GPCR heteromeric inhibitors and their uses

Abstract

To provide a method for treatment, amelioration, or prevention of a cancer in a subject having CXCR4-GPCRx heteromer.SOLUTION: The method of the present invention comprises administering to the subject a therapeutically effective amount of an inhibitor of a CXCR4-GPCRx heteromer, in which GPCRx heteromerizes with CXCR4 in the subject, the heteromerization of GPCRx with CXCR4 is accompanied by enhancement of signaling downstream of CXCR4, and the enhancement of signaling downstream of CXCR4 is suppressed by the inhibitor of the CXCR4-GPCRx heteromer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is a direct sequel to U.S. Provisional Application No. 62 / 607,876, filed December 19, 2017. and claims the benefit of priority from U.S. Provisional Application No. 62, filed June 1, 2018. / 679,598, filed September 18, 2018 This application claims the benefit of priority from U.S. Provisional Application No. 62 / 732,946 filed on Dec. 1, 2002. Each of the above related applications is incorporated herein by reference in its entirety.

[0002] Additionally, each of the references identified herein is incorporated herein by reference in its entirety. is used as a reference. [Background technology]

[0003] The inventions disclosed herein generally relate to novel inhibitors of functional GPCR heteromers, More specifically, functional heteromer formation results in enhanced signal transduction downstream of CXCR4. CXC receptor 4 (CXCR4)-G protein, which mediates signal transduction and is associated with cancer and other diseases This paper deals with inhibitors of GPCR heteromers.

[0004] G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that couple to G proteins. GPCRs are cell surface receptors that react with light, odorants, hormones, neurotransmitters, and chemokines. It responds to a variety of sensory responses by perceiving stimuli including kinases, small lipid molecules, and nucleotides. There are approximately 800 GPCR genes in the human genome. More than half of these encode sensory receptors, e.g., olfactory, visual, and taste receptors. It is predicted that (Bjarnadottir, et al., 2006). 350 GPCRs regulate embryonic development, behavior, mood, cognition, blood pressure regulation, heart rate, and digestive processes , which are physiologically important in immune system regulation and inflammation, homeostasis, and cancer growth and metastasis. plays an important role (Filmore 2004, Overington, et al., GPCRs are associated with many diseases and are the targets of approximately 40% of all prescription drugs. (Filmore 2004).

[0005] CXC receptor 4 (CXCR4) is a member of the chemokine receptor family of GPCRs. CXCR4 is expressed on most hematopoietic cell types, bone marrow stem cells, endothelial progenitor cells, and vascular endothelial cells. , expressed in neurons and neural stem cells, microglia and astrocytes (Klein a nd Rubin 2004, Griffith et al., 2014). CXCR 4 is associated with its ligand, CX-, also known as stromal cell-derived factor 1 (SDF-1). Responding to C-motif chemokine ligand 12 (CXCL12), it promotes hematopoietic, cardiovascular, and neuroprotective functions. It plays an essential role in the embryonic development of the thyroid gland (Griffith, et al., 2014 CXCR4 was discovered as a co-receptor for human immunodeficiency virus (HIV) and is involved in hematopoietic stem cells. in bone marrow homing of hematopoietic stem cells (HSCs), inflammation, tissue immune surveillance, and tissue regeneration in adults. CXCR4 plays an important role in the regulation of CXCR4 expression (Chatterjee, et al. 2014). Mutations in the C-terminus of CXCR4 cause persistent CXCR4 activation, resulting in neutropenia and WHIM syndrome (warts, hypogammaglobulinemia, This leads to a congenital immune deficiency called myelocatechus (Hernandez , et al., 2003; Kawai, 2009). CXCR4 is also involved in the regulation of inflammatory cytokines during development and It plays an essential role in the development of T and B lymphocytes in the lymphoid organs and thymus in adults. (Allen, et al., 2004; Ara, et al., 2003).

[0006] CXCR4 is involved in various immune and autoimmune diseases, such as HIV infection, ischemia, and wound healing. , rheumatoid arthritis, systemic lupus erythematosus (SLE), interstitial pneumonia, vascular disease, multiple sclerosis, It has been implicated in pulmonary fibrosis and allergic airway disease (Chu et al., 2014). 17, Debnath, et al., 2013, Domanska, et al., 2 013) The involvement of CXCR4 in rheumatoid arthritis is due to the presence of C Increased accumulation of XCR4-positive T cells and collagen-induced joint damage in CXCR4-deficient mice This was demonstrated by the reduction of flames (Buckley et al., 2000; Chung Furthermore, the CXCR4 antagonist AMD3100 has been shown to inhibit mitochondrial vasculopathy in mouse models. significantly alleviated collagen-induced arthritis in mice (De Klerck et al. , 2005). CXCR4 also mobilizes circulating fibroblasts and bone marrow-derived progenitor cells during lung injury. AMD3100 regulates pulmonary fibrosis by inhibiting bleomycin-induced mouse fibrosis. demonstrated a preventive effect in pulmonary fibrosis (Song et al., 2010). The CR4 / CXCL12 axis is also thought to be involved in the pathogenesis of SLE. Inflammatory cells, such as monocytes, neutrophils, and B cells, express CXCR4 in patients with SLE. showed an increase in CXCL12 expression and migrated toward the skin and lungs, which predominantly overexpressed CXCL12 (C hong and Mohan, 2009, Wang et al., 2009, Wan CXCR4 antagonist CTCE-9908 inhibits lupus in mouse models of lupus. The results showed that the survival time was prolonged and the disease state and nephritis were significantly improved in patients with glaucoma (Wang et al. Furthermore, CXCR4 is involved in the prevention of brain injury, stroke, myocardial infarction, and atherosclerosis. It is also involved in diseases of the brain and heart, including arteriosclerosis and injury-induced vasoconstriction (Chen g et al., 2017, Domanska, et al., 2013, Dorin g,et al.,2014).

[0007] The involvement of CXCR4 in cancer was first recognized in chronic lymphocytic leukemia (B- When B cells from CCL patients express high levels of functional CXCR4 on their surface, , enhanced calcium mobilization and actin polymerization upon CXCL12 exposure, and CXCL12 Migration to bone marrow stromal cells secreting IL-1 has been shown (Burger, et al., 1999) .

[0008] CXCR4 then mediates the expression of higher levels of CXCL12 in organs, e.g. It has been characterized as a contributor to breast cancer metastasis to the lymph nodes, bone marrow, lungs, and liver (Muller , et al., 2001). After its initial discovery, CXCR4 has been implicated in a wide variety of cancers. There is growing evidence suggesting that it has multiple potential roles in malignancies. CXCR4 is a cancer that affects breast cancer, lung cancer, brain cancer, kidney cancer (or renal cell carcinoma), pancreatic cancer, ovarian cancer, and pre-cancerous 23 types of cancer, including prostate cancer, melanoma, leukemia, multiple myeloma, gastrointestinal cancer, and soft tissue sarcoma It is overexpressed in over 100 human cancers and is considered a marker of poor prognosis (Doma nska, et al., 2013, Chatterjee, et al., 2014, Furusato et al., 2010). CXCR4 is expressed in most cancer types. It is the only chemokine receptor that expresses α-glucan (Liang et al., 2015), and is involved in the regulation of chemokine expression in cancer cells. Tumor cell growth and survival respond to CXCL12 secreted by tumor cells and surrounding cancer-related cells. stimulates proliferation and invasion (Burger and Kipps, 2006; Chatter rjee et al., 2014, Domanska et al., 2013).

[0009] Databases including PubMed, EMBASE and the Cochrane Library A systematic meta-analysis using the method was conducted on hematopoietic malignancies, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, and kidney cancer. CXCR4 in various cancers, including lung cancer, gynecological cancer, liver cancer, prostate cancer, and gallbladder cancer Overexpression of EGFR-12121 was associated with a significantly lower progression-free survival and overall survival (Du et al.,2015, Hu et al.,2015, Li et al.,201 7, Wang et al., 2016, Zhao et al., 2015).

[0010] The CXCR4 / CXCL12 axis mediates tumor growth, invasion, angiogenesis, vasculogenesis, metastasis, and drug resistance. It plays a central role in cancer cell-tumor microenvironment interactions (D'Alter io et al., 2012, Domanska et al., 2013, Guo et al., 2016).

[0011] The crucial role of CXCR4 in cancer cell proliferation and tumor growth has been demonstrated in vitro and in vivo. Various in vivo experimental models, such as orthotopic subcutaneous human xenografts and transgenic mice This has been demonstrated using CXCR4 antagonists in a model (Domanska et al ., 2013). Daoy medulloblastoma cells and U87 glioblastoma cells showed CXCR4 expression. , which exhibited a dose-dependent increase in proliferation against a CXCL12 gradient in vitro, and AMD3100 Systemic administration of inhibited the growth of intracranial U87 and Daoy cell xenografts (Rubin et al., 2003).

[0012] CXCR4 is also involved in the metastasis of cancer cells to CXCL12-expressing organs, such as the pancreas and thyroid. , melanoma, prostate, and colon cancer xenograft models (Bartolo me et al., 2009, De Falco et al., 2007, Taic hman et al., 2002, Wang et al., 2008, Zeelen berg et al., 2003).

[0013] The main mechanism of action described for small molecule or peptide antagonists of CXCR4 is their focuses on its ability to liberate malignant cells from the BM and thereby sensitize them to chemotherapy. These drugs present limitations related to their short half-lives, making their adequate administration over long periods of time difficult. In contrast, therapeutic monochromatic Clonal antibodies have the advantage of a longer half-life, making them suitable for less frequent dosing. In addition, human IgG antibodies bind to their target proteins on cancer cells. Fc on effector cells, including antibody-dependent cellular cytotoxicity / phagocytosis (ADCC / ADCP) It has the ability to induce cell death through interaction with receptors (Jiang et al. , 2011). Such a cytotoxic mechanism of action is inherent in small molecules or peptides. have not been demonstrated to play a crucial role in the clinical activity of some therapeutic antibodies. (Wang et al., 2015).

[0014] Targeting CXCR4 using neutralizing anti-CXCR4 antibodies or CXCR4-specific antagonists This has resulted in the development of primary tumors in breast cancer, colon cancer, hepatocellular carcinoma, osteosarcoma, and melanoma. Growth and metastasis to secondary organs were inhibited (De Falco et al., 2007 , Hassan et al.,2011, Huang et al.,2009, Ki m et al., 2008, Muller et al., 2001, Schiman ski et al., 2006, Smith et al., 2004, Zeelen In a transgenic mouse model of breast cancer, CTCE-9 Inhibition of CXCR4 by 908 inhibits not only primary tumor growth but also vascular endothelial growth factor (VEGF) expression. F) expression and AKT phosphorylation (Hassan et al., 2011). .

[0015] Cancer stem cells (CSCs) possess limitless self-renewal, the potential to differentiate into multiple cancer lineages, and quiescence. These include the ability to adapt to new conditions and a high intrinsic resistance to chemotherapy and radiation therapy. CSCs are a population of cancer cells with unique characteristics. They are responsible for preventing cancer relapse and recurrence after standard antiproliferative therapy. Therefore, targeting cancer stem cells is a promising approach to treat cancer. This will hopefully lead to more effective therapeutic interventions to eradicate cancer and prevent recurrence. (Batlle and Clevers, 2017, Reya et al., 2 001, Wurth, 2016). Interestingly, CSCs also express CXCR4 and XCR4 promotes the maintenance, survival, and growth of cancer stem cells through a CXCL12-rich microenvironment , promoting the trafficking and metastasis of these cells to, for example, the bone marrow and the subventricular zone of the brain. Drugs can release CSCs from these protective microenvironments to prevent the development of cancer cells, which can then be treated with conventional chemotherapy and radiation therapy, as well as chemotherapy. It has been shown to sensitize cells to anti-angiogenic therapy (Burger and Kipps, 2014). 006, Burger and Peled, 2009, Furusato et al. .,2010, Redondo-Muñoz et al.,2006, Walenka mp et al., 2017, Wurth, 2016).

[0016] In addition to the cancer cells that make up the tumor microenvironment (TME) or cancer cell niche, the tumor mass Various cell types, such as stromal fibroblasts, immune cells, endothelial cells, connective tissue and extracellular matrix There is growing evidence that the CXCR4 / CXCL12 axis is involved in the Tumor structure, growth, angiogenesis, and immune surveillance in a variety of cancers, both hematopoietic and non-hematopoietic. It plays a pivotal role in tumor cell-microenvironment interactions that support evasion. (Burger and Kipps, 2006, Burger and Peled, 2009, Wa Lenkamp et al., 2017). CXCL12 recruits endothelial cells to the TME. This is done either directly or by attracting CXCR4-positive inflammatory cells to the tumor mass. It can indirectly promote tumor angiogenesis by secreting pro-angiogenic factors. (Owen and Mohamadzadeh, 2013, Walenkamp et al., 2017).

[0017] The CXCR4 / CXCL12 axis contributes to the lack of tumor response to antiangiogenic agents Vascular endothelial growth factor (VEGF) plays a key role in cancer. VEGF is thought to be a major pro-angiogenic factor in rectal carcinoma, and anti-VEGF antibodies Targeted for antiangiogenic therapy using bevacizumab (Genentech) Surprisingly, bevacizumab suppressed the expression of CXCL12 and CXCR4 in cancer cells. and elevated plasma levels of CXCL12 in these patients are associated with rapid disease progression. and metastasis (Owen and Mohamadzadeh, 2013, Xu et al., 2009). Therefore, bevacizumab and The efficacy of combination therapy using cefotaxime and plerixafor was evaluated (Clinical Trials als.gov identifier: NCT01339039). However, the study The study was discontinued due to the lack of data (Walenkamp et al., 2017).

[0018] Inhibition of the CXCR4 / CXCL12 axis reduces the infiltration of myeloid-derived suppressor cells. by increasing the ratio of CD8+ cytotoxic T cells to Treg cells, or by inhibiting tumor growth. Disrupting the tumor microenvironment (TME) by eliminating tumor revascularization and immunizing tumor cells It has been demonstrated that exposure to attack (Burger et al., 2011, Domansk a et al., 2013, Walenkamp et al., 2017). Pancreatic ductal glands In models of cancer, advanced hepatocellular carcinoma (HCC) and CXCR4-transduced B16 melanoma Therefore, administration of the CXCR4 antagonist AMD3100 or T22 was associated with immune checkpoint inhibitor For example, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies, programmed cell death Synergistically with anti-PD-1 and anti-PD-L1 antibodies and significantly increased their antitumor activity (Chen et al., 2015, Feig et al.,2013, Lee et al.,2006, Scala,2 015, Walenkamp et al., 2017). These results suggest that CXCR4 Targeting the CXCL12 axis provides benefits over traditional immune checkpoint inhibitors This suggests that this will be the case.

[0019] Various drugs have been developed that target CXCR4 (Peled, et al., 201 2, Debnath, et al., 2013, Walenkamp, ​​et al., 2 017). CXCR4 inhibitors can be divided into five categories:

[0020] (1) Non-peptide small molecule antagonists, such as AMD3100 (Plerixafor, Moz obil (trademark), Genzyme (MA, USA), (Cashen et al., 2007, Donzella et al., 1998), AMD070 (AMD110 70, Crawford and Alan Kaller,2008, Stone e et al., 2007), AMD3465 (Genzyme Corp., Bioche m Pharmacol.2009 Oct 15;78(8):993-1000, B odart et al., 2009, Ling et al., 2013), GSK8 12397 (Jenkinson et al., 2010, Planesas et al. al., 2015), KRH-3955 (Murakami et al., 2009, Nakasone et al., 2013), KRH-1636 (Ichiyama et al., 2003), D-[Lys3]GHRP-6 (Patel et al. , 2012), TG-0054 (Brikixaphor, TaiGen Biotechnol ogy Co., Ltd., de Nigris et al., 2012, Hsu e t al., 2015), WZ811 (Zhan et al., 2007), MSX- 122 (Metastatix, Inc., Liang et al., 2012), and 508MCl (Compound26, Zhu et al., 2010);

[0021] (2) Small molecule peptide antagonists, for example, anti-HIV peptide T22 (Masuda et al., 1992), T134 and T140 (Tamamura et al., 19 98), BKT140 (a / k / a BL-8040, TF14016, 4F-benzoyl l-TN14003, Biokine Therapeutics Ltd. (Rehovot, Israel), Fahham et al., 2012, Otani et al., 2012), ALX40-4C (Canadian company Allelix Biopharmaceuticals (ON, Canada), Doranz et al., 2001), GST-NT21MP (Yang et al., 201 4), FC131 (Tanaka et al., 2009, Tanaka et al ., 2008), FC122 (Inokuchi et al., 2011), POL6 326 (de Nigris et al., 2012) and LY2510924 (Li lly) (Peng et al., 2015),

[0022] (3) Antibodies against CXCR4, such as urocupuncture (MDX1338 / BMS- 936564, Kuhne et al., 2013), PF-06747143 (Pf izer, Liu et al., 2017), 12G5 (Endres et al. ,1996), i-Body (AD-114, AD-114-6H, AD-114-Im7 -FH and AD-114-PA600-6H, AdAlta, Griffiths et al., 2016, Griffiths et al., 2018), nanobodies (2 38D2 and 238D4, Jahnichen et al., 2010, Proc.N atl Acad.Sci.2010, USA 107(47), 20565-2057 0), and ALX-0651 (Ablynx, a dual paratopic nanobody, Clini calTrials.gov identifier: NCT01374503),

[0023] (4) Ligand (CXCL12) analogs with inhibitory activity, such as CTCE-9908 (Chemokine Therapeutics (BC, Canada), Wong et al., 2014), and

[0024] (5) Radiolabeled CXCR4 ligands, such as [99mTc]O2-AMD3100(H artimath et al., 2013), [68Ga]pentixafor (Dem mer et al., 2011, Gourni et al., 2011), [177 Lu]pentixatel and [90Y]pentixatel (Herrmann et al. ,2016).

[0025] AMD3100 (JM3100, plerixafor, trade name Mozobil) CXCR4-specific CXCL12-mediated calcium mobilization and chemotaxis are inhibited in various cell types. It is a potent small molecule antagonist (Hatse, Princen et al. 2002) and Prevents tumor growth in a xenograft model (Rubin, Kung et al. 2003, Cho, Yoon et al.2013, Liao, Fu et al.2 015). AMD3100 was initially developed for HIV therapy (one of the HIV entry co-receptors). It was developed as a drug to block HIV entry by specifically antagonizing CXCR4, which is involved in HIV replication. Certified by the U.S. Food and Drug Administration for stem cell mobilization in patients with lymphoma and multiple myeloma AMD3100 for HIV infection was approved for the treatment of HIV infection (Keating 2011). During initial clinical trials, this compound was shown to induce hematopoietic stem cells (HSCs, CD34) from bone marrow to peripheral blood. + ) motion (Broxmeyer et al., 2005, De Clercq, 2003, Liles et al., 2003).

[0026] Development of AMD3100 for HIV therapy involves long-term inhibition of the CXCR4 / CXCL12 axis It was withheld due to significant side effects, including thrombocytopenia, premature ventricular contractions, and leukocytosis after (Hendrix, et al., 2004, Peled, et al., 2012) Investigations into the potential use of AMD3100 as an anti-cancer drug are ongoing, but it is orally available. It is necessary to overcome the lack of efficacy and some serious side effects associated with long-term use (Pel ed, et al., 2012). Instead, subcutaneous AMD3100 is For patients with non-Hodgkin lymphoma (NHL) or multiple myeloma, within 4 consecutive days, Patients with rheumatoid arthritis or lymphoma were treated with autologous stem cell mobilization and transplantation within 2-4 consecutive days and 7 consecutive days, respectively. It has been approved in combination with G-CSF for transplantation (DiPersio et al., 200 9a, DiPersio et al., 2009b, Keating, 2011). now To date, AMD3100 is the only CXCR4 antagonist approved by the FDA. do.

[0027] AMD3100 inhibits CXCL12 in various cancer cell lines expressing CXCR4. It has been shown to inhibit ATP-mediated calcium mobilization and chemotaxis (Hatse et al., 2002). 002), along with reduced metastasis and improved overall survival in various mouse xenograft models. demonstrated significant antitumor activity (Burger et al., 2011, Chatter jee et al., 2014, Cho et al., 2013, Debnath et al., 2013, Domanska et al., 2013, Liao et al. al., 2015, Rubin et al., 2003, Walenkamp et al. al., 2017).

[0028] However, AMD3100 exhibits partial agonism against CXCR4 in vitro. and promotes the proliferation of melanoma cells (Kim et al., 201 0, Zhang et al., 2002). AMD3100 also inhibits CXCL12. as a positive allosteric modulator of CXCR7, another chemokine receptor It acts to enhance the binding of CXCL12 to CXCR7 (Kalatskaya et al. Like CXCR4, CXCR7 is highly expressed in many types of cancer, associated with tumor metastasis (Decaillot et al., 2011, Zabel et al., 2011). The complex properties of AMD3100 make it difficult to predict the role of AMD3 in cancer. The exact role of the 100 needs to be carefully investigated.

[0029] AMD3100 is a treatment for acute myeloid leukemia (AML), MM, and myelodysplastic syndromes (MDS). In hematopoietic malignancies including CLL and small lymphocytic lymphoma (SLL), Cytotoxic drugs, e.g., mitoxantrone, etoposide, cytarabine , daunorubicin, azacitidine, lenalidomide, decitabine, clofarabine, fludarabine rabin and / or idarubicin; receptor tyrosine kinase inhibitors AC220 and Soraf HSP90 inhibitor (ganetespib); G-CSF; proteasome inhibitor (bolus) tezomib); or in combination with a monoclonal antibody (rituximab) as an anticancer drug It has been clinically evaluated (ClinicalTrials.gov identifier: NCT00 512252, NCT00694590, NCT00903968, NCT009900 54, NCT01065129, NCT01373229, NCT01352650, N CT01236144, NCT01301963, NCT01160354, NCT01 027923, NCT00943943 and NCT01435343). Relapsed or refractory AMD3100 is a potential treatment for patients with AML, ALL, and MDS, and is a potential treatment for patients with lymphomas. The tumor microenvironment signaling (NCT01610999) and combination with other anti-cancer drugs Chemosensitization was occasionally investigated (ClinicalTrials.gov identifier: NCT00906945 and NCT01319864).

[0030] AMD3100 is a treatment for high-grade glioma, Ewing's sarcoma, neuroblastoma, pancreatic, ovarian and gastrointestinal tumors. Alone or in combination with the angiogenesis inhibitor bevacizumab in solid tumors, including colon cancer have been or are currently being evaluated (ClinicalTrials.go v identifier: NCT01339039, NCT01977677, NCT01288573 , NCT02179970 and NCT03277209).

[0031] AMD3100 is a treatment for AML, MDS, neutropenia, beta-thalassemia, and sickle cell anemia. Disease, WHIMS, Fanconi anemia, Wiskott-Aldrich syndrome, systemic obesity Hematopoietic stem / progenitor / unipotent precursor cells in patients with various hematopoietic malignancies and diseases, including cytosis have been tested or are currently being evaluated for cellular recruitment (Domanska et al., 2013), NCT01058993, NCT01206075, NC T03226691, NCT00967785, NCT02678533, NCT030 19809 and NCT00001756). It is used in the treatment of diabetes, wounds, critical limb ischemia, and chronic obstructive pulmonary disease. CD34+ cells, endothelial cells in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis, and pulmonary fibrosis Mobilization of EPCs and / or CD117+ progenitor cells has been evaluated. are currently being evaluated (ClinicalTrials.gov identifier: NCT 02056210, NCT02790957, NCT01916577, NCT0318 2426).

[0032] Investigations into the potential use of AMD3100 as an anti-cancer drug are ongoing, but oral Lack of availability and some serious side effects associated with long-term use need to be overcome. (Peled,et al.,2012).

[0033] The orally available CXCR4 antagonist AMD070 (also known herein as AMD-070) , AMD11070, AMD-11070 or X4P-001; X4 Pharmac euticals) demonstrated antitumor activity in various tumor models (Morimoto et al., 2016, O'Boyle et al., 2013, Parames Waran et al., 2011), a Phase I / II clinical trial in HIV-infected subjects (ClinicalTrials.gov identifier: NCT00089466 , (Debnath et al., 2013). AMD070 is currently classified as WHIM syndrome. , alone or in combination with pembrolizumab, nivolumab, or nivolumab in patients with advanced melanoma and renal cell carcinoma. It is currently in Phase II / III clinical trials with ramucirumab or axitinib (Clinical Trials.gov identifiers: NCT03005327, NCT02823405, NC T02923531 and NCT02667886).

[0034] The small molecule peptide antagonist of CXCR4, T140, and its analogs for blocking CXCR4 TN14003, TC14012, and BKT140 (also known herein as BL-80 40, TF14016, 4F-benzoyl-TN14003, BioLineRx,Lt d.) Efficacy of stem cell mobilization, small cell lung cancer (SCLC), breast cancer, melanoma, AML, It has been demonstrated in numerous preclinical studies, including in chronic myeloid leukemia, MM, pancreatic cancer, and rheumatoid arthritis. Currently, BKT140 is involved in various hematopoietic It is currently in clinical development for mobilizing hematopoietic stem and progenitor cells in organ malignancies (ClinicalTrials.gov / clinicaltrials ... rials.gov identifiers: NCT02502968, NCT03154827, NCT 02763384, NCT02639559 and NCT02462252). In addition, B KT140 was shown to inhibit tumor microenvironment interactions in subjects with AML. It is currently undergoing Phase II clinical trials in combination with vincristine (ClinicalTrials.gov Identifier: NCT02502968). It is also used in the treatment of various cancers, such as AML, gastric cancer, Immune checkpoint inhibitors have also been shown to be effective in non-small cell lung cancer (NSCLC) and metastatic pancreatic cancer, e.g. For example, pembrolizumab (Keytruda, Merck) and atezolizumab (Tec Intriq, Genentech / Roche) are being investigated (Clinical alTrials.gov identifier: NCT03154827, NCT03281369, NCT03337698, NCT02907099, NCT03193190 and NCT 02826486).

[0035] Similarly, CTCE-9908 (Chemokine), a peptide inhibitor of CXCR4, Therapeutics Corp., Canada) is a cancer treatment for osteosarcoma and melanoma. It reduced metastasis in a mouse model (Kim et al., 2008). After a Phase I trial in cancer patients, CTCE-9908 was approved by the FDA in 2005 for the treatment of osteosarcoma. It was designated as an orphan drug for the treatment of rheumatoid arthritis. Phase I / II clinical trials were completed in 2008. There has been no further development since the project was completed (Debnath, et al., 2013).

[0036] MSX-122 (Altiris Therapeutics), a small molecule CXCR4 antagonist, ics) was abandoned after clinical trials in patients with solid tumors (ClinicalTrial s.gov identifier: NCT00591682). In 2017, CXCR4 was approved as an anticancer drug. Two major setbacks in the field of 4 antagonist development have been reported. Bristol-Myers Squibb (BMS) is a clinical trial leader in the development of urocupulumab, a fully human anti-CXCR4 antibody, for the treatment of solid tumors. Phase I / II study in patients (BMS-936564 / MDX1338, (Kuhne et al., 2013) was discontinued due to lack of validity (https: / / see kingalphacom / article / 4057548-bristol-fai lure-makes-small-dent-cxcr4-blocking-app roach?page=2). Eli Lilly is also developing a CXCR4 peptide antagonist The few anti-cancer programs, including LY2510924, have been abandoned due to the lack of solid tumors. A series of clinical trials in (ClinicalTrials.gov identifier: NCT027 37072, NCT01391130 and NCT01439568) was determined after ttp: / / wwwfiercebiotechcom / biotech / lilly- puts-two-thirds-mid-phase-cancer-pipelin e-up-for-sale-major-shake-up-rd-priorit These cases suggest that the development of CXCR4 antagonists as anticancer drugs is crucial, especially for solid tumors. This shows that cancer remains a challenging problem.

[0037] Other small molecule antagonists currently in clinical investigation include:

[0038] USL311 (Upsher-Smith) for solid tumors and glioblastoma multiforme (GBM) are in Phase I and II in patients, respectively (ClinicalTrials.g ov identifier: NCT02765165), GMI-1359 (Glycomimetic s) is currently in Phase I trials (ClinicalTrials.gov identifier: NCT02 931214).

[0039] PF-06747143 (Pfizer), a humanized anti-CXCR4 antibody, is being investigated for the treatment of AML patients. It is currently undergoing phase I clinical trials in patients with glaucoma, either alone or in combination with chemotherapy (Clin icalTrials.gov identifier: NCT02954653, (Liu et al .,2017)).

[0040] AD-114 (i-body, AdAlta, (Griffiths et al.,2 018) is a humanized shark anti-CXCR4 antibody approved by the US FDA in 2017 for the treatment of idiopathic pulmonary edema. Orphan drug designation was granted for a drug candidate used for the treatment of fibrosis ( https: / / www.reuters.com / article / idUSFWNl F70Y0). The AD-114 study is primarily aimed at treating wet age-related macular degeneration and non-alcoholic fatty liver disease. It focuses on fibrotic conditions, including liver disease (https: / / lungdisease news.com / 2017 / 08 / 23 / adalta-present-resea rch-on-investigative-therapy-ad-114-at-i pf-summit / ).

[0041] POL6326 (Valixafortide, Polypho r) were subjects with breast cancer (ClinicalTrials.gov identifier: NCT01 837095), hematopoietic malignancies (ClinicalTrials.gov identifier: NC T01413568), and stem cell mobilization and healing in patients with acute myocardial infarction (Cl Clinical evaluation in the National Trials.gov identifier: NCT01905475 It is in the middle.

[0042] Radiolabeled CXCR4 ligands, such as [99mTc]O2-AMD3100 and [68 Ga]Pentixafor was evaluated for preclinical and clinical CXCR4 expression using SPECT or PE. T imaging ( Demmer et al., 2011 , Goumi et al. al., 2011, Hartimath et al., 2013, Walenkam p et al., 2017). PET imaging of [68Ga]pentixafor , hematopoietic and solid tumors, such as leukemia, lymphoma, MM, adrenocortical carcinoma and SCLC as well as in other conditions such as splenopathy, stroke, atherosclerosis and myocardial infarction. We also demonstrated increased expression of CXCR4 in the endothelial cells (Walenkamp et al., 2012). 017).

[0043] Peptide CXCR4 ligands ([177Lu]pentixa) labeled with alpha or beta emitters [90Y]pentixatel has been shown to treat over 30 cases of hematopoietic malignancies. It has been tested alongside standard chemotherapy as CXCR4-targeted internal radiation therapy in the treatment of (Walenkamp et al., 2017).

[0044] Various CXCR4 antagonistic drugs or CXCR4-directed antibodies have been developed and are currently undergoing clinical trials. alone, in combination with conventional anti-cancer therapy, or with immune checkpoint inhibitors. However, to date, there have been limited successes (Peled et al., 2012, De bnath, et al., 2013, Walenkamp, ​​et al., 2017) Because of the pivotal role of CXCR4 in B and T lymphocyte development and immune surveillance, CXC Long-term or sustained inhibition of CXCR4 by R4 antagonists leads to dysfunction of the immune system and hematopoietic organs. This may cause cancer patients to be at risk for immunosuppression (Burger, 200 9) Hematopoietic stem cells (HSCs) are normally protected in the bone marrow niche. CXCR4 antagonists When administered with cytotoxic drugs or radiation therapy, mobilized HSCs in the periphery are converted into blood cells. Patients will be exposed to the effects of cytotoxic treatments that may exacerbate the disease. The cardiac complications observed in patients treated with CXCR4 antagonists as anticancer drugs are also related to the long-term effects of CXCR4 antagonists. This has raised general concerns about its use.

[0045] To avoid potential side effects associated with conventional CXCR4 antagonists and to target CXCR4 We are exploring new strategies for designing CXCR4 inhibitors to develop more targeted and effective anti-cancer drugs. A new paradigm is urgently needed.

[0046] In recent years, GPCR heteromers have become a novel approach to develop more specific and disease-specific therapeutics. This raises new possibilities. GPCRs have traditionally been thought to be monomers. However, monomeric GPCRs induce a conformational change in the heptahelical domain upon ligand binding. This is because the induction of G-proteins can activate them (Pin et al. al., 2008, Okada et al., 2001). However, GPCR demonstrated that can form either homo- or hetero-oligomers. There is growing evidence that GPCR heteromers are involved in the regulation of these proteins and their interactions with existing, well-defined proteins. Intrinsic properties that distinguish it from the monomeric form, e.g., signaling pathways, ligand binding It can exhibit affinity, internalization, and recycling (De Falco et al., 2007, F erre et al., 2010, Gomes et al., 2016). Therefore, GPCR oligomerization increases the diversity of GPCR entities with a limited number of genes. This may provide a way to Identification of novel GPCR heteromers will lead to the development of more effective therapeutic agents with fewer side effects. This not only provides new opportunities for the identification of GPCR heterogeneity in specific tissues and specific diseases, but also allows for the identification of GPCR heterogeneity in specific tissues and specific diseases. This will offer new possibilities for understanding the role of Romer (Milli gan 2008, Rozenfeld and Devi 2010, Gomes e t al., 2016, Farran 2017).

[0047] CXCR4 also forms heteromers with various GPCRs. The receptor family of GPCRs (CCR2 (Rodriguez-Frade et al. ,2004, Sohy et al.,2007, Sohy et al.,2009, Armando et al., 2014), CCR5 (Agrawal et al. ,2004, Rodriguez-Frade et al.,2004, Sohy e t al., 2007, Sohy et al., 2009, Martinez-Mun oz et al., 2014), CXCR3 (Watts et al., 2013) , and CXCR7 (Sierro et al., 2007, Levoye et al. .,2009, Decaillot et al.,2011)), chemerin chemokine -like receptor 1 (CMKLR1) ( de Poorter et al., 2013 ), δ- opioid receptors (OPRD) (Pello et al. 2008, Burbassi et al., 2010), and the adrenergic receptor family (ADRA1A(Tr ipathi et al., 2015), ADRA1B (Tripathi et al., 2015), ADRA1B (Tripathi et al. l., 2015), and ADRB2 (LaRocca et al. 2010, Naka i et al., 2014) have been known to interact with

[0048] The presence of CXCR4-CCR2 and CXCR4-CCR5 heteromers has been shown to be important in the study of HIV infection. Rodriguez-Frade et al. CCR2-01, a CCR2-specific monoclonal antibody, has been shown to bind to CCR2. did not compete with CCL2 or trigger CCR2 signaling, but Monocyte tropism by inducing oligomerization of CR2 with CCR5 or CXCR4 showed that it inhibited the replication of T-tropic (R5) and T-tropic (X4) HIV strains (Rodriguez et al., 2004). ez-Frade et al., 2004). Agrawal et al. R5D32 heteromerizes with CCR5 and CXCR4, Specifically inhibiting cell surface expression, thereby inhibiting CCR5 and CXC on CD4+ cells showed that R4 inhibited the HIV coreceptor activity (Agrawal et al., 2014). 004). Co-expression of CCR5 is associated with CCR5-CD4-CXCR4 oligomerization and C By inducing conformational changes in D4 and CXCR4, HIV-1 gp120 It has been shown to prevent X4 HIV-1 from binding to the cell surface and reduce X4 HIV-1 infectivity (Ma rtinez-Muñoz et al., 2014).

