Therapeutic applications based on the inhibition of G protein-coupled receptor 182 (GPR182)

Abstract

The present invention relates to an agent for inhibiting the expression or activity of G protein-coupled receptor 182 (GPR182) protein for use in the treatment or prevention of a pathological condition selected from myocardial infarction, myocardial ischemia, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, stroke, and cancer, wherein (a) the agent for inhibiting the expression of GPR182 protein is selected from siRNA, miRNA, shRNA, ribozyme, and antisense nucleic acid molecules; and / or (b) the agent for inhibiting the activity of GPR182 protein specifically binds to the GPR182 protein and inhibits the binding of one or more endogenous ligands to the GPR182 protein, and the agent is selected from an antibody, Fc fusion polypeptide, adnectin, affibody, affilin, anticalin, atrimer, avimer, evibody, Kunitz-type domain, designed ankyrin repeat protein (DARPin), finomer, peptide or peptidomimetic, aptamer, and small molecule; or any combination thereof and / or a hetero- or homo-oligomeric covalent or non-covalent complex.

Description

Technical Field

[0001] The present invention relates to an agent for inhibiting the expression or activity of G protein-coupled receptor 182 (GPR182) protein for use in the treatment or prevention of pathological conditions selected from myocardial infarction, myocardial ischemia, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, stroke, and cancer, wherein (a) the agent for inhibiting the expression of GPR182 protein is selected from siRNA, miRNA, shRNA, ribozyme, and antisense nucleic acid molecules; and / or (b) the agent for inhibiting the activity of GPR182 protein specifically binds to the GPR182 protein and inhibits the binding of one or more endogenous ligands to the GPR182 protein, and the agent is an antibody, Fc fusion polypeptide, adnectin, affibody, affilin, anticalin, atrimer, avimer, evibody, Kunitz-type domain, designed ankyrin repeat protein (DARPin), fynomer, peptide or peptidomimetic, aptamer, and small molecule; or any combination thereof and / or a hetero- or homo-oligomeric covalent or non-covalent complex selected therefrom.

[0002] Several documents are listed herein, including patent applications and manufacturer manuals. The disclosures in these documents are not considered relevant to the patentability of the present invention and are hereby incorporated by reference in their entirety. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Background Art

[0003] Myocardial infarction (MI) is one of the most common acute diseases and a cause of death worldwide. Myocardial infarction is caused by an acute and usually thrombotic occlusion of the coronary artery, resulting in ischemic myocardial necrosis. Since postnatal cardiomyocytes cannot re-enter the cell cycle and proliferate, in adult mammals, dead cells are replaced by permanent collagenous scar tissue instead of new myocardial tissue. This also causes geometric, biomechanical, and biochemical changes in the intact ventricular wall, inducing a reactive remodeling process including interstitial fibrosis and perivascular fibrosis. Early reparative fibrosis is essential to prevent rupture of the ventricular wall, but excessive fibrotic response and reactive fibrosis outside the damaged area are harmful because they cause progressive decline of cardiac function and ultimately lead to heart failure (Talman V, Ruskoaho H. Cell Tissue Res. 2016;365(3):563~581). In addition to early life-threatening complications such as cardiac arrhythmia, acute heart failure, and cardiogenic shock, late complications (i.e., so-called post-complications of MI) such as chronic heart failure, cardiac wall aneurysm, and cardiac arrhythmia also play a central role in the decline of quality of life and mean life expectancy after myocardial infarction in hospitalized patients who have overcome the acute infarction period. Any treatment means that reduces infarct size has a favorable effect on short-term and long-term prognosis.

[0004] Current therapies for acute MI are mainly based on reversing the effects of ischemia / reperfusion injury by promoting reperfusion. These measures involve the administration of fibrinolytic agents such as streptokinase or tissue plasminogen activator (tPA) variants. At the same time, anticoagulants and platelet function inhibitors are used to suppress further thrombosis. Alternatively, or simultaneously, percutaneous transluminal coronary angioplasty with balloon catheter inflation and / or stent implantation is used as part of reperfusion therapy. Reperfusion therapy is further supplemented by various symptomatic measures to improve hemodynamic parameters to some extent, such as administration of O2, nitrates, β-blockers, and / or angiotensin-converting enzyme (ACE) inhibitors. However, despite the usefulness of these current treatment options, the acute lethality of MI remains very high (3 - 9%), and the majority of patients after acute MI suffer from a poor long-term prognosis with a significantly reduced mean life expectancy (Zeymer, 2019). Therefore, these therapies are somewhat effective in improving MI symptoms but do not result in complete functional recovery of the affected tissue. More recent preclinical approaches based on experiments have focused on enhancing cardiac protection by increasing resistance to ischemia, promoting regenerative capacity, and inducing angiogenesis, but a treatment approach that can efficiently prevent or reduce myocardial scar tissue formation and / or replace it by functioning the contractile tissue after myocardial infarction has not yet been identified.

[0005] Cancer is another major cause of death globally, with the number estimated to be approximately 12.7 million worldwide and affecting both men and women equally. This number is expected to increase to 21 million by 2030. Early cancer treatment relied on surgical intervention, radiotherapy, and / or chemotherapy, while on the other hand, an increasing number of new anticancer treatments aimed at activating the patient's own immune cells to attack cancer have become available. However, the main hurdle to the effectiveness of so-called "cancer immunotherapy" (commonly also referred to as "immune-oncology") is that many cancers can evade immune recognition based on their ability to undergo immune evasion by expressing ligands on their surface that, for example, inhibit the recruitment and / or activation of immune cells, thereby inhibiting their ability to fight cancer. In the past decade, new cancer treatment strategies based on adoptive cell therapy, in particular CAR-T cell therapy that can redirect the patient's own immune cells or donor immune cells to tumor-specific surface antigens by expressing chimeric antigen receptors (CARs) on their surface, have emerged. Furthermore, so-called immune checkpoint inhibitors (ICIs) have emerged as a new class of immunotherapy drugs. These humanized monoclonal antibodies target inhibitory receptors (e.g., CTLA-4, PD-1, LAG-3, TIM-3) and ligands (PD-L1) expressed on immune cells (e.g., T lymphocytes, antigen-presenting cells) or tumor cells, and as a result, trigger an antitumor response by stimulating the immune system (see, for example, Vinay DS et al., Semin Cancer Biol. 2015 Dec;35 Suppl:S185~S1). However, despite these advances, the current understanding of the mechanisms underlying cancer immune evasion is limited, and the treatment outcomes from available cancer immunotherapies are often insufficient.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, alternative and more effective treatment strategies for the treatment and prevention of these conditions and related conditions and their post-illness complications are needed. The present invention addresses this need and several further aspects as demonstrated in the supplementary examples.

Means for Solving the Problems

[0007] Accordingly, in a first aspect, the present invention relates to an agent for inhibiting the expression or activity of G protein-coupled receptor 182 (GPR182) protein for use in the treatment or prevention of a pathological condition selected from myocardial infarction, myocardial ischemia, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, stroke, and cancer, wherein (a) the agent for inhibiting the expression of GPR182 protein is selected from siRNA, miRNA, shRNA, ribozyme, and antisense nucleic acid molecules; and / or (b) the agent for inhibiting the activity of GPR182 protein specifically binds to the GPR182 protein and inhibits the binding of one or more endogenous ligands to the GPR182 protein, and the agent is selected from antibodies, Fc fusion polypeptides, adnectins, affibodies, affilins, anticalins, atrimers, avimers, evibodies, Kunitz-type domains, designed ankyrin repeat proteins (DARPins), finomers, peptides or peptidomimetics, aptamers, and small molecules; or any combination thereof and / or heteromeric or homomeric covalent or non-covalent complexes.

Modes for Carrying Out the Invention

[0008] G protein-coupled receptors (GPCRs) represent the largest group of transmembrane receptors encoded in the genome, and they are the largest group of proteins targeted by approved drugs. GPCRs are highly versatile and can bind ligands with diverse physicochemical properties, such as ions, lipids, biogenic amines, peptides, or proteins, for example, chemokines. Through the activation of mainly heterotrimeric G proteins, typical GPCRs regulate multiple functions in essentially all cells of mammalian life forms. Despite their great physiological and pharmacological relevance, the endogenous ligands that activate the mechanisms and physiological functions of more than 100 GPCRs are still unknown, and thus these receptors are called "orphan" receptors.

[0009] "G-protein coupled receptor 182 (GPR182)" was previously known as adrenomedullin receptor (ADMR) and is a member of the "class A" G-protein coupled receptor (GPCR) family. GPR182 was previously found to be expressed on endothelial cells, but its ligand and physiological role remained unknown. However, in recent studies, particularly by the present inventors (Le Mercier A et al. Proc Natl Acad Sci U S A. 2021;118(17):e2021596118), it has been revealed that GPR182 is expressed in microvascular endothelial cells and lymphatic endothelial cells of most organs, and binds to chemokine ligands CXCL10, CXCL12, and CXCL13 with nanomolar affinity. However, in contrast to conventional chemokine receptors, binding of these ligands to GPR182 was found not to trigger typical downstream signaling processes such as Gq- and Gi-mediated signaling or β-arrestin mobilization. Instead, GPR182 was identified to have relatively high constitutive activity with respect to β-arrestin mobilization and to undergo rapid internalization in a ligand-independent manner. Further investigation based on a GPR182-deficient mouse model revealed that the absence of GPR182 expression causes a marked increase in the plasma levels of its chemokine ligands CXCL10, CXCL12, and CXCL13. Based on these findings and the observed lack of typical signaling functions, GPR182 is presumed to function as a scavenging receptor that controls the plasma levels of its chemokine ligands via receptor internalization-mediated removal.

[0010] Inhibition of the GPR182-chemokine system does not rule out the potential to create new vulnerabilities for potential therapeutic development, and the inventors conducted further studies to investigate the potential relevance of GPR182 in the pathology and recovery of myocardial infarction (MI). As reported in the examples disclosed herein, the inventors evaluated the possibility of any alteration in GPR182 expression in heart tissue as a result of acute myocardial infarction (MI), and also further explored the possibility of any effects that could result from inhibition of the GPR182 / chemokine ligand system in a mouse model of acute myocardial infarction (MI). As a result of these investigations, the inventors found that mice with global or inducible endothelial-specific deficiency of GPR182 showed a marked increase in the level of chemokine CXCL12 in their infarcted hearts. Furthermore, surprisingly and advantageously, mice lacking GPR182 expression showed a marked decrease in infarct size (which is known to be directly related to MI mortality), as well as improvements in hemodynamic parameters such as increased left ventricular ejection fraction and decreased left ventricular end-diastolic and end-systolic volumes. These observations (see, for example, Example 1 and Figures 2-4) suggest a strong therapeutic potential for inhibition of GPR182-mediated scavenging function by either an agent that inhibits the expression or activity of the GPR182 protein (in particular, an agent that interferes with the binding of one or more chemokine ligands (e.g., CXCL12)), or an agent for the treatment of (acute) myocardial infarction and their post-disease complications (in particular, for improving post-acute-phase recovery, and thus for long-term prognosis), as well as for the treatment of related additional pathological conditions.

[0011] In particular, the pathological conditions that are contemplated to be prevented and / or treated by the medical use of the present invention include myocardial infarction (and any known acute-phase or post-disease complications thereof), myocardial ischemia, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, stroke, and / or cancer.

[0012] As used herein, the term "myocardial infarction (MI)", also commonly known as "heart attack", means irreversible tissue death (infarction) of the heart muscle (myocardium) caused by ischemia, an insufficient supply of oxygen to the myocardial tissue. Myocardial infarction is a type of acute coronary syndrome and represents a sudden or short-term change in symptoms related to blood flow to the heart. Unlike unstable angina, another type of acute coronary syndrome, myocardial infarction (MI) occurs when there is cell death and can be inferred by a blood test for biomarkers (e.g., the cardiac protein troponin). If evidence of MI is present, it can be classified as ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction (NSTEMI) based on the results of an electrocardiogram (ECG). MI is different from cardiac arrest but can cause it, and if the heart is not contracting at all or is contracting insufficiently to stop all vital organs from functioning, it can result in death. It is also different from heart failure, where the pumping action of the heart is impaired. However, MI can cause heart failure. The term "MI", as used herein, also means so-called "silent myocardial infarction", which can occur without any symptoms at all. These cases can be detected later using blood enzyme tests, on an electrocardiogram, or during a postmortem autopsy. Such silent myocardial infarctions account for 22 - 64% of all infarctions and are more common in the elderly with diabetes and in the elderly after heart transplantation. In the case of people with diabetes, differences in pain thresholds, autonomic neuropathy, and psychological factors have been cited as possible explanations for the lack of symptoms. In the case of heart transplantation, the donor heart is not completely dominated by the recipient's nervous system. After MI, large-scale remodeling of the extracellular matrix contributes to scar formation. This fibrotic response, while aiming to maintain tissue integrity, is also associated with an increased risk of adverse events such as heart failure, ventricular arrhythmias, and sudden cardiac death. Cardiac fibrosis is also characterized by a large deposition of collagen and increased stiffness as a result of enhanced collagen cross-linking.Known (acute and / or late) complications of MI include, but are not limited to, sudden death, arrhythmia, heart failure (such as acute and chronic heart failure), cardiogenic shock, cardiac aneurysm, ventricular rupture, rupture of the papillary muscle with acute valvular insufficiency, and mural thrombi that may embolize, myocardial ischemia, myocardial necrosis, cardiac fibrosis, cardiac hypertrophy, and heart failure.

[0013] As used herein, the term "ischemia" (or "ischaemia") means a restriction in blood supply to any tissue, muscle group, or organ of the body that causes a deficiency of oxygen required for cellular metabolism (to keep the tissue alive). Ischemia is generally caused by problems with blood vessels, resulting in tissue damage or dysfunction, i.e., hypoxemia and microvascular dysfunction. It also means local hypoxemia in a particular part of the body, usually as a result of a decrease or interruption in blood flow (e.g., due to vasoconstriction, thrombosis, or embolism). Ischemia includes not only a lack of oxygen, but also a decrease in the availability of nutrients and an inadequate removal of metabolic waste products. Ischemia can be partial (inadequate perfusion) or complete occlusion.

[0014] As used herein, the term "myocardial ischemia" or "cardiac ischemia" means an impairment of cardiac function caused by inadequate blood flow to the muscular tissue of the heart. The decrease in blood flow can be due to, for example, stenosis of the coronary arteries (coronary atherosclerosis), occlusion by a thrombus (coronary thrombosis), or, although less common, diffuse stenosis of the arterioles and other small blood vessels within the heart. Severe interruption of the blood supply to the myocardial tissue can result in necrosis of the myocardium (myocardial infarction).

[0015] As used herein, the term "myocardial necrosis" means any myocardial cell death, regardless of cause. MI is one cause of myocardial necrosis, but many other conditions also result in necrosis. The agents, compositions, and methods of the present invention can be used to promote angiogenesis and thus reduce the lack of blood and oxygen caused by ischemia. Thus, such agents, compositions, and methods are useful in reducing myocardial injury after myocardial infarction and thus in preventing or reducing the extent of myocardial necrosis.

[0016] As used herein, the term "cardiac hypertrophy" means the process by which adult myocardial cells respond to stress by undergoing hypertrophic growth. Such growth is characterized by an increase in cell size without cell division, the assembly of additional sarcomeres within the cell to maximize force generation, and the activation of the fetal heart gene program. Cardiac hypertrophy is often associated with an increased risk of morbidity and mortality. Furthermore, cardiac hypertrophy can occur after myocardial infarction as a process for compensating for damaged myocardium and maintaining cardiac function (see, e.g., Rubin SA. J Am Coll Cardiol. 1983;1(6):1435-41).

[0017] "Cardiac fibrosis" refers to the excessive extracellular matrix (ECM) deposition by cardiac fibroblasts (CF), which is a common pathophysiological process in most heart diseases such as myocardial infarction (MI), hypertensive heart disease, and various types of myocardial disorders, and can cause cardiac dysfunction that ultimately leads to heart failure. Taking MI as an example, the sudden large-scale decrease in cardiomyocytes causes intense inflammation and replaces the dead myocardium with collagen-based scar tissue, which is important for preventing cardiac rupture. However, a long-term or excessive fibrotic response can cause excessive ECM deposition, which in turn leads to myocardial sclerosis, insufficient tissue extensibility, and deterioration of cardiac dysfunction. Depending on the location and underlying cause of the cardiac scar tissue, cardiac fibrosis can be classified into various forms. Among them, reactive interstitial fibrosis and replacement fibrosis are the most relevant types in ischemic adult hearts (see, for example, Jiang W et al. Front Cardiovasc Med. 2021 Aug 16;8:715258).

