amount of CXCR4-ADRB2 heteromers increases, tumors become more malignant. Therefore, cancers containing CXCR4-ADRB2 heteromers can be effectively treated by administering inhibitors targeting CXCR4-ADRB2 heteromers or a combination of inhibitors, such as a CXCR4 inhibitor and an ADRB2 inhibitor.

[0181] Example 15. Effect of CXCR4 inhibitors alone on tumor growth in A549 xenografted mice containing CXCR4-ADRB2 heteromers Antitumor activity was evaluated using investigational or commercially available CXCR4 inhibitors in mice implanted with A549 cells ("A549-CXCR4-ADRB2 cells"), which contain CXCR4-ADRB2 by stable overexpression of both CXCR4 and ADRB2. 7 The tumor size was approximately 50-100 mm. 3 Once the average size of the tumors reached 10 mm, mice (n = 10 / test group) were treated with CXCR4 inhibitors: AMD3100 (Figure 21A), LY2510924 (Figure 21B), AMD070 (Figure 21C), or TG-0054 (Figure 21D). The CXCR4 inhibitors were administered once daily for 4 weeks.

[0182] As shown in Figure 21A-D, most of the tested CXCR4 inhibitors showed limited inhibition of tumor growth. In addition, dose-dependent tumor reduction was not observed, and the inhibitory effect was slight. Furthermore, despite administration of higher than normal doses, tumor growth inhibition was limited. Treatment with LY2510924 at a high dose of 10 mpk resulted in the death of 3 of 10 tested mice after only 2 doses.

[0183] As described in Example 11 and Tables 3-4, co-treatment with a CXCR4 antagonist and an ADRB2 inhibitor was more effective than mono-treatment with a CXCR4 antagonist alone in cells containing CXCR4-ADRB2 heteromers (which exhibit increased Ca2+ signals). As described in cell growth assays (Example 13 and Figures 18A-18E), the CXCR4 inhibitor reduced the increase in cell proliferation resulting from CXCL12 stimulation. Increasing amounts of the CXCR4 inhibitor resulted in cytotoxicity. These results suggest that co-administration of a combination of a CXCR4 inhibitor and an ADRB2 inhibitor may be more effective than administration of a CXCR4 inhibitor alone in inhibiting the function of CXCR4-ADRB2 heteromers and inhibiting tumor growth.

[0184] Example 16. Effects of CXCR4 inhibitors and ADRB2 inhibitors, alone or in combination, on tumor growth in mice orthotopic xenografted with CXCR4-ADRB2 heteromer-containing MDA-MB-231 cells (or A549 cells). Orthotopic xenograft of CXCR4-ADRB2 heteromer-containing MDA-MB-231 cells in mice:

[0185] In mice implanted with MDA-MB-231 cells ("MDA-MB-231-CXCR4-ADRB2 cells"), which contain CXCR4-ADRB2 heteromers due to stable overexpression of both CXCR4 and ADRB2, administration of a CXCR4 inhibitor alone was not an effective antitumor treatment. As shown in Figures 22A-22C, the inhibition of tumor growth (antitumor effect) enhanced by the presence of CXCR4-ADRB2 heteromers was evaluated by comparing the administration of CXCR4 inhibitors (AMD3100 (Figure 22A), LY2510924 (Figure 22B), AMD070 (Figure 22C)) and an ADRB2 inhibitor (carvedilol) alone or in combination. Female Balb / c-nu / nu mice were orthotopically implanted with MDA-MB-231-CXCR4-ADRB2 cells (1 x 10 6(cells / animal). AMD3100 (2.5 mg / kg, 7.5 mg / kg), LY2510924 (1 mg / kg, 3 mg / kg), AMD070 (3 mg / kg, 10 mg / kg), and / or carvedilol (30 mg / kg) were administered once daily for 4 weeks (28 doses in total). AMD3100, LY2510924, and AMD070 were administered subcutaneously, and carvedilol was administered orally. Tumor size was calculated by converting the tumor size to 100 on the first day of drug administration (% tumor growth). As shown in Figure 22C, administration of the CXCR4 inhibitor AMD070 alone or the ADRB2 inhibitor carvedilol alone showed limited tumor growth inhibition. However, coadministration of AMD070 and carvedilol effectively inhibited tumor growth compared with the single administration of either drug, and when the combination (including carvedilol) was administered, tumor growth also decreased in a dose-dependent manner as the amount of the CXCR4 inhibitor AMD070 increased. These results suggest that inhibition of tumor size (anti-tumor effect) caused by CXCR4-ADRB2 heteromer formation can be achieved through coadministration of a CXCR4 inhibitor and an ADRB2 inhibitor.

[0186] CXCR4-ADRB2 heteromer-containing A549 cell xenograft mice:

[0187] To investigate the antitumor effect of CXCR4-ADRB2 heteromer inhibitors on tumor growth, A549 cells containing CXCR4-ADRB2 heteromers by stably overexpressing both CXCR4 and ADRB2 ("A549-CXCR4-ADRB2 cells") (1 × 10 7 The tumor size was 50-100 mm. 3 Once the mean age was reached, a CXCR4 inhibitor (AMD3100) or an ADRB2 inhibitor (carvedilol) was administered alone or in combination.

[0188] Figure 22D is a graph comparing tumor growth rates for the in vivo antitumor effect of the CXCR4 inhibitor AMD3100 (2.5 mg / kg, 7.5 mg / kg) and / or the ADRB2 inhibitor carvedilol (30 mg / kg) administered once daily for 4 weeks (28 doses total) via single or co-administration; AMD3100 was administered subcutaneously, and carvedilol was administered orally. Tumor growth was monitored every 3 or 4 days by measuring the length (L) and width (W) of the tumor and calculating the tumor volume based on the following formula: volume = 0.5LW 2 .

[0189] Examples 17A-17B. Detection of CXCR4-ADRB2 heteromers by ligand-assisted TR-FRET Detection of CXCR4-ADRB2 heteromers was assessed using time-resolved fluorescence energy transfer (TR-FRET). TR-FRET combines the low background aspect of time-resolved fluorescence analysis (TRF) with the homogeneous assay format of FRET. The resulting assay offers increased flexibility, reliability, and sensitivity. FRET requires two fluorophores, one donor and one acceptor. If the donor and acceptor are within a given proximity to each other, excitation of the donor by an energy source results in energy transfer to the acceptor. The acceptor then emits light at its characteristic wavelength. A549 cells were seeded into 96-well plates at a density of 20,000 cells per well. CXCR4 and ADRB2 were transiently overexpressed in A549 cells by adenoviral infection followed by 48 hours of incubation at 37°C and 5% CO2. CXCR4-expressing adenovirus was administered at MOIs of 0, 0.1, 0.5, 1.25, 2.5, 5, 10, and 20, while ADRB2-expressing adenovirus was administered at a threefold higher MOI. Adenovirus encoding HA-VC was used to adjust the total amount of transduced adenovirus. To characterize CXCR4-ADRB2 heteromers, a TR-FRET assay using labeled ligands was performed using fluorescently labeled propranolol and terbium-labeled TZ14011. Figure 23A shows that the FRET signal increased with the amount of CXCR4 and ADRB2 expression (affecting the amount of CXCR4-ADRB2 heteromers formed), and when unlabeled propranolol was used as a competitor to the ADRB2 antagonist, propranolol-g2, the FRET signal disappeared, indicating that this was a CXCR4-ADRB2-specific signal. These results demonstrate that CXCR4-ADRB2 heteromers can be quantitatively detected by the ligand-based TR-FRET method.

[0190] In addition to using ligand-fluorescent conjugates in which the fluor is conjugated to a GPCR-specific ligand, antibody-fluorescent conjugates in which the fluor is conjugated to a GPCR-specific antibody or secondary antibody may also be used for TR-FRET.

