Methods to enhance the effectiveness of immunotherapy and strengthen the host immune response

Abstract

This invention relates to the field of appropriate immunotherapy for activating the immune response in patients. More specifically, the present invention relates to negative anti-EPO function modulators useful as active ingredients in pharmaceutical compositions for immunomodulatory strategies in therapies (e.g., cancer immunotherapy, therapies for infectious and inflammatory diseases) or for activating the immune system, enhancing cell-based or pharmacological or vaccine-based immunotherapy, and stimulating the immune system response of patients as needed. In particular, the present invention also describes how to restore the immune response in pathological conditions such as cancer and refractory infections, for example, by enhancing and ensuring therapeutic access to immunotherapy strategies by products consisting of EPO pathway inhibitors that can function as active pharmaceutical ingredients in vaccine compositions that stimulate the immune response in cancer, infectious diseases, and inflammatory diseases and be delivered to patients, thereby eliminating "tolerogenic" stimulation.

Description

Technical Field

[0001] The present invention relates to immunotherapy adjuvant strategies and protocols for enhancing host immune responses and avoiding immune tolerance in cancer, infectious diseases, and inflammatory diseases. In particular, the present invention also describes how to restore immune responses in pathological conditions such as cancer and refractory infectious diseases. For example, it strengthens and ensures the therapeutic access of immunotherapy strategies by a product consisting of an EPO pathway inhibitor that can function as an active pharmaceutical ingredient of a vaccine composition stimulating immune responses in cancer, infectious diseases, and inflammatory diseases and can be delivered to patients, and excludes "tolerogenic" stimuli.

Background Art

[0002] The immune system is the body's first line of defense against anything that is foreign (non-self) to the body, whether it is a microorganism or a tumor cell. Over time, they have developed a common mechanism to make the immune system "tolerogenic" by converting the immune response from an inflammatory-like response to non-self factors (tumor or infectious pathogens) to a tolerant anti-inflammatory response by the immune system in order to avoid the body's defenses.

[0003] To induce an effective immune response capable of combating infectious diseases or cancer, cells or active molecules of the innate immune system and the adaptive immune system must cooperate and interact. Cells or active molecules involved in innate immunity recognize molecular patterns of non-self antigens. Innate immunity includes cells such as natural killer (NK) cells, macrophages, dendritic cells (DCs), and neutrophils, as well as active molecules such as the complement system in serum. Lymphocytes and antibodies involved in adaptive immunity have the ability to recognize the structure or amino acid sequence of non-self antigens in high precision and detail.

[0004] The complex interaction mechanism between inflammatory (activated immune system) and anti-inflammatory (tolerogenic) responses also involves the microbiota playing an important role.

[0005] Symbiotic microorganisms colonize the barrier surfaces of all multicellular organisms, including human microorganisms. Over more than 500 million years, symbiotic microorganisms and their hosts have co-evolved and adapted to each other. As a result, the symbiotic microbiome influences many immune and non-immune functions of the host, and in effect, the two together constitute a single meta-organism. The symbiotic microbiome communicates with the host through bioactive molecules. Imbalances in the microbiome can play a crucial role in the development of multiple diseases as part of a common mechanism, including cancer, autoimmune diseases, and increased susceptibility to infection. The gut microbiome also plays a role in the anti-cancer response; an unhealthy microbiome composition lacking immunostimulatory bacteria or containing immunosuppressive species leads to treatment failure. The gut microbiome has recently been recognized as influencing the effectiveness of PD-1-based anti-cancer immunotherapy, and a healthy gut flora is a determinant of the anti-cancer response. In fact, the regulation of the immune system with respect to the symbiotic microbiome is important for avoiding cancer development, progression, and immune evasion, as well as for several regulatory effects on cancer treatment.

[0006] One example of the relationship between gut microbiota and cancer is cervical cancer, a malignant tumor caused by persistent human papillomavirus (HPV) infection, which affects more than 500,000 women annually. Over 90% of deaths from cervical cancer occur in low- and middle-income countries. A common epidemiological feature in countries with high cervical cancer incidence is a high prevalence of intestinal parasitic infections. The ability of intestinal parasites to induce immunomodulation and create a "tolerogenic" systemic immune environment provides fertile ground for the persistence of oncogenic viruses such as HPV. While animal models have shown that intestinal parasitic infections tolerate the persistence of some viruses, there is a lack of human studies specifically focused on HPV. Large-scale, systematic studies evaluating the effects of intestinal parasitic infections on the human immune system and HPV persistence could lead to improved HPV prevention strategies in parasite-endemic regions worldwide. This invention provides strategies to counter certain ways in which intestinal parasitic infections can contribute to immunomodulation and to identify novel therapeutic targets for diseases ranging from inflammatory diseases to cancer. Negative EPO regulation counteracts parasite-induced systemic and focal immunodysregulation as a possible mechanism by which chronic intestinal parasitic infections contribute to HPV persistence. Multiple common mechanisms of immune evasion-based diseases for which the EPO pathway is a key therapeutic target are analyzable. One example of these immune evasion cases, driven by the same "tolerogenic" stimuli seen in cancer, is represented by Plasmodium falciparum, the pathogen of malaria. Malaria is one of the most important human infectious diseases, particularly affecting people living in tropical and subtropical countries. Although several antimalarial drugs are currently available, malaria remains a major public health problem, with 241 million cases and 627,000 deaths annually. This infection is caused by the protozoan parasite, Plasmodium falciparum. Five types of parasites cause malaria in humans: Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, and Plasmodium falciparum. Plasmodium vivax is the most widely distributed, while Plasmodium falciparum is responsible for almost all severe and fatal cases of malaria.Immune evasion strategies are used to avoid attack from the immune response. Plasmodium falciparum evades the mosquito's immune response to transmit to a new host. The primary and crucial gene that Plasmodium falciparum uses to evade the Anopheles mosquito's immune response is Pfs47. This inhibits JNK-mediated apoptosis by blocking the activation of multiple caspases, and the Jak-STAT pathway is a downstream pathway of the EPO / EPO-R signaling pathway. These pathways are also important in regulating macrophages and lymphocytes in responses to Mycobacterium tuberculosis, HIV, and solid tumors, suggesting that immune cells may be part of a resistance strategy to pharmacological therapies. Furthermore, abnormal erythropoiesis has been observed in malaria patients, which can contribute to anemia. The relationship between hematopoietic disorders and malarial anemia has not been extensively studied, but recent findings may provide new insights. In 2005, Tsubata et al. reported that hematopoiesis is initiated in the liver and spleen after malaria infection, and that newly produced red blood cells in the liver are essential for malaria parasite infection and its continuation.

[0007] Cancer is a disease characterized by abnormal localized cell proliferation that can spread throughout the body. In the center of the tumor, oxygen supply is limited due to abnormal angiogenesis and impaired blood flow, creating a hypoxic environment. Hypoxic conditions attract Treg cells to the tumor microenvironment (TME), which suppress the function of effector T cells and promote tumor growth. This condition upregulates the expression of hypoxia-inducible factors (HIFs), including HIF-1α and HIF-1β. Hypoxia also promotes glucose uptake by cancer cells, activates glycolysis, and exacerbates glucose deficiency in the TME. Among these factors, EPO and sphingosine-1-phosphate (S1P) play a central role in conditioning the immunosuppressive tumor microenvironment. S1P is produced from sphingosine by sphingosine kinase 1 (SphK1) during apoptosis induction. S1P expresses its function via the receptor family S1PR1-5, and mouse macrophages express only S1PR1 and S1PR2. Although S1P has been identified as a findomy signal, the circulating S1P concentration (4 mM) is higher than the concentration in tissues (low nM) and the concentration released from dead cells (400 nM), suggesting that S1P may act locally or have other functions related to the removal of apoptotic cells. The findomy signal S1P released by apoptotic cells has been shown to induce HIF-1a (hypoxia-inducible factor-1a), an oxygen-sensitive subunit of the macrophage HIF complex, and since this is a master transcription factor for EPO, it suggests that dead cell-derived S1P may activate EPO signaling in macrophages. EPO receptors such as EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, and TPRs, such as the EPOR / CD131 heterodimer (EPOR), have been identified on macrophages. Through these receptors, EPO suppresses inflammatory gene expression in macrophages, suggesting its involvement in regulating the tolerance response in macrophages. Furthermore, EPO has been shown to improve outcomes in autoimmune diseases. However, it remains unclear whether EPO is involved in dead cell removal and immune tolerance.In this specification, we have found that dead cell-derived S1P activates macrophage EPO signaling, promoting immune-silent removal of dead cells and immune tolerance in mice.

[0008] This immune tolerance mechanism of immune cells is common to lung cancer, bladder cancer, prostate cancer, pancreatic cancer, ovarian cancer, cervical cancer, brain cancer, gastric cancer, colorectal cancer, and melanoma, as well as to various cancers and infections that are refractory to therapy, including tuberculosis, malaria, HIV infection, streptococcal infections, SARS, SARS-CoV, and MERS prion diseases.

[0009] Traditionally, the most common methods for treating neoplastic cancers were surgery, radiation therapy, or chemotherapy. However, in recent years, cancer immunotherapy has demonstrated great potential as a treatment for oncology. The most fundamental challenge in tumor immunotherapy is how to activate the immune system to recognize and eliminate antigens. In this regard, a new method of genetically modifying tumor cells to secrete specific cytokines has brought about significant progress in tumor immunotherapy. The theoretical basis for immunotherapy-based genetically modified tumor vaccines is that the host possesses antigens that allow it to recognize the tumor as an exogenous factor. Human T lymphocytes and B lymphocytes can distinguish almost infinite antigenic differences in the form of antigen receptors during development. However, for tumor immunotherapy to be successful, the following two criteria must be met: First, tumor cells must express novel antigens (peptides) that are not expressed in normal cells. Second, immune cells must be properly activated to recognize these antigens. Combination therapy for cancer treatment is becoming increasingly common as the benefits of attacking the disease with multiple means are recognized. Combination therapy is also useful even when resistance to anticancer drugs is demonstrated. Furthermore, combined administration offers the advantage of reducing the dosage of anticancer drugs by enhancing their efficacy. This makes it possible to increase the anticancer effect while minimizing toxicity and side effects to various organs of the body.

[0010] In recent years, several cancer immunotherapies that train or stimulate the innate immune system to recognize, attack, and eradicate tumor cells with minimal damage to healthy cells have shown promising clinical responses. However, most of these immunotherapy strategies are effective in only a limited number of patients, and some patients experience autoimmune side effects. Immunogenic cell death (ICD) induction methods not only directly kill cancer cells but also induce an anti-tumor immune response against broad solid tumors. Strategies that generate such vaccine-like functions can be used to stimulate the “cold” tumor microenvironment into an immunogenic “hot” tumor microenvironment, thereby synergistically working with immunotherapy to increase patient response rates and lead to successful treatment outcomes.

[0011] Immunotherapy that inhibits immune checkpoints such as PD-1 or CTLA-4 (e.g., abatacept) has revolutionized the treatment paradigm for many patients with advanced tumors. Immune checkpoint inhibitors are already widely used to treat patients with many types of cancer, including melanoma, lung cancer, kidney cancer, bladder cancer, and lymphoma.

[0012] However, another form of immunotherapy, called CAR T-cell therapy, and finally tumor-infiltrating lymphocyte (TIL) therapy, are also attracting considerable interest among researchers and oncologists. These therapies have shown the ability to eradicate very advanced leukemia and lymphoma. However, metabolic constraints and soluble factors form immunosuppressive TMEs, exacerbating the functional exhaustion of tumor-infiltrating T cells, leading to poor T cell proliferation and short-term T cell persistence.

[0013] In fact, compared to hematological malignancies, CAR-T cell therapy for solid tumors is limited by the immunosuppressive tumor microenvironment, which restricts the ability of CAR-T cells to migrate and infiltrate solid tumors. One strategy to mitigate these limitations is to utilize delivery routes other than systemic delivery, such as local administration, thereby eliminating the need for CAR-T cells to migrate to the affected site and limiting on-target and off-tumor toxicity by directing the on-target activity of CAR-T cells towards tumor cells and minimizing interaction with normal tissue. In preclinical models, intracerebroventricular injection of HER2 / IL13Ra2-targeted CAR-T cells has demonstrated superior therapeutic efficacy in brain metastases of breast cancer and glioblastoma (NCT02208362, NCT03389230, NCT03696030).

[0014] Another important aspect to consider is the physical tumor barrier, such as the tumor stroma, which limits the penetration and migration of CAR-T cells. The stroma is primarily composed of the extracellular matrix, also known as the matrix, and plays a decisive role in tumor invasion, adhesion, and proliferation. In the matrix, heparin sulfate proteoglycans (HSPGs), such as glypican (GPC 1-6) and syndecan (SDC 1-4), are key components that CAR-T cells must degrade in order to invade the tumor. CAR-T cells engineered to express heparanase, an enzyme that degrades HSPGs, show enhanced tumor invasion and antitumor activity. Similarly, fibroblast-activating protein (FAP)-targeted CAR-T cells show enhanced cytotoxicity by reducing tumor fibroblasts in animal models. Furthermore, strategies to induce T cells via tumor-specific chemokines are also promising, provided that a suitable chemokine receptor is expressed on the T cells.

[0015] This specification demonstrates that EPOR is expressed on T cells, as well as monocytes and monocyte-derived macrophages. Furthermore, the inventors surprisingly show that the EPO-EPOR pathway is more active in a tolerant immune system that enables the development and maintenance of cancer and infections. Based on this evidence, the inventors decided to negatively modulate the EPO pathway to modify the pathophysiological mechanism and enhance the immune system's response to pathogenic stimuli. More specifically, for example, the inventors treated glioblastoma stem cells, breast cancer cells, melanoma cells, and colorectal cancer cells with the combined administration of CAR-T cells and anti-EPO monoclonal antibodies. Surprisingly, the results revealed that the combination therapy of anti-EPO monoclonal antibodies and CAR-T cells promoted the migration and invasion of T cells into tumor tissue, and, surprisingly, showed higher killing activity against cancer cells compared to monotherapy, even though it had previously been rarely used in solid malignancies due to its low tumor invasiveness.

[0016] Therefore, several approaches have been explored to improve and enhance the CAR-T cell response. First, combination immunotherapy with CAR-T cells and checkpoint blockade (e.g., nivolumab, reratrimab, ipilimumab, anti-CD47 antibodies) that induce CAR-T infiltration and provide PD-1 / PD-L1 blockade or SIRPα / CD47 inhibition, along with chemotherapy such as cyclophosphamide, has been investigated. In hematological malignancies, combination therapy with PD-1 blockade and CD19 CAR-T cell therapy resulted in improved CAR-T cell persistence and favorable outcomes in pediatric B-ALL patients with a severe history of prior treatment. Second, tumors, particularly solid tumors, may possess cell-specific resistance mechanisms to CAR-T cell cytotoxicity, and combination immunotherapy strategies of other forms may still be necessary to counteract the inhibitory signals present in the tumor microenvironment. Indeed, loss of IFNγR signaling in tumors has been reported in clinical studies on checkpoint blockade resistance due to downstream effects on antigen presentation. In fact, deletions of genes in the interferon-γ receptor (IFNγR) signaling pathway (IFNGR1, JAK1, or JAK2) have been demonstrated to make glioblastoma and other solid tumors more resistant to CAR-T cell killing, both in vitro and in vivo. More recently, first and second-generation bispecific antibodies have been introduced into clinical practice. In cancer immunotherapy, the ability of bispecific antibodies to form "immune synapses" between tumor cells and immune effector cells such as T cells offers the potential to precisely target the immune response against specific cancer cells. Currently, third-generation T-cell engagers are being developed. These T-cell engagers consist of two bispecific antibodies, each incorporating a module for binding to the tumor antigen and half of the domain necessary for binding to the CD3 subunit of the T-cell receptor. This strategy of splitting the anti-CD3 paratope ensures that each of the two drug components remains completely inactive until it simultaneously binds to the target antigen on the same tumor cell.