[0018] As used herein, the term "limb ischemia" (LI) generally refers to a restriction in blood supply to the extremities due to factors in the blood vessels that results in tissue damage or dysfunction. Limb ischemia is also known as a sign of peripheral arterial disease characterized by intractable pain and tissue gangrene and can occur as a result of several factors such as atherosclerosis. Peripheral arterial disease, atherosclerotic peripheral vascular disease, and embolic occlusion can occur directly due to a reduction in blood perfusion to the limb tissues by limb ischemia. Pain in the ischemic area, thickening of the toenails, skin infections, and ulcers of the extremities are considered the main symptoms of LI. Interventions such as vascular surgery and endovascular surgery are commonly used as standard approaches to assist in regulating and promoting blood circulation to the ischemic limb. Despite prescribed treatments, many patients with LI must undergo major lower limb amputations (see, e.g., Khodayari S et al. Front Cell Dev Biol. 2022 May;10:834754). Therefore, the development of safe and minimally invasive effective strategies for regenerating degenerated tissues is considered the top strategy for the treatment of LI.

[0019] As used herein, the term "ischemia-related tissue degeneration" broadly means any tissue degeneration process, such as tissue necrosis resulting from ischemia / reperfusion (IR) injury (e.g., heart necrosis in MI). CXCL12 has previously been identified as a major positive regulator of tissue repair after stroke by its interaction with the G protein-coupled receptor CXCR4 (Wang Y et al., Curr Drug Targets. 2012 Feb;13(2):166-72). Since GPR182 has also been found to be expressed in brain endothelial cells, inhibition of the GPR182 / CXCL12 interaction and the resulting increase in available CXCL12 for interaction with CXCR4, as envisioned herein, would likely provide a therapeutic benefit for the treatment of stroke, particularly by promoting and / or enhancing tissue regeneration.

[0020] As used herein, the term "stroke" means the occurrence of an interfering effect in the blood supply to the brain of a mammalian subject, resulting in a rapid loss of brain function exemplified by the inability to move one or more limbs and / or the inability to perceive sensation and / or the inability to understand or construct speech and / or blindness in one or both eyes. The neurological disorder caused by the interfering effect in the blood supply to the brain can be temporary or permanent, depending on the severity of the interfering effect or how quickly medical intervention is provided.

[0021] As used herein, the term "ischemic stroke" means a stroke caused by an insufficient blood flow to the brain due to an obstruction in the vascular system supplying the brain. The obstruction can be due to one or more of thrombosis, embolism, and / or global hypoperfusion. The term "hemorrhagic stroke" as used herein means a stroke resulting from the rapid accumulation of blood within or around the brain within the skull, and the increased pressure caused by the accumulated blood impedes blood flow to and through the brain.

[0022] Furthermore, recent studies by Torphy et al. have revealed that GPR182 also functions as a scavenging receptor for chemokines CXCL9, CXCL10, and CXCL11. The latter chemokines have previously been identified as playing a decisive role in anti-tumor immunity by regulating immune cell homing into tumors through their interactions with chemokine receptors expressed on immune cells. More specifically, chemokines CXCL9-11 have been found to be produced at the tumor site and create a gradient that induces T cells into the tumor or the tumor microenvironment (TME) by attacking glycosaminoglycans (GAGs) on endothelial cells (ECs) and within the extracellular matrix. Torphy et al. found that GPR182 is upregulated in tumor-associated endothelial cells and, in certain cases, on lymphatic endothelial cells within melanoma, and limits effector T cell infiltration and associated anti-tumor immunity by scavenging chemokine ligands, particularly CXCL9-11. Furthermore, they found that removal of GPR182 reduces tumor growth by causing an increase in intratumoral chemokine levels that induces an increase in infiltration of effector CD4+ and CD8+ T cells. Therefore, the inventors proposed that removal of GPR182 sensitizes tumors to immunotherapy (Torphy RJ et al. Nat Commun. 2022 Jan 10;13(1):97). Considering their further findings, the inventors believe it is highly plausible that inhibition of GPR182 expression and / or activity as contemplated herein also provides an effective means for enhancing anti-tumor immunity, particularly in melanoma and other cancers / tumors with lymphatic infiltration. Furthermore, corresponding inhibition of GPR182 by the agents contemplated herein is also thought to provide an effective means for sensitizing tumors with insufficient immunogenicity to immune checkpoint blockade and adoptive cell therapy.

[0023] As used herein, the term "cancer" includes any malignant tumor, such as, but not limited to, carcinomas and sarcomas. Cancer results from the uncontrolled and / or abnormal division of cells, which then invade and destroy surrounding tissues. As used herein, "proliferating" and "proliferation" mean that cells undergo mitotic nuclear division. As used herein, "metastasis" means the spread of a malignant tumor to distant sites from its original location. Cancer cells can metastasize via the bloodstream, via the lymphatic system, across body cavities, or by any combination thereof. The term "cancer cell" as provided herein includes cells that suffer from any of the cancer states provided herein. The term "carcinoma" means a malignant tumor composed of epithelial cells that tend to invade surrounding tissues and tend to metastasize.

[0024] As used herein, the term "tumor microenvironment" (TME) or "tumor stroma" means the non-cancerous and non-immune cell components of a tumor and has conventionally been regarded as the structural elements that hold tumor tissue together. The tumor stroma is composed of the extracellular matrix and specialized connective tissue cells, such as fibroblasts and mesenchymal stromal cells. All tumors have stroma and require stroma for nutritional support and waste removal.

[0025] In view of the objectives contemplated herein, it is particularly preferred that the cancer comprises cells expressing the GPR182 protein (i.e., GPR182-positive cancer). In such cancers, inhibition of the expression and / or activity of the GPR182 protein is particularly effective for increasing chemokine ligand levels (especially those of CXCL9, CXCL10, CXCL11, and / or CXCL12) within the tumor and / or the intratumoral microenvironment, and thereby enhancing antitumor immunity by promoting chemokine-mediated recruitment of tumor-infiltrating immune cells (especially effector T cells) (see Torphy et al., 2022). Various cancers are known to express the GPR182 protein. For example, GPR182 has very recently been identified as being upregulated in lymphatic endothelial cells (LECs) within human melanoma.

[0026] Accordingly, in a preferred embodiment, the cancer is a cancer that (i) comprises a tumor and / or TME comprising lymphatic endothelial cells (LECs), and / or (ii) is preferably a tumor that is insufficiently immunogenic due to GPR182 expression by tumor cells and / or the intratumoral microenvironment.

[0027] In a further preferred embodiment, the cancer is a cancer with lymphatic invasion. As used herein, the term "cancer with lymphatic invasion" is also referred to herein interchangeably as "cancer with tumor-associated lymphatics" and means a cancer having lymphatic spread / metastasis (i.e., a process also known as "lymphogenous metastasis") to distal sites such as lymph nodes. Known cancers with lymphatic invasion include, but are not intended to be limited to, melanoma, colorectal cancer, and breast cancer.

[0028] In a particularly preferred embodiment, the cancer is melanoma. As used herein, the term "melanoma" generally refers to a malignant tumor of melanocytes predominantly found in the skin but also found in the intestine and eye. "Melanocytes" refer to cells located in the basal layer of the epidermis of the skin, i.e., the basement membrane, as well as the middle layer of the eye. Thus, "melanoma metastasis" means the spread of melanoma cells to regional lymph nodes and / or distant organs (e.g., liver, brain, breast, prostate, etc.).

[0029] In an alternative preferred embodiment, the cancer is lymphoma. As used herein, when the term "lymphoma" is used interchangeably with the terms "lymphadenoma" or "lymphosarcoma", it means a cancer that originates in the lymphatic system from cancerous white blood cells, distinct from "cancer with lymphatic infiltration". Known lymphomas include, for example, but are not limited to, blood and lymphatic system tumors (e.g., Hodgkin's disease, non-Hodgkin lymphoma, Burkitt lymphoma, AIDS-related lymphoma, malignant immunoproliferative diseases, multiple myeloma and malignant plasmacytoma, lymphocytic leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other specific cell type leukemias, non-specific cell type leukemias, other malignant lymphatic system tumors of unknown details, hematopoietic agents and related tissues, such as large cell lymphoma, T cell malignant lymphoma, or cutaneous T cell lymphoma, etc.). GPR182 has been found to be expressed in endothelial cells of human lymph nodes (see, for example, Schmid CD et al., Biochem Biophys Res Commun. (2018);497(1):32 - 38). Thus, inhibition of GPR182 function (e.g., by GPR182 interference) may be useful for the treatment of lymphoma, as well as for immunotherapy against lymph node metastasis or lymphoma, for example, by enhancing anti-cancer immunity and enhancing the recruitment of immune cells (e.g., effector T cells) to the affected lymph nodes.

[0030] There may exist other cancers that have not yet been investigated with regard to the presence or absence of GPR182 expression. However, means and methods for assessing the presence or absence of expression of one or more cell surface proteins (e.g., GPR182) are known and established in the art. Accordingly, one of ordinary skill in the art will be able to identify additional cancers that are likely to benefit from the medical applications disclosed herein. For example, tumor specimens (e.g., obtained from a biopsy) can be analyzed for GPR182 expression by immunofluorescence staining / imaging using an anti-GPR182 antibody that can be detected by a conjugated fluorescent label.

[0031] The nucleotide sequences of the GPR182 coding gene and mRNA, as well as the corresponding protein sequences, are known in several species such as human GPR182, mouse GPR182, and rat GPR182, and are available from the NCBI database (https: / / www.ncbi.nlm.nih.gov). For example, the nucleotide sequence of human GPR182 mRNA is available from the NCBI database (NCBI accession ID: NM_007264.4; https: / / www.ncbi.nlm.nih.gov / nuccore / NM_007264), and is defined herein by SEQ ID NO: 1. The amino acid sequence of the full-length human GPR182 protein is available from the NCBI database (NCBI accession ID: AAH34761.1; https: / / www.ncbi.nlm.nih.gov / protein / AAH34761.1), and is defined herein by SEQ ID NO: 2. Furthermore, natural genetic variants of GPR182, including common genetic polymorphisms, have also been described. One of the natural variants of GPR182 contains a single amino acid change from cysteine to arginine at position 349 of human GPR182, which has a frequency of more than 10% in the general population. Considering the non-conserved Cys349 in GPR182 among mammals (e.g., absent in mouse GPR182 (UniProt database entry G3X9R9)), it is reasonable to expect that this variant does not affect the function of GPR182. The amino acid sequence of this natural variant (Cys349Arg) of human GPR182 is defined herein by SEQ ID NO: 3. Therefore, it is understood that the term "GPR182" as referred to herein includes natural variants of GPR182, such as those defined by SEQ ID NO: 3.

[0032] As used herein, the term "naturally occurring variant" means a variant of a GPR182 protein or GPR182-encoding nucleotide sequence that occurs naturally within a defined taxonomic group, such as mammals, for example, mice, monkeys, preferably humans. Typically, when referring to a "naturally occurring variant" of a GPR182-encoding polynucleotide, the term may include any allelic variant of the GPR182-encoding genomic DNA found in the genome by chromosomal translocation or duplication, and / or RNA, such as mRNA derived therefrom. For example, a naturally occurring variant of the human GPR182 protein includes the Cys349Arg variant defined by SEQ ID NO: 3. Naturally occurring variants may also include variants derived from alternative splicing of GPR182 mRNA. When referring to a specific protein sequence, the term may include, for example, the naturally occurring form of a protein that is processed by post-translational modifications or post-translational modifications (e.g., proteolytic cleavage, glycosylation, etc.).

[0033] As used herein, the term "endogenous ligand" means any molecule that is found endogenously in a subject (e.g., a particular mammal) and is capable of interacting (in vivo) with an endogenously expressed GPR182 protein, as contemplated for the medical / therapeutic applications disclosed herein. One of ordinary skill in the art will understand that the potential ability of a ligand to bind to the GPR182 protein can be evaluated by in vitro experiments such that the detected binding can indicate binding in vivo. Generally, such ligands can include intracellular and extracellular ligands, and can be (poly)peptides, nucleic acids, lipids, saccharides (e.g., glycans and sphingoglycolipids), metal ions, other naturally occurring molecules, and / or post-translational modifications.

[0034] In a preferred embodiment, the endogenous ligand comprises or consists of a (poly)peptide (i.e., a (poly)peptide that is endogenously expressed in the intended subject), more preferably an endogenous chemokine ligand.

[0035] As used herein, "chemokine" or "chemotactic cytokine" is a family of small cytokines or signaling proteins secreted by cells that cause the directional movement of leukocytes as well as other types of cells such as endothelial and epithelial cells. In addition to playing a major role in the activation of the host immune response, chemokines are important for biological processes such as morphogenesis and wound healing, as well as in the etiology of diseases such as cancer. Cytokine proteins are classified as chemokines according to their behavioral and structural characteristics. In addition to being known to regulate chemotaxis, all chemokines have a mass of approximately 8 - 10 kilodaltons and have four cysteine residues at conserved positions important for forming their three-dimensional shape. These proteins have historically been known by several other names, such as the SIS family of cytokines, the SIG family of cytokines, the SCY family of cytokines, the four superfamilies of platelet factor, or intercrine. Some chemokines are thought to be pro-inflammatory and can be induced to mobilize immune system cells to the site of infection during an immune response, while other chemokines are thought to be constitutive and are involved in controlling cell movement during normal processes of tissue maintenance and development. Chemokines are found in all vertebrates, some viruses, and some bacteria, but not in other invertebrates. Chemokines are classified into four major subfamilies: CXC, CC, CX3C, and C (based on the position of one or more cysteines at their N-terminus). All of these proteins exert their biological effects by interacting with G protein-coupled membrane receptors called chemokine receptors, which are selectively found on the surface of their target cells (i.e., G protein-coupled receptors (GPCRs)). Chemokines are functionally divided into two groups: constitutive and inflammatory. Constitutive chemokines are constitutively produced in certain tissues and are responsible for basal leukocyte migration.These include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, and CXCL13. This classification is not strict; for example, CCL20 can also function as a pro-inflammatory chemokine. Inflammatory chemokines are formed under pathological conditions (e.g., in pro-inflammatory stimuli such as IL-1, TNFα, LPS, or virus) and are actively involved in the inflammatory response that attracts immune cells to the site of inflammation. Examples thereof are CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10.

[0036] The term "nucleotide sequence" as used herein when used interchangeably with the term "nucleic acid sequence" or "polynucleotide sequence" means deoxyribonucleic acid (DNA) such as cDNA or genomic DNA, and, where appropriate, polynucleotides such as ribonucleic acid (RNA). It is understood that the term "RNA" as used herein encompasses all forms of RNA including mRNA.

[0037] The term "protein", when used interchangeably with the term "polypeptide" in this specification, means a linear polymer of amino acid residues linked by peptide bonds in a specific sequence. The group of "polypeptides" consists of molecules having more than 30 amino acids and is distinguished from the group of "peptides" consisting of molecules having at most 30 amino acids. The group of "peptides" also means fragments of proteins having a length of less than 30 amino acids. As used herein, the term "(poly)peptide" more generally means both "peptide" and "polypeptide". (Poly)peptides can further form dimers, trimers, and higher-order oligomers, i.e., can consist of one or more (poly)peptide molecules. Such (poly)peptide molecules forming such dimers, trimers, etc. can be the same or not the same. As a result, the corresponding higher-order structures are called homodimers or heterodimers, homotrimers or heterotrimers, etc. Homodimers or heterodimers, etc. are also included in the definition of the term "(poly)peptide". The term "(poly)peptide" also means peptides and polypeptides that have been chemically modified or post-translationally modified during translation.

[0038] The term "specifically binds" (or "binds specifically" or "specifically binds to") when used interchangeably throughout this specification in relation to an agent of the invention (e.g., an antibody) means that the agent binds to the GPR182 protein, while on the other hand, other (poly)peptides and / or other molecules present within a subject (i.e., the subject for which a therapeutic application is contemplated), for example, particularly, in the interaction with the agent, do not exhibit any undesirable effects such as inducing signal transduction that is harmful to the expected effects and the therapeutic applications contemplated herein, and / or may have any other negative impact on the health of the subject that outweighs the expected therapeutic benefit, and preferably have only a negligible binding affinity or no binding affinity at all for those other (poly)peptides or other molecules. However, this term does not exclude the fact that an agent of the invention may be cross-reactive with a GPR182 homolog, ortholog, or paralog from another mammalian species. That an agent "specifically binds" to the GPR182 protein preferably means that the binding affinity between the agent and the GPR182 protein is at least (with increasing preference) 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold, or more, compared to the binding affinity between the agent and other (poly)peptides and / or other molecules present within the subject.Preferably, the other (poly)peptides referred to in this latter embodiment are selected from one or more of C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3, also known as CXCR7). Even more preferably, the agent does not bind to CXCR4 and ACKR3 (CXCR7).

[0039] In any case, one of ordinary skill in the art can evaluate an agent (i.e., a candidate agent) for its ability to specifically bind to the GPR182 protein, using conventional (e.g., biophysical) methods such as, but not limited to, those known and routinely used in the art and described herein (see, e.g., Example 2), isothermal titration calorimetry (ITC), surface plasmon resonance, and / or (competitive) binding assays to evaluate possible binding of the agent to other (poly)peptides / molecules.