[0191] U2OS cells were seeded in 96-well plates at a density of 20,000 cells per well. Using adenovirus systems (CXCR4-bearing adenovirus at MOIs of 0.9–30 and ADRB2-bearing adenovirus at MOIs of 0.5–15), both CXCR4 and ADRB2 were transiently overexpressed after overnight incubation in a humidified 37°C incubator. To analyze the dynamic range and detection limit of antibody-mediated TR-FRET for CXCR4 and ADRB2, CXCR4-bearing and ADRB2-bearing adenoviruses (Ad-CXCR4 and Ad-ADRB2, respectively) were employed at a wide range of multiplicities of infection (MOIs). After 48 hours of infection with each adenovirus, cells were washed and fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing twice, cells were permeabilized with DPBS containing 0.1% Triton X-100 for 10 minutes at room temperature. The cells were then blocked for 30 minutes at room temperature and subsequently incubated with both rabbit anti-CXCR4 and mouse anti-ADRB2 antibodies for 3 hours at room temperature. After washing the cells four times with DPBS, they were treated with terbium cryptate-labeled goat anti-rabbit IgG and Alexa Fluor 647-labeled goat anti-mouse IgG for 1 hour at room temperature. The cells were then washed four times with DPBS and subsequently analyzed using a Varioskan LUX Multimode Microplate Reader.

[0192] The performance of antibody-mediated TR-FRET showed the highest signal at a ratio of 2 Ad-CXCR4:1 Ad-ADRB2 (Figure 23B). Therefore, Ad-CXCR4 and Ad-ADRB2 were serially diluted in half starting from 30 MOI and 15 MOI, respectively. HA-VC-carrying adenovirus was used as a negative control. FRET efficacy was successfully detected at 0.9 MOI of Ad-CXCR4 and 0.5 MOI of Ad-ADRB2, and a dose-dependent response was also observed. These results suggest that antibody-mediated TR-FRET may be a promising tool for diagnosing GPCR heteromers.

[0193] Examples 18A-18C. Detection of CXCR4-ADRB2 heteromers by PLA and quantification of CXCR4 or ADRB2 RNA expression levels by RT-qPCR in hematological cancer and solid tumor cell lines. The relationship between the amount of RNA produced by the individual GPCRs, CXCR4 and ADRB2, and the amount of CXCR4-ADRB2 heteromers detected in hematological cancer and solid tumor cell lines was examined. CXCR4 is overexpressed in a variety of human cancers, including hematological cancers such as myeloma and leukemia, and solid tumors such as lung and breast cancer. This overexpression correlates with an increased risk of recurrence and poor overall survival.

[0194] CXCR4 and ADRB2 RNA expression was observed in all three solid tumor cell lines examined by qPCR: A549 (lung cancer), U2OS (osteosarcoma), and MDA-MB-231 (breast cancer) (Figure 24A). To assess the endogenous expression levels of CXCR4-ADRB2 heteromers in A549, U2OS, and MDA-MB-231 cells, PLA was employed using two different primary antibodies for CXCR4 and ADRB2. The resulting rolling circle amplification (RCA) products were shown as red fluorescent signals, with widespread staining across the entire cell surface and covering the nucleus (stained with DAPI) and cytosol (Figure 24B). The average PLA signal for each cell was expressed as the RCA product divided by the number of cells. On average, each MDA-MB-231 cell had an RCA product value of approximately 60, followed by A549 cells with an RCA product value of approximately 35 and U2OS cells with an RCA product value of approximately 20 (Figure 24C). The RNA expression levels of CXCR4 and ADRB2 were highest in the MDA-MB-231 cell line compared with those of A549 and U2OS cells (CXCR4 / ADRB2 delta Ct: 7.4 / 8.8, 10.6 / 12.4, 12.8 / 10.5), and the PLA value was also highest in the MDA-MB-231 cell line compared with those of A549 and U2OS cells. It can be seen that there is a significant correlation between RNA expression and PLA value. These results indicate that patients with CXCR4-ADRB2 heteromers can be screened by analyzing PLA and RNA expression levels.

[0195] CXCR4 and ADRB2 RNA expression was observed in all three hematological cancer cell lines examined by qPCR: HL60 (leukemia), U937 (leukemia), and RPMI 8226 (myeloma) (Figure 25A). To assess the endogenous expression levels of CXCR4-ADRB2 heteromers in HL-60, U937, and RPMI 8226 cells, PLA was performed using two different primary antibodies for CXCR4 and ADRB2. The resulting rolling circle amplification (RCA) products were displayed as red fluorescent signals staining the entire cell surface (Figure 25B). The average PLA signal for each cell was expressed as the RCA product divided by the cell number. On average, each HL-60 cell had an RCA product value of 30, followed by both U937 and RPMI 8226 cells, which were approximately half the value of HL-60 (Figure 25C). Comparing the RNA expression levels of CXCR4 and ADRB2 by quantitative PCR, we found that ADRB2 expression was similar in these three cell lines (delta Ct: 9), whereas the CXCR4 expression level in HL60 cells was twice that of the other cell lines, U937 and RPMI 8226 (delta Ct values: 4.7, 5.9, and 5.8). These differences showed a similar pattern in the PLA values, indicating a significant correlation between the RNA expression level and the PLA values. These results demonstrate that the PLA signal of suspension cells can be successfully detected, and that HL-60 cells have twice as many CXCR4-ADRB2 heteromers as U937 and RPMI 8226 cells.

[0196] Diagnosis of CXCR4 heteromers is essential for screening subjects expressing CXCR4 heteromers and providing drugs selected according to the type of partner GPCR. PLA, a method for diagnosing CXCR4 heteromers already established in our laboratory, is a method for diagnosing tissue extracted from solid cancer subjects using antibodies in formalin-fixed, paraffin-embedded (FFPE) samples. These results provide the possibility of detecting CXCR4 heteromers in liquid biopsies, such as blood cancer samples, urine, and sediments.

[0197] Example 19. Enhanced CXCR4 downstream signaling in native cells endogenously expressing CXCR4 and ADRB2 protomers assessed by Ca2+ mobilization assay The properties of CXCR4-GPCRx heteromers, such as CXCR4-ADRB2 heteromers, were evaluated by calcium mobilization assays in U937 cells (Figure 26A) and HL-60 cells (Figure 26B), which endogenously express both CXCR4 and ADRB2, respectively, by measuring calcium signaling upon stimulation or costimulation with one or both of their respective agonists. Calcium mobilization was measured using a FlexStation 3.

[0198] In both U937 cells (FIG. 26A) and HL-60 cells (FIG. 26B), stimulation with the CXCR4 agonist CXCL12 alone (at 200 nM) induced intracellular calcium mobilization, whereas stimulation with the ADRB2 agonist formoterol alone (10 μM) did not induce a calcium response. However, costimulation with both agonists (CXCL12 and formoterol, 200 nM and 10 μM, respectively) resulted in a significant increase in calcium response in both U937 cells (FIG. 26A) and HL-60 cells (FIG. 26B) compared with the response induced by single agonist stimulation (an approximately 4-fold increase in calcium response in U937 cells compared with single stimulation with CXCL12, and an approximately 8-fold increase in calcium response in HL-60 cells compared with single stimulation with CXCL12).

