Methods for improving the efficacy of immunotherapy and enhancing host immune responses

Abstract

The present invention relates to the field of immunotherapy suitable for activating the immune response in patients. More specifically, the present invention relates to anti-EPO negative functional modulators, which can be used as active ingredients in pharmaceutical compositions for immunomodulatory strategies in therapy (e.g. cancer immunotherapy, infectious diseases, inflammatory diseases), or for immune activation, to improve cell-based, pharmacological-based or vaccine-based immunotherapies, and to stimulate the immune system response in patients in need thereof. In particular, the present invention also describes how to restore the immune response in pathological conditions (e.g. cancer and refractory infectious diseases), to enhance and ensure the therapeutic accessibility of immunotherapy strategies, and to eliminate the "tolerogenic" stimuli, by means of products containing EPO pathway inhibitors, which can be used, for example, as active pharmaceutical ingredients of vaccine compositions, which stimulate the immune response in cancer, infectious diseases and inflammatory diseases, and are transferred to patients.

Description

Technical Field

[0001] This invention relates to immunotherapy adjuvant strategies and regimens for enhancing host immune responses in cancer, infectious diseases, and inflammatory diseases to avoid immune tolerance. Specifically, the invention also describes how products containing EPO pathway inhibitors can restore immune responses in pathological states, such as cancer and refractory infectious diseases, enhance and ensure the therapeutic accessibility of immunotherapy strategies, and eliminate "tolerogenic" stimuli; these products, for example, can serve as active pharmaceutical ingredients in vaccine compositions that stimulate immune responses in cancer, infectious diseases, and inflammatory diseases and deliver them to the patient. Background Technology

[0002] The immune system is the body's first line of defense against foreign substances (not its own), whether those substances are microorganisms or tumor cells. To circumvent the body's defenses, these substances have developed a common mechanism over time to shift the immune response from an inflammatory response that targets non-self substances (tumors or infectious agents) to a tolerant anti-inflammatory response, thus making the immune system "tolerable."

[0003] For an immune response to effectively combat infectious diseases or cancer to be induced, cells or active molecules in the innate and adaptive immune systems must interact in a coordinated manner. 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 can precisely recognize the structure or amino acid sequence of non-self antigens with high accuracy.

[0004] The microbiome also plays an important role in the complex interaction between inflammation (activated immune system) and anti-inflammatory (tolerance) responses.

[0005] Symbiotic microbes colonize the barrier surfaces of all multicellular organisms, including humans. For over 500 million years, symbiotic microbes and their hosts have co-evolved and adapted to each other. Therefore, the symbiotic microbiome influences many immune and non-immune functions of their hosts; in fact, the two together constitute a macroorganism. The symbiotic microbiome communicates with the host through bioactive molecules. As part of a shared mechanism, microbial imbalance may play a crucial role in the development of various diseases, such as cancer, autoimmune diseases, and increased susceptibility to infection. The gut microbiome also plays a role in anti-cancer responses: a lack of immunostimulatory bacteria or an dysregulated microbiome composition containing immunosuppressive species can lead to treatment failure. Recently, it has been recognized that the gut microbiome affects the effectiveness of PD-1-based anti-cancer immunotherapy, and that a healthy gut microbiome is a determinant of the anti-cancer response. Indeed, the immune system's regulation of the symbiotic microbiome is crucial for preventing cancer development, progression, and immune escape, and also plays a regulatory role in cancer treatment.

[0006] A prime example of gut microbiota factors and cancer is cervical cancer, a malignant tumor caused by persistent human papillomavirus (HPV) infection that occurs in more than 500,000 women annually. More than 90% of cervical cancer deaths occur in low- and middle-income countries. A common epidemiological feature of countries with high cervical cancer incidence is a heavy burden of intestinal worm infection. Intestinal worms can trigger immune modulation, creating a "tolerogenic" systemic immune environment that provides fertile ground for the persistence of oncogenic viruses such as HPV. Animal models have shown that intestinal worm infection can lead to the persistence of some viruses; however, HPV-specific and human studies remain lacking. Large-scale, well-organized trials assessing the effects of intestinal worm infection on the human immune system and HPV persistence could help improve HPV prevention strategies in worm-endemic regions worldwide. This invention provides strategies to counteract specific immune modulatory pathways that may result from intestinal worm infection and identifies novel therapeutic targets for a variety of diseases, from inflammatory conditions to cancer. Negative EPO regulation can counteract worm-induced systemic and local immune dysregulation, which may be a mechanism by which chronic intestinal worm infection promotes HPV persistence. Common mechanisms underlying various immune escape-based diseases with the EPO pathway as a key therapeutic target can be analyzed. One example of this immune system escape driven by the same "tolerogenic" stimuli observed in cancer is Plasmodium falciparum, the causative agent of malaria. Malaria is one of the most important infectious diseases in humans, particularly affecting populations living in tropical and subtropical regions. Currently, despite the availability of some antimalarial drugs, malaria remains a major public health problem, causing 241 million cases and 627,000 deaths annually. This infection is caused by the protozoan parasite Plasmodium. Five species of Plasmodium can cause malaria in humans: *Plasmodium vivax*, *Plasmodium malariae*, *Plasmodium ovale*, *Plasmodium knowlesi*, and *Plasmodium falciparum*. Although *Plasmodium vivax* is the most widespread, almost all serious and fatal cases of malaria are caused by *Plasmodium falciparum*. Immune evasion strategies are used to circumvent immune responses. *Plasmodium falciparum* parasites evade the immune response of mosquitoes to spread to new hosts. The main and key gene in *Plasmodium falciparum* used to evade the Anopheles mosquito immune response is Pfs47. It inhibits JNK-mediated apoptosis by blocking the activation of various caspases, and the Jak-STAT pathway is a downstream pathway of EPO / EPO-R signaling. These pathways also play important roles in regulating the responses of macrophages and lymphocytes to Mycobacterium tuberculosis, HIV, and solid tumors; therefore, immune cells may be part of strategies to resist drug treatment. In addition, abnormal red blood cell production has been observed in malaria patients, which may be related to anemia.Limited research has been conducted on the relationship between erythropoiesis defects and malaria-induced anemia; however, recent findings may offer new insights. Tsubata and colleagues reported in 2005 that erythropoiesis begins in the liver and spleen after malaria infection, and that newly generated erythrocytes in the liver are crucial for the infection and persistence of Plasmodium.

[0007] Cancer is a group of diseases characterized by localized abnormal cell growth with the potential to spread throughout the body. In the core region of a tumor, abnormal angiogenesis and impaired blood flow restrict oxygen supply, creating a hypoxic environment. Hypoxia attracts regulatory T cells (Tregs) to the tumor microenvironment (TME), thereby inhibiting effector T cell function and promoting tumor growth. This condition upregulates the expression of hypoxia-inducible factors (HIFs), including HIF-1α and HIF-1β. Hypoxia also enhances glucose uptake by cancer cells and intensifies glycolysis, further exacerbating glucose depletion in the TME. Among these factors, EPO and sphingosine 1-phosphate (S1P) play a central role in regulating the immunosuppressive tumor microenvironment. S1P is generated from sphingosine via sphingosine kinase 1 (SphK1) induction of apoptosis. S1P functions through the receptor family S1PR1-5, while mouse macrophages express only S1PR1 and S1PR2. S1P has been identified as a find-me signal; however, the circulating S1P concentration (4 mM) is higher than that in tissues (low nM) and from apoptotic cells (400 nM), suggesting that S1P may act locally or have other functions in the context of apoptotic cell clearance. The find-me signal S1P released from apoptotic cells has been shown to induce HIF-1α (hypoxia-inducible factor-1α) in macrophages. HIF-1α is the oxygen-unstable subunit of the HIF complex and a major transcription factor for EPO, suggesting that S1P derived from apoptotic cells may activate EPO signaling transduction in macrophages. EPO receptors, EPOR, EPHB4, CSF2RB, CRLF3, and tissue protection factors such as the EPOR / CD131 heterodimer (EPOR) have been identified on macrophages; through these, EPO can suppress the expression of inflammatory genes in macrophages, indicating their involvement in the coordinated regulation of toxic responses in macrophages. Furthermore, previous studies have shown that EPO can improve outcomes in autoimmune diseases. However, the role of EPO in the clearance of apoptotic cells and immune tolerance remains unclear. Here, we found that S1P derived from apoptotic cells activates EPO signaling in macrophages, thereby promoting the immune-silencing clearance of apoptotic cells and immune tolerance in mice.

[0008] This immune cell tolerance mechanism is prevalent in many types of cancer and treatment-resistant infectious diseases, including lung cancer, bladder cancer, prostate cancer, pancreatic cancer, ovarian cancer, cervical cancer, brain cancer, stomach cancer, colorectal cancer, and melanoma, as well as tuberculosis, malaria, HIV infection, streptococcal infection, SARS, SARS-CoV, MERS, and prions.

[0009] In the past, the most common methods for treating neoplastic cancers were surgery, radiation therapy, or chemotherapy. However, recent studies have demonstrated the promising potential of cancer immunotherapy as a treatment approach. The most fundamental challenge in tumor immunotherapy is activating the immune system to recognize and eliminate antigens. Significant progress has been made in this area through novel methods that genetically engineer tumor cells to secrete specific cytokines. The theoretical basis for genetically modified tumor vaccines based on immunotherapy is that the host possesses antigens capable of recognizing tumors as exogenous factors. Human T lymphocytes and B lymphocytes, during development, can distinguish virtually an unlimited number of antigenic differences through antigen receptors. However, for tumor immunotherapy to succeed, two conditions must be met: First, tumor cells must express novel antigens (peptides) not expressed in normal cells. Second, immune cells must be properly activated to recognize these antigens. Due to the recognized benefits of acting on disease through multiple pathways simultaneously, combination therapy is becoming increasingly common in cancer treatment. Combination therapy remains effective even when resistance to anticancer drugs develops. Furthermore, combination therapy offers the advantage of reducing the dosage of anticancer agents by enhancing their efficacy. In this way, the toxicity and side effects on various organs of the body can be minimized while improving the efficacy of anti-cancer treatment.

[0010] In recent years, several cancer immunotherapies that train or stimulate the innate immune system to recognize, attack, and eradicate tumor cells while minimizing damage to healthy cells have shown promising clinical responses. However, most of these immunotherapy strategies benefit only a small percentage of patients and cause systemic autoimmune side effects in some. Inducing immunogenic cell death (ICD) not only directly kills cancer cells but also induces anti-tumor immune responses against various solid tumors. This vaccine-like strategy can be used to stimulate a transformation from a "cold" tumor microenvironment to an immunogenic "hot" tumor microenvironment, synergizing with immunotherapy to improve patient response rates and achieve successful treatment outcomes.

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

[0012] However, another form of immunotherapy, namely CAR-T cell therapy, and more recently, tumor-infiltrating lymphocyte (TIL) therapy, has also attracted widespread attention from researchers and oncologists. These therapies have shown the ability to eradicate very advanced leukemia and lymphoma. However, metabolic restrictions and soluble factors can form an immunosuppressive TME, exacerbating the functional exhaustion of tumor-infiltrating T cells, leading to poor T cell proliferation and shortened T cell duration.

[0013] In fact, compared to hematologic malignancies, CAR-T cell therapy for solid tumors is limited by the ability of CAR-T cells to migrate and infiltrate solid tumors due to the immunosuppressive tumor microenvironment. One strategy to mitigate these limitations is to employ delivery routes other than systemic administration, such as local administration, thereby eliminating the need to migrate CAR-T cells to the disease site and limiting on-target off-tumor toxicities. This is because the targeting activity of CAR-T cells acts directly on tumor cells, minimizing interactions with normal tissues. Preclinical models have demonstrated excellent therapeutic effects in breast cancer brain metastases and glioblastoma through intraventricular injection of CAR-T cells targeting HER2 / IL13Ra2 (NCTO2208362, NCT03389230, NCT03696030).

[0014] Another important aspect to consider is the physical barriers of the tumor, such as the tumor stroma, which restricts the penetration and migration of CAR-T cells. The stroma is primarily composed of the extracellular matrix (also known as the matrix), which helps determine tumor invasion, adhesion, and growth. Within the matrix, heparan sulfate proteoglycans (HSPGs), such as phosphatidylinositol proteoglycans (GPC 1–6) and multiligand proteoglycans (SDC 1–4), are key components that CAR-T cells must degrade to enter the tumor. Genetically engineered CAR-T cells to express heparinase (an enzyme that degrades HSPGs) exhibit enhanced tumor infiltration and antitumor activity. Similarly, in animal models, CAR-T cells targeting fibroblast activation protein (FAP) exhibit enhanced cytotoxicity by reducing tumor fibroblasts. Furthermore, a promising strategy is to guide T cell migration via tumor-specific chemokines, provided that the T cells express corresponding matching chemokine receptors.

[0015] Here, we demonstrate that EPOR is expressed on T cells, as well as monocytes and macrophages derived from monocytes. We further demonstrate, unexpectedly, that the EPO-EPOR pathway is more active in the immune system's toxic state, thereby allowing the development and maintenance of cancer and infectious diseases. Based on this evidence, we decided to negatively modulate the EPO pathway to alter pathophysiological mechanisms and enhance the immune system's response to pathogenic stimuli. More specifically, for example, we treated glioblastoma stem cells, breast cancer cells, melanoma cells, and colon cancer cells with a combination of CAR-T cell therapy and anti-EPO monoclonal antibody. Unexpectedly, the results showed that the combination therapy of anti-EPO monoclonal antibody and CAR-T promoted T cell migration and penetration into tumor tissue; this therapy has been less used in solid malignancies due to poor tumor penetration. Furthermore, we unexpectedly observed that this combination therapy had higher killing activity against cancer cells compared to monotherapy.

[0016] Therefore, various approaches have been investigated to improve and enhance CAR-T cell responses. First, combined immunotherapy with CAR-T cells and checkpoint inhibitors (e.g., nivolumab, relatlimab, ipilimumab, anti-CD47; checkpoint inhibitors can induce CAR-T infiltration and provide PD-1 / PD-L1 blockade or SIRPα / CD47) and chemotherapy (e.g., cyclophosphamide). In hematologic malignancies, combination therapy with PD-1 blockade and CD19 CAR-T cells in children with multiple prior treatments for B-ALL improved CAR-T cell persistence and achieved better outcomes. Second, tumors, especially solid tumors, may possess intrinsic cellular tolerance mechanisms to CAR-T cell cytotoxicity, thus requiring the integration of other forms of immunotherapy strategies to counteract inhibitory signals present in the tumor microenvironment. In fact, the loss of IFNγR signaling in tumors has been reported in clinical studies of checkpoint blockade tolerance due to its downstream effects on antigen presentation. In fact, it has been confirmed that the absence of genes (IFNGR1, JAK1, or JAK2) in the interferon-γ receptor (IFNγR) signaling pathway leads to stronger tolerance of glioblastoma and other solid tumors to the killing effect of CAR-T cells, both in vitro and in vivo. Recently, first- and second-generation bispecific antibodies have entered clinical practice. In cancer immunotherapy, bispecific antibodies can form "immune synapses" between tumor cells and immune effector cells (such as T cells), making it possible for the immune response to precisely target specific cancer cells. Currently, a third-generation T-cell connector has been developed. This T-cell connector consists of two bispecific antibodies, each containing a tumor antigen-binding module and half of the domain required to bind to the CD3 subunit of the T-cell receptor. This strategy of splitting the anti-CD3 antigen-binding paratope ensures that both drug components are completely inactive until they simultaneously bind to the target antigen on the same tumor cell.