[0049] Sohy et al. There is negative binding cooperativity between the two subunits, i.e., both in recombinant cell lines and primary Ligands for one receptor compete for binding of a specific tracer for the other in leukocytes reported that (Sohy et al., 2007, Sohy et al., 2 009). They also found that CCR2 and CXCR4, or CCR5 and CXCR4, co-express. In cells expressing CXCR4, TAK-779, a CCR2 and CCR5 antagonist, inhibits CXCR4 expression. Prevented calcium mobilization and chemotaxis initiated by the CR4 agonist CXCL12 (Sohy et al., 2007, Sohy et al., 2009) Armando et al. reported that CXCR4 and CCR2 are homo- and hetero-oligomers. and human monocyte chemotactic protein 1 (MCP-1) and Coactivation of CCR2 and CXCR4 by CXCL12 synergistically enhances calcium mobilization (Armando et al., 2014).

[0050] Watts et al. The endogenous agonist and the small molecule CXCR3 agonist VUF10661 showed negative results. CXCR3 antagonist VUF10085 and CXCR4 antagonist AMD310, which are synergistic 0 showed that this was not the case (Watts et al., 2013).

[0051] CXCR4 is a member of the chemokine family GPC that binds to CXCL12 and CXCL11. R and also interacts with ACKR3, also known as CXCR7, but binds agonists and cannot bind to G proteins. Sierro et al. 3 cells, showed enhanced CXCL12-induced calcium signaling and upregulated ERK1 / 2 signaling. We identified the CXCR4-ACKR3 heteromer as a functional heteromer that alters signal transmission. (Sierro et al., 2007). Lovoye et al. CXCR4-ACKR3 heteromers are formed in HO-K1 cells upon exposure to CXCL12. reported opposite results showing that αi and calcium responses were reduced (Levo ye et al., 2009). Decaillot et al. Co-expression of CXCR4 and ACKR3 constitutively binds β-arrestin to CXCR4 / ACKR3 heteromers. CXCL12-mediated β-arrestin-dependent recruitment of ERK1 / 2 and p38MAPK This enhanced downstream signaling pathways and enhanced cell migration upon exposure to CXCL12. acknowledged (Decaillot et al., 2011).

[0052] CXCR4 is a chemokine-like receptor 1 (CMKLR1, also known as ChemR23) It also interacts with CXCR4-C (de Poorter et al., 2013). MKLR1 heteromers were synthesized as described by Sohy et al. (Sohy et al., 2007 , Sohy et al., 2009) It exhibits negative agonist binding cooperativity similar to that of the XCR4-CCR5 heteromer, but AMD31 00 did not cross-inhibit chemerin binding or CXCL12-induced did not inhibit calcium mobilization ( de Poorter et al., 2013 ).

[0053] CXCR4 and δ-opioid receptors (DOR) are widely distributed in brain tissue and immune cells. Pello et al. reported that CXCR4 and DOR form heteromers. Although individual agonists can elicit robust Gαi signaling, Co-stimulation with both agonists suppresses activation of Gαi signaling and promotes activation of CXCL12. reported that it prevented cell migration in Burton et al. (Pello et al., 2008). Bassi et al. also used brain tissue from mice lacking the μ-opioid receptor (MOR). In cultured glial cells, the increase in CXCR4-DOR heteromers and the interaction between CXCR4 and G proteins Decreased binding to proteins was observed (Burbassi et al., 2010). 4 function was rescued by a DOR antagonist, suggesting that DOR regulates CX in glial cells. These results suggest that the formation of CXCR4-DOR heteromers is involved in the suppression of CXCR4. (Burbassi et al., 2010).

[0054] CXCR4 interacts with α- and β-adrenergic receptors (α-AR and β-AR) It is also known that the vasoconstriction of the vasoconstriction occurs in adult rat ventricular myocytes. In this study, the stimulation of CXCR4 by CXCL12 was suppressed by the selective β-AR agonist isoproterenol. Negatively regulating β-AR-induced cAMP accumulation and PKA-dependent phosphorylation of phospholamban using reported that co-immunoprecipitation of β-lactamase inhibitors (LaRocca et al., 2010) CXCR4 and ADRB2(β) on cardiomyocytes using ion channels and bioluminescence resonance energy transfer (BRE) 2-AR) and physical association with CXCR4 and ADRB2, These results suggest the CXCR4-ADRB2 heteromer as a potential therapeutic agent.

[0055] Nakai et al. investigated the function of ADRB2 in lymphocytes. Stimulation of lymphocytes with a 2-selective agonist inhibits lymphocyte egress from lymph nodes and induces lymphocyte release in mice. ADRB2 caused lymphopenia in the thyroid gland (Nakai et al., 2014). ADRB2 physically interacts with CCR7 and CXCR4, and activation of ADRB2 is mediated by CCR7-ADR B2 and CXCR4-ADRB2 heteromers enhance the retention-promoting signal, which then leads to reactivation. It reduced lymphocyte egress from lymph nodes.

[0056] Tripathi et al. reported that CXCR4 on the surface of vascular smooth muscle cells (VSMCs) -ADRA1A (α1A-AR) and CXCR4-ADRA1B (α1B-AR) heterozygotes (Tripathi et al., 2015). Peptides derived from the second transmembrane helix of XCR4 bind to ADRA1A / B and CXCR4 It disrupts the interaction with α1-AR, inhibiting calcium mobilization and VSMC concentration upon α1-AR stimulation. CXCR4 activation by CXCL12 significantly altered α1-AR agonism on blood pressure responses in rats. This may enhance the efficacy of drugs and suggest that CXCR4-ADRA1A / B heteromers may be a novel therapeutic option for blood pressure regulation. This suggests that it may be a pharmacological target.

[0057] CXCR4 interacts with CNR2 (cannabinoid receptor 2, also known as CB2) have been reported (Coke et al., 2016, Scarlett et al., 2018). Coactivation of CXCR4 and CB2 by both agonists results in the upregulation of ERK1. α / 2 activation, calcium mobilization, and cell chemotaxis were reduced. These results suggest that cannabinoids These results indicate that the id system can negatively regulate CXCR4 function and tumor progression.

[0058] As mentioned above, GPCRs that form heteromers with CXCR4 are limited in number. Within the PCR family, e.g., chemokine, adrenergic, and opioid receptor families The key role of CXCR4 and its increased expression in various pathologies have been investigated. Considering this, various CXCR4-GPCRx confer unique characteristics for specific diseases. It is highly likely that heteromers exist. However, due to the large number of GPCR genes ( Approximately 800), as well as high-throughput proximity-based screening techniques and functional assays. Identification of new CXCR4-GPCRx heteromers has been largely hampered by the difficulty of establishing them. It was a difficult problem. Summary of the Invention

[0059] technical challenges Thus, the art provides functional GPCR heteromers, e.g., CXCR4-GPCR Identifying Rx and developing GPCR heteromer-directed cancer treatments with higher efficacy and fewer side effects There is a need to develop inhibitors of these compounds for use as medicines. The present invention addresses this need. and provide related benefits.

[0060] Means to solve the problem The present inventors have demonstrated the ability to rapidly and efficiently generate multiple recombinant adenoviruses simultaneously. Adenovirus high-throughput system (AdHTS) (Choi, et al., 2012), and adenovirus-based bimolecular fluorescence complementation (BiFC) assay (patent, Song, 2014) was used to detect the activity of many GPCRs in U-2 OS cells. We screened for those that have an association with CXCR4.

[0061] In one aspect, the present invention provides a method for treating or ameliorating cancer in a subject having a CXCR4-GPCRx heteromer. A method for improving, preventing or diagnosing a CXCR4-GPCRx comprising administering to a subject a therapeutically effective amount of CXCR4-GPCRx. administering an inhibitor of a telomer thereof, wherein GPCRx is heterodimerized with CXCR4 in the subject. Heteromerization of GPCRx with CXCR4 leads to downstream signaling of CXCR4. Enhanced signaling occurs, and enhanced signaling downstream of CXCR4 leads to CXCR4-G Such methods are provided herein, which are inhibited by inhibitors of PCRx heteromers.

[0062] In another embodiment, the cancer in a patient having cells containing CXCR4-GPCRx heteromers. Provided herein are methods for treating a disease, the methods comprising administering an inhibitor of CXCR4, an inhibitor of GPCRx, an inhibitor or inhibitors selected from the group consisting of inhibitors of CXCR4-GPCRx heteromers; or a combination of inhibitors,

[0063] i) CXCR4-GPCRx heteromers have enhanced downstream signaling and

[0064] ii) The administered inhibitor or combination of inhibitors is effective in preventing the above-mentioned CXCR4- Suppresses enhanced downstream signaling from GPCRx heteromers.

[0065] In another embodiment, a CXCR4-GPCRx heterodimer in cells of a patient suffering from cancer is Provided herein are methods for suppressing enhanced downstream signaling from a marker, the methods comprising: , CXCR4 inhibitors, GPCRx inhibitors, and CXCR4-GPCRx heteromer inhibitors and administering to the patient an inhibitor or combination of inhibitors selected from the group consisting of: ,

[0066] i) CXCR4-GPCRx heteromers have enhanced downstream signaling and

[0067] ii) The administered inhibitor or combination of inhibitors is effective in preventing the above-mentioned CXCR4- Suppresses enhanced downstream signaling from GPCRx heteromers.

[0068] In another embodiment, the cancer in a patient having cells containing CXCR4-GPCRx heteromers. A pharmaceutical kit for use in the treatment of a patient, comprising: a CXCR4 inhibitor; a GPCRx inhibitor; and an inhibitor of CXCR4-GPCRx heteromers, or CXCR4-GPCRx heteromers, including combinations of inhibitors, enhance downstream signaling Provided herein are pharmaceutical kits comprising:

[0069] In another embodiment, the cancer in a patient having cells containing CXCR4-GPCRx heteromers. Provided herein is a pharmaceutical composition for use in the treatment of

[0070] i) CXCR4 inhibitors, GPCRx inhibitors and CXCR4-GPCRx heteromers an inhibitor or combination of inhibitors selected from the group consisting of:

[0071] ii) comprising a pharmaceutically acceptable carrier;

[0072] CXCR4-GPCRx heteromers have enhanced downstream signaling do.

[0073] In another embodiment, the method comprises administering to a subject having cancer cells containing CXCR4-GPCRx heteromers. A method of treating cancer, comprising administering to a subject a CXCR4-GPCRx heteromer, an antibody to ...

[0013] Provided herein is a method for the treatment of a pulmonary artery disease, the method comprising:

[0074] 1) CXCR4-GPCRx heteromers with enhanced downstream signaling The determination of whether a patient has cancer cells is carried out by obtaining or having obtained a biological sample from the patient. and for biological samples

[0075] i) whether the patient's cancer cells contain the above-mentioned CXCR4-GPCRx heteromer; and teeth

[0076] ii) CXCR4-GPCRx heteromer-selective reagents: The heteromer-specific properties or functions of the CXCR4-GPCRx heteromer are altered. whether or not to use patient-derived cell(s) containing CXCR4-GPCRx heteromers; Whether or not to alter telomer-specific properties; or contain CXCR4-GPCRx heteromers and performing an assay to determine whether the antibody reduces cell proliferation of the patient-derived cell(s). and

[0077] 2) When a patient has cancer cells containing the above-mentioned CXCR4-GPCRx heteromer, , CXCR4 inhibitors, GPCRx inhibitors, and CXCR4-GPCRx heteromer inhibitors and administering to a cancer patient an inhibitor or a combination of inhibitors selected from the group consisting of Includes:

[0078] In another aspect, Methods for treating cancer in patients with cancer cells containing CXCR4-GPCRx heteromers A method is provided herein, the method comprising:

[0079] 1) determining whether the patient's cancer cells contain CXCR4-GPCRx heteromers; Obtaining or obtaining a biological sample from a patient and injecting the above-mentioned CXCR4-GPC into the patient's cancer cells An assay to determine whether the Rx heteromer is present is performed on the biological sample. including by doing or performing,

[0080] a) The GPCRx in the CXCR4-GPCRx heteromer is ADCYAP1R1, ADO RA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNR B, HRH1, MLNR, NTSR1 and TACR3;

[0081] b) The assays performed on the biological sample include: co-internalization assay, co-localization assay, Sei, in situ hybridization, immunohistochemistry, immunoelectron microscopy, proximity one or more of the following: a fluorescence-based assay, a co-immunoprecipitation assay, or a fluorescent animal assay and

[0082] 2) When the patient's cancer cells contain the above CXCR4-GPCRx heteromer, CXC R4 inhibitors, GPCRx inhibitors, and CXCR4-GPCRx heteromer inhibitors The method comprises administering to a patient an inhibitor or combination of inhibitors selected from the group consisting of:

[0083] In another embodiment, a CXCR4-GPCRx heteromer with enhanced downstream signaling is provided. Provided herein are methods for treating cancer in a patient having cancer cells containing teeth,

[0084] 1) CXCR4-GPCRx heteromers with enhanced downstream signaling The determination of whether a patient has cancer cells is made by obtaining or following obtaining a biological sample from the patient. , for biological samples

[0085] i) whether the patient's cancer cells contain the above-mentioned CXCR4-GPCRx heteromer; and teeth

[0086] ii) CXCR4-GPCRx heteromer-selective reagents: The heteromer-specific properties or functions of the CXCR4-GPCRx heteromer are altered. or whether or not to inject the patient-derived cell(s) containing the CXCR4-GPCRx heteromer. whether or not to alter the heteromer-specific properties of the CXCR4-GPCRx heteromer; an assay to determine whether a patient-derived cell(s) containing the compound reduces cell proliferation and further including by performing or having performed:

[0087] 2) When a patient has cancer cells containing the above-mentioned CXCR4-GPCRx heteromer, , CXCR4 inhibitors, GPCRx inhibitors, and CXCR4-GPCRx heteromer inhibitors administering to a cancer patient a combination of inhibitors selected from the group consisting of:

[0088] 3) The patient does not have cancer cells containing the above CXCR4-GPCRx heteromer. In these cases, either a CXCR4 inhibitor or a GPCRx inhibitor can be used as a single inhibitor to treat cancer patients. This includes administering the compound to a subject.

[0089] In another embodiment, the method comprises administering to a subject having cancer cells containing CXCR4-GPCRx heteromers. Provided herein are methods of treating cancer, the methods comprising:

[0090] 1) determining whether the patient's cancer cells contain CXCR4-GPCRx heteromers; Obtaining or obtaining a biological sample from a patient and injecting the above-mentioned CXCR4-GPC into the patient's cancer cells An assay to determine whether the Rx heteromer is present is performed on the biological sample. including by doing or performing,

[0091] a) The GPCRx in the CXCR4-GPCRx heteromer is ADCYAP1R1, ADO RA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNR B, HRH1, MLNR, NTSR1 and TACR3;

[0092] b) The assays performed on the biological sample include: co-internalization assay, co-localization assay, Sei, in situ hybridization, immunohistochemistry, immunoelectron microscopy, proximity degree-based assay, co-immunoprecipitation assay, enzyme-linked immunosorbent assay (ELISA), Flow cytometry, RNAseq, qRT-PCR, microarrays or fluorescent animals assays; and

[0093] 2) When the patient's cancer cells contain the above CXCR4-GPCRx heteromer, CXC R4 inhibitors, GPCRx inhibitors, and CXCR4-GPCRx heteromer inhibitors administering to a cancer patient a combination of inhibitors selected from the group consisting of:

[0094] 3) When the patient's cancer cells do not contain the above CXCR4-GPCRx heteromers In addition, we are considering administering either a CXCR4 inhibitor or a GPCRx inhibitor as a single inhibitor to cancer patients. This includes administering the drug intravenously.

[0095] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this context, heteromerization of CXCR4 with GPCRx was demonstrated by proximity-based assays. and is evaluated accordingly.

[0096] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In general, proximity-based assays include bimolecular fluorescence complementation (BiFC), proximity ligation, and fluorescence resonance energy transfer (FRET), bioluminescence resonance energy assay (PLA) Transfer (BRET), cysteine ​​cross-linking and co-immunoprecipitation, and TR-FRET and SNAP The combination of the tag and the It is selected.

[0097] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this study, heteromerization of CXCR4 with GPCRx was assessed by co-internalization assays. can be.

[0098] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, the enhancement of cell signaling downstream of CXCR4 was confirmed by intracellular Ca2+ assay. It is evaluated as follows.

[0099] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, APLNR, C5AR1, CALCR, CCR5, CHRM1, GALR1, ED NRB, HRH1, MLNR, NTSR1, PTGER2, PTGER3, SSTR2 and and TACR3.

[0100] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTS R1, PTGER2 and TACR3.

[0101] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTS R1 and TACR3.

[0102] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TA CR3.

[0103] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3 The compound is selected from the group consisting of:

[0104] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, CHRM1, EDNRB, HRH1, NTSR1 and TACR3 It is selected.

[0105] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADCYAP1R1, ADORA2B, ADORA3, ADR B2, CHRM1, HRH1, NTSR1 and TACR3.

[0106] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx are ADORA2B, ADORA3, ADRB2, CHRM1, E It is selected from the group consisting of DNRB, HRH1, NTSR1 and TACR3.

[0107] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, GPCRx include ADRB2, CHRM1, EDNRB, HRH1, and NTSR1. and TACR3.

[0108] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, GPCRx include ADRB2, CHRM1, EDNRB, HRH1, and TACR 3.

[0109] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the GPCRx is a member of the group consisting of ADRB2, CHRM1, HRH1, and TACR3. is selected from.

[0110] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the GPCRx is a member of the group consisting of ADRB2, EDNRB, HRH1, and TACR3. is selected from.

[0111] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the GPCRx is selected from the group consisting of ADRB2, EDNRB, and HRH1. do.

[0112] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the GPCRx is selected from the group consisting of ADRB2, CHRM1, and HRH1. do.

[0113] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the GPCRx is selected from the group consisting of ADRB2, HRH1, and TACR3. do.

[0114] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the GPCRx is selected from the group consisting of ADRB2 and HRH1.

[0115] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, an inhibitor of the CXCR4-GPCRx heteromer is an inhibitor of CXCR4, or or includes it.

[0116] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of CXCR4 inhibitors, AD-114, AD-114-6H, AD-114- Im7-FH, AD-114-PA600-6H, ALX-0651, ALX40-4C , AMD070 (AMD11070, X4P-001), AMD3100 (Prelixaf ), AMD3465, ATI2341, BKT140(BL-8040, TF140 16, 4F-benzoyl-TN14003), CTCE-9908, CX549, D-[ Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK8123 97, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t ), KRH-1636, KRH-3955, LY2510924, LY2624587, MSX-122, N-[ 11 C]methyl-AMD3465, PF-06747143, P OL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T 140, T22, TC14012, TG-0054 (Binxaphor), USL311, Ulocuplumab (MDX1338 / BMS-936564), viral macrophage inflammation virulence protein-II (vMIP-II), WZ811, 12G5, 238D2, ​​238 D4, [ 64 Cu]-AMD3100,[ 64 Cu]-AMD3465, [ 68 Ga]pe Ntixafor, [ 90 Y]Pentixatel,[ 99m Tc]O2-AMD3100,[ 177 Lu]pentixatel, and 508MCl (Compound 26). do.

[0117] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, inhibitors of CXCR4-GPCRx heteromers are antagonists of CXCR4, or or includes it.

[0118] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, CXCR4 antagonists include ALX40-4C, AMD070 (AMD11070) and , X4P-001), AMD3100 (Plerixafor), AMD3465, ATI2 341, BKT140(BL-8040, TF14016, 4F-benzoyl-TN14 003), CTCE-9908, CX549, D-[Lys3]GHRP-6, FC12 2, FC131, GMI-1359, GSK812397, GST-NT21MP, Iso Thiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3 955, LY2510924, MSX-122, N-[11C]methyl-AMD3465 , POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134 , T140, T22, TC14012, TG-0054 (Binxaphor), USL31 1, viral macrophage inflammatory protein-II (vMIP-II), WZ811; [64Cu]-AMD3100, [64Cu]-AMD3465, [68Ga] Penchiki Saffol, [90Y]pentixatel, [99mTc]O2-AMD3100, [177 Lu] pentixatel, and 508MCl (Compound 26).

[0119] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of the CXCR4-GPCRx heteromer is an antibody to CXCR4; includes it.

[0120] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of form, CXCR4 antibodies are AD-114, AD-114-6H, and AD-114-I. m7-FH, AD-114-PA600-6H, ALX-0651, LY2624587 , PF-06747143, urocupulumab (MDX1338 / BMS-936564) , 12G5, 238D2 and 238D4.

[0121] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of a CXCR4-GPCRx heteromer is an inhibitor of GPCRx, Or further includes it.

[0122] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, the inhibitor of GPCRx is administered concurrently or sequentially with the inhibitor of CXCR4. can be.

[0123] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some forms, inhibitors of CXCR4-GPCRx heteromers act as antagonists or inverse agonists of GPCRx. , an allosteric modulator, an antibody or binding portion thereof, a ligand, or any combination thereof Being or containing.

[0124] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, inhibitors of GPCRx include ADCYAP1R1, ADORA2B, and ADORA3. , ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR , an inhibitor of a molecule selected from the group consisting of NTSR1, PTGER2 and TACR3. do.

[0125] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADCYAP1R1, ADORA2B, and ADORA3. , ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR , NTSR1 and TACR3.

[0126] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADCYAP1R1, ADORA2B, and ADORA3. , ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3.

[0127] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADCYAP1R1, ADORA2B, and ADORA3. , ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR 3.

[0128] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADCYAP1R1, ADORA2B, and ADORA3. , ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3 The inhibitor is a molecule selected from the group:

[0129] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADCYAP1R1, ADORA2B, and ADORA3. , ADRB2, CHRM1, HRH1, NTSR1, and TACR3. It is an inhibitor of a molecule that

[0130] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, inhibitors of GPCRx include ADORA2B, ADORA3, ADRB2, and CHR. a molecule selected from the group consisting of M1, EDNRB, HRH1, NTSR1, and TACR3 is an inhibitor of

[0131] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADRB2, CHRM1, EDNRB, HRH1, and N It is an inhibitor of a molecule selected from the group consisting of TSR1 and TACR3.

[0132] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx include ADRB2, CHRM1, EDNRB, HRH1, and and TACR3 inhibitors.

[0133] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, inhibitors of GPCRx are ADRB2, CHRM1, HRH1, and TACR3. The inhibitor is a molecule selected from the group consisting of:

[0134] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In particular, inhibitors of GPCRx are ADRB2, EDNRB, HRH1, and TACR3. The inhibitor is a molecule selected from the group consisting of:

[0135] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of GPCRx is selected from the group consisting of ADRB2, EDNRB, and HRH1. It is an inhibitor of the molecule of choice.

[0136] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of GPCRx is selected from the group consisting of ADRB2, CHRM1, and HRH1. It is an inhibitor of the molecule of choice.

[0137] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of GPCRx is selected from the group consisting of ADRB2, HRH1, and TACR3. It is an inhibitor of the molecule of choice.

[0138] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of GPCRx is a molecule selected from the group consisting of ADRB2 and HRH1. It is an inhibitor of the offspring.

[0139] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of form, inhibitors of GPCRx include M65, Max.d.4, MK-0893, and N-stearyl-2-phosphate dehydrogenase (N-STD). Allyl-[Nle 17 ]Neurotensin-(6-11) / VIP-(7-28), PA ADCYAP1 selected from the group consisting of CAP-(6-38), and PG97-269 It is an inhibitor of R1.

[0140] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of inhibitors of GPCRx, 3-isobutyl-8-pyrrolidinoxanthine, aloxal Sajin, AS16, AS70, AS74, AS94, AS95, AS96, AS99, A S100, AS101, ATL802, BW-A1433, caffeine, CGS1594 3, CPX, CSC, CVT-6883, DAX, DEPX, delenophylline (dere nofylline), DPCPX, FK-453, I-ABOPX, Istradefylline N, KF26777, LAS38096, LUF5981, MRE2029F20, MR E3008F20, MRS1191, MRS1220, MRS1523, MRS1706 , MRS1754, MSX-2, OSIP339391, Pentoxifylline, Prelade Nantes, PSB-10, PSB-11, PSB36, PSB603, PSB-0788, PSB1115, rolofylline, SCH58261, SCH442416, ST-153 5, theophylline, tonapofylline, bipadenant, xanthine amines, XCC, and and ZM-241385, an inhibitor of ADORA2B selected from the group consisting of:

[0141] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of form, inhibitors of GPCRx include ATL802, BW-A1433, caffeine, and CG S15943, CSC, CVT-6883, delenophilin, dexniguldipine, DP CPX, FK-453, flavanone, flavone, galangin, I-ABOPX, Istra Defylline, KF26777, LAS38096, LUF5981, MRE2029F2 0, MRE3008F20, MRE3010F20, MRS1041, MRS1042, MRS1067, MRS1088, MRS1093, MRS1097, MRS1177, MRS1186, MRS1191, MRS1191, MRS1220, MRS1476, MRS1486, MRS1505, MRS1523, MRS1754, MRS928, M SX-2, nicardipine, preladenant, PSB-10, PSB-11, PSB36, PSB603, PSB1115, rolofylline, sakuranetin, SCH58261, SC H442416, ST-1535, Theophylline, Tonapofylline, Bipadenant, Bis Nagin, VUF5574, VUF8504, VUF8507, xanthine amines and Z and M-241385, an inhibitor of ADORA3.

[0142] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of inhibitors of GPCRx, alprenolol, atenolol, betaxolol, Bupranolol, butoxamine, carazolol, carvedilol, CGP12177, Cloprolol, ICI118551, ICYP, labetalol, levobetaxolol, Levobunolol, LK204-545, metoprolol, nadolol, NIHP, NIP , propafenone, propranolol, sotalol, SR59230A and timolol and an inhibitor of ADRB2 selected from the group consisting of:

[0143] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of ADRB2 is carvedilol.

[0144] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of GPCRx is an inhibitor of C5AR1, and the inhibitor is A8 Δ71 -73 , AcPhe-Orn-Pro-D-Cha-Trp-Arg, Avacopan, C0 89, CHIPS, DF2593A, JPE1375, L-156,602, NDT95 20492, N-methyl-Phe-Lys-Pro-D-Cha-Trp-D-Arg- A group consisting of CO2H, PMX205, PMX53, RPR121154 and W54011 is selected from.

[0145] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of inhibitors of GPCRx, α-CGRP-(8-37) (human), AC187, CALCR selected from the group consisting of CT-(8-32) (salmon) and olcegepant It is an inhibitor.

[0146] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of CALCR is AC187.

[0147] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of inhibitors of GPCRx, 3-quinuclidinyl benzilate (QNB), 4-DA MP, aclidinium, AE9C90CB, AFDX384, amitriptyline, AQ- RA741, atropine, benzatropine, biperiden, darifenacin, dicyclomine , dosulepin, ethopropazine, glycopyrrolate, guanylpirenzepine, hexahydride Lodifenidol, hexahydrosiladifenidol, hexocyclium, himbacine, Ipratropium, lithocholylcholine, methoctramine, ML381, muscarinic toxin 1 , Muscarinic toxin 2, Muscarinic toxin 3, N-methylscopolamine, Otenzepado, Oxytocin Sibutinin, pF-HHSiD, pirenzepine, propantheline, (R,R)-quinucline Lysinyl-4-fluoromethyl-benzilate, scopolamine, silahexocyclium, Solifenacin, telenzepine, tiotropium, tolterodine, trihexyphenidyl , tripitramine, UH-AH37, umeclidinium, and VU0255035 The inhibitor of CHRM1 is selected from the group:

[0148] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of CHRM1 inhibitors, oxybutynin, umeclidinium, and VU0255 The number is 035.

[0149] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of form, inhibitors of GPCRx include A192621, ambrisentan, and atrasentan. , bosentan (RO470203, Tracleer), BQ788, IRL2500, K-8 794, Macitentan, RES7011, Ro46-8443, SB209670, SB 217242 (enrasentan), TAK044 and tezosentan (RO610612) The inhibitor of EDNRB is selected from the group consisting of:

[0150] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of EDNRB is bosentan.

[0151] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of inhibitors of GPCRx, (-)-chlorpheniramine, (+)-chlorpheniramine, Lamin, (-)-trans-H2-PAT, (+)-cis-H2-PAT, (+)-trans ance-H2-PAT, (±)-cis-H2-PAT, (±)-trans-H2-PAT, (R)-Cetirizine, (S)-Cetirizine, 9-OH-Risperidone, A-317920 , A-349821, ABT-239, Alimemazine, Amitriptyline, Aripiprazo ol, apromidine, asenapine, astemizole, AZD3778, azelastine, BU-E47, cetirizine, chlorpheniramine, chlorpromazine, ciproxifan , clemastine, clobenpropit, clozapine, conessine, cyclizine, cyproheptane Putadine, desloratadine, diphenhydramine, dosulepin, doxepin, epinastine fexofenadine, fluphenazine, fluspirilene, haloperidol, hydrochloride Cidin, Impromidine, INCB-38579, JNJ-39758979, Ketotif phenanthrene, loratadine, loxapine, MK-0249, molindone, olanzapine, perfecund Nadine, pimozide, pipamperon, pitolisant, promethazine, pyrilamine, quetia pine, risperidone, sertindole, terfenadine, thioridazine, thiothixene, Trifluoperazine, tripelennamine, triprolidine, ziprasidone and zotepine The inhibitor is an HRH1 inhibitor selected from the group consisting of:

[0152] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some forms, inhibitors of HRH1 are cetirizine, pyrilamine, hydroxyzine or loratadine. It's gin.

[0153] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of GPCRx inhibitors, GM-109, MA-2029, and OHM-1152 6.

[0154] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of MLNR is MA-2029.

[0155] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the form of GPCRx inhibitors, meclineltant, SR48527, SR48692 and SR142948A.

[0156] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In its form, the inhibitor of NTSR1 is mercurinertant. be.

[0157] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In the morphology, inhibitors of GPCRx are [Trp 7 , β-Ala 8 ]Neurokinin A-(4 -10), AZD2624, FK224, GR138676, GSK172981, GS K256471, N',2-diphenylquinoline-4-carbohydrazide 8m, N',2 -Diphenylquinoline-4-carbohydrazide, Osanetant, PD154740, PD 161182, PD157672, Saledutant, SB218795, SB22220 0, SB235375, SCH206272, SSR146977 and talnetant The TACR3 inhibitor is selected from the group consisting of:

[0158] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor of TACR3 is SSR146977.

[0159] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this context, inhibitors of CXCR4-GPCRx heteromers are known to inhibit protein-protein interactions (PPIs). ) inhibitor.

[0160] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of form, cancers include breast cancer, lung cancer, brain cancer, kidney cancer, pancreatic cancer, ovarian cancer, prostate cancer, melanoma multiple myeloma, gastrointestinal cancer, renal cell carcinoma, soft tissue sarcoma, hepatocellular carcinoma, gastric cancer, colon cancer, esophageal cancer The cancer is selected from the group consisting of thyroid cancer and leukemia.

[0161] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, the inhibitor of the CXCR4-GCPRx heteromer is in a pharmaceutical composition. It is administered to elephants.

[0162] Further provided herein are CXCR4-GPCRx inhibitors for the treatment, amelioration, or prevention of cancer. A method for assessing a response or potential response of a subject having a heteromer, comprising: obtaining a sample; detecting heteromerization of CXCR4 and GPCRx in the sample; and methods for treating cancer based at least in part on the detection of heteromerization between CXCR4 and GPCRx. the method, including assessing a subject's response or potential response to treatment, amelioration, or prevention. Disclose the law.

[0163] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this morphology, heteromerization of CXCR4 with GPCRx leads to downstream signaling of CXCR4. Enhancement of neural transmission occurs.

[0164] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, the enhancement of cell signaling downstream of CXCR4 was confirmed by intracellular Ca2+ assay. It is evaluated as follows.

[0165] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this context, heteromerization of CXCR4 with GPCRx was demonstrated by proximity-based assays. and is evaluated accordingly.

[0166] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In general, proximity-based assays include bimolecular fluorescence complementation (BiFC), proximity ligation, and fluorescence resonance energy transfer (FRET), bioluminescence resonance energy assay (PLA) transfer (BRET), cysteine ​​bridging and co-immunoprecipitation.

[0167] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this study, heteromerization of CXCR4 with GPCRx was assessed by co-internalization assays. can be.

[0168] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, CXCR4-GPCRx heteromers have enhanced downstream signaling To cause, bring about, or bring about.

[0169] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this morphology, enhanced downstream signaling is attributed to CXCR4-GPCRx heteromers. For example, it is due to the agonistic effect of CXCR4-GPCRx heteromers. This is due to the CXCR4 agonism of the CXCR4-GPCRx heteromer. Or it is due to the GPCRx agonistic action of CXCR4-GPCRx heteromers.

[0170] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this case, enhanced downstream signaling is observed in CXCR4, each GPCRx or CXC downstream of the R4-GPCRx heteromer.

[0171] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, an inhibitor or combination of inhibitors is administered to treat the above-mentioned CXCR4-GPCR in cancer patients. x heteromers, e.g., to suppress downstream signaling in cancer cells of patients. Enhanced downstream signaling from the CXCR4-GPCRx heteromer in cells Suppress the spread of

[0172] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, enhanced downstream signaling from CXCR4-GPCRx heteromers is associated with cell proliferation. Determined by intracellular Ca2+ assay.

[0173] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the enhanced downstream signaling is the amount of enhanced calcium mobilization.

[0174] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the amount of enhanced calcium mobilization is the amount of synergistic calcium mobilization.

[0175] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the CXCR4-GPCRx heteromer has two or more of the following characteristics: :

[0176] 1) Determine the following: co-internalization assay, co-localization assay, in situ hybridization immunoprecipitation assays, immunohistochemistry, immunoelectron microscopy, proximity-based assays, and co-immunoprecipitation assays. Intracellular CXCR4 as measured by one or more of the following assays: -GPCRx heteromeric components colocalize to mediate direct or allosteric interactions. that they physically interact through intermediate proteins that act as stages;

[0177] 2) As determined by calcium mobilization assay, a) CXCL12 and each GPC Upon costimulation with Rx, CXCR4 or each of the individual protomers in the context of the cells Either CXCL12 or the respective selective GPCRx agonist The amount of calcium mobilization resulting from single agonist stimulation by b) inhibiting CXCL12 and each of the selective GPCRs Upon costimulation with agonists, CXCR4-GPCRx heteromers bind to CXCL12 or each The amount of calcium mobilization resulting from single agonist stimulation with either selective GPCRx agonists and the enhanced calcium mobilization of the ATP-dependent ATPase, which exhibits enhanced calcium mobilization compared to the sum of both the ATP-dependent ATPase and the ATP-dependent ATPase. the amount of mobilization; or

[0178] 3) CXCR4-GPCRx heteromer-selective reagents are capable of: i) detecting CXCR4 in patient-derived cells; ii) alter the heteromer-specific properties of CR4-GPCRx heteromers; and ii) patient-derived cells. Altering the heteromer-specific function of CXCR4-GPCRx heteromers in ) Alter the heteromer-specific properties of patient-derived cells containing CXCR4-GPCRx heteromers or iv) patient-derived cells containing CXCR4-GPCRx heteromers ( Reducing cell proliferation of multiple cells.

[0179] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein Morphology was assessed using the following methods: co-internalization assay, co-localization assay, and in situ hybridization. lysis, immunohistochemistry, immunoelectron microscopy, proximity-based assays, co-immunoprecipitation Intracellular CXC activity as measured by one or more of the following assays: R4-GPCRx heteromeric components colocalize to mediate direct or allosteric signaling. They physically interact with each other through an intermediate protein that acts as a vehicle for delivery.

[0180] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein Proximity-based assays include resonance energy transfer (RET), bioluminescence RET, and (BRET), fluorescent RET (FRET), time-resolved fluorescent RET (TR-FRET), antibody FRET, ligand-assisted FRET, bimolecular fluorescence complementation (BiFC) or proximity ligation The present invention relates to a polymerase chain reaction (PLA) assay.