[0199] Example 20. Ca2+ Mobilization Inhibition of CXCR4 Downstream Signaling Enhanced upon CXCR4-ADRB2 Heteromer Formation in U937 and HL-60 Cell Lines - Comparison of Combination Versus Single Inhibitor Treatment The efficacy of CXCR4 inhibitors in suppressing the increased calcium response of CXCR4-ADRB2 heteromers in both U937 and HL-60 cells (Example 19) was measured in the presence or absence of carvedilol, a representative ADRB2 inhibitor. As discussed in Example 19, increased calcium response of CXCR4-ADRB2 heteromers was induced by the agonists CXCL12 (200 nM) and formoterol (10 μM). CXCR4 inhibitors (in the presence or absence of carvedilol, an ADRB2 inhibitor) were added 2 hours before treating the cells with the agonists. Calcium mobilization was measured using a FlexStation 3, and the IC50 of the resulting Ca2+ response was calculated using GraphPad Prism software. The data are shown in Table 5. [Table 5]

[0200] As shown in Table 5, the IC values ​​of each CXCR4 inhibitor in U937 cells were 50 As shown by the decreased IC50 values, the CXCR4 inhibitors AMD3100, urocupulumab, and TG-0054 inhibited CXCR4-ADRB2 signaling more efficiently in the presence of 10 μM carvedilol. Notably, the IC50 value of TG-0054 was significantly decreased in the presence of carvedilol. Cotreatment of carvedilol with AMD3100, urocupulumab, or TG-0054, respectively, reduced the IC50 values ​​of the Ca2+ response by approximately 6.4-fold (from 3.34 nM to 0.52 nM), approximately 4.8-fold (from 1.25 nM to 0.26 nM), and approximately 69-fold (from 24 nM to 0.35 nM).

[0201] As shown in Table 5, in HL-60 cells, each of the CXCR4 inhibitors more effectively suppressed CXCR4-ADRB2 signaling in the presence of 10 μM carvedilol. Upon cotreatment of carvedilol with AMD3100, urocupulumab, AMD070, and TG-0054, respectively, the IC50 values ​​of the Ca2+ response decreased by approximately 4.4-fold (from 1.50 nM to 0.34 nM), approximately 3.3-fold (from 0.02 nM to 0.006 nM), approximately 12.7-fold (from 3.56 nM to 0.28 nM), and approximately 12.5-fold (from 4 nM to 0.32 nM).

[0202] These results suggest that in cells containing CXCR4-ADRB2 heteromers, co-treatment with a CXCR4 inhibitor and an ADRB2 inhibitor simultaneously inhibits the increased Ca2+ response more effectively than mono-treatment with a CXCR4 inhibitor alone.

[0203] Example 21. Inhibition of Ca2+ mobilization of CXCR4 downstream signaling enhanced upon CXCR4-ADRB2 heteromer formation in MDA-MB-231 cell line - Comparison of combined inhibitor treatments versus single inhibitor treatments The efficacy of ADRB2 inhibitors in suppressing the calcium response of ADRB2 was measured in MDA-MB-231 cells transduced with adenovirus encoding ADRB2 alone, and signaling was measured by stimulating the cells with salmeterol (1 μM). The data are shown in Table 6. Additionally, the efficacy of ADRB2 inhibitors in suppressing the calcium response (enhanced signaling) of CXCR4-ADRB2 heteromers, which is enhanced in MDA-MB-231 cells co-transduced with adenovirus encoding CXCR4 and ADRB2, was measured in the presence or absence of AMD3100, a representative CXCR4 inhibitor. Ca2+ flux was measured by costimulating MDA-MB-231 cells with CXCL12 (20 nM) and salmeterol (1 μM), and the ADRB2 inhibitor (with or without the CXCR4 inhibitor AMD3100) was added 2 hours before treating the cells with the agonist. Calcium mobilization was measured using FlexStation3 and IC50 values ​​of the resulting calcium responses were calculated using GraphPad Prism software. The data are shown in Table 6. [Table 6]

[0204] As shown in Table 6, in MDA-MB-231 cells, single treatment with bupranolol inhibited ADRB2 signaling with an IC50 value of 0.82 nM, and single treatment with labetalol inhibited ADRB2 signaling with an IC50 value of 24.81 nM. The ADRB2 inhibitors bupranolol or labetalol inhibited CXCR4-ADRB2 signaling more efficiently and with higher potency in the presence of AMD3100 at 650 nM (IC90), as indicated by the decreased IC50 values, compared to the IC50 values ​​of the ADRB2 inhibitors in the absence of AMD3100. The IC50 value of bupranolol was reduced approximately 33-fold in combination with AMD3100 compared to single treatment (from 3.12 nM to 0.10 nM), while the IC50 of labetalol was reduced approximately 107-fold in combination with AMD3100 compared to single treatment (from 57.98 nM to 0.54 nM). These results suggest that in cells containing CXCR4-ADRB2 heteromers, co-treatment with a CXCR4 inhibitor and an ADRB2 inhibitor simultaneously inhibits the increased Ca2+ response more effectively than single treatment with an ADRB2 inhibitor alone.

[0205] Example 22. Ca2+ Mobilization Inhibition of CXCR4 Downstream Signaling Enhanced upon CXCR4-ADRB2 Heteromer Formation in MDA-MB-231 Cell Line - Comparison of Combined Inhibitor Treatments with TG-0054 Single Treatment The efficacy of the CXCR4 inhibitor TG-0054 in suppressing CXCR4 responses was measured in MDA-MB-231 cells transduced with adenovirus encoding CXCR4 alone, and signaling was measured by stimulating the cells with CXCL12 (20 nM). In MDA-MB-231 cells (expressing CXCR4), TG-0054 was determined to have an IC value (nM) of 65.78 ± 1.34. The efficacy of TG-0054 in suppressing the enhanced signaling of CXCR4-ADRB2 heteromers was then measured in MDA-MB-231 cells cotransduced with adenovirus encoding CXCR4 and ADRB2, in the presence or absence of an ADRB2 inhibitor (10 μM). The data are shown in Table 7. Ca2+ flux was measured by costimulating MDA-MB-231 cells with CXCL12 (20 nM) and salmeterol (1 μM), and the CXCR4 inhibitor TG-0054 (with or without an ADRB2 inhibitor) was added 2 hours before treating the cells with agonists. Calcium mobilization was measured using FlexStation 3, and IC50 values ​​of the resulting calcium responses were calculated using GraphPad Prism software. The data are shown in Table 7. [Table 7]

[0206] As described above, in MDA-MB-231 cells, single treatment with TG-0054 inhibited CXCR4 signaling with an IC50 value of 65.78 nM in MDA-MB-231 cells transduced with CXCR4 alone. As shown in Table 7, TG-0054 inhibited CXCR4-ADRB2 signaling more efficiently and with higher potency in the presence of an ADRB2 inhibitor than single treatment in the absence of an ADRB2 inhibitor, as indicated by the reduced IC50 value. The IC50 value of TG-0054 decreased approximately 4645-fold when combined with carvedilol (from 92.89 nM to 0.02 nM), approximately 3573-fold when combined with labetalol (from 92.89 nM to 0.026 nM), and approximately 10-fold or more when combined with any one of the ADRB2 inhibitors alprenolol, carazolol, propafenone, or timolol. These results suggest that in cells containing CXCR4-ADRB2 heteromers, simultaneous co-treatment with TG-0054 and an ADRB2 inhibitor inhibited the increased Ca2+ response more effectively than mono-treatment with TG-0054 alone.

[0207] Illustrative Embodiments Embodiment A1. A method of inhibiting enhanced downstream signaling due to CXCR4-ADRB2 heteromers in cells of a subject suffering from cancer, comprising administering to the subject: a) a CXCR4 inhibitor that is blixafor; and b) ADRB2 inhibitors administering At that time, i) the enhanced downstream signaling is due to the CXCR4-ADRB2 heteromer; and ii) the method, wherein the administered blixafor and ADRB2 inhibitor suppress downstream signaling enhanced from the CXCR4-ADRB2 heteromer in the cancer subject.

[0208] Embodiment A2. A method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising administering to the subject: a) a CXCR4 inhibitor that is blixafor; and b) ADRB2 inhibitors administering At that time, i) enhanced downstream signaling results from the CXCR4-ADRB2 heteromer; and ii) the method, wherein the administered blixafor and ADRB2 inhibitor suppress downstream signaling enhanced from the CXCR4-ADRB2 heteromer in the cancer subject.