[0017] In solid tumors, such as brain tumors, the immunosuppressive microenvironment is formed by mutations in the IDH1 and IDH2 genes of isocitrate dehydrogenase (IDH) in glioma cells. These mutations suppress STAT1 expression and reduce the production of CD8 T cells, type 1-related effector molecules, and chemokines such as CXCL10. Consistent with these findings, T cell infiltration is significantly lower in IDH-mutant gliomas compared to IDH wild-type gliomas.

[0018] One challenge in targeting solid tumor antigens is that these antigens are often also expressed at varying levels in normal tissues. Therefore, antigen selection is crucial in CAR design, not only to ensure therapeutic efficacy but also to limit "non-tumor-targeting" toxicity. To overcome the fact that solid tumor antigens are also targeted in normal tissues, one potential approach is to target tumor-restricted post-translational modifications, such as truncated O-glycans overexpressed in solid tumors, such as Tn (GalNAca1-O-Ser / Thr) and sialyl-Tn (STn) (NeuAca2-6-GalNAca1-O-Ser / Thr). Based on this consideration, we found that anti-EPO antibodies that specifically bind to human EPO splice variants (EV-3 isoforms, highly expressed in tumor cells) can enhance immune cell responses without affecting normal cells. Simultaneously, inhibitors of EPO and EV-3 can induce ferroptosis-based cell death mechanisms. Ferroptosis, along with apoptosis, copper death, autophagy, and pyroptosis, is a key cell death pathway involved in many human diseases, including cancer. Another important characteristic of CAR-T cell products is the CD4+ / CD8+ subset ratio. Although CD8+ T cells have long been considered the dominant 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 both mediate direct cytotoxicity against tumor cells and provide helper functions to induce endogenous antitumor immune responses. In particular, the presence of CD4+ subsets was associated with the persistence of CAR-T cells in solid tumors. In summary, these findings suggest that CD4+-mediated immune responses play a crucial role in antitumor processes, a point highlighted by several ongoing clinical studies with precise dosage control of CD4+. We demonstrate that administration of an anti-EPO monoclonal antibody effectively induces CD4+ activation and migration to tumor tissue, thereby suppressing tolerogenic behavior in the immune response.

[0019] However, one of the most challenging limitations of CAR-T cell therapy is the development of resistance in tumors to single-antigen-targeting CAR constructs, a phenomenon known as antigen escape, which occurs particularly in solid tumors. For example, case reports of CAR-T cell therapy targeting IL13Ra2 in glioblastoma have shown that tumor recurrence is characterized by decreased IL13Ra2 expression. To reduce the recurrence rate of CAR-T cell therapy in hematologic malignancies and solid tumors, many current strategies rely on targeting multiple antigens. These strategies employ dual-CAR constructs or tandem CARs; a tandem CAR is a single CAR construct containing two scFvs that can simultaneously target multiple target tumor antigens; for example, in hematologic malignancies (ALL and diffuse large B-cell lymphoma), it can simultaneously target CD19 / CD20, CD19 / CD22, or CD19 / BCMA; or in solid tumors (such as glioblastoma, breast cancer, and colon cancer), it can simultaneously target HER2 / IL13Ra2, as well as HER2 / MUC1, TAG7228, B7-H3, MUC1, MUC16, αvβ6, CXCR1, or CXCR2. The importance of optimizing target antigen selection lies not only in improving antitumor responses but also in reducing antigen escape mechanisms to prevent relapse.

[0020] Furthermore, recent research has focused on modifying CARs to resist immunosuppressive factors in the adverse tumor microenvironment, such as TGF-β, IL-12, IL-15, IL-4, IL-10, arginase 1, and indoleamine 2,3-dioxygenase (IDO)-mediated inhibitory signals. Based on these premises, many studies have investigated various cytokines to create such "armored CARs." Therefore, an interesting strategy is to engineer CAR-T cells to provide immunostimulatory signals in the form of stimulating cytokines, thereby increasing T cell survival, proliferation, and anti-tumor activity, and rebalancing the tumor microenvironment. In recent years, RNA CAR-T technology has also been proposed for cancer therapy. However, current CAR-T cell engineering methods use viral delivery vectors, which can induce permanent CAR expression and may lead to serious adverse reactions. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate adverse reactions associated with viral vectors; however, it typically requires electroporation for T cell mRNA delivery, which can be cytotoxic. Currently, ionizable lipid nanoparticles (LNPs) have been engineered for mRNA delivery to human T cells. In addition to novel CAR-T cell-based immunotherapies, research on CAR-engineered macrophages (CAR-M), CAR-engineered natural killer (NK) cells (CAR-NK), or CAR-γ / δ is also establishing its place in the field of immuno-oncology.

[0021] In summary, the use of negative functional modulators of EPO is of great significance for overcoming host immune tolerance to microbial agents. It can induce a shift in the inflammatory response, activate macrophages and other immune system cells, thereby clearing infection. It can also be used in combination with antimicrobial agents, or to enhance vaccine efficacy, or to develop novel delivery systems.

[0022] New methods currently being reported, such as trojan horses, can destroy tumor cells by not only acting at the injection site but also inducing a systemic immune response, thereby clearing circulating and metastatic tumor cells.

[0023] Furthermore, controlled-release systems based on functionalized microparticles, microcapsules, and microspheres can ensure long-term therapeutic drug levels while minimizing the number of repeated injections and continuously influencing the pathological microenvironment, such as that of degenerative diseases or solid tumors.

[0024] Furthermore, new frontiers in cancer treatment are based on innovative nanomedicine and nanodelivery systems, such as tissue guns or probes, viral and nonviral vectors, nanomaterials for targeted delivery of bioactive drugs, nanoparticle-based approaches that combine imaging and therapy for cancer diagnosis and treatment, and lipid systems (such as liposomes and micelles), gold nanoparticles, or magnetic nanoparticles, which can also be used in combination with natural products.

[0025] When the target tissue is the central nervous system, strategies to overcome the blood-brain barrier (BBB) ​​can be employed, such as physical stimulation (e.g., focused ultrasound (FUS) and MRI fields) or Trojan molecules (e.g., IL13 / IL 13R, transferrin, natural or synthetic microvesicles, resveratrol). Furthermore, nicotine can increase the permeability of the blood-brain barrier in vivo. This suggests that nicotinic acetylcholine receptors may play a role in regulating brain microvascular permeability. Additionally, using drug-eluting embolization (DEE) microspheres as drug delivery carriers for local immunotherapy via direct perfusion into the tumor-feeding arteries may increase and prolong tumor drug concentrations, reducing systemic drug exposure and potentially improving the risk-benefit ratio of these drugs.

[0026] Furthermore, negative function modulators have advantages in anti-tumor treatment regimens and can be used in combination with other natural or synthetic drugs. Flavonoids, particularly luteolin (an inhibitor of S1P kinase), have been shown to play beneficial roles in tumors by regulating the activity of reactive oxygen species (ROS) scavenging enzymes, participating in cell cycle arrest, inducing apoptosis and autophagy, and inhibiting cancer cell proliferation and invasion, thus exhibiting a synergistic effect with anti-EPO therapy. Rapamycin and its derivatives are promising therapeutic agents with both immunosuppressive and anti-tumor properties. Rapamycin's action is mediated by specific inhibition of mTOR protein kinase. mTOR, as part of an evolutionarily conserved signaling pathway, controls the cell cycle in response to changes in nutrient levels. Another molecule controlling cellular nutrient levels is metformin. Metformin is a widely prescribed drug by physicians today due to its specific efficacy in treating and curing type II diabetes. Metformin inhibits mTOR activity by activating ATM (ataxia-telangiectasia mutation) and LKB1 (hepatic kinase B1), which in turn activates adenosine monophosphate-activated protein kinase (AMPK), thereby preventing protein synthesis and cell growth. Furthermore, one mechanism by which metformin exerts its effect is by activating AMPK, an intracellular enzyme that lowers blood glucose by promoting energy utilization. AMPK activation has broad effects, extending far beyond blood glucose control itself. Studies have shown that increasing AMPK activity can prevent or even reverse age-related lifespan-shortening effects, such as cardiovascular disease, diabetes, neurodegenerative diseases, and cancer. Moreover, strong evidence suggests that AMPK negatively regulates the mTOR pathway, thus suggesting that metformin mediates a close interaction between mTOR and AMPK. We demonstrate that the synergistic effect of metformin and anti-EPO antibodies on cancer cell viability, reducing PD-1 gene expression and increasing IFNγ and IL-1B gene overexpression, promotes a pro-inflammatory shift in the tumor and infection site microenvironment, and has the beneficial effect of interrupting pathophysiological mechanisms and eradicating pathogenic stimuli.

[0027] Glucagon-like peptide-1 (GLP-1) receptor agonists are widely used to treat type 2 diabetes due to their glucose-lowering effects and low risk of hypoglycemia. GLP-1 binds to its specific G protein-coupled receptor, activating downstream pathways, including the cAMP / protein kinase A (PKA), cAMP / guanine nucleotide exchange factor (Epac), or phosphatidylinositol-3 kinase / PKC pathways. It has been reported that GLP-1 may be involved in carcinogenesis due to its nutritional role.

[0028] Glycoprotein nonmetastatic melanoma protein B (GPNMB), whose extracellular domain detaches from the cell surface and interacts with integrins, promotes the recruitment of immunosuppressive and pro-angiogenic cells to the tumor microenvironment, thereby enhancing tumor migration and invasion. The effects of GPNMB-mediated cell signaling within the tumor, combined with GPNMB's ability to influence the primary and metastatic microenvironments in a non-cell-autonomous manner, collectively enhance the malignant tumor phenotype. Furthermore, GPNMB is frequently overexpressed in various cancers, making it an attractive therapeutic target. In this regard, the antibody-drug conjugate (ADC) glembatumumab vedotin targeting GPNMB is currently under investigation. In addition, GPNMB has shown anti-inflammatory effects in various neurological disorders. Based on these premises, the regulation of GPNMB also appears promising in cancer and infectious diseases.

[0029] In summary, this approach can function like a vaccine, stimulating the transformation of a "cold" site microenvironment into an immunogenic "hot" site microenvironment, a common feature of cancer, infection, and inflammatory diseases. It works synergistically with immunotherapy to improve patient response rates and achieve successful treatment outcomes.

[0030] The present invention also provides the possibility of combating infectious agents (such as TBC, malaria, HIV, SARS, drug-resistant bacteria, fungi and viruses) by stimulating the host's inflammatory response, enhancing the efficacy of conventional vaccines, recombinant vaccines and peptide, lipid, DNA or mRNA-based vaccines, and enhancing the efficacy of antimicrobial therapy.

[0031] Furthermore, the technical solutions proposed in this paper can serve as a useful tool to combat the negative associated mechanisms and effects of 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, hypoxia, cosmic and UV radiation exposure (e.g. during spaceflight missions).

[0032] Beneficial effects EPO specifically participates in the regulation of the immune system and serves as a pathogenesis mechanism for various diseases, such as cancer, infectious diseases, and autoimmune diseases.

[0033] The cancer immunotherapy adjuvant of the present invention, when administered in combination with or alone with a cancer immunotherapy agent, can activate the function of immune factors without producing side effects in the body, thereby demonstrating the effect of a kit that enhances the anticancer effect 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 treatment.

[0034] For example, the tumor microenvironment (TME) is a major barrier to T lymphocyte infiltration and inhibits their function. Several immune checkpoint proteins have been identified that can interfere with ligand / receptor interactions and impair T cell anti-tumor responses. Immunotherapy that blocks immune checkpoints has revolutionized the treatment paradigm for many patients with advanced cancer. However, metabolic restrictions and soluble factors present in the TME exacerbate the functional exhaustion of tumor-infiltrating T cells. Using negative modulators of EPO offers an option to enhance immunotherapy strategies and improve the effectiveness of current treatments. Similar characteristics have been observed in some refractory infectious diseases, such as malaria and HIV; paradoxically, in these diseases, EPO is sometimes used to combat iatrogenic anemia associated with antimicrobial and antiviral therapy. In this case, EPO induces a reduced immune response, leading to immune tolerance in macrophages and lymphocytes. This invention can enhance the efficacy of antimicrobial therapies, such as antibiotics (penicillins, macrolides, cephalosporins, fluoroquinolones, enhanced-activity β-lactams, tetracyclines, trimethoprim-sulfamethoxazole, urinary tract anti-infectives, lincosamides), antiviral drugs (reverse transcriptase inhibitors, protease inhibitors, and other antiviral drug classes), antifungals (polyenes, azoles, allylamines, and echinocandins), and antimalarial therapies, as combination therapy for prion diseases. Summary of the Invention

[0035] The inventors unexpectedly discovered that by blocking EPO, the innate and adaptive immune response regulation mechanisms common to tumors, infectious diseases, and inflammatory diseases can be overcome, thereby developing new therapeutic strategies to inhibit drug resistance and progression of pathological processes and eliminate "tolerance-inducing" stimulation of the immune system. This invention relates to negative functional modulators of EPO for use in methods of activating innate and adaptive immune responses in patients in need. Unexpectedly, the compounds appear to be active in inducing immune cell migration, inducing immune cell implantation into tumor tissue, enhancing the immunostimulatory effects and cytotoxic capabilities of immune cells, and also in combating infection processes and enhancing vaccine efficacy.

[0036] EPO plays a specific role in the regulation of the immune system as a pathogenesis of various diseases, such as cancer, infectious diseases, and autoimmune diseases.

[0037] In a first aspect, the present invention relates to an anti-EPO negative functional modulator or an anti-EPO antigen-binding fragment selected from the group consisting of Fab, -F(ab')2, single-chain antibodies, biantibodies, triantibodies, tetraantibodies, repeatbodies, or domain antibodies, for use in a method of activating an immune response in a patient in need.

[0038] The immunotherapy adjuvant of the present invention, when administered in combination with or alone with an immunotherapy agent, activates the function of immune factors in cancer, infectious diseases, and inflammatory diseases without producing side effects in the body, and is therefore effective as an immunotherapy adjuvant.