[0181] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this configuration, CXCR4-GPCRx heteromers exhibit enhanced calcium mobilization. ,this is,

[0182] a) Individual intracellular responses upon co-stimulation with CXCL12 and each selective GPCRx agonist Either CXCR4 or each GPCRx in the context of the protomers of CXCL1 Calcium production resulting from single agonist stimulation by either two or each of the selective GPCRx agonists and resulting in calcium mobilization equal to or less than the sum of the ammonium mobilization and the phosphate ion transporter.

[0183] b) CXCR4- upon co-stimulation with CXCL12 and each selective GPCRx agonist GPCRx heteromers react with either CXCL12 or each selective GPCRx agonist. The calcium mobilization caused by single agonist stimulation by α- and β-glucan-1-phosphate dehydrogenase (AGD) was enhanced compared to the sum of both α- and β-glucan-1-phosphate dehydrogenase (AGD) agonists. It is like showing sodium mobilization,

[0184] As determined by calcium mobilization assay.

[0185] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the CXCR4-GPCRx heteromer-selective reagent is capable of: i) detecting CXCR4-GPCRx heteromers in patient-derived cells; ii) patient-derived, altering the heteromer-specific properties of CXCR4-GPCRx heteromers; Altering the heteromer-specific function of CXCR4-GPCRx heteromers in cells, i ii) Heteromer-specific properties of patient-derived cells containing CXCR4-GPCRx heteromers or iv) patient-derived cells containing CXCR4-GPCRx heteromers. reduces cell proliferation.

[0186] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, the CXCR4-GPCRx heteromer selective reagent is a CXCR4 inhibitor, GPC It is an Rx inhibitor or a CXCR4-GPCRx heteromeric inhibitor.

[0187] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, the CXCR4-GPCRx heteromer selective reagent is a CXCR4 antagonist, GPC It is an Rx antagonist or a CXCR4-GPCRx heteromer antagonist.

[0188] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the method includes administering to a subject a CXCR4 inhibitor, a GPCRx inhibitor, or a CXCR4-GPCRx inhibitor. The present invention provides for the administration of an inhibitor selected from the group consisting of heteromeric inhibitors.

[0189] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, the inhibitor is administered as a pharmaceutical composition.

[0190] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the inhibitor administered is a CXCR4 inhibitor.

[0191] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the inhibitor administered is a GPCRx inhibitor.

[0192] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the administered inhibitor is an inhibitor of the CXCR4-GPCRx heteromer.

[0193] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the method includes administering a CXCR4 inhibitor, a GPCRx inhibitor, and a CXCR4-GPCRx inhibitor. In one embodiment, a combination of inhibitors selected from the group consisting of heteromeric inhibitors is administered.

[0194] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the combination of inhibitors may be administered sequentially, concurrently, or simultaneously.

[0195] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some forms, CXCR4 inhibitors are CXCR4 antagonists, CXCR4 inverse agonists, CXCR4 inhibitors, CXCR4 partial antagonists, CXCR4 allosteric modulators, CXCR4 antibodies, CXCR4 an antibody fragment, a ligand for CXCR4, or an antibody-drug conjugate for CXCR4.

[0196] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some forms, GPCRx inhibitors are antagonists of GPCRx, inverse agonists of GPCRx, and inhibitors of GPCR. x partial antagonists, GPCRx allosteric modulators, GPCRx antibodies, GPCRx an antibody fragment, a ligand for a GPCRx, or an antibody-drug conjugate for a GPCRx.

[0197] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, inhibitors of CXCR4-GPCRx heteromers are antagonist of CXCR4-GPCR heteromers, inverse agonist of CXCR4-GPCR heteromers, CXCR4-GPCR Partial antagonist of x heteromer, allosteric modulator of CXCR4-GPCRx heteromer , CXCR4-GPCRx heteromer antibody, CXCR4-GPCRx heteromer antibody Fragments, ligands of CXCR4-GPCRx heteromers, CXCR4-GPCRx heteromers -protein-protein interaction (PPI) inhibitors, or CXCR4-GPCRx heteromers It is an antibody-drug conjugate.

[0198] In certain embodiments of the methods of treatment or suppression disclosed herein, the method further comprises: This involves detecting CXCR4-GPCRx heteromers in cancer patients.

[0199] In certain embodiments of the methods of treatment or suppression disclosed herein, the method further comprises: This involves identifying CXCR4-GPCRx heteromers in cancer patients.

[0200] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the method further includes:

[0201] i) obtaining or having obtained a biological sample from a cancer patient;

[0202] ii) CXCR4-GPCRx heteromers in biological samples obtained from cancer patients Performing or having performed a diagnostic assay to determine the presence, identity, or presence and identity; Beauty

[0203] iii) Suppression of enhanced downstream signaling from CXCR4-GPCRx heteromers Select an inhibitor or combination of inhibitors that

[0204] In certain embodiments of the methods of treatment or suppression disclosed herein, a patient biological sample is a biological fluid sample or a biological tissue sample.

[0205] In certain embodiments of the methods of treatment or suppression disclosed herein, a biological fluid sample or A liquid biopsy is performed on a biological tissue sample.

[0206] In certain embodiments, the biological fluid sample may be prepared using methods such as those described in Campbell et al., which are incorporated herein by reference in their entirety. os CDM et al., “Molecular Profiling of Li quid Biopsy Samples for Precision Medici ne,”Cancer J.2018 Mar / Apr;24(2):93-103 Circulating tumor cells (CTCs), tumor-derived cell-free DNA (cfD) from body fluids as indicated These include extracellular vesicles (e.g., NAs), circulating small RNAs, and exosomes.

[0207] In certain embodiments of the methods of treatment or suppression disclosed herein, the biological fluid sample comprises: The sample may be a blood sample, a plasma sample, a saliva sample, a cerebrospinal fluid sample, an intraocular fluid sample or a urine sample.

[0208] In certain embodiments of the methods of treatment or suppression disclosed herein, the biological tissue sample comprises The tissue sample may be an organ tissue sample, a bone tissue sample or a tumor tissue sample.

[0209] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, cancer cells contain CXCR4-GPCRx heteromers.

[0210] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein Morphology: Normal, non-cancerous cells do not contain CXCR4-GPCRx heteromers.

[0211] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, upon administration of the inhibitor combination, the compound contains a CXCR4-GPCRx heteromer. Cancer progression in patients with these cancer cells is due to the use of C as a single inhibitor in these patients. 5-100% more effective than XCR4 inhibitors or GPCRx inhibitors Decreases.

[0212] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this case, patients with the above cancer cells containing CXCR4-GPCRx heteromers were treated with GPCRs. The efficacy of CXCR4 inhibitors when administered in combination with PCRx inhibitors is comparable to that of single inhibitors. The efficacy of CXCR4 inhibitors when administered as anti-inflammatory drugs ranges from 5 to 2000% and improves.

[0213] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this case, patients with the above cancer cells containing CXCR4-GPCRx heteromers were treated with C The efficacy of GPCRx inhibitors when administered in combination with XCR4 inhibitors is comparable to that of single inhibitors. Range of 5-2000% efficacy compared to GPCRx inhibitors when administered as anti-inflammatory agents and improves.

[0214] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, upon administration of the inhibitor combination, the compound contains a CXCR4-GPCRx heteromer. Cancer progression in patients with these cancer cells is due to the use of C as a single inhibitor in these patients. 5-100% more effective than XCR4 inhibitors or GPCRx inhibitors Decreases.

[0215] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this case, patients with the above cancer cells containing CXCR4-GPCRx heteromers were treated with GPCRs. The efficacy of CXCR4 inhibitors when administered in combination with PCRx inhibitors is comparable to that of single inhibitors. The efficacy of CXCR4 inhibitors when administered as anti-inflammatory drugs ranges from 5 to 2000% and improves.

[0216] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this case, patients with the above cancer cells containing CXCR4-GPCRx heteromers were treated with C The efficacy of GPCRx inhibitors when administered in combination with XCR4 inhibitors is comparable to that of single inhibitors. Range of 5-2000% efficacy compared to GPCRx inhibitors when administered as anti-inflammatory agents and improves.

[0217] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the method includes administering to a subject a CXCR4 inhibitor, a GPCRx inhibitor, and a CXCR4-GPCR inhibitor. x heteromeric inhibitors.

[0218] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the method involves administering a combination of a CXCR4 inhibitor and a GPCRx inhibitor. do.

[0219] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the method involves administering a CXCR4-GPCRx heteromeric inhibitor.

[0220] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, administration of the inhibitor combination is effective in preventing or alleviating the above-mentioned CXCR4-GPCRx inhibitors in cancer patients. Enhanced downstream signaling from agonists was observed after 5-200 cycles compared to administration of a single inhibitor. Suppressed within the 0x range.

[0221] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, administration of the inhibitor combination is effective in preventing or alleviating the above-mentioned CXCR4-GPCRx inhibitors in cancer patients. Enhanced downstream signaling from agonists was observed after 5-150 min compared to administration of a single inhibitor. Suppressed within the 0x range.

[0222] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, administration of the inhibitor combination is effective in preventing or alleviating the above-mentioned CXCR4-GPCRx inhibitors in cancer patients. Enhanced downstream signaling from agonists compared to single inhibitor administration Inhibits in the range of 500 times.

[0223] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, administration of an inhibitor or a combination of inhibitors is directed to the treatment of cancer patients with CXCR4-G Enhanced downstream signaling from PCRx heteromers in the context of each individual protomer downstream signaling from either CXCR4 protomers or GPCRx protomers It inhibits in the range of 5 to 2000 times compared to inhibition.

[0224] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, administration of an inhibitor or a combination of inhibitors is directed to the treatment of cancer patients with CXCR4-G Enhanced downstream signaling from PCRx heteromers in the context of each individual protomer downstream signaling from either CXCR4 protomers or GPCRx protomers It inhibits in the range of 5 to 1500 times compared to inhibition.

[0225] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, administration of an inhibitor or a combination of inhibitors is directed to the treatment of cancer patients with CXCR4-G Enhanced downstream signaling from PCRx heteromers in the context of each individual protomer downstream signaling from either CXCR4 protomers or GPCRx protomers It inhibits in the range of 500 to 1500 times compared to inhibition.

[0226] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, the patient's cancer cells are more concentrated than normal non-cancerous cells from the patient. It contains CXCR4-GPCRx heteromers at high concentrations.

[0227] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In morphology, the presence of CXCR4-GPCRx heteromers in patients' cancer cells indicates C Subpopulations of patients with XCR4-mediated cancers are identified.

[0228] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein Morphologically, the presence of CXCR4-GPCRx heteromers in patients' cancer cells is 4 is a biomarker for a subpopulation of patients with mediated cancer.

[0229] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In terms of morphology, biomarkers for subpopulations of patients with CXCR4-mediated cancers are important for precision medicine, patient demographics, and This allows for classification or patient classification.

[0230] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this study, GPCR-based biomarkers for subpopulations of patients with CXCR4-mediated cancers were identified. Enabling precision cancer treatment.

[0231] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the inhibitor is an antibody.

[0232] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In one embodiment, the CXCR4-GPCRx heteromer-selective reagent is an antibody.

[0233] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the antibody is a CXCR4-GPCRx heteromeric bispecific antibody.

[0234] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the antibody is a heteromer-specific antibody for a CXCR4-GPCRx heteromer.

[0235] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In this form, the inhibitor acts as a dual specific ligand(s) of the CXCR4-GPCRx heteromer. is.

[0236] Specific implementations of the methods of treatment, suppression, pharmaceutical compositions, or pharmaceutical kits disclosed herein In some embodiments, the antibodies may be those described, for example, in Beck et al., each of which is incorporated herein by reference in its entirety. l.,“Strategies and challenges for the ne xt generation of antibody-drug conjugate s”, Nature Reviews Drug Discovery,16:315 -337,(2017) and Lambert,et al.,“Antibody-Dr ug Conjugates for Cancer Treatment”, Annu Review of Medicine, 69:191-207 (2018) As shown, it is an antibody-drug conjugate (ADC). [Brief explanation of the drawings]

[0237]

[0238] [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.

[0239] [Figure 2]Identification of CXCR4-interacting GPCRs using BiFC assays. Representative images showing negative BiFC signals are CXCR4-VN and HA-VC (Figure 2a) and CXCR4-VN and GCGR-VC (Figure 2c). Representative images of CXCR4 and GPCRx showing positive BiFC signals are CXCR4-VN and CXCR4-VC (Figure 2b), CXCR4-VN and ADCYAP1R1-VC (Figure 2d), CXCR4-VC and ADORA2B-VN (Figure 2e), CXCR4-VN and ADORA3-VC (Figure 2f), CXCR4-VN and ADRB2-VC (Figure 2g), CXCR4-VN and APLNR-VC (Figure 2h), CXCR4-VN and C5AR1-VC (Figure 2i), CXCR4-VN and CALCR-VC (Figure 2j), and CXCR4-VN and CCR5-VC (Figure 2j). 2k), CXCR4-VN and CHRM1-VC (Fig. 2l), CXCR4-VN and GALR1-VC (Fig. 2m), CXCR4-VN and EDNRB-VC (Fig. 2n), CXCR4-VN and HRH1-VC (Fig. 2o), CXCR4-VN and MLNR-VC (Fig. 2p), CXCR4-VN and NTSR1-VC (Fig. 2q), CXCR4-VN and PTGER2-VC (Fig. 2r), CXCR4-VC and PTGER3-VN (Fig. 2s), CXCR4-VN and SSTR2-VC (Fig. 2t), and CXCR4-VN and TACR3-VC (Fig. 2u).

[0240] [Figure 3] Figures a and b show the principle of the GPCR co-internalization assay. Cells co-expressing CXCR4-GFP and GPCRx are treated with a GPCRx-specific agonist. (Figure 3a) CXCR4 and GPCRx do not physically interact with each other. The GPCRx agonist induces the internalization of GPCRx but not CXCR4-GFP. (Figure 3b) CXCR4 and GPCRx physically interact, forming a heteromer. The GPCRx agonist induces the internalization of GPCRx, and CXCR4-GFP is co-internalized with GPCRx.

[0241] [Figure 4]a-q show the co-internalization of CXCR4-EGFP upon stimulation of GPCRx with its corresponding agonist (control: CXCR4-GFP (FIG. 4a)). 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 to induce co-internalization of CXCR4-EGFP: GPCRx expressed ADCYAP1R1 (Figure 4b), ADORA2B (Figure 4c), ADORA3 (Figure 4d), ADRB2 (Figure 4e), APLNR (Figure 4f), C5AR1 (Figure 4g), CCR5 (Figure 4h), CHRM1 (Figure 4i), GALR1 (Figure 4j), EDNRB (Figure 4k), HRH1 (Figure 4l), MLNR (Figure 4m), NTSR1 (Figure 4n), PTGER3 (Figure 4o), SSTR2 (Figure 4p), and TACR3 (Figure 4q).

[0242] [Figure 5]Figures 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 (Figure 5a), ADRB2 and HA-VC (Figure 5b), or CXCR4 and ADRB2 (Figure 5c). 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-520AM 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 multimode microplate reader. Results were normalized to baseline activity. Data represent three independent experiments (mean ± standard error). (Fig. 5d) 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.

[0243] [Figure 6]Panels a–l show the enhanced calcium response in cells coexpressing CXCR4 and GPCRx upon costimulation with their respective selective agonists, similar to panels a–c in Figure 5. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and HA-VC, GPCRx and HA-VC, or CXCR4 and GPCRx. GPCRx express ADCYAP1R1 (Figure 6a), ADORA2B (Figure 6b), ADORA3 (Figure 6c), C5AR1 (Figure 6d), CALCR (Figure 6e), CHRM1 (Figure 6f), EDNRB (Figure 6g), HRH1 (Figure 6h), MLNR (Figure 6i), NTSR1 (Figure 6j), PTGER2 (Figure 6k), and TACR3 (Figure 6l). Cells were incubated with Cal-520AM and treated with CXCL12, GPCRx agonists, or CXCL12 and GPCRx agonists. Calcium responses were quantified as described in Figure 5. Data represent three independent experiments (mean ± standard error). *P<0.05, **P<0.01, ***P<0.001, Student's t-test.

[0244] [Figure 7] Figures a–e show examples of CXCR4-GPCRx heteromers that failed to show enhanced calcium signaling in the presence of agonists for both GPCRs in cells coexpressing CXCR4 and GPCRx. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and HA-VC, GPCRx and HA-VC, or CXCR4 and GPCRx. GPCRx expressed APLNR (Figure 7a), CCR5 (Figure 7b), GALR1 (Figure 7c), PTGER3 (Figure 7d), and SSTR2 (Figure 7e). Cells were incubated with Cal-520AM and treated with CXCL12, GPCRx agonists, or CXCL12 and GPCRx agonists. Calcium mobilization was quantified as described in Figure 5. Data represent three independent experiments (mean ± standard error). Student's t-test. ns: non-significant.

[0245] [Figure 8](a)–(l) show that the enhanced calcium response observed when cells expressing CXCR4-GPCRx heteromers were simultaneously stimulated with CXCL12 and a GPCRx agonist was efficiently suppressed by cotreatment with both antagonists. MDA-MB-231 cells were cotransduced with adenoviruses encoding CXCR4 and GPCRx. GPCRx express ADRB2 (Figure 8a), CHRM1 (Figures 8b–8d), EDNRB (Figure 8e), HRH1 (endogenous) (Figures 8f and 8g), HRH1 (Figures 8h and 8i), MLNR (Figure 8j), NTSR1 (Figure 8k), and TACR3 (Figure 8l). (F) and (G) show that cells were transduced with adenovirus encoding CXCR4 and the endogenous HRH1 response was examined by 50 nM histamine. Cells were incubated with Cal-520AM for 2 hours, incubated with a GPCR antagonist or vehicle for 30 minutes, and stimulated with the indicated amounts of CXCL12, a GPCR agonist, or CXCL12 and a GPCR agonist. Calcium mobilization was quantified as described in Figure 5d. Data represent three independent experiments (mean ± standard error). *P<0.05, **P<0.01, ***P<0.001, Student's t-test.

[0246] [Figure 9] The principle of the internalization inhibition assay is shown below. Cells co-expressing CXCR4-GFP and GPCRx are 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.

[0247] [Figure 10]Figures a–c show inhibition of internalization by GPCRx antagonists of heteromers: (Figure 10a) CXCR4-ADRB2, (Figure 10b) CXCR4-CHRM1, and (Figure 10c) CXCR4-HRH1. (Figure 10a) CXCR4-GFP-expressing U-2 OS cells were transduced with adenovirus encoding ADRB2. Treatment with the CXCR4 agonist CXCL12 (SDF-1) induced CXCR4-ADRB2 internalization. However, the ADRB2 antagonist carvedilol did not induce internalization. Cotreatment with CXCL12 and carvedilol partially inhibited CXCL12-induced CXCR4-ADRB2 internalization. (Figure 10b) CXCR4-GFP-expressing U-2 OS cells were transduced with adenovirus encoding CHRM1. Treatment with CXCL12 induced CXCR4-CHRM1 internalization. However, the CHRM1 antagonists oxybutynin and umeclidinium did not induce internalization. Cotreatment of CXCL12 with oxybutynin or umeclidinium inhibited CXCL12-induced CXCR4-CHRM1 internalization (Fig. 10c). CXCR4-GFP-expressing U-2 OS cells were transduced with an adenovirus encoding HRH1. Treatment with CXCL12 induced CXCR4-HRH1 internalization. However, the HRH1 antagonists promethazine, hydroxyzine, or loratadine did not induce internalization. Cotreatment of CXCL12 with promethazine, hydroxyzine, or loratadine inhibited CXCL12-induced CXCR4-HRH1 internalization.

[0248] [Figure 11]Figures 11a-c show the effects of GPCRx antagonists on the viability of patient-derived cells (PDCs) from cancer patients. (Figure 11a) The effect of an ADRB2 antagonist (carvedilol) on PDC viability. Carvedilol induced a significant decrease in cell viability (IC50 = 11.69 μM). (Figure 11b) The effect of CHRM1 antagonists (oxybutynin, umeclidinium) on PDC viability. Oxybutynin and umeclidinium each showed a significant decrease in cell viability with IC50 = 3.04 μM and 4.03 μM, respectively. (Figure 11c) The effect of HRH1 antagonists (promethazine, hydroxyzine, and loratadine) on PDC viability. Promethazine, hydroxyzine and loratadine each showed a decrease in PDC viability with IC50 = 18.39 μM, 12.79 μM and 5.29 μM, respectively.

[0249] [Figure 12] Figures 12a-c show the detection of CXCR4-ADRB2 heterodimers in U-2 OS cells overexpressing CXCR4 and ADRB2 by PLA and qRT-PCR. CXCR4-GFP-expressing U-2 OS cells were transduced with adenovirus encoding ADRB2 at various MOIs for 2 days. Then, PLA was performed on CXCR4-ADRB2-coexpressing U-2 OS cells. (Figure 12a) Images of CXCR4-ADRB2 heterodimer detection by PLA. (Figure 12b) The PLA signal increased in a dose-dependent manner proportional to the expression level of ADRB2. (Figure 12c) Data from qRT-PCR showing the endogenous ADRB2 expression level in U-2 OS cells.

[0250] [Figure 13]Figures 13a-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. (Figure 13a) Images of CXCR4-ADRB2 heteromer detection. Nuclei were visualized with DAPI staining, and CXCR4-ADRB2 heteromers are indicated by small dots. (Figure 13b) Percentage of CXCR4-ADRB2 heteromers in PDCs.

[0251] [Figure 14] Figures 14a and 14b show the detection of CXCR4-CHRM1 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-CHRM1 heteromers were detected by PLA using antibodies specific for CXCR4 and CHRM1. (Figure 14a) Images of CXCR4-CHRM1 heteromer detection. Nuclei were visualized with DAPI staining, and CXCR4-ADRB2 heteromers are indicated by small dots. (Figure 14b) Percentage of CXCR4-CHRM1 heteromers in PDCs.

[0252] [Figure 15] Figures 15a-b show results from the detection of CXCR4-GPCRx heteromers in PDX. CXCR4-ADRB2 heteromers were detected by PLA in PDXs originating from GBM (sample IDs: 777T, 783T, 948T, and 559T) using antibodies specific for CXCR4 and ADRB2. (Figure 15a) Images of CXCR4-ADRB2 heteromer detection. Nuclei were visualized with DAPI staining, and CXCR4-ADRB2 heteromers were indicated by small dots. (Figure 15b) Percentage of CXCR4-ADRB2 heteromers in PDX.

[0253] [Figure 16]Figures 16a-b show the enhanced calcium response in cells coexpressing CXCR4 and GPCRx upon costimulation with their respective agonists. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and ADRB2 (Figure 16a) or CXCR4 and HRH1 (Figure 16b). Cells were cultured for 3 days, stained with Cal-520AM, and treated with either CXCL12 (30 nM) alone, increasing doses of histamine or salmeterol alone, or increasing doses of histamine or salmeterol in combination with 30 nM CXCL12. Calcium mobilization was measured using FlexStation 3. Total represents the sum of the responses evoked by 30 nM CXCL12 alone (open squares) and the indicated doses of GPCRx ligand alone (filled circles). The sum graph is depicted by inverted triangles and dashed lines. Statistical significance of differences between the total (inverted triangles) and co-treatment (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).

[0254] [Figure 17]Figures 17a-c show that cells coexpressing CXCR4 and GPCRx show no enhancement of calcium responses upon costimulation with their respective agonists. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and APLNR (Figure 17a), CXCR4 and PTGER3 (Figure 17b), or CXCR4 and SSTR2 (Figure 17c). Cells were cultured for 3 days, stained with Cal6 dye, and treated with either CXCL12 (20 nM) alone, increasing doses of apelin-13, PGE2, or octreotide alone, or increasing doses of apelin-13, PGE2, or octreotide in combination with 20 nM CXCL12. Calcium mobilization was measured using a FlexStation 3. Total represents the sum of the responses evoked by 20 nM CXCL12 alone (open squares) and the indicated doses of GPCRx ligand alone (filled circles). The sum graph is depicted by inverted triangles and dashed lines. Statistical significance between the sum (inverted triangles) and co-treatment (filled squares) at each point was determined by Student's t-test. No statistical differences were observed at any point. Data represent the mean ± standard deviation (n = 3).

[0255] [Figure 18] Figures 18a-b show enhanced calcium responses in wild-type MDA-MB-231 cells upon costimulation with CXCL12 and histamine. (Figure 18a) MDA-MB-231 cells were stimulated with CXCL12 (100 nM), histamine (10 nM), or CXCL12 and histamine, and the Ca2+ responses elicited by endogenous CXCR4 and HRH1 were measured. (Figure 18b) Calcium mobilization was quantified by calculating the area under the curve (AUC) for each graph. The sum represents the sum of the responses evoked by each agonist. Data represent three independent experiments performed in triplicate (mean ± standard error). Statistical significance between the sum and cotreatment was determined by Student's t-test. *P<0.05.

[0256] [Figure 19]Figures 19a-b show enhanced MDA-MB-231 cell migration in response to costimulation with CXCL12 (30 nM) and histamine (50 nM). MDA-MB-231 cells were transduced with a lentivirus encoding CXCR4 at 1 MOI, and chemotactic migration of cells toward CXCL12, histamine, or CXCL12 and histamine was assessed in the presence or absence of the HRH1-selective inverse agonist pyrilamine (1 μM). Chemotaxis was quantified by counting the number of migrated cells present on the underside of the membrane in 10 fields per chamber. (Figure 19a) Representative photographs for each group. (Figure 19b) Histamine itself did not induce MDA-MB-231 cell migration, but it significantly enhanced cell migration when costimulated with CXCL12. The enhanced MDA-MB-231 cell migration was due to endogenous HRH1, because addition of pyrilamine (1 μM), an HRH1-selective inverse agonist, completely abolished the enhanced response. Data represent mean ± standard error (n = 3 or 5). Statistical significance was determined by Student's t-test. *P < 0.05.

[0257] [Figure 20] Figure 1 shows that cotreatment with an anti-CXCR4 antibody and an ADRB2 antagonist effectively suppressed the enhanced calcium response observed in cells expressing CXCR4-ADRB2 heteromers when simultaneously stimulated with CXCL12 and an ADRB2 agonist. MDA-MB-231 cells were cotransfected 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 Cal6 for 2 hours. Cells were then stimulated with the indicated amounts of CXCL12, an ADRB2 agonist (salmeterol), or CXCL12 and an ADRB2 agonist.

[0258] [Figure 21]Figures 21a-b show the detection of CXCR4-GPCR heteromers by PLA in U-2 OS cells overexpressing CXCR4 and GPCRx. CXCR4-GFP-expressing U-2 OS cells were transduced with adenovirus encoding CHRM1 or HRH1 at various MOIs for 2 days. Subsequently, PLA was performed on U-2 OS cells coexpressing CXCR4 and GPCRx using antibodies specific for CXCR4 and GPCRx. The PLA signal increased in a dose-dependent manner proportional to the expression levels of CHRM1 (Figure 21a) and HRH1 (Figure 21b).

[0259] [Figure 22a] Shown are images of three mice 28 days after transplantation, each of which was transplanted with parental A549 cells, A549-CXCR4 cells stably overexpressing CXCR4, or A549-CXCR4-ADRB2 cells stably overexpressing CXCR4-ADRB2 heteromers. [Figure 22b] Graphs comparing tumor growth of transplanted cells from Figure 22a are shown, and tumor growth was followed 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 triplicates.

[0260] [Figure 23] Figures 23a-g show calcium responses in MDAMB231 cells coexpressing CXCR4 and GPCRx upon costimulation with CXCL12 and the endogenous ligands of GPCRx. MDAMB231 cells were transduced with adenoviruses encoding CXCR4 and HA-VC, GPCRx and HA-VC, or CXCR4 and GPCRx, where GPCRx expresses ADCYAP1R1 (PAC1) (Figure 23a), ADORA2B (Figure 23b), ADORA3 (Figure 23c), CHRM1 (Figure 23d), EDNRB (Figure 23e), MLNR (Figure 23f), and TACR3 (Figure 23g).

[0261] [Figure 24]Figures 24a-b show calcium responses in MDA-MB-231 cells coexpressing CXCR4 and ADCYAP1R1 upon costimulation with their selective endogenous ligands. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and ADCYAP1R1. (Figure 24a) Cells were treated with PACAP38 (1 nM, the ADCYAP1R1-selective endogenous ligand) alone, increasing doses of CXCL12 alone, or increasing doses of CXCL12 in combination with 1 nM PACAP38. (Figure 24b) Cells were treated with CXCL12 alone, increasing doses of PACAP38 alone, or increasing doses of PACAP38 in combination with 15 nM CXCL12.

[0262] [Figure 25] Figures 25a-b show calcium responses in MDA-MB-231 cells coexpressing CXCR4 and TACR3 upon costimulation with their selective endogenous ligands. MDA-MB-231 cells were transduced with adenoviruses encoding CXCR4 and TACR3. (Figure 25a) Cells were treated with neurokinin B (0.4 nM, a TACR3-selective endogenous ligand) alone, increasing doses of CXCL12 alone, or increasing doses of CXCL12 combined with 0.4 nM neurokinin B. (Figure 25b) Cells were treated with CXCL12 alone (30 nM), increasing doses of neurokinin B alone, or increasing doses of neurokinin B combined with 30 nM CXCL12.

[0263] [Figure 26]Figures 26a-b show validation of CRISPR / Cas9-mediated CXCR4 and HRH1 gene editing in MDA-MB-231 cells. (Figure 26a) MDA-MB-231 cells stably expressing CXCR4 (MDACXCR4+,HRH- cells) were generated by disrupting the HRH1 gene through transduction with a lentivirus encoding Cas9 and a guide RNA targeting HRH1. The absence of functional HRH1 was confirmed by measuring the calcium response upon exposure to histamine. (Figure 26b) The CRISPR / Cas-9 system was used to edit the CXCR4 gene in MDA-MB-231 cells, and CXCR4 expression was detected by immunoblotting.

[0264] [Figure 27] Figures 27a-b show that the enhanced calcium response in MDA-MB-231 cells co-treated with CXCL12 and histamine is abolished in the absence of HRH1. (Figure 27a) MDA-MB-231 cells stably overexpressing CXCR4 (MDACXCR4+) or MDACXCR4+ cells in which HRH1 had been ablated using the CRISPR / Cas9 system (MDACXCR4+,HRH1-) were treated with CXCL12 alone (50 nM), increasing concentrations of histamine, or histamine and CXCL12. (Figure 27b) MDACXCR4+ or MDACXCR4+,HRH1- cells were treated with histamine alone (15 nM), increasing concentrations of CXCL12, or CXCL12 and histamine.

[0265] [Figure 28]Figures 28a-b show that the enhanced calcium response in MDA-MB-231 cells co-treated with CXCL12 and histamine is abolished in the absence of CXCR4. (Figure 28a) MDA-MB-231 cells (MDAWT) or MDAWT cells in which CXCR4 had been ablated using the CRISPR / Cas9 system (MDACXCR 4-) were treated with histamine alone (15 nM), increasing concentrations of CXCL12, or histamine and CXCL12. (Figure 28b) MDA-MB-231 cells or MDACXCR 4+ cells were treated with CXCL12 alone (100 nM), increasing concentrations of histamine alone, or CXCL12 and histamine, and the Ca2+ response was measured.

[0266] [Figure 29] Figures a and b show the antitumor effects of CXCR4 inhibitors, ADRB2 inhibitors, or CXCR4 and ADRB2 inhibitors in mice implanted with the A549-CXCR4-ADRB2 cell line, which stably overexpresses CXCR4-ADRB2 heteromers. Figure a shows a graph comparing tumor growth rates in vivo with the antitumor effects of the CXCR4 inhibitor LY2510924, the ADRB2 inhibitor carvedilol, or the combination of LY2510924 and carvedilol. Figure b shows a graph comparing tumor growth rates in vivo with the antitumor effects of the CXCR4 inhibitor AMD070, the ADRB2 inhibitor carvedilol, or the combination of AMD070 and carvedilol. Tumor growth was monitored every 3 or 4 days by measuring the length (L) and width (W) of the tumor and calculating tumor volume according to the following formula: volume = 0.5LW². Results are expressed as the mean ± standard deviation of 10 tumors. DETAILED DESCRIPTION OF THE INVENTION

[0267] Unless otherwise specified, the following include abbreviations for terms disclosed herein: acute myeloid leukemia (AML), adenosine A3 receptor (ADORA3), adenosine receptor A2b (ADO RA2B), adenovirus high throughput system (AdHTS), adenylate cyclase activity Adrenergic receptor type 1 (ADCYAP1R1), pituitary receptor Adrenergic receptor alpha 1A (ADRA1A), adrenergic receptor beta 2 (ADRB2), apelin receptor (APLNR), atypical chemokine receptor 3 (ACKR3), bimolecular fluorescence complementation (B iFC), bioluminescence resonance energy transfer (BRET), bovine serum albumin (BSA), Calcitonin receptor (CALCR), cancer stem cell (CSC), CC chemokine receptor 2 type (CCR2), chemokine chemokine-like receptor 1 (CMKLR1), muscarinic cholinergic agonist receptor 1 (CHRM1), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia ( CML), chronic obstructive pulmonary disease (COPD), complement C5a receptor 1 (C5AR1), Ven C-terminal fragment of us (VC), C-X-C motif chemokine ligand 12 (CXCL1 2), CXC receptor 4 (CXCR4), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) ), δ-opioid receptor (OPRD), endothelin receptor type B (EDNRB), enzyme ELISA, formalin-fixed paraffin-embedded (FFPE), fluorescent Photoresonance energy transfer (FRET), G protein-coupled receptor (GPCR), galanin receptor Glucagon receptor 1 (GALR1), glioblastoma multiforme (GBM), glucagon receptor (GCGR), G PCR heteromer identification technology (GPCR-HIT), granulocyte colony stimulating factor (G-CSF) ), hematopoietic stem cells (HSCs), hepatocellular carcinoma (HCC), histamine receptor H1 (HRH1) , Human Immunodeficiency Virus (HIV), Committee on Receptor Nomenclature of the International Union of Pharmacology (NC-IUPHA) R), μ-opioid receptor (MOR), motilin receptor (MLNR), multiple myeloma ( MM), multiplicity of infection (MOI), myelodysplastic syndrome (MDS), neurotensin receptor 1 (NTSR1), non-Hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), Ve N-terminal fragment of nus (VN), patient-derived cells (PDC), patient-derived xenografts (PDX) ), positron emission tomography (PET), computed tomography (CT), programmed cell death Ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), prostaglandin Prostaglandin E receptor 2 (PTGER2), prostaglandin E receptor 3 (PTGER3), Proximity ligation assay (PLA), quantitative reverse transcription polymerase chain reaction (RT-q PCR), single photon emission computed tomography (SPECT), small lymphocytic lymphoma ( SLL), small cell lung cancer (SCLC), somatostatin receptor 2 (SSTR2), stromal cells derived factor 1 (SDF-1), systemic lupus erythematosus (SLE), tachykinin receptor 3 (TAC R3), threshold cycle (Ct), time-resolved FRET (TR-FRET), tumor microenvironment ( TME), vascular endothelial growth factor (VEGF), vascular smooth muscle cells (VSMC), WHIM syndrome Group (warts, hypogammaglobulinemia, immunocompromise, myelocatechus), green fluorescent protein green fluorescent protein (GFP), and yellow fluorescent protein (YFP).