[0209] Embodiment A3. A method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising: a) determining whether the subject's cells contain CXCR4-ADRB2 heteromers and, if so, whether enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromers; and b) if the subject's cells contain the CXCR4-ADRB2 heteromer, administering to the cancer subject: i) a CXCR4 inhibitor that is blixafor; and ii) ADRB2 inhibitors The method comprises administering

[0210] Embodiment A4. A method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to said CXCR4-ADRB2 heteromers, comprising: 1) obtaining or obtaining a biological sample from the subject to determine whether the subject has cells containing the CXCR4-ADRB2 heteromer; and i) whether the subject's cells contain the CXCR4-ADRB2 heteromer; or ii) whether the combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; alters the heteromer-specific properties of a cell(s) derived from the subject containing the CXCR4-ADRB2 heteromer; or reduces the progression of cancer in the subject having cells containing the CXCR4-ADRB2 heteromer. performing or having performed an assay on the biological sample to determine 2) administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, if the subject has cells containing the CXCR4-ADRB2 heteromer, wherein the CXCR4 inhibitor is blixafor; and 3) administering to the cancer subject a single inhibitor, either the blixafor or the ADRB2 inhibitor, if the subject does not have cells containing the CXCR4-ADRB2 heteromer.

[0211] Embodiment A5. A method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to said CXCR4-ADRB2 heteromers, comprising: 1) obtaining or obtaining a biological sample from the subject to determine whether the subject has cells containing the CXCR4-ADRB2 heteromer; and i) whether the subject's cells contain the CXCR4-ADRB2 heteromer; or ii) whether the combination of a CXCR4 inhibitor and an ADRB2 inhibitor: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; alters the heteromer-specific properties of a cell(s) derived from the subject containing the CXCR4-ADRB2 heteromer; or reduces the progression of cancer in the subject having cells containing the CXCR4-ADRB2 heteromer. performing or having performed an assay on the biological sample to determine 2) administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor, if the subject has cells containing the CXCR4-ADRB2 heteromer, wherein the CXCR4 inhibitor is blixafor; and 3) administering to the cancer subject a single inhibitor of either blixafor or the ADRB2 inhibitor, if the subject does not have cells containing the CXCR4-ADRB2 heteromer, wherein: a) the progression of the cancer in the subject having the cells containing the CXCR4-ADRB2 heteromer is reduced by 5% to 100% or more upon administration of the combination of blixafor and the ADRB2 inhibitor to the cancer subject compared to administration of either the blixafor or the ADRB2 inhibitor alone; b) the efficacy of the blixafor, when administered in combination with the ADRB2 inhibitor to the subject having cells containing the CXCR4-ADRB2 heteromer, is increased by 5% to 2000% compared to the efficacy of the blixafor when administered as a single inhibitor; and / or c) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to the subject having cells containing the CXCR4-ADRB2 heteromer, is increased by 5% to 2000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor.

[0212] Embodiment A6. A method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to said CXCR4-ADRB2 heteromers, comprising: 1) determining whether cells of the subject contain the CXCR4-ADRB2 heteromer by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromer is present in cells of the subject; wherein the assay performed on the biological sample is or includes one or more of a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, proximity-based assay, co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNA sequencing, RT-qPCR, CXCR4 expression level, ADRB2 expression level, CXCR4 and ADRB2 expression level, microarray, or fluorescent animal assay; 2) administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor if the subject's cells contain the CXCR4-ADRB2 heteromer, wherein the CXCR4 inhibitor is blixafor; and 3) administering to the cancer subject a single inhibitor, either the blixafor or the ADRB2 inhibitor, if the subject's cells do not contain the CXCR4-ADRB2 heteromer.

[0213] Embodiment A7. A method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, wherein enhanced downstream signaling is attributable to said CXCR4-ADRB2 heteromers, comprising: 1) determining whether cells of the subject contain the CXCR4-ADRB2 heteromer by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine whether the CXCR4-ADRB2 heteromer is present in cells of the subject; wherein the assay performed on the biological sample is or includes one or more of a co-internalization assay, a co-localization assay, in situ hybridization, immunohistochemistry, immunoelectron microscopy, proximity-based assay, co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, RNA sequencing, RT-qPCR, CXCR4 expression level, ADRB2 expression level, CXCR4 and ADRB2 expression level, microarray, or fluorescent animal assay; 2) administering to the cancer subject a combination of a CXCR4 inhibitor and an ADRB2 inhibitor if the subject's cells contain the CXCR4-ADRB2 heteromer, wherein the CXCR4 inhibitor is blixafor; and 3) if the subject's cells do not contain the CXCR4-ADRB2 heteromer, administering to the cancer subject a single inhibitor of either the blixafor or the ADRB2 inhibitor, wherein: a) the progression of the cancer in the subject having the cells containing the CXCR4-ADRB2 heteromer is reduced by 5% to 100% or more upon administration of the combination of blixafor and the ADRB2 inhibitor to the cancer subject compared to administration of either the blixafor or the ADRB2 inhibitor alone; b) the efficacy of the blixafor, when administered in combination with the ADRB2 inhibitor to the subject having cells containing the CXCR4-ADRB2 heteromer, is increased by 5% to 2000% compared to the efficacy of the blixafor when administered as a single inhibitor; and / or c) the efficacy of the ADRB2 inhibitor, when administered in combination with blixafor to the subject having cells containing the CXCR4-ADRB2 heteromer, is increased by 5% to 2000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor.

[0214] Embodiment A8. A pharmaceutical kit for use in treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising: a) a CXCR4 inhibitor that is blixafor; and b) contains an ADRB2 inhibitor; In this case, the enhanced downstream signaling is due to the CXCR4-ADRB2 heteromer.

[0215] Embodiment A9. A pharmaceutical composition for use in treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising: a) blixafor, a CXCR4 inhibitor; b) an ADRB2 inhibitor; and c) containing a pharmaceutically acceptable carrier; wherein the enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromer.

[0216] Embodiment A10. A method of inhibiting enhanced downstream signaling due to CXCR4-ADRB2 heteromers in a cell, comprising administering to the cell: a) a CXCR4 inhibitor that is blixafor; and b) ADRB2 inhibitors administering At that time, i) the enhanced downstream signaling is due to the CXCR4-ADRB2 heteromer; and ii) the method, wherein contacting the blixafor and the ADRB2 inhibitor suppresses downstream signaling enhanced from the CXCR4-ADRB2 heteromer in the cell.

[0217] Embodiment A11. The method of embodiment A10, further comprising determining whether the cells contain the CXCR4-ADRB2 heteromer.

[0218] Embodiment A12. A pharmaceutical kit for use in inhibiting enhanced downstream signaling due to CXCR4-ADRB2 heteromers in a cell, comprising: a) a CXCR4 inhibitor that is blixafor; and b) ADRB2 inhibitors Includes; In this case, the enhanced downstream signaling is due to the CXCR4-ADRB2 heteromer.

[0219] Embodiment A13. A pharmaceutical composition for use in inhibiting enhanced downstream signaling due to CXCR4-ADRB2 heteromers in a cell, comprising: a) blixafor, a CXCR4 inhibitor; b) an ADRB2 inhibitor; and c) a pharmaceutically acceptable carrier Includes; wherein the enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromer.

[0220] Embodiment A14. The method of any one of embodiments A10 to A13, wherein the cell is a cell of interest.

[0221] Embodiment A15. The method of embodiment A14, wherein the cells of the subject are cancer cells.

[0222] Embodiment A16. The method of any one of Embodiments A1 to A13, wherein the cells are cancer cells.

[0223] Embodiment A17. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9, wherein the method further comprises detecting the presence of the CXCR4-ADRB2 heteromer in the cancer subject.