[0017] In solid tumors such as brain tumors, mutations in the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2 in gliomas create an immunosuppressive microenvironment. This inhibits STAT1 expression and reduces the production of CD8 T cells, type 1-related effector molecules, and chemokines, such as CXCL10. Consistent with these findings, IDH-mutated gliomas showed significantly lower T cell infiltration compared to IDH wild-type gliomas.

[0018] One of the challenges in targeting solid tumor antigens is that they are often expressed at various levels in normal tissues as well. Therefore, antigen selection in CAR design is crucial not only for ensuring therapeutic efficacy but also for limiting "on-target, off-tumor" toxicity. One possible way to overcome the challenge of targeting solid tumor antigens present in normal tissues is to target tumor-specific post-translational modifications, such as cleaved O-glycans overexpressed in solid tumors, e.g., Tn(GalNAca1-O-Ser / Thr) and sialyl-Tn(STn)(NeuAca2-6-GalNAca1-O-Ser / Thr). In line with this consideration, we have shown that an anti-EPO antibody that specifically binds to the EV-3 isoform, a splice variant of human EPO highly expressed in tumor cells, can enhance the immune cell response without affecting normal cells. In parallel, inhibitors of EPO and EV-3 can induce ferroptosis-based cell death mechanisms. Ferroptosis, along with apoptosis, cyproptosis, autophagy, and pyroptosis, is a major cell death pathway associated with multiple human diseases, including cancer. Another important feature of CAR-T cell products is the CD4+ / CD8+ subset ratio. While CD8+ T cells have long been considered the major cytotoxic population, CD4-mediated antitumor effects have also been observed. When administered to tumor-bearing mice, CD4+ T cells with tumor-specific TCRs were found to mediate direct cytotoxicity against tumor cells while simultaneously providing a helper function to elicit an intrinsic antitumor immune response. In particular, the presence of the CD4+ subset correlated with the persistence of CAR-T cells in solid tumors. Taken together, these findings suggest an essential role in the CD4-mediated immune response against tumors, which is also highlighted in several ongoing clinical studies where CD4+ doses are strictly controlled. We have demonstrated that administration of an anti-EPO monoclonal antibody is effective in inducing CD4+ activation and migration to tumor specimens, and in suppressing tolerogenic behavior in the immune response.

[0019] However, one of the most challenging limitations of CAR-T cell therapy is the development of tumor resistance to CAR constructs targeting a single antigen, a phenomenon known as "antigen evasion," which occurs particularly in solid tumors. For example, a case report of CAR-T cell therapy targeting IL13Ra2 in glioblastoma suggested that tumor recurrence was accompanied by decreased IL13Ra2 expression. To reduce recurrence rates in CAR-T cell therapy in both hematological malignancies and solid tumors, many strategies now rely on targeting multiple antigens. These employ dual CAR constructs, or tandem CARs, to target multiple target tumor antigens within a single CAR construct. For example, in hematological malignancies (ALL and diffuse large B-cell lymphoma), these include CD19 / CD20, CD19 / CD22, or CD19 / BCMA, while in solid tumors (glioblastoma, breast cancer, colorectal cancer), they include HER2 / IL13Ra2 and two scFv such as HER2 / MUC1, TAG7228, B7-H3, MUC1, and MUC16, αvβ6, CXCR1, or CXCR2. Optimizing the selection of target antigens is important not only for improving the antitumor response but also for reducing antigen escape mechanisms and preventing recurrence.

[0020] Furthermore, recent research has focused on manipulating CARs to be resistant to immunosuppressive factors in the adversarial tumor microenvironment, such as TGFβ, IL-12, IL-15, IL-4, IL-10, arginase 1, and indoleamine 2,3-dioxygenase (IDO)-mediated inhibitory signals. Given this premise, many studies have explored numerous cytokines for creating these "armed CARs." Therefore, an interesting strategy is the manipulation of CAR-T cells to provide immunostimulatory signals in the form of stimulating cytokines that enhance T cell survival, proliferation, and antitumor activity, and re-modify the tumor microenvironment. In recent years, RNA CAR-T technology has also been proposed as a tumor therapy. In fact, current CAR-T cell manipulation methods use viral delivery vectors, which can induce persistent CAR expression and cause serious side effects. Messenger RNA (mRNA) has been explored as a promising strategy to induce transient CAR expression in T cells to mitigate viral vector-related adverse effects; however, mRNA delivery to T cells typically requires electroporation, which can be cytotoxic. Recently, ionizable lipid nanoparticles (LNPs) for mRNA delivery to human T cells have been designed. In addition to novel immunotherapy strategies based on CAR-T cells, research based on CAR-manipulated macrophages (CAR-M) and CAR-manipulated natural killer (NK) cells (CAR-NK), or CAR-GAMMA / Delta, is becoming established in the field of immuno-oncology.

[0021] Overall, the use of negative EPO function modulators is crucial for overcoming host immune tolerance to microorganisms, inducing a shift in the inflammatory response to activate macrophages and other immune system cells, and eradicating infection, in conjunction with enhanced antimicrobial or vaccine efficacy or the development of new delivery systems.

[0022] A new approach, described as a Trojan horse, has been reported that not only destroys tumor cells but also spreads a systemic immune response to destroy circulating and metastatic tumor cells.

[0023] Furthermore, sustained-release systems based on functional microparticles, microcapsules, and microspheres, for example, can ensure the achievement of long-term therapeutic levels while minimizing the number of repeated injections that affect the pathological microenvironment, such as in degenerative diseases or solid tumors.

[0024] Furthermore, the forefront of new cancer treatment approaches includes innovative nanomedicines and nanodelivery systems as tissue guns or probes, viral and nonviral vectors, nanomaterials for the delivery of bioactive drugs for targeted delivery, nanoparticle-based methods combining both therapeutic and imaging techniques for cancer diagnosis and therapy, lipid systems such as liposomes and micelles, and those based on gold or magnetic nanoparticles, as well as those combined with natural products.

[0025] When the target tissue is the central nervous system, blood-brain barrier (BBB) ​​crossing strategies such as physical stimulation (e.g., focused ultrasound (FUS) and MRI magnetic fields) or Trojan horse molecules (e.g., IL13 / IL13R, transferrin, natural or synthetic microvesicles, resveratrol) can be employed. Furthermore, nicotine increases blood-brain barrier permeability in vivo. This means that nicotinic acetylcholine receptors may be involved in regulating cerebral microvascular permeability. In addition, local administration of immunotherapy using drug-eluting embolization (DEE) microspheres as a drug delivery vehicle for direct injection into tumor nutrient arteries may increase and sustain tumor drug concentrations, reduce systemic drug exposure, and improve the risk / benefit ratio of these drugs.

[0026] Furthermore, negative functional modifiers may offer advantages in antitumor treatment regimens, even when used in combination with other natural or synthetic drugs. Flavonoids, particularly luteolin, an S1P kinase inhibitor, have been demonstrated to play a beneficial role in tumors by regulating reactive oxygen species (ROS) scavenging enzyme activity, participating in cell cycle arrest, inducing apoptosis and autophagy, and suppressing cancer cell proliferation and invasion, thus providing a synergistic additional effect to anti-EPO therapy. Rapamycin and its derivatives are promising therapeutic agents possessing both immunosuppressive and antitumor properties. Rapamycin's action is mediated through the specific inhibition of mTOR protein kinase. mTOR functions as part of an evolutionarily conserved signaling pathway that regulates the cell cycle in response to changes in trophic levels. Another molecule that regulates cellular trophic levels is metformin. Metformin is a drug widely prescribed by modern physicians due to its specific effects in the treatment and cure of type II diabetes. Metformin inhibits mTOR activity by activating ATM (ataxia telangiectasia mutated) and LKB1 (liver kinase B1), followed by activation of adenosine monophosphate-activated kinase (AMPK), thereby suppressing protein synthesis and cell proliferation. Furthermore, one of the mechanisms by which metformin acts is through the activation of AMPK, an intracellular enzyme that lowers blood glucose levels by promoting energy utilization. AMPK activation has a wide range of effects far beyond blood glucose control. Studies suggest that enhanced AMPK activity may prevent, and even reverse, the effects of aging that shorten lifespan, such as cardiovascular disease, diabetes, neurodegenerative diseases, and cancer. Moreover, strong evidence has been shown that AMPK negatively modulates the mTOR pathway, suggesting a strict interaction between mTOR and AMPK mediated by metformin. The inventors have demonstrated that metformin and anti-EPO antibodies synergistically improve cancer cell survival, reduce PD-1 gene expression, and enhance the overexpression of IFNγ and IL-1β genes. This promotes pro-inflammatory changes in the tumor and infection site microenvironment, disrupting physiological and pathological mechanisms and resulting in beneficial effects that eradicate pathogenic stimuli.

[0027] Glucagon-like peptide 1 (GLP1) agonists are widely used in the treatment of type 2 diabetes because of their low risk of hypoglycemia and blood glucose-lowering ability. GLP-1 binds to specific G protein-coupled receptors and activates downstream pathways including the cAMP / protein kinase A (PKA), cAMP / guanine nucleotide exchange factor (Epac), or phosphatidylinositol 3 kinase / PKC pathways. Also, it has just been reported that GLP-1 may be involved in carcinogenesis due to its trophic effect.

[0028] Glycoprotein non-metastatic melanoma protein B (GPNMB) interacts with integrins via its extracellular domain shed from the cell surface, and enhances tumor migration and invasion by promoting the recruitment of immunosuppressive and angiogenesis-promoting cells into the tumor microenvironment. The combination of the action on cell signaling via tumor-specific GPNMB and the ability of GPNMB to non-cell-autonomously affect the primary tumor and metastatic microenvironment enhances the malignant cancer phenotype. Furthermore, GPNMB is often overexpressed in various cancers and has become an attractive therapeutic target. From this perspective, glembatumumab vedotin, an antibody-drug conjugate (ADC) targeting GPNMB, is currently under investigation. Furthermore, GPNMB has been shown to have anti-inflammatory effects in various neurological diseases. Based on these premises, the regulation of GPNMB is considered to be promising in cancer and infectious diseases.

[0029] Overall, this solution exhibits a vaccine-like function and can be used to stimulate the microenvironment of "cold" sites, which is a common feature of cancer, infectious diseases, and inflammatory diseases, to become the immunogenic microenvironment of "hot" sites, thereby synergizing with immunotherapy to increase the response rate of patients and leading to effective treatment outcomes.

[0030] Furthermore, the present invention provides the possibility of stimulating an inflammatory host response against infectious pathogens (e.g., TBC, malaria, HIV, SARS, drug-resistant bacteria, fungi, and viruses) and enhancing the efficacy of conventional vaccines, recombinant vaccines, peptide or lipid or DNA or mRNA-based vaccines, and antimicrobial therapies.

[0031] Furthermore, the solutions presented herein may be useful means of counteracting the negative associated mechanisms and effects caused by atmospheric flight or space exploration, orbital flight and suborbital flight, as well as the suppression of the host immune system caused by extreme environmental conditions such as microgravity, high altitude, low oxygen, cosmic radiation and ultraviolet exposure during space flight missions.

[0032] These beneficial effects are based on the fact that EPO is particularly involved in the modulation of the immune system as a pathogenesis mechanism in several diseases, such as cancer, infectious diseases, and autoimmune diseases.

[0033] The cancer immunotherapy adjuvant according to the present invention, when administered in combination with or alone to a cancer immunotherapy agent, activates the function of immune factors without causing in vivo side effects and enhances the anticancer action of the cancer immunotherapy agent. Therefore, it can be effectively used as a cancer immunotherapy adjuvant or as an adjuvant in vaccine-based prevention or therapy.

[0034] The tumor microenvironment (TME) acts as a major barrier to infiltrating T lymphocytes, for example, suppressing their function. Several immune checkpoint proteins have been identified that interfere with ligand / receptor interactions and inhibit the antitumor response of T cells. Immunotherapy that blocks immune checkpoints has revolutionized the treatment paradigm for many patients with advanced tumors. However, metabolic constraints and soluble factors present within the TME exacerbate the functional exhaustion of tumor-infiltrating T cells. Using negative modulators of EPO offers a strategy to enhance immunotherapy strategies and improve the efficacy of current therapies. Similar characteristics are observed in several refractory infections, such as malaria and HIV, where, paradoxically, EPO is sometimes used to counteract iatrogenic anemia associated with antimicrobial and antiviral therapy. In this context, EPO induces a deficiency in the immune response, leading macrophages and lymphocytes to immune tolerance. The present invention can enhance the effectiveness of antimicrobial therapies such as antibiotics (penicillin, macrolides, cephalosporins, fluoroquinolones, enhanced β-lactams, tetracyclines, trimethoprim-sulfamethoxazole, urinary tract antiinfectives, lincosamide antivirals (reverse transcriptase inhibitors, protease inhibitors, and antiviral agents)), antibacterial / antifungal agents (polyenes, azoles, allylamines, and echinocandins), antimalarial therapy, and combination therapy in prion diseases. [Overview of the project]

[0035] The inventors have surprisingly found that blocking EPO can overcome the regulatory mechanisms of innate and adaptive immune responses common to tumors, infections, and inflammatory diseases, thereby inhibiting disease resistance and progression, and enabling the development of novel therapeutic strategies to eliminate "tolerogenic" stimuli of the immune system. The present invention relates to a negative functional modulator of EPO for use in methods of activating the innate and adaptive immune responses of patients as needed. Surprisingly, the compound has been found to be active in inducing the migration of immune cells, inducing engraftment in tumor tissue, as well as in immune stimulation of immune cells, enhancing the cytotoxicity of immune cells, and counteracting infectious processes and enhancing vaccine efficacy.

[0036] EPO is particularly involved in regulating the immune system as a pathogenesis mechanism in several diseases, such as cancer, infectious diseases, and autoimmune diseases.

[0037] In a first embodiment, the present invention relates to a negative anti-EPO function modulator, or an anti-EPO antigen-binding fragment selected from the group consisting of Fab, -F(ab')2, single-chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies, for use in a method of activating a patient's immune response as needed.

[0038] The immunotherapy adjuvant according to the present invention can be effectively used as an immunotherapy adjuvant because, when administered in combination with an immunotherapy agent or alone, it activates the function of immune factors in cancer, infection, and inflammatory diseases without causing in vivo side effects.