[0039] In other respects, the present invention relates to a method for activating or enhancing an immune response in a patient in need, the method comprising using an anti-EPO negative functional modulator or an anti-EPO antigen-binding fragment alone, or in combination with the following: - Checkpoint inhibitor or immunomodulatory agent therapy (e.g., anti-PDL1 antibody, nivolumab, ipilimumab, abatacept, glembatumumab vedotin); and / or - Cell-based immunotherapies (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, activated dendritic cells targeting tumor-associated antigens, and / or antigen-presenting cells, tumor-associated peptides, therapies based on engineered mononuclear-macrophages or polymorphonuclear cells, and / or therapies for enhancing and reprogramming tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM) responses). - Antimicrobial therapy (e.g., antibiotics, antiviral drugs, antifungal agents, antifungal drugs, antipruritic drugs); - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Prophylactic or therapeutic vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids (e.g., anti-HPV vaccines, anti-EBV vaccines, or anti-HIV vaccines). - Immunotherapy based on oncolytic viruses; - Chemotherapy agents; - Anticancer drugs; - Enzymes that degrade heparan sulfate proteoglycans (e.g., heparinase); - A negative regulator of the sphingosine phosphate-1 (S1P) signaling pathway; or - EPO mimics that retain erythropoiesis function; The anti-EPO antigen-binding fragment is selected from the group including Fab, F(ab')2, single-chain antibodies, biantibodies, triantibodies, tetraantibodies, repeat antibodies, or domain antibodies; the anti-EPO negative functional regulator is selected from the group consisting of monospecific or multispecific anti-EPO antibodies, gene therapy, DNA decoys, RNA decoys, ribozymes, antagomiR, shRNA, LNA, siRNA, antisense oligonucleotides, or anti-EPO receptors; the anti-EPO receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR, and EPOR / CD131 heterodimers.

[0040] Secondly, this article describes a pharmaceutical kit comprising negative functional modulators of EPO / EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protectant, TPR (such as EPOR / CD131 heterodimer)) and / or their natural or synthetic variants; and: - Checkpoint inhibitor or immunomodulatory agent therapy (e.g., anti-PDL1 antibody, nivolumab, ipilimumab, abatacept, glembatumumab vedotin); and / or - Cell-based immunotherapies (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, activated dendritic cells targeting tumor-associated antigens), therapies based on engineered mononuclear-macrophages or polymorphonuclear cells, and / or therapies for enhancing and reprogramming tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM) responses; and one or more components, said components being selected from the group consisting of: - Peptides or antibodies, biantibodies, and nanobodies targeting natural and synthetic variants, including physiological and pathological splice variants and post-translational modified forms of erythropoietin; - Antimicrobial therapy (e.g., antibiotics, antiviral drugs, antifungal agents, antifungal drugs, antipruritic drugs); - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Prophylactic or therapeutic vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids (e.g., anti-HPV vaccines, anti-EBV vaccines, or anti-HIV vaccines). - Immunotherapy based on oncolytic viruses; - Chemotherapy drugs; - Anticancer drugs; - Enzymes that degrade heparan sulfate proteoglycans (e.g., heparinase); - A negative regulator of the sphingosine phosphate-1 (S1P) signaling pathway; or - An EPO mimic that retains the function of erythropoiesis.

[0041] Thirdly, the present invention relates to a diagnostic or prognostic method for assessing EPO and its somatic mutations or variants thereof, EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protectant, TPR, EPOR / CD131 heterodimer) and their somatic mutations and / or their variants and / or C4 mAb. The expression of ligands, a method that can predict responses to treatments targeting negative regulation of EPO / EPOR, to enable personalized treatment in immunotherapy or prevention of cancer and infectious diseases, and to stratify patients to optimize patient response; the method includes the steps of: determining the amount / detection of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants, as diagnostic or prognostic markers, in tissues, cells or human body fluids (saliva, blood, cerebrospinal fluid, sweat or derived extracellular vesicles) / 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 tissues, cells or human body fluids (saliva, blood, cerebrospinal fluid, sweat or derived extracellular vesicles).

[0042] Fourthly, the present invention relates to a diagnostic method for assessing the methylation of promoters of genes for EPO and EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR (e.g., EPOR / CD131 heterodimer)), which can predict increased expression of EPO / EPO-R and their negative role in suppressing the immune system's clearance of cancer cells or microbial factors, thereby enabling personalized immunotherapy or prevention and for prognostic purposes; the method includes the step of determining the methylation of EPO and EPO receptor genes as diagnostic or prognostic markers in tissues, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or derived extracellular vesicles).

[0043] In a further aspect, the present invention relates to an EPO / EPOR negative modulator delivery system, which can be administered clinically in various formulations via oral, parenteral, intralesional (intratumoral and intracavitary), intraventricular, intrathecal, intranasal, or local administration; the system is based on innovative nanomedicine and nanodelivery systems, such as tissue guns or probes; viral and nonviral vectors; nanomaterials for targeted delivery of bioactive drugs; plant-based vesicles; nanoparticle-based methods that allow simultaneous therapeutic and in vivo imaging for diagnostic and therapeutic purposes; lipid systems, such as liposomes and micelles; gold nanoparticles or magnetic nanoparticles, and functionalized nanoparticles, microspheres, and biomaterials, such as PEG, used in combination with natural products; and Trojan horse methods, such as micropumps for releasing therapeutic drugs in tissues to enhance the modulation of the immune system in the local pathological microenvironment or attract and increase homing of cell-based immunotherapies or vaccines.

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

[0045] In yet another embodiment, the present invention relates to a method based on EPO / EPOR negative regulation for improving the efficacy of CAR-T and CAR-γ / δ T cell therapy for solid tumors.

[0046] In another aspect, the present invention relates to inhibitors of EPO / EPO-R and their variants, which are capable of stimulating CTL infiltration and inhibiting the recruitment of immunosuppressive cells in tumors and infectious diseases, thereby increasing tissue penetration of inflammatory and immune cells.

[0047] In another aspect, the present invention relates to inhibitors of EPO / EPO-R and their variants, which are capable of inducing inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferroptosis, autophagy, copper death and immunostimulatory cell death, thereby enhancing the immunogenicity of tumors.

[0048] Another aspect describes a product selected from EPO and their natural and synthetic variants as inhibitors that can activate immune responses against cancer and infectious agents, reprogram the tumor microenvironment, and enhance immunotherapy and immunomodulatory strategies. Attached Figure Description

[0049] The features and advantages of the present invention will be described in detail below, through embodiments given for illustrative purposes and not for limiting purposes, and through the accompanying drawings. Figures 1-19 It becomes obvious that: Figure 1Anti-EPO treatment increases the migration of peripheral blood mononuclear cells (PBMCs) in the presence of GBM tissue. Figure 1 A is a schematic diagram of a chemotaxis assay (modified Boyden chamber) used for migration testing, showing GBM tissue at the bottom of the lower chamber. GBM tissue in CTR medium (CTR, Figure 1 B) 100 μg / mL anti-EPO (C4, Figure 1 C) and recombinant human EPO (rhEPO, Figure 1 D) After 48 hours, migration tests were performed on PBMCs stained with Hoechst (blue). Figure 1 E shows the total number of migrating cells, counted using the AnalyzeParticle plugin in ImageJ. Data are the mean ± SD of at least three replicates. *P < 0.05 compared to CTR for all treatment groups.

[0050] Figure 2 Anti-EPO treatment induced differentiation, activation, and expression of natural killer cell markers in PBMCs. Immunophenotypic characteristics of PBMCs were assessed using flow cytometry. Figure 2 Report A presents the proportions of biomarker expression in PBMCs after the following treatments: CTR, anti-EPO (C4), and rhEPO. Data are reported in comparison to the CTR condition. Figure 2 In section B, data represent the analysis of biomarkers used in PBMCs treated with C4 compared to those treated with rhEPO. Data are the mean ± SD of at least three replicates. *P < 0.05 compared to CTR; **P < 0.01 for all treatment groups compared to CTR.

[0051] Figure 3 Anti-EPO treatment induces EPOR-dependent PBMC migration.

[0052] Figure 3 A shows a schematic diagram of a modified Boyden chamber used for migration testing, illustrating GBM tissue at the bottom of the lower chamber. A red arrow indicates the lower surface of the photograph.

[0053] Figure 3 Image B shows representative images of immunofluorescence analysis of CD8 (green), CD14 (red), and EPOR (grey). Cell nuclei were counterstained with Hoechst. Figure 3 C reported the total number of all cells present in the stained sample at the bottom of the well 48 hours after transmembrane migration, particularly all CD8+ T cells (green bars) and CD14+ monocytes (red bars). Figure 3The total number of D); among which, the number of double-positive EPOR+CD8+ cells (green bars) and EPOR+CD14+ cells (red bars) for specific subpopulations is calculated. Figure 3 E). At 63x magnification, n=10 fields of view (FOV) were acquired, and the average total number of cells analyzed for each sample was n=500.

[0054] Figure 4 Molecular signatures of PBMC activation induced by anti-EPO treatment and blockade of exhaustion. Gene expression of markers associated with inflammation and exhaustion. This analysis was performed by assessing the gene expression profile of human PBMCs using real-time PCR. L-1b ( Figure 4 A), IL-6 ( Figure 4 B), IFNg ( Figure 4 C), IL-10 ( Figure 4 D), TGFb ( Figure 4 E), IFNa2 ( Figure 4 F), PD1 ( Figure 4 G), LAG-3 ( Figure 4 H), CTLA4 ( Figure 4 I) Genes were analyzed. Data are the mean ± SD of at least three replicates. *P < 0.05 compared to CTR for all treatment groups.

[0055] Figure 5 Anti-EPO treatment induces monocyte migration and their activation.

[0056] Monocytes were studied in a modified Boyden chamber to assess their migration ability after anti-EPO (C4) treatment. Figure 5 Cell migration assays were performed on cells stained with calcein (green) for 6 days, and the number of migrating cells at the bottom of the wells was counted after treatment with the following conditions: CTR medium (CTR, Figure 5 A), 10 μg / mL C4 ( Figure 5 B) and recombinant human EPO (rhEPO, Figure 5 C). Figure 5 D reported the total number of migrating cells, counted using the Analyze Particle plugin in ImageJ. The immunophenotype of infiltrating macrophage-monocytes was assessed by evaluating CD86+ / HLA-DR+ expression under the following conditions: CTR, TMZ, C4, TMZ+C4, and rhEPO (…). Figure 5 E), thereby assessing differentiation towards the M1 type. Data are the mean ± SD of at least 3 replicates. *P < 0.05 compared to CTR for all treatment groups. Data are the mean ± SD of at least 3 replicates. *P < 0.05 compared to CTR for all treatment groups.

[0057] Figure 6 Anti-EPO treatment induces macrophage infiltration into GBM tissues and their differentiation into M1 type.

[0058] The immunophenotypic characteristics of infiltrating macrophages were assessed under the following conditions by the expression of CD86 and HLA-DR: CTR, TMZ, C4, TMZ+C4, and rhEPO. Figure 6 A). Figure 6 The histogram in section B shows the quantitative distribution of infiltrating macrophages in GBM tissue. The histogram also shows the CD86+ / HLA-DR+ ratio in the treated macrophages. Figure 6 C) and CD86+ Figure 6 Co-expression of D) was evaluated, demonstrating differentiation toward the M1 type. Data are mean ± SD of at least three replicates. *P < 0.05 compared to CTR in all treatment groups.

[0059] Figure 7 Anti-EPO treatment induces differentiation of PBMC-derived monocytes into macrophages through EPOR surface expression.

[0060] Cells were fixed and immunolabeled to detect phalloidin (A, B) and EPOR (C, D) in PBMC-derived macrophages. Figures E and DF show the colocalization of phalloidin and EPOR. Cell nuclei were stained with DAPI (blue) (scale bar = 100 μm). Representative images of C4-treated cells (Figures A, C, E) and rhEPO-treated cells (Figures B, D, F) are shown, respectively.

[0061] Figure 8 Anti-EPO administration induces the migration of naive T cells.

[0062] Naïve T cells were analyzed to assess their migration ability after anti-EPO treatment. A modified Boyden chamber was used. The following conditions were observed: CTR medium (CTR, Figure 8 A), 10 μg / mLC4 ( Figure 8 B) and recombinant human EPO (rhEPO, Figure 8 C) and C4+rhEPO ( Figure 8 D) After 48 hours of treatment, chemotaxis experiments were performed on cells stained with calcein (green). Figure 8 E reported the total number of migrating cells, counted using the Analyze Particle plugin in ImageJ. CD8+ migration was also observed in the presence of several EPO negative regulators. Figure 8F). For this purpose, we tested the commercial antibodies B4 (Santa Cruz Biotechnology) and 16FH11 (Stem Cell Technologies). Data are mean ± SD of at least 3 replicates. *P < 0.05 compared to CTR for all treatment groups.

[0063] Figure 9 Anti-EPO treatment induces T cell activation through EPOR expression in GBMTME.

[0064] Initial CD4+ T cells were cultured in GSC conditioned medium with the following conditions: CTR ( Figure 9 A) Temozolomide (TMZ, Figure 9 B), rhEPO ( Figure 9 C), Anti-EPO (C4) Figure 9 D), TMZ+C4 Figure 9 E) and rhEPO+C4 ( Figure 9 F). Immunophenotypic characteristics were assessed by evaluating the expression of EPOR and CD69+ under the following conditions: CTR, TMZ, C4, TMZ+C4, rhEPO, and rhEPO+C4. Figure 9 G). Data are the mean ± SD of at least 3 replicates. **P < 0.01, ***P < 0.001 compared to CTR for all treatment groups.

[0065] Figure 10 Anti-EPO treatment induces CD4+ CTL lymphocytes to migrate to the TME.

[0066] Cytotoxic CD4+ T cells were studied to assess their migration ability. A modified Boyden chamber was used. Figure 10 A). Cells stained with calcein (green) for 48 hours were subjected to a migration assay, and the number of migrating cells at the bottom of the wells was counted after treatment with conditioned medium collected from GSCs treated under the following conditions. Figure 10 B): CTR medium (CTR, Figure 10 C), 100 μM TMZ ( Figure 10 D), 10 μg / mL C4 ( Figure 10 E), TMZ+C4 ( Figure 10 F) and recombinant human EPO (rhEPO, Figure 10 G). Figure 10B reported the total number of migrating cells, counted using the Analyze Particle plugin in ImageJ. Data are the mean ± SD of at least three replicates. **P < 0.01, ***P < 0.001 compared to CTR for all treatment groups.

[0067] Figure 11 Anti-EPO treatment induces deep penetration of CTL lymphocytes into GBM tissue.