[0224] Embodiment A18. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17, wherein the method further comprises identifying the CXCR4-ADRB2 heteromer in the cancer subject.

[0225] Embodiment A19. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A18, wherein the method further comprises obtaining a biological sample from the subject.

[0226] Embodiment A20. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A19, wherein the method further comprises performing an assay on the biological sample obtained from the subject.

[0227] Embodiment A21. The method comprises: i) obtaining or having obtained a biological sample from said cancer subject; ii) performing or having performed a diagnostic assay to determine the presence, identity, or presence and identity of CXCR4-ADRB2 heteromers in the biological sample obtained from the cancer subject; and iii) selecting an ADRB2 inhibitor for administration in combination with blixafor to suppress enhanced downstream signaling resulting from the CXCR4-ADRB2 heteromer; The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A17 to A20, further comprising:

[0228] Embodiment A22. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A21, further comprising determining whether cells of the subject contain the CXCR4-ADRB2 heteromer, wherein the determining comprises performing an assay on a biological sample obtained from the subject.

[0229] Embodiment A23. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A22, further comprising determining whether cells of the subject contain the CXCR4-ADRB2 heteromer, wherein the determining comprises obtaining a biological sample from the subject and performing an assay on the biological sample obtained from the subject.

[0230] Embodiment A24. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A9 or A17-A23, wherein the biological sample obtained from the subject contains the CXCR4-ADRB2 heteromer.

[0231] Embodiment A25. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A14-A24, wherein cells of the subject contain the CXCR4-ADRB2 heteromer.

[0232] Embodiment A26. The CXCR4-ADRB2 heteromer has the following characteristics: 1) the CXCR4-ADRB2 heteromeric components within the cell colocalize and physically interact, either directly or through an intermediate protein that acts as a vehicle for allosterism; 2) enhanced downstream signaling results from the CXCR4-ADRB2 heteromer; and / or 3) The combination of blixafor and an ADRB2 inhibitor: i) altering the heteromer-specific properties of the CXCR4-ADRB2 heteromer in the cell; ii) altering the heteromer-specific function of the CXCR4-ADRB2 heteromer in the cell; and / or iii) altering the heteromer-specific properties of the cells containing the CXCR4-ADRB2 heteromers. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use according to any one of embodiments A1 to A25, comprising two or more of:

[0233] Embodiment A27. The CXCR4-ADRB2 heteromer has the following characteristics: 1) the CXCR4-ADRB2 heteromeric components within the cell colocalize and physically interact, either directly or through an intermediate protein that acts as a vehicle for allosterism; 2) enhanced downstream signaling results from the CXCR4-ADRB2 heteromer; and / or 3) The combination of blixafor and an ADRB2 inhibitor: i) altering the heteromer-specific properties of the CXCR4-ADRB2 heteromers in a cell(s) derived from the subject; ii) altering a heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; iii) altering the heteromer-specific properties of the cell(s) derived from the subject that contain the CXCR4-ADRB2 heteromer; and / or iv) reducing cancer progression in said subject having cells containing said CXCR4-ADRB2 heteromers. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A14 to A25, comprising having two or more of:

[0234] Embodiment A28. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of any one of Embodiments A1 to A27, wherein the CXCR4-ADRB2 heteromer is characterized by the following characteristic: the CXCR4-ADRB2 heteromeric components within the cell are colocalized and physically interact with each other, either directly or through an intermediate protein that acts as a vehicle for allosterism.

[0235] Embodiment A29. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A28, wherein the CXCR4-ADRB2 heteromeric components within the cell co-localize and physically interact directly or through an intermediate protein that acts as a vehicle for allosterism, as determined by one or more of co-internalization assays, co-localization assays, in situ hybridization, immunohistochemistry, immunoelectron microscopy, proximity-based assays, co-immunoprecipitation assays, enzyme-linked immunosorbent assays (ELISAs), flow cytometry, RNAseq, RT-qPCR, CXCR4 expression levels, ADRB2 expression levels, CXCR4 and ADRB2 expression levels, microarrays, or fluorescent animal assays.

[0236] Embodiment A30. The method of inhibition, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A29, wherein the proximity-based assay is or comprises resonance energy transfer (RET), bioluminescence RET (BRET), fluorescence RET (FRET), time-resolved fluorescence RET (TR-FRET), antibody-assisted FRET, ligand-assisted FRET, bimolecular fluorescence complementation (BiFC), or proximity ligation assay (PLA).

[0237] Embodiment A31. The method of inhibition, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A30, wherein the TR-FRET is ligand-assisted TR-FRET.

[0238] Embodiment A32. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A30, wherein the TR-FRET is antibody-mediated TR-FRET.

[0239] Embodiment A33. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A30, wherein the CXCR4-ADRB2 heteromeric components within the cell co-localize and physically interact directly or through an intermediate protein that acts as a vehicle for allosterism, as determined by one or more of a co-internalization assay, bimolecular fluorescence complementation (BiFC), RT-PCR, RT-qPCR, CXCR4 expression level, ADRB2 expression level, CXCR4 and ADRB2 expression level, or proximity ligation assay (PLA).

[0240] Embodiment A34. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of embodiment A33, wherein the CXCR4-ADRB2 heteromeric components within the cell co-localize and physically interact, either directly or through an intermediate protein that acts as a vehicle for allosterism, as determined by a co-internalization assay.

[0241] Embodiment A35. The method of inhibition, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A33, wherein the CXCR4-ADRB2 heteromeric components in the cell co-localize and physically interact, either directly or through an intermediate protein that acts as a vehicle for allosterism, as determined by bimolecular fluorescence complementation (BiFC).

[0242] Embodiment A36. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of embodiment A33, wherein the CXCR4-ADRB2 heteromeric components in the cell co-localize and physically interact directly or through an intermediate protein that acts as a vehicle for allosterism, as determined by proximity ligation assay (PLA).

[0243] Embodiment A37. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A17 to A36, wherein the assay determines the presence of the CXCR4-ADRB2 heteromer in the biological sample obtained from the subject.

[0244] Embodiment A38. The method of inhibition, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A37, wherein the assay determines colocalization and interaction of the CXCR4 and ADRB2 components of the CXCR4-ADRB2 heteromer.

[0245] Embodiment A39. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A14-A38, wherein the assay determines the presence of the CXCR4-ADRB2 heteromer in cells of the subject.

[0246] Embodiment A40. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A39, wherein the assay determines the presence of the CXCR4-ADRB2 heteromer in the biological sample obtained from the subject.

[0247] Embodiment A41. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A40, wherein the assay determines the presence of the CXCR4-ADRB2 heteromer in the subject.

[0248] Embodiment A42. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A41, wherein the CXCR4-ADRB2 heteromer is characterized by the following characteristic: enhanced downstream signaling is attributable to the CXCR4-ADRB2 heteromer.

[0249] Embodiment A43. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A14-A42, wherein the enhanced downstream signaling is due to the CXCR4-ADRB2 heteromers in cells of the subject.

[0250] Embodiment A44. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A14-A43, wherein the enhanced downstream signaling results from the presence of the CXCR4-ADRB2 heteromer in cells of the subject.

[0251] Embodiment A45. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A44, wherein the CXCR4-ADRB2 heteromers result in said enhanced downstream signaling.

[0252] Embodiment A46. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A14-A45, wherein the CXCR4-ADRB2 heteromers result in said enhanced downstream signaling in cells of said subject.

[0253] Embodiment A47. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A46, wherein the enhanced downstream signaling results from agonism of the CXCR4-ADRB2 heteromer.

[0254] Embodiment A48. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A47, wherein the enhanced downstream signaling results from CXCR4 agonism of the CXCR4-ADRB2 heteromer.

[0255] Embodiment A49. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A47, wherein the enhanced downstream signaling results from ADRB2 agonism of the CXCR4-ADRB2 heteromer.