[0040] The term "inhibiting binding" in the context of the agent of the present invention (i.e., of one or more endogenous ligands to the GPR182 protein) means the ability of said agent to prevent (e.g., inhibit) the binding of one or more endogenous ligands (i.e., ligands of the GPR182 protein) based on its ability to specifically bind to the GPR182 protein. For example, the agent may specifically bind to the GPR182 protein at or near the binding site of one or more endogenous ligands, and as a result, for example, by providing steric hindrance and / or electrostatic repulsive forces against the binding of one or more endogenous ligands to the GPR182 protein, make the binding site of the GPR182 protein inaccessible or thermodynamically / energetically less favorable. Thus, the agent may compete with one or more endogenous ligands for binding to the GPR182 protein. Alternatively, the agent may bind to an allosteric site of the GPR182 protein, which may be a site that is distinct and remote from the binding site of one or more endogenous ligands on the GPR182 protein, and the binding of the agent to the GPR182 protein may cause a conformational change in the GPR182 protein, which causes distortion of the binding site of one or more endogenous ligands, such that one or more endogenous ligands cannot (or are at least negatively affected by) bind to the distorted GPR182 protein accordingly.

[0041] It is generally understood by those skilled in the art, considering the objectives contemplated herein, that agents that do not bind to one or more endogenous ligands (i.e., have no binding affinity) are preferred. On the other hand, in any case, if the agent has some binding affinity for one or more endogenous ligands, it should be understood that the agent has a higher binding affinity for the GPR182 protein compared to the binding affinity for one or more endogenous ligands. "Higher binding affinity" preferably means that the binding affinity is at least (with increasing preference) 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, or 500-fold, or more, compared to the binding affinity between the agent and one or more endogenous ligands.

[0042] Furthermore, generally, agents that non-covalently bind to the GPR182 protein (i.e., reversibly bind) are preferred. However, alternatively, agents that can undergo a covalent (i.e., irreversible) interaction with the GPR182 protein under physiological conditions are also contemplated herein.

[0043] The ability of an agent to inhibit (or prevent or interfere with) the binding of one or more endogenous ligands means that the binding of the latter to the GPR182 protein is preferably inhibited (i.e., prevented and / or interfered with) by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, at least in part, with an increase in preference. Those skilled in the art will understand that (at least partial) inhibition of the binding of one or more endogenous ligands to the GPR182 protein, i.e., by adjusting the dose / concentration of the agent to shift the binding equilibrium to the complex of the agent and the GPR182 protein, can also be achieved by an agent having a weaker binding affinity for the GPR182 protein compared to the binding affinity between one or more endogenous agents and the GPR182 protein. On the other hand, compared to the binding affinity between one or more endogenous ligands and the GPR182 protein, it is generally preferred that the agent has a higher affinity for the GPR182 protein (i.e., a lower dissociation constant (K D ))). The strength of inhibition is typically measured by assessing the concentration of the maximum half-maximal inhibition (IC 50 ). The terms of inhibition and assessment of IC 50 values are well established in the art.

[0044] According to the present invention, an "agent" that specifically binds to the GPR182 protein is selected from antibodies, Fc fusion polypeptides, adnectins, affibodies, affilins, anticalins, atrimers, avimers, designed ankyrin repeat proteins (DARPins), evobodies, finomers, Kunitz-type domains, peptides or peptidomimetics, aptamers, and small molecules.

[0045] As used herein, the term "antibody" includes, for example, polyclonal or monoclonal antibodies. Further included within the term "antibody" are derivatives or fragments thereof that still maintain binding specificity for the target, in this case the GPR182 protein or a particular portion thereof. Antibody fragments or derivatives include, inter alia, Fab or Fab' fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains, such as VhH or V-NAR-domains, etc., as well as multimeric forms, such as minibodies, diabodies, tribodies, or triplebodies, tetrabody, or chemically conjugated Fab' multimers (see, e.g., Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 198; Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol. 75(13), 1584; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 1126). In particular, multimeric forms include bispecific antibodies that can bind simultaneously to two different types of antigens. For example, the first antigen can be found on the GPR182 protein and the second antigen can be, for example, a tumor marker that is specifically expressed on cancer cells or a particular type of cancer cell.Non-limiting examples of bispecific antibody formats are Biclonics (bispecific full-length human IgG antibodies), DART (Dual-affinity Re-targeting Antibody), and BiTE (composed of two single-chain variable region fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann (2015), Drug Discovery Today, 20(7):838-847).

[0046] The term "antibody" as used herein also means chimeric (human constant domain, non-human variable domain), single-chain, and humanized (human antibody except for non-human CDRs) antibodies.

[0047] A variety of techniques for the production of antibodies are well known in the art, for example, as described in Harlow and Lane (1988) and (1999), and Altshuler EP et al., Biochemistry (Mosc). 2010 Dec;75(13):1584 - 605. Thus, polyclonal antibodies can be obtained from the blood of an animal after immunization with an antigen in a mixture of an additive and an adjuvant, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line culture. Examples of such techniques are described, for example, in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, and such techniques include the hybridoma technique originally described by Kohler and Milstein in 1975, the trioma technique, the human B cell hybridoma technique (see, for example, Kozbor D, 1983, Immunology Today, vol.4, 7; Li J et al. 2006, PNAS, vol. 103(10), 3557), and the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc, 77 - 96). Furthermore, recombinant antibodies can be obtained from monoclonal antibodies or newly prepared using various display methods, such as phage, ribosome, mRNA, or cell display (see, for example, Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol. 2005 Sep;23(9):1105 - 16).Suitable systems for the expression of recombinant (humanized) antibodies can be selected, for example, from bacteria, yeast, insects, mammalian cell lines, or transgenic animals or plants (see, for example, US patent 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., vol. 23(9), 11265). Furthermore, the techniques described for the production of single-chain antibodies (see especially US patent No. 4,946,778) can be adapted to produce single-chain antibodies specific for epitopes on the GPR182 protein.

[0048] As used herein, the term "antibody" also means "nanobody (Nb)". The term "nanobody" is also known as "single domain antibody (sdAb)" and is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domain antibodies were engineered from heavy chain antibodies found in camelids; these are called VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR, "novel antigen receptor of immunoglobulin") from which single domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domain derived from common immunoglobulin G (IgG) from humans or mice into monomers. Most research on single domain antibodies is currently based on the heavy chain variable domain, but nanobodies derived from the light chain have also been found to specifically bind to target epitopes. Nanobodies can be efficiently selected from large (semi)synthetic / naive or immunized cDNA libraries using well-established display technologies such as phage display or yeast display. The simple single gene format enables the production of purified nanobodies in the mg to g range per liter of culture, thereby enabling an unlimited supply of defined binding molecules. Furthermore, nanobodies can be easily genetically or chemically manipulated. Nanobodies are characterized by high affinity and specificity, a robust structure, e.g., stable soluble behavior in a hydrophilic environment and excellent accessibility to potential clefts, low off-target accumulation, and deep tissue penetration. To date, many nanobodies have been developed for research in various fields, and the list of diagnostic tools and therapeutic nanobodies applied in clinical trials is constantly growing. Nanobody-derived formats include the nanobody itself, homo- or heteromultimers, nanobody-coated nanoparticles or matrices, nanobody-displaying bacteriophages, or nanobodies labeled with enzymes, fluorophores, or radionuclides.All of these formats have been successfully applied to basic biomedical research, cell and molecular imaging, diagnostics, or targeted drug delivery and therapy (see, for example, Chames P, Rothbauer U. Special Issue: Nanobody. Antibodies (Basel). 2020 Mar 6;9(1):6). Methods for the generation and target-specific selection of nanobodies are well established in the art and can be found, for example, in Salema V et al., Escherichia coli surface display for the selection of nanobodies. Microb Biotechnol. 2017 Nov;10(6):1468-1484. Embodiments in which the agent that specifically binds to the GPR182 protein is a "nanobody" are particularly preferred.

[0049] According to the present invention, the "agent" can be an "Fc fusion polypeptide". The latter term means a fusion construct of a binding domain with an antibody Fc region (e.g., a single-chain antibody or an antigen-binding protein of any alternative scaffold as mentioned herein).

[0050] Furthermore, the term "agent", as used herein, also encompasses antibody mimetics, i.e., molecules that can specifically bind to an antigen, such as in this case the GPR182 protein, in a manner similar to an antibody but are not structurally related to an antibody, i.e., antigen-binding molecules derived from alternative structural scaffolds. A number of such alternative (e.g., non-antibody) binding protein scaffolds have been developed in just the last few decades (e.g., as reviewed in Hosse RJ et al. Protein Sci. 2006;15(1):14-27; Weidle UH et al., (2013), Cancer Genomics & Proteomics; 10(4):155-68), and it is contemplated that any of these can be suitably used as an agent for the purposes of the present invention.

[0051] Exemplary antibody mimetics particularly contemplated herein include, but are not intended to be limited to, adnectins, affibodies, affilins, anticalins, atrimers, avimers, designed ankyrin repeat proteins (DARPins), evobodies, finomers, and Kunitz-type domains.

[0052] As used herein, "adnectin" means a class of binding proteins having a scaffold consisting of the backbone of the native amino acid sequence of the tenth domain of 15 repeat units of human fibronectin type III (FN3). The molecule has a β-sandwich fold with seven strands that are connected by six loops similar to immunoglobulin domains but do not have disulfide bonds. Three loops at one end of the β-sandwich can be engineered to allow the adnectin to specifically recognize a target therapeutic target. It has also been found that non-loop residues can expand the available binding footprint. Ligand-binding adnectin variants with binding affinities in the nanomolar to picomolar range have been selected via mRNA, phage, and yeast display (Hackel BJ et al. (2008) J Mol Biol 381:1238-1252).

[0053] As used herein, "affibody" means a class of binding proteins derived from the Z domain of staphylococcal protein A. Affibodies are structurally based on a domain of three-helix bundles. Affibodies have a molecular mass of approximately 6 kDa and are stable under high temperature and acidic or alkaline conditions. Target specificity is obtained by randomization of the amino acids located in two α-helices involved in the binding activity of the parent protein domain (Feldwisch, J & Tolmachev, V. (2012) Methods Mol. Biol. 899:103-126). Methods for generating affibodies are known in the art and are described in Wikman M et al., Protein Eng Des Sel. (2004);17(5):455-62.

[0054] As used herein, "affilin" refers to a class of binding proteins developed by using either γ-B crystallin or ubiquitin as a scaffold and modifying the amino acids on the surface of these proteins by random mutagenesis. The selection of an affilin with the desired target specificity, i.e., specificity for GPR182, is achieved, for example, by phage display or ribosome display technology. Depending on the scaffold, an affilin has a molecular weight of about 10-20 kDa. As used herein, the term affilin also means the dimeric or multimeric form of an affilin (Weidle UH et al., (2013), Cancer Genomics & Proteomics; 10(4):155-68).

[0055] As used herein, "antikarin" refers to a class of engineered proteins derived from lipocalin. Antikarins have eight twisted β-barrels that form a highly conserved core unit among lipocalins and naturally form a binding site for a ligand by four structurally variable loops at the open ends. Antikarins are not homologous to the IgG superfamily but show features that were considered to be typical for the binding site of an antibody to some extent: (i) high structural plasticity as a result of sequence variation, and (ii) high conformational flexibility that allows induced fit to targets with different shapes (see, for example, Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci U S A. 96(5):1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255); Rothe C & Skerra A., BioDrugs. (2018);32(3):233-243; Gebauer M & Skerra A, Curr Opin Biotechnol. (2019); 60:230-241).

[0056] As used herein, "atrimer" means a binding molecule in which the basic scaffold is derived from the protein tetranectin. Tetranectin is a member of the C-type lectin family characterized by a C-type lectin domain (CTLD). It is composed of three identical chains with a C-terminal trimerization coiled-coil region. The formation of the homotrimer causes an apparent 100-fold increase in affinity for cognate ligands, probably due to an avidity effect based on the trimeric clustering of the CTLD. Each CTLD has five loop regions 6-9 amino acids in length that mediate binding specificity. The sequences of these loops can be varied without disrupting the overall structure. Thus, monomeric CTLDs can be displayed on phage libraries for the selection of binders to specific targets, while the trimeric version has been used for the latter application. An atrimer having a molecular weight in the range of 60-70 kDa is much smaller than an antibody, is not glycosylated, and can confer better tissue penetration compared to an antibody (Weidle UH et al., (2013), Cancer Genomics & Proteomics; 10(4):155-68).

[0057] "Abimer" (short for avidity multimer) is, as used herein, a class of artificial multi - domain proteins that specifically bind to a particular antigen by multiple binding sites. This protein is also known as "maxibody" or low - density lipoprotein receptor (LDLR) domain A. It consists of two or more (poly)peptide sequences based on the A domain. The A domain is a 30 - 35 amino acid scaffold (about 4 kDa) derived from an extracellular cysteine - rich cell - surface receptor protein and is stabilized by disulfide - bond formation and calcium - ion complex formation. The scaffold structure is maintained by 12 conserved amino acids, leaving all remaining non - conserved residues randomized and suitable for ligand binding. Abimers are very thermostable. Due to their small size, abimers often consist of multiple A domains that each bind to different sites on the target, resulting in an increase in affinity due to avidity (Silverman J et al. (2005), Nat Biotechnol 23:1556 - 1561).

[0058] "DARPin", as used herein, is a designed ankyrin repeat domain based on tightly packed ankyrin repeats that each form a β - sheet and two antiparallel α - helices. DARPin typically carries three repeat sequences corresponding to an artificial consensus sequence, whereby a single repeat typically consists of 33 amino acids, six of which form the binding surface. These sites are used to introduce random amino - acid codons during the design of recombinant libraries. DARPin is typically formed by two or three binding motifs contained between an N - terminal motif and a C - terminal motif that block hydrophobic regions. DARPin is a small protein (about 14 - 18 kDa) that is very thermostable and resistant to proteases and denaturants (Pluckthun A., Annu Rev Pharmacol Toxicol. (2015); 55:489 - 511).

[0059] As used herein, "evibody" is an engineered binding protein derived from the variable (V) set Ig-like scaffold of the cytotoxic T lymphocyte antigen 4 (CTLA-4), a T cell surface receptor. The loops corresponding to the CDRs of the antibody can be substituted with heterologous sequences to confer different binding properties. Methods for making evibodies are known in the art and are described, for example, in U.S. Patent No. 7,166,697.

[0060] The term "finomer" (or "Fynomer®") as used herein means a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Polypeptides derived from Fyn SH3 are well known in the art and are described, for example, in Grabulovski et al. (2007) JBC, 282, pp. 3196-3204, International Publication No. 2008 / 022759, Bertschinger et al. (2007) Protein Eng Des Sel 20(2):57-68, Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255, or Schlatter et al. (2012), MAbs 4:4, 1-12.

[0061] The term "Kunitz-type domain" or "Kunitz domain-based binder" means a class of binding proteins derived from the active motif of Kunitz-type protease inhibitors. The latter proteins contain loop regions that can be mutated without destabilizing the structural backbone. The hydrophobic core of the Kunitz domain consists of a twisted antiparallel β-sheet stabilized by three sets of disulfide bonds and two α-helices (Hosse RJ et al. (2006). Protein Sci 15:14-27; Simeon R. & Chen Z. Protein Cell. (2018);9(1):3-14).

[0062] Means and methods for the development, screening, and identification of suitable antigen-binding molecules (e.g., (poly)peptides of various scaffolds, such as, but not limited to, those described above in the present specification) against a desired target structure (e.g., GPR182 protein or certain portions thereof) are known and routinely employed in the art. Today, examples of routinely practiced methods include, but are not limited to, high-throughput (HT) combinatorial library-based display and selection methods, such as phage display, ribosome display, mRNA display, and cell surface display (e.g., yeast display); see, for example, Davydova et al. Biochemistry Moscow 87, S146-S167 (2022); Frei JC, Lai JR. Protein and Antibody Engineering by Phage Display. Methods Enzymol. 2016;580:45-87.

[0063] According to the present invention, an "agent" that specifically binds to the GPR182 protein can also be a "peptidomimetic". As used herein, the term "peptidomimetic" means a molecule that contains non-peptidic structural elements and can mimic or antagonize the biological action of a natural peptide. Thus, as used herein, the term "peptidomimetic" can mean any sequence that is designed to mimic peptide structure and / or function but whose backbone is not based solely on α-amino acid residues. To discover potent peptidomimetic-based drug leads that exhibit high cell selectivity and improved bioavailability, numerous strategies have been explored to date (ranging from the incorporation of a single unnatural amino acid residue into an alternative backbone structure). Peptidomimetics can include, for example, N-alkylated glycine (peptoid), β-peptoid (N-alkylated β-alanine oligomer), β-peptide, α-peptide / β-peptoid, α / γ N-acylated N-aminoethyl peptide (AApeptide), and oligoacyl lysine (OAK). The advantage of such peptidomimetics is that they allow for the facile preparation of various analogs by solid-phase synthesis and by the simple substitution / insertion of alternative residues with commercially available and / or synthetic amino acid analogs. Furthermore, these peptide analogs often have enhanced in vivo stability (see, for example, Vagner J, Qu H, Hruby VJ. Peptidomimetics, a synthetic tool of drug discovery. Curr Opin Chem Biol. 2008;12(3):292-296).