[0068] CTL T lymphocytes were studied to evaluate their anti-EPO (C4) migration ability in the presence of GBM tissue. A modified Boyden chamber was used. A large piece of GBM tissue was placed at the bottom of the well. Figure 11 A). Cells stained with calcein (green) for 48 hours were subjected to migration assays, and the number of migrated cells at the bottom of the wells was counted after treatment under the following conditions (A). Figure 11 B): CTR medium (CTR, Figure 11 C), 100μM TMZ ( Figure 11 D), 10 μg / mL C4 ( Figure 11 E), TMZ+C4 ( Figure 11 F), Recombinant human EPO (rhEPO, Figure 11 G) and rhEPO+C4 ( Figure 11 H). Figure 11 B reports the total number of migrating cells, counted at the bottom of the wells using the Analyze Particle plugin in ImageJ. Data are the mean ± SD of at least three replicates. **P < 0.01, ***P < 0.001 compared to CTR; #P < 0.05 compared to rhEPO.

[0069] Figure 12 Anti-EPO treatment induced CTL lymphocytes to migrate to GBM tissue.

[0070] Migration assays were performed to assess the infiltration capacity of CD4+ CTL T cells into GBM tissue. Cells were stained with calcein and migrated in modified Boyden chambers for 48 hours. Figure 12 A); Subsequently, GBM tissue was collected, digested with trypsin, and filtered. Single-cell suspensions were analyzed using flow cytometry, and infiltrating cells were counted as event numbers. Figure 12 B reported the total number of infiltrating cells. Data are the mean ± SD of at least three replicates. ***P < 0.01 compared with CTR in all treatment groups.

[0071] Figure 13 Expression of EPO-related targets in GBM cells.

[0072] The expression patterns of EPO-related genes, transferrin-related genes, and IL-13-related genes in CTR and GBM mRNA extracted from cells were analyzed by real-time PCR. A) Expression of EGFR, EPOR, EPHB4, CSF2RB, and CRLF3; B) Expression of IL-13, IL-13R1, and IL-13R2; C) Expression of transferrin receptor 1 (TfR1), transferrin receptor 2 (TfR2), and folic acid receptor in the control group and GBM. Data are the mean ± SD of at least three replicates. *P < 0.05 compared to CTR in all treatment groups.

[0073] Figure 14 Anti-EPO treatment enhances the cytotoxic effect of CAR-T cells on GBM cells.

[0074] To test the cytotoxic activity of T cells, WT T cells, CAR-T cells alone, or WT T cells, CAR-T cells combined with C4, were co-cultured with glioblastoma stem cells for 48 hours. Cytotoxicity was measured by co-culturing immune T cells with GSCs at effector-to-target ratios (E:T) of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Figure 14 A). TNFα ( ) was obtained from the supernatant after CAR-T cells were co-cultured with GSCs for 48 hours. Figure 14 B) and IFNγ Figure 14 C) Cytokine quantification. Data are the mean ± SD of at least three replicates. *P < 0.05 compared with CTR for all treatment groups.

[0075] Figure 15 Anti-EPO treatment enhances the cytotoxicity of CAR-T cells against breast cancer cells, pancreatic cancer cells, and melanoma cancer cells.

[0076] To test the cytotoxic activity of T cells, WT cells, CAR-T cells alone, or WT cells, CAR-T cells combined with C4, were used with breast cancer cells (MCF7, Figure 15 A) Melanoma cancer cells ( Figure 15 B) and colon cancer cells (DLD1, Figure 15 C) Co-culture for 48 hours. Cytotoxicity was measured by co-culturing immune T cells with breast cancer cells, melanoma cancer cells, and colon cancer cells at effector-to-target ratios (E:T) of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Figure 15 A- Figure 15C). Data are the mean ± SD of at least three replicates. *P < 0.05 compared to CTR for all treatment groups.

[0077] Figure 16 By combining anti-EPO and metformin treatment, the cytotoxicity of CAR-T cells against glioblastoma cancer cells was enhanced in a synergistic manner.

[0078] The combined treatment with metformin was also tested for its effectiveness against EPO (C4) treatment and cytotoxic activity against T cells. WT cells, CAR-T cells alone, or WT cells, CAR-T cells in combination with C4 and / or metformin, were co-cultured with GSCs for 48 hours. Figure 16 A). Cytotoxicity was measured by co-culturing immune T cells with glioblastoma cancer cells at effector-to-target ratios (E:T) of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. Figure 16 A). After combined administration of anti-EPO (C4) and metformin (MET), the effect on IL-1β ( Figure 16 B), IFNγ ( Figure 16 C) and PD-1 ( Figure 16 The gene expression of D) was evaluated. Figure 16 B. Data are the mean ± SD of at least three replicates. *P < 0.05 compared to CTR for all treatment groups.

[0079] Figure 17 Anti-EPO treatment stimulated immune cell infiltration into a GBM subcutaneous PDX mouse model, serving as an example of solid tumor penetration and potency. Representative images of intratumoral inflammatory cell infiltration are shown by hematoxylin-eosin staining. In placebo-treated PDX tumors, the density of inflammatory cell infiltration was lower (a: × 100; b: × 400). Figure 17 A, Figure 17 B). In PDX tumors treated with 10 mg / kg anti-EPO antibody, the density of inflammatory cell infiltration was high (c: × 100; d: × 400). Figure 17 C Figure 17 D).

[0080] Figure 18 Anti-EPO treatment reduces parasitemia in blood cells.

[0081] Parasitemia curves of infected erythrocytes after exposure to placebo (WT) or anti-EPO antibody. Data are mean ± SD of at least three replicates. *P < 0.05 compared with CTR in all treatment groups.

[0082] Figure 19Anti-EPO treatment increases the migration of PBMCs in infectious diseases. Figure 19 A is a schematic diagram of a chemotactic assay (modified Boyden chamber) used for migration testing, showing the migration ability of PBMCs after anti-EPO (C4) treatment in the presence of lipopolysaccharide (LPS). The treatment involved administering 10 μg / mL LPS for 48 hours. Figure 19 B), and administration of anti-EPO (C4) (B ...) Figure 19 C) and rhEPO ( Figure 19 D), treated for 48 hours. PBMC migration was assessed using a modified Boyden chamber, and the number of migrating cells was counted using the Analyze Particle plugin in ImageJ (D). Figure 19 E). Data are the mean ± SD of at least 3 replicates. *P < 0.05 compared to CTR for all treatment groups.

[0083] Figure 20 In an in vivo orthotopic rodent brain cancer model, compared with a healthy contralateral cerebral hemisphere ( Figure 20 Compared to A), administration of anti-EPO significantly induced activation of the immune system and migration of T lymphocytes. Figure 20 B). Surprisingly, anti-EPO treatment induced ferroptosis in cancer cells as a marker of cell death (B). Figure 20 C). Detailed Implementation

[0084] This invention provides methods and uses for modulating the immune system based on the negative regulation of EPO and EPO receptors, as well as their natural splice variants and synthetic variants. Pharmaceutical compositions and treatment methods are also provided.

[0085] In another form, the present invention provides methods and uses thereof for modulating the immune system based on negative regulation of typical and alternative EPO receptors (EPO-R; EPHB4; CSF2RB; CD131; CRLF3; soluble EPO-R; tissue protectant; TPR, such as EPOR / CD131 heterodimer) or their splice variants, somatic mutations or variants thereof.

[0086] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are typically implemented using conventional methods, such as the widely used hybridoma method and phage display technology.

[0087] This invention describes a method for activating the immune system to fight tumors or infectious agents, which can be performed alone or in combination with standard therapies.

[0088] In a first aspect, the present invention relates to an anti-EPO negative functional modulator or an anti-EPO antigen-binding fragment selected from the group consisting of Fab, -F(ab')2, single-chain antibodies, biantibodies, triantibodies, tetraantibodies, repeat antibodies, or domain antibodies, for use in a method of activating an immune response in a patient in need.

[0089] In a further aspect, the present invention relates to a method for activating or enhancing an immune response in a patient in need, the method comprising using an anti-EPO negative functional modulator or an anti-EPO antigen-binding fragment alone, or in combination with the following: - Checkpoint inhibitor or immunomodulatory agent therapy (e.g., anti-PDL1 antibody, nivolumab, ipilimumab, abatacept, glembatumumab vedotin); and / or - Cell-based immunotherapies (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, activated dendritic cells targeting tumor-associated antigens, and / or antigen-presenting cells, tumor-associated peptides, therapies based on engineered mononuclear-macrophages or polymorphonuclear cells, and / or therapies for enhancing and reprogramming tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM) responses). - Antimicrobial therapy (e.g., antibiotics, antiviral drugs, antifungal agents, antifungal drugs, antipruritic drugs); - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Prophylactic or therapeutic vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids (e.g., anti-HPV vaccines, anti-EBV vaccines, or anti-HIV vaccines). - Immunotherapy based on oncolytic viruses; - Chemotherapy agents; - Anticancer drugs; - Enzymes that degrade heparan sulfate proteoglycans (e.g., heparinase); - A negative regulator of the sphingosine phosphate-1 (S1P) signaling pathway; or - EPO mimics that retain erythropoiesis function; The anti-EPO antigen-binding fragment is selected from the group including Fab, F(ab')2, single-chain antibodies, biantibodies, triantibodies, tetraantibodies, repeat antibodies, or domain antibodies; the anti-EPO negative functional regulator is selected from the group consisting of monospecific or multispecific anti-EPO antibodies, gene therapy, DNA decoys, RNA decoys, ribozymes, antagomiR, shRNA, LNA, siRNA, antisense oligonucleotides, or anti-EPO receptors; the anti-EPO receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR, and EPOR / CD131 heterodimers.

[0090] In a preferred aspect, the anti-EPO negative function modulator or anti-EPO antigen-binding fragment used according to the invention binds to EPO, wherein the EPO receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR, EPOR / CD131 heterodimer and their variants or products of their somatic mutations.

[0091] Preferably, the modulator or anti-EPO antigen-binding fragment is an immunotherapeutic adjuvant and / or induces an immune cell response (antigen-presenting cells activated by T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells), wherein the cell response is an antitumor T cell response or an antimicrobial cell response in a patient affected by cancer or an infectious disease.

[0092] In a further preferred aspect, in the use of the negative functional modulator or anti-EPO antigen-binding fragment according to the invention, or in a method for activating or enhancing the immune response of a patient in need, the anti-tumor T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells are immune cells derived from PBMCs.

[0093] Preferably, in the use of a negative functional modulator or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response of a patient in need, the anti-tumor T cell response is a CD8+ T cell response and a CD4+ T cell response.

[0094] Preferably, in the use of a negative functional modulator or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing an immune response in a patient in need, the anti-tumor T cell response is attributed to the expression of CD14, CD69, and EPOR in migrating PBMCs.

[0095] In a preferred aspect, in the use of a negative functional modulator or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing an immune response in a patient in need, the anti-EPO antigen-binding fragment is a neutralizing antibody that binds to EPO, an EPO variant, or an EPO receptor and restores activation of T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells.

[0096] In yet another preferred aspect, in the use of a negative functional modulator or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response of a patient in need, the anti-EPO antigen-binding fragment is a neutralizing antibody selected from the group consisting of C4, B4, and 16F1H11.

[0097] One example described in this invention relates to a purified anti-erythropoietin (EPO) antibody, also known as a "C4 antibody" or "C4", wherein the antibody comprises: a. A light chain variable region (VL) having the amino acid sequence SEQ ID NO: 6; and b. Heavy chain variable region (VH) having the amino acid sequence SEQ ID NO: 14.

[0098] The hybridoma cells that produce C4 antibodies according to the present invention were deposited at Leibniz-Institute DSMZ on September 9, 2021, with accession number DSM ACC 3370. The antibody comprises a light chain variable region (VL) having the amino acid sequence SEQ ID NO: 6 and a heavy chain variable region (VH) having the amino acid sequence SEQ ID NO: 14.

[0099] In a preferred aspect, the antibody of the present invention is an isolated anti-EPO antibody, wherein the antibody comprises six CDR regions, the CDR regions being: a. VL-CDR1 having the amino acid sequence SEQ ID NO: 4; b. VL-CDR2 with the GAS (Gly-Ala-Ser) amino acid sequence; c. VL-CDR3 having the amino acid sequence SEQ ID NO: 5; d. VH-CDR1 having the amino acid sequence SEQ ID NO: 11; e. VH-CDR2 having the amino acid sequence SEQ ID NO: 12; and f. VH-CDR3 having the amino acid sequence SEQ ID NO: 13.

[0100] For the purposes of this disclosure, the SEQ ID NO. corresponding to each sequence is as follows: DNA sequence of the CDR1 region (VL-CDR1) of the light chain variable region corresponding to SEQ ID NO: 1 of the anti-EPO antibody: GAAAGTGTTGACTATTATGGCACAGGTTTA.

[0101] GGTGCATCC corresponds to the DNA sequence of the CDR2 region (VL-CDR2) of the light chain variable region of the anti-EPO antibody.

[0102] DNA sequence of the CDR3 region (VL-CDR3) of the light chain variable region corresponding to SEQ ID NO: 2 of the anti-EPO antibody: CAGCAAACTAGGAAGGTTCCTTCGACG.

[0103] DNA sequence of the light chain variable region of SEQ ID NO: 3 (333bp, CDRs are in bold: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): GATATCGTTCTCACTCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGACTATTATGGCACAGGTTTAATGCAGTGGTACCAACAGAGACCAGGACAGCCACCCAAACTCCTCATCTATGGTGCATC CAACGTAGGATCTGGGGTCCCTGCCAGGTTTAGCGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGGGGATGATATTGCAATGTATTTCTGTCAGCAAACTAGGAAGGTTCCTTCGACGTTCGGTGGAGGCACCAAGTTGGAAATCAAA.

[0104] SEQ ID NO: 4 corresponds to the amino acid sequence of the CDR1 region (VL-CDR1) of the light chain variable region of the anti-EPO antibody: ESVDYYGTGL.

[0105] GAS (Gly-Ala-Ser) corresponds to the amino acid sequence of the CDR2 region (VL-CDR2) of the light chain variable region of the anti-EPO antibody.

[0106] SEQ ID NO: 5 corresponds to the amino acid sequence of the CDR3 region (VL-CDR3) of the light chain variable region of the anti-EPO antibody: QQTRKVPST.

[0107] SEQ ID NO: 6 is the amino acid sequence of the light chain variable region (111aa, CDRs are shown in yellow: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): DIVLTQSPASLAVSLGQRATISCRASESVDYYGTGLMQWYQQRPGQPPKLLIYGASNVGSGVPARFSGSGSGTDFSLNIHPVEGDDIAMYFCQQTRKVPSTFGGGTKLEIK.

[0108] The DNA sequence of the CDR1 region (VH-CDR1) of the heavy chain variable region corresponding to SEQ ID NO: 7 of the anti-EPO antibody: GGATTCACTTTCAGTACCTATACC.

[0109] The DNA sequence of SEQ ID NO: 8 corresponding to the CDR2 region (VH-CDR2) of the heavy chain variable region of the anti-EPO antibody: ATTAGTAATGGTGGTGATAGAACC.