[0268] CXCR4 plays an important role in tumor formation and progression, and CXCR4 as an anti-cancer drug The development of 4 antagonists has likely been hampered by side effects and lack of efficacy within acceptable dose ranges. In recent years, various CXCR4 antibodies with distinctive physiological and pharmacological properties have been identified. -GPCR heteromers have been reported, but their role in cancer biology, or C.X.C. The potential for developing anti-cancer therapeutics targeting R4-GPCR heteromers is not clearly understood. It has not been done.

[0269] Traditionally, GPCRs are monomeric G proteins that interact with heterotrimeric G proteins upon ligand binding. Drugs are being developed based on monomeric or homomeric GPCRs, which are thought to function as (Milligan 2008). This idea is based on the idea that GPCRs form heteromers. The discovery that heteromerization is essential for some GPCRs has significantly improved GPCR heteromerization is a key factor in GPCR maturation and cell surface delivery, as well as ligand binding affinity. known to alter receptor activity, signaling strength and pathways, and receptor desensitization and regeneration (Terrillon and Bouvier 2004, Ferre et al., 2010, Rozenfeld and Devi 2010, Gomes et al., 2016, Farran 2017). Different GPCR heteromers are distinct. GPCR heteromerization exhibits diverse functional and pharmacological properties, and GPCR heteromerization is highly dependent on cell types, tissues, and diseases. may vary depending on the disease state (Terrillon and Bouvier 2004, Ferre et al., 2010, Rozenfeld and Devi 201 0, Gomes et al., 2016, Farran 2017). Currently, GPCR Heteromerization is considered a general phenomenon, and elucidating GPCR heteromerization is crucial for understanding receptor function. It opens new avenues for understanding the role of ATP in function, physiology, disease, and pathology. Therefore, elucidating GPCR heteromers and their functional properties will lead to fewer side effects and To develop new drugs with higher efficacy and improved tissue selectivity or to ... This provides new opportunities for finding new uses for 0, Rozenfeld and Devi 2010, Farran 2017).

[0270] Identification of true GPCR heteromers requires thorough and critical evaluation. To distinguish between simple association of GPCRs with ATP and GPCRs, researchers in this field and international pharmacological associations have The Committee on Nomenclature of GPCRs (NC-IUPHAR) defines GPCR heteromers as "a group of at least two (functional) receptor units [protomers], the biochemical properties of which are not clearly defined. These heteromers are macromolecular complexes with distinctly different biochemical properties, and these heteromers are naturally occurring. It has been stated that it exists in tissues (Ferre et al., 2009, Gomes et al., 2016, Pin et al., 2007). We proposed three criteria to demonstrate a marker: (1) co-immunoprecipitation, in si tu hybridization, or proximity-based assays, including proximity ligation assays Using techniques similar to those described above, heteromers are allosteric in cells / tissues where both receptors are expressed. Cells / tissues lacking one receptor exhibiting the appropriate colocalization and interaction that allows rhythmicity (2) heteromers must not exhibit this property in both receptors; signal transduction, ligand binding and / or transport only in cells / tissues where the These receptors exhibit distinct properties such as changes in the receptors, but do not exhibit these properties in cells / tissues lacking one of the receptors. Furthermore, (3) heteromer-selective reagents must possess heteromer-specific properties. Heteromer-selective reagents include heteromer-selective antibodies, membranes, and These include penetrating peptides, and bivalent / bifunctional ligands (Gomes et al. ,2016, Pin et al.,2007). Many GPCR heteromers are expressed in heterologous cells. Although many have been identified in vitro using recombinant receptors expressed in cells, only a few have been novel However, due to technical challenges, few studies have shown evidence of GPCR heteromerization in native tissues. (Gomes et al., 2016). To be considered a valid GPCR heteromer, at least two of the three criteria above must be met. (Pin et al., 2007) .

[0271] As disclosed herein, determining the presence / existence of CXCR4-GPCRx heteromers When determining whether heteromeric components colocalize directly or allogeneically, one of three criteria must be met. They physically interact with each other through intermediate proteins that act as vehicles for sterism. To obtain certainty about whether a product meets the criteria, a number of methods may be used, including: One or more of these may be utilized, including, but not limited to: co-internalization assay; co-localization assay; Assays (which determine the colocalization of receptor protomers within cellular compartments), e.g., in in situ hybridization, immunohistochemistry, or immunoelectron microscopy; proximity-based Assays, e.g., proximity-based biophysical techniques, e.g., resonance energy transfer (RET) RET), bioluminescence RET (BRET), fluorescence resonance RET (FRET), time-resolved fluorescence R ET (TR-FRET), antibody-mediated FRET, ligand-mediated FRET, bimolecular fluorescence complementation ( BiFC) and proximity ligation assay (PLA); co-immunoprecipitation assay; or fluorescent For example, BiFC, co-internalization assays, or PLA were used to detect CXCR4-GP CRx heteromers were assessed to see if they met criterion 1 of 3.

[0272] As disclosed herein, criterion 2 of the three (heteromers are not necessarily identical to individual protomers) whether it satisfies the requirements (whether it exhibits properties distinct from those of the signal transduction, e.g., enhanced calcium mobilization (as determined by, e.g., calcium mobilization assays) We have confirmed whether the CXCR4-GPCRx heteromer is responsible for the To achieve this, we need CXCR4-GPCRx heteromers that meet criterion 1 of the three above. We used a two-step approach to: (1) enhance downstream sequences in the context of individual protomers; To determine the presence / absence of signal transmission, e.g., enhanced calcium mobilization (potentiation). Co-expression of HA-VC and one of the protomers (either CXCR4 or GPCRx) Calcium mobilization in cells expressing CXCL12 and its selective agonists was investigated. (b) with either CXCL12 alone or with each selective agonist alone and (2) in the context of CXCR4-GPCRx heteromers, enhanced expression of GPCRs. The presence / absence of enhanced downstream signaling, e.g., enhanced calcium mobilization (potentiation) To determine calcium mobilization in cells co-expressing CXCR4 and GPCRx (a) When co-stimulated with CXCL12 and each selective agonist, and (b) when CXCL12 The stimulation by either the agonist alone or each selective agonist alone is compared with the sum of both. As disclosed herein, criteria 2 of 3 are met, and enhanced downstream signaling is achieved. For example, CXCR4-GPCRx heteromers, which result in enhanced calcium mobilization, have been identified. To be recognized, (1) either protomer context (i.e., CXCR4 and HA-VC) Enhanced downstream signaling, e.g., in the context of GPCRx and HA-VC (2) the absence of enhanced calcium mobilization, and (3) the presence of CXCR4-GPCRx heteromers. In the absence of enhanced downstream signaling in the context, e.g., enhanced calcium mobilization, In both the protomer context and the CXCR4-GPCRx heteromeric context, In this study, we used the GPCRs to stimulate cells (either as single agents or in combination with each other). The concentrations of CXCL12 (in combination with x agonists) and CXCL12 (alone) used to stimulate the cells Selective GPCRx agonists (as a single agent or in combination with CXCL12) The concentration of either an endogenous agonist or a known selective agonist for CRx independently affects the EC For example, the concentration of the compound used to stimulate cells (as a single agent) is 100 times or less than the 50 concentration. The concentration of CXCL12 (as a single agent or in combination with each selective GPCRx agonist) , at a concentration of 15 nM (approximately the EC50 concentration for CXCR4).

[0273] As disclosed herein, determining the presence / existence of CXCR4-GPCRx heteromers When determining whether a heteromer-selective reagent changes heteromer-specific properties, we must consider criterion 3 of the three. To be certain whether a product meets the criteria (whether it is a product or not), the first and second criteria of the three Patient-derived cells that meet criteria 2 of 3 will be treated with antagonists (CXCR4 antagonist, GPCRx antagonist). In the presence of a CXCR4-GPCRx heteromer antagonist, e.g., Patient-derived cells containing R4-GPCRx heteromers are expanded.

[0274] In some embodiments, the methods of treatment, suppression, pharmaceutical compositions, or The pharmaceutical kit detects the association of CXCR4 with GPCRx in cells as CXCR4-GPCRx. At least two of the criteria or characteristics to be considered an Rx heteromer include: 1) determining whether the following criteria are met: assay, colocalization assay, in situ hybridization, immunohistochemistry, immunoassay immunoelectron microscopy, proximity-based assays, co-immunoprecipitation assays or fluorescent animal assays Intracellular CXCR4-GPCRx heteromeric structure when performed by one or more of the following The elements colocalize and act directly or as a vehicle for allosterism through an intermediate protein. 2) by calcium mobilization assays; When determined, a) upon costimulation with CXCL12 and each selective GPCRx agonist, Either CXCR4 or each GPCRx in the context of individual protomers within the cell , induced by single agonist stimulation with either CXCL12 or each selective GPCRx agonist. Calcium mobilization equal to or less than the sum of both the calcium mobilization due to b) upon co-stimulation with CXCL12 and each selective GPCRx agonist, CXCR4-GPCRx heteromers are either CXCL12 or each selective GPCRx agonist The amount of calcium mobilization resulting from single agonist stimulation by either or 3) the amount of enhanced calcium mobilization, such that C XCR4-GPCRx heteromer-selective reagents are used to: i) detect CXCR4- in patient-derived cells; ii) alter the heteromer-specific properties of GPCRx heteromers in patient-derived cells altering the heteromer-specific function of CXCR4-GPCRx heteromers; iii) CXC Altering heteromer-specific properties of patient-derived cells containing R4-GPCRx heteromers or iv) patient-derived cell(s) containing CXCR4-GPCRx heteromers. In some embodiments, the method comprises at least one of the following steps: CXCR4-GPCRx heteromer-selective reagents are effective against patient-derived cells as determined by one Altering heteromer-specific properties of CXCR4-GPCRx heteromers in vesicles: PL A, radioligand binding assay, [35S]GTP-γS binding assay, calcium adenosine triphosphate (Ca2+) binding assay assay, cAMP assay, GTPase assay, PKA activation, ERK1 / 2 and / or or Akt / PKB phosphorylation assay, Src and STAT3 phosphorylation assay, CR E reporter assay, NFAT-RE reporter assay, SRE reporter assay , SRF-RE reporter assay, NF-kB-RE reporter assay, secreted Al Potassium phosphatase assay, inositol 1-phosphate production assay, adenylyl cyclase enzyme activity assay, target gene expression by RT-PCR, RT-qPCR, by Aseq, by next-generation sequencing (NGS), or by microarray In some embodiments, the assay is performed by at least one of the following methods: CXCR4-GPCRx heteromer-selective reagents can be used to detect CXCR4 in patient-derived cells. - Altering heteromer-specific functions of GPCRx heteromers: PLA, radioligand binding Binding assay, [35S]GTP-γS binding assay, calcium assay, cAMP uptake SEQ ID NO: 1, 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 NF-kB-RE reporter assay, secreted alkaline phosphatase assay assay, inositol 1-phosphate production assay, adenylyl cyclase activity assay, Gene expression analysis by RT-PCR, RT-qPCR, RNAseq, and In some embodiments, the method comprises at least one of the following steps: A CXCR4-GPCRx heteromer-selective reagent is a CXCR4-GPCRx heteromer-selective reagent, as determined by one Altering heteromer-specific properties of patient-derived cells containing 4-GPCRx heteromers: Assays for cancer cell proliferation, migration, invasion, and drug resistance (survival), and investigation of immune cell function Nodes, angiogenesis, vasculogenesis, metastasis, drug resistance, tissue microarrays (TMA) and cancer cells - Tumor microenvironment (TME) interactions. For example, in some embodiments, The therapeutic method, the suppression method, the pharmaceutical composition, and the pharmaceutical kit are The association with GPCRx includes the following to be considered a CXCR4-GPCRx heteromer: It includes or includes establishing that the product satisfies at least two of the criteria or characteristics set forth in This is based on: 1) determination by the following: co-internalization assay, bimolecular fluorescence complementation (BiFC) or Intracellular C as measured by one or more of the proximity ligation assays (PLA) XCR4-GPCRx heteromeric components colocalize directly or through allostery that they physically interact with each other through intermediate proteins that act as vehicles for communication; 2) As determined by calcium mobilization assay, a) CXCL12 and each of the selective G CXCR in the context of individual protomers within cells upon costimulation with PCRx agonists 4 or each GPCRx, CXCL12 or each selective GPCRx agonist The amount of calcium mobilization resulting from single agonist stimulation by either b) CXCL12 and each of the selective G Upon costimulation with a PCRx agonist, CXCR4-GPCRx heteromers bind to CXCL1 Calcium production resulting from single agonist stimulation by either two or each of the selective GPCRx agonists The enhanced calcium mobilization is compared to the sum of both the or 3) the amount of calcium mobilization achieved by the CXCR4-GPCRx heteromer-selective reagent. ) Altering the heteromer-specific properties of CXCR4-GPCRx heteromers in patient-derived cells ii) promote heteromeric fixation of CXCR4-GPCRx heteromers in patient-derived cells; iii) patient-derived cells containing CXCR4-GPCRx heteromers or iv) alter the heteromeric properties of the cytoplasm; or In some embodiments, the patient-derived cell(s) containing the antibody or antibody fragments are mutated. The therapeutic method, suppression method, pharmaceutical composition, and pharmaceutical kit disclosed herein are directed to a method for treating a cell The association of CXCR4 with GPCRx in the CXCR4-GPCRx complex is considered to be a CXCR4-GPCRx heteromer. This includes or relies on establishing that criteria 1 and 2 are met to be considered a regulated entity. In some embodiments, the methods of treatment, suppression, pharmaceutical compositions or The pharmaceutical kit is a kit for detecting the association of CXCR4 with GPCRx in cells. x heteromers, and In some embodiments, the methods of treatment and suppression disclosed herein are The pharmaceutical composition or pharmaceutical kit is characterized in that the association of CXCR4 with GPCRx in cells is C Establishing that criteria 2 and 3 are met to be considered XCR4-GPCRx heteromers In some embodiments, the method includes or relies on the The treatment method, the suppression method, the pharmaceutical composition, or the pharmaceutical kit is a method for inhibiting CXCR4 and GPC in cells. The association with Rx satisfies criteria 1, 2 and 3 for being considered a CXCR4-GPCRx heteromer. 3.

[0275] As disclosed herein, CXCR4 antibodies that meet at least two of the three criteria -GPCRx heteromers may have or cause enhanced downstream signaling, Enhanced downstream signaling may occur through CXCR4-GPCRx heteromers. -, for example, by agonism of CXCR4-GPCRx heteromers, CXCR4-GPCRx heteromers due to the CXCR4 agonism of GPCRx heteromers by GPCRx agonism and / or CXC of CXCR4-GPCRx heteromers This may be the result of R4 agonism and GPCRx agonism. Enhanced downstream signaling may be due to the activation of CXCR4, the respective GPCRx, or CXCR4-G In some embodiments, the enhanced downstream Current signaling occurs via the CXCR4 protomer in the context of each individual protomer or each G CXCR4-GPCRx in comparison with downstream signaling from PCRx protomers In some embodiments, the enhanced downstream signal transduction These results suggest that the downstream signaling from CXCR4 protomers is related to the context of individual protomers. In comparison, it may be from a CXCR4-GPCRx heteromer. In this configuration, enhanced downstream signaling occurs due to the activation of each GPCR in the context of individual protomers. CXCR4-GPCRx heterodimers compared with downstream signaling from x protomers In some embodiments, the enhanced downstream signaling can be from a From the CXCR4 protomer and each GPCRx protomer in the context of each individual protomer In comparison with downstream signaling from CXCR4-GPCRx heteromers, Enhanced downstream signaling from the CXCR4-GPCRx heteromers. In some embodiments, the expression of α-glucan can be inhibited in cancer patients, e.g., by inducing α-glucan in cancer cells of the patient. In some embodiments, the CXCR4-GPCRx heteromers can be inhibited. Enhanced downstream signaling from The amount of calcium mobilization can be measured using an intracellular Ca2+ assay, e.g., calcium ion transport assay. This can be determined by a neutrophil recruitment assay.

[0276] As disclosed herein, CXCR4 antibodies that meet at least two of the three criteria -GPCRx heteromers may have or cause enhanced downstream signaling, or may result in enhanced downstream signaling, such as enhanced calcium mobilization. The amount of enhanced calcium mobilization from CXCR4-GPCRx heteromers is , calcium mobilization activity upon costimulation with CXCL12 and each selective GPCRx agonist. Either CXCL12 or each selective GPCRx agonist as determined by assay The amount of calcium mobilization resulting from single agonist stimulation of the cells by α- and β-actin is less than the sum of both the amounts of calcium mobilization resulting from single agonist stimulation of the cells by α- and β-actin. In some embodiments, CXCR4-G The amount of enhanced calcium mobilization from PCRx heteromers was significantly increased with CXCL12 and each of the selected as determined by calcium mobilization assays upon costimulation with specific GPCRx agonists Single agonism of the above cells with either CXCL12 or each selective GPCRx agonist A synergistic calcium mobilization that is at least 10% greater than the sum of both stimuli For example, the amount of calcium mobilization from CXCR4-GPCRx heteromers may be increased. The amount of calcium mobilization achieved (or the amount of synergistic calcium mobilization) was determined by the combination of CXCL12 and each Determined by calcium mobilization assay upon costimulation with selective GPCRx agonists When the cells were monocultured with either CXCL12 or each selective GPCRx agonist, At least 20% more or less than the sum of the calcium mobilization caused by agonist stimulation and the 30% more, at least 40% more, at least 50% more, at least 75% more , at least 90% more, at least 100% more, at least 150% more, or less In some embodiments, CXCR The amount of enhanced calcium mobilization from 4-GPCRx heteromers (or synergistic calcium The amount of CXCL12 mobilization was significantly increased upon co-stimulation with each selective GPCRx agonist. CXCL12 or each of the selective GPCRs as determined by calcium mobilization assays The amount of calcium mobilization resulting from single agonist stimulation of these cells with either agonists or The calcium mobilization amount may be 10 to 100% greater than the sum of CXCL12 and CXCL12. Determined by calcium mobilization assay upon costimulation with selective GPCRx agonists When the cells were stimulated with either CXCL12 or each selective GPCRx agonist, 25-100% greater than the combined calcium mobilization caused by agonist stimulation, and 50-100% greater than the combined calcium mobilization caused by agonist stimulation. Calcium, which can be 100% more, 75-100% more, or 100-200% more. It may be the amount of um mobilization.

[0277] In some embodiments, the methods of treatment, suppression, pharmaceutical compositions, or According to the drug kit, CXCR4-GPCRx heteromers meet at least three criteria. and (b) satisfying at least two of the above conditions, thereby resulting in enhanced downstream signaling. or causes enhanced downstream signaling, as measured in calcium mobilization assays. Thus, when determined, a) co-stimulation with CXCL12 and each selective GPCRx agonist At the time of the cell, either CXCR4 or each GPCRx is expressed in the context of individual protomers. These results suggest that monoagonist stimulation with either CXCL12 or each selective GPCRx agonist may be beneficial. The calcium mobilization amount due to the stimulation is equal to or less than the sum of both b) co-stimulation with CXCL12 and each selective GPCRx agonist At this time, CXCR4-GPCRx heteromers bind to CXCL12 or each selective GPCRx. The amount of calcium mobilization resulting from single agonist stimulation by either an agonist or a pharmacokinetic agent was compared with the sum of both. The amount of enhanced calcium mobilization is such that the amount of enhanced calcium mobilization exhibits enhanced calcium mobilization. For example, in some embodiments, enhanced expression of CXCR4-GPCRx heteromers Downstream signaling is determined by i) individual intracellular calcium mobilization assays; Calcium mobilization from protomer CXCR4 or GPCRx in the context of protomer is non- and ii) CXC in cells as determined by calcium mobilization assays. Enhanced calcium mobilization from R4-GPCRx heteromers is synergistic. In some embodiments, the context of an individual protomer is the amount of calcium mobilization. The effects of CXCL12 and each selective GPCRx agonist on the cellular cellular responses were determined by a lysine mobilization assay. Upon co-stimulation with a) each individual GPCRx in the cell in the absence of each individual protomer protomer CXCR4, or b) in cells in the absence of individual protomer CXCR4. Each individual protomer GPCRx is either CXCL12 or a selective GPCRx agonist. The calcium mobilization resulting from single agonist stimulation by either In some embodiments, the amount of calcium mobilized may be less than that of the amount of calcium mobilized. The context of each individual protomer can be determined independently by: a) determining the GPCR activity in the absence of each individual protomer; individual protomer CXCR4 in cells, and b) in the absence of individual protomer CXCR4 Each individual protomer of GPCRx in cells at 1000 kJ / s is determined by calcium mobilization assays. When co-stimulation with CXCL12 and each selective GPCRx agonist was performed, CXC The cAMP response resulting from single agonist stimulation with either L12 or each of the selective GPCRx agonists was The calcium mobilization amount is equal to or less than the sum of both For example, in some embodiments, calcium mobilization assays can When determined, CXC12 was co-stimulated with each selective GPCRx agonist. R4-GPCRx heteromers are either CXCL12 or each selective GPCRx agonist. The amount of calcium mobilization resulting from single agonist stimulation of the cells by either It may result in calcium mobilization.

[0278] As used herein, the term "CXCR4" refers to a CXC motif chemokine receptor. It refers to condition 4 and is also identified by its unique database identifier (ID) and alternative names shown in Table 1. (Chatterjee et al., 2014, Debnath et al. .,2013, Domanska et al.,2013, Guo et al.,2 016, Peled et al.,2012, Roccaro et al.,201 4, Walenkamp et al., 2017).

[0279] As used herein, the term "GPCRx" refers to the GPCRs that bind to CXCR4. They interact with ADCYAP1R1 (ADCYAP receptor type I), ADORA2B (adeno adenosine A2b receptor), ADORA3 (adenosine A3 receptor), ADRB2 (adrenergic receptor) APLNR (apelin receptor beta 2), C5AR1 (complement C5a receptor 1) ), CALCR (calcitonin receptor), CCR5 (chemokine (CC motif) receptor) 5), CHRM1 (muscarinic cholinergic receptor 1), GALR1 (galanin receptor Endothelin receptor type 1), EDNRB (endothelin receptor type B), HRH1 (histamine receptor H1), MLNR (motilin receptor), NTSR1 (neurotensin receptor 1), PTGER2 (prostaglandin E receptor 2), PTGER3 (prostaglandin E receptor 3), Including SSTR2 (somatostatin receptor 2) and TACR3 (tachykinin receptor 3) The proteins used in this study were used to investigate whether they exhibit properties different from those of the individual protomers. The GPCRs identified in Tables 1 and 2 are identified by their unique database identifiers (IDs) and alternatives. It is also identified by its name.

[0280] Table 1 below shows that CXCR4 forms heteromers and is co-stimulated by both agonists. Table 2 below provides a nomenclature for GPCRx that synergistically enhance Ca2+ responses to C Forms heteromers with XCR4 and synergizes Ca2+ responses upon costimulation with both agonists This provides a nomenclature for GPCRxs that do not specifically enhance GPCR activity.

[0281] [Table 1] TIFF0007818648000002.tif248170TIFF0007818648000003.tif248170TIFF0007818648000004.tif188170

[0282] * GCID:Genecards identification

[0283] HGNC: HUGO Gene Nomenclature Committee

[0284]

[0285] [Table 2] TIFF0007818648000006.tif214170

[0286] * GCID:Genecards identification

[0287] HGNC: HUGO Gene Nomenclature Committee

[0288]

[0289] Further details regarding the GPCRs evaluated herein as heteromers with CXCR4 are provided. The information detailed below:

[0290] (1) Pituitary adenylate cyclase activity, also known as ADCYAP1R1-PAC1 The activating polypeptide type I receptor is encoded by the ADCYAP1R1 gene in humans. This receptor is a protein that binds to pituitary adenylate cyclase-activating peptide. ADCYAP1R1 is a membrane-bound protein that mediates the G protein-coupled class B glucanase. It shares considerable homology with members of the gonadotropin / secretin receptor family. Mediates the diverse biological effects of adenylate cyclase-activating polypeptide 1 and adenylate There is a positive association with acid cyclase (Vaudry et al., 2000). is a receptor for PACAP-27 and PACAP-38. The activity of this receptor is It is mediated by a G protein that activates adenylyl cyclase, ADCYAP1. R1 is a hormone that regulates the production of adrenocorticotropic hormone, luteinizing hormone, growth hormone, prolactin, and epinephrine. It may regulate the release of nephrin and catecholamines. ADCYAP1R1 is involved in spermatogenesis and ADCYAP1R1 may play a role in smooth muscle relaxation and in the gastrointestinal tract. It causes secretion in the blood (Ogi et al., 1993).

[0291] ADCYAP1R1 is expressed in the adrenal medulla, pancreatic acini, uterus, myenteric plexus, and brain (R Reubi, 2000; Reubi et al., 2000). It also It is also expressed in the otic ganglion and superior cervical ganglion (presynaptic), and in the cerebral artery (postsynaptic). (Knutsson and Edvinsson, 2002). ADCYAP1R1 Related disorders include post-traumatic stress disorder (Lowe et al., 2015) and and anxiety disorders. The pathways involved include G alpha(s) signaling These include transduction events and RET signaling (Cooper et al., 2013 ).

[0292] (2) ADORA2B - The adenosine A2B receptor, also known as ADORA2B, G protein-coupled adenosine receptor and its encoding human adenosine A2 Adenosine binds to four distinct adenosine receptors—one each for the A and B receptor genes. ADORA1, ADORA2A, ADORA 1, also known as A2A, A2B, and A3 It functions as a signaling molecule through activation of 2B and ADORA3. The protein stimulates adenylate cyclase activity in the presence of adenosine. These receptors also interact with netrin-1, which is involved in axon outgrowth. It has been implicated in several biological functions, both physiological and pathological. Both ORA2A and ADORA2B receptors are important in cancer immunology. The microenvironment is hypoxic, which promotes the expression of CD39 and CD73 by immune cells. CD39 and CD73 convert ATP to adenosine and regulate the adenosine concentration locally. In cancer, adenosine is an ectonucleotidase that increases the level of It is an essential mediator of the activity of ADORA2A and ADOR on lymphocytes. It binds to the A2B receptor and silences the anti-tumor immune response (Chen et al. .,2013).

[0293] ADORA2B regulates CXCR4 expression in vivo and plays a role in preventing vascular lesion formation. (Yang et al., 2008) and promotes the progression of human oral cancer (Kasam a et al., 2015). Pharmacologically tractable Fra-1 / Identification of the ADORA2B axis (Desmet et al., 2013). The cells exhibit high expression levels in the cecum, colon, and bladder, and lower levels in the lungs, blood vessels, and eyes. exhibit high levels of oxidative stress (Fagerberg et al., 2014).

[0294] (3) ADORA3 - Adenosine A3 receptor, also known as ADORA3, The term "ADORA3 receptor" refers to the human receptor, but also to the human gene that encodes it. It is a G protein-coupled receptor that is coupled to Gi / Gq and is involved in various intracellular signal transduction pathways and It mediates sustained cardioprotection during cardiac ischemia. It is also involved in the inhibition of neutrophil degranulation during neutrophil-mediated tissue injury and It has been implicated in both neuroprotective and neurodegenerative effects, and it It may also mediate both cell proliferation and cell death (Gao et al., 2008, Miwatashi et al., 2008).

[0295] Cordycepin inhibits apoptosis in human bladder cancer cells by activating the ADORA3 receptor. Cao et al., 2017). Jafari et al. ADORA3 receptor agonist inhibits breast cancer stem cell proliferation via GLI-1 and ERK1 / 2 pathways ADORA3 inhibited the proliferation of GABA receptors (Jafari et al., 2017). , adrenal gland (RPKM4.4), small intestine (RPKM2.9), as well as the brain, bladder, lymph nodes and It is widely expressed in 20 other tissues, including the colon (Fagerberg et al. .,2014).

[0296] (4) ADRB2 - also known as ADRB2, beta-2 adrenergic receptor (β2 adrenergic receptor 2) Adrenergic receptors) are receptors for hormones and neurotransmitters (ligand synonym, adrenaline) It is a beta-adrenergic receptor that spans the cell membrane and interacts with epinephrine, Signal transduction induces smooth muscle relaxation and bronchodilation through downstream L-type calcium channel interactions ADRB mediates physiological responses such as hypertension (Gregorio et al., 2017). 2 has various functions in the muscular system, such as smooth muscle relaxation, motor nerve endings, and glycogenolysis, and in myocardial contraction. In normal eyes, salbutamol reduces blood flow to the eye, and increases cardiac output. ADRB2 stimulation increases intraocular pressure via the retina. In the digestive system, ADRB2 stimulates the liver and Induces lycogenolysis and glycogen production, as well as insulin secretion from the pancreas ( Fitzpatrick, 2004).

[0297] ADRB2 signaling in cardiomyocytes is regulated by interaction with CXCR4 (LaRocca et al., 2010). Norepinephrine is activated via ADRB2. to decrease CXCR4 expression and corresponding invasion of MDA-MB-231 breast cancer cells (Wa ng et al., 2015a). ADRB2 is a key regulator of several cancers, e.g., pancreatic, Standing gland (Braadland et al., 2014, Xu et al., 2017) , and is expressed in kidney and breast cancer ( Choy et al., 2016 ).

[0298] (5) C5AR1 - Complement component 5a receptor 1 (C5AR1) or CD88 (surface antigen component) The C5a receptor, also known as C5a class 88, is a G protein-coupled receptor for C5a. It functions as a complement receptor. C5AR1 is involved in inflammatory responses, obesity, development, and cancer. Regulate (Gerard and Gerard, 1994).

[0299] C5AR1 is expressed on granulocytes, monocytes, dendritic cells, the hepatoma-derived cell line HepG2, and apoptosis. It is expressed on strocytes and microglia (Klos et al., 2013). 5AR1 is involved in several diseases, such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, and experimental allergies. It has been associated with encephalomyelitis, multiple sclerosis, meningitis, and brain trauma (Lee et al. .,2008).

[0300] (6) CALCR - Calcitonin receptor (CT) is a receptor for the peptide hormone calcitonin. It is a G protein-coupled receptor that binds to calcium ions and is involved in the maintenance of calcium homeostasis, particularly in bone formation and metabolism. involved in the understanding of gratitude (Dacquin et al., 2004, Davey et al., CT has been shown to be effective on osteoclasts, on cells in the kidney, and on cells in multiple regions of the brain. They work by activating the G proteins Gs and Gq, which are often found on the surface of cells. It may also affect the ovaries in women and the testes in men. Calcitonin is mediated by G proteins that activate adenylyl cyclase. The cholera toxin-sensitive heterotrimeric guanosine triphosphate-binding protein (GTP-binding protein) is a cholera toxin-sensitive protein. The physiological actions of CT, e.g., those mediated by osteoclasts, are thought to be coupled. The inhibition of bone resorption or increased Ca2+ secretion by the kidney is mediated by high-affinity CT. (Albrandt et al., 1995).

[0301] Loss-of-function mutations in CT equate to highly aggressive glioblastoma with poor outcome. (Pal et al., 2018). CTR expression in mammary gland, cartilage, nose, and ear. The regulation of CALCR gene expression in various tissues mentioned above during development is This suggests a role for CTR in the morphogenesis of tissues (Pondel, 2000).

[0302] (7) CHRM1-muscarinic cholinergic receptor is a G protein-coupled receptor These receptors belong to a large family. The functional diversity of these receptors is due to their binding of acetylcholine. This determines the cellular response, e.g., adenylate cyclase inhibition, phosphoinositides Muscarinic receptors are found in the central and peripheral nervous systems. Muscarinic acid affects many of the actions of acetylcholine in the nervous system (Rang, 2003). Cholinergic receptor 1 is involved in mediating vagally induced bronchoconstriction and gastrointestinal acid secretion. It is believed that the signal transduction pathway involves the activation of phospholipase C and therefore inositol triphosphates. coupled to G proteins of class Gq with upregulation of phosphate and intracellular calcium It is mainly found in (Qin et al., 2011).

[0303] Muscarinic receptors are widely distributed throughout the body and vary in location and subtype ( They control distinct functions depending on the neurotransmitter CHRM1-CHRM5. They are primarily parasympathetic. This receptor is expressed in the cerebrospinal fluid and exerts both inhibitory and excitatory actions. It has been found to mediate slow excitatory postsynaptic potentials in the ganglia, and is known to be involved in exocrine glands and the CNS. CHRM1-related diseases are common in , asthma and heart block, congenital disorders and Pelizaeus-Merzbacher disease. Activators of CHRM1 muscarinic acetylcholine receptors (mAChRs) are effective in treating schizophrenia and may provide novel treatments for Alzheimer's disease (Marlo et al. al., 2009).

[0304] (8) EDNRB - Endothelin receptor type B (ETB) receptor is an endothelin receptor (ED It was cloned shortly after EDNRA (BRA). Like EDNRA, it is a G protein-coupled It belongs to the class A of putahelical receptors and has an outer amino terminus, an inner carboxy terminus, and The binding site is specific to the heptahelical portion of the receptor. Like EDNRA, endogenous The agonist ET-1 has high affinity for EDNRB. The pharmacology of the EDNRB receptor is as follows: Multiple agonists of EDNRB, such as sarafotoxin 6c (S6c) and IRL1620 It is somewhat richer than EDNRA in that it is recognized as a Selective antagonists of BQ788, A192621, RES7011, and IRL25 00. EDNRB is found in cardiovascular tissue, the pulmonary system, neurons, bone, pancreas, and kidney. It is localized (Watts, 2010).

[0305] EDNRB activates the phosphatidylinositol-calcium second messenger system Its ligand, endothelin, is a G protein-coupled receptor that binds to three powerful blood Consists of a family of duct-dilating peptides: ET1, ET2, and ET3. The EDNRB gene is regulated by the microphthalmia-associated transcription factor. Mutations in this gene are associated with Waardenburg syndrome (Sato-Jin et al. Hirschsprung disease type 2, a polygenic disorder, is caused by the EDNRB gene. Caused by a mutation in the offspring (Tanaka et al., 1998). Related to EDNRB Diseases that affect this condition include Waardenburg syndrome, type 4A, and Abcd syndrome. Related pathways include calcium signaling pathways, prostaglandin synthesis, and and Waardenburg syndrome (Sato-Jin et al., 2014). 08).

[0306] (9) The HRH1-H1 receptor belongs to the family of rhodopsin-like G protein-coupled receptors. This receptor is activated by the biogenic amine histamine. HRH1 is activated by phospholipase C and inositol triphosphate (IP3) synthase. It is associated with intracellular G protein (Gq) which activates signal transduction pathways. Anti-allergic drugs act on this receptor and are used as anti-allergic drugs. The crystal structure of the receptor has been determined and has been shown to be effective in structure-based virtual screening studies. It has been used to discover new HRH1 ligands (de Graaf et al. , 2011). The G protein subunits subsequently dissociate, releasing cyclic AMP, cyclic GMP, and catecholamines. Downstream signaling is achieved through various intermediaries, including Calcium and Nuclear Factor Kappa B It affects intracellular signaling, including immune-cell chemotaxis, pro-inflammatory cytokines, and It functions in inflammatory cytokine production, cell adhesion molecule expression, and other allergic and inflammatory conditions. Canonica and Blaiss, 2011 ).

[0307] In peripheral tissues, HRH1 mediates neurotransmission in the central nervous system as well as smooth muscle Constriction of the adrenal glands, increased capillary permeability due to constriction of peripheral veins, and catecholamines from the adrenal medulla It also mediates the release of hydroxylase inhibitors (HAs), which are known to be effective in treating allergic diseases such as atopic dermatitis, Diseases of asthma, anaphylaxis and allergic rhinitis (Xie and He, 2005) It is also known to cause vasoconstriction in smooth muscle, on vascular endothelial cells, in the heart, and It is expressed in the central nervous system (de Graaf et al., 2011).