[0064] According to the present invention, a "drug" that specifically binds to the GPR182 protein can also be an "aptamer". An "aptamer" is a nucleic acid molecule or peptide molecule that can bind to a specific target with high affinity and specificity. Aptamers are usually created by selecting them from large random sequence pools, although natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecules. These compound molecules have further research, industrial, and clinical applications (Osborne et al. (1997), Current Opinion in Chemical Biology, 1:5-9; Stull & Szoka (1995), Pharmaceutical Research, 12, 4:465-483).

[0065] Nucleic acid aptamers are nucleic acid species usually consisting of (usually short) strands of oligonucleotides (e.g., DNA or RNA 15 - 100 nt in length). Aptamers against almost any molecular target (e.g., small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, etc.) can be obtained using an in vitro evolutionary approach called SELEX (systematic evolution of ligands by exponential enrichment) (see, for example, Ellington A.D., Szostak J.W. Nature. 1990; 346:818 - 822; Santosh B, Yadava PK. Nucleic acid aptamers: research tools in disease diagnostics and therapeutics. Biomed Res Int. 2014;2014:540451). SOMAmer (slow off - rate modified aptamer) is a DNA aptamer in which the nucleobases are modified with several hydrophobic moieties. These modifications expand the range of aptamer - target interactions with the hydrophobic moieties and are not present in conventional DNA and RNA aptamers. This results in higher affinity of SOMAmer for their targets (see, for example, Elskens et al., Int. J. Mol. Sci. 2020; 21:4522).

[0066] Peptide aptamers are peptides or proteins that are usually designed to interfere with other protein interactions within cells. They consist of variable peptide loops that bind to a protein scaffold at both ends. This dual structural constraint greatly enhances the binding affinity of peptide aptamers to levels comparable to antibodies (nanomolar range). The variable peptide loops typically contain 10 - 20 amino acids, and the scaffold can be any protein with good solubility properties. Currently, the bacterial protein thioredoxin-A (TrxA) is the most commonly used scaffold protein. TrxA has an active site composed of a Cys-Gly-Pro-Cys stretch that can accept long peptide insertions despite the accompanying loss of enzymatic function. The selection of peptide aptamers can be generated using various systems, but the most widely used is currently the yeast two-hybrid system (see Reverdatto S et al Peptide aptamers: development and applications. Curr Top Med Chem. 2015;15(12):1082 - 101).

[0067] Since aptamers provide molecular recognition properties comparable to those of commonly used biomolecules, especially antibodies, they offer utility for biotechnological and therapeutic applications. In addition to their discriminatory recognition, aptamers can be designed entirely in vitro, are readily produced by chemical synthesis, have desirable storage properties, and offer advantages over antibodies such as causing little or no immunogenicity in therapeutic applications. Unmodified aptamers rapidly disappear from the bloodstream with a half-life of minutes to hours, mainly due to nuclease degradation by the kidneys and removal from the body as a result of the aptamer's originally low molecular weight. The uses of unmodified aptamers currently focus on the treatment of transient conditions such as blood clotting or the treatment of organs such as the eye where local delivery is possible. This rapid disappearance can be advantageous in applications such as in vivo diagnostic imaging. Some modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) conjugation, fusion to albumin or other proteins with extended half-lives, etc., can extend the half-life of the aptamer from days to even weeks, which is advantageous for scientists.

[0068] As used herein, the term "small molecule" preferably refers to an organic molecule. An organic molecule relates to or belongs to a class of chemical compounds having a carbon group in which carbon atoms are linked together by carbon-carbon bonds. The original definition of the term "organic" relates to the source of the chemical compound, where an organic compound is a carbon-containing compound obtained from a plant, animal, or microbial source, while an inorganic compound is obtained from a mineral source. Organic compounds can be natural compounds or synthetic compounds. Organic molecules are preferably aromatic molecules, more preferably heteroaromatic ring molecules. In organic chemistry, the term "aromaticity" is used to describe cyclic (ring-shaped), planar (flat) molecules having a ring of resonance bonds that exhibit higher stability than other geometric or bonding arrangements of the same set of atoms. Aromatic molecules are very stable and do not readily break apart upon reaction with other substances. In a heteroaromatic ring molecule, at least one of the atoms of the aromatic ring is an atom other than carbon, such as N, S, or O. In all of the organic molecules described above, the molecular weight preferably ranges from 200 Da to 1500 Da, more preferably from 300 Da to 1000 Da. Alternatively, the "small molecule" according to the present invention can be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds that do not have carbon atoms (except for carbon dioxide, carbon monoxide, and carbonic acid). Preferably, the small molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da, such as less than about 500 Da, and even more preferably less than about 250 Da. The size of the small molecule can be determined by methods well known in the art, such as spectroscopy.Small molecules can be rationally designed based on, for example, a prerequisite structural model (for the structural model of human GPR182, see https: / / alphafold.ebi.ac.uk / entry / O15218), or structural information obtained from a high-resolution structure of a target molecule identified experimentally (e.g., by X-ray crystallography, NMR spectroscopy, or Cryo-EM). In this case, sites that presumably carry biological activity, such as ligand-binding sites on receptors, can be identified and verified in in vivo assays such as in vivo high-throughput screening (HTS) assays.

[0069] According to the present invention, the "agent" that inhibits the expression of GPR182 protein is selected from siRNA, miRNA, shRNA, ribozyme, and / or antisense nucleic acid molecules.

[0070] As used herein, the term "small interfering RNA (siRNA)" is also known as "short interfering RNA" or "silencing RNA" and refers to a class of double-stranded RNA molecules 18 - 30, preferably 19 - 25, most preferably 21 - 23, or even more preferably desirably 21 nucleotides in length that play various roles in biology. Notably, siRNA is involved in the RNA interference (RNAi) pathway by which siRNA inhibits the expression of a specific gene. In addition to their role in the RNAi pathway, siRNA also functions in RNAi-related pathways, for example, as an antiviral mechanism or in the formation of the chromatin structure of the genome. Naturally occurring siRNA in nature has a well-defined structure: a short double-stranded (dsRNA) of RNA with 2-nt 3' overhangs at either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group.

[0071] This structure is the result of processing by Dicer, an enzyme that converts either long double-stranded RNA or small hairpin-shaped RNA into siRNAs. siRNAs can also be introduced exogenously (artificially) into cells to cause specific knockdown of a target gene. Thus, essentially, any gene with a known sequence can be targeted based on sequence complementarity with an appropriately matched siRNA. Double-stranded RNA molecules or their metabolic processing products can mediate target-specific nucleic acid modifications, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNAs can be derived from overhangs at their 3' and 5' ends, although it is preferred that at least one RNA strand has a 5' and / or 3' overhang. Preferably, one end of the double strand has a 3' overhang of 1 to 5 nucleotides, more preferably 1 to 3 nucleotides, and most preferably 2 nucleotides. The other end can be a blunt end or have a 3' overhang up to 6 nucleotides in length. Generally, any RNA molecule suitable for functioning as an siRNA is contemplated in the present invention. The most efficient silencing has so far been obtained with siRNA duplexes composed of a 21-nt sense strand and a 21-nt antisense strand paired to have a 2-nt 3' overhang. The sequence of the 2-nt 3' overhang contributes slightly to the specificity of target recognition restricted to unpaired nucleotides adjacent to the first base pair (Elbashir et al. Nature. 2001;411(6836):494-8). 2'-Deoxynucleotides in the 3' overhang are as efficient as ribonucleotides, but are often less expensive to synthesize and are perhaps more nuclease-resistant. Methods (e.g., siRNA design software) for designing and preparing siRNAs suitable for efficiently knocking down the expression of a selected target gene have been established and can be routinely used by those skilled in the art (see, e.g., Naito Y et al. Front Genet. 2012; and the review by Fakhr E et al., Cancer Gene Ther. 2016;23(4):73-82).Furthermore, various companies (e.g., ThermoFisher, https: / / www.thermofisher.com) offer online design tools and manufacturing services for siRNA. Delivery of siRNA can be achieved using any of the methods known in the art. For example, two of the most popular methods for delivering siRNA therapies are liposome-based systems and nanoparticle-based systems. siRNA can be combined with saline, and the combination can be administered intravenously or intranasally, or by formulating the siRNA in glucose (e.g., 5% glucose, etc.), or cationic lipids and polymers can be used for in vivo siRNA delivery via either an intravenous (IV) or intraperitoneal (IP) systemic route (Fougerolles et al. (2008), Current Opinion in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular Biology, vol. 437: Drug Delivery Systems - Chapter 3: Delivering Small Interfering RNA for Novel Therapeutics; Tatiparti K et al. siRNA Delivery Strategies: A Comprehensive Review of Recent Developments. Nanomaterials (Basel). 2017;7(4):77; Paunovska, K et al., Drug delivery systems for RNA therapeutics. Nat Rev Genet 23, 265-280 (2022)).

[0072] "Small hairpin RNA (shRNA)" is a sequence of RNA that forms a sharp hairpin curve and can be used to stop gene expression by RNA interference. shRNA typically uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is constantly expressed. This vector is usually inherited to daughter cells, enabling the inhibition of gene silencing. The shRNA hairpin structure is cleaved by cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves the mRNA that matches the bound siRNA. The si / shRNA used in the present invention is preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers.

[0073] Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). Most conveniently, siRNA or shRNA can be obtained from commercial RNA oligo synthesis suppliers that sell RNA synthesis products of different qualities and costs. Generally, the RNA applicable in the present invention is customarily synthesized and readily available in a quality suitable for RNAi.

[0074] Further molecules that act on RNAi include, for example, "microRNA (miRNA)". The said RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to complementary mRNA transcripts and causing the degradation of the said mRNA transcripts by a process similar to RNA interference. Therefore, exogenous miRNA can be used as an inhibitor of GPR182 expression after introduction into each cell.

[0075] A "ribozyme" (derived from ribonucleic acid enzymes and also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes chemical reactions. Many natural ribozymes catalyze either their own cleavage or that of other RNAs, although they have also been shown to catalyze the amino transferase activity of ribosomes. Non-limiting examples of well-characterized small self-cleaving RNAs are the hammerhead hairpin hepatitis D virus and in vitro selected lead-dependent ribozymes, while type I introns are examples of larger ribozymes. The principle of catalyzed self-cleavage has been well established in recent years. The hammerhead ribozyme is the best characterized among RNA molecules with ribozyme activity. Since the hammerhead structure has been shown to be able to integrate into non-homologous RNA sequences and thereby transfer ribozyme activity to these molecules, it is thought that a catalytic antisense sequence can be created for almost any target sequence as long as the target sequence has potential identity to the cleavage site. The basic principle for constructing a hammerhead ribozyme is as follows: A region of interest in the RNA containing the GUC (or CUC) triplet is selected. A catalytic hammerhead sequence is inserted between two oligonucleotide strands, usually each 6 - 8 nucleotides long. Usually, the best results are obtained with small ribozymes and target sequences.

[0076] A more recent development that is also useful according to the present invention is a combination of aptamers that recognize small molecules by a hammerhead ribozyme. The conformational change induced in the aptamer upon binding to the target molecule can modulate the catalytic function of the ribozyme.

[0077] The term "antisense oligonucleotide (ASO)", as used herein, refers to a single-stranded DNA or RNA molecule that is complementary to a selected target nucleic acid sequence. Antisense nucleic acid molecules can interact with target nucleic acid molecules according to the present invention, and more particularly, they can hybridize with target nucleic acids. By forming hybrids, transcription of the target gene and / or translation of the target mRNA are reduced or inhibited. For example, antisense RNA is a single-stranded RNA molecule that is complementary to the protein-coding mRNA (mRNA) to which it hybridizes, thereby being able to inhibit translation into protein. Standard methods related to antisense technology have been described (see, for example, Melani et al., Cancer Res. (1991) 51:2897-2901).

[0078] Furthermore, in an alternative preferred embodiment, the "agent" that inhibits the expression of the GPR182 protein can be a nucleotide-based inhibitor, and the nucleotide-based inhibitor is (a) a polynucleotide sequence comprising or consisting of a polynucleotide sequence that is complementary to at least 12 consecutive nucleotides of the nucleic acid sequence encoding the GPR182 protein (preferably SEQ ID NO: 1); (b) a polynucleotide sequence comprising or consisting of a nucleic acid sequence that is at least 70% identical to the complementary strand of one or more nucleic acid sequences encoding the GPR182 protein (preferably SEQ ID NO: 1); (c) a polynucleotide sequence comprising or consisting of a polynucleotide sequence according to (a) or (b), which is a polynucleotide sequence that is DNA or RNA; (d) an expression vector that expresses a polynucleotide sequence as defined in any one of (a) to (c) preferably under the control of a tissue-specific promoter (preferably a heart tissue-specific promoter if the target medical condition is a heart condition (e.g., MI)); or (e) a host containing the expression vector of (d) comprises or consists of.

[0079] A "promoter" is a nucleic acid sequence that initiates transcription of a particular gene or polynucleotide (e.g., a nucleotide-based inhibitor as referred to in the above embodiments). The promoter can be a constitutively active promoter, a tissue-specific or development-stage-specific promoter, an inducible promoter, or a synthetic promoter. A constitutively active promoter directs expression in almost all tissues and is almost independent of environmental and developmental factors, although not completely. Since the expression of a constitutively active promoter is usually not affected by endogenous factors, constitutively active promoters are usually active across species and even across fields. Tissue-specific or development-stage-specific promoters direct the expression of a gene in a particular tissue or at a particular stage of development. The activity of an inducible promoter is induced by the presence or absence of a biotic or abiotic factor. Inducible promoters are very powerful tools in genetic engineering because they can turn the expression of a gene operably linked to them on or off as needed. Synthetic promoters are constructed by combining the major elements of promoter regions from different origins. In embodiments where the target medical condition is heart disease (e.g., MI), the expression vector preferably comprises a heart-specific promoter. Heart-specific promoters are known in the art, for example, by Boecker et al. (2004), Mol Imagin.; 3(2):69-75. The use of a heart-specific promoter ensures that the nucleic acid sequence is expressed only in the heart and can avoid unwanted potential side effects due to expression in other organs.

[0080] According to the present invention, the term "percent (%) sequence identity" describes the number of identical nucleotide / amino acid matches ("hits") of two or more aligned nucleic acid or amino acid sequences when compared to the number of nucleotides or amino acid residues that make up the entire length of the template nucleic acid or amino acid sequence. In other words, when a (partial) sequence is compared and aligned with maximum identity in a comparison window or specified region measured using sequence comparison algorithms known in the art, or when aligned manually and visually confirmed, the percentage of amino acid residues or nucleotides that are identical (e.g., 70%, 75%, 80%, 85%, 90%, or 95% identity) can be determined for two or more sequences or subsequences. This definition also applies to the complement of any sequence being aligned.

[0081] Nucleotide and amino acid sequence analysis and alignment related to the present invention are preferably performed using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389 - 3402). BLAST can be used for nucleotide sequences (Nucleotide BLAST) and amino acid sequences (Protein BLAST). Those skilled in the art know additional suitable programs for aligning nucleic acid sequences.

[0082] As defined herein, at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity is contemplated by the present invention. However, with increasing preference, at least 97.5%, at least 98.5%, at least 99%, at least 99.5%, at least 99.8%, and 100% sequence identity are also contemplated by the present invention.

[0083] It will be understood that the medical uses and applications disclosed herein are useful in the fields of human and veterinary medicine. Thus, as used herein, the term "subject" or "patient" means, for example, but not limited to, humans and other primates (e.g., chimpanzees and other anthropoid and ape species), farm animals (e.g., cows, sheep, pigs, goats, and horses), domesticated mammalian pets (e.g., dogs and cats), laboratory animals (e.g., rodents, e.g., mice, rats, rabbits, guinea pigs, and hamsters), and birds (e.g., domesticated birds, wild birds, and game birds, e.g., chickens, turkeys and other gallinaceous birds, ducks, geese, etc.), and any vertebrate animal such as these. In a preferred embodiment, the subject is a mammal. In an even more preferred embodiment, the subject is a human. Preferably, the term "subject" means a mammal that endogenously expresses GPR182, while on the other hand, such a mammal that heterologously expresses the GPR182 gene / protein derived from another mammal, e.g., a mouse expressing the human GPR182 protein as used in the examples disclosed herein, is also intended to be encompassed by the term subject.