[0256] Embodiment A50. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A47, wherein the enhanced downstream signaling results from CXCR4 agonism and ADRB2 agonism of the CXCR4-ADRB2 heteromer.

[0257] Embodiment A51. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A50, wherein the enhanced downstream signaling is downstream of the CXCR4, the ADRB2, or the CXCR4-ADRB2 heteromer.

[0258] Embodiment A52. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A51, wherein the enhanced downstream signaling is downstream of CXCR4.

[0259] Embodiment A53. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A51, wherein the enhanced downstream signaling is downstream of ADRB2.

[0260] Embodiment A54. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A51, wherein the enhanced downstream signaling is downstream of the CXCR4-ADRB2 heteromer.

[0261] Embodiment A55. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A50, wherein the enhanced downstream signaling from the CXCR4-ADRB2 heteromer is compared to downstream signaling from CXCR4 protomers or ADRB2 protomers in the context of each individual protomer.

[0262] Embodiment A56. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of embodiment A55, wherein the enhanced downstream signaling from the CXCR4-ADRB2 heteromer is compared to downstream signaling from CXCR4 protomers in the context of the individual protomers.

[0263] Embodiment A57. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A55, wherein the enhanced downstream signaling from the CXCR4-ADRB2 heteromer is compared to downstream signaling from ADRB2 protomers in the context of the individual protomers.

[0264] Embodiment A58. The method of inhibiting, the method of treatment, the pharmaceutical kit for use, or the pharmaceutical composition for use of embodiment A55, wherein the enhanced downstream signaling from the CXCR4-ADRB2 heteromers is compared to downstream signaling from CXCR4 protomers and ADRB2 protomers in the context of each individual protomer.

[0265] Embodiment A59. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A58, wherein the enhanced downstream signaling is enhanced calcium mobilization.

[0266] Embodiment A60. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A59, wherein the amount of enhanced calcium mobilization is determined by an intracellular Ca2+ assay.

[0267] Embodiment A61. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A60, wherein enhanced downstream signaling from the CXCR4-ADRB2 heteromers is determined by an intracellular Ca2+ assay.

[0268] Embodiment A62. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A61, wherein the intracellular Ca2+ assay is a calcium mobilization assay.

[0269] Embodiment A63. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A62, wherein the calcium mobilization assay determines that the CXCR4-ADRB2 heteromer results in said enhanced downstream signaling.

[0270] Embodiment A64. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A62 or A63, wherein the calcium mobilization assay determines that the enhanced downstream signaling is due to the presence of the CXCR4-ADRB2 heteromer.

[0271] Embodiment A65. The CXCR4-ADRB2 heteromer, as determined by a calcium mobilization assay: a) upon costimulation with CXCL12 and an ADRB2 agonist, either the CXCR4 or the ADRB2 in the context of their individual protomers in the cell results in calcium mobilization that is equal to or less than the sum of the calcium mobilization that results from single agonist stimulation with either the CXCL12 or the ADRB2 agonist; and b) The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A64, wherein upon costimulation with the CXCL12 and the ADRB2 agonist, the CXCR4-ADRB2 heteromer exhibits enhanced calcium mobilization compared to the sum of the calcium mobilization resulting from single agonist stimulation with either the CXCL12 or the ADRB2 agonist.

[0272] Embodiment A66. i) the calcium mobilization from the protomer CXCR4 or ADRB2 in the context of the individual protomers in the cell is non-synergistic, as determined by a calcium mobilization assay; and ii) the calcium mobilization from the CXCR4-ADRB2 heteromer in the cell is synergistic as determined by a calcium mobilization assay; A method of suppression, a method of treatment, a pharmaceutical kit for use, or a pharmaceutical composition for use according to any one of embodiments A1 to A65.

[0273] Embodiment A67. In the context of said individual protomers, as determined by a calcium mobilization assay: a) the individual protomer CXCR4 in the cell in the absence of the individual protomer ADRB2; or b) said individual protomer ADRB2 in said cells in the absence of said individual protomer CXCR4; The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A66, wherein upon co-stimulation with CXCL12 and an ADRB2 agonist, results in an amount of calcium mobilization that is equal to or less than the sum of the amount of calcium mobilization resulting from single agonist stimulation with either the CXCL12 or the ADRB2 agonist.

[0274] Embodiment A68. Independently in the context of each said protomer, as determined by a calcium mobilization assay: a) the individual protomer CXCR4 in the cell in the absence of the individual protomer ADRB2, and b) said individual protomer ADRB2 in said cells in the absence of said individual protomer CXCR4; The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A67, wherein upon co-stimulation with CXCL12 and an ADRB2 agonist, results in an amount of calcium mobilization that is equal to or less than the sum of the amount of calcium mobilization resulting from single agonist stimulation with either the CXCL12 or the ADRB2 agonist.

[0275] Embodiment A69. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of any one of Embodiments A1-A68, wherein the CXCR4-ADRB2 heteromer, upon costimulation with the CXCL12 and the ADRB2 agonist, results in an amount of calcium mobilization that is greater than the sum of the amounts of calcium mobilization that result from single-agonist stimulation of the cells with either the CXCL12 or the ADRB2 agonist, as determined by a calcium mobilization assay.

[0276] Embodiment A70. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of any one of Embodiments A1 to A69, wherein the amount of calcium mobilization resulting from costimulation of the CXCR4-ADRB2 heteromers is enhanced compared to the sum of calcium mobilization resulting from single agonist stimulation of the CXCR4-ADRB2 heteromers, as determined by a calcium mobilization assay.

[0277] Embodiment A71. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of any one of Embodiments A1-A70, wherein the enhanced calcium mobilization caused by the CXCR4-ADRB2 heteromers upon costimulation with CXCL12 and an ADRB2 agonist is greater than the sum of the calcium mobilization caused by monoagonist stimulation of the cells with either the CXCL12 or the ADRB2 agonist, as determined by a calcium mobilization assay.

[0278] Embodiment A72. The method of inhibiting, treating, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A71, wherein the enhanced calcium mobilization caused by the CXCR4-ADRB2 heteromer, upon costimulation with the CXCL12 and the ADRB2 agonist, is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, or at least 90% greater than the sum of the calcium mobilization caused by single-agonist stimulation of the cells with either the CXCL12 or the ADRB2 agonist, as determined by a calcium mobilization assay.

[0279] Embodiment A73. The enhanced calcium mobilization caused by the CXCR4-ADRB2 heteromer is greater than or equal to the CXCL12 agonist upon costimulation with the CXCL12 and the ADRB2 agonist, as determined by a calcium mobilization assay. Agonist or the amount of calcium mobilization resulting from single agonist stimulation of said cells with any of said ADRB2 agonists is at least 100% greater than the sum of the amounts of calcium mobilization resulting from single agonist stimulation of said cells with any of said ADRB2 agonists. thing.

[0280] Embodiment A74. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A71 to A73, wherein the amount of enhanced calcium mobilization is an amount of synergistic calcium mobilization.

[0281] Embodiment A75. The method of inhibiting, the method of treating, the pharmaceutical kit for use, or the pharmaceutical composition for use of embodiment A74, wherein the amount of synergistic calcium mobilization from the cells containing the CXCR4-ADRB2 heteromers upon costimulation with the CXCL12 and the ADRB2 agonist is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, or at least 90% greater than the sum of the amounts of calcium mobilization resulting from single-agonist stimulation of the cells with either the CXCL12 or the ADRB2 agonist, as determined by a calcium mobilization assay.