[0110] The DNA sequence of SEQ ID NO: 9 corresponding to the CDR3 region (VH-CDR3) of the heavy chain variable region of the anti-EPO antibody: GCAAGACATAATATTACTACGGTTCCCTTTACTATGGACTAC.

[0111] SEQ ID NO: 10 is the DNA sequence of the heavy chain variable region (363 bp, CDRs are shown in bold: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): GAGGTGAAGCTGCAGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATACCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGATAGAACCTACTATCC AGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACGATGCCAAGAACACCCTGTTCCTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGACATAATATTACTACGGTTCCCTTTACTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0112] SEQ ID NO: 11 corresponds to the amino acid sequence of the CDR1 region (VH-CDR1) of the heavy chain variable region of the anti-EPO antibody: GFTFSTYT.

[0113] SEQ ID NO: 12 corresponds to the amino acid sequence of the CDR2 region (VH-CDR2) of the heavy chain variable region of the anti-EPO antibody: ISNGGDRT.

[0114] SEQ ID NO: 13 corresponds to the amino acid sequence of the CDR3 region (VH-CDR3) of the heavy chain variable region of the anti-EPO antibody: ARHNITTVPFTMDY.

[0115] SEQ ID NO: 14 is the amino acid sequence of the heavy chain variable region (VH) (121aa, CDRs are in bold: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4): EVKLQESGGGLVQPGGSLKLSCAASGFTFSTYTMSWVRQTPEKRLEWVAYISNGGDRTYYPDTVKGRFTISRDDAKNTLFLQMSSLKSEDTAMYYCARHNITTVPFTMDYWGQGTSVTVSS.

[0116] SEQ ID NO: 15: EPO amino acid sequence (N-terminal signal peptide + protein chain), aa1-193, MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR. SEQ ID NO: 16 is the amino acid sequence of the mature EPO peptide (aa 28-193).

[0117] APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR.

[0118] SEQ ID NO: 17 is the EPO gene sequence.

[0119] For the purposes of this invention, the phrases “EPO negative function regulator,” “EPO / EPO-R negative regulator,” or “anti-EPO negative function regulator” are intended to refer to preparations that inhibit EPO function.

[0120] For the purposes of this invention, the "antibody" or "monoclonal antibody" is an "EPO negative function regulator" targeting human EPO. Specifically, the antibody targets the mature form of EPO, corresponding to amino acids 28-193 (AA) of the complete EPO amino acid sequence (SEQ ID NO: 15). The mature EPO amino acid sequence (AA 28-193) is described in SEQ ID NO: 16. Human EPO is encoded by the gene sequence SEQ ID NO: 17.

[0121] An anti-EPO antibody is a molecule that recognizes and binds to the amino acid sequence contained in erythropoietin, and can interact directly or indirectly with EPO, and / or directly or indirectly with the biosynthetic pathway of EPO, wherein the interaction results in a decrease in EPO levels, rather than a decrease in stimulation of the signal transduction cascade in which EPO participates. In a further embodiment, the EPO negative functional regulator acts on post-translational modified EPO, such as EV-3.

[0122] In a more preferred aspect, 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, which is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv or (Fab')2 fragments; more preferably, it also includes a frame sequence, at least a portion of which is a human common frame sequence.

[0123] The primary goal of the humanization process is to reduce the immunogenicity of antibodies to improve human tolerance and enhance their biophysical properties. In short, variable region sequence information is generated by reverse transcription of total RNA extracted from hybridoma cell lines. The variable regions of the heavy chain (VH) and light chain (VL) are amplified by PCR and cloned into shuttle vectors for sequencing. A total of five independent clones for each variable chain are sequenced. The hybridoma sequence is determined based on the sequencing results of VH and VL. Chimeric constructs are designed and expressed by combining mouse VH and VL variable regions with human IgG1 constant regions to confirm the affinity / binding ability and biological function associated with the parental mouse hybridoma. The antibody sequence is humanized by inserting 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 inserted into a human VH germline that is as homologous as possible to the mouse antibody VH. Furthermore, because different framework backgrounds can add value to the resulting antibodies, CDRs were implanted into human VH and VL germlines known to have good overall biophysical properties, even if their homology was low. A total of 9–18 VH / VL combinations were generated among CDR-implanted VHs, CDR-implanted VLs, and chimeric versions of VH and VL. (via XtenCHO) TMThe platform instantaneously produces proof-of-concept quantities of each recombinant humanized antibody and compares it with chimeric versions from parental mouse hybridomas to assess its binding affinity, biological activity, and biophysical properties. Comprehensive antibody affinity maturation services can be provided using phage display technology, leveraging custom libraries generated through random or targeted mutagenesis. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprising the population are identical, except for the possibility of a small number of possible mutations (e.g., naturally occurring mutations). Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies typically comprise an antibody containing a polypeptide sequence capable of binding to a target, wherein the process of obtaining the target-binding polypeptide sequence involves selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, this selection process may be to select a unique clone from multiple clones, such as a hybridoma clone pool, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, for example, to improve the affinity for the target, humanize the target-binding sequence, improve its yield in cell culture, reduce its immunogenicity in vivo, construct multispecific antibodies, etc., and antibodies containing modified target-binding sequences are also monoclonal antibodies of the present invention.

[0124] Furthermore, antibodies can be prepared using various techniques. For example, monoclonal antibodies can be purified in cells that naturally express them (such as hybridoma cells) or produced in recombinant expression systems in mammalian systems or prokaryotes (such as *E. coli*). Recently, antibody fragments have been introduced into clinical practice. In fact, antibody fragments have become important tools in imaging and diagnostics 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, biantibodies, triantibodies, tetraantibodies, and domain antibodies. They can be readily linked to radioisotopes, fluorescent molecules, or enzymes to label specific biomarkers in a patient's body. They also have short half-lives in vivo, allowing for faster clearance and potentially reducing the risk of side effects from potentially invasive diagnostic reagents. If necessary, the affinity of the monoclonal antibody or antibody fragment comprising one or more of the above-described CDRs according to the present invention can be enhanced through an affinity maturation procedure.

[0125] Preferably, the antibody described herein is a full-length monoclonal antibody and / or may be a bispecific anti-EPO antibody. The amino acid sequence of this anti-EPO antibody is identical to that of VL in SEQ ID NO: 6 and VH in SEQ ID NO: 14, or contains 0, 1, 2, or 3 amino acid residue substitutions relative to VL in SEQ ID NO: 6 and VH in SEQ ID NO: 14.

[0126] The advantageous properties of the EPO negative regulation of the present invention will become apparent in the experimental section.

[0127] In yet another preferred aspect, in the use of the negative function modulator or the anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response in a patient in need, the negative function modulator or the anti-EPO antigen-binding fragment induces T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells and / or antigen-presenting cells; said cellular response is further enhanced by a therapy based on CAR-T, CAR-M, CAR-NK, engineered monocyte-macrophages or polymorphonuclear cells, or by enhancing and reprogramming the response of tumor-associated lymphoid tissue (TIL) or tumor-associated macrophages (TAM).

[0128] In another preferred aspect, in the use of a negative functional modulator or an anti-EPO antigen-binding fragment, or in a method for activating or enhancing the immune response of a patient in need, the patient in need suffers from cancer, proliferative lesions, chronic inflammatory diseases based on autoimmunity and non-autoimmunity, neurodegenerative diseases, Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1, endometriosis, Crohn's disease, ulcerative colitis, neuroinflammatory diseases, and infectious diseases, or the patient in need is a patient who has undergone organ or tissue transplantation, wherein the cancer is selected from cerebral astrocytoma, cerebellar astrocytoma, pineal astrocytoma, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, glioblastoma multiforme, glioblastoma grade II fibrous astrocytoma, protoplasmic astrocytoma, grade III obese astrocytoma. IIIgemistocytic, anaplastic astrocytoma (including gliomatosis), pituitary adenoma, ependymoma, medulloblastoma, neuroectodermal tumor, neuroblastoma, hypothalamic glioma, breast cancer, lung cancer, colon cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, esophageal cancer, basal cell carcinoma, bile duct cancer, spleen cancer, osteosarcoma, intraocular melanoma, retinoblastoma, gastric cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal and paranasal sinus cancer, salivary gland cancer. The group consists of nasopharyngeal carcinoma, laryngeal carcinoma, thyroid cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, stomach and liver cancer, colorectal cancer, rectal cancer, testicular cancer, renal cell carcinoma, melanoma, sarcoma, mesothelioma, pheochromocytoma, hematologic malignancies or chronic myeloid leukemia, diffuse midline glioma (DMG), diffuse endophytic pontine glioma (DIPG), embryonal tumors, brainstem glioma, pineal blastoma, choroid plexus carcinoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, and hemangioblastoma.

[0129] In a more preferred aspect, the cancer is glioblastoma multiforme.

[0130] In a further aspect, the negative functional modulator or anti-EPO antigen-binding fragment promotes the immune system response in patients affected by refractory or persistent infectious diseases, including tuberculosis, malaria, HIV, EBV, or HPV-induced precancerous lesions and their prevention.

[0131] Preferably, the negative functional modulator or anti-EPO antigen-binding fragment promotes an immune system response in patients affected by streptococcal, staphylococcal, fungal, viral, Sars-Cov2, SARS, MERS, or prion pathogens, said patients being tolerant to antimicrobial therapy and / or having immune system tolerance.

[0132] More preferably, negative functional modulators or anti-EPO antigen-binding fragments are used to promote immune system responses and enhance the efficacy of prophylactic and therapeutic DNA and / or RNA or peptide and / or lipid-based vaccines in the immunotherapy of infectious diseases and / or cancer.

[0133] Secondly, this article describes a pharmaceutical kit comprising negative functional modulators of EPO / EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protectant, TPR (e.g., EPOR / CD131 heterodimer)) and / or their natural or synthetic variants; and: - Checkpoint inhibitor or immunomodulatory agent therapy (e.g., anti-PDL1 antibody, nivolumab, ipilimumab, abatacept, glembatumumab vedotin); and / or - Cell-based immunotherapies (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, activated dendritic cells and / or antigen-presenting cells targeting tumor-associated antigens), therapies based on engineered mononuclear-macrophages or polymorphonuclear cells, and / or therapies for enhancing and reprogramming tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM) responses. and one or more components, said components being selected from the group consisting of: - Peptides or antibodies, biantibodies, and nanobodies targeting natural and synthetic variants, including physiological and pathological splice variants and post-translational modified forms of erythropoietin; - Antimicrobial therapy (e.g., antibiotics, antiviral drugs, antifungal agents, antifungal drugs, antipruritic drugs); - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Prophylactic or therapeutic vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids (e.g., anti-HPV vaccines, anti-EBV vaccines, or anti-HIV vaccines). - Immunotherapy based on oncolytic viruses; - Chemotherapy drugs; - Anticancer drugs; - Enzymes that degrade heparan sulfate proteoglycans (e.g., heparinase); - A negative regulator of the sphingosine phosphate-1 (S1P) signaling pathway; or - An EPO mimic that retains the function of erythropoiesis.

[0134] Thirdly, the present invention relates to a diagnostic or prognostic method for assessing EPO and its somatic mutations or variants thereof, EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protectant, TPR, EPOR / CD131 heterodimer) and somatic mutations and / or their variants, C4 The method predicts the response to therapy targeting negative regulation of EPO / EPO-R to enable personalized treatment in immunotherapy or prevention of cancer and infectious diseases, and to stratify patients to optimize patient response; the method includes the steps of: determining the amount / detection of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants, as diagnostic or prognostic markers, in tissues, cells or human body fluids (saliva, blood, cerebrospinal fluid, sweat or derived extracellular vesicles) / 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 tissues, cells or human body fluids (saliva, blood, cerebrospinal fluid, sweat or derived extracellular vesicles).

[0135] Fourthly, the present invention relates to a diagnostic method for assessing promoter methylation of genes for EPO and EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR (e.g., EPOR / CD131 heterodimer)), which can predict increased expression of EPO / EPO-R and C4 ligands and their negative effects in suppressing the immune system's clearance of cancer cells or microbial factors, thereby enabling personalized immunotherapy or prevention and for prognostic purposes; the method includes the step of detecting methylation of EPO and EPO receptor genes as diagnostic or prognostic markers in tissues, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or derived extracellular vesicles).

[0136] As an immunotherapy or immunotherapy adjuvant regimen according to the present invention, compounds represented by EPO and its variants and / or negative modulators of EPO receptors and their variants (such as compounds shown in Formula 1) or pharmaceutically acceptable salts thereof can be administered clinically in various formulations via oral, parenteral, intralesional (intratumoral and intracavitary), intraventricular, intrathecal, intranasal, or local administration. More preferably, they can be parenteral formulations. The methods described in this invention may also include preparing compounds shown in Formula 1 or pharmaceutically acceptable salts thereof for oral or parenteral administration by mixing with commonly used diluents or excipients (such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants). Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid dosage forms are prepared by mixing compounds shown in Formula 1 of the present invention or pharmaceutically acceptable salts thereof with one or more suitable excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups; these preparations, besides commonly used simple diluents such as water and liquid paraffin, can also contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, water-insoluble excipients, suspensions, and emulsions. Besides the active compound, water-insoluble excipients and suspensions can also contain propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable esters (such as ethyl oleate).

[0137] The cancer immunotherapy adjuvants described herein may include compounds of Formula 1 or pharmaceutically acceptable salts thereof as active ingredients, which may be administered via parenteral administration, including subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection.

[0138] To prepare a formulation of the compound of Formula 1 or a pharmaceutically acceptable salt thereof for parenteral administration, the compound of 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 into ampoules or vials. The compositions herein may be sterilized and may additionally contain preservatives, stabilizers, wettable powders or emulsifiers, salts and / or buffers for adjusting osmotic pressure, and other materials with therapeutic uses; the compositions may be prepared by conventional mixing, granulation, or coating methods.

[0139] Examples of formulations for oral administration include 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, glucose, 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 carboxymethyl cellulose, and / or polyvinylpyrrolidone; and, where necessary, may additionally contain disintegrants such as starch, agarose, alginate, or its sodium salt or azeotropic mixtures, and / or adsorbents, colorants, flavorings, and sweeteners.

[0140] This cancer immunotherapy adjuvant can enhance the efficacy of cancer immunotherapies. More specifically, it can enhance the efficacy of cancer immunotherapies by activating immune factors to assist the anti-cancer activity of cancer immunotherapies. Immune factors can 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.

[0141] Cancer immunotherapy adjuvants can be administered simultaneously with or sequentially with cancer immunotherapy agents. When administered sequentially, the adjuvant can be administered after or after the cancer immunotherapy agent. However, this administration method is merely an example, and can be modified to enhance the anti-cancer immune effect. In embodiments of the present invention, the cancer immunotherapy adjuvant is administered daily via intravenous injection, while the cancer immunotherapy agent is administered three times weekly via intraperitoneal injection, but the administration method is not limited to these.