[0084] It has been found by the inventors that GPR182 undergoes constitutive internalization (endocytosis) in a ligand-independent manner and that this causes local removal of its endogenous chemokine ligand from the plasma at the site of GPR182 expression. Thus, GPR182 is thought to function as a scavenging receptor that acts in the control of the plasma and / or tissue levels of its endogenous chemokine ligand. Given the further finding that lack of GPR182 functionality causes an increase in the plasma availability of its endogenous chemokine ligand (particularly CXCL12) and the observed beneficial effects thereby caused, inhibition of the binding of one or more endogenous ligands affected by a drug to prevent GPR182-mediated internalization of one or more endogenous ligands is particularly contemplated herein. Thus, in a preferred embodiment, binding of a drug to the GPR182 protein inhibits GPR182-mediated internalization of one or more endogenous GPR182-ligands. Further, considering the possibility that the GPR182 protein can return to the cell surface after internalization and as a result its scavenging ability can be restored, it is even more preferred that binding of a drug to the GPR182 protein prevents internalization of GPR182. Means and methods for assessing the potential inhibition of receptor-mediated ligand internalization are known in the art and are also described, for example, in Le Mercier et al 2021.

[0085] Furthermore, in an alternative preferred embodiment, the agent promotes internalization and lysosome-dependent degradation of the GPR182 protein by directing the GPR182 protein to the endosome-lysosome degradation pathway upon binding to the GPR182 protein. In a further alternative preferred embodiment, the agent promotes internalization and proteasome-dependent degradation of the GPR182 protein by directing the GPR182 protein to the ubiquitin proteasome system (UPS) upon binding to the GPR182 protein. The feasibility of such an approach to cause targeted proteolysis in GPCRs has been demonstrated in recent studies by Li et al. using proteolysis-inducing chimeric molecules (PROTACs) (demonstrated in the case of the α1A-adrenergic receptor); see, for example, Li, Z. Z et al., Acta Pharm. Sin. B. 10, 1669-1679 (2020); and review by He M et al., Signal Transduct Target Ther. 2022 Jun 9;7(1):181.

[0086] As described above herein, GPR182 has been found to act as a receptor for various CXC-type chemokines (see Le Mercier et al., 2021; Torphy et al., 2022). Thus, in a preferred embodiment, the one or more endogenous ligands are one or more C-X-C chemokine ligands. For example, the one or more endogenous ligands can be selected from C-X-C motif chemokine ligand 9 (CXCL9), CXCL10, CXCL11, CXCL12, and / or CXCL13.

[0087] In an even more preferred embodiment, the one or more endogenous ligands are selected from one or more of C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 12 (CXCL12), and / or C-X-C motif chemokine ligand 13 (CXCL13). In an even more preferred embodiment, the one or more endogenous ligands are (i) CXCL12; or (ii) CXCL10 and CXCL12; or (iii) CXCL12 and CXCL13; or (iv) CXCL10 and CXCL13; or (v) CXCL10, CXCL12, and CXCL13. The latter two embodiments are particularly preferred in embodiments where the target pathological condition is myocardial infarction (MI) (or any post-disease complication thereof), myocardial ischemia, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, and / or any other cardiovascular condition.

[0088] In other embodiments, the one or more endogenous ligands are selected from one or more of C-X-C motif CXCL9, CXCL10, and / or CXCL11. In preferred embodiments of the latter embodiment, the one or more endogenous ligands are (i) CXCL9; or (ii) CXCL10; or (iii) CXCL11; or (iv) CXCL9 and CXCL10; or (v) CXCL9 and CXCL11; or (vi) CXCL10 and CXCL11; or (vii) CXCL9, CXCL10, and CXCL11. The latter two embodiments are particularly preferred in embodiments where the target pathological condition is cancer.

[0089] Structure prediction suggests that the GPR182 protein has a typical GPCR topology with an extracellular N-terminus, seven transmembrane helices, and an intracellular C-terminus (see, for example, the corresponding prediction provided in the UniProt database accession no.: O15218 for human GPR182; https: / / www.uniprot.org / uniprot / O15218).

[0090] In a preferred embodiment, an agent that specifically binds to the GPR182 protein specifically binds to one or more epitopes within one or more of the extracellular portions of the GPR182 protein.

[0091] According to the structural prediction, the extracellular portion of the human GPR182 protein consists of amino acids 1-57, 114-127, 194-217, and 281-299 of the amino acid sequence defined by SEQ ID NO: 2 (see UniProt entry: O15218; https: / / www.uniprot.org / uniprot / O15218).

[0092] When the terms "epitope" or "binding site" are used interchangeably herein, they relate to a particular binding member, i.e., a macromolecule, in this case a part of the GPR182 protein, to which an agent that specifically binds to the GPR182 protein as referred to herein can bind. An epitope generally consists of chemically active surface groups and has specific three-dimensional structural characteristics as well as specific charge characteristics. The epitopes referred to herein are "functional epitopes", which is understood to mean that the binding of an agent to the epitope affects the function of GPR182. In particular, the binding of an agent to an epitope typically antagonizes the biological activity of the GPR182 protein to interact with one or more of its endogenous ligands. Thus, an epitope is known as an "inhibiting" epitope or an "inhibitory" epitope. An "inhibiting" or "inhibitory" epitope means an epitope present on the GPR182 protein that, when bound by an agent, results in the loss or decrease of the biological activity of GPR182.

[0093] An epitope can be an adjacent or non - adjacent sequence of amino acid residues contained within a polypeptide sequence. The term "contiguous epitope" defines an epitope composed of a linear series of amino acid residues within the polypeptide that defines the epitope. A "non - contiguous epitope" can also be referred to as a conformational epitope or a discontinuous epitope and is an epitope composed of a series of amino acid residues that are not linearly aligned, i.e., the residues are spaced along the length of the polypeptide sequence or grouped discontinuously. A discontinuous epitope can be a discontinuous epitope in which the amino acid residues are grouped into two linear sequences, or alternatively, a discontinuous epitope can be a discontinuous scattered epitope in which the residues contributing to the epitope are provided by three or more groups of linear amino acid sequences arranged along the length of the polypeptide.

[0094] In a particularly preferred embodiment, one or more epitopes to which the agent specifically binds are contained in (i) the N - terminal extracellular domain; and / or (ii) the second extracellular loop (ECL2) of the GPR182 protein.

[0095] As described above herein, according to the prediction provided in the UniProt database (see UniProt accession no.: O15218; https: / / www.uniprot.org / uniprot / O15218), the N - terminal extracellular domain of human GPR182 (defined by SEQ ID NO: 2) corresponds to amino acids 1 - 57 of the amino acid sequence of SEQ ID NO: 2. The second extracellular loop (ECL2) of human GPR182 defined by SEQ ID NO: 2 corresponds to amino acids 194 - 217 of the amino acid sequence defined by SEQ ID NO: 2.

[0096] One of ordinary skill in the art could identify the amino acid sequences corresponding to the N-terminal extracellular domain and ECL2 of homologs of human GPR182 derived from other species, such as mouse and rat GPR182, and of further native GPR182 variants, by using methods known in the art, such as topology and / or structure prediction tools available in the art. For example, structural models of human, mouse, and rat GPR182 proteins are searchable from the AlphaFold database (https: / / alphafold.ebi.ac.uk / search / text / GPR182).

[0097] In view of the objects contemplated herein, it is particularly preferred that the agents of the invention specifically interact with the GPR182 protein (and thereby inhibit the binding of one or more of its endogenous ligands to GPR182), while on the other hand not significantly binding to other receptors for those ligands (such as CXCR4 and / or ACKR3 (CXCR7)), and provide a signal transduction function that is known to be responsible for (or at least contribute to) any of the therapeutically beneficial effects envisioned herein during ligand engagement, without specifically binding to receptors that provide the signal transduction function contemplated herein for the local increase in chemokine ligand levels to inhibit the scavenging function of GPR182 and thereby make them available for interaction with their signal transduction receptors (such as CXCR4 and / or ACKR3 (CXCR7)).

[0098] In this regard, it should be noted that according to the latest model of the CXCL12-CXCR4 interaction by Stephens et al., the following epitopes of CXCR4: CRS0.5, CRS1, CRS1.5, and CRS2 of the chemokine recognition site (CRS) have been identified to be involved in CXCL12 binding. CRS0.5, CRS1, and CRS1.5 are all contained in the N-terminal extracellular domain of CXCR4, while CRS2 is part of the transmembrane domain pocket and ECL2 (see Stephens BS et al., Sci Signal. 2020;13(640):eaay5024). Gustavsson M. et al. investigated the structural basis of the ligand interaction with ACKR3 (also known as C-X-C chemokine receptor type 7 (CXCR7) or G protein-coupled receptor 159 (GPR159)), and similarly predicted the involvement of the N-terminal extracellular domain and the CRS2 interaction site of ACKR3 in ligand binding (such as CXCL12) (see Gustavsson M. et al., Nat Commun. 2017;8:14135).

[0099] The N-terminal extracellular domain and ECL2 of GPR182 are significantly different from the N-terminal extracellular domain and ECL2 of CXCR4 and ACKR3 (CXCR7), respectively, and no significant cross-reactivity of the agents of the present invention that specifically bind to GPR182 is expected with respect to binding to CXCR4 and / or ACKR3 (CXCR7). For example, sequence comparison between human GPR182 and human CXCR4 reveals only 14% and 12.5% inadequate amino acid sequence identity with respect to their N-terminal extracellular regions (amino acid residues 1-57) and their ECL2, respectively. In a similar manner, sequence comparison between human GPR182 and human ACKR3 (CXCR7) reveals only 12.3% and 16.7% low sequence identity in their N-terminal extracellular regions (amino acid residues 1-57) and their ECL2, respectively. Thus, an agent that specifically interacts with the N-terminal extracellular domain of GPR182 or with the ECL2 of GPR182 is not expected to also have significant binding affinity for these or other portions of CXCR4 or ACKR3 (CXCR7). In any case, one of ordinary skill in the art will be able to evaluate the binding of any candidate agent to GPR182, CXCR4, ACKR3 (CXCR7), or other receptors using methods known in the art as described herein, and thus, if desired, select only those such agents (and / or only those having significant binding activity) that have favorable binding activity for GPR182.

[0100] In a recent study by the inventors (Le Mercier et al., 2021), the chemokines CXCL10, CXCL12, and CXCL13 were identified as endogenous (i.e., cognate) ligands of human GPR182. Saturation binding experiments using fluorescently labeled chemokine ligands with HEK293 cells recombinantly expressing human GPR182 showed that human GPR182 had K values of 19 nM and 41 nM, respectively. DIt was revealed to be bound by CXCL10 and CXCL12. Furthermore, competitive binding experiments showed inhibition constants (K i ) of 9 nM and 10 nM for CXCL10 and CXCL13, respectively, and K i values of 31 nM and 19 nM for the two CXCL12 isoforms α and β, respectively, indicating a slightly lower affinity. Evaluation of other chemokine ligands revealed some interaction with CXCL19 and CXCL16, but these binding affinities were determined to be either much lower (Ki: 260 nM) or even below the limit of analysis for CXCL19 and CXCL16, suggesting a lack of physiological relevance of the latter two chemokines to GPR182-mediated functions.

[0101] Considering the findings of the inventors in the context of the present invention, the increased levels of CXCL12 in the detected heart tissue are expected to be due to the observed beneficial therapeutic effects in MI. Thus, preferably, in view of the objectives contemplated herein, an agent that specifically binds to the GPR182 protein has a binding affinity for the GPR182 protein that is at least equal to or more preferably exceeds the binding affinity of CXCL12 for the GPR182 protein. A person skilled in the art can evaluate and compare the respective binding affinities of any candidate agent for the GPR182 protein and CXCL12 (or other endogenous ligand) by using methods known in the art and some described herein (e.g., see Example 2).

[0102] Thus, in a particularly preferred embodiment, an agent that specifically binds to the GPR182 protein: (i) exhibits a higher binding affinity for the GPR182 protein, preferably at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, compared to the binding affinity between one or more endogenous GPR182-ligands and the GPR182 protein; and / or (ii) 10 -6 M, 10 -7M or 10 -8 less than M, preferably less than 41 nM, more preferably 10 -9 M or 10 -10 with a dissociation constant (K D ) that binds to the GPR182 protein.

[0103] The therapeutic benefits provided by the medical applications disclosed herein that are dependent on inhibition of the expression or activity of the GPR182 protein may be further enhanced by the simultaneous application and / or additional application of one or more therapeutic means that are currently available and routinely used in the disease states contemplated herein. Accordingly, each of these embodiments is also specifically contemplated herein, some of which are illustrated below but are not necessarily limited thereto.

[0104] According to a particular embodiment of the first aspect of the invention, the agent is employed for use in the treatment or prevention of myocardial infarction (MI) and / or one or more of their post-disease complications. In a preferred embodiment of the latter embodiment, a subject (e.g., a subject at risk of myocardial infarction or a patient diagnosed with myocardial infarction) is administered a therapeutically effective amount of the agent disclosed herein and additionally (i.e., simultaneously with, before, or after it), is subjected to further therapeutic means that may enhance the therapeutic effect. For example, a patient diagnosed with myocardial infarction, in addition to administration of the agent according to the first aspect of the invention, may be subjected to a percutaneous coronary intervention (i.e., a non-surgical procedure using a catheter to position a stent to open a blood vessel of the heart narrowed by plaque accumulation) and / or a surgical procedure such as coronary artery bypass (CAB); and / or the agent may be administered in combination with one or more additional agents, such as those currently routinely used in MI therapy. Preferred additional agents for such purposes are defined in the embodiments illustrated below. However, it should be understood that the following embodiments may also be applied in connection with the use of the agent for the treatment or prevention of any other disease state contemplated herein.

[0105] According to a preferred embodiment, the agent is (i) preferably - Tissue plasminogen activator; preferably alteplase, reteplase, and / or tenecteplase; - Streptokinase; - Anistreplase; and - Urokinase a thrombolytic agent selected from (ii) preferably - Heparin; preferably unfractionated heparin (UFH) and / or low molecular weight heparin (LMWH); - Factor Xa inhibitor; preferably apixaban and / or rivaroxaban; and - Hirudin and / or bivalirudin an anticoagulant selected from (iii) preferably - Irreversible cyclooxygenase inhibitor; preferably acetylsalicylic acid (ASA) and / or triflusal; - Adenosine diphosphate (ADP) receptor inhibitor; preferably cangrelol, clopidogrel, prasugrel, ticagrelor, and / or ticlopidine; - Phosphodiesterase inhibitor; preferably cilostazol; - Protease-activated receptor-1 (PAR-1) antagonist; preferably vorapaxar; - Glycoprotein IIb / IIIa inhibitor; preferably abciximab, eptifibatide, and / or tirofiban; - Adenosine reuptake inhibitor; preferably dipyridamole; - Thromboxane inhibitor; - Thromboxane synthase inhibitor; - Thromboxane receptor antagonist; preferably terutroban a platelet aggregation inhibitor selected from (iv) Preferably, a β-blocker selected from propranolol, alprenolol, bufetolol, carteolol, carvedilol, labetalol, levobunolol, medroxalol, mepindolol, metipranolol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, and nebivolol; and (v) Preferably, an angiotensin-converting enzyme inhibitor (ACE inhibitor) selected from benazepril, zofenopril, perindopril,trandolapril, captopril, enalapril, lisinopril, and ramipril; (vi) Nitrate; preferably isosorbide dinitrate, isosorbide mononitrate, and / or pentrinitrol; (vii) Oxygen; (viii) One or more C-X-C chemokine receptor type 4 (CXCR4) agonists, preferably CXCL12 and / or one or more synthetic CXCR4 agonists; (ix) One or more C-X-C motif chemokine receptor type 7 (CXCR7) agonists, preferably CXCL11, CXCL12, and / or one or more synthetic CXCR7 agonists; (x) C-X-C motif chemokine ligand 10 (CXCL10); and / or (xi) C-X-C motif chemokine ligand 13 (CXCL13) is administered in combination with or sequentially with one or more of the foregoing, preferably, the pathological condition is selected from myocardial infarction (and / or one or more of their post-disease complications) and stroke.

[0106] According to a specific embodiment of the first aspect of the present invention, the agent is used for the treatment or prevention of cancer. In an exemplary embodiment of the latter embodiment, a subject (e.g., a subject at risk of developing cancer or a patient suffering from cancer) is administered a therapeutically effective amount of the agent disclosed herein, and additionally (i.e., simultaneously, before, or after), radiation therapy and / or surgical procedures can be performed for the removal of cancer cells or tissues, and / or the agent can be administered in combination with one or more additional anti-cancer agents, such as, but not limited to, one or more chemotherapeutic agents, etc., and / or in combination with immunotherapy. Preferred agents / therapies for combination with the agent of the present invention are defined in the following exemplary embodiments. However, the following embodiments may alternatively be applied in connection with the use of the agent for the treatment or prevention of any other pathological condition contemplated herein.