[0282] Embodiment A76. The amount of calcium mobilization that is enhanced, as determined by a calcium mobilization assay, is: a) either the CXCR4 or the ADRB2 in the context of their respective protomers in a cell, upon costimulation with CXCL12 and an ADRB2 agonist, results in calcium mobilization that is equal to or less than the sum of the calcium mobilization that results from single agonist stimulation with either the CXCL12 or the ADRB2 agonist; and b) the CXCR4-ADRB2 heteromer exhibits an enhanced amount of calcium mobilization upon costimulation with the CXCL12 and the ADRB2 agonist, such that the CXCR4-ADRB2 heteromer exhibits enhanced calcium mobilization compared to the sum of the amounts of calcium mobilization resulting from single agonist stimulation with either the CXCL12 or the ADRB2 agonist. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use according to any one of embodiments A1 to A75, characterized in that:

[0283] Embodiment A77. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A76, wherein the CXCR4-ADRB2 heteromer is characterized by the following characteristic: an increased amount of downstream ERK signaling results from costimulation of the CXCR4-ADRB2 heteromer with the CXCR4 agonist and the ADRB2 agonist compared to the amount of downstream ERK signaling that results from stimulation of the CXCR4-ADRB2 heteromer with either the CXCR4 agonist or the ADRB2 agonist alone.

[0284] Embodiment A78. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A77, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is greater than the amount resulting from stimulation with the CXCR4 agonist alone.

[0285] Embodiment A79. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A78, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is at least 5% greater than the amount resulting from stimulation with the CXCR4 agonist alone.

[0286] Embodiment A80. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A78, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% greater than the amount resulting from stimulation with the CXCR4 agonist alone.

[0287] Embodiment A81. The amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is 5-15%, 10-25%, 20-50%, 40-75%, or 60-100% greater than the amount resulting from stimulation with the CXCR4 agonist alone. stomach,A method of suppression, a method of treatment, a pharmaceutical kit for use, or a pharmaceutical composition for use according to any one of embodiments A78 to A80.

[0288] Embodiment A82. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A77, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is greater than the amount resulting from stimulation with the ADRB2 agonist alone.

[0289] Embodiment A83. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A82, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is at least 5% greater than the amount resulting from stimulation with the ADRB2 agonist alone.

[0290] Embodiment A84. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A82, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is at least 10%, at least 25%, at least 50%, at least 75%, or at least 90% greater than the amount resulting from stimulation with the ADRB2 agonist alone.

[0291] Embodiment A85. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A82 to A84, wherein the amount of downstream ERK signaling resulting from costimulation with the CXCR4 agonist and the ADRB2 agonist is in the range of 5-15%, 10-25%, 20-50%, 40-75%, or 60-100% greater than the amount resulting from stimulation with the ADRB2 agonist alone.

[0292] Embodiment A86. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A14 to A85, wherein the CXCR4-ADRB2 heteromer is characterized as having the following feature: the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific properties of the CXCR4-ADRB2 heteromer in the cell(s) derived from the subject.

[0293] Embodiment A87. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A14 to A86, wherein the CXCR4-ADRB2 heteromer is characterized as having the following feature: the combination of blixafor and an ADRB2 inhibitor alters a heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject.

[0294] Embodiment A88. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A14 to A87, wherein the CXCR4-ADRB2 heteromer is characterized as having the following feature: the combination of blixafor and an ADRB2 inhibitor alters the heteromer-specific properties of the subject-derived cell(s) containing the CXCR4-ADRB2 heteromer.

[0295] Embodiment A89. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1 to A9 or A14 to A88, wherein the CXCR4-ADRB2 heteromer is characterized as having the following feature: the combination of blixafor and an ADRB2 inhibitor reduces cancer progression in cell(s) derived from the subject containing the CXCR4-ADRB2 heteromer.

[0296] Embodiment A90. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1 to A89, wherein the administered combination of blixafor and an ADRB2 inhibitor suppresses enhanced downstream signaling from the CXCR4-ADRB2 heteromer.

[0297] Embodiment A91. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A90, wherein the administered combination of blixafor and an ADRB2 inhibitor suppresses enhanced downstream signaling from the CXCR4-ADRB2 heteromer in the cell.

[0298] Embodiment A92. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A1-A9 or A17-A91, wherein the administered combination of blixafor and an ADRB2 inhibitor suppresses enhanced downstream signaling from the CXCR4-ADRB2 heteromer in the cancer subject.

[0299] Embodiment A93. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A9 or A17-A92, wherein the administered combination of blixafor and an ADRB2 inhibitor suppresses enhanced downstream signaling from the CXCR4-ADRB2 heteromer in the cells of the cancer subject.

[0300] Embodiment A94. The method of inhibiting, the method of treatment, the pharmaceutical kit for use, or the pharmaceutical composition for use of any one of Embodiments A1 to A93, wherein the CXCR4-ADRB2 heteromeric component comprises individual protomers of CXCR4 and ADRB2.

[0301] Embodiment A95. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A94, wherein the cells containing the CXCR4 in the context of an individual protomer comprise the individual protomer CXCR4 in the presence or absence of the individual protomer ADRB2.

[0302] Embodiment A96. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A95, wherein the cells containing the CXCR4 in the context of an individual protomer comprise the individual protomer CXCR4 in the absence of the individual protomer ADRB2.

[0303] Embodiment A97. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A95, wherein the cells containing the CXCR4 in the context of an individual protomer comprise the individual protomer CXCR4 in the presence of the individual protomer ADRB2.

[0304] Embodiment A98. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A1-A94, wherein the cells containing the ADRB2 in the context of individual protomers comprise the individual protomer ADRB2 in the presence or absence of the individual protomer CXCR4.

[0305] Embodiment A99. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A98, wherein the cells containing said ADRB2 in the context of individual protomers comprise said individual protomer ADRB2 in the absence of said individual protomer CXCR4.

[0306] Embodiment A100. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A98, wherein the cells containing said ADRB2 in the context of individual protomers comprise said individual protomer ADRB2 in the presence of said individual protomer CXCR4.

[0307] Embodiment A101. The administered combination of blixafor and an ADRB2 inhibitor comprises: i) altering the heteromer-specific properties of the CXCR4-ADRB2 heteromer in the cell; ii) altering the heteromer-specific function of the CXCR4-ADRB2 heteromer in the cell; and / or iii) altering the heteromer-specific properties of the cells containing the CXCR4-ADRB2 heteromers; A method of suppression, a method of treatment, a pharmaceutical kit for use, or a pharmaceutical composition for use according to any one of embodiments A1 to A100.

[0308] Embodiment A102. The combination of blixafor and the ADRB2 inhibitor administered is: i) altering the heteromer-specific properties of the CXCR4-ADRB2 heteromers in a cell(s) derived from the subject; ii) altering a heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; iii) altering the heteromer-specific properties of the cell(s) from the subject that contain the CXCR4-ADRB2 heteromer; or iv) reducing cancer progression in said subject having cells containing said CXCR4-ADRB2 heteromers. A method of suppression, a method of treatment, a pharmaceutical kit for use, or a pharmaceutical composition for use according to any one of embodiments A1 to A9 or A14 to A101.

[0309] Embodiment A103. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A102, wherein the administered combination of the blixafor and the ADRB2 inhibitor alters the heteromer-specific properties of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject.

[0310] Embodiment A104. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of embodiment A102 or 103, wherein the administered combination of the blixafor and the ADRB2 inhibitor alters a heteromer-specific function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject.

[0311] Embodiment A105. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A104, wherein the administered combination of the blixafor and the ADRB2 inhibitor alters the heteromer-specific properties of the subject-derived cell(s) containing the CXCR4-ADRB2 heteromer.

[0312] Embodiment A106. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of Embodiments A102 to A105, wherein the administered combination of the blixafor and the ADRB2 inhibitor reduces cancer progression in the subject having cells containing the CXCR4-ADRB2 heteromer.

[0313] Embodiment A107. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A106, wherein the CXCR4-ADRB2 heteromer is characterized as having the following feature: the combination of blixafor and an ADRB2 inhibitor reduces cancer progression in cell(s) derived from the subject containing the CXCR4-ADRB2 heteromer.