[0039] In a further embodiment, the present invention relates to a method for activating or enhancing the immune response of a patient as needed, wherein the method involves the use of a negative anti-EPO function modulator or an anti-EPO antigen-binding fragment alone or - Checkpoint inhibitor or immunomodulatory agent (e.g., anti-PDL1 antibody; nivolumab; ipilimumab, abatacept, glenbatumumab vedotin) therapy; and / or - Cell-based immunotherapy (therapies based on CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic cells activated against tumor-associated antigens, and / or antigen-presenting cells, tumor-associated peptides, engineered monocyte-macrophages, or polymorphonuclear cells), and / or therapies that enhance and reprogram the response of tumor-associated lymphocytes (TILs) and / or tumor-associated macrophages (TAMs); - Antimicrobial therapy (e.g., antibiotics; antiviral agents, antifungal agents, antiprion agents) -Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists, - Vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids for prophylactic or therapeutic purposes (e.g., anti-HPV, anti-EBV, or anti-HIV vaccines); - Immunotherapy based on oncolytic viruses; - Chemotherapy agents; - Anticancer drugs; - Enzymes that break down heparin sulfate proteoglycans (e.g., heparanase); - Negative functional modulator of the sphingosine-1-phosphate (S1P) signaling pathway, or - EPO mimetic agents that maintain red blood cell production function Including use in combination with, The anti-EPO antigen-binding fragment is selected from the group consisting of Fab, -F(ab')2, single-chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies; the negative anti-EPO function modifier is selected from the group consisting of anti-EPO monospecific or multispecific antibodies, gene therapy, DNA decoys, RNA decoys, ribozymes, antagonist miRs, shRNA, LNA, siRNA, antisense oligonucleotides, or anti-Epo receptors; and the anti-Epo receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, and EPOR / CD131 heterodimers.

[0040] In a second aspect of this specification, negative functional modulators of EPO / EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, e.g., EPOR / CD131 heterodimer) and / or their natural or synthetic variants, - Checkpoint inhibitor or immunomodulatory agent (e.g., anti-PDL1 antibody; nivolumab; ipilimumab, abatacept, glenbatumumab vedotin) therapy; and / or - Cell-based immunotherapy (therapy based on CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic cells activated against tumor-associated antigens, engineered monocyte-macrophages or polymorphonuclear cells), and / or therapies that enhance and reprogram the response of tumor-associated lymphocytes (TILs) and / or tumor-associated macrophages (TAMs), - Peptides or antibodies, diabodies, and nanobodies against natural and synthetic variants of erythropoietin, including physiological and pathological splicing variants and post-translational modifications; - Antimicrobial therapy (e.g., antibiotics; antiviral agents, antifungal agents, antiprion agents) -Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids for prophylactic or therapeutic purposes (e.g., anti-HPV, anti-EBV, or anti-HIV vaccines); - Immunotherapy based on oncolytic viruses; - Chemotherapy drugs; - Anticancer drugs; - Enzymes that break down heparin sulfate proteoglycans (e.g., heparanase); - A negative functional modulator of the sphingosine-1-phosphate (S1P) signaling pathway; or - EPO mimetic agents that maintain red blood cell production function One or more components selected from the group consisting of and A pharmaceutical kit containing [the specified ingredient] is listed.

[0041] In a third embodiment, the present invention relates to a diagnostic or prognostic method for evaluating the expression of EPO and its somatic mutations or variants, EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factor, TPR, EPOR / CD131 heterodimer) and their somatic mutations and / or variants and / or C4 mAb ligands to predict a response to negatively targeted EPO / EPOR modulated therapy, for the purpose of individualizing therapy, stratifying patients, and optimizing patient responses in immunotherapy or prophylaxis for cancer and infectious diseases, the method comprising the step of measuring / detecting the presence of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants as diagnostic or prognostic markers in tissue, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or induced extracellular vesicles).

[0042] In a fourth aspect, the present invention relates to a diagnostic method for evaluating promoter methylation of EPO and EPO receptor genes (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factor, TPR, e.g., EPOR / CD131 heterodimer) that can predict increased EPO / EPO-R expression and their negative role in immune system inhibition in the eradication of cancer cells or microbial pathogens, for the purpose of individualizing immunotherapy or prevention and prognostic purposes, comprising the step of detecting methylation of EPO and EPO receptor genes as a diagnostic marker or prognostic marker in tissue, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or induced extracellular vesicles).

[0043] In further embodiments, the present invention relates to innovative nanomedicines and nanodelivery systems as tissue guns or probes, viral and nonviral vectors, nanomaterials for the delivery of bioactive drugs for targeted delivery, plant-based vesicles, nanoparticle-based methods enabling both therapeutic and in vivo imaging for diagnostic and therapeutic purposes, lipid systems such as liposomes and micelles, gold or magnetic nanoparticles, as well as combinations with natural products, functional nanoparticles, biomaterials such as microspheres and PEGs, and negative EPO / EPOR modulator delivery systems that are administered orally, parenterally, intra-tumor and intra-cavity, intraventricular, intraarachnoid, intranasal or topically at clinical administration, based on a Trojan horse approach as micropumps for releasing therapeutic agents into tissues, for enhancing modulatory effects on the immune system in local pathological microenvironments or for inducing and enhancing homing of cell-based immunotherapy or vaccines.

[0044] In a further embodiment, the present invention relates to a negative EPO / EPOR-based method for reprogramming tumor-associated immune cells to avoid immune system exhaustion and immune tolerance.

[0045] In further embodiments, the present invention relates to a method based on negative EPO / EPOR modulation for improving the efficacy of CAR-T and CAR-γ / δT cell therapy for solid tumors.

[0046] In a further embodiment, the present invention relates to EPO / EPO-R and their variant inhibitors, which enable stimulation of CTL infiltration and suppression of immunosuppressive cell recruitment in tumors and infectious diseases, and increase tissue infiltration of inflammatory cells and immune cells.

[0047] In a further embodiment, the present invention relates to EPO / EPO-R and their variant inhibitors that can induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferroptosis, autophagy, cyproptosis, and immunostimulatory cell death that enhances tumor immunogenicity.

[0048] Further embodiments describe products selected from EPOs and their natural and synthetic mutant inhibitors that can activate immune responses against cancer and infectious pathogens, reprogram the tumor microenvironment, and enhance immunotherapy and immunomodulatory strategies. [Brief explanation of the drawing]

[0049] The features and advantages of the present invention will become apparent from the detailed description below, the examples shown for illustrative purposes and not intended to limit, and the accompanying drawings 1 to 19.

[0050] [Figure 1] Anti-EPO treatment increases the migration of peripheral blood mononuclear cells (PBMCs) in the presence of GBM tissue. Figure 1A is a schematic diagram of the modified Boyden chamber used in the chemotactic assay for the migration test, with GBM tissue shown at the bottom of the lower compartment. The migration test was performed on PBMCs stained with Hoechst (blue) after 48 hours of incubation under the following conditions: GBM tissue in CTR medium (CTR, Figure 1B), 100 μg / mL anti-EPO antibody (C4) (Figure 1C), and recombinant human EPO (rhEPO, Figure 1D). Figure 1E shows the total number of migrating cells counted using the ImageJ Analyze Particle plugin. Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05; compared to CTR for all treatment groups. [Figure 2] Anti-EPO treatment induces differentiation, activation, and expression of natural killer markers in PBMCs. The immunophenotypic profiles of PBMCs were evaluated by flow cytometry. Figure 2A shows the marker expression ratios in PBMCs after the following treatments: CTR, anti-EPO (C4), and rhEPO. Data are shown in comparison to the CTR condition. Figure 2B shows the analysis of markers used, comparing PBMCs treated with C4 and those treated with rhEPO. Data are mean ± SD across 3 replicates from at least 3 experiments. For all treatment groups, *P<0.05 (compared to control); **P<0.01 (compared to control). [Figure 3]Anti-EPO treatment induces EPOR-dependent PBMC migration. Figure 3A shows a schematic diagram of the modified Boyden chamber used in the migration study, with GBM tissue shown at the bottom of the lower compartment. The red arrow indicates the lower surface where the photographs were taken. Figure 3B shows representative images of immunofluorescence analysis for CD8 (green), CD14 (red), and EPOR (gray). The nuclei were counterstained with Hoechst. Figure 3C shows the total number of cells present in the stained sample from the lower well 48 hours after migration, particularly the total number of all CD8+ T cells (green bars) and CD14+ monocytes (red bars, Figure 3D), from which specific subpopulations of double-positive cells, EPOR+CD8+ cells (green bars) and EPOR+CD14+ cells (red bars), were calculated (Figure 3E). n=10. Fields of view (FOV) were acquired at 63x magnification, with an average total number of analyzed cells n=500 / sample. [Figure 4] Anti-EPO treatment induces a molecular signature of PBMC activation and blocks exhaustion. Gene expression of markers related to inflammation and exhaustion was analyzed. Analysis was performed by evaluating gene expression profiles in human PBMCs using real-time PCR. Analysis was performed on the L-1b (Figure 4A), IL-6 (Figure 4B), IFNg (Figure 4C), IL-10 (Figure 4D), TGFb (Figure 4E), IFNa2 (Figure 4F), PD1 (Figure 4G), LAG-3 (Figure 4H), and CTLA4 (Figure 4I) genes. Data are mean ± SD over 3 replicates from at least 3 experiments. P<0.05 (compared to control) for all treatment groups. [Figure 5]Anti-EPO treatment induces monocyte migration and activation. For monocytes, we investigated the migratory ability after anti-EPO (C4) treatment in a modified Boyden chamber (Figure 5). A migration test was performed after 6 days using cells stained with calcein (green), and the number of cells that migrated to the bottom of the well was counted under the following conditions: CTR medium (CTR, Figure 5A), C4 10 μg / mL (Figure 5B), and recombinant human EPO (rhEPO, Figure 5C). Figure 5D shows the total number of migrating cells counted using the ImageJ Analyze Particle plugin. To evaluate the immunophenotypic profile of infiltrating macrophage monocyte-derived cells, CD86+ / HLA-DR+ expression was evaluated under the following conditions, and differentiation to the M1 phenotype was assessed: CTR, TMZ, C4, TMZ+C4, and rhEPO (Figure 5E). Data are mean ± SD across 3 replicates from at least 3 experiments. *P<0.05; Compared to CTR for all treatment groups. Data are the mean ± SD of 3 trials in at least 3 experiments. *P<0.05; Compared to CTR for all treatment groups. [Figure 6] Anti-EPO treatment induces macrophage infiltration into GBM tissue and their differentiation into the M1 phenotype. Immunophenotypic profiles of infiltrating macrophages were evaluated by CD86 and HLA-DR expression under the following conditions: CTR, TMZ, C4, TMZ+C4, and rhEPO (Figure 6A). The histogram in Figure 6B shows the quantification of infiltrating macrophages in GBM tissue. Co-expression of CD86+ / HLA-DR+ (Figure 6C) and CD86+ (Figure 6D) was evaluated in treated macrophage cells, demonstrating differentiation into the M1 phenotype. Data are mean ± SD across 3 replicates from at least 3 experiments. *P<0.05; compared to CTR for all treatment groups. [Figure 7]Anti-EPO treatment induces differentiation of PBMC-derived monocytes into macrophages via EPOR surface expression. Cells were fixed, labeled with a liquid solution, and (A, B) phalloidin and EPOR (C, D) were detected in PBMC-derived macrophages. Figures E and DF show the co-localization of phalloidin and EPOR. Nuclei were stained with DAPI (blue) (scale = 100 μm). Representative images of C4 are shown (Figures A, C, E) and rhEPO (B, D, F) administration. [Figure 8] Anti-EPO administration induces naive T cell induction. Naive T cells were analyzed to evaluate their migratory ability after anti-EPO treatment. A modified Boyden chamber was used. Chemotaxis assays were performed on cells stained with calcein (green) after 48 hours under the following conditions: CTR medium (CTR, Figure 8A), C4 10 μg / mL (Figure 8B), recombinant human EPO (rhEPO, Figure 8C), and C4+rhEPO (Figure 8D). Figure 8E shows the total number of migrating cells counted using the ImageJ Analyze Particle plugin. CD8+ migration was also performed in the presence of several negative functional modifiers of EPO (Figure 8F). For this purpose, we tested commercially available antibodies B4 (Santa Cruz Biotechnology) and 16FH11 (StemCell Technologies). Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05; Compared with CTR for all treatment groups. [Figure 9] Anti-EPO treatment induces T cell activation in GBM TMEs through EPOR expression. Naive CD4+ T cells were cultured in GSC-conditioned medium treated under the following conditions: CTR (Figure 9A), temozolomide (TMZ, Figure 9B), rhEPO (Figure 9C), anti-EPO (C4) (Figure 9D), TMZ+C4 (Figure 9E), and rhEPO+C4 (Figure 9F). Immunophenotypic profiles were determined by evaluating EPOR and CD69+ expression under the following conditions: CTR, TMZ, C4, TMZ+C4, rhEPO, and rhEPO+C4 (Figure 9G). Data are mean ± SD across 3 replicates from at least 3 experiments. **P<0.01, ***P<0.001 (compared to CTR for all treatment groups). [Figure 10] Anti-EPO treatment induces CD4+ migration of CTL lymphocytes in TMEs. We investigated the migratory ability of cytotoxic CD4+ T cells. A modified Boyden chamber was used (Figure 10A). The migration test was performed on cells stained with calcein (green) after 48 hours. After treatment with conditioned medium recovered from GSCs treated under the following conditions, the number of migrating cells at the bottom of the well was counted (Figure 10B): CTR medium (CTR, Figure 10C), TMZ 100μ (Figure 10D), C4 10μg / mL (Figure 10E), TMZ+C4 (Figure 10F), and recombinant human EPO (rhEPO, Figure 10G). Figure 10B shows the total number of migrating cells counted using the ImageJ Analyze Particle plugin. Data are mean ± SD across 3 replicates from at least 3 experiments. **P<0.01, ***P<0.001; compared with CTR for all treatment groups. [Figure 11] Anti-EPO treatment induces deep infiltration of CTL lymphocytes into GBM tissue. We investigated the anti-EPO (C4) migration ability of CTL T lymphocytes in the presence of GBM tissue. A modified Boyden chamber was used. GBM tissue fragments were placed at the bottom of the wells (Figure 11A). Migration tests were performed 48 hours later on cells stained with calcein (green). Analysis was performed by counting the number of cells that migrated to the bottom of the wells after treatment under the following conditions (Figure 11B): CTR medium (CTR, Figure 11C), TMZ 100μg (Figure 11D), C4 10μg / mL (Figure 11E), TMZ+C4 (Figure 11F), recombinant human EPO (rhEPO, Figure 11G), and rhEPO+C4 (Figure 11H). Figure 11B shows the total number of migrating cells counted at the bottom of the wells using the ImageJ Analyze Particle plugin. Data are mean ± SD across 3 replicates of at least 3 experiments. **P<0.01, ***P<0.001 (compared to CTR for all treatment groups), and #P<0.05 (compared to rhEPO). [Figure 12]Anti-EPO treatment induces migration of CTL lymphocytes to GBM tissue. A migration assay was performed to evaluate the ability of CD4+ CTL T cells to infiltrate GBM tissue. Cells were stained with calcein and migrated in a modified Boyden chamber for 48 hours (Figure 12A). GBM tissue was then collected, digested by trypsin, and filtered. Single-cell suspensions were analyzed using a flow cytometer, and infiltrating cells were counted as events. Figure 12B shows the total number of infiltrating cells. Data are mean ± SD over 3 replicates from at least 3 experiments ***P<0.01; compared to CTR for all treatment groups. [Figure 13] EPO-related target expression on GBM cells. Expression patterns of EPO-related genes, transferrin-related genes, and IL-13-related genes in CTR and GBM mRNA extracted from cells, as determined by real-time PCR. A) Expression of EGFR, EPOR, EPHB4, CSF2RB, and CRLF3 in control and GBM; B) Expression of IL-13, IL-13R1, and IL-13R2; c) Expression of transferrin receptor 1 (TfR1), transferrin receptor 2 (TfR2), and folate receptor in control and GBM. Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05, compared to CTR for all treatment groups. [Figure 14] CAR-T cell toxicity is enhanced by anti-EPO treatment of GBM cells. To investigate the cytotoxic activity of T cells, WT T cells and CAR-T cells were co-cultured with glioblastoma stem cells for 48 hours, either alone or in combination with C4. Immuno-T cells and GSCs were co-cultured at effector / target (E:T) ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64, and cytotoxicity was measured (Figure 14A). Cytokines, specifically TNFα (Figure 14B) and IFNγ (Figure 14C), were obtained in the supernatant from 48-hour co-cultures of CAR-T cells and GSCs. Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05; compared to CTR for all treatment groups. [Figure 15]CAR-T cell toxicity is enhanced by anti-EPO treatment of breast cancer, pancreatic cancer, and melanoma cells. To investigate T cell cytotoxicity, WT cells, CAR-T cells, and C4 were co-cultured for 48 hours with breast cancer (MCF7, Figure 15A), melanoma (Figure 15B), and colorectal cancer cells (DLD1, Figure 15C). Cytotoxicity was measured by co-culturing immune T cells with breast cancer, melanoma, and colorectal cancer cells at effector / target (E:T) ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 (Figures 15A-C). Data are mean ± SD over 3 replicates of at least 3 experiments. *P<0.05; compared to CTR for all treatment groups. [Figure 16] CAR-T cell toxicity is synergistically enhanced in glioblastoma cancer cells by co-treatment with anti-EPO and metformin. The combined treatment of anti-EPO (C4) and T cell cytotoxicity was investigated even when metformin was administered concomitantly. WT cells and CAR-T cells were co-cultured with GSCs for 48 hours with either C4 alone or in combination with C4 and / or metformin (Figure 16A). Cytotoxicity was measured by co-culturing immune T cells and glioblastoma cancer cells with effector / target (E:T) ratios of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32 (Figure 16A). Gene expression of IL-1β (Figure 16B), IFNγ (Figure 16C), and PD-1 (Figure 16D) was evaluated after co-administration of anti-EPO (C4) and metformin (MET), Figure 16B. Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05; Compared with CTR for all treatment groups. [Figure 17] Anti-EPO treatment stimulates solid tumor invasion and immune cell infiltration into a GBM subcutaneous PDX mouse model as an example of efficacy. Representative images of intratumoral inflammatory cell infiltration by hematoxylin and eosin staining. Low-density inflammatory cell infiltration in placebo-treated PDX tumors (a: ×100; b: ×400, Figure 17A, B). High-density inflammatory cell infiltration in PDX tumors treated with 10 mg / Kg anti-EPO antibody (c: ×100, d: ×400, Figure 17C, D). [Figure 18]Anti-EPO treatment reduces parasites in blood cells. Parasitemia curves of infected red blood cells after placebo (wild-type) or anti-EPO antibody exposure. Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05; compared to CTR for all treatment groups. [Figure 19] Anti-EPO treatment increases PBMC migration in infections. Figure 19A is a schematic diagram of the chemotactic assay and modified Boyden chamber used in the migration test, showing the migratory ability of PBMCs after anti-EPO(C4) treatment in the presence of lipopolysaccharide (LPS). 10 ug / mL of LPS (Figure 19B), as well as anti-EPO(C4) (Figure 19c) and rhEPO (Figure 19D) were administered for 48 hours. PBMC migration was evaluated using the modified Boyden chamber, and the number of migrating cells was counted using the ImageJ Analyze Particle plugin (Figure 19E). Data are mean ± SD over 3 replicates from at least 3 experiments. *P<0.05; compared to CTR for all treatment groups. [Figure 20] In an in vivo orthotopic rodent brain tumor model, anti-EPO administration significantly induced immune system activation accompanied by T lymphocyte migration compared to the healthy contralateral hemisphere (Figure 20A) (Figure 20B). Surprisingly, anti-EPO treatment induced ferroptosis in cancer cells, serving as a marker of cell death (Figure 20C). [Modes for carrying out the invention]