[0107] Thus, according to a preferred embodiment, the agent is (i) one or more chemotherapeutic agents; (ii) adoptive cell therapy, preferably adoptive T cell therapy; (iii) one or more immune checkpoint inhibitors preferably selected from antibodies against cytotoxic T lymphocyte-associated antigen 4 (CTLA4), programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2), or combinations thereof; and / or (iv) one or more C-X-C motif chemokine receptor type 3 (CXCR3) agonists, preferably CXCL9, CXCL10, or CXCL11, and / or one or more synthetic CXCR7 agonists administered in combination with or sequentially with one or more of the foregoing, preferably the pathological condition is cancer, more preferably melanoma.

[0108] As used herein, the term "chemotherapeutic agent" means a compound that may be useful in the treatment of a disease (e.g., cancer). Exemplary chemotherapeutic agents effective against cancer include, but are not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, docetaxel, vincristine, vinblastine, etoposide, teniposide, cisplatin, and diethylstilbestrol (DES).

[0109] As used herein, the term "immunotherapy" generally means a means for the treatment of a disease or condition (e.g., cancer) by inducing, enhancing, or suppressing an immune response. The term "immunotherapy" also encompasses adoptive cell therapy.

[0110] For the purposes of the present disclosure, the terms "adoptive cellular therapy (ACT)", "cellular adoptive immunotherapy", or "adoptive immunotherapy" mean cell-based therapies by various lymphocytes and / or antigen-presenting cells. For example, in some cases, the therapy involves collecting T cells from a patient and culturing the collected T cells in a laboratory to increase the number of T cells. These T cells are then returned to the same patient or another patient to assist the immune system in fighting a disease, e.g., cancer. Further, as used herein, the term "adoptive cellular therapy (ACT)" also means an immunotherapy in which (a) genetically modified lymphocytes are administered to a patient or (b) a target gene therapy vector that modifies lymphocytes is administered to a patient.

[0111] Examples of adoptive cell therapies include, but are not limited to, CAR-T therapy. For the purposes of the present disclosure, the term "CAR T therapy" or "CAR T cell therapy" means treating a disease (e.g., cancer) using CAR-T cells. For the purposes of the present disclosure, the term "CAR-T" or the term "CAR T cell" means a genetically engineered T cell that produces a "chimeric antigen receptor (CAR)" on its cell surface. A chimeric antigen receptor (also known as CAR, chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is a receptor protein that has been engineered to give T cells the new ability to target specific proteins. The receptor is chimeric because it combines both an antigen-binding function and a T cell activation function into a single receptor. The backbone of a CAR-T or CAR T cell is a T cell, which is often collected and isolated from the subject, such as a patient, who is to receive the CAR-T or CAR T cells. CAR T cells are engineered immune cells of the patient that are typically used to treat the patient's cancer. For example, CAR T therapy can be an immunotherapy that utilizes the subject's or patient's own immune cells that have been engineered to be able to produce a specific chimeric antigen receptor on their surface. In some situations, T cells are collected from the subject's body by apheresis, which is a process of removing one or more blood components (e.g., plasma, platelets, or white blood cells) from the blood withdrawn from the body. The T cells collected from the body are then genetically engineered to produce a specific chimeric antigen receptor on their surface. After the engineering, the T cells are known as chimeric antigen receptor (CAR) T cells. CAR T cells are grown by culturing in the laboratory and then administered to the subject or patient, or another subject or patient. CAR T cells recognize and cause the death of cancer cells that express the target antigen on their surface. For the purposes of the present disclosure, the terms "chimeric antigen receptor" and "CAR" mean an engineered receptor that recognizes a target antigen expressed on the surface of target cells, such as an antigen expressed on cancer cells.

[0112] As used herein, the term "immune checkpoint protein" has its ordinary meaning in the art and refers to a molecule expressed by T cells and / or NK cells that either enhances a signal (stimulatory checkpoint molecule) or weakens a signal (inhibitory checkpoint molecule). It is recognized in the art that immune checkpoint molecules constitute immune checkpoint pathways similar to the CTLA-4 and PD-1-dependent pathways (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489). Examples of inhibitory checkpoint molecules include B7-H3, B7-H4, BTLA, CTLA-4, CD277, KIR, PD-1, LAG-3, TIM-3, TIGIT, and VISTA. B7-H3, also called CD276, was originally thought to be a co-stimulatory molecule but is now considered co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor evasion. B and T Lymphocyte Attenuator (BTLA), also called CD272, is a ligand for HVEM (Herpesvirus Entry Mediator). Cell surface expression of BTLA is gradually downregulated during the differentiation of human CD8+ T cells from naive to effector cell phenotypes; however, tumor-specific human CD8+ T cells express BTLA at high levels. Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), also called CD152, is overexpressed on Treg cells and functions to control T cell proliferation. Killer-cell Immunoglobulin-like Receptor (KIR) is a receptor for MHC class I molecules on natural killer cells.Lymphocyte Activation Gene-3 (LAG3) functions to suppress the immune response by acting on Tregs and having a direct effect on CD8+ T cells. T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 functions as a negative regulator of Th1 / Tc1 function by inducing cell death in its interaction with its ligand galectin-9. V-domain Ig suppressor of T cell activation (VISTA) is mainly expressed on hematopoietic cells, and thus, constitutive expression of VISTA on leukocytes within tumors may render VISTA blockade effective against a wide range of solid tumors. Programmed cell death protein 1 (PD-1), also known as CD279, acts as an immune checkpoint, and in the binding of one of its ligands, programmed cell death ligand 1 (PD-L1) or programmed cell death ligand 2 (PD-L2), enables Shp2 to dephosphorylate CD28, inhibiting T cell activation.

[0113] As used herein, the term "immune checkpoint inhibitor" has its ordinary meaning in the art and means any compound that inhibits the function of an immune inhibitory checkpoint protein. Inhibition in this context includes both reduction and complete abrogation of function. In particular, immune checkpoint inhibitors are particularly suitable for enhancing the proliferation, migration, survival, and / or cytotoxic activity of CD8+ T cells in a patient, particularly the tumor-infiltrating ability of the patient's CD8+ T cells. As used herein, the term "CD8+ T cells" has its ordinary meaning in the art and means a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8+ T cells" are also referred to as cytotoxic T lymphocytes (CTLs), T-killer cells, cytotoxic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is an associative recognition element in major histocompatibility complex class I-restricted interactions. As used herein, the term "tumor-infiltrating CD8+ T cells" means the pool of a patient's CD8+ T cells that have migrated from the bloodstream into the tumor.

[0114] Non-limiting examples of other anti-cancer agents include, but are not limited to, angiogenesis inhibitors, anti-proliferative agents, other kinase inhibitors, other receptor tyrosine kinase inhibitors, aurora kinase inhibitors, polo-like kinase inhibitors, bcr-abl kinase inhibitors, growth factor inhibitors, anti-mitotic agents, alkylating agents, anti-metabolites, platinum-containing agents, growth factor inhibitors, ionizing radiation, cell cycle inhibitors, topoisomerase inhibitors, biological response modifiers, immunomodulators, immunologicals, antibodies, hormonal therapy, retinoids / deltinoid plant alkaloids, proteasome inhibitors, HSP-90 inhibitors, histone deacetylase (HDAC) inhibitors, purine analogs, pyrimidine analogs, MEK inhibitors, CDK inhibitors, ErbB2 receptor inhibitors, mTOR inhibitors, Bcl inhibitors, Mcl inhibitors, and combinations thereof, as well as other anti-cancer agents. Angiogenesis inhibitors include, but are not limited to, EGFR inhibitors, PDGFR inhibitors, VEGFR inhibitors, TIE2 inhibitors, IGFIR inhibitors, matrix metalloprotease 2 (MMP-2) inhibitors, matrix metalloproteinase 9 (MMP-9) inhibitors, thrombospondin analogs such as thrombospondin-1, etc.

[0115] In another preferred embodiment, the agent that specifically binds to the GPR182 protein is a (poly)peptide and is administered in the form of a polynucleotide encoding the (poly)peptide; More preferably, the polynucleotide is: (i) contained in a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector; or (ii) RNA, preferably mRNA.

[0116] In a preferred embodiment, the agent has no substantial cytotoxic and / or immunogenic activity. In a preferred embodiment, the agent that specifically binds to the GPR182 protein is an antibody or an Fc fusion polypeptide, and does not cause substantial antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0117] In a preferred embodiment of the first aspect of the present invention, or in a further aspect of the present invention, the agent as defined herein is (i) promoting tissue repair, preferably angiogenesis and / or neovascularization, after (acute) myocardial infarction; (ii) reducing the size and / or volume of the infarct after (acute) myocardial infarction; (iii) enhancing perfusion and / or reducing the risk or severity of heart failure, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, and / or ventricular arrhythmia after (acute) myocardial infarction; (iv) promoting angiogenesis and / or neovascularization in ischemic diseases; (v) enhancing anti-tumor immunity; (vi) enhancing tumor infiltration by T cells, preferably CD8+ cytotoxic T cells and / or CAR T cells; and / or (vii) enhancing the efficacy of adoptive cell therapy and can be used for use in.

[0118] In a further preferred embodiment, the agent as defined herein is an agent for use in the treatment of myocardial infarction by promoting the formation of collateral coronary arteries. In a preferred embodiment, the agent that specifically binds to the GPR182 protein is: (i) different from CXCL10, CXCL12, and / or CXCL13; (ii) different from CXCL9, CXCL10, and / or CXCL11; (iii) different from CXCL9, CXCL10, CXCL11, CXCL12, and / or CXCL13; and / or (iv) It is heterologous to the subject to which the agent is administered.

[0119] For the medical uses contemplated herein, it will be understood by those skilled in the art that the agent is administered to the subject in a therapeutically effective amount. The terms "therapeutically effective amount", "pharmaceutically effective amount", or "effective amount", which are used interchangeably herein, mean an amount having a therapeutic effect or an amount necessary to produce a therapeutic effect in a subject. For example, the therapeutically effective amount of an agent or its pharmaceutical composition is the amount of the agent or pharmaceutical composition necessary to produce the desired therapeutic effect, which can be determined by the results of clinical trials, studies in model animals, and / or in vitro studies. The pharmaceutically effective amount depends on several factors, such as, but not limited to, the particular pathological condition targeted, the characteristics of the subject (e.g., body weight, sex, age, and medical history), and the particular type of agent used. For prophylactic (i.e., preventive) use (or treatment), the therapeutically effective amount or prophylactically effective amount will be an amount effective to prevent the respective pathological condition or to prevent further complications that may arise as a result of symptoms not yet present or of that pathological condition.

[0120] The agent can be formulated for administration according to the present invention as part of a pharmaceutical composition. The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier or excipient. "Pharmaceutically acceptable carrier or excipient" means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation aid (see also Handbook of Pharmaceutical Excipients 6ed. 2010, published by Pharmaceutical Press). Examples of suitable pharmaceutical carriers and excipients are well known in the art and include, for example, phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and organic solvents such as DMSO. Compositions containing such carriers or excipients can be formulated by well-known conventional methods. The pharmaceutical composition can be administered, in dosage unit formulations optionally containing conventional pharmaceutically acceptable carriers or excipients, for example, orally, parenterally such as subcutaneously, intravenously, intramuscularly, intraperitoneally, intrathecal, transdermally, transmucosally, subdermally, topically, or locally by iontophoresis, sublingually, by inhalation spray, aerosol, or rectally, etc.

[0121] In the case of oral administration, the drug or the pharmaceutical composition containing the drug can be formulated into solid dosage forms, such as tablets, pills, powders, granules, or capsules, etc. These solid dosage forms can be prepared by mixing the drug of the present invention with one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Further, in addition to simple excipients, lubricants, such as magnesium stearate, talc, etc. can be used. Further, the pharmaceutical composition can be formulated into liquid dosage forms, such as suspensions, oral liquids, emulsions, syrups, etc. In addition to water and liquids, paraffins, various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc. can be used for the formulation of liquid dosage forms. In the case of parenteral administration, the pharmaceutical composition generally contains the drug of the present invention in an injectable form (solution, suspension, or emulsion) of a unit dosage amount at a desired degree of purity, and is formulated by mixing with a pharmaceutically acceptable carrier, that is, a carrier that is non-toxic to the recipient at the dosage amount and concentration used and is compatible with other raw materials of the formulation.

[0122] Generally, formulations are prepared by uniformly and intimately contacting the components of the pharmaceutical composition with a liquid carrier or a finely divided solid carrier or both. Then, if necessary, the product is formed into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the recipient's blood. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes. The carrier preferably contains small amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to the recipient at the dosage and concentrations employed, and include buffering agents such as phosphates, citrates, succinates, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides such as polyarginine or tripeptides; serum albumin, gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates such as cellulose or its derivatives, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbate, poloxamer, or PEG.

[0123] The agent according to the present invention (or the pharmaceutical composition comprising said agent) can be administered systemically (e.g., orally, rectally, parenterally (e.g., intravenously), intramuscularly, intraperitoneally, transdermally (e.g., by patch), topically (such as by powder, ointment, drops, or transdermal patch), buccally, or as an oral or nasal spray, by inhalation, subcutaneously, etc.), into the central nervous system (e.g., into the brain (e.g., intracerebrally, intraventricularly, or intrathecal) or into the spinal cord or cerebrospinal fluid), or by any combination thereof.

[0124] In a preferred embodiment, the agent or polynucleotide encoding the agent is administered intravenously, intramuscularly, and / or orally. The agent or pharmaceutical composition comprising the agent may be administered at least once a day, for one day or for several days (e.g., 1, 2, 3, 4, 5, 6, 7 days; 2, 3, 4 weeks; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months; 2 years, 3 years, 4 years, or more) in one or multiple doses of administration. However, those skilled in the art will understand that the duration of administration can, as appropriate, be continued for the remainder of the subject's life in accordance with the present invention. The agent or pharmaceutical composition comprising the agent may be administered daily, or at a less frequent rate, e.g., twice a week every other day, weekly, every other week, monthly, every other month, etc.

[0125] The agent of the present invention (or the pharmaceutical composition comprising the agent) can be administered to a subject in a suitable dose and / or a therapeutically effective amount. The dosage of the agent of the present invention required will vary somewhat depending on the species, age, weight, and general condition of the subject, the particular active agent and pharmaceutical carrier used, the mode of administration, etc. For example, the dosage of the agent when administered parenterally can range from about 0.01 to 1000 mg / kg of the patient's body weight (bd weight), but as noted above, this will be subject to the discretion of the treatment. More preferably, this dosage is at least 0.5 mg / kg / day, and most preferably, in the case of humans, about 5 to 100 mg / kg / day. When administered continuously, the pharmaceutical composition is typically administered either by 1 to 4 injections per day at a dosage ratio of from about 1 mg / kg / hour to about 50 mg / kg / hour or by continuous subcutaneous injection using, for example, a minipump. An intravenous bag solution can also be used. The duration of treatment required to observe a change and the period after treatment for a response to occur would seem to vary depending on the desired effect. The exact amount can be determined by conventional tests well known to those skilled in the art.

[0126] To identify a therapeutically effective dosage of a drug or a particular pharmaceutical formulation containing the drug, for example, an animal model (e.g., a mouse) expressing the human GPR182 protein can be used to monitor the level of response. Exemplary methods that can also be utilized to identify suitable dosages and dosing regimens are the methods described in connection with the fourth and fifth aspects of the present invention.

[0127] Furthermore, in a third aspect, the present invention provides an in vitro method for identifying a drug suitable for use according to the first aspect of the present invention, the method comprising evaluating the binding of one or more endogenous GPR182-ligands to cells expressing the GPR182 protein in the presence of a candidate drug, wherein a decrease in the binding of one or more endogenous GPR182-ligands in the presence of the candidate drug as compared to the absence of the candidate ligand indicates that the candidate drug is suitable for use according to the first aspect of the present invention.

[0128] In a preferred embodiment of the method according to the third aspect of the present invention, either the endogenous ligand or the candidate drug is fluorescently labeled and the binding is evaluated by assessing the fluorescence signal in a fluorescence competition assay; and / or the cells expressing the GPR182 protein are recombinant eukaryotic cells; more preferably HEK293T cells.

[0129] Furthermore, in a fourth aspect, the present invention provides a method for evaluating the efficacy of a drug defined in any of the preceding claims in the treatment of myocardial infarction, the method comprising, before and / or simultaneously with and after administration of the drug: (a) the size and volume of the infarct area; (b) the left ventricular ejection fraction and / or the left ventricular end-diastolic volume and / or the left ventricular end-systolic volume; (c) the concentration of CXCL12 in the heart and / or blood; and / or (d) the plasma levels of one or more biological markers of myocardial injury, preferably troponin-I and / or troponin-T including the step of evaluating one or more of whereby, a decrease in the volume of the infarct area, an improvement in the left ventricular ejection fraction, a decrease in the left ventricular end-diastolic volume, a decrease in the left ventricular end-systolic volume, an increase in the CXCL12 concentration in the heart and / or blood, and / or a decrease in the plasma level of one or more biological markers of myocardial injury, determined after administration of the agent, indicates that the agent is effective in the treatment of myocardial infarction, relates to a method.