[0308] (10) The MLNR-motilin receptor is a G protein-coupled receptor that binds to motilin. Motilin is an intestinal peptide that stimulates the contraction of gastrointestinal smooth muscles. MLNR mediates gastrointestinal motility promoting effects. MLNR is important for the treatment of hypomotility disorders. It is a promising therapeutic target (Depoortere, 2001).

[0309] It is found in highest concentrations in the nerves of the gastric antral wall and throughout the smooth muscle of the upper gastrointestinal tract. It is found at significant levels throughout the world (Kitazawa et al., 1995). Diseases associated with MLNR include gastroparesis and diabetic autonomic neuropathy. (Kitazawa et al., 1997).

[0310] (11) NTSR1 - Neurotensin receptor 1 is a large G protein-coupled receptor. It belongs to the neurotensin superfamily. Neurotensin was originally derived from the hypothalamus of cattle and It is a 13 amino acid peptide that was later isolated from the intestine. In the brain, neurotensin is primarily Although found in nerve cells, fibers, and terminals, the majority of peripheral neurotensin is located in the intestine. Central and peripheral injections of neurotensin have been shown to induce the This suggests that they do not cross the blood-brain barrier, resulting in completely different pharmacological effects. The multiple functions of urotensin, e.g., hypotension, hyperglycemia, hypothermia, antinociception, and mediates the regulation of intestinal motility and secretion (Vincent, 1995).

[0311] Signal transduction activates the phosphatidylinositol-calcium second messenger system. Signal transduction is mediated by G proteins that activate downstream MAP kinases. and protect cells from apoptosis (Heakal et al., 2011 Swift et al. reported that altered expression of neurotensin receptors is a key factor in prostate cancer. showed that cognitive function is related to cognitive status (Swift et al., 2010).

[0312] (12) PTGER2 - Prostaglandin E2 receptor 2, also known as EP2, The human gene PTGER2 encodes the prostaglandin E2 (PGE2) PTGER2 is a prostaglandin receptor that relaxes certain types of smooth muscle upon activation. It is classified as a relaxant-type prostanoid receptor based on its ability to When first bound to PGE2 or any of its agonists, it contains the Gas-Gβγ complex. The Gαs-Gβγ complex recruits G proteins with their Gαs and Gβγ subunits. They dissociate into units that in turn regulate cell signaling pathways. stimulates adenyl cyclase to increase cellular levels of cAMP, thereby inducing PKA PKA activates various signaling molecules, e.g., various signaling molecules depending on the cell type. Activates the transcription factor CREB, which leads to functional responses of the following types (Markovic et al., 2017, Woodward et al., 2011). PTGER2 also demonstrated that the cellular migration response and the production of pro-inflammatory cytokines and interleukins were involved. Activates the GSK-3 pathway, which regulates innate immune responses and also regulates cell-cell adhesion It activates the beta-catenin pathway, which not only activates the α-catenin pathway but also activates the Wnt signaling pathway. , which in turn stimulates the transcription of genes responsible for regulating cell migration and proliferation (Woodwa rd et al., 2011).

[0313] The PTGER2 receptor may act as a tumor promoter. Gene knockout mice develop fewer lung, skin, and colon cancers after exposure to carcinogens. Knockout of this gene in mice with the intestinal adenomatous polyposis mutation also resulted in animals developing These effects are generally associated with a reduction in the size and number of precancerous intestinal polyps. TGER2 mediates: reduced vascular endothelial growth factor production and therefore reduced tumor angiogenesis Decreased regulation of endothelial cell motility and survival; Interference with the anti-proliferative activity of transforming growth factor beta and, more recently, decreased regulation of the host antitumor immune response have been implicated. (O'Callaghan and Houston, 2015).

[0314] PTGER2 is widely distributed in humans. The protein is expressed in the human small intestine, lung, and , the tunica media of the renal arteries and arterioles, the thymus, the uterus, the cerebral cortex of the brain, the striatum of the brain, the hippocampus of the brain, the cornea Membrane epithelium, choriocapillaris of the cornea, myometrial cells, eosinophils, sclera of the eye, articular cartilage, spongy cavern of the penis It is expressed in somatic and airway smooth muscle cells, and its mRNA is expressed in gingival fibroblasts, monocyte-derived dendritic cells, and It is expressed in the aorta, corpus cavernosum of the penis, articular cartilage, airway smooth muscle, and airway epithelial cells. Receptor proteins and / or mRNA are expressed in the lung, spleen, intestine, skin, kidney, liver, Found in long bones and somewhat widely throughout the brain and other parts of the central nervous system It has been found (Yagami et al., 2016).

[0315] Preclinical studies have shown that PTGER2 may be involved in the treatment of allergic diseases, such as asthma (certain aspirin and and nonsteroidal inflammatory drug-induced asthma syndrome) and rhinitis (Machado-C arvalho et al., 2014), glaucoma (Doucette and Wa lter, 2017), and various diseases of the nervous system (Yagami et al., 2016). , fractures, osteoporosis and other bone abnormalities (Li et al., 2007), pulmonary fibrosis, specific forms Malignant diseases of the colon, including those caused by adenomatous polyposis coli mutations for the treatment and / or prevention of certain human disorders, including (Yang and Du, 2012) This suggests that this receptor could be a target for contraception. It has been suggested that this is the case (Sugimoto et al., 2015).

[0316] (13) TACR3 - Tachykinin receptor 3, also known as TACR3, is a receptor for tachykinins. Tachykinins function as receptors for substance P (SP), neurokinin A (N KA), neurokinin B (NKB), and species-specific endokinins, e.g., human These tachykinins are a family of peptides that includes endokinin B (EKB). Preprotachykinin 1 (TAC1), which encodes SP and NKA, and NKB, which encodes NKB. TAC3, which encodes EKB, and TAC4, which encodes EKB. The three tachykinin receptors, ACR1, TACR2, and TACR3, are NK1 receptors, respectively. , NK2, and NK3 have been identified and interact with these tachykinins. SP and EKB act on TACR1, and TACR2. NKA shows the highest potency against TACR3, and NKB shows the highest potency against TACR3. Tachykinin receptor 3 (TACR3) is defined by mutations at the 5' end of the sequence. It is a mediator of biological actions encoded by the C-terminal sequence of tachykinins, and therefore Neurokinin B is a more potent agonist than neurokinin A or substance P (Page et al., 2003).

[0317] Four human pedigrees with severe congenital gonadotropin deficiency and pubertal failure were affected. All individuals expressing TAC3 (encoding neurokinin B) or its receptor TACR3 reported to be homozygous for loss-of-function mutations in NK3R (encoding NK3R) It has been reported that NKB is a P-substance related A member of the kikinin family, but also a recently identified gonadotropin-releasing hormone In hypothalamic neurons that also express kisspeptin, a regulator of secretion, It is known that the expression of β-glucan is high in β-glucan (Gianetti and Seminara, 2014). 08).

[0318] (14) APLNR-apelin receptor (also known as APJ receptor) is a receptor that binds apelin. and G protein-coupled receptors that bind to Apela / ELABELA / Toddler. (Medhurst et al., 2003). Inhibits adenylate cyclase activity. G protein-coupled apelin receptor early endogenous ligand (APELA) and APLNR is a receptor for the hormone apelin (APLN). It plays a key role in early development, such as gastrulation and cardiac morphogenesis, by acting as a receptor for APLNR also acts as a receptor for the hormone APLN. Its use in adults has been shown to affect various processes, such as angiogenesis, blood pressure, cardiac contractility, and It also plays a role in regulating heart failure. A decade of research has revealed that the apelin system of action is widespread. It has a number of biological functions, and plays an important role in maintaining homeostasis of the cardiovascular system and fluid metabolism. Activation of APLNR is associated with cAMP suppression, protein kinase C activation, and phosphorylation of Akt, ERK1 / 25, and p70S6K, calcium mobilization, It induces a wide spectrum of biochemical changes, including oxidative stress and nitric oxide synthase (NOS). However, the specificity linking these biochemical activities to specific biological functions has yet to be discovered. has not been reported (Wang et al., 2015c).

[0319] Furthermore, it has been shown to be effective in embryonic and tumor angiogenesis (Wu et al., 2017) and as a human immunodeficiency virus (HIV-1) coreceptor (Zhou et al., 200 3) Function. Both APLNR and apelin are involved in the regulation of the immune system, including the heart, lungs, endothelium, kidneys, and brain. It is expressed in many tissues (Wang et al., 2015c).

[0320] (15) CCR5 - CC chemokine receptor, also known as CCR5 or CD195 Type 5 chemokine is involved in the immune system when it acts as a receptor for chemokines. A protein on the surface of blood cells that attracts T cells to specific tissue and organ targets. Many strains of HIV, the virus that causes AIDS, initially They use CCR5 to enter and infect host cells. Certain individuals have CCR5-Δ32 and They carry a mutation in the CCR5 gene known as Certain populations inherit the Delta 32 mutation, which protects against HIV strains. This mutation results in a partial genetic deletion of the CCR5 gene. Homozygous carriers of this mutation , resistant to M-tropic strains of HIV-1 infection (Hutter et al., 2009). The cognate ligands of CCR5 are CCL3 and CCL4 (MIPs, respectively). CCR5 also includes CCL3L1, CCL4L2, CCL5L3, CCL5L4, CCL5L5, CCL5L6, CCL5L7, CCL5L8, CCL5L9, CCL5L10, CCL5L11, CCL5L12, CCL5L13, CCL5L14, CCL5L15, CCL5L16, CCL5L17, CCL5L18, CCL5L1 It interacts with CCL5 ( Struyf et al., 2001 ).

[0321] CCR5 plays a key role in the defense against infection, although its exact role in normal immune function is unclear. This region of the protein may play a role in inflammatory responses. It is also essential for HIV-ligand binding, functional receptor responses, and HIV coreceptor activity (Bar Mania and Pepper, 2013). CCR5 is mainly expressed on T cells and macrophages. Subpopulations of phage, dendritic cells, eosinophils, microglia, and ether breast or prostate cancer cells It is expressed in the nucleus (Sicoli et al., 2014, Velasco-Velazq uez et al., 2012).

[0322] (16) GALR1 - Galanin receptor 1 (GAL1) is encoded by the GALR1 gene. Galanin is a ubiquitous neuropeptide that mediates many functions. These functions of galanin include endocrine secretion, intestinal motility, and behavioral activity. The modulatory effect is mediated by interaction with specific membrane receptors, including pertussis toxin-sensitive guanine The nucleotide-binding proteins Gi / Go are involved as transduction elements (Habert-Or toli et al., 1994).

[0323] GALR1 has antiproliferative effects in oral squamous cell carcinoma. Expression of proteins and mRNA, and GAL secretion were measured in immortalized human oral keratinocytes and human oropharynx. It was detected at variable levels in squamous cell carcinoma cell lines and in immortalized and malignant keratinocytes. In mice, proliferation was upregulated upon competitive inhibition of GALR1 (Henson et al. .,2005).

[0324] Stevenson et al. reported that the stem cell marker and regulator galanin and and GALR1 as crucial determinants of drug resistance and potential therapeutics to combat drug resistance. Regarding the mechanism, they reported that GALR1, a receptor for galanin, acts as a therapeutic target. The endothelial axis as a key upstream regulator of the expression of the anti-apoptotic protein FLIPL Clinically, galanin mRNA is overexpressed in colorectal tumors. Notably, high galanin mRNA expression was found in early-stage disease. It correlates with poor disease-free survival (Stevenson et al., 2012). R1 is widely expressed in the brain and spinal cord as well as peripheral sites such as the small intestine and heart (Fager berg et al., 2014).

[0325] (17) PTGER3 - Prostaglandin EP3 receptor (53 kDa) binds EP3 Prostaglandin E2 (PG), also known as prostaglandin E2 (PG), is encoded by the human gene PTGER3. E2), which is one of the four identified prostaglandin receptors. It is one of the three receptors, the others being PTGER1, PTGER2, and PTGER4. These are also called EP1, EP2, and EP4, respectively, and all of them are PGE2-dependent. and also to certain other prostanoids, although usually with lower affinity and response. PTGER3 activation mediates cellular responses to PTGER3 even in mice. promotes duodenal secretion in humans, a function mediated by PTGER3 activation EP receptor function can vary between species, and most of the functional studies cited here These animal and tissue models do not replace humans (Moreno, 2017 ).

[0326] Studies of the direct effects of PTGER3 activation on cancer in animal and tissue models have Conflicting results have been obtained, suggesting that this receptor does not play an important role in carcinogenesis. However, some studies have suggested that PTGER3 plays an indirect role in promoting tumorigenesis. This suggests that the PTGER3-deficient mice were treated with transplanted mouse lung cancer cell lines. The growth and metastasis of Lewis lung carcinoma cells were inhibited. This effect was attributed to the vascular endothelium in the tumor stroma. Growth factors and matrix metalloproteinase 9 expression, lymphangiogenic growth factor VEGF Expression of VEGF-C and its receptor VEGFR3, and tumor-associated angiogenesis and lymphangiogenesis was associated with lower raw levels (O'Callaghan and Houston, 2015).

[0327] PTGER3 is widely distributed in humans. Its protein and / or mRNA , the kidney (i.e., Tamm-Horsfall protein-negative late distal convoluted tubules, connective segments, cortical and medullary collecting ducts, media and endothelial cells of arteries and arterioles); stomach (vascular smooth muscularis and fundus mucosal cells; thalamus (anterior, ventromedial, dorsolateral, paraventricular and medial central nuclei); crypt crests intestinal mucosal epithelium in the uterine muscle (stromal cells, endothelial cells, and placenta, chorion, and amnion during pregnancy) ); oral gingival fibroblasts; and eye (corneal endothelium and keratocytes, trabecular cells, supraciliary body cells) It is expressed in the skin, as well as in conjunctival and iris stromal cells, and retinal Müller cells (Nore l,2016).

[0328] (18)SSTR2-Somatostatin receptor type 2 is located in the SSTR2 gene in humans. Somatostatin acts at many sites and inhibits many hormones. It inhibits the release of somatostatin and other secretory proteins. It is mediated by a family of G protein-coupled receptors that are expressed in a tissue-specific manner. STR2 has been shown to interact with shank2 (Zitzer et al. l.,1999).

[0329] SSTR2 encodes somatostatin receptors that can inhibit cell proliferation in solid tumors. Hypermethylation of the SSTR2 promoter is associated with male laryngeal squamous cell carcinoma Most of the cases are associated with the risk and progression of ptosis (Shen et al., 2016). Although somatostatin adenomas express SSTR2, other somatostatin receptors have also been found (Mi ller et al., 1995). Somatostatin analogs (i.e., octreotide) The drugs lanreotide and lanreotide stimulate this receptor, thus further inhibiting tumor growth. SSTR2 is used to It is expressed at the highest levels in the brain and kidney (Fagerberg et al., 2014 ).

[0330] As used herein, the term "inhibitor" refers to an inhibitor of the CXCR4-GPCRx heteromer. This refers to molecules that inhibit or suppress enhanced functions. Treatment and improvement of cancer or related symptoms Non-limiting examples of inhibitors of the present invention that can be used for the prevention or prophylaxis include GP CRx antagonists, GPCRx inverse agonists, positive and negative allosteric modulators of GPCRx, C XCR4-GPCRx heteromer-specific antibodies or antibodies thereof containing single-domain antibody-like scaffolds The original binding moiety is linked to a GPCRx-selective pharmacophore by a spacer arm. Bivalent ligands with a CXCR4-selective pharmacophore, CXCR4 and GPC Bispecific antibodies against Rx and radiolabeled CXCR4 ligands linked to GPCRx ligands and small molecule ligands that inhibit heteromer-selective signaling. It forms heteromers with CXCR4 and enhances Ca2+ responses upon costimulation with both agonists. Some examples of inhibitors of GPCRxs that are potent are listed in Table 3.

[0331] As used herein, the term "antagonist" refers to an agent that acts by binding to and blocking a receptor. A class of receptor ligands or drugs that block or attenuate biological responses, also known as blockers Antagonists have affinity for their cognate receptors but lack potency. Their binding disrupts the interaction and inhibits the function of agonists or inverse agonists at their cognate receptors. It forms heteromers with CXCR4 and inhibits Ca2+ responses upon costimulation by both agonists. Some examples of antagonists to GPCRx that enhance response are listed in Table 3.

[0332] Table 3. Ca2+ response upon co-stimulation with both agonists by forming heteromers with CXCR4. Example of an inhibitor of a GPCRx that enhances response.

[0333] [Table 3] TIFF0007818648000008.tif248170TIFF0007818648000009.tif248170TIFF0007818648000010.tif210170

[0334] In some embodiments, the CXCR4-GPCRx heteromers disclosed herein A method of treating cancer in a patient having cells containing the compound or a method of treating cancer in a patient having cells containing the compound Suppresses enhanced downstream signaling from CXCR4-GPCRx heteromers in vesicles The method comprises combining an inhibitor of CXCR4, an inhibitor of GPCRx, and a CXCR4-GPCRx inhibitor. and administering to the patient an inhibitor or combination of inhibitors selected from the group consisting of: i) CXCR4-GPCRx heteromers induce enhanced downstream signaling; ii) the administered inhibitor or combination of inhibitors has a therapeutic effect in cancer patients suppresses enhanced downstream signaling from the CXCR4-GPCRx heteromers In some embodiments, the CXCR4-GPCRx heteromers disclosed herein Pharmaceutical kit or pharmaceutical composition for use in treating cancer in a patient having cells containing CXCR4 inhibitors, GPCRx inhibitors, and CXCR4-GPCRx heteromer inhibitors The inhibitor or combination of inhibitors may be selected from the group consisting of CXCR4-G PCRx heteromers are those that have enhanced downstream signaling. For example, some In some embodiments, the CXCR4 inhibitor is an antagonist of CXCR4, an inverse agonist of CXCR4, or , CXCR4 partial antagonists, CXCR4 allosteric modulators, CXCR4 antibodies, C CXCR4 antibody fragment, CXCR4 ligand, or CXCR4 antibody-drug conjugate. For example, in some embodiments, a GPCRx inhibitor is an antagonist of GPCRx, Inverse agonists of CRx, partial antagonists of GPCRx, allosteric modulators of GPCRx, GP CRx antibodies, GPCRx antibody fragments, GPCRx ligands, GPCRx antibody-drugs For example, in some embodiments, a CXCR4-GPCRx heteromeric complex The inhibitors are antagonists of the CXCR4-GPCRx heteromer, Inverse agonist of CXCR4-GPCRx heteromer, partial antagonist of CXCR4-GPCRx heteromer, CXCR4-GPC Allosteric modulators of Rx heteromers, antibodies to CXCR4-GPCRx heteromers, C Antibody fragments of XCR4-GPCRx heteromers, ligation of CXCR4-GPCRx heteromers and inhibitors of protein-protein interactions (PPIs) of CXCR4-GPCRx heteromers, or CXCR4-GPCRx heteromeric antibody-drug conjugates.

[0335] In some embodiments, the methods of treatment, suppression, pharmaceutical kits or The pharmaceutical composition includes a CXCR4 inhibitor, a GPCRx inhibitor, and a CXCR4-GPCRx inhibitor. and combinations of inhibitors selected from the group consisting of inhibitors of telomeres, Each individual inhibitor may be contained in a separate pharmaceutical composition, or each individual inhibitor may be contained in a separate pharmaceutical composition. In some embodiments, the combination of inhibitors may include a CXCR4 inhibitor and a GPCRx inhibitor. In some embodiments, the inhibitor combination comprises a CXCR4 In some embodiments, the inhibitor comprises an inhibitor of CXCR4-GPCRx heteromers. The inhibitor combinations include a GPCRx inhibitor and an inhibitor of the CXCR4-GPCRx heteromer. In some embodiments, the combination of inhibitors is sequential, concurrent, or simultaneous. In some embodiments, the combination of inhibitors is administered in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the combination of inhibitors is administered as a pharmaceutical composition comprising: The separate pharmaceutical compositions are administered as separate pharmaceutical compositions, each of which independently contains a pharmaceutically acceptable carrier. In some embodiments, the methods of treatment, suppression, and medicine disclosed herein include a carrier. The pharmaceutical kit or composition may comprise a therapeutically effective or sub-therapeutically effective amount of a CXCR4 inhibitor. For example, administering a therapeutically effective amount of a CXCR4 inhibitor to a patient. In some embodiments, the therapeutic methods, suppressive methods, pharmaceutical kits or pharmaceutical compositions disclosed herein The composition may comprise a therapeutically effective amount or a sub-therapeutically effective amount of a GPCRx inhibitor, e.g., In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a GPCRx inhibitor. The methods of treatment, suppression, pharmaceutical kits, and pharmaceutical compositions disclosed herein may be administered in a therapeutically effective amount. or a subtherapeutic effective amount of an inhibitor of a CXCR4-GPCRx heteromer, e.g. administering to the patient a therapeutically effective amount of an inhibitor of the CXCR4-GPCRx heteromer. .

[0336] In some embodiments, the CXCR4-GPCRx heteromers disclosed herein The method of treating cancer in a patient having cancer cells containing: 1) an enhanced downstream signal; Whether or not the patient has cancer cells containing CXCR4-GPCRx heteromers that are transduced The determination of whether or not a biological sample is obtained from a patient is made by obtaining or obtaining a biological sample from the patient and performing the following on the biological sample: whether the cancer cells contain the above CXCR4-GPCRx heteromer, or ii) CX CR4-GPCRx heteromer-selective reagents: whether it alters the heteromer-specific properties or function of CR4-GPCRx heteromers; Heteromer-specificity of patient-derived cell(s) containing CXCR4-GPCRx heteromers or patient-derived CXCR4-GPCR heteromers, whether or not they alter their properties. performing or conducting an assay to determine whether cell proliferation of the cell(s) is reduced 2) performing the above-described CXCR4-GPCRx heterotransferase; In patients with cancer cells containing CXCR4, inhibitors of GPCRx an inhibitor selected from the group consisting of an inhibitor of CXCR4-GPCRx heteromers; may include administering a combination of inhibitors to a cancer patient internally. having cancer cells containing CXCR4-GPCRx heteromers as disclosed herein. The method for treating a patient's cancer comprises: 1) determining whether the patient's cancer cells are CXCR4-GPCRx heteromers; The determination of whether or not a biological sample contains a cancer cell of the patient is based on the fact that the biological sample was obtained or acquired from the patient and the fact that the patient's cancer cell An assay was performed to determine whether the CXCR4-GPCRx heteromer was present in the cells. may include performing or having performed on a biological sample, The GPCRx in the R4-GPCRx heteromer is ADCYAP1R1, ADORA2B, and A DORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1 , MLNR, NTSR1, and TACR3; and b) to a biological sample. The assays performed include: co-internalization assay, co-localization assay, in situ hybridization assay, and co-localization assay. hybridization, immunohistochemistry, immunoelectron microscopy, proximity-based assays, co- immunoprecipitation assay, or fluorescent animal assay, , e.g., co-internalization assays, bimolecular fluorescence complementation (BiFC) or proximity ligation assays. 2) the patient's cancer cells are CXCR4-G In the case of containing PCRx heteromers, a CXCR4 inhibitor, a GPCRx inhibitor and a CXCRx an inhibitor or inhibitors selected from the group consisting of inhibitors of CR4-GPCRx heteromers In some embodiments, the combination may be administered to a patient using a biological sample from the patient. is a biological fluid sample or a biological tissue sample. In some embodiments, the liquid biopsy It is performed on a biological fluid sample or on a biological tissue sample. In some embodiments, the biological fluid sample is a blood sample, a plasma sample, a saliva sample, a cerebrospinal fluid sample, an intraocular fluid sample, or In certain embodiments, the biological fluid sample includes, for example, a urine sample. Campos CDM et al., “Molecular Profiling of Liquid Biopsy Samples for Precision Medicine,”Cancer J.2018 Mar / Apr;24(2):93 Circulating tumor cells (CTCs) from body fluids, tumor-derived acellular D NA (cfDNA), circulating small RNAs, and extracellular vesicles including exosomes In some embodiments, the biological tissue sample is an organ tissue sample, a bone tissue sample, or tumor tissue. This is a sample.

[0337] As used herein, the term "heteromer" refers to a heteromer consisting of at least two GPCR units [ It consists of protomers with biochemical properties that are distinct from those of the individual components. Heteromerization refers to the formation of macromolecular complexes. Immunohistochemistry, RNAseq, reverse transcription quantitative PCR (RT-qPCR, real-time PCR) ), microarray, proximity ligation assay (PLA), time-resolved FRET (TR -FRET), whole-body single-photon emission computed tomography (SPECT), or positron emission tomography (PET) This can be assessed by PET / CT.

[0338] As used herein, the phrase "effective amount" means an amount sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications or dosages. Such delivery may depend on the duration for which the individual dosage units are used, the bioavailability of the drug, and the route of administration. It depends on several variables, including

[0339] As used herein, the phrase "therapeutically effective amount" refers to a therapeutically effective amount for treating cancer and / or related conditions. Treatment sufficient to reduce, ameliorate, and / or prevent the severity and / or duration of symptoms refers to the amount of an agent (e.g., an inhibitor, antagonist, or any other therapeutic agent provided herein) A therapeutically effective amount of a therapeutic agent is an amount that reduces, improves, or prevents the progression or progression of cancer, or prevents or alleviates or prevents the progression or progression of cancer. Therapeutic use may include reducing, ameliorating, or preventing recurrence, progression, or onset, and / or preventing the progression of a disease associated with another therapy (e.g., inhibitor). prophylactic treatment of a patient with a rheumatoid arthritis (other than administration of an inhibitor, antagonist, or any other therapeutic agent provided herein) Alternatively, it may be the amount required to enhance or increase the therapeutic effect.

[0340] The term "therapeutic agent" refers to a substance that treats, ameliorates, prevents, or reduces cancer and / or its related symptoms. In certain embodiments, a therapeutic agent refers to any agent that can be used in the management or treatment of a cancer. Therapeutic agents are inhibitors of XCR4-GPCRx heteromers. are well known to be used in the treatment, amelioration, prevention or management of the symptoms involved, or It may be a drug that has been used or is currently being used for it.

[0341] As used herein, the terms "intracellular Ca2+ assay" and "calcium mobilization assay" refer to The phrase "calcium ion transporter" or variations thereof refers to calcium ion transporters associated with GPCR activation or inhibition. This refers to a cell-based assay for measuring the flow of ATP into the cytoplasm of most cells. The dye is released from intracellular stores and then absorbed into the cells. The change in fluorescence intensity indicates the activity of the receptor of interest. This directly correlates with the amount of intracellular calcium released into the cytoplasm in response to ligand activation.

[0342] As used herein, the phrase "proximity-based assay" refers to an in vitro (cell lysis) assay. Track and assess the proximity and / or binding of two protein molecules in living organisms and living cells Bioluminescence resonance energy transfer (BRI) refers to a biophysical and biochemical technique that can RET), Fluorescence Resonance Energy Transfer (FRET), Bimolecular Fluorescence Complementation (BiFC), Proximity Labeling These include immunoprecipitation assays (PLA), cysteine ​​cross-linking, and co-immunoprecipitation (Ferre et al., 2009, Gomes et al., 2016.

[0343] An alternative method for detecting heteromer formation is (Bushlin et al. ,2012, Decaillot et al.,2008); Immunoelectron microscopy (Fe rnandez-Duenas et al.,2015);BRET(Pfleger and Eidne, 2006); time-resolved FRET assay (Fernandez- Duenas et al., 2015); In Situ Hybridization (H e et al.,2011);FRET(Lohse et al.,2012);B RET, FRET, BiFC, bimolecular emission complementation, enzyme fragmentation assays and Tango T Ango GPCR Assay System (Thermo Fisher Scientific) GPCR heteromer identification technology (GPCR-ic) (Mustafa, 2010) HIT, Dimerix Bioscience)(Mustafa and Pfle 3-arrestin recruitment assay using PRESTO-Tango system (ger, 2011) Stem (Kroeze et al., 2015); Adjusted Secretion / Aggregation Technology (ARIAD) Pharmaceuticals) (Hansen et al., 2009); receptor Selection amplification technology (ACADIA Pharmaceuticals) (Hansen et al. al.,2009);DimerScreen(Cara Therapeutics ) (Mustafa, 2010); dimer / interacting protein translocation assay (Pato bios) (Mustafa, 2010); co-immunoprecipitation (Abd Alla et al .,2009); surface enzyme-linked immunosorbent assay (ELISA) (Decaillot et al., 2008) or flow cytometry (Law et al., 200 GPCR internalization assay using a whole-cell phosphorylation assay (Pfeiffer et al., 2015); t al., 2002); and proximity ligation assay (PLA) (Frederi ck et al., 2015), but are not limited to these.

[0344] Pharmacological properties and signal transduction in cells expressing both CXCR4 and GPCRx An alternative method for detecting changes in delivery and / or transport properties is the use of radioligands. Binding assays ( Bushlin et al., 2012 , Pfeiffer et al. l., 2002); cell surface biotinylation and immunoblotting (He et al., 2011); immunostaining (Bushlin et al., 2012, Decaillot et al., 2008); Immunoelectron microscopy (Fernandez-Duenas et al., 2015); [35S]GTP S binding assay (Bushlin et al. al., 2012); Fura 2-acetomethoxy ester (Molecular P robes), Fluo-4 NW calcium dye (Thermo Fisher Scientific ientific) or FLIPR5 dye (Molecular Devices) Which dyes to use for calcium imaging or assay; radioimmunoassay kit (Ame cAMP assay using AlphaScr (Brothersham Biosciences); een (PerkinElmer Life Sciences); Parameter Cycling cAMP assay (R&D Systems); Femto cAMP kit (Cisb cAMP direct immunoassay kit (Calbiochem) or GloSen sor cAMP assay (Promega); GTPase assay (Pello t al., 2008);PKA activation (Stefan et al., 2007);E RK1 / 2 and / or Akt / PKB phosphorylation assays (Callen et al. , 2012); Src and STAT3 phosphorylation assays (Rios et al., 20 06); reporter assays, e.g., cAMP response element (CRE); activated T cell nuclear factor NFAT-RE; Serum Response Element (SRE); Serum Response Factor Response Element (SR F-RE); and NF-κB response element luciferase reporter assay; secreted calcium phosphatase assay ( Decaillot et al., 2011 ); TR -FRET or [3H]myo-inositol for inositol 1-phosphate production Measurement of downstream target gene expression (Mustafa et al., 2012). RT-qPCR for adenylyltransferase (Mustafa et al., 2012); cyclase activity (George et al., 2000); next-generation sequencing ( NGS); as well as detecting changes in receptor function as a result of receptor heterodimerization. Examples of assays that can be used include, but are not limited to, any other assay that can

[0345] As used herein, the term "protein-protein interaction inhibitor" or "PPI inhibitor" or variations thereof, any molecule capable of interfering with protein-protein interactions. Unlike enzyme-substrate interactions, which involve distinct binding pockets, protein-protein interactions The use of proteins is a transient interaction or association between proteins over a relatively large area, and Facilitated by electrostatic interactions, hydrophobic interactions, hydrogen bonding and / or van der Waals forces PPI inhibitors often target the GPCR heteromeric interface, e.g., the transmembrane domain of GPCRx. Membrane-transmitting peptides fused to peptide sequences that disrupt helices, intracellular loops, or C-terminal tails. These may include, but are not limited to, transient peptides or lipids. x heteromeric PPI inhibitors, for example, target the CXCR4-GPCRx heteromeric interface (multiple Membrane-penetrating or cell-penetrating peptides conjugated to peptides that target the targeting CPP) or the CXCR4-GPCRx heteromeric interface(s) The peptide may be a cell-permeable lipidated peptide that targets the

[0346] For example, the membrane-permeable peptide or cell-permeable peptide is an HIV-1 TAT peptide. Petite, e.g. TAT 48-60 and TAT 49-57 penetratins, e.g., pAnt p(43-58); polyarginine (Rn, e.g., R5-R12); Diatos peptide Dovector 1047 (DPV1047, Vectocell®); MPG (S HIV gp41 fused to the nuclear localization signal (NLS) of V40 large T antigen); Pe p-1 (tryptophan-rich cluster fused to the NLS of SV40 large T antigen); pVEC peptide (vascular endothelial cadherin); p14 alternative reading frame (ARF) Protein-based ARF(1-22); the N-terminus of unprocessed bovine prion protein End BPrPr(1-28); Model amphipathic peptide (MAP); Transportan; p28 peptide derived from durin; amphipathic beta-sheet peptides, e.g., VT5; proline-rich CPPs, such as Bac7 (Bac1-24); hydrophobic CPPs, such as α1-antithrombin C105Y derived from peptic ulcer; PFVYLI derived from synthetic C105Y; Pep-7 peptidase peptide phage clones; and modified hydrophobic CPPs, e.g., Stapled and prenylated peptides (Guidotti et al., 201 7, Kristensen et al., 2016). Membrane-penetrating peptides Alternatively, the cell-penetrating peptide may further include, for example, a TAT-derived cell-penetrating peptide, a signal peptide, or the like. cell-penetrating peptides based on amino acid sequences (e.g., NLS), hydrophobic membrane translocating sequence (MTS) peptides, These may include ribosomal, ribosomal, and arginine-rich molecular transporters. Examples of peptides include pepducins, such as ICL1 / 2 / 3, C-tail short-chain palmitoyl lysed peptides (Covic et al., 2002; O'Callaghan et al. al., 2012).

[0347] Peptide(s) targeting the CXCR4-GPCRx heteromeric interface may be, for example: CXCR4 transmembrane domain, GPCRx transmembrane domain, CXCR4 intracellular loop , intracellular loop of GPCRx, C-terminal domain of CXCR4 or C-terminus of GPCRx lateral domain, extracellular loop of CXCR4, extracellular loop of GPCRx, N-terminus of CXCR4 The GPCR may be a terminal region or an N-terminal region of GPCRx.

[0348] In some embodiments, the present invention provides a CXCR4-GPCRx heteromer of the present invention. A pharmaceutical composition (sometimes referred to herein as a "pharmaceutical composition") having an inhibitor and a pharmaceutically acceptable carrier. Pharmaceutically acceptable pharmaceutical compositions that can be used in the pharmaceutical compositions of the present invention are provided. The carrier may be any of the standard pharmaceutical carriers known in the art, e.g., physiological saline. a physiologically acceptable carrier, excipient, or stabilizer, such as phosphate buffered saline, water, and These include emulsions, such as oil-water emulsions, and various types of wetting agents. The pharmaceutical composition may be administered in a liquid unit dosage form or in a CXCR4-containing formulation of the present invention to a target area of ​​a subject in need of treatment. or prepared in any other dosage form sufficient to deliver the inhibitor of 4-GPCRx heteromers. For example, the pharmaceutical composition may be administered in a selected manner, e.g., intravenously, intramuscularly, subcutaneously, or intradermally. The composition may be prepared in any suitable manner for intrathecal administration, intrathecal administration, or intrathecal administration. , pharmaceutical grade stabilizers, buffers, preservatives, excipients, etc. can be readily selected by one of ordinary skill in the art. It is necessary to adjust pharmaceutical compositions with due consideration given to pH, isotonicity, stability, etc. Included in the level of skill in a technical field.