[0051] The present invention provides, as herein, methods and uses for modulating the immune system based on the negative regulation of EPO and EPO receptors, as well as their natural splicing and synthetic variants. Pharmaceutical compositions and therapeutic methods are also provided.

[0052] In another embodiment, the present invention provides, as herein, methods and uses for modulating the immune system based on canonical EPO receptors and alternative receptors (EPO-R; EPBH4; CSF2RB; CD131; CRLF3, soluble EPO-R, tissue protective factors, TPR, e.g., EPOR / CD131 heterodimers) or splicing variants or somatic variants thereof or negative modulation of such variants.

[0053] The techniques and procedures described or referenced herein are generally well understood and widely adopted by those skilled in the art using conventional methods, such as the widely used hybridoma method and phage display technique.

[0054] This invention describes a method for activating the immune system against tumors and infectious pathogens, either alone or in combination with standard therapy.

[0055] In a first embodiment, the present invention relates to anti-EPO negative functional modifiers, or anti-EPO antigen-binding fragments selected from the group consisting of Fab, -F(ab')2, single-chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies, for use in a method of activating a patient's immune response as needed.

[0056] In a further embodiment, the present invention relates to a method for activating or enhancing the immune response of a patient as needed, using a negative anti-EPO function modulator or an anti-EPO antigen-binding fragment alone or - Checkpoint inhibitor or immunomodulatory agent (e.g., anti-PDL1 antibody; nivolumab; ipilimumab, abatacept, glenbatumumab vedotin) therapy; and / or - Cell-based immunotherapy (therapies based on CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic cells activated against tumor-associated antigens, and / or antigen-presenting cells, tumor-associated peptides, engineered monocyte-macrophages, or polymorphonuclear cells), and / or therapies that enhance and reprogram the response of tumor-associated lymphocytes (TILs) and / or tumor-associated macrophages (TAMs); - Antimicrobial therapy (e.g., antibiotics; antiviral agents, antifungal agents, antiprion agents) -Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists, - Vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids for prophylactic or therapeutic purposes (e.g., anti-HPV, anti-EBV, or anti-HIV vaccines); - Immunotherapy based on oncolytic viruses; - Chemotherapy agents; - Anticancer drugs; - Enzymes that break down heparin sulfate proteoglycans (e.g., heparanase); - Negative functional modulator of the sphingosine-1-phosphate (S1P) signaling pathway, or - EPO mimetic agents that maintain red blood cell production function Including use in combination with, The present invention provides a method in which the anti-EPO antigen-binding fragment is selected from the group consisting of Fab, -F(ab')2, single-chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies, the negative anti-EPO function modifier is selected from the group consisting of anti-EPO monospecific or multispecific antibodies, gene therapy, DNA decoys, RNA decoys, ribozymes, antagonist miRs, shRNA, LNA, siRNA, antisense oligonucleotides, or anti-Epo receptors, and the anti-Epo receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, and EPOR / CD131 heterodimers.

[0057] In a preferred embodiment, a negative anti-EPO function modulator or anti-EPO antigen-binding fragment for use according to the present invention binds to an EPO receptor selected from the group consisting of EPO, EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, EPOR / CD131 heterodimers, and their variants or somatic mutations.

[0058] Preferably, the modulator or anti-EPO antigen-binding fragment is an immunotherapy adjuvant and / or elicits an immune cell (T helper lymphocytes and / or T cytotoxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or activated dendritic cells, antigen-presenting cells) response, the cell response being an antitumor T cell response or antimicrobial cell response in a patient suffering from cancer or infection.

[0059] In a more preferred embodiment, in the use of a negative functional modifier or anti-EPO antigen-binding fragment according to the present invention, or in a method for activating or enhancing the immune response of a patient as needed, the antitumor T helper lymphocytes and / or T cell-toxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells are immune cells derived from PBMCs.

[0060] Preferably, in the use of a negative functional modifier or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response of a patient as needed, the antitumor T cell response is a CD8+ and CD4+ T cell response.

[0061] Preferably, in the use of negative functional modulodies or anti-EPO antigen-binding fragments, or in methods for activating or enhancing the immune response of a patient as needed, the antitumor T cell response is due to the expression of CD14, CD69, and EPOR in migrating PBMCs.

[0062] In a preferred embodiment, in the use of a negative functional modifier or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response of a patient as needed, the anti-EPO antigen-binding fragment is a neutralizing antibody that binds to EPO, an EPO variant, or an EPO receptor and restores T helper lymphocytes and / or T cytotoxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or activated dendritic cells.

[0063] In a more preferred embodiment, in a negative functional modifier or anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response of a patient as needed, the anti-EPO antigen-binding fragment is a neutralizing antibody selected from the group consisting of C4, B4, and 16F1H11.

[0064] One example described in the present invention relates to a purified anti-erythropoietin (EPO) antibody (sometimes referred to as "C4 antibody" or "C4"), wherein the antibody is a. Variable domain of the light chain (VL) having the amino acid sequence of SEQ ID NO: 6; and b. Variable domain of the heavy chain (VH) having the amino acid sequence of SEQ ID NO: 14 Includes.

[0065] A hybridoma producing the C4 antibody according to the present invention, comprising a variable domain of the light chain (VL) having the amino acid sequence of SEQ ID NO: 6, and a variable domain of the heavy chain (VH) having the amino acid sequence of SEQ ID NO: 14, was deposited with the Leibniz-Institute DSMZ on September 9, 2021, under accession number DSM ACC 3370.

[0066] In a preferred embodiment, the antibody of the present invention is an isolated anti-EPO antibody, the antibody comprising six CDR regions, the CDR regions being a. VL-CDR1 having the amino acid sequence of SEQ ID NO: 4; b. VL-CDR2 having the amino acid sequence (Gly-Ala-Ser) of GAS; c. VL-CDR3 having the amino acid sequence of SEQ ID NO: 5; d. VH-CDR1 having the amino acid sequence of SEQ ID NO: 11; e. VH-CDR2 having the amino acid sequence of SEQ ID NO: 12; and H-CDR3 with the amino acid sequence of fV SEQ ID NO: 13 That is the case.

[0067] For the purposes of this disclosure, each sequence has the following corresponding sequence number: Sequence ID 1 is the DNA sequence of the variable light chain CDR1 region (VL-CDR1) of the anti-EPO antibody: GAAAGTGTTGACTATTATGGCACAGGTTTA It corresponds to this. GGTGCATCC corresponds to the DNA sequence of the variable light chain CDR2 region (VL-CDR2) of the anti-EPO antibody. Sequence ID 2 is the DNA sequence of the variable light chain CDR3 region (VL-CDR3) of the anti-EPO antibody: CAGCAAACTAGGAAGGTTCCTTCGACG It corresponds to this. Variable light chain DNA sequence of Sequence ID No. 3 (333 bp, CDR is underlined: FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4): GATATCGTTCTCACTCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGT GAAAGTGTTGACTATTATGGCACAGGTTTA ATGCAGTGGTACCAACAGAGACCAGGACAGCCACCCAAACTCCTCATCTAT GGTGCATCCAACGTAGGATCTGGGGTCCCTGCCAGGTTTAGCGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGGGGATGATATTGCAATGTATTTCTGT CAGCAAACTAGGAAGGTTCCTTCGACG TTCGGTGGAGGCACCAAGTTGGAAATCAAA Sequence ID 4 is the amino acid sequence of the CDR1 region (VL-CDR1) of the variable light chain of the anti-EPO antibody: ESVDYYGTGL It corresponds to this. GAS (Gly-Ala-Ser) corresponds to the amino acid sequence of the CDR2 region (VL-CDR2) of the variable light chain of an anti-EPO antibody. Sequence ID 5 is the amino acid sequence of the CDR3 region (VL-CDR3) of the variable light chain of the anti-EPO antibody: QQTRKVPST It corresponds to this. Variable light chain amino acid sequence of SEQ ID NO: 6 (111aa, CDR is underlined: FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4): DIVLTQSPASLAVSLGQRATISCRAS ESVDYYGTGL MQWYQQRPGQPPKLLIY GAS NVGSGVPARFSGSGSGTDFSLNIHPVEGDDIAMYFC QQTRKVPST FGGGTKLEIK Sequence ID 7 is the DNA sequence of the CDR1 region (VH-CDR1) of the variable heavy chain of the anti-EPO antibody: GGATTCACTTTCAGTACCTATACC It corresponds to this. Sequence ID 8 is the DNA sequence of the CDR2 region (VH-CDR2) of the variable heavy chain of the anti-EPO antibody: ATTAGTAATGGTGGTGATAGAACC It corresponds to this. Sequence ID 9 is the DNA sequence of the CDR3 region (VH-CDR3) of the variable heavy chain of the anti-EPO antibody: GCAAGACATAATATTACTACGGTTCCCTTTACTATGGACTAC It corresponds to this. Variable heavy chain DNA sequence of Sequence ID No. 10 (363 bp, CDR is underlined: FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4): GAGGTGAAGCTGCAGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCT GGATTCACTTTCAGTACCTATACC ATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATAC ATTAGTAATGGTGGTGATAGAACC TACTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACGATGCCAAGAACACCCTGTTCTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCATGTATTACTGT GCAAGACATAATATTACTACGGTTCCCTTTACTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA Sequence ID 11 is the amino acid sequence of the CDR1 region (VH-CDR1) of the variable heavy chain of the anti-EPO antibody: GFTFSTYT It corresponds to this. Sequence ID 12 is the amino acid sequence of the CDR2 region (VH-CDR2) of the variable heavy chain of the anti-EPO antibody: ISNGGDRT It corresponds to this. Sequence ID 13 is the amino acid sequence of the CDR3 region (VH-CDR3) of the variable heavy chain of the anti-EPO antibody: ARHNITTVPFTMDY It corresponds to this. Variable heavy chain amino acid sequence (VH) of sequence number 14 (121aa, CDR is underlined: FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4): EVKLQESGGGLVQPGGSLKLSCAAS GFTFSTYT MSWVRQTPEKRLEWVAY ISNGGDRT YYPDTVKGRFTISRDDAKNTLFLQMSSLKSEDTAMYYC ARHNITTVPFTMDY WGQGTSVTVSS Sequence ID 15: EPO amino acid sequence (N-terminal signal peptide + protein chain) aa1~193 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR Sequence ID No. 16 EPO mature peptide amino acid sequence (aa28~193) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR Sequence ID 17 EPO gene sequence.