[0130] Means and methods for identifying the size or volume of the infarct area are known and commonly used in the art, such as echocardiography, and magnetic resonance imaging (MRI) as used in the attached examples (see, for example, Lee DC et al. J Am Heart Assoc. 2020;9(3):e014205). Other approaches commonly used in the art for these purposes are based on contrast-enhanced computed tomography (CECT) (see, for example, textbook ”Computed Tomography of the Cardiovascular System” by Thomas C. Gerber, Birgit Kantor, Eric E. Williamson, CRC Press; 1st Ed. 2007).

[0131] The left ventricular ejection fraction and / or the left ventricular end-diastolic volume and / or the left ventricular end-systolic volume can be identified by using a plurality of non-invasive imaging technique modalities, such as echocardiography, cardiac magnetic resonance (CMR) imaging, and single photon emission computed tomography (SPECT) imaging. All of these methods are commonly used for clinical decision-making and research study registration.

[0132] Furthermore, in a fifth aspect, the present invention is a method for evaluating the efficacy of an agent as defined in any of the preceding claims in the treatment of cancer, comprising, before and / or simultaneously with and after administration of the agent to a subject, the following: (a) The level of CXCL9 and / or CXCL10 in cancer and / or blood; (b) The level of T cells, preferably CD8+ cytotoxic T cells, in cancer and / or blood; and / or (c) The size of the cancer comprising the step of evaluating one or more of: whereby an increase in the concentration of CXCL9 and / or CXCL10 in cancer and / or blood, and / or an increase in the level of T cells, preferably CD8+ cytotoxic T cells, in cancer and / or blood, and / or a decrease in the size of the cancer, determined after administration of the agent, indicates that the agent is effective in the treatment of cancer.

[0133] In a sixth aspect, the present invention relates to a method of treating and / or preventing any of the pathological conditions as referred to herein in connection with the medical use according to the first or other aspect of the present invention, the method comprising administering a therapeutically effective amount of an agent to a subject in need thereof.

[0134] It is understood that the definitions and embodiments described above in connection with the first aspect of the present invention also apply, to the extent possible, to the second, third, fourth, fifth, and sixth aspects of the present invention, and any further disclosed aspects.

[0135] The present invention is described herein by way of example only, with reference to the accompanying drawings which illustrate preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0136]

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Example

[0137] The examples illustrate the present invention. Example 1: Inhibition of scavenging of GPR182-mediated chemokine ligands improves clinical outcomes in an animal model of acute myocardial infarction (MI) 1.1 Method Animal model All mice were backcrossed at least 8–10 times onto the C57BL / 6J background, and littermates were used as controls for the experiments. Unless otherwise specified, mice were housed under specific pathogen-free conditions with free access to food and water under a 12-hour light–dark cycle.

[0138] GPR182-deficient mice (Gpr182 gKO) and endothelium-specific inducible GPR182-deficient mice (EC-Gpr182-KO) were generated as previously described (Le Mercier et al., 2021). For induction of Cre-mediated recombination in EC-Gpr182-KO mice (Cdh5CreERT2, Gpr182f / f), 1 mg of tamoxifen diluted in Miglyol was intraperitoneally injected into adult mice at 8–10 weeks of age for 5 consecutive days. Experiments were performed 2 weeks after the last tamoxifen injection. Myocardial infarction Coronary artery ligation was performed in male mice at 10–12 weeks of age. Briefly, after anesthesia (isoflurane inhalation) and tracheal intubation, the chest was opened through the fourth intercostal space. Under microscopic control, the left ascending coronary artery was tightly ligated with an 8-0 suture. Myocardial ischemia was confirmed by observing color changes in the segments of the left ventricle where coronary blood flow was occluded. The incision wound was closed with a 5-0 suture.

[0139] After surgery, mice were monitored daily for 3 days and treated twice a day by intraperitoneal injection of buprenorphine (0.1 mg / kg). Mice that died within 24 hours after surgery were excluded from the analysis. Surgery was performed by individuals blinded to the mouse genotype. Echocardiography Cardiac function was evaluated at the indicated time points before and after surgery using a Vevo2100 ultrasound system (VisualSonics, Fujifilm) equipped with an MS550d transducer. Left ventricular ejection fraction (LVEF), LV fractional area change, and LV systolic and diastolic volumes were measured by the Simpson's method. Magnetic resonance imaging Cardiac MRI measurements were performed on a 7.0 T Bruker Pharmascan equipped with a 300 mT / m gradient system using a custom-built circularly polarized birdcage resonator and an Early Access Package (Bruker, Ettlingen, Germany) for self-gated cardiac imaging. Mice were measured under volatile isoflurane (2.0%) anesthesia. Measurements were based on the gradient echo method (repetition time = 6.2 ms; echo time = 1.6 ms; field of view = 2.20 α 2.20 cm; slice thickness = 1.0 mm; matrix = 128 × 128; repetitions = 100). Scout images showing two-chamber and four-chamber views of the heart were used to localize the acquisition plane, followed by acquisition at orthogonal short-axis views on the septum of both scouts. To completely cover the left ventricle, multiple consecutive short-axis slices consisting of 9 or 10 slices were acquired. Magnetic resonance imaging data were analyzed using Qmass digital imaging software (Medis, Leiden, the Netherlands). Histological analysis At the indicated time points after coronary artery ligation, heart tissue was harvested. Heart tissue from the apex was perfused with cold PBS and fixed in 4% PFA at 4°C overnight. Samples were embedded in paraffin. Serial 5-μm-thick sections of the short axis of the heart were deparaffinized and rehydrated. Masson trichrome staining was performed according to the manufacturer's instructions (Sigma-Aldrich, HT15). For CXCL12 fluorescence immunostaining, after rehydration of tissue sections, endogenous peroxidase was inactivated using 3% H2O2 in H2O for 30 minutes. Subsequently, microwave-assisted antigen retrieval was applied to the slides at 1000 W for 5 minutes and then at 500 W for 10 minutes in a pH 6.0 solution of 10 mM sodium citrate and 0.05% tween 20. After cooling and removal of the antigen retrieval solution, sections were blocked and permeabilized in an antibody diluent (PBS, 5% horse serum, 0.1% Triton) for 1 hour at room temperature, and then avidin / biotin blocking was performed using an avidin / biotin blocking kit from Vector Laboratories (SP-2001). Sections were then incubated overnight at 4°C with anti-SDF1 (CXCL12) (Abcam, ab25117, 1:200) diluted in the antibody diluent. After washing, slides were incubated for 30 minutes at room temperature with a goat anti-rabbit-IgG biotinylated secondary antibody (Vector Laboratories, PK-6101, 1:500), and then amplified at room temperature for 30 minutes using the VECTASTAIN® Elite ABC-HRP kit, peroxidase (rabbit IgG) (Vector Laboratories, PK-6101). Signals were visualized and enhanced with a TSA-Cy3 signal amplification kit (AKOYA BIOSCIENCES, SAT704A001EA). The cell membrane and nucleus were visualized with AF647-labeled WGA (Thermo Fisher Scientific, W32466 5 μg / ml) and DAPI (LifeTechnologies D3571), respectively. Sections were mounted with FluoroMount, covered with a glass coverslip, and images were acquired by confocal microscopy using a Leica TCS SP8 confocal microscope. Bioimage analysis All microscopic images were analyzed using Image J or Fiji distribution of Image J. Infarct size was calculated as the mean ratio of scar tissue length to the LV circumference.

[0140] CXCL12 fluorescence intensity was measured using the mean signal intensity of the area outlined by a line drawn by hand in the remote zone, border zone, or the area corresponding to the infarct. Measurement of the thickness of the healthy myocardium on the endocardial side of the infarct: The inventors manually selected the area of the healthy myocardium region directly adjacent to the endocardium in the infarct region of hematoxylin and eosin (H&E)-stained paraffin sections (using the selection brush tool). This represents Selection #1. A mask was created from Selection #1, duplicated, and the Local Thickness (entire process) plugin was applied to the duplicated mask. The skeletonization function was applied to the first mask to create a new selection (#2). By using this Selection #2, which represents the inner line along the healthy myocardium selection, in the output image from the Local Thickness plugin, the mean pixel intensity from Selection #2 was measured. This measurement represents the mean thickness along Selection #1 (healthy myocardium). Small trabecular muscles from the left ventricle were excluded from the analysis. Statistics Statistical analysis was performed using GraphPad Prism software v.8.3.0 from GraphPad Software Inc. (La Jolla, California, USA). Values are presented as mean ± SEM. For two groups, if appropriate, statistical analysis was performed by unpaired two-sided Student's t-test or nonparametric Mann-Whitney U test. The results of repeated measurements were evaluated by two-way repeated measures ANOVA followed by post hoc Sidak or Tukey multiple comparison tests. A p-value less than 0.05 was considered statistically significant. 1.2 Introduction GPR182 has recently been shown to be expressed in the endothelial cells of the microvasculature and lymphatic system of mammalian species such as humans (Le Mercier et al., 2021; Schmid et al., 2018; Torphy et al., 2022; Xiao et al., 2014), and to function as an atypical chemokine receptor, i.e., it has been shown to bind chemokines with a rather high affinity. However, the ligands that bind to GPR182 do not induce any downstream signaling (Le Mercier et al., 2021). At the same time, GPR182 has a relatively high basal constitutive activity with respect to β-arrestin recruitment (Kroeze et al., 2015; Le Mercier et al., 2021). This constitutive activity follows a relatively high constitutive β-arrestin-dependent internalization rate (Le Mercier et al., 2021). Thus, the binding of chemokines to GPR182 causes their receptor-dependent internalization, and as a result, the receptors function as chemokine scavengers. The spectrum of chemokines that bind to GPR182 is broad; at physiological concentrations, they bind to CXCL10, CXCL12, and CXCL13 (Le Mercier et al., 2021). Studies in constitutive and inducible GPR182 knockout mice have shown that the receptor plays a role in maintaining hematopoietic stem cells in the bone marrow, and that the deficiency of GPR182 results in a decrease in the maintenance of HSCs in the bone marrow (Le Mercier et al., 2021). 1.3 Results and Explanation In this study, the inventors attempted to investigate the potential relevance of GPR182 in the pathology and recovery of myocardial infarction (MI).

[0141] By further analyzing the global transcriptome profiling data (obtained from single-cell RNA sequencing) of the animal model of myocardial infarction made available by Tombor et al., the inventors surprisingly found that the expression of GPR182 is upregulated in the endothelial cells of the myocardium after myocardial infarction. In particular, in a mouse model (ligation of the left anterior descending coronary artery (LAD)), an increase in GPR182 expression was found within the first week after myocardial infarction (Figure 1).

[0142] Next, the inventors attempted to investigate the potential effects of inhibition of GPR182 in a mouse model of myocardial infarction. Surprisingly and advantageously, it was found that in mice constitutively lacking GPR182 as well as in mice in which endothelial-specific deletion of GPR182 was induced, the infarct area was significantly reduced compared to control animals (Figure 2).

[0143] An increase in the mass of the intact myocardium was also observed on the endocardial side within the infarct area (Figure 3). By both echocardiography and magnetic resonance imaging (MRI), the inventors found that GPR182-KO mice starting from the first week after infarction showed improvement in cardiac parameters such as an increase in left ventricular ejection fraction and a decrease in left ventricular diastolic and systolic volumes compared to wild-type littermate controls (Figure 4).

[0144] Absence of GPR182 does not affect normal cardiac function. Three days after myocardial infarction, the concentration of the chemokine CXCL12 in cardiac tissue was significantly increased in GPR182-deficient mice (Figure 5).

[0145] Therefore, lack of GPR182 function causes a decrease in scavenging of CXCL12 and an increase in CXCL12 levels. This then increases angiogenesis after myocardial infarction, resulting in a decrease in infarct size and improvement in cardiac function after infarction.

[0146] Taking these results into account, pharmacological inhibition of the scavenging function of GPR182, for example, by an antibody, a nanobody, or other agents envisioned herein, to increase the concentration of CXCL12 in the heart would provide a novel and effective therapeutic means for the treatment of acute myocardial infarction (MI) and related post-acute MI complications, as it would particularly improve post-infarct angiogenesis and / or collateral growth, resulting in improved perfusion, reduced infarct size, and reduced heart failure.

[0147] When developing agents envisioned herein (e.g., antagonists or inhibitors of GPR182), care must be taken with respect to specificity. In particular, such agents should preferably specifically inhibit the expression or activity of the GPR182 protein without affecting the expression or activity of other proteins that function as signaling receptors for one or more endogenous GPR182-ligands. For example, the structurally related receptor ACKR3 (CXCR7) also functions as an atypical chemokine receptor (ACKR) for CXCL12, but in contrast to GPR182, they induce some downstream signaling by β-arrestin mobilization (Rajagopal et al., 2010) (Figure 6) and appear to have an opposing function after myocardial infarction. Endothelial-specific deletion of ACKR3 (CXCR7) results in an increase in infarct size and a decrease in cardiac function after myocardial infarction, and cardiomyocyte-specific deletion of ACKR3 (CXCR7) also reduces cardiac function after myocardial infarction. This is most likely due to a decrease in β-arrestin-mediated signaling (Hao et al., 2017; Ishizuka et al., 2021).

[0148] Therefore, the antagonist / inhibitor should preferably be selective for GPR182 and should not interfere with ACKR3 (CXCR7) either. Advantages of the approach compared to other therapeutic approaches Despite significant progress in the treatment of myocardial infarction (MI) resulting mainly from the development of drugs and technical methods aimed at reperfusion of the coronary arteries in thrombosis, a significant portion of MI patients still do not survive acute myocardial infarction. Furthermore, the surviving patients also suffer from long-term complications such as arrhythmia or heart failure. In these cases, improvement of CXCL12-mediated angiogenesis as an additional or alternative means must have an additive or synergistic effect on reducing the infarct area and the risk of secondary diseases. Improvement of neovascularization after myocardial infarction is a new approach that has not been used in the clinical treatment of acute myocardial infarction so far.