[0142] This cancer immunotherapy adjuvant can activate one or more immune factors selected from a group consisting of helper T cells, cytotoxic T cells, natural killer cells (NK cells), and cytokines. By activating these immune factors, this cancer immunotherapy adjuvant enhances the anticancer effects of cancer immunotherapies.

[0143] At this point, cancer immunotherapy adjuvants can be used to prevent or treat cancer by being administered in combination with cancer immunotherapy agents.

[0144] The cancer may be at least one selected from the group consisting of 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 astrocytoma, grade III obese astrocytoma, anaplastic astrocytoma (including cerebral gliomatosis), ependymoma, medulloblastoma, neuroectodermal tumor, neuroblastoma, hypothalamic glioma, 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, colon cancer, colorectal cancer, ovarian cancer, ovarian epithelial cancer, gestational villous disease. Diseases, cervical cancer, endometrial cancer, uterine cancer, ovarian germ cell cancer, esophageal cancer, basal cell carcinoma, bile duct cancer, choroidal melanoma, choroid plexus carcinoma, spleen cancer, osteosarcoma, intraocular melanoma, malignant melanoma, retinoblastoma, stomach cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cavity 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 bowel cancer, gastrointestinal stromal tumor, testicular cancer, renal cell carcinoma, adrenal cancer, renal pelvis cancer, malignant mesothelioma, mesothelioma, pheochromocytoma, blood cancer or chronic myeloid leukemia, lip cancer, tonsil cancer, squamous cell carcinoma, ampullary carcinoma. Cancer, peritoneal cancer, tongue cancer, pseudomyxoma, intrahepatic hepatoblastoma, myelodysplastic syndrome, Wilms' cancer, penile cancer, pharyngeal cancer, juvenile lymphoma, juvenile leukemia, Paget's disease, skin cancer, anal cancer, pleural cancer, blood cancer, acute myeloid leukemia, acute lymphoblastic leukemia, myeloma, duodenal cancer, malignant soft tissue cancer, malignant lymphoma, chronic myeloid leukemia. Leukemia, gallbladder cancer, bile duct cancer, chronic lymphocytic leukemia, malignant bone cancer, metastatic bone cancer, eye cancer, vulvar cancer, ureteral cancer, mediastinal cancer, urethral cancer, cancer of unknown primary origin, vaginal cancer, spinal cord cancer, vestibular schwannoma, diffuse midline glioma (DMG), diffuse endophytic pontine glioma (DIPG), embryonal tumors, pineal blastoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, and hemangioblastoma.And at least one of the following groups: proliferative disorders, chronic inflammatory diseases based on autoimmune and non-autoimmune factors, 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 HIV-2, sickle cell disease, SARS, SARS-CoV, and MERS.

[0145] Cancer immunotherapy adjuvants may be administered in combination with cancer immunotherapy agents that are conventionally known and well-known to those skilled in the art, and are not limited thereto. For example, cancer immunotherapy adjuvants may 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, but are not limited thereto.

[0146] In another aspect of the invention, the invention provides a combination drug for cancer immunotherapy.

[0147] Specifically, the present invention provides a combination drug for cancer immunotherapy, the drug composition comprising a cancer immunotherapy agent and a cancer immunotherapy adjuvant.

[0148] The specific descriptions of cancer immunotherapy agents, cancer immunotherapy adjuvants, and combination drugs are the same as those of cancer immunotherapy adjuvants.

[0149] In another aspect of the invention, a pharmaceutical composition for enhancing the potency of cancer immunotherapy agents is provided.

[0150] Specifically, the present invention provides a pharmaceutical composition for enhancing the efficacy of cancer immunotherapy agents, the pharmaceutical composition comprising the compound shown in Sequence 1 as an active ingredient, its isomer, its solvate, its hydrate or a pharmaceutically acceptable salt thereof.

[0151] Furthermore, the specific description of the pharmaceutical composition used to enhance the efficacy of cancer immunotherapy agents is the same as the specific description of cancer immunotherapy adjuvants.

[0152] In another aspect of the invention, a pharmaceutical composition for enhancing immunity is provided.

[0153] Specifically, the present invention provides a pharmaceutical composition for enhancing immunity, the pharmaceutical composition comprising the compound shown in Sequence 1 as an active ingredient, its isomer, its solvate, its hydrate or a pharmaceutically acceptable salt thereof.

[0154] Furthermore, the specific description of this pharmaceutical composition for enhancing immunity is the same as that of adjuvants for cancer immunotherapy.

[0155] In another aspect of the invention, the 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.

[0156] Cancer immunotherapy adjuvants and cancer immunotherapy agents can be administered in combination or at different times.

[0157] In another aspect of the invention, the invention provides the use of cancer immunotherapy adjuvants and immunotherapy agents in the prevention or treatment of cancer.

[0158] In another aspect of the invention, the invention provides a combination therapy for treating cancer, comprising the step of administering a cancer immunotherapy adjuvant and a cancer immunotherapy agent to a subject in need.

[0159] In another aspect, the present invention provides a kit for the prevention or treatment of cancer, the kit comprising a cancer immunotherapy agent and a cancer immunotherapy adjuvant as active ingredients. Furthermore, the present invention includes the use of a negative regulator of the EPO-EPO receptor pathway in the treatment of autoimmune diseases and infectious diseases, such as HIV, tuberculosis, malaria, streptococcal infection, staphylococcal infection, fungal infection, viral infection (e.g., SARS-CoV-2, MERS), prion diseases, and parasitic infections, which can be administered as a single dose or in combination with antimicrobial agents or vaccines and recombinant cytokines. Additionally, the present invention describes a method for enhancing the efficacy of prophylactic and therapeutic vaccines by stimulating an inflammatory response of the immune system in the immunotherapy of infectious diseases and cancer.

[0160] In a further aspect, the present invention relates to an EPO / EPO-R negative modulator delivery system, which can be administered clinically in various formulations via oral, parenteral, intralesional (intratumoral and intracavitary), intraventricular, intrathecal, intranasal, or local administration. The system is based on innovative nanomedicine and nanodelivery systems, such as tissue guns or probes; viral and nonviral vectors; nanomaterials for targeted delivery of bioactive drugs; plant-based vesicles; nanoparticle-based methods that allow for simultaneous therapy and in vivo imaging for diagnosis and treatment; lipid systems, such as liposomes and micelles; gold nanoparticles or magnetic nanoparticles, and functionalized nanoparticles, microspheres, and biomaterials, such as PEG, used in combination with natural products; and Trojan horse methods, such as micropumps for releasing therapeutic drugs in tissues to enhance the modulation of the immune system in the local pathological microenvironment or attract and increase homing of cell-based immunotherapies or vaccines.

[0161] In a further embodiment, the present invention relates to a method based on EPO / EPO-R negative regulation for reprogramming tumor-associated immune cells to avoid exhaustion and tolerance of the immune system.

[0162] In yet another embodiment, the present invention relates to a method based on EPO / EPO-R negative regulation for improving the efficacy of CAR T and CAR-γ / δ T cell therapy for solid tumors.

[0163] In another aspect, the present invention relates to inhibitors of EPO / EPO-R and variants thereof, which are capable of stimulating CTL infiltration and inhibiting the recruitment of immunosuppressive cells in tumors and infectious diseases, thereby increasing tissue penetration of inflammatory and immune cells.

[0164] Another aspect involves EPO / EPO-R and their variant inhibitors, which can induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferroptosis, autophagy, copper death, and immunostimulatory cell death, thereby enhancing the immunogenicity of tumors.

[0165] Another aspect describes a product selected from EPO and their natural and synthetic variants as inhibitors that can activate immune responses against cancer and infectious agents, reprogram the tumor microenvironment, and enhance immunotherapy and immunomodulatory strategies.

[0166] The present invention will be described in detail below through the following embodiments and experimental examples.

[0167] Example The following embodiments, in conjunction with the above description, illustrate some implementations of the invention.

[0168] Example 1. Anti-EPO increases the migration of peripheral blood mononuclear cells (PBMCs) in the presence of GBM tissue.

[0169] Figure 1 This illustrates the effect of anti-EPO antibody and rhEPO treatment on PBMC migration when GBM tissue blocks are placed in the lower chamber and anti-EPO antibody and rhEPO are added to the Transwell membrane. Figure 1 A). On the day of surgery, the excised GBM tissue was cut under aseptic conditions and placed at the bottom of a 24-well plate for the following processing: CTR ( Figure 1 B), 10 μg / mL anti-EPO antibody ( Figure 1 C) and 100 ng / mL rhEPO ( Figure 1 D). The migration capability of PBMCs was evaluated at T0 h, T24 h, and T48 h. 2 × 10 4 One PBMC (per well) was resuspended in 100 μL of cell culture medium per well, and the PBMC suspension was placed in the upper chamber. In the lower chamber of the Transwell plate, the PBMCs were treated with C4 or rhEPO in 500 μL of medium containing GBM tissue. After incubation at 37°C for 48 hours in 5% CO2, the upper chamber was removed, and the PBMCs were stained with Hoechst (1:1000 diluted in PBS, 37°C for 15 min). The number of PBMCs in the lower chamber was counted under a microscope. Figure 1 E), and analyzed using ImageJ and its Analyze Particle plugin. Immunolabel acquisition was performed using a NikonCrest system (NikonTi + Andor Du888 + Zyla 4.6 + 16-led CoolLED + 4 lasercubes LDI + Crest Optics Spinnig Disk and VCS-sim XlightV2 / VCS), using the Nis-Elements V.5.3.2 software module for acquisition and the GA3 module for analysis.

[0170] Analysis showed that a significantly higher number of migrating PBMCs were recorded in the presence of anti-EPO antibodies. Figure 1 E). Interestingly, the analysis also showed that rhEPO has an inhibitory effect on PBMC migration.

[0171] Example 2. Anti-EPO induces differentiation, activation, and expression of natural killer cell markers in PBMCs.

[0172] PBMCs were analyzed by flow cytometry 48 hours after migration to assess the expression of CD56, CD86, and CD69. For analysis, indirect binding of primary and secondary antibodies was performed first, followed by the addition of a mixture of conjugated antibodies. Specifically, EPOR was added to the PBMCs as AB-I, and the cells were incubated at RT for 1 h in a blocking solution containing PBS + 5% BSA + 2% donkey serum. AB-I was removed, and the cells were washed three times with PBS + 0.1% BSA, followed by the addition of AB-II and incubation at RT for 1 h. The PBMCs were then washed with PBS, and a mixture of the following conjugated antibodies was added: CD56-APC, CD86-PE-Cy7, and CD69-APC-Cy7. Analysis was performed using a FACS Canto II flow cytometer and FACSSDiva software (BD Bioscience, version 5.0). In CTR, gates were used for forward scattering vs. side scattering (FSC-A vs. SSC-A) to identify intact PBMCs based on size and granularity. Figure 2 The results showed that, compared with untreated PBMCs under CTR conditions, anti-EPO administration induced significant overexpression of CD56, CD86, CD69, and EPOR. Figure 2 A). Surprisingly, when compared with the rhEPO group, anti-EPO administration induced strong expression of CD56, CD86, CD69, and EPOR in migrating PBMCs. Figure 2 B).

[0173] Example 3. Anti-EPO treatment induces EPOR-dependent PBMC migration.

[0174] PBMCs collected from n=3 GBM patients were used for migration analysis in the Boyden chamber for 48 h. Figure 3 A). 2×10 4One PBMC (per well) was resuspended in 100 μL of cell culture medium per well, and the PBMC suspension was placed in the upper chamber. The lower chamber of the Transwell plate was treated with anti-EPO or rhEPO in 500 μL of medium containing GBM tissue. After incubation at 37°C and 5% CO2 for 48 h, the upper chamber was removed, and the insert was fixed on ice with 4% PFA for 10 min. The insert was then removed and inverted onto a glass slide for immunolabeling of CD14, CD8, and EPOR targets. PBMCs were blocked at RT in PBS + 5% BSA + 2% donkey serum for 30 min. Primary antibodies (AB-I) diluted in blocking buffer were incubated 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-I was removed, and the cells were washed three times with PBS + 0.1% BSA. AB-II was then added, and the cells were incubated at RT for 1 h. PBMCs were treated with PBS + 0.5% Triton X-100 to permeate the cell membrane and then cultured directly with DAPI. Immunolabeling was obtained using a high-resolution SP5 confocal microscope. Interestingly, CD8+ cells exhibited a larger and rounder morphology compared to those treated with CTR and rhEPO. Figure 3 B).

[0175] Furthermore, analysis showed that, compared with CTR and rhEPO administration, anti-EPO treatment induced significant migration of CD14+ and CD8+ PBMC-derived cells. Figure 3 C). Surprisingly, the microscopic images and related quantitative results ( Figure 3 D- Figure 3 E) showed that when anti-EPO was added to the culture medium, migrating CD8+ and CD14+ cells exhibited higher EPOR expression. These data demonstrate that the anti-EPO antibody is a potent immunomodulatory stimulant with a triggering effect on PBMCs from GBM patients. Unexpectedly, the stimulatory effect was mediated by the overexpression of EPOR on migrating PBMCs.

[0176] Example 4. Molecular characteristics against EPO-induced activation and blocking depletion.

[0177] Gene expression analysis of PBMCs cultured for 48 h under CTR, anti-EPO, and rhEPO conditions was performed using real-time PCR. PBMCs were collected according to the aforementioned method, and PBMC migration was assessed using the Boyden chamber method. After 48 h of migration, PBMCs were collected from the bottom of the wells, centrifuged at 300 g × 10 min, and the precipitate was resuspended in Tri reagent according to the manufacturer's instructions for RNA extraction. RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the TranScriba kit (A&A Biotechnology) according to the manufacturer's instructions, with 1 μg of RNA (A260 / A280 > 1.8) loaded. 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 using TBP expression as an internal control. The relative expression level of the gene was determined using the 2-ΔΔCt method. Figure 4 The results showed that anti-EPO treatment induced PBMC activation, and importantly, it blocked the depletion phenomenon and regulated IL-1β (…). Figure 4 A), IL-6 ( Figure 4 B), IFNg ( Figure 4 C), IL-10 ( Figure 4 D), TGFb ( Figure 4 E), IFNa2 ( Figure 4 F), PD1 ( Figure 4 G), LAG-3 ( Figure 4 H), CTLA4 ( Figure 4 I) Gene expression. Data are the mean ± SD of at least three replicates. *P < 0.05 compared with CTR for all treatment groups.

[0178] Example 5. Anti-EPO induces monocyte migration and their activation.