[0068] For the purposes of this invention, the expressions "negative EPO function modifier," "negative EPO / EPO-R modifier," or "negative function modifier anti-EPO" are intended to refer to agents that inhibit the function of EPO.

[0069] For the purposes of this invention, "antibody" or "monoclonal antibody" is a "negative functional regulator of human EPO." In particular, this antibody is for the mature form of EPO corresponding to amino acids (AA) 28-193 of the entire EPO amino acid sequence (SEQ ID NO: 15). The mature EPO amino acid sequence (AA28-193) is described in SEQ ID NO: 16. Human EPO is encoded by the gene sequence of SEQ ID NO: 17.

[0070] Anti-EPO antibodies are molecules capable of recognizing and binding to amino acid sequences contained in erythropoietin that are capable of direct or indirect interaction with EPO and / or direct or indirect interaction with the EPO biosynthetic pathway, and such interaction results in a decrease in EPO levels rather than a decrease in the stimulation of signaling cascades involving EPO. In further embodiments, the negative functional modifier of EPO acts on post-translationally modified EPO such as EV-3.

[0071] In a more preferred embodiment, the isolated anti-EPO antibody of the present invention is a monoclonal antibody, a chimeric antibody and / or a humanized antibody or a human antibody, an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment, and more preferably further comprises a framework sequence, at least a portion of which is a human consensus framework sequence.

[0072] The primary objective of the humanization process is to reduce the immunogenicity of antibodies in order to improve immune tolerance in humans and enhance their biophysical properties. Briefly, sequence information for the variable regions is generated by reverse transcription of total RNA extracts obtained from hybridoma cell lines. The variable regions of the heavy chain (VH) and light chain (VL) are amplified by PCR and cloned into a shuttle vector for sequencing. The sequences of each variable chain of a total of five independent clones are determined. The hybridoma sequence is determined from the sequencing results of VH and VL. To confirm affinity / binding and biological functions related to the parental mouse hybridoma, chimeric constructs combining mouse VH and VL variable regions with the human IgG1 constant region are designed and expressed. Antibody sequences are humanized by transplanting three CDRs of the light chain variable region (VL) into a human VL germline that is as homologous as possible to the mouse antibody VL. Similarly, three CDRs of the heavy chain variable region (VH) are transplanted into a human VH progenitor cell line that is as homologous as possible to the mouse antibody VH. Furthermore, since different framework contexts may add value to the resulting antibodies, CDRs are also transplanted into human VH and VL germ cell lines, which are well known to exhibit superior overall biophysical properties even with low homology. A total of 9 to 18 VH / VL combinations are generated among CDR-transplanted VH, CDR-transplanted VL, and chimeric types of both VH and VL. The XtenCHOTM platform is used to transiently produce proof-of-concept levels of each recombinant humanized antibody and evaluate its binding / biological activity / biophysical properties compared to chimeric types of parental mouse hybridomas. Comprehensive antibody affinity maturation services can be performed by phage display using custom libraries generated by random or targeted mutagenesis. The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous antibody population; that is, the individual antibodies constituting the population are identical except for any possible trace mutations, such as spontaneous mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of individual antibodies.In certain embodiments, such monoclonal antibodies typically comprise an antibody containing a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may involve selecting a unique clone from a pool of clones, such as a hybridoma clone, a phage clone, or a recombinant DNA clone. The selected target-binding sequence can be further modified, for example, to improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce immunogenicity in vivo, or create a multispecific antibody, and it should be understood that antibodies containing the modified target-binding sequence are also monoclonal antibodies of the present invention.

[0073] Furthermore, antibodies may be produced using different techniques. For example, monoclonal antibodies may be purified from cells that naturally express them, such as hybridoma cells, or they may be produced in recombinant expression systems derived from both mammalian and prokaryotes (e.g., Escherichia coli). More recently, fragment antibodies have been introduced into clinical practice. In fact, fragment antibodies have emerged as excellent tools for imaging and diagnosis because they can detect cellular proteins with high affinity and specificity. Antibody fragments include, but are not limited to, Fab, F(ab')2, single-chain antibodies, nanobodies, diabodies, triabodies, tetrabodies, and domain antibodies. These can be readily bound to radioisotopes, fluorescent molecules, or enzymes that label specific biomarkers of a patient. Also, because they have a short half-life in the body, they have rapid clearance, which is expected to reduce the risk of side effects from potentially invasive diagnostic agents. If necessary, the affinity of the monoclonal or fragment antibody of the present invention, which contains one or more of the above CDRs, can be improved by an affinity maturation process.

[0074] Preferably, the antibodies described herein are full-length monoclonal antibodies and / or bispecific anti-EPO antibodies. The anti-EPO antibody has an amino acid sequence in which the VL of SEQ ID NO: 6 and the VH of SEQ ID NO: 14 are identical, or which contain 0, 1, 2, or 3 amino acid residue substitutions.

[0075] The advantageous characteristics of the negative EPO regulation of the present invention are shown in the examples.

[0076] In a more preferred embodiment, the use of a negative functional modifier or anti-EPO antigen-binding fragment in a method for activating or enhancing a patient's immune response as needed, wherein the negative functional modifier or anti-EPO antigen-binding fragment elicits T helper lymphocytes and / or T cytotoxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells and / or antigen-presenting cells, the cellular response being further enhanced by therapies based on CAR-T, CAR-M, CAR-NK, engineered monocyte-macrophage or polymorphonuclear cells, or for enhancing and reprogramming the response of tumor-associated lymphocytes (TILs) or tumor-associated macrophages (TAMs).

[0077] In a more preferred embodiment, in the use of a negative functional modifier or an anti-EPO antigen binding fragment, or in a method for activating or enhancing the immune response of a patient requiring such action, the patient requiring action is cancer, proliferative conditions, autoimmune and non-autoimmune chronic inflammatory diseases, neurodegenerative diseases, Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN). 2) The patient has type 1 neurofibromatosis, endometriosis, Crohn's disease, ulcerative colitis, neuroinflammatory or infectious disease, or has undergone organ or tissue transplantation, and the cancers include cerebral astrocytoma, cerebellar astrocytoma, pineal astrocytoma, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, glioblastoma multiforme, glioblastoma, grade II fibrous astrocytoma, protoplasmic, grade III hypertrophic, anaplastic astrocytoma, for example, cerebral glioma, pituitary adenoma, ventriculependymal cell tumor, medulloblastoma, neuroectodermal tumor, neuroblastoma, hypothalamic glioma, breast cancer, lung cancer, colorectal cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, The following are selected from the group consisting of esophageal cancer, basal cell carcinoma, bile duct cancer, splenic cancer, osteosarcoma, intraocular melanoma, retinoblastoma, gastric cancer, cardiac cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cavity / paranasal sinus cancer, salivary gland cancer, nasopharyngeal cancer, pharyngeal cancer, thyroid cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, gastric / liver cancer, colorectal cancer, rectal cancer, testicular cancer, renal cell carcinoma, melanoma, sarcoma, mesothelioma, chromaffin cell tumor, hematological malignancy or chronic myeloid leukemia, diffuse median glioma (DMG), diffuse endogenous pontine glioma (DIPG), germ cell tumor, brainstem glioma, pineal blastoma, choroid plexus papilloma, germ cell tumor, acoustic neuroma, schwannoma, meningioma, and hemangioblastoma.

[0078] In a more preferred embodiment, the cancer is glioblastoma multiforme.

[0079] In a further embodiment, the negative functional modifier or anti-EPO antigen binding fragment promotes the immune system response and prevention thereof in patients suffering from refractory or persistent infections, the persistent infections including tuberculosis, malaria, HIV, EBV, or HPV-induced precancerous lesions.

[0080] Preferably, negative functional modifiers or anti-EPO antigen-binding fragments promote the immune response in patients infected with antimicrobial therapy-resistant streptococci, staphylococci, fungi, viruses; SARS-CoV-2, SARS, MERS, prion pathogens, and / or in whom immune system tolerance exists.

[0081] More preferably, negative functional modifiers or anti-EPO antigen-binding fragments are intended for use in promoting immune system responses and enhancing the efficacy of prophylactic and therapeutic DNA and / or RNA or peptide and / or lipid-based vaccines in infectious disease and / or cancer immunotherapy.

[0082] In a second embodiment, this specification refers to negative functional modifiers of EPO / EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, e.g., EPOR / CD131 heterodimer) and / or their natural or synthetic variants, - Checkpoint inhibitor or immunomodulatory agent (e.g., anti-PDL1 antibody; nivolumab; ipilimumab, abatacept, glenbatumumab vedotin) therapy; and / or - Cell-based immunotherapy (therapy based on CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic cells and / or antigen-presenting cells activated against tumor-associated antigens, engineered monocyte-macrophages or polymorphonuclear cells, and / or therapies that enhance and reprogram the response of tumor-associated lymphocytes (TILs) or tumor-associated macrophages (TAMs)), - Peptides or antibodies, diabodies, and nanobodies against natural and synthetic variants of erythropoietin, including physiological and pathological splicing variants and post-translational modifications; - Antimicrobial therapy (e.g., antibiotics; antiviral agents, antifungal agents, antiprion agents) -Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids for prophylactic or therapeutic purposes (e.g., anti-HPV, anti-EBV, or anti-HIV vaccines); - Immunotherapy based on oncolytic viruses; - Chemotherapy drugs; - Anticancer drugs; - Enzymes that break down heparin sulfate proteoglycans (e.g., heparanase); - A negative functional modulator of the sphingosine-1-phosphate (S1P) signaling pathway; or - EPO mimetic agents that maintain red blood cell production function One or more components selected from the group consisting of and A pharmaceutical kit containing [the specified ingredient] is listed.

[0083] In a third aspect, the present invention relates to a diagnostic or prognostic method for evaluating the expression of EPO and its somatic mutations or variants, EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factor, TPR, EPOR / CD131 heterodimer) and somatic mutations and / or variants thereof, and C4 mAb ligands, for the purpose of individualizing therapy, stratifying patients, and optimizing patient responses in immunotherapy or prophylaxis for cancer and infectious diseases, the method comprising the step of measuring / detecting the presence of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants thereof, as diagnostic or prognostic markers, in tissue, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or induced extracellular vesicles).

[0084] In a fourth aspect, the present invention relates to a diagnostic method for evaluating promoter methylation of EPO and EPO receptor genes (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factor, TPR, e.g., EPOR / CD131 heterodimer) that can predict increased expression of EPO / EPO-R, C4 ligand and their negative role in immune system inhibition in the eradication of cancer cells or microbial pathogens, for the purpose of individualizing immunotherapy or prophylaxis and for prognostic purposes, comprising the step of detecting methylation of EPO and EPO receptor genes as a diagnostic marker or prognostic marker in tissue, cell, or human bodily fluid (saliva, blood, cerebrospinal fluid, sweat, or induced extracellular vesicles).

[0085] As an immunotherapy or immunotherapy adjuvant protocol according to the present invention, compounds represented by EPO and its variants and / or EPO receptors and negative modulators of their variants, as shown in Formula 1, or pharmaceutically acceptable salts thereof, can be administered orally, parenterally, intra-tumorally (intra-cavity, intra-cavity), intraventricularly, intraarachnoidally, intranasally, or topically in various formulations at the time of clinical administration. More preferably, these may be parenteral formulations. The methods described in the present invention may also include the preparation of compounds represented by Formula 1 or pharmaceutically acceptable salts thereof for oral or parenteral administration by mixing them with commonly used diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. These solid formulations are prepared by mixing compounds represented by Formula 1 or pharmaceutically acceptable salts thereof with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, and gelatin. Aside from simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid formulations for oral administration include suspensions, solutions, emulsions, and syrups, and these formulations may contain various excipients such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble excipients, suspensions, and emulsions. Water-insoluble excipients and suspensions may contain one or more active compounds, as well as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injection esters such as ethylolate.

[0086] The cancer immunotherapy adjuvants described herein may contain a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and may be administered by parenteral administration, which includes subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection.

[0087] To prepare a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as a parenteral administration formulation, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is mixed with a stabilizer or buffer in water to form a solution or suspension, which is then formulated as an ampoule or vial. The compositions herein can be sterilized. Further comprising preservatives, stabilizers, hydrates or emulsifiers, salts and / or buffers for osmotic pressure adjustment, and other therapeutically useful materials, the compositions can be prepared by conventional mixing, granulation, or coating methods.

[0088] Oral formulations are exemplified by tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, and lozenges. These formulations may contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine) and lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone, and may optionally contain disintegrants or azeotropes such as starch, agarose, alginic acid, or its sodium salts, and / or adsorbents, colorants, flavorings, and sweeteners.

[0089] Cancer immunotherapy adjuvants can enhance the effectiveness of cancer immunotherapy agents, more specifically, by activating immune factors to support the anticancer activity of cancer immunotherapy agents. Immune factors may be at least one selected from the group consisting of helper T cells, cytotoxic T cells, natural killer cells (NK cells), CAR-T cells, CAR-M cells, tumor-infiltrating lymphocytes (TILs), and cytokines.

[0090] Cancer immunotherapy adjuvants can be administered simultaneously or sequentially with cancer immunotherapy agents. When administered sequentially, the cancer immunotherapy adjuvant can be administered after the cancer immunotherapy agent, or vice versa. However, this method of administration is merely an example, and the method of administration can be modified to enhance the anti-cancer immune effect. In one embodiment of the present invention, the cancer immunotherapy adjuvant was administered daily by intravenous injection, and the cancer immunotherapy agent was administered three times a week by intraperitoneal injection, but this is not always the case.

[0091] Cancer immunotherapy adjuvants can activate one or more immune factors selected from the group consisting of helper T cells, cytotoxic T cells, natural killer cells (NK cells), and cytokines. By activating these immune factors, cancer immunotherapy adjuvants enhance the anti-cancer effects of cancer immunotherapy agents.

[0092] At this point, cancer immunotherapy adjuvants can prevent or treat cancer when administered in combination with cancer immunotherapy agents.