[0349] One or more of the inhibitors of CXCR4-GPCRx heteromers of the invention provided herein The pharmaceutical preparation containing the inhibitor with the desired purity is prepared in a physiologically acceptable form for storage. Suitable carriers, excipients or stabilizers (Remington's Pharmaceuticals cal Sciences(1990)Mack Publishing Co.,Ea It can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing with ston, PA. Acceptable carriers, excipients, or stabilizers are those that are acceptable at the dosages and concentrations employed. Non-toxic to the recipient and does not contain buffers, e.g., phosphates, citrates and other organic acids; aspartame Antioxidants, including corbic acid and methionine; preservatives, such as octadecyldimethyl chloride Benzylammonium chloride; Hexamethonium chloride; Benzalkonium chloride, Benzethonium chloride phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methylparabens methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3 -pentanol; and m-cresol; low molecular weight polypeptides of less than about 10 residues; proteins Proteins, e.g., serum albumin, gelatin or immunoglobulins; hydrophilic polymers, e.g. for example, polyvinylpyrrolidone; amino acids, for example, glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, e.g., glutamic acid ... glucose, mannose or dextrin; chelating agents such as EDTA; sugars, e.g. sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, e.g. sodium; metal complexes, e.g., Zn-protein complexes; and / or non-ionic interfaces Active agents, such as TWEEN™, PLURONICS™, or polyethylene Contains PEG (polyethylene glycol).

[0350] Thus, in some embodiments, the present invention provides a method for treating, ameliorating, or preventing a disease. The methods of the present invention provide methods for administering the therapeutic agent provided herein to a subject in need thereof. For example, the pharmaceutical composition may comprise administering to the subject a therapeutically effective amount of the pharmaceutical composition. The present invention may include one or more inhibitors of CXCR4-GPCRx heteromers provided in Diseases that can be treated or prevented using the methods include cancer, tumors, metastases and / or Specifically, the methods of the present invention include the treatment of cancer, tumors, and / or microcirculation. Treating cancers or related conditions in which cells in the periphery express CXCR4-GPCRx heteromers The methods of the present invention can be used to treat, ameliorate, or prevent Non-limiting examples of cancers or tumors include tumors of the gastrointestinal tract, e.g., breast cancer, lung cancer, small cell lung cancer, carcinoma, hepatocellular carcinoma, brain tumor, kidney cancer, pancreatic cancer or pancreatic adenocarcinoma, ovarian cancer, prostate cancer, melanoma tumors, lymphomas, leukemia, multiple myeloma, renal cell carcinoma, soft tissue sarcomas, gastrointestinal cancers, gastric cancer, Bowel cancer, colorectal cancer, colorectal adenocarcinoma, bladder adenocarcinoma, esophageal cancer, and adenocarcinoma of the stomach, esophagus, throat, and genitourinary tract Examples include:

[0351] In some embodiments, the present invention provides cells containing CXCR4-GPCRx heteromers. and a method for treating cancer in a patient having a cancer cell. Suppressing enhanced downstream signaling from CXCR4-GPCRx heteromers in The method comprises administering to a subject a CXCR4 inhibitor, a GPCRx inhibitor, and a CXCR4 - an inhibitor or combination of inhibitors selected from the group consisting of inhibitors of GPCRx heteromers to a patient, wherein i) the CXCR4-GPCRx heteromer exhibits enhanced immune responses. ii) the administered inhibitor or combination of inhibitors Enhanced downstream signaling from CXCR4-GPCRx heteromers in cancer patients Inhibit transmission.

[0352] In some embodiments, the present invention provides cells containing CXCR4-GPCRx heteromers. A pharmaceutical kit for use in treating cancer in a patient having a tumor, comprising a CXCR4 inhibitor. an inhibitor of GPCRx and an inhibitor of CXCR4-GPCRx heteromers; and a combination of selected inhibitors or inhibitors, wherein the CXCR4-GPCRx heteromer is increased. The pharmaceutical kit also provides a pharmaceutical kit having enhanced downstream signaling. In an embodiment, the present invention relates to a method for treating a patient having cells containing CXCR4-GPCRx heteromers. 1. A pharmaceutical composition for use in treating cancer in a subject, comprising: i) an inhibitor of CXCR4, GPC an inhibitor of Rx and an inhibitor of CXCR4-GPCRx heteromers; an inhibitor or combination of inhibitors, and ii) a pharmaceutically acceptable carrier, The 4-GPCRx heteromer has enhanced downstream signaling. Pharmaceutical compositions are provided.

[0353] In some embodiments, the methods of treatment, methods of suppression, pharmaceutical kits or According to the pharmaceutical composition, the above-mentioned cancer cells containing CXCR4-GPCRx heteromers are The progression of cancer in patients receiving a combination of inhibitors is significantly greater than that of a single inhibitor administered to said patients. in the range of 5-100% compared with administration of CXCR4 inhibitors or GPCRx inhibitors as a therapeutic agent. The range is reduced more than when a single inhibitor is administered to the patient, for example, when a combination of inhibitors is administered. 5-100%, 10% compared with administration of a CXCR4 inhibitor or GPCRx inhibitor as ~100%, 20~100%, 30~100%, 40~100%, 50~100%, 60 ~100%, 75-100%, 5-75%, 5-50%, or 5-25% In some embodiments, the GPCR of the CXCR4-GPCRx heteromer is significantly reduced. x represents ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3 For example, ADCYAP1R1, ADORA2B, ADORA3 , ADRB2, C5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3; ADCYAP1R1, ADORA2B, ADO RA3, ADRB2, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and T ACR3; ADCYAP1R1, ADORA2B, ADORA3 , ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3 Selected from the group consisting of ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C selected from the group consisting of HRM1, HRH1, NTSR1, and TACR3; ADORA2 B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NTSR1 and T ACR3; ADRB2, CHRM1, EDNRB, HRH1, N Selected from the group consisting of TSR1 and TACR3; ADRB2, CHRM1, EDNRB , HRH1 and TACR3; and TACR3; ADRB2, EDNRB, HRH1 and TAC R3; selected from the group consisting of ADRB2, EDNRB, and HRH1 selected from the group consisting of ADRB2, CHRM1 and HRH1; or 2, HRH1, and TACR3. In some embodiments, GP CRx inhibitors include ADCYAP1R1 inhibitors, ADORA2B inhibitors, and ADORA3 inhibitors. agents, ADRB2 inhibitors, C5AR1 inhibitors, CALCR inhibitors, CHRM1 inhibitors, ED NRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and TACR3 inhibitors agents; for example, ADCYAP1R1 inhibitors, ADORA2B inhibitors , ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CHRM1 inhibitor, ED NRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and TACR3 inhibitors an ADCYAP1R1 inhibitor, an ADORA2B inhibitor, an ADO RA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor an MLNR inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, NTSR1 inhibitors and T ACR3 inhibitors; ADCYAP1R1 inhibitors, ADORA2B inhibitors inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CHRM1 inhibitors, HRH1 inhibitors, N an inhibitor selected from the group consisting of a TSR1 inhibitor and a TACR3 inhibitor; an ADORA2B inhibitor; ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH 1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor; inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, NTSR1 inhibitors and TACR3 inhibitors; ADRB2 inhibitors, CHRM1 inhibitors, ED an NRB inhibitor, an HRH1 inhibitor, and a TACR3 inhibitor; 2 inhibitor, CHRM1 inhibitor, HRH1 inhibitor, and TACR3 inhibitor ADRB2 inhibitors, EDNRB inhibitors, HRH1 inhibitors and TACR3 inhibitors an ADRB2 inhibitor, an EDNRB inhibitor, and an HRH1 inhibitor; selected from the group consisting of: an ADRB2 inhibitor, a CHRM1 inhibitor, and an HRH1 inhibitor; or an ADRB2 inhibitor, an HRH1 inhibitor, and a TACR3 inhibitor. The compound is selected from the group consisting of:

[0354] In some embodiments, the methods of treatment, methods of suppression, pharmaceutical kits or According to the pharmaceutical composition, the above-mentioned cancer cells containing CXCR4-GPCRx heteromers are Efficacy of CXCR4 inhibitors when administered in combination with GPCRx inhibitors in patients with is 5-20 times more effective than CXCR4 inhibitors when administered as a single inhibitor. 00%, for example, the above cancers containing CXCR4-GPCRx heteromers. When administered in combination with a GPCRx inhibitor to patients with HER2-positive cells, 5-1750% and 5-150% compared to the efficacy of CXCR4 inhibitors when administered as a single agent. 0%, 5~1250%, 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%, 100~2000%, 200~2 000%, 300~2000%, 500~2000%, 750~2000%, 1000~ 2000%, 1250~2000%, 1500~2000%, 5~1500%, 25~1 500%, 50-1500%, 75-1500%, 100-1500%, 200-150 0%, 300-1500%, 500-1500%, 750-1500%, 1000-15 In some embodiments, the CX The GPCRx in the CR4-GPCRx heteromer is ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH 1, MLNR, NTSR1, and TACR3; e.g., ADCYA P1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, ED selected from the group consisting of NRB, HRH1, MLNR, NTSR1, and TACR3; AD CYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB , HRH1, MLNR, NTSR1, and TACR3; P1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HR H1, NTSR1, and TACR3; ADCYAP1R1, ADO RA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1 and TACR 3; ADORA2B, ADORA3, ADRB2, CHRM1, Selected from the group consisting of EDNRB, HRH1, NTSR1 and TACR3; ADRB2 , CHRM1, EDNRB, HRH1, NTSR1 and TACR3. selected from the group consisting of ADRB2, CHRM1, EDNRB, HRH1, and TACR3 selected from the group consisting of ADRB2, CHRM1, HRH1, and TACR3; AD RB2, EDNRB, HRH1, and TACR3; selected from the group consisting of DNRB and HRH1; or selected from the group consisting of ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of an ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, C ALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor an anti-cancer drug, an anti-inflammatory ... AP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C 5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor an ADCYAP1R inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; 1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and T ACR3 inhibitors; ADCYAP1R1 inhibitors, ADORA2B inhibitors inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, an HRH1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; CYAP1R1 inhibitors, ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors , consisting of a CHRM1 inhibitor, an HRH1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CH RM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor and TACR3 inhibitor an inhibitor selected from the group consisting of an ADRB2 inhibitor, a CHRM1 inhibitor, and an EDNRB inhibitor A is selected from the group consisting of an HRH1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; DRB2 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors and TACR3 inhibitors; ADRB2 inhibitors, CHRM1 inhibitors, HRH1 inhibitors and TACR3 inhibitors; ADRB2 inhibitors, EDNRB inhibitors, an HRH1 inhibitor and a TACR3 inhibitor; an ADRB2 inhibitor, an ED an NRB inhibitor and an HRH1 inhibitor; an ADRB2 inhibitor, a CHRM inhibitor or an ADRB2 inhibitor, an HR inhibitor, and an HRH1 inhibitor; H1 inhibitors and TACR3 inhibitors.

[0355] In some embodiments, the methods of treatment, methods of suppression, pharmaceutical kits or According to the pharmaceutical composition, the above-mentioned cancer cells containing CXCR4-GPCRx heteromers are Efficacy of GPCRx inhibitors when administered in combination with CXCR4 inhibitors in patients with is 5-20 times more effective than GPCRx inhibitors when administered as a single inhibitor. 00%, for example, the above cancers containing CXCR4-GPCRx heteromers. When administered in combination with a CXCR4 inhibitor to patients with BRCA1-positive BRCA1 cells, 5-1750% and 5-150% greater efficacy compared to GPCRx inhibitors administered as a single agent 0%, 5~1250%, 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%, 100~2000%, 200~2 000%, 300~2000%, 500~2000%, 750~2000%, 1000~ 2000%, 1250~2000%, 1500~2000%, 5~1500%, 25~1 500%, 50-1500%, 75-1500%, 100-1500%, 200-150 0%, 300-1500%, 500-1500%, 750-1500%, 1000-15 In some embodiments, the CX The GPCRx in the CR4-GPCRx heteromer is ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH 1, MLNR, NTSR1, and TACR3; e.g., ADCYA P1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, ED selected from the group consisting of NRB, HRH1, MLNR, NTSR1, and TACR3; AD CYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB , HRH1, MLNR, NTSR1, and TACR3; P1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HR H1, NTSR1, and TACR3; ADCYAP1R1, ADO RA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1 and TACR 3; ADORA2B, ADORA3, ADRB2, CHRM1, Selected from the group consisting of EDNRB, HRH1, NTSR1 and TACR3; ADRB2 , CHRM1, EDNRB, HRH1, NTSR1 and TACR3. selected from the group consisting of ADRB2, CHRM1, EDNRB, HRH1, and TACR3 selected from the group consisting of ADRB2, CHRM1, HRH1, and TACR3; AD RB2, EDNRB, HRH1, and TACR3; selected from the group consisting of DNRB and HRH1; or selected from the group consisting of ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is selected from the group consisting of an ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, C ALCR inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor an anti-cancer drug, an anti-inflammatory ... AP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C 5AR1 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor an ADCYAP1R inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; 1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and T ACR3 inhibitors; ADCYAP1R1 inhibitors, ADORA2B inhibitors inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, an HRH1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; CYAP1R1 inhibitors, ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors , consisting of a CHRM1 inhibitor, an HRH1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CH RM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor and TACR3 inhibitor an inhibitor selected from the group consisting of an ADRB2 inhibitor, a CHRM1 inhibitor, and an EDNRB inhibitor A is selected from the group consisting of an HRH1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; DRB2 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors and TACR3 inhibitors; ADRB2 inhibitors, CHRM1 inhibitors, HRH1 inhibitors and TACR3 inhibitors; ADRB2 inhibitors, EDNRB inhibitors, an HRH1 inhibitor and a TACR3 inhibitor; an ADRB2 inhibitor, an ED an NRB inhibitor and an HRH1 inhibitor; an ADRB2 inhibitor, a CHRM inhibitor or an ADRB2 inhibitor, an HR inhibitor, and an HRH1 inhibitor; H1 inhibitors and TACR3 inhibitors.

[0356] In some embodiments, the methods of treatment, methods of suppression, pharmaceutical kits or According to the pharmaceutical composition, the method comprises combining an inhibitor of CXCR4, an inhibitor of GPCRx, and a CXC and administering a combination of inhibitors selected from the group consisting of inhibitors of R4-GPCRx heteromers. or the pharmaceutical kit or pharmaceutical composition is a CXCR4 inhibitor, GPC an inhibitor of CXCR4-GPCRx heteromer; In some embodiments, the combination of inhibitors includes a combination of inhibitors that inhibit CXCR4. a group consisting of inhibitors of GPCRx, inhibitors of CXCR4-GPCRx heteromers, In some embodiments, the combination of two inhibitors is selected from the group consisting of: According to the method for treatment, the method for suppression, the pharmaceutical kit or the pharmaceutical composition, the method comprises: or a pharmaceutical kit or In some embodiments, the pharmaceutical composition comprises a combination of a CXCR4 inhibitor and a GPCRx inhibitor. In embodiments, the methods of treatment, suppression, pharmaceutical kits, or pharmaceutical compositions provided herein include According to the method, the method comprises administering a CXCR4-GPCRx heteromeric inhibitor; and The pharmaceutical kit or pharmaceutical composition includes a CXCR4-GPCRx heteromeric inhibitor. .

[0357] In some embodiments, the methods of treatment or suppression provided herein include: Alternatively, by using the pharmaceutical kit or pharmaceutical composition provided herein, a combination of inhibitors can be administered. The combined administration of these compounds demonstrated enhanced expression of the CXCR4-GPCRx heteromer in cancer patients. The downstream signaling pathway was inhibited by 5- to 2000-fold compared to the administration of a single inhibitor. For example, enhanced downstream activity from the CXCR4-GPCRx heteromers in cancer patients signaling by 5-1750-fold, 5-1500-fold, and 5-1800-fold compared to administration of a single inhibitor. 1250x, 5~1000x, 5~900x, 5~800x, 5~700x, 5~500 times, 5~400 times, 5~250 times, 5~200 times, 5~100 times, 5~75 times, 5~50 times, 5~40 times, 5~30 times, 5~25 times, 100~2000 times, 200~2000 times, 300~2000x, 500~2000x, 750~2000x, 1000~2000x , 1250~2000x, 1500~2000x, 5~1500x, 25~1500x, 50~1500x, 75~1500x, 100~1500x, 200~1500x, 30 0-1500 times, 500-1500 times, 750-1500 times, 1000-1500 times, In some embodiments, CXCR4-G is inhibited by 1250 to 1500 times. The GPCRx in the PCRx heteromer are ADCYAP1R1, ADORA2B, and ADORA 3, ADRB2, C5AR1, CALCR, CHRM1, EDNRB, HRH1, MLN R, NTSR1 and TACR3; e.g., ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CHRM1, EDNRB, H selected from the group consisting of RH1, MLNR, NTSR1 and TACR3; ADCYAP1 R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1 , MLNR, NTSR1 and TACR3; ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB, HRH1, NT SR1 and TACR3; ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH1, NTSR1, and TACR3 ADORA2B, ADORA3, ADRB2, CHRM1, EDNRB , HRH1, NTSR1 and TACR3; ADRB2, CHRM 1, EDNRB, HRH1, NTSR1, and TACR3; ADR B2, CHRM1, EDNRB, HRH1, and TACR3; AD RB2, CHRM1, HRH1, and TACR3; ADRB2, E selected from the group consisting of DNRB, HRH1, and TACR3; ADRB2, EDNRB, and and HRH1; selected from the group consisting of ADRB2, CHRM1 and HRH1; or selected from the group consisting of ADRB2, HRH1, and TACR3. In some embodiments, the GPCRx inhibitor is an ADCYAP1R1 inhibitor, ADORA 2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, C5AR1 inhibitor, CALCR inhibitor inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTS R1 inhibitors and TACR3 inhibitors; e.g., ADCYAP1R1 inhibitors, ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, C5AR1 inhibitors inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTS R1 inhibitors and TACR3 inhibitors; ADCYAP1R1 inhibitors, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitor, E DNRB inhibitors, HRH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and TACR3 inhibitors an ADCYAP1R1 inhibitor, an ADORA2B inhibitor, an AD ORA3 inhibitors, ADRB2 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors an ADCYAP1 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; R1 inhibitors, ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CHRM 1 inhibitor, HRH1 inhibitor, NTSR1 inhibitor, and TACR3 inhibitor ADORA2B inhibitors, ADORA3 inhibitors, ADRB2 inhibitors, CHRM1 inhibitors agents, EDNRB inhibitors, HRH1 inhibitors, NTSR1 inhibitors and TACR3 inhibitors an ADRB2 inhibitor, a CHRM1 inhibitor, an EDNRB inhibitor, an HRH1 inhibitor, an ADRB2 inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors and TACR3 inhibitors an ADRB2 inhibitor, a CHRM1 inhibitor, an HRH1 inhibitor, and a TAC inhibitor; R3 inhibitors; ADRB2 inhibitors, EDNRB inhibitors, HRH1 inhibitors an ADRB2 inhibitor, an EDNRB inhibitor, and a TACR3 inhibitor; and HRH1 inhibitors; ADRB2 inhibitors, CHRM1 inhibitors, and and an HRH1 inhibitor; or an ADRB2 inhibitor, an HRH1 inhibitor and TACR3 inhibitors.

[0358] In some embodiments, the methods of treatment or suppression provided herein include: Alternatively, by using the pharmaceutical kit or pharmaceutical composition provided herein, an inhibitor or The administration of the inhibitor combination may be effective in preventing the above-mentioned CXCR4-GPCRx heteromers in cancer patients. These enhanced downstream signaling were observed in the CXCR4 protomer in the context of each individual protomer. 5-2 compared to the inhibition of downstream signaling from either GPCR or GPCRx protomers. 000-fold suppression, for example, in the above-mentioned CXCR4-GPCRx heterozygotes in cancer patients. Enhanced downstream signaling from CXCR4 promoters in the context of each individual promoter was observed. compared with the inhibition of downstream signaling from either rotomers or GPCRx protomers. 5~1750x, 5~1500x, 5~1250x, 5~1000x, 5~900x, 5 ~800x, 5~700x, 5~500x, 5~400x, 5~250x, 5~200x , 5~100x, 5~75x, 5~50x, 5~40x, 5~30x, 5~25x, 10 0~2000x, 200~2000x, 300~2000x, 500~2000x, 75 0~2000x, 1000~2000x, 1250~2000x, 1500~2000x , 5~1500x, 25~1500x, 50~1500x, 75~1500x, 100~ 1500x, 200~1500x, 300~1500x, 500~1500x, 750~ Inhibits in the range of 1500-fold, 1000-1500-fold, or 1250-1500-fold. In some embodiments, the GPCRx of the CXCR4-GPCRx heteromer is ADCYA P1R1, ADORA2B, ADORA3, ADRB2, C5AR1, CALCR, CH RM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3 For example, ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C 5AR1, CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3 and selected from the group consisting of: ADCYAP1R1, ADORA2B, ADORA3, ADRB 2, consisting of CHRM1, EDNRB, HRH1, MLNR, NTSR1 and TACR3 Selected from the group consisting of ADCYAP1R1, ADORA2B, ADORA3, ADRB2, C selected from the group consisting of HRM1, EDNRB, HRH1, NTSR1 and TACR3; ADCYAP1R1, ADORA2B, ADORA3, ADRB2, CHRM1, HRH 1, NTSR1, and TACR3; ADORA2B, ADORA3 , ADRB2, CHRM1, EDNRB, HRH1, NTSR1, and TACR3 Selected from the group: ADRB2, CHRM1, EDNRB, HRH1, NTSR1 and TA CR3; ADRB2, CHRM1, EDNRB, HRH1, and T ACR3; ADRB2, CHRM1, HRH1, and TACR3 selected from the group consisting of ADRB2, EDNRB, HRH1, and TACR3; selected from the group consisting of ADRB2, EDNRB, and HRH1; , CHRM1 and HRH1; or ADRB2, HRH1 and In some embodiments, the GPCRx inhibitor is selected from the group consisting of: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor inhibitors, C5AR1 inhibitors, CALCR inhibitors, CHRM1 inhibitors, EDNRB inhibitors, H From the group consisting of RH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and TACR3 inhibitors For example, an ADCYAP1R1 inhibitor, an ADORA2B inhibitor, an ADORA3 inhibitor inhibitors, ADRB2 inhibitors, C5AR1 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, H From the group consisting of RH1 inhibitors, MLNR inhibitors, NTSR1 inhibitors and TACR3 inhibitors Selected: ADCYAP1R1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, A DRB2 inhibitor, CHRM1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, MLNR inhibitor an ADCYAP1R inhibitor, an NTSR1 inhibitor, and a TACR3 inhibitor; 1 inhibitor, ADORA2B inhibitor, ADORA3 inhibitor, ADRB2 inhibitor, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, NTSR1 inhibitors and TACR3 inhibitors an ADCYAP1R1 inhibitor, an ADORA2B inhibitor, an ADORA 3 inhibitors, ADRB2 inhibitors, CHRM1 inhibitors, HRH1 inhibitors, NTSR1 inhibitors and and TACR3 inhibitors; ADORA2B inhibitors, ADORA3 inhibitors agents, ADRB2 inhibitors, CHRM1 inhibitors, EDNRB inhibitors, HRH1 inhibitors, NTS R1 inhibitors and TACR3 inhibitors; ADRB2 inhibitors, CHRM 1 inhibitor, EDNRB inhibitor, HRH1 inhibitor, NTSR1 inhibitor and TACR3 inhibitor an ADRB2 inhibitor, a CHRM1 inhibitor, an EDNRB inhibitor, a H an RH1 inhibitor and a TACR3 inhibitor; an ADRB2 inhibitor, a CHR an ADRB2 inhibitor selected from the group consisting of an M1 inhibitor, an HRH1 inhibitor, and a TACR3 inhibitor; inhibitors, EDNRB inhibitors, HRH1 inhibitors and TACR3 inhibitors. selected from the group consisting of an ADRB2 inhibitor, an EDNRB inhibitor, and an HRH1 inhibitor; selected from the group consisting of an ADRB2 inhibitor, a CHRM1 inhibitor, and an HRH1 inhibitor; and is selected from the group consisting of an ADRB2 inhibitor, an HRH1 inhibitor, and a TACR3 inhibitor. do.

[0359] Enhanced expression of CXCR4-GPCRx heteromers according to the methods disclosed herein in inhibiting downstream signaling and / or according to the assays disclosed herein. Inhibitors or inhibitors that are effective or therapeutically effective in determining IC50 values Initial testing and evaluation of inhibitor combinations involves inhibitor concentrations ranging from 1 to 10 μM (or Concentrations ranging from 1 to 10 μM of each inhibitor in the combination), e.g., 1, 2, 3, 4, 5, 6, 7 Concentrations of 1, 8, 9, 10 μM may be used. If the measurement is not sufficiently clear for a determinable measurement result, e.g. For example, higher concentrations of inhibitors can be used to better assess IC50 values. If the suppression of the signal is too strong, the determinable measurement, e.g., IC50 value, will be Lower concentrations of inhibitors may be used to better assess.

[0360] Modifications that do not substantially affect the activity of the various embodiments of this invention are also provided herein. It is understood that the following examples are included within the definition of the invention set forth herein. It is intended to illustrate but not limit the invention disclosed herein.

[0361]

[0362] [Example]

[0363] Example 1. Assessment of CXCR4-GPCRx heteromer formation by BiFC assay.

[0364] To identify novel CXCR4-GPCRx heteromers, we performed a multi-step analysis using Song et al. (Song et al., 2014, SNU patent, Song, paper) As previously reported, 1 was fused to the N-terminal fragment of the yellow fluorescent protein Venus (VN). 43 GPCRs and 147 GPCRs fused to the C-terminal fragment of Venus (VC) We generated recombinant adenovirus encoding R. bimolecular fluorescence complementation (BiFC) assay. We identified CXCR4-GPCRx heteromers using the CXCR4-GPCRx complex (Fig. 1), which is a heteromeric complex of Venus. Between two different proteins to which two complementary VN and VC fragments are fused The fluorescence signal is reconstituted only when the two fragments are sufficiently close to each other due to the interaction of the two fragments. There is (Hu et al., 2002).

[0365] U-2 OS cells were seeded in 96-well plates and cultured with CXCR4-VN and GPCRx-V. C, or adenoviruses encoding CXCR4-VC and GPCRx-VN, respectively, were administered in a 30-well plate. The cells were co-transfected with 1000kJ / mL IgG1 at an MOI of 1000kJ / mL and allowed to express GPCRs for 2 days. After staining, cells were collected in triplicate per well using an IN Cell Analyzer 1000. BiFC and nuclear images were acquired from approximately 200 cells from each well. Cell Developer ToolBox(GE Healthcare,Wau Cell boundaries were analyzed using the multi-target analysis software in the BioScanner app (Berkeley, WI). The signal was highlighted and the fluorescence intensity per cell was measured. Cells with higher fluorescence intensity than the control were considered BiFC-positive cells. Dead cells were excluded from the cell count. Positive cells were determined and the positive cell count ratio (BiFC) was calculated. The "score" was calculated as (positive cells / total cells) x 100 (see Table 4 below). ).

[0366] CXCR4-VN interacts with HA-VC (Fig. 2a) or with G, which encodes the glucagon receptor. When co-expressed with GCGR-VC (Figure 2c), a PCR product, yellow fluorescent protein (YFP) In contrast, no BiFC signal was observed. When co-expressed with -VC (Fig. 2b), BiFC signals were observed in the plasma membrane and cytoplasm. CXCR4-VN was transfected with ADCYAP1R1-VC (Fig. 2d) and ADORA3-VC ( Figure 2f), ADRB2-VC (Figure 2g), APLNR-VC (Figure 2h), C5AR1-V C (Fig. 2i), CALCR-VC (Fig. 2j), CCR5-VC (Fig. 2k), CHRM1- VC (Figure 2l), GALR1-VC (Figure 2m), EDNRB-VC (Figure 2n), HRH1 -VC (Fig. 2o), MLNR-VC (Fig. 2p), NTSR1-VC (Fig. 2q), PTGE R2-VC (Fig. 2r), SSTR2-VC (Fig. 2t), and TACR3-VC (Fig. 2u) When co-transfected with β-lactamase, a strong BiFC signal was observed at the plasma membrane and in the cytoplasm. Robust BiFC signals were observed in cells expressing CXCR4-VC and ADORA2B-VN (Figure 2e) or This was also observed when co-transfected with PTGER3-VN (Fig. 2s). Cells that showed a BiFC fluorescent signal higher than the baseline level were counted as BiFC-positive cells. The iFC score was calculated.

[0367] Protein-protein interactions are mediated by fluorescent protein fragments in BiFC or retinal lucidation in BRET. The effect of fusion tags such as acetyltransferase on the expression, folding, or localization of partner proteins Partner proteins can also be affected by the expression or localization of fusion tags. This can impair folding and affect the results of proximity-based assays. Therefore, the absence of a signal between two proteins in a particular combination does not necessarily mean that the signal is present. This is not necessarily an indication that the proteins do not interact, but simply that the bound donor and antigen This suggests that the receptor molecule is in a specific conformation that does not allow the interaction to occur. (Eidne et al., 2002, Kerppola, 2006).

[0368] Therefore, CXCR4-VN and GPCRx-VC or CXCR4-VC and GPCRx CXCR4 and GPCRx showed BiFC signals in either combination with -VN. It is believed that the protein that is involved in this function is the well-known homomer CXCR. The BiFC score for the pair of 4-VN and CXCR4-VC was 9.9. As shown in 4, CXCR showed a BiFC score equal to or higher than 10. The 4-GPCRx pair was selected as a candidate for the CXCR-GPCRx heteromer.

[0369]

[0370] Table 4. BiFC sequences equal to or higher than 10 when co-expressed with CXCR4 GPCR with a core.

[0371] [Table 4]

[0372]

[0373] As shown in Table 4, the combination of CXCR4-VN and GPCRx-VC inhibited ADCY AP1R1, ADORA3, ADRB2, APLNR, C5AR1, CALCR, CCR 5, CHRM1, GALR1, EDNRB, HRH1, MLNR, NTSR1, PTGE R2, SSTR2, and TACR3 were identified as CXCR4-interacting GPCRs. The combination of CXCR4-VC and GPCRx-VN inhibited ADORA2B and Two GPCRs, PTGER3 and PTGER4, showed BiFC scores higher than 10. However, ADRB2 and CCR5 have been reported to form heteromers with CXCR4. (LaRocca 2010, Nakai 2014, (Agrawal et al. .,2004, LaRocca et al.,2010, Rodriguez-Fra de et al., 2004, Sohy et al., 2007, Sohy et al. al., 2009, Martinez-Muñoz et al., 2014)), rest The 16 GPCRs include a novel CXCR4 that has never been reported to the best of our knowledge. -GPCRx heteromers.

[0374] Among the GPCRs known to interact with CXCR4 are ADRA1A and C XCR3 was also included in our BiFC assay. , and was excluded from further investigation because the BiFC signal with CXCR4 was less than 10:A DRA1A (CXCR4-VN-ADRA1A-VC: BiFC score 7.85) and C XCR3 (BiFC score 1.16).

[0375] Cannabinoid receptor 2 (CB2, sometimes called CNR2) is the receptor for the cannabinoids that are the most important receptors for the cannabinoids in our Biology. FC assay (CNR2-VN-CXCR4-VC construct: BiFC score 4.25, CN R2-VC-CXCR4-VN configuration: BiFC score 38.1) and Fig. 3 a and b and CXCR4-interacting GP in co-internalization assays as described in Figure 4a-q. However, CNR2 was not increased in calcium mobilization assays. were not selected as final candidates because they failed to exhibit enhanced downstream signaling. (See Example 3 below).

[0376]

[0377] Example 2. Assessment of CXCR4-GPCRx heteromer formation by co-internalization assay.

[0378] Some GPCR heteromers exhibit changes in transport properties as a result of heteromerization, e.g. , maturation of partner GPCRs (GABA(B) receptors) (White, 1998), and activation Drug-mediated internalization of partner GPCRs from the cell surface (DOR-GRPR, A2A-D2R)(Hillion et al.,2002, Liu et al., 2011, Torvinen et al., 2005), and the cellularity of partner GPCRs Change in localization from the intracellular compartment to the cell surface (DOR-CB1) (Rozenfeld et al., 2012).

[0379] Using co-internalization of a pair of co-expressed GPCRs in response to agonists selective for only one of the pair, GPCR heteromerization was confirmed (Milligan, 2008). Whether or not CXCR4 trafficking is regulated when expressed and CXCR4-GPCRx heteromers To investigate whether or not CX forms a CX receptor, further confirmation was made using the BiFC assay. To confirm the physical interaction between CR4 and GPCRx, CXCR4-GFP and Co-transduction of cells with adenovirus encoding GPCRx and induction of GPCRx agonists GFP images were taken before and 30 minutes after stimulation. The appearance of GFP granules inside the cells was correlated with co-internalization of CXCR4-GFP with GPCRx. This was considered (Figure 3a-b).

[0380] In Figure 4a-q, CXCR4 (Figure 4a), ADCYAP1R1 (Figure 4b), ADOR A2B (Fig. 4c), ADORA3 (Fig. 4d), ADRB2 (Fig. 4e), APLNR (Fig. 4 f), C5AR1 (Fig. 4g), CCR5 (Fig. 4h), CHRM1 (Fig. 4i), GALR1 (Fig. 4j), EDNRB (Fig. 4k), HRH1 (Fig. 4l), MLNR (Fig. 4m), NTS R1 (Fig. 4n), PTGER3 (Fig. 4o), SSTR2 (Fig. 4p) and TACR3 (Fig. 4 q) Cells were stimulated with the following GPCRx agonists specific for each: 10 nM CXCL; 12 (Fig. 4a), 1 μM vasoactive intestinal peptide (VIP) (Fig. 4b), 1 μM BA Y60-6583 (Fig. 4c), 1 μM CGS21680 (Fig. 4d), 100 nM phosphatase lumoterol (Fig. 4e), 1 μM apelin-13 (Fig. 4f), 100 nM C5a (Fig. 4g), 100 nM CCL2 (Fig. 4h), 1 μM acetylcholine (Fig. 4i), 100 nM galanin (Fig. 4j), 1 μM endothelin-1 (Fig. 4k), 1 μM histamine ( 4l), 100 nM motilin (Fig. 4m), 1 μM neurotensin (Fig. 4n), 1 μM PGE2 (Fig. 4o), 1 μM SRIF-14 (Fig. 4p) and 1 μM senktide (Fig. 4q). Images were taken before and 30 min after agonist stimulation. The concentrations of these GPCRx agonists were initially selected for each The EC50 concentration (alternatively, Ki or Kd concentration) of each specific GPCRx was determined and the results were compared. If the resulting signal is too strong, reduce the concentration below the EC50 and If the signal was too weak, the concentration was increased to 10,000 times or less of the EC50 concentration.

[0381] Plasma membrane cytoplasmic changes as a result of CXCL12 stimulation of cells expressing CXCR4-GFP. GFP relocated from the surface to distinct intracellular granules, and surface CXCR4-GFP was transferred into the cytoplasm. Internalization was demonstrated (Fig. 4a). 18 CXCR4 species identified by BiFC assay Among the GPCRx heteromers, 16 heteromers containing the following GPCRx are C Co-expression of CXCR4-GFP and GPCRx induced the internalization of XCR4. This was evident by an increase in intracellular GFP granules and a decrease in the GFP signal at the plasma membrane in cells with This confirmed heteromeric co-internalization of ADCYAP1R1, ADORA2B, ADORA3, ADRB2, APLNR, C5AR1, CCR5, CHRM1, GALR 1, EDNRB, HRH1, MLNR, NTSR1, PTGER3, SSTR2 and TA CR3 (b to q in Figure 4). On the other hand, CXCR4-G containing CALCR and PTGER2 PCRx heteromers did not show co-internalization of the heteromers upon GPCRx agonist treatment. These results suggest that BiFC is a promising candidate for cellular transporters, as evidenced by the altered cellular transport properties of the heteromers. The specific GPCRx partner identified in the assay is the GPCRx partner of a GPCR co-expressed with both GPCRs. We demonstrate that CXCR4-GPCRx heteromers do not form in cells.