[0179] Monocyte migration was assessed using 24-well Transwell plates (8.0 μm wells; Corning, NY). Briefly, monocytes were washed once with RPMI 1640 medium and the cell number was readjusted (5 × 10⁶ cells / well) in RPMI 1640 + 10% FBS. 5Mononuclear cells / mL were stained with calcein (1:1000 diluted in RPMI medium, for 15 min), washed with PBS, and aliquots (100 μL) of the cell suspension were placed in the upper chamber. The lower chamber of the Transwell plate received 500 μL of specially treated medium. After culturing at 37°C and 5% CO2 for 48 h, the upper chamber was removed, and the number of cells at the bottom of the chamber was counted under a microscope and analyzed using ImageJ and its AnalyzeParticle plugin under the following conditions: CTR (… Figure 5 A), anti-EPO ( Figure 5 B), rhEPO ( Figure 5 C). Interestingly, 6 days after migration, anti-EPO treatment increased monocyte migration compared to CTR conditions. Notably, migration was reduced in the presence of rhEPO and all of the above rhEPOs. Figure 5 D). Surprisingly, phenotypic analysis of migrating monocytes showed that C4 treatment significantly induced overexpression of CD86 and HLA-DR. Figure 5 E).

[0180] When monocyte migration was performed with GBM fragments present at the bottom of the Transwell, tissue was collected from the bottom of the well after 6 days of experimental conditions. The tissue was digested with 0.25% trypsin for 30 min, filtered through a 70 μm pore size filter, and analyzed by flow cytometry to count green-positive infiltrating cells. The results showed that administration of anti-EPO antibody significantly increased the number of monocytes derived from infiltrating macrophages. Figure 6 A, Figure 6 B). Notably, immunophenotypic analysis of infiltrating macrophages showed a significant increase in CD86+ / HLA-DR+ expression, indicating M1 macrophage polarization. Figure 6 C), and it is worth noting that infiltrating macrophages overexpress EPOR ( Figure 6 D).

[0181] Interestingly, immunofluorescence analysis of infiltrating macrophages showed higher expression of EPOR, as highlighted in the red-marked cells. Figure 7 A- Figure 7 F).

[0182] In summary, these data demonstrate that anti-EPO exhibits strong chemotactic activity, inducing monocyte migration. Unexpectedly, anti-EPO antibodies increased the infiltration of macrophage-derived monocytes into tumor tissue; more importantly, they polarized macrophages towards the M1 phenotype. Interestingly, this mechanism is mediated by EPOR expression on macrophages.

[0183] Example 6. Anti-EPO is a potent migration stimulant for T cells. Migration assays, or "chemotaxis analyses," are performed to highlight the migratory capacity of naïve T cells, a phenomenon observed in immune responses. For this purpose, Transwell multiwell plates equipped with polycarbonate membrane inserts are used. The 8 μm diameter pores in the membrane trap cells and culture medium but allow cells to actively migrate across the membrane to the next well. 2 × 10⁶ cells are then placed in the membrane. 4 One initial CD4+ T cell per well was stained with calcein (diluted 1:1000 in PBS) for 10 min, and then resuspended in 100 μL of cell culture medium per well. Cell resuscitation was performed under the following conditions: the lower chamber of the Transwell plate was treated with CTR in 500 μL of culture medium. Figure 8 A), C4 (10 μg / mL), Figure 8 B), rhEPO (100 ng / mL), Figure 8 C) or rhEPO + anti-EPO Ab combination ( Figure 8 D) Treatment. After culturing at 37℃ and 5% CO2 for 48 h, the upper chamber was removed, and the migrating T cells at the bottom of the chamber were counted under a microscope and analyzed using ImageJ and its Analyze Particle plugin. Figure 8 E). Immunolabeling was acquired using Leica time-lapse microscopy. All stained wells were imaged at high resolution using whole-well imaging; during high-magnification acquisition, large-mosaic rendering was employed to obtain the complete field of view (FOV) for each IF-labeled instance. Results showed that T cells were chemotactically attracted by anti-EPO antibodies, but this effect was significantly reduced in the presence of rhEPO. Interestingly, a significantly higher number of migrating T cells were recorded when anti-EPO was co-administered with rhEPO. Therefore, it can be inferred that anti-EPO treatment is a potent stimulant that significantly increases T cell migration. Conversely, rhEPO treatment blocked T cell migration, indicating that erythropoietin has an immunosuppressive effect. *P<0.05 compared to CTR treatment.

[0184] Immunophenotypic analysis of T cells after C4 administration was performed by evaluating EPOR expression in the CD69+ subset under the following conditions: CTR ( Figure 9 ), temozolomide (TMZ, Figure 9 B), rhEPO ( Figure 9 C), C4 ( Figure 9 D), TMZ+C4 Figure 9 E) and rhEPO+C4 ( Figure 9F). The results showed that EPOR expression was significantly increased in activated CD69+ positive cells under all anti-EPO antibody conditions. In fact, migrating T cells showed higher levels of CD69 and EPOR expression compared to CTR and rhEPO conditions. Figure 9 G). Data are the mean ± SD of at least 3 replicates. **P < 0.01, ***P < 0.001 compared to CTR for all treatment groups.

[0185] Example 7. Anti-EPO induces CD4+ CTL lymphocytes to migrate and deeply penetrate GBM tissue in the TME. Lymphocyte migration was assessed using 24-well Transwell plates (8.0 μm wells; Corning, NY). Briefly, T cells were washed once with RPMI 1640 medium and the cell number was readjusted to 5 × 10⁶ cells in T cell medium (RPMI 1640 + 10% FBS). 5 T cells / mL were stained with calcein (1:1000 diluted in RPMI medium, for 15 min), washed with PBS, and aliquots (100 μL) of the T cell suspension were placed in the upper chamber. The lower chamber of the Transwell plate received 500 μL of conditioned medium (CM) containing chemokines. Figure 10 A). After culturing at 37°C and 5% CO2 for 48 h, 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 ImageJ and its Analyze Particle plugin. Figure 10 B shows the number of migrating T cells at the bottom of the pore. Data demonstrate that anti-EPO induces higher levels of T cell migration, whether administered alone or in combination with TMZ. Figure 10 B), as shown in the micrograph of the hole under the following conditions: CTR ( Figure 10 C), TMZ Figure 10 D), Anti-EPO ( Figure 10 E), TMZ+C4 ( Figure 10 F) and rhEPO ( Figure 10 G).

[0186] Surprisingly, in the presence of GBM tissue fragments ( Figure 11 A), the number of migrating T cells significantly increased after C4 administration or combined administration with TMZ. Figure 11 B). Figure 11 C- Figure 11 The micrograph in H shows the overall condition of the bottom of the hole under the following conditions: CTR ( Figure 11 C), TMZ Figure 11D), C4 Figure 11 E), TMZ+C4 ( Figure 11 F), rhEPO ( Figure 11 G) and rhEPO+C4 ( Figure 11 H). After migration analysis, GBM tissue fragments were collected from the bottom of the wells. Figure 12 A), digested with 0.25% trypsin for 30 min, filtered through a 70 μm pore size filter, and analyzed by flow cytometry to count green-positive infiltrating cells. The results showed that administration of anti-EPO antibody alone, or in the presence of TMZ or rhEPO, significantly increased the number of infiltrating T cells (A). Figure 12 B). In summary, these data demonstrate that anti-EPO exhibits a strong chemotactic capacity to induce T cell migration. Unexpectedly, the anti-EPO antibody was able to induce T cell infiltration into GBM tissues, revealing its powerful ability as a chemokine.

[0187] Example 8. Expression of EPO-related targets in GBM cells The expression patterns of EPO-related genes, transferrin-related genes, and IL-13-related genes in CTR and GBM mRNA were analyzed by real-time PCR to identify the specific characteristics of GBM, thereby developing CAR constructs to guide the immune system to attack tumors.

[0188] Real-time PCR was performed to obtain expression information for specific genes. Specifically, gene expression analysis was performed on GBM cells and CTR mRNA (Takara). Cells were collected, centrifuged at 300 g for 10 min, and the pellet was resuspended in Tri reagent according to the manufacturer's instructions for RNA extraction. RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the TranScriba kit (A&ABiotechnology) according to the manufacturer's instructions, with 1 μg of RNA (A260 / A280 > 1.8) loaded. 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 using 18S gene expression as an internal control. The relative expression level of the gene was determined using the 2-ΔΔCt method. Figure 13 ). Figure 13The gene expression results of EGFR, EPOR, EPHB4, CSF2RB, and CRLF3 in tumor cells and commercial CTR mRNA are shown. Figure 13 A). In addition, IL-13, IL-13R1, and IL-13R2 are also shown. Figure 13 B) and TfR1, TfR2 and folate receptor (B) Figure 13 C) Gene expression status. The results showed that, compared with healthy cells, GBM cells had higher expression levels of EGFR, EPHB4, CSF2RB and CRLF3, as well as higher expression levels of IL-13, IL-13R1, IL-13R2, TfR1, TfR2 and folate receptor, indicating that these molecules could serve as potential targets for the development of CAR constructs.

[0189] Example 9. The CAR-T therapeutic effect of anti-EPO treatment on cancer cells Because EGFR amplification, overexpression, or mutation is present in approximately half of glioblastomas and other malignant CNS tumors (including ependymomas and medulloblastomas in children and adults), EGFR CAR-T cells (EGFR scFv-4-1BB-CD3ζ CAR T cells, Promab) were used. To examine the effects of T cells (WT) and EGFR-CAR-T cells in the presence or absence of anti-EPO on glioblastoma stem cells (… Figure 14 ) and killing tests were conducted on cancer cells from the colon, breast, and melanoma. Figure 14 ). Target cells were spaced at 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a concentration of [number] cells per well in 96-well plates, with three replicates. The culture medium was DMEM:F12 supplemented with 10% FBS (ThermoFisher Scientific). Target cells were cultured alone with T cells or CAR-T cells for 24 hours at 37°C and 5% CO2, or in the presence of anti-EPO at a series of effector-to-target ratios (E:T) of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 for 24 hours. Figure 14 A). Each well was stained with crystal violet (ThermoFisher Scientific). After staining, the cells were observed and photographed under an inverted microscope (TS100, NIKON Instruments Inc). Data are expressed as percentage of viable cells (%). Figure 14 A). Under these conditions, significant differences were observed when T cells were cultured in the presence of anti-EPO treatment. In fact, T cells exhibited better control over target cell numbers. Therefore, CAR-T cells combined with anti-EPO maintained extremely high cytotoxicity even at lower effector cell abundance.

[0190] In addition, to determine whether the observed enhanced cell lysis activity was accompanied by a similarly significant increase in TNF-α and IFN-γ secretion, GSCs were co-cultured with EGFR-CAR-T cells alone for 24 h, or with GSCs and EGFR-CAR-T cells co-cultured with C4 antibody for 24 h, and then cytokines were measured using an ELISA kit (R&D System) according to the manufacturer's instructions. Figure 14 As shown, individual EGFR-CAR-T cells spontaneously produce detectable levels of TNF-α (… Figure 14 B) and IFN-γ Figure 14 C). When these cells were co-cultured with CAR-T and anti-EPO antibodies, TNF-α and IFN-γ were induced, and the levels of these cytokines were significantly higher. These results are consistent with the cytotoxicity data above, and together they indicate that the co-administration of anti-EPO antibodies with EGFR-CAR-T significantly enhances the effector function of T cells against EGFR+ glioma cells.

[0191] In addition, the cytotoxicity of EGFR-CAR-T cells was evaluated in the following cell models: i) Breast cancer cell line (MCF-7); breast cancer cells. Previous studies have shown that this cell line is responsive to human erythropoietin (rHuEPO) treatment, exhibiting increased cell proliferation. Furthermore, the MCF7 cell line has been reported to express EGFR (…). Figure 15 A).

[0192] ii) Prostate cancer cell line (LNCAP); human prostate cancer cells are used for cancer research and drug development. It has been reported that the human hepatocyte receptor (Eph), which produces erythropoietin, is overexpressed in prostate cancer patients and is associated with poor prognosis and reduced survival, thus being considered a predictive biomarker for the aggressive behavior of prostate cancer. Furthermore, recent studies have shown that co-overexpression of EPO and EPOR plays an important role in disease progression in LNCAP cells of drug-resistant prostate cancer and is associated with the formation of a neuroendocrine phenotype; interestingly, it has been reported that the LNCAP cell line expresses EGFR (… Figure 15 B).

[0193] iii) Melanoma cell line derived from primary amelanoma A375. Elevated EGFR expression levels were detected. Based on these premises, we decided to use the A375 cell line as a model for the cytotoxic activity of EGFR-CAR-T cells. Figure 15 C). Under these conditions, when T cells were cultured in the presence of anti-EPO treatment, in the MCF7 breast cancer cell line ( Figure 15A) LNCAP prostate cancer cell line ( Figure 15 B) and the A375 melanoma cell line ( Figure 15 Significant differences were observed in C). In fact, T cells have better control over the number of target cells. Therefore, even at lower effector cell abundance, CAR-T cells plus anti-EPO still exhibited significantly higher cytotoxicity.

[0194] Furthermore, the results showed that in glioblastoma cancer cells, combined treatment with anti-EPO and metformin (MET) enhanced the cytotoxicity of CAR-T cells. Figure 16 A). Furthermore, gene expression analysis was performed on PBMCs treated with the following conditions: CTR, anti-EPO, MET, and anti-EPO+MET. The results showed that combined administration of anti-EPO and MET induced IL-1β (CTR, anti-EPO, anti-MET) in PBMCs. Figure 16 B) and IFNγ overexpression ( Figure 16 C), while downregulating PD-1 expression ( Figure 16 D).

[0195] Example 10. After anti-EPO treatment, immune cells were injected into a GBM subcutaneous PDX mouse model. SCID mice (n=10) were subcutaneously inoculated with the GBM fragment and monitored until the tumor volume reached 50-100 mm. 3 Mice were then randomly assigned to two groups: CTRL (tumor irradiation with PBS) and anti-EPO (tumor irradiation with intravenous injection of 15 mg / kg three times a week). The weight of the tumor mass was measured using calipers before each administration. Mouse weight was monitored throughout the experiment. On the day of sacrifice, the tumor mass was excised and stained with hematoxylin and eosin. Immunohistochemical evaluation was performed by two independent pathologists unaware of the clinical information. In the anti-EPO treatment (… Figure 17 A, Figure 17 B) after, with CTR condition ( Figure 17 C Figure 17 Compared to D), increased polymorphonuclear leukocyte infiltration was observed in GBM tissue blocks on day 21 post-injection.

[0196] Example 11. Anti-EPO treatment improves malaria with infected red blood cells Human erythrocytes, after leukocyte removal, were processed. Following processing, the erythrocytes were washed with filtered RPMI 1640 medium (containing 25 mM HEPES and 50 μg / mL hypoxanthine). Plasmodium parasites were cultured in complete 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) at a hematocrit of 5%, 37°C, and a mixed atmosphere of 90% N2, 5% CO2, and 5% O2. For monitoring parasitemia, erythrocytes were prepared on slides, fixed with 100% methanol, and stained with 20% Giemsa stain (Sigma-Aldrich, St. Louis, MO) for 15 min. Red blood cells were counted using a bright-field microscope, and the number of parasitic cells was used to estimate the percentage of parasitemia under CTR conditions or after treatment with anti-EPO antibodies. When anti-EPO antibodies were administered to infected red blood cells, all cells showed a reduction in parasitemia and recovery from infection. Figure 18 A). When monocytes were co-cultured with infected cells, immunophenotypic analysis showed increased CD86+ / HLA-DR+ positive expression, indicating M1 polarization (A). Figure 18 B). These results suggest that the use of anti-EPO antibodies after malaria infection may have therapeutic value in severe cases of malaria. Figure 18 ).