[0149] In contrast to the systemic application of CXCL12, the blockade of the scavenger receptor GPR182 is a much more selective means of increasing the local CXCL12 effect precisely by reducing the local degradation of CXCL12. Thus, this approach results in a much more selective and specific CXCL12-mediated effect, which is likely to lead to a reduction in unwanted effects. Further contemplated indications Recent publications have confirmed the role of GPR182 as a chemokine scavenging receptor (Torphy et al., 2022). In this study, the authors showed that GPR182 is upregulated in lymphatic endothelial cells of human melanoma and that GPR182 contributes to immunotherapy resistance in cancer by scavenging chemokines that are important for lymphocyte recruitment to tumors. In GPR182-deficient mice, increased T cell infiltration into transplanted melanoma was shown, leading to enhanced effector T cell function and improved antitumor immunity. This was accompanied by an increase in the intratumoral concentration of chemokines, causing sensitization of poorly immunogenic tumors to immune checkpoint blockade and adoptive cell therapy. Therefore, inhibition of GPR182 by the agents contemplated herein is likely to be used to sensitize melanoma with resistance to insufficient immunogenicity and immune checkpoint blockade. Example 2: Competitive binding assay A single-cell suspension of HEK293 cells stably expressing human or mouse GPR182 was prepared by trypsin / EDTA treatment. After inactivation of trypsin by dilution in DMEM (Gibco, 10938025) containing 10% fetal bovine serum (FBS), the cells were passed through a 70-μm cell strainer to remove clumps and counted using a Neubauer chamber. The required cells were then centrifuged at 200 g for 5 minutes at 4 °C and subsequently washed once with binding medium (serum-free DMEM, 10 mM HEPES (Gibco, 15630080), 0.5% BSA, 4 °C). The cells were then incubated for 30 minutes at 4 °C without internalization or recycling with a defined concentration of unlabeled drug in 100 μL of binding medium, with gentle rocking to allow binding, and then washed three times with binding medium. The cells were then resuspended in 100 μL of binding medium containing AlexaFluor-647 fluorescently labeled chemokine CXCL10, CXCL12, or CXCL13 (Almac). Each sample was then incubated for 1 hour at 4 °C with gentle rocking in a 96-well plate to allow binding without internalization or recycling. The cells were then washed three times with binding medium and incubated with DAPI immediately before analysis as a counterstain for dead cells, and the cells were analyzed by flow cytometry. The cells were gated to include only live singlets for analysis. The mean fluorescence intensity (MFI) of the channel detecting the signal from the AlexaFluor-647 fluorescently labeled chemokine was used as a readout of binding strength. The Ki value of the drug was then calculated using standard equations well known in the art. Further references Das S, Goldstone AB, Wang H, Farry J, D'Amato G, Paulsen MJ, Eskandari A, Hironaka CE, Phansalkar R, Sharma B, Rhee S, Shamskhou EA, Agalliu D, de Jesus Perez V, Woo YJ, Red-Horse K (2019) A Unique Collateral Artery Development Program Promotes Neonatal Heart Regeneration. Cell 176: 1128-1142 e1118 Hao H, Hu S, Chen H, Bu D, Zhu L, Xu C, Chu F, Huo X, Tang Y, Sun X, Ding BS, Liu DP, Hu S, Wang M (2017) Loss of Endothelial CXCR7 Impairs Vascular Homeostasis and Cardiac Remodeling After Myocardial Infarction: Implications for Cardiovascular Drug Discovery. Circulation 135: 1253-1264 Ishizuka M, Harada M, Nomura S, Ko T, Ikeda Y, Guo J, Bujo S, Yanagisawa-Murakami H, Satoh M, Yamada S, Kumagai H, Motozawa Y, Hara H, Fujiwara T, Sato T, Takeda N, Takeda N, Otsu K, Morita H, Toko H, Komuro I (2021) CXCR7 ameliorates myocardial infarction as a beta-arrestin-biased receptor. Sci Rep 11: 3426 Korf-Klingebiel M, Reboll MR, Grote K, Schleiner H, Wang Y, Wu X, Klede S, Mikhed Y, Bauersachs J, Klintschar M, Rudat C, Kispert A, Niessen HW, Lubke T, Dierks T, Wollert KC (2019) Heparan Sulfate-Editing Extracellular Sulfatases Enhance VEGF Bioavailability for Ischemic Heart Repair. Circ Res 125: 787-801 Kroeze WK, Sassano MF, Huang XP, Lansu K, McCorvy JD, Giguere PM, Sciaky N, Roth BL (2015) PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol 22: 362-369 Le Mercier A, Bonnavion R, Yu W, Alnouri MW, Ramas S, Zhang Y, Jager Y, Roquid KA, Jeong HW, Sivaraj KK, Cho H, Chen X, Strilic B, Sijmonsma T, Adams R, Schroeder T, Rieger MA, Offermanns S (2021) GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis. Proc Natl Acad Sci U S A 118 Macarthur JW, Jr., Cohen JE, McGarvey JR, Shudo Y, Patel JB, Trubelja A, Fairman AS, Edwards BB, Hung G, Hiesinger W, Goldstone AB, Atluri P, Wilensky RL, Pilla JJ, Gorman JH, 3rd, Gorman RC, Woo YJ (2014) Preclinical evaluation of the engineered stem cell chemokine stromal cell-derived factor 1alpha analog in a translational ovine myocardial infarction model. Circ Res 114: 650-659 Rajagopal S, Kim J, Ahn S, Craig S, Lam CM, Gerard NP, Gerard C, Lefkowitz RJ (2010) Beta-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7. Proc Natl Acad Sci U S A 107: 628-632 Saxena A, Fish JE, White MD, Yu S, Smyth JW, Shaw RM, DiMaio JM, Srivastava D (2008) Stromal cell-derived factor-1alpha is cardioprotective after myocardial infarction. Circulation 117: 2224-2231 Schmid CD, Schledzewski K, Mogler C, Waldburger N, Kalna V, Marx A, Randi AM, Geraud C, Goerdt S, Koch PS (2018) GPR182 is a novel marker for sinusoidal endothelial differentiation with distinct GPCR signaling activity in vitro. Biochem Biophys Res Commun 497: 32-38 Tombor LS, John D, Glaser SF, Luxan G, Forte E, Furtado M, Rosenthal N, Baumgarten N, Schulz MH, Wittig J, Rogg EM, Manavski Y, Fischer A, Muhly-Reinholz M, Klee K, Looso M, Selignow C, Acker T, Bibli SI, Fleming I, Patrick R, Harvey RP, Abplanalp WT, Dimmeler S (2021) Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction. Nat Commun 12: 681 Torphy RJ, Sun Y, Lin R, Caffrey-Carr A, Fujiwara Y, Ho F, Miller EN, McCarter MD, Lyons TR, Schulick RD, Kedl RM, Zhu Y (2022) GPR182 limits antitumor immunity via chemokine scavenging in mouse melanoma models. Nat Commun 13: 97 Xiao L, Harrell JC, Perou CM, Dudley AC (2014) Identification of a stable molecular signature in mammary tumor endothelial cells that persists in vitro. Angiogenesis 17: 511-518 Zeymer U (2019) Herzinfarkt - Was kommt in den Jahren danach. Dtsch Arztebl 116(40): 22-26 Zhang M, Qiu L, Zhang Y, Xu D, Zheng JC, Jiang L (2017) CXCL12 enhances angiogenesis through CXCR7 activation in human umbilical vein endothelial cells. Sci Rep 7: 8289 In connection with the first aspect of the present invention, it is understood that the definitions and embodiments as described above are, to the extent possible, also applied mutatis mutandis to the second, third, fourth, fifth, and sixth aspects of the present invention.

[0150] The present invention will be described, by way of example, with reference to the accompanying drawings which are for the purpose of illustrating exemplary embodiments of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of contradiction, this patent specification, including definitions, will prevail.

[0151] Regarding the embodiments characterized in this specification, especially in the claims, each embodiment mentioned in a dependent claim is intended to be combined with each embodiment of each claim (independent or dependent claim) on which the said dependent claim depends. For example, in the case of independent claim 1 listing three options A, B, and C, dependent claim 2 listing three options D, E, and F, and claim 3 depending on claims 1 and 2 and listing three options G, H, and I, unless specifically mentioned otherwise, the specification is understood to clearly disclose embodiments corresponding to the combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I.

[0152] Similarly, even when an independent claim and / or a dependent claim do not list options, when a dependent claim refers back to multiple preceding claims, any combination of the subject matter covered by them is also considered to be clearly disclosed. For example, in the case of independent claim 1, dependent claim 2 referring back to claim 1, and dependent claim 3 referring back to both claims 2 and 1, the combination of the subject matter of claim 3 and 1 will be clearly and explicitly disclosed as being the combination of the subject matter of claims 3, 2, and 1. If a further dependent claim 4 is presented that refers to any one of claims 1 - 3, the combinations of the subject matter of claim 4 and 1, claim 4 and 2 and 1, claim 4 and 3 and 1, and claim 4 and 3 and 2 and 1 will be clearly and explicitly disclosed.

[0153] The above considerations apply mutatis mutandis to all of the appended claims. The entire contents of each patent, patent application, publication, and document referred to herein are hereby incorporated by reference into this specification. The citation of the above patents, patent applications, publications, and documents is not an admission that any of them are appropriate prior art, nor does it constitute any admission as to the content or date of these publications or documents. Their citation is not evidence of a search of related disclosures. All statements as to the date or content of such documents are based on available information and are not an admission as to their accuracy or validity.

[0154] Modifications may be made to the foregoing without departing from the basic interpretation of the technology. Although the technology has been described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that changes may be made to the embodiments disclosed in detail herein, and that such modifications and improvements remain within the scope and spirit of the technology.

[0155] The technology described as an example herein can be suitably practiced in the absence of any element not specifically disclosed herein. Thus, for example, in each instance herein, the terms "comprising", "consisting essentially of", and "consisting of" may be replaced by either of the other two terms. The terms and expressions used are used as terms of explanation and not of limitation, and the use of such terms and expressions does not exclude any equivalents of the diagrams shown and described or portions thereof, and various modifications are possible within the scope of the claimed technology. The terms "method" and "process" are used interchangeably herein. The term "a" or "an" can mean one or more of the elements it modifies unless the context clearly dictates that one or more of the elements are being described (e.g., "a cell" can mean "one or more cells").

[0156] As used herein, the term "about" means a value within 10% of a basic parameter (i.e., ±10%), and the use of the term "about" at the beginning of a series of values modifies each of those values (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). For example, a weight of "about 100 grams" can encompass weights between 90 grams and 110 grams. Further, when a list of values is recited herein (e.g., about 50, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values and decimal values thereof (e.g., 54%, 85.4%). Accordingly, although the technology is specifically disclosed by representative embodiments and optional features, it should be understood that variations and modifications of the concepts disclosed herein may be reclassified by those skilled in the art and such variations and modifications are considered to be within the scope of this technology.

[0157] Certain embodiments of the technology are described in the following claims.

Claims

1. A drug for use in the treatment or prevention of pathological conditions selected from myocardial infarction, myocardial ischemia, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, stroke, and cancer, which inhibits the expression or activity of the G protein-coupled receptor 182 (GPR182) protein, (a) A drug that inhibits the expression of the GPR182 protein is selected from siRNA, miRNA, shRNA, ribozymes, and antisense nucleic acid molecules; and / or (b) A drug that inhibits the activity of the GPR182 protein specifically binds to the GPR182 protein and inhibits the binding of one or more endogenous ligands to the GPR182 protein, wherein the drug is selected from antibodies, Fc fusion polypeptides, adonectin, afibody, affin, antikalin, atrimers, avimers, everybodys, Kunitz-type domains, designed ankyrin repeat proteins (DARPin), finomers, peptides or peptide mimes, aptamers, and small molecules; or any combination thereof and / or hetero- or homo-oligomeric covalent or non-covalent complexes.

2. The agent for use according to claim 1, wherein the binding of the agent to the GPR182 protein according to claim 1(b) inhibits the GPR182-mediated internalization of one or more endogenous GPR182-ligands.

3. The agent for use according to claim 1 or 2, wherein the one or more endogenous ligands according to claim 1(b) are one or more C-X-C chemokine ligands, more preferably one or more C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 12 (CXCL12), and / or C-X-C motif chemokine ligand 13 (CXCL13).

4. The agent according to claim 1(b) specifically binds to one or more epitopes in one or more extracellular portions of the GPR182 protein, Preferably, one or more epitopes are associated with the GPR182 protein. (i) the extracellular portion of the N-terminal domain; and / or (ii) Second extracellular loop (ECL2) A drug for use according to claim 1 or 2, which is included in the above.

5. The drug according to claim 1(b) (i) exhibiting a higher binding affinity to the GPR182 protein, preferably at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more, compared to the binding affinity between one or more endogenous GPR182-ligands and the GPR182 protein; and / or (ii) 10 －６ M, 10 －７ M, or 10 －８ M or less, preferably less than 41 nM, more preferably 10 －９ M or 10 －１０ Dissociation constant less than M (K Ｄ A drug for use according to claim 1 or 2, which binds to the GPR182 protein.

6. The drug, (i) Preferably, - Tissue plasminogen activators; preferably alteplase, leteplase, and / or tenecteplase; - Streptokinase; - Anistreplasm; and - Urokinase Thrombolytic agents selected from: (ii) Preferably, - Heparin; preferably unfractionated heparin (UFH) and / or low molecular weight heparin (LMWH); - Factor Xa inhibitors; preferably apixaban and / or rivaroxaban; Furthermore - Hirudine and / or bivalirudine Anticoagulants selected from: (iii) Preferably, - Irreversible cyclooxygenase inhibitors; preferably acetylsalicylic acid (ASA) and / or triflusar; - Adenosine diphosphate (ADP) receptor inhibitors; preferably cangrelol, clopidogrel, prasugrel, ticagrelor, and / or ticlopidine; - Phosphodiesterase inhibitors; preferably cilostazol; - Protease-activated receptor-1 (PAR-1) antagonist; preferably borapaxal; - Glycoprotein IIB / IIIA inhibitors; preferably absiximab, eptifivatide, and / or tyrofiban; - Adenosine reuptake inhibitors; preferably dipyridamole; - Thromboxane inhibitors; - Thromboxane synthase inhibitors; - Thromboxane receptor antagonist; preferably tertroban Platelet aggregation inhibitors selected from: (iv) Preferably a β-blocker selected from propranolol, alprenolol, bucindolol, carteolol, carvedilol, labetalol, levobunolol, medroxalol, mepindolol, metipranolol, nadolol, oxprenolol, penbutrol, pindolol, sotalol, timolol, acebutrol, atenolol, betaxolol, bisoprolol, ceriprolol, esmolol, metoprolol, and neviborol; and (v) Preferably an angiotensin-converting enzyme inhibitor (ACE inhibitor) selected from benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril, and ramipril; (vi) Nitrate; preferably isosorbide dinitrate, isosorbide mononitrate, and / or penslit; (vii) oxygen; (viiii) One or more C-X-C chemokine receptor type 4 (CXCR4) agonists, preferably CXCL12 and / or one or more synthetic CXCR4 agonists; (ix) One or more C-X-C motif chemokine receptor type 7 (CXCR7) agonists, preferably CXCL11, CXCL12, and / or one or more synthetic CXCR7 agonists; (x) C-X-C motif chemokine ligand 10 (CXCL10); and / or (xi) C-X-C motif chemokine ligand 13 (CXCL13); A drug for use according to claim 1 or 2, which is administered in combination with or in succession to one or more of the following.

7. The drug, (i) One or more chemotherapeutic agents; (ii) Adoptive cell therapy, preferably adoptive T cell therapy; (iii) Preferably, one or more immune checkpoint inhibitors selected from an antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA4), programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2), or a combination thereof; and / or (iv) One or more C-X-C motif chemokine receptor type 3 (CXCR3) agonists, preferably CXCL9, CXCL10, or CXCL11, and / or one or more synthetic CXCR7 agonists. A drug for use according to claim 1 or 2, which is administered in combination with or in succession to one or more of the following.

8. The drug is a (poly)peptide, and is administered in the form of a polynucleotide encoding the (poly)peptide. Preferably, polynucleotides (i) contained in a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector; or (ii) A drug for use according to claim 1 or 2, wherein RNA, preferably mRNA.

9. An agent for use according to claim 1 or 2, wherein the agent does not have substantial cytotoxic and / or immunogenic activity.

10. The agent for use according to claim 1 or 2, wherein the agent is an antibody or an Fc fusion polypeptide and does not cause substantial antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC).

11. (i) To promote tissue repair, preferably angiogenesis and / or neovascularization, after (acute) myocardial infarction; (ii) Reducing the size and / or volume of an infarct following an (acute) myocardial infarction; (iii) To increase perfusion and / or reduce the risk or severity of heart failure, myocardial necrosis, cardiac hypertrophy, cardiac fibrosis, limb ischemia, ischemia-related tissue degeneration, and / or ventricular arrhythmias following (acute) myocardial infarction; (iv) To promote angiogenesis and / or neovascularization in ischemic diseases; (v) Enhancing antitumor immunity; (vi) Enhancing tumor infiltration by T cells, preferably CD8+ cytotoxic T cells and / or CAR T cells; and / or (vii) To enhance the efficacy of adoptive cell therapy. The agent according to claim 1 or 2 for use in [the specified area].

12. The drug, (i) Unlike CXCL12, CXCL10, and / or CXCL13; and / or (ii) The agent for use according to claim 1 or 2, which is heterogeneous to the subject to which the agent is administered.

13. An in vitro method for identifying a suitable agent for use according to claim 1 or 2, comprising the step of evaluating the binding of one or more endogenous GPR182-ligands to cells expressing the GPR182 protein in the presence of a candidate agent, wherein a decrease in the binding of one or more endogenous GPR182-ligands in the presence of the candidate agent, compared to the absence of the candidate ligands, indicates that the candidate agent is suitable for use according to claim 1 or 2.

14. A method for evaluating the efficacy of the drug described in claim 1 or 2 in the treatment of myocardial infarction, wherein before and / or simultaneously with the administration of the drug, and after the administration, the following: (a) Size and volume of the infarcted area; (b) Left ventricular ejection fraction and / or left ventricular end-diastolic volume and / or left ventricular end-systolic volume; (c) Levels of CXCL12 in the heart and / or blood; and / or (d) Plasma levels of one or more biological markers of myocardial injury, preferably troponin-I and / or troponin-T This includes a step of evaluating one or more of the following: A method in which a decrease in the volume of the infarcted area, an improvement in the left ventricular ejection fraction, a decrease in the left ventricular end-diastolic volume, a decrease in the left ventricular end-systolic volume, an increase in CXCL12 concentration in the heart and / or blood, and / or a decrease in plasma levels of one or more biological markers of myocardial injury, as determined after administration of the drug, indicates that the drug is effective in treating myocardial infarction.

15. A method for evaluating the efficacy of a drug according to claim 1 or 2 in the treatment of cancer, wherein before and / or simultaneously with administration of the drug to a subject, and after administration, the following: (a) cancer and / or blood levels of CXCL9 and / or CXCL10; (b) levels of cancer and / or T cells in the blood, preferably CD8+ cytotoxic T cells; and / or (c) Size of the cancer This includes a step of evaluating one or more of the following: A method by which an increase in the concentration of CXCL9 and / or CXCL10 in the cancer and / or blood, and / or an increase in the level of T cells, preferably CD8+ cytotoxic T cells, and / or a decrease in the size of the cancer, as determined after administration of the drug, indicates that the drug is effective in treating cancer.

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