[0093] Cancers include brain cancer, metastatic brain cancer, brainstem glioma, cerebral astrocytoma, cerebellar astrocytoma, pineal astrocytoma, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, uterine sarcoma, rhabdomyosarcoma, Kaposi's sarcoma, glioma, glioblastoma multiforme, glioblastoma, grade II fibrous astrocytoma, protoplasmic, grade III hypertrophic, undifferentiated astrocytoma, for example, cerebral gliomatosis, ependymal cell tumor, medulloblastoma, neuroectodermal tumor, neuroblastoma, hypothalamic glioma, breast cancer, triple-negative breast cancer, lung adenocarcinoma, lung cancer, lung pharyngitis Squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, colorectal cancer, colorectal cancer, ovarian cancer, ovarian epithelial cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine cancer, ovarian germ cell cancer, esophageal cancer, basal cell carcinoma, cholangiocarcinoma, choroidal melanoma, choroid plexus papilloma, splenic cancer, bone cancer cancer, intraocular melanoma, malignant melanoma, retinoblastoma, gastric cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, oral cavity cancer, nasal cavity / sinus cancer, salivary gland cancer, nasopharyngeal cancer, pharyngeal cancer, thyroid cancer, parathyroid cancer, thymic cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, stomach / liver cancer, Gastric lymphoma, colorectal cancer, rectal cancer, rectal carcinoma, small intestine cancer, gastrointestinal stromal cancer, testicular cancer, renal cell carcinoma, adrenal cancer, renal pelvis cancer, malignant mesothelioma, mesothelioma, chromaffin cell carcinoma, hematological cancer or chronic myeloid leukemia, lip cancer, tonsil cancer, squamous cell carcinoma, ampulla of Vater cancer, peritoneal cancer, tongue cancer, pseudomyxoma, intrahepatic hepatoblastoma, myelodysplastic syndrome, Vilmus' cancer, penile cancer, pharyngeal cancer, juvenile lymphoma, juvenile leukemia, Paget's disease, skin cancer, anal cancer, thoracic cancer, hematological cancer, acute myeloid leukemia, acute lymphoblastic leukemia It may be at least one selected from the group consisting of hematological cancers, myeloma, duodenal cancer, malignant soft tissue cancer, malignant lymphoma, chronic myeloid leukemia, bladder cancer, biliary tract cancer, chronic lymphocytic leukemia, malignant bone cancer, metastatic bone cancer, ocular cancer, vulvar cancer, ureteral cancer, mediastinal cancer, urethral cancer, cancer of unknown primary site, vaginal cancer, spinal cord cancer, vestibular schwannoma, diffuse median glioma (DMG), diffuse endogenous pontine glioma (DIPG), germ cell tumors, pineal blastoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, and hemangioblastoma.Furthermore, the present invention is also applicable to other diseases or conditions including proliferative conditions, autoimmune and non-autoimmune chronic inflammatory diseases, neurodegenerative diseases, Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1, endometriosis, Crohn's disease, ulcerative colitis, neuroinflammatory and infectious diseases, mycosis fungoides, and infectious diseases such as malaria, tuberculosis, HIV 1 and 2, sickle cell anemia, SARS, SARS-CoV, and MERS.

[0094] Cancer immunotherapy adjuvants can be administered in combination with conventionally known cancer immunotherapy agents that are well-known to those skilled in the art, although this is not limited to them. For example, cancer immunotherapy adjuvants can be administered in combination with one or more cancer immunotherapy agents selected from the group consisting of anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, anti-VISTA, anti-BTLA, anti-TIM3, anti-HVEM, anti-CD27, anti-CD137, anti-OX40, anti-CD28, anti-PDL2, anti-GITR, anti-ICOS, anti-SIRPα, anti-ILT2, anti-ILT3, anti-ILT4, anti-ILT5, anti-EGFR, anti-CD19, and anti-TIGIT, although this is not always limited to them.

[0095] In another aspect of the present invention, the present invention provides combination drugs for cancer immunotherapy.

[0096] In particular, the present invention provides a combination drug for cancer immunotherapy comprising an immunotherapy agent and a cancer immunotherapy adjuvant.

[0097] The specific descriptions of cancer immunotherapy agents and combination drugs are the same as the specific descriptions of cancer immunotherapy adjuvants.

[0098] In another aspect of the present invention, the present invention provides a pharmaceutical composition for use in enhancing the efficacy of cancer immunotherapy agents.

[0099] In particular, the present invention provides a pharmaceutical composition for use in enhancing the efficacy of cancer immunotherapy agents, comprising as an active ingredient the compound represented by Sequence ID No. 1, its isomer, its solvate, its hydrate, or a pharmaceutically acceptable salt thereof.

[0100] Furthermore, the specific description of pharmaceutical compositions used to enhance the efficacy of cancer immunotherapy agents is the same as the specific description of cancer immunotherapy adjuvants.

[0101] In another aspect of the present invention, the present invention provides a pharmaceutical composition for use in enhancing immunity.

[0102] In particular, the present invention provides a pharmaceutical composition for use in enhancing immunity, comprising as an active ingredient the compound represented by Sequence ID No. 1, its isomer, its solvate, its hydrate, or a pharmaceutically acceptable salt thereof.

[0103] Furthermore, the specific description of the pharmaceutical composition used for immune enhancement is the same as the specific description of the adjuvant for cancer immunotherapy.

[0104] In another aspect of the present invention, the present invention provides a method for preventing or treating cancer, comprising the step of administering a cancer immunotherapy agent and a cancer immunotherapy adjuvant to a subject in need.

[0105] Cancer immunotherapy adjuvants and cancer immunotherapy agents can be administered in combination or at different time points.

[0106] In another aspect of the present invention, the present invention provides the use of cancer immunotherapy adjuvants and immunotherapy agents in the prevention or treatment of cancer.

[0107] In another aspect of the present invention, the present invention provides a combination therapy for the treatment of cancer, comprising the step of administering a cancer immunotherapy adjuvant and a cancer immunotherapy agent to a subject in need.

[0108] In another aspect of the present invention, the present invention provides a kit for preventing or treating cancer, comprising a cancer immunotherapy agent and a cancer immunotherapy adjuvant as active ingredients. The present invention also includes the use of negative modulators of the EPO-EPO receptor pathway as monotherapy or in combination with antimicrobial agents or vaccines and recombinant cytokines in the treatment of autoimmune conditions and infections, where said conditions may be HIV, tuberculosis, malaria, streptococcus, staphylococcus, fungi, viruses such as SARS-CoV-2 and MERS, prion diseases, and parasitic infections. Furthermore, the present invention describes methods for enhancing the efficacy of prophylactic and therapeutic vaccines that stimulate the inflammatory response of the immune system in infections and cancer immunotherapy.

[0109] In further embodiments, the present invention relates to innovative nanomedicines and nanodelivery systems as tissue guns or probes, viral and nonviral vectors, nanomaterials for the delivery of bioactive drugs for targeted delivery, plant-based vesicles, nanoparticle-based methods enabling both therapeutic and in vivo imaging for diagnostic and therapeutic purposes, lipid systems such as liposomes and micelles, gold or magnetic nanoparticles, as well as combinations with natural products, functional nanoparticles, biomaterials such as microspheres and PEGs, and negative EPO / EPO-R modulator delivery systems that are administered orally, parenterally, intra-tumor and intra-cavity, intraventricular, intraarachnoid, intranasal or topically at clinical administration, based on a Trojan horse approach as micropumps for releasing therapeutic agents into tissues, for enhancing modulatory effects on the immune system in local pathological microenvironments or for inducing and enhancing homing of cell-based immunotherapy or vaccines.

[0110] In a further embodiment, the present invention relates to a negative EPO / EPO-R modulation-based method for reprogramming tumor-associated immune cells to avoid immune system exhaustion and immune tolerance.

[0111] In a further embodiment, the present invention relates to a method based on negative EPO / EPOR modulation for improving the efficacy of CAR T and CAR-γ / δ T cell therapy for solid tumors.

[0112] In a further embodiment, the present invention relates to EPO / EPO-R and their variant inhibitors, which enable stimulation of CTL infiltration and suppression of immunosuppressive cell recruitment in tumors and infectious diseases, and increase tissue infiltration of inflammatory cells and immune cells.

[0113] In a further embodiment, the present invention relates to EPO / EPO-R and their variant inhibitors that can induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferroptosis, autophagy, cyproptosis, and immunostimulatory cell death that enhances tumor immunogenicity.

[0114] Further embodiments describe products selected from EPOs and their natural and synthetic mutant inhibitors that can activate immune responses against cancer and infectious pathogens, reprogram the tumor microenvironment, and enhance immunotherapy and immunomodulatory strategies.

[0115] The present invention will be described in detail below with reference to the following examples and experimental examples. [Examples]

[0116] The following examples illustrate several embodiments of the present invention, in conjunction with the above description.

[0117] Example 1. Anti-EPO increases the migration of peripheral blood mononuclear cells (PBMCs) in the presence of GBM tissue. Figure 1 shows the effect of treatment with anti-EPO antibody and rhEPO on the migration of PBMCs added to a Transwell membrane with GBM fragments placed in the lower chamber (Figure 1A). On the day of surgery, after resection, GBM tissue was cut under sterile conditions and placed at the bottom of a 24-well plate treated as follows: CTR (Figure 1B), 10 μg / mL anti-EPO antibody (Figure 1C), and 100 ng / mL rhEPO (Figure 1D). PBMC migration was evaluated at T0, T24, and T48 hours. 2 × 10^4 PBMCs (each well) were resuspended in cell medium to 100 μL per well and added to the upper chamber. In the lower chamber of the Transwell plate, C4 or rhEPO-treated PBMCs were placed in 500 μL of medium containing GBM tissue. After incubation at 37°C in a 5% CO2 atmosphere for 48 hours, the upper chamber was removed, and the PBMCs were stained with Hoechst (1:1000 in PBS, 15 minutes at 37°C). The number of PBMCs on the lower chamber (Figure 1E) was counted under a microscope and analyzed using the ImageJ analyze particle plugin. Immunolabeling was performed using Nikon Crest (NikonTi + Andor Du888 + Zyla 4.6 + 16-led CoolLed + 4 laser cube LDI + Crest Optics spinning disk and VCS-sim XlightV2 / VCS), with the Nis-Elements V.5.3.2 software module used for acquisition and the GA3 module for analysis.

[0118] Analysis revealed a significantly higher number of migrating PBMCs in the presence of anti-EPO antibodies (Figure 1E). Interestingly, this analysis demonstrated an inhibitory effect of rhEPO on PBMC migration.

[0119] Example 2. Anti-EPO induces differentiation, activation, and expression of natural killer markers in PBMCs. To evaluate the expression of CD56, CD86, and CD69, flow cytometry analysis was performed on PBMCs after 48 hours of migration. For the analysis, indirect conjugation was first performed using primary and secondary antibodies, followed by the addition of a mixture of the conjugated antibodies. Specifically, EPOR was added to PBMCs as AB-I and incubated at room temperature for 1 hour in a blocking solution containing PBS + 5% BSA + 2% donkey serum. After removing AB-I and washing three times with PBS + 0.1% BSA, AB-II was added at room temperature for 1 hour. Subsequently, the PBMCs were washed with PBS and administered the following mixtures containing conjugated antibodies: CD56-APC, CD86-PE-Cy7, and CD69-APC-Cy7. Analysis was performed using a FACS Canto II flow cytometer and FACSDiva software (BD Bioscience, version 5.0). In the CTR group, forward scatter versus side scatter (FSC-A vs SSC-A) gating was used to identify complete PBMCs based on size and granularity. The results in Figure 2 show that anti-EPO administration induced significant overexpression of CD56, CD86, CD69, and EPOR compared to the CTR condition (untreated PBMCs) (Figure 2A). Surprisingly, when analyzed in comparison with rhEPO, anti-EPO administration induced strong expression of CD56, CD86, CD69, and EPOR in migrating PBMCs (Figure 2B).

[0120] Example 3. Anti-EPO treatment induces PBMC migration in an EPOR-dependent manner. PBMCs collected from n=3 GBM patients were subjected to 48-hour migration analysis in a Boyden chamber (Figure 3A). 2 × 10^4 PBMCs (each well) were resuspended in cell medium to 100 μL per well, and the PBMC suspension was placed in the upper chamber. In the lower chamber of the Transwell plate, 500 μL of medium containing GBM tissue was placed, along with anti-EPO or rhEPO treatment. After incubation at 37°C under a 5% CO2 atmosphere for 48 hours, the upper chamber was removed, and the insert was fixed on ice with 4% PFA for 10 minutes. Subsequently, the insert was removed, inverted onto a glass slide, and immunolabeled for CD14, CD8, and EPOR targets. PBMCs were blocked in PBS + 5% BSA + 2% donkey serum at room temperature for 30 minutes. Incubation with primary antibody (AB-I) diluted in blocking buffer was performed overnight at 4°C. The following AB-Is were used: anti-CD14 (ThermoFisher Scientific), anti-CD8 (Abcam, Cambridge, UK), and anti-EPOR (SantaCruz Technology). The following day, AB-Is were removed, washed three times with PBS + 0.1% BSA, and then AB-II was added at room temperature for 1 hour. PBMCs were permeabilized by treating the cell membrane with PBS + 0.5% Triton X-100 and directly incubated with DAPI. Immunolabeling was acquired using a high-resolution SP5 confocal microscope. Interestingly, CD8+ cells showed a larger, more rounded morphology compared to the CTR and rhEPO-treated groups (Figure 3B).

[0121] Furthermore, analysis revealed that anti-EPO treatment significantly induced migration in both CD14+ and CD8+ PBMC-derived cells compared to CTR and rhEPO administration (Figure 3C). Surprisingly, microscopic images and relative quantification (Figures 3D-E) revealed that migrating CD8+ and CD14+ cells showed higher EPOR expression when anti-EPO was added to the culture medium. These data indicate that anti-EPO antibodies are a potent immunomodulatory stimulus that induces effects on PBMCs from GBM patients. Surprisingly, the effect of the stimulus is mediated by the overexpression of EPOR in migrating PBMCs.

[0122] Example 4. Anti-EPO induces a molecular signature of activation and blocks the exhaustion phenomenon. Gene expression analysis was performed by real-time PCR on PBMCs cultured for 48 hours under CTR, anti-EPO, and rhEPO conditions. PBMCs were harvested as described above, and migration was evaluated by Boyden chamber assay. After 48 hours of migration, PBMCs were harvested from the bottom of the well, centrifuged at 300g for 10 minutes, the pellet was resuspended in Tri-Reagent, and RNA was extracted according to the manufacturer's instructions. RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcriptase reactions were performed using a TranScriba kit (A&A Biotechnology) with 1 μg of RNA (A260 / A280 > 1.8) loaded, according to the manufacturer's instructions. qRT-PCR was performed using StepOnePlus® (Thermo Fisher Scientific), 1 μg of cDNA, forward and reverse primers (250 nM each), and Titan HotTaq EvaGreen® qPCR Mix (Bioatlas). Data were normalized to TBP expression used as an internal control. Relative gene expression was determined using the 2-ΔΔCt method (Figure 4). The results showed that anti-EPO treatment induces PBMC activation, and importantly, blocks exhaustion, and modulates the expression of IL-1b (Figure 4A), IL-6 (Figure 4B), IFNg (Figure 4C), IL-10 (Figure 4D), TGFb (Figure 4E), IFNa2 (Figure 4F), PD1 (Figure 4G), LAG-3 (Figure 4H), and CTLA4 (Figure 4I) genes. Data are mean ± SD across 3 replicates from at least 3 experiments. * P<0.05, compared with CTR for all treatment groups.

[0123] Example 5. Anti-EPO induces monocyte migration and their activation. Monocyte migration was evaluated using a 24-well Transwell plate (pore size 8.0 μm; Corning, NY). Briefly, monocytes were washed once with RPMI1640 medium and readjusted in RPMI1640 + 10% FBS (5 × 10^5 cells / mL). Monocytes were stained with calcein (1:1000 dilution in RPMI medium, 15 minutes), washed with PBS, and a portion of the cell suspension (100 μL) was placed in the upper chamber. A specific treatment agent was placed in 500 μL of medium in the lower chamber of the Transwell plate. After incubation at 37°C under a 5% CO2 atmosphere for 48 hours, the upper chamber was removed, and the number of cells at the bottom of the chamber was counted under a microscope and analyzed using the ImageJ analyze particle plugin under the following conditions: CTR (Figure 5A), anti-EPO (Figure 5B), rhEPO (Figure 5C). Interestingly, after 6 days of migration, anti-EPO treatment increased monocyte migration compared to CTR conditions. In particular, migration decreased in the presence of rhEPO, especially with rhEPO (Figure 5D). Surprisingly, phenotypic analysis of migrating monocytes revealed that C4 treatment could significantly induce overexpression of CD86 and HLA-DR (Figure 5E).