[0197] Example 12. PBMC migration: a chemotactic model in infectious diseases under anti-EPO stimulation. Figure 19 The effect of lipopolysaccharide (LPS) combined with anti-EPO antibody and rhEPO treatment on PBMC migration was demonstrated. LPS is a molecule present on the membranes of Gram-negative bacteria. Migration assays, or "chemotaxis assays," are performed to highlight the migratory capacity of PBMCs, a phenomenon observed in immune responses. For this purpose, Transwell multi-well plates (24 wells) equipped with polycarbonate membrane inserts were used. The 8 μm diameter pores in the membrane can trap cells and culture medium but allow cells to actively migrate across the membrane to the next well. 2 × 10⁶ cells were then placed in the membrane. 4 One PBMC (per well) was resuspended in 100 μL of cell culture medium per well, and the PBMC suspension was placed in the upper chamber ( Figure 19 A). The lower chamber of the Transwell plate was treated with CTR+LPS in 500 μL of culture medium ( Figure 19 B), 10 μg / mL anti-EPO+LPS ( Figure 19 C) or 100 ng / mL rhEPO + LPS ( Figure 19 D) Treatment. After incubation at 37℃ and 5% CO2 for 48 h, the upper chamber was removed, and PBMCs were stained with Hoechst (1:1000 diluted in PBS, stained at 37℃ for 15 min). The PBMCs were washed with PBS, and the number of PBMCs at the bottom of the chamber was counted under a microscope. Analysis was performed using ImageJ and its AnalyzeParticle plugin. Figure 19 E). Immunolabeling was acquired using a high-resolution Nikon Ti rotating disk confocal microscope (Nikon Instruments, Florence, Italy), equipped with a CREST-Optics rotating disk scanning head, a VCS structured illumination module for super-resolution imaging (CREST-Optics, Rome, Italy), and an Andor camera for resolution (Andor Zyla, Andor Technology, Oxford Instruments, Oxford, UK) and quantum efficiency (Andor Technology, Oxford Instruments, Oxford, UK). The rotating disk confocal images were deconvolved using NIS-Elements V.5.3.2, and the correct structured illumination images were reconstructed using a specific VCS-Studio algorithm (CRESTOptics, Rome, Italy). High-resolution imaging of all stained wells was performed using whole-well imaging; during high-magnification acquisition, large-scale stitching was used to obtain the full field of view (FOV) of each IF-labeled instance. Interestingly, PBMCs were chemotactic with anti-EPO antibodies, but this effect was significantly reduced in the presence of recombinant human EPO (rhEPO). Therefore, it can be inferred that in the presence of LPS (simulating infectious disease conditions), anti-EPO treatment significantly increases PBMC migration due to potent stimulation. Conversely, in the presence of LPS, rhEPO treatment blocks PBMC migration, indicating that EPO has an immunosuppressive effect. Compared with CTR treatment, *P<0.05.

[0198] Example 13. In an orthotopic rodent GBM model, administration of anti-EPO induced increased immune cell migration and promoted tumor penetration by T lymphocytes.

[0199] In the in vivo in situ GBM model, compared with the healthy contralateral cerebral hemisphere ( Figure 20 In contrast, administration of anti-EPO unexpectedly induced activation of the immune system, accompanied by T lymphocyte migration. Figure 20B (white arrow). Interestingly, ferroptosis was assessed by histological analysis after administration of anti-EPO, which showed the presence of iron deposition, a hallmark of cell death.

Claims

1. A method for activating or enhancing the immune response in a patient in need, the method comprising using an anti-EPO negative function modulator or an anti-EPO antigen-binding fragment alone, or in combination with the following: - Checkpoint inhibitor or immunomodulatory agent therapy (e.g., anti-PDL1 antibody, nivolumab, ipilimumab, abatacept; glembatumumab vedotin); and / or - Cell-based immunotherapies (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, activated dendritic cells targeting tumor-associated antigens, and / or antigen-presenting cells, tumor-associated peptides, therapies based on engineered mononuclear-macrophages or polymorphonuclear cells, and / or therapies for enhancing and reprogramming tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM) responses). - Antimicrobial therapy (e.g., antibiotics, antiviral drugs, antifungal agents, antifungal drugs, antipruritic drugs); - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Prophylactic or therapeutic vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids (e.g., anti-HPV vaccines, anti-EBV vaccines, or anti-HIV vaccines). - Immunotherapy based on oncolytic viruses; - Chemotherapy agents; - Anticancer drugs; - Enzymes that degrade heparan sulfate proteoglycans (e.g., heparinase); - A negative regulator of the sphingosine phosphate-1 (S1P) signaling pathway; or - EPO mimics that retain erythropoiesis function; in, The anti-EPO antigen-binding fragment is selected from the group including Fab, -F(ab')2, single-chain antibodies, biantibodies, triantibodies, tetraantibodies, repeat antibodies, or domain antibodies; the anti-EPO negative functional regulator is selected from the group consisting of monospecific or multispecific anti-EPO antibodies, gene therapy, DNA decoys, RNA decoys, ribozymes, antagomiR, shRNA, LNA, siRNA, antisense oligonucleotides, or anti-EPO receptors; the anti-EPO receptor is selected from the group consisting of EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR, and EPOR / CD131 heterodimers.

2. The method according to claim 1, wherein, The negative functional modulator or anti-EPO antigen-binding fragment induces an immune cell response (activation of T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells, antigen-presenting cells), wherein the cell response is an antitumor T cell response or an antimicrobial cell response in a patient affected by cancer or an infectious disease.

3. The method according to claim 2, wherein, The anti-tumor T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells are immune cells derived from PBMCs.

4. The method according to any one of claims 2 or 3, wherein, The anti-tumor T cell response is a CD8+ and CD4+ T cell response.

5. The method according to any one of claims 2 to 4, wherein, The anti-tumor T cell response was attributed to the expression of CD69 and EPOR in the migrating PBMCs.

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

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

8. The method according to any one of claims 1 to 7, wherein, The negative functional modulator or anti-EPO antigen-binding fragment induces T helper cells and / or cytotoxic T cells and / or B lymphocytes and / or NK lymphocytes and / or macrophages and / or neutrophils and / or dendritic cells and / or antigen-presenting cells; wherein the cellular response is further enhanced by CAR-T, CAR-M, CAR-NK, engineered monocyte-macrophage or polymorphonuclear cell-based therapies, or by enhancing and reprogramming the response of tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM).

9. The method according to any one of claims 1 to 8, wherein, The patients in need are diagnosed with cancer, proliferative disorders, chronic inflammatory diseases based on autoimmune and non-autoimmune factors, 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 HIV-2, sickle cell disease, SARS, and SARS. CoV, MERS; or the patients in need are those who have undergone organ or tissue transplantation, wherein the cancer is selected from 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 astrocytoma, grade III obese astrocytoma, anaplastic astrocytoma, including cerebral gliomatosis, ependymoma, medulloblastoma, neurosurgical Germ cell tumors, neuroblastoma, hypothalamic glioma, 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, colon cancer, colorectal cancer, ovarian cancer, ovarian epithelial cancer, choriocarcinoma of pregnancy, cervical cancer, endometrial cancer, uterine cancer, ovarian germ cell cancer, esophageal cancer, basal cell carcinoma, bile duct cancer, choroidal melanoma, choroid plexus carcinoma, spleen cancer, osteosarcoma, intraocular melanoma, malignant melanoma, retinoblastoma, gastric cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasal cavity and paranasal sinus cancer, salivary gland cancer, nasopharyngeal cancer, pharyngeal cancer, thyroid 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 epithelial cancer, small bowel cancer, gastrointestinal stromal tumor, testicular cancer, renal cell carcinoma, adrenal cancer, renal pelvis cancer, malignant mesothelioma, mesothelioma, pheochromocytoma, blood cancers or chronic myeloid leukemia, lip cancer, tonsil cancer, squamous cell carcinoma, ampullary cancer, peritoneal cancer, tongue cancer, pseudomyxoma peritoneum, intrahepatic hepatoblastoma, myelodysplastic syndrome, Wilms' tumor, penile cancer, pharyngeal cancer, juvenile lymphoma, juvenile leukemia, Paget's disease, skin cancer, anal cancer. This group comprises pleural carcinoma, leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, myeloma, duodenal cancer, malignant soft tissue cancer, malignant lymphoma, chronic myeloid leukemia, gallbladder cancer, bile duct cancer, chronic lymphocytic leukemia, malignant bone cancer, metastatic bone cancer, eye cancer, vulvar cancer, ureteral cancer, mediastinal cancer, urethral cancer, cancer of unknown primary origin, vaginal cancer, spinal cord cancer, vestibular schwannoma, diffuse midline glioma (DMG), diffuse endophytic pontine glioma (DIPG), embryonal tumors, pineal blastoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, and hemangioblastoma.

10. The method according to any one of claims 1 to 9, wherein, The cancer in question is glioblastoma multiforme.

11. The method according to any one of claims 1 to 10, wherein, The negative functional modulator or anti-EPO antigen-binding fragment promotes the immune system response in patients affected by refractory or persistent infectious diseases; wherein the persistent infectious diseases include tuberculosis, malaria, HIV, EBV or HPV-induced precancerous lesions and their prevention.

12. The method according to any one of claims 1 to 11, wherein, The negative functional modulator or anti-EPO antigen-binding fragment promotes an immune system response in patients affected by streptococcal, staphylococcal, fungal, viral, Sars-Cov2, SARS, MERS, or prion pathogens, who are tolerant to antimicrobial therapy and / or have immune system tolerance.

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

14. A pharmaceutical kit comprising a negative functional modulator of EPO / EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protectant, TPR, such as EPOR / CD131 heterodimer) and / or their natural or synthetic variants, and - Checkpoint inhibitor or immunomodulatory agent therapy (e.g., anti-PDL1 antibody, nivolumab, ipilimumab, abatacept, glembatumumab vedotin); and / or - Cell-based immunotherapies (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, activated dendritic cells targeting tumor-associated antigens), therapies based on engineered mononuclear-macrophages or polymorphonuclear cells, and / or therapies for enhancing and reprogramming tumor-associated lymphoid tissue (TIL) or tumor-associated macrophage (TAM) responses. and one or more components, said one or more components being selected from the group consisting of: - Peptides or antibodies, biantibodies, and nanobodies targeting natural and synthetic variants, including physiological and pathological splice variants and post-translational modified forms of erythropoietin; - Antimicrobial therapies (e.g., antibiotics, antiviral drugs, antifungal agents, antifungal drugs, antipruritics); - Flavonoid molecules; - Metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonists; - Prophylactic or therapeutic vaccines based on DNA and / or RNA and / or peptides or carbohydrates or lipids (e.g., anti-HPV vaccines, anti-EBV vaccines, or anti-HIV vaccines). - Immunotherapy drugs based on oncolytic viruses; - Chemotherapy; - Anticancer drugs; - Enzymes that degrade heparan sulfate proteoglycans (e.g., heparinase); - A negative regulator of the sphingosine phosphate-1 (S1P) signaling pathway; or - An EPO mimic that retains the function of erythropoiesis.

15. A diagnostic or prognostic method for assessing EPO and its somatic mutations or variants thereof, EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protectant, TPR, EPOR / CD131 heterodimer) and their somatic mutations and / or variants and / or C4. The method for predicting response to therapy targeting negative regulation of EPO / EPOR, based on mAb ligand expression, aims to enable personalized treatment in immunotherapy or prevention of cancer and infectious diseases, and to stratify patients to optimize patient response. The method includes the steps of: determining the amount / detection of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants, as diagnostic or prognostic markers, in tissues, cells, or human body fluids (saliva, blood, cerebrospinal fluid, sweat, or derived extracellular vesicles); and determining the presence of EPO and its somatic mutations or variants, EPO receptors and their somatic mutations and variants, as diagnostic or prognostic markers, in tissues, cells, or human body fluids (saliva, blood, cerebrospinal fluid, sweat, or derived extracellular vesicles).

16. A diagnostic method for assessing promoter methylation of genes for EPO and EPO receptors (EPOR, EPHB4, CSF2RB, CRLF3, tissue protection factor, TPR, such as EPOR / CD131 heterodimer), said method being able to predict increased expression of EPO / EPO-R and their negative role in suppressing the immune system's clearance of cancer cells or microbial agents, thereby enabling personalized immunotherapy or prophylaxis, and for prognostic purposes; said method comprising the step of: detecting methylation of EPO and EPO receptor genes as diagnostic or prognostic markers in tissues, cells, or human bodily fluids (saliva, blood, cerebrospinal fluid, sweat, or derived extracellular vesicles).

17. An EPO / EPO-R negative modulator delivery system, wherein the system, in clinical administration, is administered in various formulations via oral, parenteral, intralesional (intratumoral and intracavitary), intraventricular, intrathecal, intranasal, or local administration; said system is based on innovative nanomedicine and nanodelivery systems, such as tissue guns or probes; viral and nonviral vectors; nanomaterials for targeted delivery of bioactive drugs; plant-based vesicles; nanoparticle-based methods that allow simultaneous therapeutic and in vivo imaging for diagnostic and therapeutic purposes; lipid systems, such as liposomes and micelles; gold nanoparticles or magnetic nanoparticles, and functionalized nanoparticles, microspheres, and biomaterials, such as PEG, used in combination with natural products; Trojan horse methods, such as micropumps for releasing therapeutic drugs in tissues to enhance the modulation of the immune system in the local pathological microenvironment or attract and increase homing of cell-based immunotherapies or vaccines.

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

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

20. Inhibitors of EPO / EPO-R and their variants, wherein the inhibitors of EPO / EPO-R and their variants are capable of stimulating CTL infiltration and inhibiting the recruitment of immunosuppressive cells in tumors and infectious diseases, thereby increasing tissue penetration of inflammatory cells and immune cells.

21. Inhibitors of EPO / EPO-R and their variants, wherein the inhibitors of EPO / EPO-R and their variants are capable of inducing inflammation, associated pyroptosis, immunogenic cell death, necrotizing apoptosis, ferroptosis, autophagy, copper death and immunostimulatory cell death, thereby enhancing the immunogenicity of tumors.

22. A product selected from inhibitors of EPO and their natural and synthetic variants, the product being able to activate immune responses against cancer and infectious agents, reprogram the tumor microenvironment, and enhance immunotherapy and immunomodulatory strategies.

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