[0382]

[0383] Example 3. Enhanced CXCR4 downstream signaling upon CXCR4-GPCRx heteromer formation Inhibition of null transmission and enhanced signaling assessed by Ca 2+ mobilization assay.

[0384] These CXCR4-GPCRx heteromers (in cells lacking one receptor) To further investigate whether the protomers exhibit properties distinct from those of the individual protomers, we In cells expressing either or both GPCRs, studied calcium signaling in the presence of both agonists. As described in Example 2, the selection of concentrations of these GPCRx agonists was initially performed based on the specific GPCR activity of each individual GPCR. The EC50 concentration (or Ki or Kd concentration) of CRx is determined, and the resulting C If the Ca2+ signal is too strong, reduce the concentration below the EC50 and the resulting Ca2+ If the signal was too weak, the concentration was increased to 100-fold below the EC50 concentration.

[0385] MDA-MB-231 human breast cancer cells were transduced with adenovirus encoding CXCR4. When administered intracellularly, CXCL12 treatment induced intracellular calcium mobilization (Fig. 5a). Stimulation of cells with salmeterol, a selective DRB2 agonist, induces a calcium response. This suggests that the calcium response induced by CXCL12 is mediated by CXCR4. It was demonstrated that the co-treatment of cells with CXCL12 and salmeterol is mediated by the The calcium response induced by CXCL12 was significantly higher than that induced by CXCL12 alone. In cells overexpressing ADRB2 alone, CXCL1 2 did not induce a calcium response, whereas salmeterol did. showed that salmeterol induces calcium responses via ADRB2 ( The calcium response induced by co-treatment with both agonists was significantly greater than that induced by salmeterol (Fig. 5b). The calcium response was comparable to that stimulated by acetaminophen alone.

[0386] In cells overexpressing both CXCR4 and ADRB2, stimulation by each agonist was induced. The calcium response induced by CXCR4 was observed in cells expressing either CXCR4 or ADRB2 alone. The calcium response was comparable to that observed in the control group (Fig. 5c vs. a and b). Co-treatment with agonists simultaneously suppresses calcium responses induced by individual agonists. The calcium signal was significantly increased compared to the normal calcium response (Fig. 5c and d). CXCR4 and ADRB2 are expressed in cells expressing both CXCR4 and ADRB2. This was not observed in cells expressing either CR4 or ADRB2 alone. These results clearly demonstrate that the properties of CXCR4-ADRB2 heteromers are distinct from those of individual GPCRs. This demonstrates that the property is distinct from the property.

[0387]

[0388] Other GPs identified by BiFC and co-internalization assays to interact with CXCR4 To investigate whether CR also exhibits distinct properties in calcium signaling, we investigated other The experiments shown in Figure 5a-d were carried out for the identified GPCRx partners. As shown in Fig. 6a and 6d and Fig. 6a to 6l, CXCR4 and ADCYAP1R1 (Fig. 6a) , ADORA2B (Fig. 6b), ADORA3 (Fig. 6c), C5AR1 (Fig. 6d), CAL CR (Fig. 6e), CHRM1 (Fig. 6f), EDNRB (Fig. 6g), HRH1 (Fig. 6h), MLNR (Fig. 6i), NTSR1 (Fig. 6j), PTGER2 (Fig. 6k) or TACR3 CXCL12 and individual GPCRs in cells co-expressing either CXCL12 or any of the GPCRs (Figure 6l). Co-treatment with x agonists reduces calcium responses compared to the sum of the individual agonist-induced calcium responses. significantly increased calcium responses as determined by calcium mobilization assay. XCR4 and GPCRx (ADCYAP1R1 (Figure 6a), ADORA2B (Figure 6b), A DORA3 (Fig. 6c), C5AR1 (Fig. 6d), CALCR (Fig. 6e), CHRM1 (Fig. 6f), EDNRB (Fig. 6g), HRH1 (Fig. 6h), MLNR (Fig. 6i), NTSR1 (Fig. 6j), co-expressed with either PTGER2 (Fig. 6k) or TACR3 (Fig. 6l) The cells expressing CXCL12 were co-stimulated with each selective GPCRx agonist (or When treated with either CXCL12 or each selective GPCRx agonist alone (or co-treatment), Enhanced calcium mobilization compared to the sum of both agonist stimulations Notably, in cells co-expressing CXCR4 and CNR2, CXCL12 alone or together with JWH-133, a selective CNR2 agonist, Therefore, the calcium response induced by CXCL12 was not observed when CXCR4 was expressed alone. CXCL12-mediated calcium responses are not enhanced compared to those observed in cells containing was significantly reduced (data not shown).

[0389] Among the above co-expression systems, in cells involving CXCR4 and PTGER2 (Fig. 6k), , the context of individual protomers (e.g., the absence or presence of each GPCRx) CXCR4 expressed in the presence of CXCR4 or individual GPCRx expressed in the absence of CXCR4. In either case, co-expression of CXCL12 with each selective GPCRx agonist enhanced It was also shown that the amount of calcium mobilization increased. For (Fig. 6k), in the context of individual protomers, in the absence of each GPCRx, CXC Each GPCRx (PTGER2) is expressed when R4 is expressed and in the absence of CXCR4. In both cases, CXCL12 and each selective GPCRx agonist (5μ Upon co-stimulation with M (PGE2), enhanced calcium mobilization was observed, and individual The amount of calcium mobilization resulting from the protomer context of the system is determined by the amount of CXCL12 or each selective G The calcification caused by single agonist stimulation with either PCRx agonist (5 μM PGE2) The amount of sodium mobilized was greater than the total of both.

[0390] Therefore, these observations suggest that CXCR4 and ADCYAP1R1 (Fig. 6a), ADORA2B (Fig. 6b), ADORA3 (Fig. 6c), C5AR1 (Fig. 6d), CALC R (Fig. 6e), CHRM1 (Fig. 6f), EDNRB (Fig. 6g), HRH1 (Fig. 6h), M Selected from the group consisting of LNR (Fig. 6i), NTSR1 (Fig. 6j) and TACR3 (Fig. 6l) The system in which the GPCRx is co-expressed with the target GPCRx is determined by (i) calcium mobilization assay. When the CXCL12-dependent GPCRx agonists were administered, co-stimulation (or co-treatment) of the CXCL12-dependent GPCRx agonists was observed. ) single agonist with either CXCL12 or each selective GPCRx agonist The calcium mobilization caused by the stimulation was enhanced compared to the total calcium mobilization caused by both the stimulation and the calcium mobilization caused by the stimulation. and (ii) CXCL12 or each selective GPCRx agonist in the context of individual protomers. Is the calcium mobilization resulting from single agonist stimulation by either or less calcium mobilization, and thus (iii) the characteristics of individual GPCRs. It is thought that a CXCR4-GPCRx heteromer was formed that exhibits characteristics distinct from sex. The enhanced calcium response was not observed in cells expressing individual GPCRs. This allows these CXCR4-GPCRx heteromers to differentiate from the properties of individual GPCRs. It was demonstrated that the material exhibits unique properties.

[0391] The enhanced calcium signaling induced by small amounts of individual agonists together is XCR4-GPCRx heteromers are able to bind these receptors at a constrained ligand concentration in vivo. Increased sensitivity and dynamic range of the body may be associated with poor prognosis. This clearly suggested that CXCR4-GPCRx heteromers enhance The improved calcium signaling will change current diagnoses based on the level of CXCR4 expression alone. Furthermore, it is also suggested that the expression levels of CXCR4 and GPCRx should be considered. .

[0392] In contrast, GPCRs such as APLNR (Fig. 7a), CCR5 (Fig. 7b), and GALR 1 (Fig. 7c), PTGER3 (Fig. 7d), and SSTR2 (Fig. 7e) were identified as markers of oxidative stress responses to BiFC (Table 5). These GPCRs interacted with CXCR4 in a CXCR4-GFP co-internalization assay. Although both receptors were shown to act together, cells expressing both receptors were not treated with either agonist. Costimulation with drugs did not result in an enhanced calcium response. The synergistic increase in calcium response shown in Figures 5a-d and 6a-l was due to the CXCR4-AP LNR, CXCR4-CCR5, CXCR4-GALR1, CXCR4-PTGER3 and and CXCR4-SSTR2, and is not shared by other CXCR4-GPCRx heteromers This demonstrates that this is a unique feature of CXCR4-GPCRx heteromers.

[0393] Table 5. BiFC sequences equal to or higher than 10 when co-expressed with CXCR4 showed no enhanced Ca2+ signaling upon co-treatment with both agonists. GPCR.

[0394] [Table 5]

[0395] Furthermore, CXCL12 and GPCRx were expressed in cells co-expressing CXCR4 and GPCRx. Enhanced calcium responses upon costimulation with CRx ligands were observed with GPCRx antagonists. We investigated whether CXCR4, ADRB2 (Fig. 8a), and CHRM1 (Fig. 8b) were inhibited by CXCR4. 8 b and c), HRH1 (Fig. 8 f–i), MLNR (Fig. 8 j), or NTSR1 (Fig. In cells co-expressing either 1 or 8k, 10 μM of each ADRB2 antagonist was administered. Rubedilol (Figure 8a), 1 μM of the CHRM1 selective antagonist VU0255035 (Figure 8b ), 10 μM of the CHRM1 antagonist oxybutynin (Fig. 8c), 1 μM of the HRH1-selective antagonist The anti-cancer drug cetirizine (Fig. 8f), 1 μM of the HRH1 selective antagonist pyrilamine (Fig. 8g), 10 10 μM of the HRH1 antagonist hydroxyzine (Figure 8h), 10 μM of the HRH1 selective antagonist loraxol Tajine (Fig. 8i), 1 μM of the MLNR-selective antagonist MA-2029 (Fig. 8j) and 1 μM Treatment with meclineltant (Fig. 8k), a selective NTSR1 antagonist, enhanced calcium Furthermore, co-treatment with both antagonists significantly suppressed signal transduction. These results suggest that GPCRx regulates ADRB2. CX GPCR antagonists expressing CHRM1, HRH1, MLNR, and NTSR1 It can be used as an effective therapeutic agent against CR4-GPCRx heteromers. was demonstrated.

[0396] In cells co-expressing CXCR4 and CHRM1, 10 μM muscarinic acetylcholine The cholinergic receptor antagonist umeclidinium induces enhanced calcium signaling in the statin In these cells, AMD3100 and Co-treatment with umeclidinium induces CXCL12 and bethanechol simultaneously. The calcium response was almost completely inhibited by ATP.

[0397] CXCR4 was co-expressed with either EDNRB (Fig. 8e) or TACR3 (Fig. 8l). In cells with endothelial cells, 1 μM AMD3100 alone (Fig. 8e and l) or 1 μM endothelial cells The serine receptor antagonist bosentan (Fig. 8e) or 1 μM of the TACR3 selective antagonist SSR Treatment with either 146977 or 146977 alone (Fig. 8l) also produced an enhanced calcium response. However, AMD3100 and bosentan (Fig. 8e ), or when cells were simultaneously co-treated with AMD3100 and SSR146977 (Fig. 8l). In this case, the enhanced calcium response was significantly inhibited.

[0398] CXCR4 and CHRM1 (Fig. 8b), EDNRB (Fig. 8e), HRH1 (Fig. 8f and In cells co-expressing either MLNR (Fig. 8j) or MLNR (Fig. 8f), enhanced colorimetric activity was observed. Although 1 μM AMD3100 alone was unable to inhibit calcium signaling, Regardless, simultaneous co-treatment with 1 μM of both antagonists resulted in enhanced calcium synapses. Signal transmission was significantly suppressed.

[0399] These results suggest that co-treatment with small doses of antagonists targeting each protomer significantly reduces the effects of high doses of each individual antagonist. Efficiently enhance CXCR4-GPCRx heteromeric responses while avoiding the side effects associated with antagonists of CXCR4. These results clearly demonstrate that this provides a novel therapeutic tool for effectively inhibiting leukemia.

[0400]

[0401] Example 4. Inhibition of internalization by GPCRx antagonists.

[0402] Co-internalization of CXCR4 heterodimers is blocked by partner GPCRx antagonists To further investigate whether or not this is the case, we performed an internalization inhibition assay. As shown, when cells were co-transduced with CXCR4 and GPCRx, CXCR4- GFP-expressing U-2 OS cells were co-internalized with partner GPCRx-specific agonists (Control: CXCR4-GFP (Fig. 4a)). CXCR4 forms a heterodimer with GPCRx. When co-internalized by a partner GPCRx, it is inhibited by a GPCRx-specific antagonist. You can block it by doing so.

[0403] Adenoviruses encoding GPCRx (ADRB2, CHRM1, HRH1) were isolated from C XCR4-GFP was transduced into stably expressing U-2 OS cells. After 2 days, C XCR4-specific agonist CXCL12 (SDF-1) (20 nM) and / or GPCRx Images were acquired before and 20 min after stimulation of the cells with specific antagonists (10 μM). Using a Cell Analyzer2500, internalization of CXCR4-GFP was confirmed by GFP The decrease in GFP expression on the cell surface or the G The appearance of FP granules was considered to be CXCR4-GFP co-internalization. 2 induced the internalization of CXCR4-GFP with GPCRx (Figure 10a-c, first column) The following GPCRx antagonists treated had no effect on the internalization of the heteromer. (Fig. 10a-c, second row): carvedilol, an ADRB2 antagonist (Fig. 10a); xybutynin and umeclidinium, CHRM1 antagonists (Fig. 10b); promethazine, hyaluronan Droxidine and loratadine, HRH1 antagonists (Fig. 10c). Stimulated by CXCL12 The internalization of CXCR4-GFP with GPCRx was inhibited by a GPCRx-specific antagonist. were damaged (Fig. 10a-c, third column).

[0404] These data demonstrate that CXCR4-GPCRx co-internalization is a heteromer-specific event. These data demonstrate that CXCR4 and GPCRx form heterodimers. Furthermore, we have demonstrated that CXCR4-GP receptors, such as those in cancer, can be inhibited by inhibiting the internalization of CXCR4 heteromers. Therapeutic blocking of aberrant downstream signals in CRx heteromer-overexpressing cells It proves that it can be done.

[0405]

[0406] Example 5. Effect of inhibitors of CXCR4-GPCRx heteromeric signaling on tumor growth Evaluation of phenotype-related effects by cell proliferation assay.

[0407] Several CXCR4 antagonists have been developed, but so far none have been approved as anticancer drugs. We are working to overcome the limitations of CXCR4 inhibitors and develop therapeutic agents based on CXCR4 heteromers. To develop this, the present inventors tested the effects of GPCRx antagonists on cell proliferation.

[0408] We prepared single cell suspensions of dissociated glioblastoma tissue excised from patients. (Provided by Samsung Seoul Hospital, Seoul, South Korea). The cells were cultured under conditions optimal for proliferation and non-differentiation of normal neural stem cells. It consisted of serum-free Neurobasal medium supplemented with EGF.

[0409] The effect of GPCRx antagonists on the survival of patient-derived cells (PDCs) was investigated using ATPlite ( The assay was performed using PerkinElmer (Cat. No. 6016739) reagents. Plite is a firefly luciferase-based adenosine triphosphate tracking assay system. This luminescent assay is useful for quantitative assessment of proliferation and cytotoxicity in cultured mammalian cells. It is an alternative to colorimetric, fluorometric and radioisotopic assays for detecting chromatographic changes in cells. Well plates were seeded at 500 cells / well in 40 μl of culture medium. 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 plates were incubated. The mixture was shaken at 700 rpm for 5 minutes on an orbital shaker. Luminescent signals were detected within 30 minutes using a TopCount detector. Equation: Cell viability (%) = Cell viability was calculated using (OD of antagonist treatment / OD of DMSO treatment) x 100%. Ta.

[0410] As shown in Figure 11 a to c, PDCs expressing CXCR4 and ADRB2 were treated with AD Treatment with carvedilol, an RB2-specific antagonist, significantly inhibited cell growth. (IC50 = 11.69 μM, Figure 11a). Oxybutynin, a CHRM1 antagonist, Oxybutynin and umeclidinium also inhibited the survival of PDc. Each of the compounds significantly reduced cell proliferation with IC50 values ​​of 3.04 μM and 4.03 μM, respectively. The survival rate was shown (Fig. 11b). Promethazine and hydroxyzine are HRH1 antagonists. and or loratadine, each with IC50 = 18.39uM and 12.79uM, respectively. At 5.29 μM or 5.29 μM, the viability of PDc was reduced (Fig. 11c).

[0411] These results suggest that CXCR4-GPCRx heteromer-expressing cells Abnormal cell proliferation induced by telomers is prevented by specific antagonists of GPCRx partners It has been demonstrated that it can block CXCR4-GPCRx heteromers in patients Inhibition of cancer cell growth using partner GPCRx antagonists as cancer therapeutics This suggests that this method may be able to overcome the limitations of CXCR4 inhibitors alone.

[0412]

[0413] Example 6. CXCR4-GPCRx heteromer formation in patient-derived cells (PDCs) Evaluation using proximity ligation assay (PLA).

[0414] To investigate the presence of GPCR complexes in native tissues, atomic force microscopy (AFM) was used. iadis et al., 2006), co-immunoprecipitation (Gomes et al., 20 04), and binding or functional assays (Wreggett and Wells, 1999 5) have been used. The most common method for tracking and evaluating interactions is The method is based on resonance energy transfer, which is performed using labeled proteins. The labels can be antibodies or This can be achieved by selective probes such as fluorescent ligands (Roess et al. ., 2000, Patel et al., 2002).

[0415] Bazin et al. reported a time-resolved fluorescence coherence tomography (TIMF) assay that exhibited a much higher signal-to-noise ratio than the conventional TIMF assay. We employed a method based on resonance energy transfer (FRET) (Bazin et al. ., 2002). FRET occurs between two fluorophores, a donor and an acceptor, in close proximity. The intermolecular interactions between biomolecules are based on the transfer of energy between the two molecules. and detecting the level of energy transfer. By introducing a time delay of approximately 50-150 μs between excitation and fluorescence measurement, the signal It is possible to eliminate non-specific short-lived luminescence from the signal.

[0416] Proximity ligation assays (PLA) extend the capabilities of traditional immunoassays to Techniques include direct detection of proteins, protein interactions and modifications with high specificity and sensitivity. (Gullberg et al., 2004). The antibodies recognize the target antigen on the protein of interest. They are directed against the constant region of a different primary antibody. A secondary antibody, called a PLA probe, binds to the primary antibody. When a unique short DNA strand is bound and the PLA probe is in close proximity (i.e., as shown in the figure), The original two proteins of interest are in close proximity or in a protein complex, as shown If the DNA strand is part of a rolling circle, it will form a rolling circle when the appropriate substrate and enzyme are added. It can be incorporated into DNA synthesis, which results in the DNA circle being amplified several hundred times. Next, fluorescently labeled complementary oligonucleotide probes are added, which are then used to identify the amplified DNA. The resulting high intensity of fluorescence is clearly visible under a fluorescence microscope. These can be easily seen as bright spots (Gustafsdottir et al. ,2005).

[0417] CXCR4-overexpressing cell line U2OS-CXCR4 at MOIs of 0, 2.5, 10, and 40 The cells were infected with a large amount of ADRB2-expressing adenovirus Ad-ADRB2 for 2 days. The study was performed as previously described (Brueggemann et al., 2012). 014, Tripathi et al., 2014). Cells were fixed in 4% paraformaldehyde (PFA) on 16-well tissue culture slides. The slides were blocked with the blocking solution provided by Duolink. , mouse anti-CXCR4 (1:200, Santacruz, Sc-53534), rabbit Anti-ADRB2 (1:200, Thermoscientific, PA5-33333) , along with rabbit anti-CHRM1 (1:200, Ls bio, Ls-C313301) The slides were then washed and incubated in a humidified chamber at 37°C for 1 hour. Secondary anti-rabbit and anti-mouse antibodies conjugated to and minus Duolink II PLA probes The slides were washed again and then incubated with the antibody (1 hour at 37°C). Incubation with ligation-ligase solution (30 min at 37°C) followed by amplification The slides were incubated with the polymerase solution (2 hours at 37°C). A minimal volume of Duolink II plating medium was added to 4',6-diamidino-2-phenylindole. The cells were then mounted with DAPI and left for 15-30 minutes. PLA signal [Duolink In Situ Detection Reagent Green (λ excitation)] under ER2500 Fluorescent spot with red (λ excitation / emission 495 / 527 nm) or red (λ excitation / emission 575 / 623 nm) and identified.

[0418] As shown in Figure 12a-b, the PLA signal correlated with the dose as the expression level of ADRB2. Figure 12a: CXCR4-ADRB2 expression at a range of MOIs (multiplicity of infection). Figure 12b: PLA signal image from telomer-expressing U2OS cells. The PLA signal was calculated by counting and normalizing to the negative control. Figure 12c: To examine endogenous ADRB2 expression, qRT-PCR was performed using ADRB2-specific primers. As shown, the endogenous ADRB2 expression level in U2OS cells is extremely high, and it is not observed in the absence of viral infection ( This suggests that PLA signals can be detected even in areas where ADRB2 is at 0 MOI. there was.

[0419] Traditionally, glioblastoma (GBM) is the most common and deadly primary brain tumor. Cancer biology is largely based on the use of human cancer cell lines in vitro and the differentiation of these established cell lines. However, the process of establishing conventional cell lines is complex. This leads to irreversible loss of essential biological properties, and as a result, xenograft tumor models are unable to replicate the original tumor. It does not maintain the genomic and phenotypic characteristics that were present in

[0420] Patient-derived cells (PDCs) derived directly from glioblastoma cells show extensive similarities to normal neural stem cells The genotype, gene expression patterns, and biology of human glioblastoma are similar to those of other tumors. Reproduce the biology within.

[0421] To perform PLA on pDC samples, patient-derived cells were plated onto 16-well tissue culture slides. The cells were seeded onto 100 wells and fixed in 4% PFA. Block the slides with blocking solution and then use mouse anti-CXCR4 (1:200, Santa Cruz z, Sc-53534), rabbit anti-ADRB2 (1:200, Thermo Scientific tific, PA5-33333), rabbit anti-CHRM1 (1:200, Lsbio, L The mixture was incubated with 100 μg of PBS (s-C313301) at 37°C for 1 hour in a humidified chamber. Afterwards, the slides were washed and probed with a Duolink II PLA probe. Incubation with conjugated secondary anti-rabbit and anti-mouse antibodies was performed (1 hour at 37°C). The slides were washed again and then incubated with ligation-ligase solution ( 30 min at 37°C), followed by incubation with the amplification polymerase solution (at 37°C Afterwards, apply a minimum volume of Duolink II mounting medium to the slide for 4 minutes. The cells were then loaded with 6-diamidino-2-phenylindole (DAPI) for 15 to 30 minutes. In Cell analyzer 2500, the PLA signal [Duolink I In situ detection reagents green (λ excitation / emission 495 / 527 nm) or red (λ excitation / emission Light 575 / 623 nm) was identified as a fluorescent spot.

[0422] As shown in Figures 13a to 13b and Figures 14a to 14b, the PLA ratio was The frequency of heteromer formation varies among patients. was calculated as the number of fluorescent puncta in the PDC sample / the number of fluorescent puncta in the negative control. represents the background fluorescence signal, which is due to the presence of the primary antibody (mouse anti-C) in the PLA treatment. XCR4, rabbit anti-ADRB2, rabbit anti-CHRM1) without treatment, plus and minus D The number of points treated with only the secondary antibody conjugated to the uolink II PLA probe These data demonstrate that CXCR4-ADRB expression in cancer patient samples Quantitative analysis of the two heteromers is demonstrated.

[0423] Example 7. In vivo CXCR4-GPCRx heteromer formation using a PDX model Rating.

[0424] To perform PLA on PDX samples, we used a 100-well plate at Samsung Medical Center, Seoul, Korea. FFPE samples from glioblastoma patients (provided by the hospital) were used. After finning, heat-induced antigen retrieval was performed at 100°C for 15 minutes. Slides were blocked with the blocking solution provided by the manufacturer and then incubated with rabbit anti-CXCR4 ( 1:200, Thermoscientific, PA3305), mouse anti-ADRB2 (1:200, Santacruz, Sc-271322) at 37°C for 1 hour in a humidified atmosphere. The incubation was carried out in the chamber. The other processes were the same as above (PLA in PDC). It was.

[0425] In Figure 15a, nuclei were visualized by DAPI staining, and CXCR4-ADRB4 heteromers were identified by P As shown in Figure 15b, the PLA ratio varied among patients. Companion diagnostics can be used to implement personalized medicine. This suggests that it is possible.

[0426]

[0427] Example 8. Enhanced CXCR4 downstream signaling upon CXCR4-GPCRx heteromer formation Assessment of nuclear transmission by Ca 2+ mobilization assay.

[0428] In cells overexpressing both CXCR4 and ADRB2, salmeterol Stimulation with either alone did not elicit calcium mobilization in a dose-dependent manner (Fig. 16a). However, when cells were costimulated with salmeterol in the presence of CXCL12, a wide range of Lumeterol concentrations, for example, in the range of 10 to 300 nM, inhibit calcium signal transduction. have been greatly enhanced.

[0429] Similarly, histamine was expressed in cells overexpressing both CXCR4 and HRH1. Costimulation with histamine and CXCL12 also significantly increased histamine concentrations over a wide range (0.3–300 nM ), and thus, histamine concentrations that did not induce calcium responses when administered alone At high concentrations (0.3 nM and 1 nM), it significantly enhanced the calcium response (Fig. 16b). Considering that the concentrations of histamine in the plasma and glomerulus are less than 10 nM and 2 μM, respectively, Considering this (Sedor and Abboud, 1984), this result is consistent with the Enhanced calcium signaling by HRH1 heteromers increases histamine levels in vivo This suggests that this may occur at physiological levels.

[0430] CXCR4-APLNR (Figure 17a), CXCR4-PTGER3 (Figure 17b) or In cells expressing CXCR4-SSTR2 (Fig. 17c), GPCRx agonists (i.e., Stimulation with apelin-13, PGE2, or octreotide alone increased calcium signaling. However, CXCR4-ADRB2 or CXCR Unlike in cells expressing 4-HRH1, CXCR4-APLNR, CXC Any of the cells tested expressing R4-PTGER3 or CXCR4-SSTR2 Calcitriol was also observed upon costimulation with a range of doses of both agonists (CXCL12 and GPCRx agonists). The sodium signal did not further increase. These results are shown in Figure 7a and 7b, respectively. Consistent with the results shown in d, e, CXCR4-APLNR or CXCR4-SSTR The lack of signal enhancement upon costimulation in cells expressing GPCR2 was observed at a specific dose. This was not due to the use of animal drugs but rather to the inherent properties of these heteromers. These results further demonstrate the enhancement shown in Figures 6a-l and 16a-b. The calcium responses measured were compared with those of CXCR4-AD cells as shown in Fig. 5c-d and Fig. 6h, respectively. The unique characteristics of CXCR4-GPCRx heteromers, such as RB2 or CXCR4-HRH1, This clearly demonstrates that it is a sign.

[0431]

[0432] Example 9. Endogenous CXCR4-HRH1 heteromers in MDA-MB-231 cells Assessment of enhanced Ca 2+ mobilization upon costimulation of .

[0433] CXCR4 and HRH1 expression in MDA-MB-231 cells using RT-qPCR Endogenous expression levels were measured. When 12.5 ng of total RNA was analyzed, CXCR4 and The threshold cycles (Ct) of HRH1 were 28.5 and 27.7, respectively. MDA-MB-23 with either 2 (100 nM) or histamine (10 nM) alone Stimulation of CXCL1 cells elicited a weak calcium response (Fig. 18a). When cells were co-stimulated with 12 and histamine, CXCR4 and HRH1 were expressed together. A significantly enhanced calcium response was observed, similar to the enhancement observed in cells overexpressing The results clearly show that both agonists Enhanced calcium responses upon costimulation were observed when both CXCR4 and HRH1 were expressed together. This suggests that this may occur in natural cells expressing

[0434]

[0435] Example 10. CXCR4-HRH1 heteromers upon costimulation with both ligands Assessment of enhanced cancer cell migration by IFN-γ.

[0436] MDA-MB-231 cells express CXCR4 mRNA as measured by RT-qPCR. Because they express twice as much HRH1 mRNA as CXCR4, MDA-MB-231 cells were transduced with lentivirus (1 MOI) and CXCL12 and The chemotactic migration of cells toward HRH1 and HRH1 was measured (Fig. 19a and b). Histamine (5%) was sufficient to produce calcium signaling as shown in Fig. 1. 0 nM) alone did not induce cell migration. On the other hand, when treated with CXCL12, Histamine significantly enhanced the migration of MDA-MB-231 cells toward CXCL12. However, in the presence of pyrilamine, an HRH1-selective inverse agonist, CXCL1 Costimulation of cells with 2 and histamine inhibits CXCL12-induced cell migration. The results clearly showed that the observed enhancement of cancer cell migration was specific. It was primarily induced by HRH1, but not by other histamine receptor subtypes. This proves that they were not led.

[0437]

[0438] Example 11. Enhanced CXCR4 downstream signaling upon CXCR4-GPCRx heteromer formation Inhibition of signal transmission and enhanced signal transduction assessed by Ca 2+ mobilization assay.

[0439] In cells where both CXCR4 and ADRB2 are overexpressed, CXCR4 agonism Co-treatment of both CXCL12 and ADRB2 selective agonists, salmeterol, significantly improved the efficacy of CXCL12- and ADRB2-selective agonists. The calcium response was significantly higher than that evoked by the individual agonists. These results clearly demonstrate that CXCR4-ADRB2 heteromers It has been demonstrated that they exhibit properties that differ from those of individual GPCRs.

[0440] CXCL12 and ADRB2 linkage in cells co-expressing CXCR4 and ADRB2. Anti-C as a CXCR4 antagonist enhanced calcium responses upon co-stimulation with ATP. As shown in Figure 20, the expression of CXCR4 and A Treatment of cells co-expressing DRB2 with 2 μg of the anti-CXCR4 antibody 12G5 Both antagonists (ADRB2 antagonists, Carbenezer, and Benzyl Alcohol-Induced Rhodopsin) suppressed the enhanced calcium signaling. Co-treatment of zirol with the CXCR4 antagonist 12G5 resulted in more significant inhibition. The results showed that anti-CXCR4 antibodies and ADRB2 antagonists inhibited the CXCR4-ADRB2 heteromerization. demonstrated that it could be used as an effective treatment for

[0441] Using the calcium mobilization assay, 20,000 cells per well were cultured in 10% F Include MDA-MB-231 in 100 μL of RPMI 1640 supplemented with BS. Human breast cancer cells were cultured in black clear-bottom 96-well plates (Corning Costar, #33 The next day, CXCR4 at 10 MOI and GPCRx at 30 MOI were added to the cells. Two days later, the indicated doses of the ADRB2 antagonist carvedilol (Tocr is), anti-CXCR4 antibody 12G5 (Thermo Scientific, 35-88 00), and then the cells were treated with Cal6 (Molecular Devices' FLIPR ( Incubate for 2 hours with the registered trademark Calcium 6 Assay Kit, catalog R8191 This was followed by the indicated doses of CXCL12, ADRB2 agonists, or CXCL Cells were stimulated with 12 and ADRB2 agonists. Calcium mobilization was measured using a plate reader. Results were compared against baseline activity. The area under the curve (AUC) of each graph was calculated to determine calcium mobilization. The data were compared to CXCL12-stimulated calcification in cells expressing only CXCR4. Normalized to sodium response. Data represent three independent experiments (mean ± standard error). . * P<0.05, Student's t test.

[0442]

[0443] Example 12. CXCR4-GPCRx heterozygotes in CXCR4-GPCRx-expressing cells Evaluation of polymer formation using PLA.

[0444] The process for screening CXCR4-GPCRx heteromers is CXCR4 and G are essential for the processing of anti-cancer drugs that target GPCRx. 3. Quantitative Detection of PCRx Heteromers with PLA-CXCR4-GPCRx-Expressing Cells The CXCR4-overexpressing cell line U2OS-CXCR4 was treated with 0, 2.5, 10, and 40 The cells were infected with the GPCRx-expressing adenovirus Ad-GPCRx at an MOI of 100 for 2 days. Afterwards, the transformed cells were fixed with 4% paraformaldehyde and PLA was performed. The number of signal averages quantitatively indicates the formation of CXCR4-GPCRx heteromers.

[0445] As shown in Figure 21a-b, the PLA signal is CHRM1 (Figure 21a) and HRH1 (Figure 21b). These results were obtained from cancer patient samples. Detection of various types of CXCR4-GPCR heteromers and CXCR4-GP Quantitative analysis of CRx heteromers is demonstrated.

[0446] PLA was used to inoculate the CXCR4-overexpressing cell line U2OS-CXCR4 with 0, 2.5 Infect the cells with GPCRx-expressing adenovirus Ad-GPCRx at doses of 10, 40, or 10 MOI for 2 days. PLA was performed as previously described (Brueggema et al. nn et al., 2014, Tripathi et al., 2014). PLA To perform this, infected cells were fixed with 4% PFA on 16-well tissue culture slides. Block the slides with the blocking solution provided by Duolink. Mouse anti-CXCR4 (Santacruz, Sc-53534), rabbit anti-CHRM1 ( LS Bio, Ls-C313301) or rabbit anti-HRH1 (Thermoscie Incubate with 100% Fibrinogen (Pfizer, Inc., PA5-27817) at 37°C for 1 hour in a humidified chamber. The slides were then washed and stained with Duolink II P The LA probe was incubated with secondary anti-rabbit and anti-mouse antibodies ( 37 (1 hour at 37°C). The slides were washed again and then inoculated with ligation-ligase solution. Incubate (30 min at 37°C) followed by incubation with the polymerase solution for amplification. The slides were then placed in a minimal volume of Duolink II. The culture medium was then plated with 4',6-diamidino-2-phenylindole (DAPI) for 15-20 min. After 30 minutes, the PLA signal [Duo link In Situ Detection Reagents Green (λ excitation / emission 495 / 527 nm) or Red (λ excitation / emission 575 / 623 nm) was identified as the fluorescent spot.

[0447]

[0448] Example 13. Enhanced CXCR4 downstream signaling upon CXCR4-GPCRx heteromer formation Ca2+ mobilization inhibition of signal transmission - comparison of single and combined inhibitor treatments.

[0449] The data presented above (see Example 3, Figure 8a) demonstrate that CXCR4-ADRB2 The increased Ca2+ signal caused by telomer formation was mediated by CXCR4 inhibitors and ADRB2 inhibitors. We demonstrated that the expression of CXCR4 was significantly reduced when the cells were treated with the inhibitors. As a single treatment in cells containing CXCR4-ADRB (in the context of individual protomers), As a single treatment in CXCR4-ADRB2 heteromer-expressing cells and in CXCR4-ADRB2 heteromer-expressing cells Evaluated as a co-treatment with an ADRB2 inhibitor (carvedilol, 10 μM) in expressing cells The inhibitory effect (Ca) of a series of CXCR4 inhibitors was investigated. 2+ Response measured as IC50 value We compared the results of the two methods.