[0314] Embodiment A108. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A107, wherein said decreasing cancer progression comprises decreasing cancer progression in a cell(s) derived from said subject that contains said CXCR4-ADRB2 heteromer.

[0315] Embodiment A109. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A108, wherein said decreasing cancer progression comprises decreasing cell proliferation of a cell or cells derived from the subject that contain said CXCR4-ADRB2 heteromer.

[0316] Embodiment A110. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A109, wherein said decreasing cancer progression comprises decreasing cell migration of said subject-derived cell(s) containing said CXCR4-ADRB2 heteromer.

[0317] Embodiment A111. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A110, wherein said reduced cancer progression comprises reduced metastasis of cell(s) derived from said subject containing said CXCR4-ADRB2 heteromer.

[0318] Embodiment A112. The method of suppression, method of treatment, pharmaceutical kit for use, or pharmaceutical composition for use of any one of embodiments A102 to A111, wherein said decreasing cancer progression comprises decreasing ang...

Claims

1. 1. A pharmaceutical composition for use in a method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising a CXCR4 inhibitor, the method further comprising administering to the subject an ADRB2 inhibitor, wherein i) the CXCR4-ADRB2 heteromers result in enhanced downstream signaling of CXCR4 or ADRB2; and ii) the CXCR4 inhibitor and the ADRB2 inhibitor suppress the enhanced downstream signaling of CXCR4 or ADRB2 from the CXCR4-ADRB2 heteromer in the cancer subject; the efficacy of the CXCR4 inhibitor, when administered in combination with an ADRB2 inhibitor to the subject having cancer cells containing the CXCR4-ADRB2 heteromer, is increased in the range of 3000-5000% compared to the efficacy of the CXCR4 inhibitor when administered as a single inhibitor; and The pharmaceutical composition, wherein the CXCR4 inhibitor is BKT140 and the ADRB2 inhibitor is carvedilol.

2. 2. The pharmaceutical composition of claim 1, wherein the combination of the CXCR4 inhibitor and the ADRB2 inhibitor is administered as a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

3. The pharmaceutical composition of claim 1 or 2, wherein the CXCR4 inhibitor and the ADRB2 inhibitor are administered sequentially, in combination, or simultaneously.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cancer is a hematological cancer or a solid tumor.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the enhanced downstream signaling results in enhanced cancer progression in the subject having cell(s) containing the CXCR4-ADRB2 heteromer.

6. 6. The pharmaceutical composition of claim 5, wherein the enhanced cancer progression is enhanced cell migration, enhanced metastasis, or enhanced tumor growth in the subject having cell(s) containing the CXCR4-ADRB2 heteromer.

7. The method comprises: i) whether or not the cells of said subject contain said CXCR4-ADRB2 heteromers; or ii) the CXCR4 inhibitor and the ADRB2 inhibitor are: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; alters the heteromer-specific properties of the subject-derived cell(s) containing the CXCR4-ADRB2 heteromer; or whether or not the progression of cancer is reduced in said subject having cells containing said CXCR4-ADRB2 heteromers The pharmaceutical composition of any one of claims 1 to 6, further comprising determining:

8. 1. A pharmaceutical composition for use in a method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising a CXCR4 inhibitor, the method further comprising administering to the subject an ADRB2 inhibitor, wherein i) the CXCR4-ADRB2 heteromers result in enhanced downstream signaling of CXCR4 or ADRB2; and ii) the CXCR4 inhibitor and the ADRB2 inhibitor suppress the enhanced downstream signaling of CXCR4 or ADRB2 from the CXCR4-ADRB2 heteromer in the cancer subject; the efficacy of the CXCR4 inhibitor, when administered in combination with an ADRB2 inhibitor to the subject having cancer cells containing the CXCR4-ADRB2 heteromer, is increased in the range of 2000-4000% compared to the efficacy of the CXCR4 inhibitor when administered as a single inhibitor; and The pharmaceutical composition, wherein the CXCR4 inhibitor is urocuplumab and the ADRB2 inhibitor is carvedilol.

9. 9. The pharmaceutical composition of claim 8, wherein the combination of the CXCR4 inhibitor and the ADRB2 inhibitor is administered as a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

10. The pharmaceutical composition of claim 8 or 9, wherein the CXCR4 inhibitor and the ADRB2 inhibitor are administered sequentially, in combination, or simultaneously.

11. The pharmaceutical composition according to any one of claims 8 to 10, wherein the cancer is a hematological cancer or a solid tumor.

12. The pharmaceutical composition of any one of claims 8 to 11, wherein the enhanced downstream signaling results in enhanced cancer progression in the subject having cell(s) containing the CXCR4-ADRB2 heteromer.

13. 13. The pharmaceutical composition of claim 12, wherein the enhanced cancer progression is enhanced cell migration, enhanced metastasis, or enhanced tumor growth in the subject having cell(s) containing the CXCR4-ADRB2 heteromer.

14. The method comprises: i) whether or not the cells of said subject contain said CXCR4-ADRB2 heteromers; or ii) the CXCR4 inhibitor and the ADRB2 inhibitor are: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; alters the heteromer-specific properties of the subject-derived cell(s) containing the CXCR4-ADRB2 heteromer; or whether or not the progression of cancer is reduced in said subject having cells containing said CXCR4-ADRB2 heteromers The pharmaceutical composition of any one of claims 8 to 13, further comprising determining:

15. 1. A pharmaceutical composition for use in a method of treating cancer in a subject having cells containing CXCR4-ADRB2 heteromers, comprising a CXCR4 inhibitor, the method further comprising administering to the subject an ADRB2 inhibitor, wherein i) the CXCR4-ADRB2 heteromers result in enhanced downstream signaling of CXCR4 or ADRB2; and ii) the CXCR4 inhibitor and the ADRB2 inhibitor suppress the enhanced downstream signaling of CXCR4 or ADRB2 from the CXCR4-ADRB2 heteromer in the cancer subject; the efficacy of the ADRB2 inhibitor, when administered in combination with a CXCR4 inhibitor to the subject having cancer cells containing the CXCR4-ADRB2 heteromer, is increased in the range of 3000-5000% compared to the efficacy of the ADRB2 inhibitor when administered as a single inhibitor; and The pharmaceutical composition, wherein the CXCR4 inhibitor is AMD3100 and the ADRB2 inhibitor is bupranolol.

16. 16. The pharmaceutical composition of claim 15, wherein the combination of the CXCR4 inhibitor and the ADRB2 inhibitor is administered as a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

17. 17. The pharmaceutical composition of claim 15 or 16, wherein the CXCR4 inhibitor and the ADRB2 inhibitor are administered sequentially, in combination, or simultaneously.

18. The pharmaceutical composition according to any one of claims 15 to 17, wherein the cancer is a hematological cancer or a solid tumor.

19. The pharmaceutical composition of any one of claims 15 to 18, wherein the enhanced downstream signaling results in enhanced cancer progression in the subject having cell(s) containing the CXCR4-ADRB2 heteromer.

20. 20. The pharmaceutical composition of claim 19, wherein the enhanced cancer progression is enhanced cell migration, enhanced metastasis, or enhanced tumor growth in the subject having cell(s) containing the CXCR4-ADRB2 heteromer.

21. The method comprises: i) whether or not the cells of said subject contain said CXCR4-ADRB2 heteromers; or ii) the CXCR4 inhibitor and the ADRB2 inhibitor are: alters the heteromer-specific properties or function of the CXCR4-ADRB2 heteromer in a cell(s) derived from the subject; alters the heteromer-specific properties of the subject-derived cell(s) containing the CXCR4-ADRB2 heteromer; or whether or not the progression of cancer is reduced in said subject having cells containing said CXCR4-ADRB2 heteromers The pharmaceutical composition of any one of claims 15 to 20, further comprising determining:

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