[0124] Monocyte migration was performed with GBM fragments present at the bottom of the transwells. After 6 days of experimental conditions, tissue was collected from the bottom of the wells, digested with 0.25% trypsin for 30 minutes, filtered through a 70 μm pore mesh, and analyzed by flow cytometry to count the number of green-positive infiltrating cells. The results showed that administration of anti-EPO antibody significantly increased the number of monocytes derived from infiltrating macrophages (Figure 6A, B). Interestingly, the immunophenotypic profile of the infiltrating macrophages showed significantly high expression of CD86+ / HLA-DR+, indicating polarization of M1 macrophages (Figure 6C). Furthermore, it was revealed that the infiltrating macrophages overexpressed EPOR (Figure 6D).

[0125] Interestingly, immunofluorescence analysis performed on infiltrating macrophages showed higher EPOR expression, as highlighted by the red-stained cells (Figure 7A-F).

[0126] Overall, these data revealed that anti-EPO exhibits potent chemotaxis, inducing monocyte migration. Surprisingly, anti-EPO antibodies increased the infiltration of macrophage-derived monocytes into tumor tissue and, more importantly, polarized macrophages into the M1 phenotype. Interestingly, this mechanism is mediated by EPOR expression on macrophages.

[0127] Example 6. Anti-EPO is the most effective migratory stimulus for T cells. A migration test, or "chemotaxis assay," was performed to elucidate the migratory ability of naive T cells, a phenomenon observed in response to an immune response. For this purpose, a Transwell multiwell plate fitted with a polycarbonate membrane insert was used. The 8 μm diameter pores of the membrane could hold cells and culture medium, and allowed for active migration of cells through the membrane to the lower wells. 2 × 10^4 naive CD4+ T cells (each well) were stained with calcein (1:1000 in PBS) for 10 minutes, and then resuspended in cell medium to 100 μL in each well. The cells were resuspended in the presence of the following treatments: in the lower chamber of the Transwell plate, 500 μL of medium contained either CTR (Figure 8A), C4 (10 μg / mL, Figure 8B), rhEPO (100 ng / mL, Figure 8C), or a combination of rhEPO + anti-EPO Ab (Figure 8D). After incubation at 37°C and 5% CO2 for 48 hours, the upper chamber was removed, and the T cells that had migrated to the bottom of the chamber were counted under a microscope and analyzed using the ImageJ analyze particle plugin (Figure 8E). Immunolabeling was acquired using a Leica Time-Lapse microscope. High-resolution imaging was performed on all stained wells using a full-well approach with large-scale mosaic rendering at high magnification, and the full field of view (FOV) of all wells was obtained for each case of IF labeling. The results show that T cells are chemoattracted by anti-EPO antibodies, and this effect is significantly reduced in the presence of rhEPO. Interestingly, a significantly higher number of migrating T cells was observed when anti-EPO and rhEPO were administered together. Therefore, it can be concluded that anti-EPO treatment is a potent stimulus and significantly increases T cell migration. In contrast, treatment with rhEPO inhibits T cell migration, suggesting the immunosuppressive role of erythropoietin. * P < 0.05 (compared to CTR processing).

[0128] The immunophenotypic profiles of T cells after C4 administration were assessed by evaluating EPOR expression in CD69+ subpopulations under the following conditions: CTR (Figure 9), temozolomide (TMZ, Figure 9B), rhEPO (Figure 9C), C4 (Figure 9D), TMZ+C4 (Figure 9E), and rhEPO+C4 (Figure 9F). The results showed a significant increase in EPOR expression in activated CD69+-positive cells under all conditions in which anti-EPO antibody was added. Indeed, migrating T cells showed higher expression of CD69 and EPOR compared to the CTR and rhEPO conditions (Figure 9G). Data are mean ± SD across 3 replicates from at least 3 experiments. ** P<0.01, *** P<0.001, * P<0.05, compared with CTR for all treatment groups.

[0129] Example 7. Anti-EPO induces migration of CTL lymphocytes (CD4+) and deep infiltration into GBM tissue in TME. Lymphocyte migration was evaluated using a 24-well Transwell plate (pore size 8.0 μm; Corning, NY). Briefly, T cells were washed once with RPMI1640 medium and renumbered in T cell medium (RPMI1640 + 10% FBS) (5 × 10^5 cells / mL). The T cells were stained with calcein (1:1000 in RPMI medium, 15 min), washed with PBS, and a portion of the T cell suspension (100 μL) was placed in the upper chamber. Chemokines in 500 μL of conditioned medium (CM) were placed in the lower chamber of the Transwell plate (Figure 10A). After incubation at 37°C in a 5% CO2 atmosphere for 48 hours, the upper chamber was removed, and the number of T cells at the bottom of the chamber was counted under a microscope and analyzed using the ImageJ analyze particle plugin. Figure 10B shows the number of T cells that migrated to the bottom of the wells. The data showed that anti-EPO, when administered alone or in combination with TMZ, could induce higher T cell induction, as shown in the micrographs of the wells under the following conditions: CTR (Figure 10C), TMZ (Figure 10D), anti-EPO (Figure 10E), TMZ + C4 (Figure 10F), and rhEPO (Figure 10G) (Figure 10B).

[0130] Surprisingly, the number of migrating T cells was significantly higher in the presence of GBM tissue fragments (Figure 11A), after C4 administration, and in combination with TMZ (Figure 11B). Micrographs in Figures 11C-H represent the entire well bottom under the following conditions: CTR (Figure 11C), TMZ (Figure 11D), C4 (Figure 11E), TMZ+C4 (Figure 11F), rhEPO (Figure 11G), and rhEPO+C4 (Figure 11H). At the end of the migration analysis, GBM tissue fragments (Figure 12A) were collected from the bottom of the wells, digested with 0.25% trypsin for 30 minutes, filtered through a 70 μm mesh, and analyzed by flow cytometry to count the number of green-positive infiltrating cells. The results showed that administration of anti-EPO antibody significantly increased the number of infiltrating T cells, both alone and in the presence of TMZ or rhEPO (Figure 12B). Overall, these data revealed that anti-EPO exhibits potent chemotaxis, inducing T cell migration. Surprisingly, anti-EPO antibodies were able to induce T cell infiltration into GBM tissue, demonstrating their potent capabilities as chemotactic molecules.

[0131] Example 8. Expression of EPO-related targets on GBM cells To develop CAR constructs that utilize the immune system to attack tumors, we analyzed the expression patterns of EPO-related, transferrin-related, and IL-13-related genes in CTR and GBM mRNA using real-time PCR to identify specific GBM signatures.

[0132] Real-time PCR was performed to obtain information on the expression of specific genes. Specifically, gene expression analysis was performed using GBM cells and CTR mRNA (Takara). Cells were harvested, centrifuged at 300g for 10 minutes, and the pellet was resuspended in Tri-Reagent. RNA was extracted according to the manufacturer's instructions. RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcriptase reactions were performed using a TranScriba kit (A&A Biotechnology) with 1 μg of RNA (A260 / A280 > 1.8) loaded, according to the manufacturer's instructions. qRT-PCR was performed using StepOnePlus® (Thermo Fisher Scientific), 1 μg of cDNA, forward and reverse primers (250 nM each), and Titan HotTaq EvaGreen® qPCR Mix (Bioatlas). Data were normalized to the expression of the 18S gene used as an internal control. Relative gene expression was determined using the 2-ΔΔCt method (Figure 13). Figure 13 shows the gene expression results for EGFR, EPOR, EPHB4, CSF2RB, and CRLF3 in tumor cells and commercially available CTR mRNA (Figure 13A). Furthermore, gene expression for IL-13, IL-13R1, IL-13R2 (Figure 13B), and TfR1, TfR2, and folate receptor (Figure 13C) is also shown. The results reveal that GBM cells have higher expression levels of EGFR, EPHB4, CSF2RB, and CRLF3, as well as IL-13, IL-13R1, IL-13R2, TfR1, TfR2, and folate receptor compared to healthy cells, suggesting that these could be targets for developing CAR constructs.

[0133] Example 9. CAR-T efficacy of anti-EPO treatment in cancer cells Since EGFR amplification, overexpression, or mutations are found in approximately half of glioblastomas in both children and adults, as well as other malignant CNS tumors including ependymomas and medulloblastomas, EGFR CAR-T cells (EGFR scFv-4-1BB-CD3ζ CAR T cells, Promab) were used. To investigate the effects of T cells (WT) and EGFR-CAR-T cells in the presence and absence of anti-EPO, killing tests were performed against glioblastoma stem cells (Figure 14) and cancer cells derived from colorectal cancer, breast cancer, and melanoma (Figure 14). Target cells were seeded in 96-well plates in triple rows at a density of 1 × 10^4 cells per well in DMEM:F12 (ThermoFisher Scientific) medium supplemented with 10% FBS. Target cells were cultured for 24 hours at 37°C under 5% CO2 in a series of E:T (effector:target) ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64, either as T cells, CAR-T cells alone, or in the presence of anti-EPO (Figure 14A). Each well was stained with crystal violet (ThermoFisher Scientific). After staining, images were observed and captured under an inverted microscope (TS100, NIKON Instruments Inc). Data are shown as viability (%) (Figure 14A). Under these conditions, a significant difference was observed when T cells were cultured in the presence of anti-EPO. In fact, T cells mediated better control of the target cell number. Therefore, the cytotoxic activity of CAR-T cells + anti-EPO showed high efficiency even at low effector levels.

[0134] Furthermore, to determine whether the observed enhancement of cytolytic activity was accompanied by a similar significant increase in TNF-α and IFN-γ secretion, GSCs were co-cultured for 24 hours with EGFR-CAR-T cells alone or in combination with a C4 antibody, and cytokines were measured according to the instructions using an ELISA kit (R&D Systems). As shown in Figure 14, both EGFR-CAR-T cells spontaneously produced detectable levels of TNF-α (Figure 14B) and IFN-γ (Figure 14C). When these cells were cultured with CAR-T cells in combination with an anti-EPO antibody, TNF-α and IFN-γ were induced, and significantly higher levels of cytokines were produced. These results are consistent with the cytotoxic data described above, and based on these findings, it is suggested that the combined administration of an anti-EPO antibody and EGFR-CAR-T cells significantly enhances the effector function of T cells responding to EGFR+ glioblastoma cells.

[0135] Furthermore, the cytotoxic effects of EGFR-CAR-T cells were evaluated using the following cell models. i) Breast cancer cell line (MCF-7); breast cancer cells. This cell line has been shown to be responsive to human erythropoietin (rHuEPO) treatment in terms of increased cell proliferation. Furthermore, the MCF7 cell line has also been reported to express EGFR (Figure 15A). ii) LNCAP (Long-Natural Prostate Cancer Cell Line); Human prostate cancer cells used in cancer research and drug development. Human hepatocyte receptors (Ephs), which produce erythropoietin, have been reported to be overexpressed in prostate cancer patients and associated with poor prognosis and reduced survival rates, and are considered a predictive marker of invasive prostate cancer behavior. Furthermore, recent studies have shown that simultaneous overexpression of EPO and EPOR in drug-resistant prostate cancers in LNCAP cells plays a crucial role in disease progression and is involved in the development of neuroendocrine phenotypes. Interestingly, EGFR has been reported to be expressed in LNCAP cell lines (Figure 15B). iii) The melanoma cell line was derived from primary achromatic tumor A375. Elevated EGFR expression levels were confirmed. Based on these premises, the inventors decided to use the A375 cell line as a model for the cytotoxic activity of EGFR-CAR-T cells (Figure 15C). Under these conditions, significant differences were observed in the MCF7 breast cancer cell line (Figure 15A), LNCAP prostate cancer cell line (Figure 15B), and A375 melanoma cell line (Figure 15C) when T cells were cultured in the presence of anti-EPO treatment. In fact, T cells mediated better control of the target cell number. Therefore, the cytotoxic activity of CAR-T cells + anti-EPO showed high efficiency even at low effector doses.

[0136] Furthermore, the results showed that combined treatment with anti-EPO and metformin (MET) enhanced the cytotoxic effect of CAR-T cells against glioblastoma cancer cells (Figure 16A). In addition, gene expression analysis was performed on PBMCs treated under the following conditions: CTR, anti-EPO, MET, and anti-EPO+MET. The results revealed that combined administration of anti-EPO+MET induced the expression of IL-1β (Figure 16B) and IFNγ (Figure 16C) in PBMCs, and downregulated PD-1 expression (Figure 16D).

[0137] Example 10. Infiltration of immune cells into a GBM subcutaneous PDX mouse model after anti-EPO treatment. SCID mice (n=10) were subcutaneously inoculated with GBM fragments and observed until the tumor mass reached 50-100 mm3. The mice were then randomly assigned to two groups: a CTRL group treated with PBS and an anti-EPO group treated with 15 mg / kg intravenous (IV) injections three times weekly. Tumor mass weight was measured with a caliber before each administration. Mouse body weight was monitored throughout the entire experiment. On the day of sacrificial death, the tumor mass was excised and hematoxylin-eosin staining was performed. Immunohistochemical evaluation was performed by two independent pathologists blinded to clinical information. After anti-EPO treatment (Figure 17A, B), increased polymorphonuclear leukocyte infiltration was observed in GBM masses 21 days post-injection compared to the CTR condition (Figure 17C, D).

[0138] Example 11. Anti-EPO treatment improves malaria in infected red blood cells. Human erythrocytes, from which leukocytes had been removed, were processed. Upon arrival, the erythrocytes were washed with filtered RPMI 1640 medium (containing 25 mM HEPES and 50 μg / ml hypoxanthine). Malaria parasites were grown in complete culture medium (cRPMI) containing 1% AlbuMax II (ThermoFisher Scientific), 0.21% sodium bicarbonate (Gibco), and 20 μg / ml gentamicin (ThermoFisher Scientific) in RPMI 1640 (ThermoFisher Scientific) under a hematocrit of 5%, at 37°C, and under a gas mixture containing 90% N2, 5% CO2, and 5% O2. To monitor for parasitemia, erythrocytes were prepared on glass slides, fixed with 100% methanol, and stained with 20% Giemsa stain (Sigma-Aldrich, St. Louis, MO) for 15 minutes. Red blood cells were counted using a bright-field microscope, and the parasitic rate was evaluated based on the number of parasitic cells under CTR conditions or the following anti-EPO antibody conditions. Administration of anti-EPO antibodies to infected red blood cells resulted in a decrease in parasitic rate and recovery from infection in all cells (Figure 18A). When monocytes were cultured with infected cells, the immunophenotypic profile showed higher expression of CD86+ / HLA-DR+ positivity, suggesting M1 polarization (Figure 18B). These results suggest that the use of anti-EPO antibodies after malaria infection may have therapeutic value in severe malaria cases (Figure 18).