[0450] Adenoviruses encoding CXCR4 alone or both CXCR4 and ADRB were administered to MDA- MB-231 cells were transduced with CXCR4 inhibitors. The cells were cultured for 2 days and then treated with CXCR4 inhibitors. Or co-treatment with a CXCR4 inhibitor and an ADRB2 inhibitor (carvedilol, 10 μM) The cells were then stained with Cal-6 for 2 hours and treated with CXCR4 agonists (CXCL12, 20 The cells were stimulated with 100 nM of ATP and an ADRB2 agonist (salmeterol, 1 μM). The results of calcium mobilization measured using ion3 are shown in Table 6, including: (1) C MDA-MB-231 cells expressing only CXCR4 were treated with only an XCR4 inhibitor. (2) CXCR4 inhibitors and ADRB2 inhibitors (individual protomer context) (column 2); Co-treated with carvedilol, expressing CXCR4-ADRB2 heteromers MDA-MB-231 cells (column 3); and (3) treated with CXCR4 inhibitor only, C In MDA-MB-231 cells expressing XCR4-ADRB2 heteromers (column 4), Ca2 in + The IC50 of the response is shown.

[0451]

[0452] [Table 6]

[0453]

[0454] In Table 6, Ca 2+ The IC50 value of the response was 0.01 for MDA-MB- In the context of single treatment of 231 cells, as well as in the context of expression of CXCR4-ADRB2 heteromers, In the context of treatment of MDA-MB-231 cells alone and in the context of co-treatment with ADRB2 inhibitors Depending on the type of CXCR4 inhibitor, When treated in combination with an RB2 inhibitor (column 3), CXCR4-ADRB2 heteromers Ca in the context of 2+ The IC50 values ​​of the responses were obtained after single treatment with the CXCR4 inhibitor alone (column 4). For example, the CXCR4-ADRB2 heterozygote Ca in the context of 2+ The IC50 values ​​of the responses were measured using carvedilol, AMD3100, and Urocco. AMD3100, uropulma, and uropulma when co-treated with pulumab or TZ14011, respectively Approximately 540-fold (28.65 nM to 10.25 nM) 0.053nM), approximately 1400 times lower (1.12nM to 0.0008nM) or approximately 8 These results suggest that CXCR In cells expressing 4-ADRB2 heteromers, CXCR4 inhibitors and ADRB2 inhibitors Co-treatment with 2+ The response was greater than that of the CXCR4 inhibitor alone. This suggests that it effectively inhibits

[0455] CXCR4-ADRB2 heteromerization induces conformational changes and / or CXCR4 To determine whether this could alter the binding affinity of the inhibitor, we investigated the effect of CXCR4-only expression on the CXCR4-dependent markers. CXCR4 on cells expressing CXCR4-ADRB2 heteromers and on cells expressing CXCR4-ADRB2 heteromers. Treatment with the inhibitor alone increased Ca 2+ The IC50 values ​​of the responses were compared. CXCR4-ADRB2 heteromers were expressed compared to cells expressing CXCR4-ADRB2 (column 2). In cells containing CXCR4 (column 4), treatment with a CXCR4 inhibitor alone increased the IC50 by approximately 0.4-5 times. These results indicate that treatment with a CXCR4 inhibitor alone only altered the expression of CXCR4. Compared with co-treatment with a CXCR4 inhibitor and an ADRB2 inhibitor, The toxic effects of these agents dramatically increased the expression of CXCR4-ADRB2 heteromers in patients and / or patient cells / tissues. This suggests that the therapeutic efficacy of steroids against steroid-resistant steroids may be improved.

[0456]

[0457] Example 14. Effect of CXCR4-ADRB2 heteromers on tumor growth.

[0458] To investigate the effect of CXCR4-ADRB2 heteromers on tumor growth, CXC Cell lines stably overexpressing R4 alone or CXCR4-ADRB2 heteromers were A549 lung cancer cells were used, and the same amount of cells (1 × 10 7 cells / animal) into nude mice subcutaneously As shown in Figure 22a-b, the tumor growth rate on day 28 after the injection was The size of A549 is 351.4±214.7mm 3 , 726 for A549-CXCR4 0.9±259.6mm 3 , and 1012.2±5 for A549-CXCR4-ADRB2. 56.1mm 3 A549-CXCR4, which overexpresses CXCR4, was transplanted. The tumor growth rate in mice bearing the parental A549 gene was faster than that in mice bearing only the parental A549 gene. However, in mice transplanted with cells overexpressing CXCR4-ADRB2 heteromers, These results suggest that CXCR4-ADRB2 heteromers were associated with the fastest tumor growth. The formation of Ca 2+have been shown to enhance tumor response and therefore promote tumor growth. It is suggesting.

[0459] The parental A549 cells, A549-CXCR4 cells stably overexpressing CXCR4, and A549-CXCR4, a mouse model stably overexpressing CXCR4-ADRB2 heteromers, was used. Figure 22a shows images of three mice transplanted with either α-ADRB2 or α-ADRB2 at 28 days after transplantation. These images show that CXCR4-ADRB2 heteromers are expressed among them. The tumor size was the largest (the most accelerated) in mice carrying the cells. The tumor growth rate over time for three different mice is shown graphically in Figure 22b. The length (L) and width (W) of the tumor were measured and the tumor volume was calculated based on the following formula: Tumor growth was followed and assessed every 3 or 4 days using a 100-well platelet count (volume = 0.5LW). 2 . Parent cell A5 Mice carrying CXCR4-expressing cells showed a relatively rapid tumor growth compared to mice carrying 49. The tumor growth was observed in the grafts, and cells overexpressing CXCR4-ADRB2 heteromers were transplanted. Tumor growth is most rapid in mice.

[0460]

[0461] Example 15. Co-stimulation of CXCR4-GPCRx heteromers by endogenous ligands Assessment of enhanced CXCR4 downstream signaling by Ca 2+ mobilization assay.

[0462] CXC in MDA-MB-231 cells co-expressing CXCR4 and GPCRx Enhancement of calcium responses upon costimulation with L12 and endogenous ligands for GPCRx The results are shown in Figure 23a to g. Adenoviruses encoding XCR4 and GPCRx were transduced into MDA-MB-231 cells. The GPCRx was expressed as ADCYAP1R1 (Fig. 23a), ADORA2B ( Figure 23b), ADORA3 (Figure 23c), CHRM1 (Figure 23d), EDNRB (Figure 23 e), MLNR (Fig. 23f) and TACR3 (Fig. 23g). Cells were treated with either the endogenous GPCRx ligand alone or with CXCL12 and a GPCRx ligand. Calcium mobilization was calculated as described in Figure 5d. Total (spotted squares) and co-treatment Statistical significance of differences from control (black squares) was determined by Student's t-test. * P<0. 05. ** P<0.01, *** P<0.001, mean ± standard error (n=3).

[0463] In Figure 6a, CXCL12 and the nonselective endogenous ADCYAP1R1 ligand, CXCR4 and ADCYAP1R1 are co-expressed upon co-treatment with VIP In cells, enhanced Ca 2+ Signaling was observed, but only individual GPCRs were expressed. CXCL12 and ADCYAP1R1-selective endogenous receptors were not observed in cells containing CXCL12. Co-treatment of cells with PACAP-38, a phosphodiesterase inhibitor, also enhanced Ca 2+ Sig The results showed that CXCR4-ADCYAP1R1 heterozygotes were significantly transduced (Fig. 23a). Enhanced signaling of CXCL12 is limited by VIP upon costimulation with CXCL12 We demonstrate that this is not only limited by the specificity of ADCYAP1R1 but also by selective natural ligands. It is further shown that in cells where CXCR4 and ADCYAP1R1 are co-expressed, These results suggest that this enhancement may occur in vivo via their natural ligands. are.

[0464] CXCL12 and BAY60-6583 (ADORA2B selective agonist), or CXC Co-treatment with L12 and Cl-IB-MECA (an ADORA3 selective agonist) CXCR4 and ADORA2B (Fig. 6b) or CXCR4 and ADORA3 (Fig. 6c) Co-expressing cells showed enhanced Ca 2+ Signal transduction was observed, but individual G This was not observed in cells expressing only PCR. When co-treated with adenosine, the endogenous ligand for all adenosine receptors Also, enhanced Ca 2+ Signal transduction was observed (Fig. 23b and c). Not only the natural agonist but also the natural endogenous ligand CXCR4 activates CXCR4 during costimulation with CXCL12. -Enhance downstream signaling of ADORA2B and CXCR4-ADORA3 heteromers It further demonstrates that CXCR4 and ADORA2B, or CXCR4 In cells where ADORA3 and its natural ligands co-express, downstream activation of ADORA3 was induced. These findings suggest that synergistic enhancement of signal transmission may occur in vivo.

[0465] In Figure 6f, the effects of CXCL12 and the synthetic CHRM1 agonist bethanechol were Upon co-treatment, cells co-expressing CXCR4 and CHRM1 showed enhanced Ca 2+ Signaling was observed, but not in cells expressing individual GPCRs alone. CXCL12, an endogenous ligand for all acetylcholine receptors, Co-treatment of cells with acetylcholine also resulted in enhanced Ca 2+ Signal transduction is recognized The results showed that not only synthetic agonists but also natural endogenous ligands acted on CXC receptors. Enhancement of CXCR4-CHRM1 heteromeric signaling upon costimulation with L12 It further demonstrates that cells expressing CXCR4 and CHRM1 together synergistic enhancement of downstream signaling by their natural ligands in vivo suggests that it is possible.

[0466] In Figure 6g, the effect of CXCL12 and BQ3020, a selective EDNRB agonist, was observed. Upon co-treatment with CXCR4, cells expressing CXCR4 and EDNRB showed enhanced C a 2+ Signal transduction was observed, but not in cells expressing individual GPCRs alone. CXCL12 and endothelin, an endogenous ligand for the endothelin receptor, were not detected. Co-treating cells with serine-1 also resulted in enhanced Ca 2+ Signal transduction was observed The results showed that not only synthetic agonists but also natural endogenous ligands inhibited CXCL12. We demonstrated that co-stimulation with CXCR4 enhances signal transduction of CXCR4-EDNRB heteromers. It further demonstrates that in cells where CXCR4 and EDNRB are co-expressed, These natural ligands may synergistically enhance downstream signaling in vivo. This suggests that there is.

[0467] In Figure 6i, CXCL12 and Loki, an antibiotic and full MLNR agonist, were compared. Upon co-treatment with cystomycin, cells expressing CXCR4 and MLNR were In the vesicles, enhanced Ca2+ Signaling was observed, but only individual GPCRs were expressed. CXCL12 and selective endogenous ligands for MLNR were not observed in cells containing CXCL12. Co-treatment of cells with motilin, a Ca receptor, also resulted in enhanced Ca 2+ Signal transduction is recognized The results showed that not only synthetic agonists but also natural endogenous ligands acted on CXC receptors. Enhancement of CXCR4-MLNR heteromeric signaling upon costimulation with L12 It further demonstrates that in cells where CXCR4 and MLNR are co-expressed, It is possible that synergistic enhancement of downstream signaling occurs in vivo with their natural ligands. This suggests that there is a gender.

[0468] In Figure 6l, the effect of CXCL12 and senktide, a selective TACR3 agonist, on Upon co-treatment, cells co-expressing CXCR4 and TACR3 showed enhanced Ca 2+ Signaling was observed, but not in cells expressing individual GPCRs alone. CXCL12 and Neurokinase, a selective endogenous ligand for TACR3, were Enhanced Ca was also observed when cells were co-treated with nin B. 2+ Signal transduction was observed ( The results showed that not only synthetic agonists but also natural endogenous ligands interacted with CXCL12. We demonstrated that co-stimulation enhances CXCR4-TACR3 heteromeric signaling. It was further shown that CXCR4 and EDNRB are expressed together in cells. These natural ligands may synergistically enhance downstream signaling in vivo. This suggests that

[0469]

[0470] Example 16. Enhanced CXCR4 expression upon CXCR4-GPCRx heteromeric co-stimulation Assessment of current signaling by Ca 2+ mobilization assay.

[0471] In MDA-MB-231 cells co-expressing CXCR4 and ADCYAP1R1, and selective endogenous ligands CXCL12 or PACAP38, respectively. Stimulation with CX induced calcium signaling in a dose-dependent manner (Figure 24a-b). Adenoviruses encoding CR4 and ADCYAP1R1 were transfected into MDA-MB-231 cells. As shown in Figure 24a, a small amount of ADCYAP1R1-selective endogenous ligand was transduced into Addition of PACAP38 (1 nM) suppressed the activity of PACAP38 alone at the doses shown. By adding the responses obtained with CXCL12 alone to the responses obtained with CXCL12 alone, The calcium response was significantly enhanced over a wide range of CXCL12 concentrations compared with the total value calculated by The maximum Ca induced by CXCL12 alone was 2+ 100% response The total was calculated using 1 nM PACAP38 and the indicated doses of CXCL12. Similarly, as shown in Figure 24b, This indicates that a wide range of PACAP38 concentrations, even when treated alone, did not induce any response. CXCR4-selective endogenous ligand (CXCR4) at concentrations (0.03-0.3 nM) The addition of CXCL12 (15 nM) was comparable to the effect achieved by CXCL12 alone at the doses shown. The response was calculated by adding the response obtained with PACAP38 alone to the response obtained with PACAP38 alone. PACAP38 alone significantly enhanced downstream responses compared to the total values ​​(Fig. 24b). The maximum Ca induced by 2+ The response was taken as 100%. The sum of the responses induced by α-glucan alone and PACAP38 alone at the doses indicated was calculated. The statistical significance of the difference between the total and the co-treatment at each point was determined using Student's t-test. was determined by assay. * P<0.05, ** P<0.01, *** P<0.001, flat Mean ± standard deviation (n = 3). These results suggest that small amounts of CXCL12 and ADCYAP1R1 In the presence of ATP, cells co-expressing CXCR4 and ADCYAP1R1 responded This suggests that the activity of the ATP-dependent agonist is enhanced in vivo.

[0472] CXCR4 and TACR3 co-expressing cells show cytotoxicity across a wide range of ligand concentrations. The enhanced calcium response is shown in Figure 25a-b. MDA-MB-231 cells were transduced with adenovirus. CXCL12 alone or nucleoside-dependently expressed CXCL12 was transduced. Stimulation of cells with acetylcholinesterase B alone induced calcium signaling in a dose-dependent manner. As shown in Figure 25a, a small amount of neurokinin B (0.4 nM, TACR3-selective endogenous ligand) was added to the neurokinin receptor at the doses indicated. The responses obtained with CXCL12 alone were added together to those obtained with CXCL12 alone. The calcium response was measured over a wide range of CXCL12 concentrations compared with the total value calculated by The maximum calcium response evoked by CXCL12 alone was significantly enhanced by 100 The total was calculated based on 0.4 nM of neurokinin alone and the indicated dose of CXCL1. Similarly, Fig. 25b shows the sum of the responses caused by only As shown, the addition of CXCL12 significantly reduced the effects of CXCL12 alone at the doses shown. By adding the responses obtained with neurokinin B alone, A wide range of neurokinin B concentrations significantly enhanced downstream responses compared with the calculated total values The maximum response evoked by neurokinin B alone was taken as 100%. Elicited by 0 nM CXCL12 alone and the indicated doses of neurokinin B alone The sum of the responses was calculated and the statistical significance of the sum and co-treatment at each point was calculated. Differences were determined by Student's t-test. * P<0.05, ** P<0.01, flat Mean ± standard deviation (n = 3). These results suggest that the presence of small amounts of CXCL12 and neurokinin B In vivo, the response was enhanced in cells co-expressing CXCR4 and TACR3. This suggests that

[0473]

[0474] Example 17. Confirmation of loss of heteromeric specific properties upon deletion of one protomer.

[0475] In Figure 8 f and g, MDA-MB-231 cells overexpressing only CXCR4 In cells, co-treatment with CXCL12 and histamine resulted in enhanced calcium signaling. Using RT-qPCR, we confirmed that MDA-MB-231 cells express HRH1 and and low levels of CXCR4 mRNA, and HRH1 mRNA increased CXCR4 mRNA. As shown in Figure 8 f and g, the expression of NA was approximately twice as high as that of NA (Example 9). The enhanced calcium signaling observed in the IL-16 mice is due to the presence of endogenous HRH1 expression, whereas other To confirm whether this is not due to the presence of histamine receptors, CRIS MDA using the PR / Cas9 system CXCR4+ The HRH1 gene in cells is deleted. A lentivirus encoding Cas9 and a guide RNA targeting HRH1 was then transfected into C MDA-MB-231 cells stably expressing XCR4 (MDA CXCR4+ ) in the shape Functional H was assessed by measuring calcium responses upon exposure to histamine. The presence of RH1 was detected. MDA CXCR4+ Cells respond to histamine in a dose-dependent manner showed a significant increase in calcium signaling, but MDA CXCR4+,HRH1- Cells Even at 1 μM histamine, no calcium response was observed (Fig. 26a). The result is MDA CXCR4+,HRH1- There is almost no functional HRH1 in the cells. It implies that there isn't.

[0476] MDA-MB-231 cells stably overexpressing CXCR4 (MDA CXCR4 + cells) were treated with histamine, and a dose-dependent increase in calcium signaling was observed. When cells were treated with histamine in the presence of CXCL12 (50 nM), Significantly enhanced calcium responses were observed across a wide range of histamine concentrations, and EC 50 and E max However, the CRISPR / MDA with HRH1 removed using the Cas9 system CXCR4+ Cells (MDA CX CR4+,HRH1- In the absence or presence of CXCL12 (50 nM), No stamin-mediated response was obtained, reaffirming the absence of functional HRH1 in these cells MDA CXCR4+ The maximum potentiation induced by histamine alone in cells The calcium response was taken as 100%. The total was calculated using 50 nM CXCL12 alone and the indicated The sum of the responses evoked by the dose of histamine alone is calculated.

[0477] MDA CXCR4+ CXCL12 stimulation induces a dose-dependent calcium response in cells. The addition of a non-signaling concentration of histamine (15 nM) produced a wide range of responses (Fig. 27b). CXCL12 significantly enhanced calcium responses across a range of concentrations, resulting in EC 50 and E max Value change Evidence of improved potency and efficacy has been obtained. CXCR4+,HRH1- cell CXCL12-mediated calcium response in MDA CXCR4+ Responses observed in cells However, MDA CXCR4+,HRH1- Histamine in cells Addition of hydroxybenzoates failed to augment CXCL12-mediated calcium responses, and deletion of HRH1 The loss of heteromeric specific properties due to MDA was demonstrated. CXCR4+ CXC in cells The maximum r...

Claims

1. A pharmaceutical composition for use in a method for treating cancer in a patient having cancer cells containing a CXCR4-GPCRx heteromer, the pharmaceutical composition comprising a CXCR4 inhibitor, the pharmaceutical composition being administered in combination with a GPCRx inhibitor that targets the same GPCRx as the GPCRx of the CXCR4-GPCRx heteromer, the GPCRx of the CXCR4-GPCRx heteromer being selected from the group consisting of ADRB2, CHRM1 and HRH1, The CXCR4 inhibitor is AD-114, AD-114-6H, AD-114-Im7-FH, AD-114-PA600-6H, ALX-0651, ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI2341, BKT140 (BL-8040; TF14016; 4F-benzyl benzoate), Zoyl-TN14003), CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, LY2624587, MSX-122, N-[ [ 11 C]methyl-AMD3465, PF-06747143, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC14012, TG-0054 (Blixafor), USL311, urocuplumab (MDX1338 / BMS-936564), viral macrophage inflammatory protein-II (vMIP-II), WZ811, 12G5, 238D2, ​​238D4, [ 64 Cu]-AMD3100, [ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]pentixatel, [ 99m Tc]O 2 - selected from the group consisting of AMD3100, [ 177 Lu]pentixatel, and 508MCl (compound 26), and The GPCRx inhibitor is an ADRB2 inhibitor selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP12177, cycloprolol, ICI118551, ICYP, labetalol, levobetaxolol, levobunolol, LK204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A and timolol; 3-quinuclidinyl benzilate (QNB), 4-DAMP, aclidinium, AE9C90CB, AFDX384, amitriptyline, AQ-RA741, atropine, benzatropine, biperiden, darifenacin, dicyclomine, dosulepin, ethopropazine, glycopyrrolate, guanylpirenzepine, hexahydrodiphenidol, hexahydrosilazifenidol, hexocyclium, himbacine, ipratropium, lithocholylcholine, methoctramine, ML381, muscarine A CHRM1 inhibitor selected from the group consisting of Toxin 1, Muscarinic Toxin 2, Muscarinic Toxin 3, N-methylscopolamine, otenzepad, oxybutynin, p-F-HHSiD, pirenzepine, propantheline, (R,R)-quinuclidinyl-4-fluoromethyl-benzilate, scopolamine, silahexocyclium, solifenacin, telenzepine, tiotropium, tolterodine, trihexyphenidyl, tripitramine, UH-AH37, umeclidinium, and VU0255035, and (-)-Chlorpheniramine, (+)-Chlorpheniramine, (-)-trans-H 2 -PAT, (+)-cis-H 2 -PAT, (+)-trans-H 2 -PAT, (±)-cis-H 2 -PAT, (±)-trans-H 2 -PAT, (R)-cetirizine, (S)-cetirizine, 9-OH-risperidone, A-317920, A-349821, ABT-239, alimemazine, amitriptyline, aripiprazole, apromidine, asenapine, astemizole, AZD3778, azelastine, BU-E47, cetirizine, chlorpheniramine, chlorpromazine, ciproxifan, clemastine, clobenpropit, clozapine, conessine, cyclizine, cyproheptadine, desloratadine, diphenhydramine, dosulepin, doxepin, epinastine, fexof an HRH1 inhibitor selected from the group consisting of phenazine, fluphenazine, fluspirilene, haloperidol, hydroxyzine, impromidine, INCB-38579, JNJ-39758979, ketotifen, loratadine, loxapine, MK-0249, molindone, olanzapine, perphenazine, pimozide, pipamperon, pitolisant, promethazine, pyrilamine, quetiapine, risperidone, sertindole, terfenadine, thioridazine, thiothixene, trifluoperazine, tripelennamine, triprolidine, ziprasidone and zotepine; The pharmaceutical composition is selected from the group consisting of:

2. A pharmaceutical composition for use in a method for treating cancer in a patient having cancer cells containing a CXCR4-GPCRx heteromer, the pharmaceutical composition comprising a GPCRx inhibitor that targets the same GPCRx as the GPCRx of the CXCR4-GPCRx heteromer, the pharmaceutical composition being used such that the GPCRx inhibitor is administered in combination with a CXCR4 inhibitor, and the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of ADRB2, CHRM1, and HRH1; The CXCR4 inhibitor is AD-114, AD-114-6H, AD-114-Im7-FH, AD-114-PA600-6H, ALX-0651, ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI2341, BKT140 (BL-8040; TF14016; 4F-benzyl benzoate), Zoyl-TN14003), CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, LY2624587, MSX-122, N-[ [ 11 C]methyl-AMD3465, PF-06747143, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC14012, TG-0054 (Blixafor), USL311, urocuplumab (MDX1338 / BMS-936564), viral macrophage inflammatory protein-II (vMIP-II), WZ811, 12G5, 238D2, ​​238D4, [ 64 Cu]-AMD3100, [ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]pentixatel, [ 99m Tc]O 2 - selected from the group consisting of AMD3100, [ 177 Lu]pentixatel, and 508MCl (compound 26), and The GPCRx inhibitor is an ADRB2 inhibitor selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP12177, cycloprolol, ICI118551, ICYP, labetalol, levobetaxolol, levobunolol, LK204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A and timolol; 3-quinuclidinyl benzilate (QNB), 4-DAMP, aclidinium, AE9C90CB, AFDX384, amitriptyline, AQ-RA741, atropine, benzatropine, biperiden, darifenacin, dicyclomine, dosulepin, ethopropazine, glycopyrrolate, guanylpirenzepine, hexahydrodiphenidol, hexahydrosilazifenidol, hexocyclium, himbacine, ipratropium, lithocholylcholine, methoctramine, ML381, muscarine A CHRM1 inhibitor selected from the group consisting of Toxin 1, Muscarinic Toxin 2, Muscarinic Toxin 3, N-methylscopolamine, otenzepad, oxybutynin, p-F-HHSiD, pirenzepine, propantheline, (R,R)-quinuclidinyl-4-fluoromethyl-benzilate, scopolamine, silahexocyclium, solifenacin, telenzepine, tiotropium, tolterodine, trihexyphenidyl, tripitramine, UH-AH37, umeclidinium, and VU0255035, and (-)-Chlorpheniramine, (+)-Chlorpheniramine, (-)-trans-H 2 -PAT, (+)-cis-H 2 -PAT, (+)-trans-H 2 -PAT, (±)-cis-H 2 -PAT, (±)-trans-H 2 -PAT, (R)-cetirizine, (S)-cetirizine, 9-OH-risperidone, A-317920, A-349821, ABT-239, alimemazine, amitriptyline, aripiprazole, apromidine, asenapine, astemizole, AZD3778, azelastine, BU-E47, cetirizine, chlorpheniramine, chlorpromazine, ciproxifan, clemastine, clobenpropit, clozapine, conessine, cyclizine, cyproheptadine, desloratadine, diphenhydramine, dosulepin, doxepin, epinastine, fexof an HRH1 inhibitor selected from the group consisting of phenazine, fluphenazine, fluspirilene, haloperidol, hydroxyzine, impromidine, INCB-38579, JNJ-39758979, ketotifen, loratadine, loxapine, MK-0249, molindone, olanzapine, perphenazine, pimozide, pipamperon, pitolisant, promethazine, pyrilamine, quetiapine, risperidone, sertindole, terfenadine, thioridazine, thiothixene, trifluoperazine, tripelennamine, triprolidine, ziprasidone and zotepine; The pharmaceutical composition is selected from the group consisting of:

3. A pharmaceutical composition for use in a method for suppressing enhanced downstream signaling from a CXCR4-GPCRx heteromer in cancer cells of a patient suffering from cancer, the pharmaceutical composition comprising a CXCR4 inhibitor, the pharmaceutical composition being used such that the CXCR4 inhibitor is administered in combination with a GPCRx inhibitor that targets the same GPCRx as the GPCRx of the CXCR4-GPCRx heteromer, the GPCRx of the CXCR4-GPCRx heteromer being selected from the group consisting of ADRB2, CHRM1 and HRH1, The CXCR4 inhibitor is AD-114, AD-114-6H, AD-114-Im7-FH, AD-114-PA600-6H, ALX-0651, ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI2341, BKT140 (BL-8040; TF14016; 4F-benzyl benzoate), Zoyl-TN14003), CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, LY2624587, MSX-122, N-[ [ 11 C]methyl-AMD3465, PF-06747143, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC14012, TG-0054 (Blixafor), USL311, urocuplumab (MDX1338 / BMS-936564), viral macrophage inflammatory protein-II (vMIP-II), WZ811, 12G5, 238D2, ​​238D4, [ 64 Cu]-AMD3100, [ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]pentixatel, [ 99m Tc]O 2 - selected from the group consisting of AMD3100, [ 177 Lu]pentixatel, and 508MCl (compound 26), and The GPCRx inhibitor is an ADRB2 inhibitor selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP12177, cycloprolol, ICI118551, ICYP, labetalol, levobetaxolol, levobunolol, LK204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A and timolol; 3-quinuclidinyl benzilate (QNB), 4-DAMP, aclidinium, AE9C90CB, AFDX384, amitriptyline, AQ-RA741, atropine, benzatropine, biperiden, darifenacin, dicyclomine, dosulepin, ethopropazine, glycopyrrolate, guanylpirenzepine, hexahydrodiphenidol, hexahydrosilazifenidol, hexocyclium, himbacine, ipratropium, lithocholylcholine, methoctramine, ML381, muscarine A CHRM1 inhibitor selected from the group consisting of Toxin 1, Muscarinic Toxin 2, Muscarinic Toxin 3, N-methylscopolamine, otenzepad, oxybutynin, p-F-HHSiD, pirenzepine, propantheline, (R,R)-quinuclidinyl-4-fluoromethyl-benzilate, scopolamine, silahexocyclium, solifenacin, telenzepine, tiotropium, tolterodine, trihexyphenidyl, tripitramine, UH-AH37, umeclidinium, and VU0255035, and (-)-Chlorpheniramine, (+)-Chlorpheniramine, (-)-trans-H 2 -PAT, (+)-cis-H 2 -PAT, (+)-trans-H 2 -PAT, (±)-cis-H 2 -PAT, (±)-trans-H 2 -PAT, (R)-cetirizine, (S)-cetirizine, 9-OH-risperidone, A-317920, A-349821, ABT-239, alimemazine, amitriptyline, aripiprazole, apromidine, asenapine, astemizole, AZD3778, azelastine, BU-E47, cetirizine, chlorpheniramine, chlorpromazine, ciproxifan, clemastine, clobenpropit, clozapine, conessine, cyclizine, cyproheptadine, desloratadine, diphenhydramine, dosulepin, doxepin, epinastine, fexof an HRH1 inhibitor selected from the group consisting of phenazine, fluphenazine, fluspirilene, haloperidol, hydroxyzine, impromidine, INCB-38579, JNJ-39758979, ketotifen, loratadine, loxapine, MK-0249, molindone, olanzapine, perphenazine, pimozide, pipamperon, pitolisant, promethazine, pyrilamine, quetiapine, risperidone, sertindole, terfenadine, thioridazine, thiothixene, trifluoperazine, tripelennamine, triprolidine, ziprasidone and zotepine; The pharmaceutical composition is selected from the group consisting of:

4. A pharmaceutical composition for use in a method for suppressing enhanced downstream signaling from a CXCR4-GPCRx heteromer in cancer cells of a patient suffering from cancer, the pharmaceutical composition comprising a GPCRx inhibitor that targets the same GPCRx as the GPCRx of the CXCR4-GPCRx heteromer, the pharmaceutical composition being used such that the GPCRx inhibitor is administered in combination with a CXCR4 inhibitor, and the GPCRx of the CXCR4-GPCRx heteromer is selected from the group consisting of ADRB2, CHRM1 and HRH1, The CXCR4 inhibitor is AD-114, AD-114-6H, AD-114-Im7-FH, AD-114-PA600-6H, ALX-0651, ALX40-4C, AMD070 (AMD11070, X4P-001), AMD3100 (plerixafor), AMD3465, ATI2341, BKT140 (BL-8040; TF14016; 4F-benzyl benzoate), Zoyl-TN14003), CTCE-9908, CX549, D-[Lys3]GHRP-6, FC122, FC131, GMI-1359, GSK812397, GST-NT21MP, isothiourea-1a, isothiourea-1t (IT1t), KRH-1636, KRH-3955, LY2510924, LY2624587, MSX-122, N-[ [ 11 C]methyl-AMD3465, PF-06747143, POL6326, SDF-1 1-9[P2G] dimer, SDF1 P2G, T134, T140, T22, TC14012, TG-0054 (Blixafor), USL311, urocuplumab (MDX1338 / BMS-936564), viral macrophage inflammatory protein-II (vMIP-II), WZ811, 12G5, 238D2, ​​238D4, [ 64 Cu]-AMD3100, [ 64 Cu]-AMD3465, [ 68 Ga]pentixafor, [ 90 Y]pentixatel, [ 99m Tc]O 2 - selected from the group consisting of AMD3100, [ 177 Lu]pentixatel, and 508MCl (compound 26), and The GPCRx inhibitor is an ADRB2 inhibitor selected from the group consisting of alprenolol, atenolol, betaxolol, bupranolol, butoxamine, carazolol, carvedilol, CGP12177, cycloprolol, ICI118551, ICYP, labetalol, levobetaxolol, levobunolol, LK204-545, metoprolol, nadolol, NIHP, NIP, propafenone, propranolol, sotalol, SR59230A and timolol; 3-quinuclidinyl benzilate (QNB), 4-DAMP, aclidinium, AE9C90CB, AFDX384, amitriptyline, AQ-RA741, atropine, benzatropine, biperiden, darifenacin, dicyclomine, dosulepin, ethopropazine, glycopyrrolate, guanylpirenzepine, hexahydrodiphenidol, hexahydrosilazifenidol, hexocyclium, himbacine, ipratropium, lithocholylcholine, methoctramine, ML381, muscarine A CHRM1 inhibitor selected from the group consisting of Toxin 1, Muscarinic Toxin 2, Muscarinic Toxin 3, N-methylscopolamine, otenzepad, oxybutynin, p-F-HHSiD, pirenzepine, propantheline, (R,R)-quinuclidinyl-4-fluoromethyl-benzilate, scopolamine, silahexocyclium, solifenacin, telenzepine, tiotropium, tolterodine, trihexyphenidyl, tripitramine, UH-AH37, umeclidinium, and VU0255035, and (-)-Chlorpheniramine, (+)-Chlorpheniramine, (-)-trans-H 2 -PAT, (+)-cis-H 2 -PAT, (+)-trans-H 2 -PAT, (±)-cis-H 2 -PAT, (±)-trans-H 2 -PAT, (R)-cetirizine, (S)-cetirizine, 9-OH-risperidone, A-317920, A-349821, ABT-239, alimemazine, amitriptyline, aripiprazole, apromidine, asenapine, astemizole, AZD3778, azelastine, BU-E47, cetirizine, chlorpheniramine, chlorpromazine, ciproxifan, clemastine, clobenpropit, clozapine, conessine, cyclizine, cyproheptadine, desloratadine, diphenhydramine, dosulepin, doxepin, epinastine, fexof an HRH1 inhibitor selected from the group consisting of phenazine, fluphenazine, fluspirilene, haloperidol, hydroxyzine, impromidine, INCB-38579, JNJ-39758979, ketotifen, loratadine, loxapine, MK-0249, molindone, olanzapine, perphenazine, pimozide, pipamperon, pitolisant, promethazine, pyrilamine, quetiapine, risperidone, sertindole, terfenadine, thioridazine, thiothixene, trifluoperazine, tripelennamine, triprolidine, ziprasidone and zotepine; The pharmaceutical composition is selected from the group consisting of:

5. i) the CXCR4-GPCRx heteromer has enhanced downstream signaling; and ii) The pharmaceutical composition of any one of claims 1 to 4, wherein the combination of the CXCR4 inhibitor and the GPCRx inhibitor suppresses the enhanced downstream signaling from the CXCR4-GPCRx heteromer in the cancer patient.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the combination of the CXCR4 inhibitor and the GPCRx inhibitor is administered sequentially, concurrently or simultaneously.

7. The pharmaceutical composition of any one of claims 1 to 6, further comprising a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the combination of the CXCR4 inhibitor and the GPCRx inhibitor is administered as separate formulations.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the combination of the CXCR4 inhibitor and the GPCRx inhibitor is administered as a single formulation.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the method comprises detecting or identifying the CXCR4-GPCRx heteromer in the cancer patient.

11. The method comprising: i) obtaining or having obtained a biological sample from said cancer patient; ii) performing or having performed a diagnostic assay that determines the presence, identity, or presence and identity of a CXCR4-GPCRx heteromer in the obtained biological sample from the cancer patient; and iii) selecting said inhibitor combinations that suppress enhanced downstream signaling from said CXCR4-GPCRx heteromers; The pharmaceutical composition according to any one of claims 1 to 10, comprising:

12. 12. The pharmaceutical composition of claim 11, wherein the biological sample is a biological fluid sample, the biological fluid sample being selected from a blood sample, a plasma sample, a saliva sample, a cerebrospinal fluid sample, an intraocular fluid sample, and a urine sample.

13. The pharmaceutical composition of claim 11 , wherein the biological sample is a biological tissue sample, and the biological tissue sample is selected from an organ tissue sample, a bone tissue sample, and a tumor tissue sample.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, brain tumor, kidney cancer, pancreatic cancer, ovarian cancer, prostate cancer, melanoma, multiple myeloma, gastrointestinal cancer, renal cell carcinoma, soft tissue sarcoma, hepatocellular carcinoma, gastric cancer, colon cancer, esophageal cancer, and leukemia.

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