[0139] Example 12. Migration of PBMCs: A model of chemotaxis in infections stimulated by anti-EPO. Figure 19 shows the effects of lipopolysaccharide (LPS) treatment with anti-EPO antibody and rhEPO on PBMC migration. LPS is a molecule present on the membrane of Gram-negative bacteria. To clarify the migratory ability of PBMCs (a phenomenon observed in response to an immune response), a migration test, i.e., a chemotaxis assay, was performed. For this purpose, a Transwell multiwell plate (24 wells) was used and fitted with an insert equipped with a polycarbonate membrane. The 8 μm diameter pores of the membrane can hold cells and culture medium, and allow for active migration of cells through the membrane to the lower wells. 2 × 10^4 PBMCs (each well) were resuspended in cell medium to 100 μL per well, and the PBMC suspension was placed in the upper chamber (Figure 19A). In the lower chamber of the Transwell plate, 500 μL of culture medium was placed containing either CTR+LPS (Figure 19B), anti-EPO+LPS 10 μg / mL (Figure 19C), or rhEPO+LPS 100 ng / mL (Figure 19D). After incubation at 37°C under a 5% CO2 atmosphere for 48 hours, the upper chamber was removed, and the PBMCs were stained with Hoechst (1:1000 in PBS, 37°C for 15 minutes), washed with PBS, and the number of PBMCs on the lower chamber was counted under a microscope and analyzed using the analyze particle plugin in ImageJ (Figure 19E). Immunolabeling was acquired using a high-resolution Nikon Ti spinning disk microscope (Nikon Instruments, Florence, Italy) equipped with a CREST-optics spinning disk head, a VCS structural illumination module for super-resolution (CREST-Optics, Rome, Italy), and an Andor camera for resolution (Andor Zyla, Andor Technology, Oxford Instruments, Oxford, UK) and an Andor camera for quantum efficiency (Andor Technology, Oxford Instruments, Oxford, UK). Deconvolution of the spinning disk confocal images was performed using NIS-Elements V.5.3.2, and a specific VCS-Studio algorithm (CREST Optics, Rome, Italy) was employed to correct the reconstruction of the structural illumination.High-resolution imaging was performed on all stained wells using a full-well approach with large-scale mosaic rendering at high magnification, and the full field of view (FOV) of all wells was obtained for each case of IF labeling. Interestingly, PBMCs were chemoattracted by anti-EPO antibodies, and this effect was significantly reduced in the presence of recombinant human EPO (rhEPO). Therefore, it can be concluded that in the presence of LPS, a condition that mimics infection, strong stimulation by anti-EPO treatment significantly increases PBMC migration. Conversely, rhEPO treatment in the presence of LPS inhibited PBMC migration, suggesting an immunosuppressive role of EPO. * P<0.05 Compared to CTR treatment.

[0140] Example 13. Administration of anti-EPO in a rodent orthotopic GBM model induces increased immune cell migration accompanied by T lymphocyte tumor infiltration. In an in vivo orthotopic GBM model, anti-EPO administration surprisingly induced immune system activation accompanied by T lymphocyte migration (Figure 20B, white arrows) compared to a healthy contralateral hemisphere (Figure 20A). Interestingly, ferroptosis was assessed by histological analysis after anti-EPO administration, and iron deposition, a marker of cell death, was observed.

Claims

1. A method for activating or enhancing the immune response of a patient in need, Use of a negative anti-EPO function modulator or anti-EPO antigen-binding fragment alone, or - Checkpoint inhibitor or immunomodulatory agent (e.g., anti-PDL1 antibody; nivolumab; ipilimumab, abatacept, glenbatumumab vedotin) therapy; and / or, - Cell-based immunotherapy (therapies based on CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic cells activated against tumor-associated antigens, and / or antigen-presenting cells, tumor-associated peptides, engineered monocyte-macrophages, or polymorphonuclear cells), and / or therapies that enhance and reprogram the response of tumor-associated lymphocytes (TILs) or tumor-associated macrophages (TAMs); - Antimicrobial therapy (e.g., antibiotics; antiviral agents, antifungal agents, antiprion agents) - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists, - Vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids for prophylactic or therapeutic purposes (e.g., anti-HPV, anti-EBV, or anti-HIV vaccines); - Immunotherapy based on oncolytic viruses; - Chemotherapy agents; - Anticancer drugs; - Enzymes that break down heparin sulfate proteoglycans (e.g., heparanase); - A negative functional modulator of the sphingosine-1-phosphate (S1P) signaling pathway, or - EPO mimetic agents that maintain erythrocyte production; Including use in combination with, The anti-EPO antigen-binding fragment is selected from the group consisting of Fab, -F(ab')2, single-chain antibody, diabody, triabody, tetrabody, repebody, or domain antibody. The negative anti-EPO function modifier is selected from the group consisting of anti-EPO monospecific or multispecific antibodies, gene therapy, DNA decoys, RNA decoys, ribozymes, antagonist miR, shRNA, LNA, siRNA, antisense oligonucleotides, or anti-Epo receptors, and the anti-Epo receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, and EPOR / CD131 heterodimers. method.

2. The negative functional modifier or anti-EPO antigen-binding fragment elicits an immune cell response (T helper lymphocytes and / or T cytotoxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or activated dendritic cells, antigen-presenting cells), and the cellular response is an antitumor T cell response or antimicrobial cell response in patients suffering from cancer or infection. The method according to claim 1.

3. The antitumor T helper lymphocytes and / or T cell-toxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells are PBMC-derived immune cells. The method according to claim 2.

4. The antitumor T cell response is a CD8+ T cell response and a CD4+ T cell response. The method according to claim 2 or 3.

5. The aforementioned antitumor T cell response is due to the expression of CD69 and EPOR in migrating PBMCs. The method according to any one of claims 2 to 4.

6. The anti-EPO antigen-binding fragment is a neutralizing antibody that binds to EPO, EPO variants, or EPO receptors and restores T helper lymphocytes and / or T-cytotoxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or activated dendritic cells. The method according to any one of claims 1 to 5.

7. The anti-EPO antigen-binding fragment is a neutralizing antibody selected from the group consisting of C4, B4, and 16F1H11. The method according to any one of claims 1 to 6.

8. The negative functional modifier or anti-EPO antigen-binding fragment elicits T helper lymphocytes and / or T cytotoxic lymphocytes and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells and / or antigen-presenting cells, and the cellular response is further enhanced by therapies based on CAR-T, CAR-M, CAR-NK, engineered monocyte-macrophages or polymorphonuclear cells, or therapies that enhance and reprogram the response of tumor-associated lymphocytes (TILs) or tumor-associated macrophages (TAMs). The method according to any one of claims 1 to 7.

9. The aforementioned patient has cancer, proliferative conditions, autoimmune and non-autoimmune chronic inflammatory diseases, neurodegenerative diseases, Hippel-Lindau disease (VHL), and multiple endocrine neoplasia type 2 (MEN).2) Patients suffering from neurofibromatosis type 1, endometriosis, Crohn's disease, ulcerative colitis, neuroinflammatory and infectious diseases, mycosis fungoides, and infectious diseases such as malaria, tuberculosis, HIV 1 and 2, sickle cell anemia, SARS, SARS-CoV, and MERS, or who have undergone organ or tissue transplantation, and the cancers include brain cancer, metastatic brain cancer, brainstem glioma, cerebral astrocytoma, cerebellar astrocytoma, pineal astrocytoma, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, uterine sarcoma, rhabdomyosarcoma, Kaposi's sarcoma, glioma, glioblastoma multiforme, glioblastoma, and grade II fibrous sarcoma. Cell tumors, protoplasmic, grade III hypertrophic cell tumors, undifferentiated astrocytomas, e.g., gliomas, ependymal cell tumors, medulloblastomas, neuroectodermal tumors, neuroblastomas, hypothalamic gliomas, breast cancer, triple-negative breast cancer, lung adenocarcinoma, lung cancer, squamous cell carcinoma of the lung, small cell lung cancer, non-small cell lung cancer, colorectal cancer, colorectal cancer, ovarian cancer, ovarian epithelial carcinoma, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine cancer, ovarian germ cell carcinoma, esophageal cancer, basal cell carcinoma, cholangiocarcinoma, choroidal melanoma, choroid plexus papilloma, splenic cancer, osteosarcoma, intraocular melanoma, malignant melanoma, retinoblastoma, gastric cancer, cardiac cancer, liver cancer Cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal and paranasal sinus cancer, salivary gland cancer, nasopharyngeal cancer, pharyngeal cancer, thyroid cancer, parathyroid cancer, thymic cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, stomach and liver cancer, gastric lymphoma, colorectal cancer, rectal cancer, rectal carcinoma, small intestine cancer, gastrointestinal stromal cancer, testicular cancer, renal cell carcinoma, adrenal cancer, renal pelvis cancer, malignant mesothelioma, mesothelioma, chromaffin cell carcinoma, hematological cancer or chronic myeloid leukemia, lip cancer, tonsil cancer, squamous cell carcinoma, ampulla of Vater cancer, peritoneal cancer, tongue cancer, pseudomyxoma, intrahepatic hepatoblastoma, myelodysplastic syndrome, Pyrmus' cancer, penile cancer, pharyngeal cancer, juvenile lymphoma, juvenile leukemia A selection from the group consisting of hematological malignancies, Paget's disease, skin cancer, anal cancer, thoracic cancer, blood cancer, acute myeloid leukemia, acute lymphoblastic leukemia, myeloma, duodenal cancer, malignant soft tissue cancer, malignant lymphoma, chronic myeloid leukemia, bladder cancer, biliary tract cancer, chronic lymphocytic leukemia, malignant bone cancer, metastatic bone cancer, eye cancer, vulvar cancer, ureteral cancer, mediastinal cancer, urethral cancer, cancer of unknown primary site, vaginal cancer, spinal cord cancer, vestibular schwannoma, diffuse median glioma (DMG), diffuse endogenous pontine glioma (DIPG), germ cell tumor, pineal blastoma, germ cell tumor, acoustic neuroma, schwannoma, meningioma, and hemangioblastoma. The method according to any one of claims 1 to 8.

10. The aforementioned cancer is glioblastoma multiforme. The method according to any one of claims 1 to 9.

11. The aforementioned negative functional modifier or anti-EPO antigen binding fragment promotes an immune system response in patients suffering from refractory or persistent infections, the persistent infections including precancerous lesions induced by tuberculosis, malaria, HIV, EBV, or HPV, and their prevention. The method according to any one of claims 1 to 10.

12. The aforementioned negative functional modifier or anti-EPO antigen-binding fragment promotes the immune system response in patients infected with antimicrobial therapy-resistant streptococci, staphylococci, fungi, viruses; SARS-CoV-2, SARS, MERS, prion pathogens, and / or in whom immune system tolerance exists. The method according to any one of claims 1 to 11.

13. The aforementioned negative functional modifier or anti-EPO antigen binding fragment promotes the immune system response and enhances the efficacy of prophylactic and therapeutic DNA and / or RNA or peptide and / or lipid-based vaccines in infectious disease and / or cancer immunotherapy. The method according to any one of claims 1 to 12.

14. Negative functional modifiers of EPO / EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, e.g., EPOR / CD131 heterodimer) and / or their natural or synthetic variants, - Checkpoint inhibitor or immunomodulatory agent (e.g., anti-PDL1 antibody; nivolumab; ipilimumab, abatacept, glenbatumumab vedotin) therapy; and / or - Cell-based immunotherapy (therapy based on CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic cells activated against tumor-associated antigens, engineered monocyte-macrophages or polymorphonuclear cells), and / or therapy to enhance and reprogram the response of tumor-associated lymphocytes (TILs) or tumor-associated macrophages (TAMs), - Peptides or antibodies, diabodies, and nanobodies against natural and synthetic variants of erythropoietin, including physiological and pathological splicing variants and post-translational modifications; - Antimicrobial therapy (e.g., antibiotics; antiviral agents, antifungal agents, antiprion agents) - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids for prophylactic or therapeutic purposes (e.g., anti-HPV, anti-EBV, or anti-HIV vaccines); - Immunotherapy drugs based on oncolytic viruses; -Chemotherapy; - Anticancer drugs; - Enzymes that break down heparin sulfate proteoglycans (e.g., heparanase); - A negative functional modulator of the sphingosine-1-phosphate (S1P) signaling pathway; or - EPO mimetic agents that maintain erythrocyte production function It comprises one or more components selected from the group consisting of, Medical kit.

15. A diagnostic or prognostic method for evaluating the expression of EPO and its somatic mutations or variants, EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, EPOR / CD131 heterodimer) and their somatic mutations and / or variants and / or C4 mAb ligands, in order to individualize therapy, stratify patients, and optimize patient response in immunotherapy or prophylaxis for cancer and infectious diseases, A method comprising the step of measuring / detecting the amount of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants, as diagnostic markers or prognostic markers, in tissue, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or induced extracellular vesicles).

16. A diagnostic method for evaluating promoter methylation of EPO and EPO receptor (EPOR, EPHB4, CSF2RB, CRLF3, tissue protective factors, TPR, e.g., EPOR / CD131 heterodimer) genes, which can predict increased EPO / EPO-R expression and their negative role in inhibiting the immune system in eradicating cancer cells or microbial pathogens, for the purpose of individualizing immunotherapy or prophylaxis and for prognostic purposes, A method comprising the step of detecting methylation of EPO and EPO receptor genes as diagnostic or prognostic markers in tissue, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or induced extracellular vesicles).

17. Innovative nanomedicines and nanodelivery systems as tissue guns or probes, viral and nonviral vectors, nanomaterials for the delivery of bioactive drugs for targeted delivery, plant-based vesicles, nanoparticle-based methods enabling both therapeutic and in vivo imaging for diagnostic and therapeutic purposes, lipid systems such as liposomes and micelles, gold or magnetic nanoparticles, as well as combinations with natural products, functional nanoparticles, biomaterials such as microspheres and PEGs, and negative EPO / EPO-R modifier delivery systems based on a Trojan horse approach as micropumps for releasing therapeutic agents into tissues, which are administered orally, parenterally, intra-focal (intratumoral and intracavitary), intraventricular, intraarachnoid, intranasal or topically at clinical administration in various formulations, based on a Trojan horse approach as micropumps for releasing therapeutic agents into tissues.

18. A negative EPO / EPO-R modulation-based method for reprogramming tumor-associated immune cells to avoid immune system exhaustion and immune tolerance.

19. A method based on negative EPO / EPO-R modulation to improve the efficacy of CAR T and CAR-γ / δ T cell therapy for solid tumors.

20. This enables stimulation of CTL infiltration and suppression of immunosuppressive cell recruitment in tumors and infections, and increases tissue infiltration of inflammatory and immune cells. EPO / EPO-R and their variant inhibitors.

21. It can induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferroptosis, autophagy, cuproptosis, and immunostimulatory cell death that enhances tumor immunogenicity. EPO / EPO-R and their variant inhibitors.

22. Products selected from EPOs and their natural and synthetic mutant inhibitors that can activate immune responses against cancer and infectious pathogens, reprogram the tumor microenvironment, and enhance immunotherapy and immunomodulatory strategies.

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