Method for boosting the efficacy of immunotherapy and enhancing the host immune response

Abstract

The present invention concerns the field of immunotherapies suitable for activating the immune response in a patient. More in detail the present invention relates to anti-EPO negative functional modulators, useful as active ingredients in a pharmaceutical composition for immunomodulating strategies in therapy (eg cancer immunotherapies, infectious, inflammatory diseases) or a pharmaceutical composition for immuno-activation, boosting cell based or pharmacological or vaccine based immunotherapies and stimulating the immune system response of a patient in need thereof. In particular, the invention also describes how to restore the immune response in pathological conditions such as cancer and refractory infectious diseases enhancing and assuring the therapeutic access of immunotherapies strategies and abolishing the "tolerogenic" stimuli through products consisting in inhibitors of EPO pathway that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate immune responses in cancer, infectious and inflammatory diseases and transfer into patients..

Description

[0001] METHOD FOR BOOSTING THE EFFICACY OF IMMUNOTHERAPY AND ENHANCING THE HOST IMMUNE RESPONSE

[0002] ***** ***** *****

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to an immunotherapy adjuvant strategy and a protocol to enhance host immune response in cancer, infectious and inflammatory diseases to avoid immunotolerance. In particular, the invention also describes how to restore the immune response in pathological conditions such as cancer and refractory infectious diseases enhancing and assuring the therapeutic access of immunotherapies strategies and abolishing the “tolerogenic” stimuli through products consisting in inhibitors of EPO pathway that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate immune responses in cancer, infectious and inflammatory diseases and transfer into patients.

[0005] STATE OF THE ART

[0006] The immune system is the body's first line of defense against what is foreign to the body (not self), be it a microbe or a tumor cell. In order to evade the body's defenses over time, these have developed common mechanisms to make the immune system "tolerogenic" by creating a shift in the immune response from inflammatory-like, capable of providing a response towards the not self-agent (tumor or infectious agent) towards a tolerant anti-inflammatory response by the immune system.

[0007] In order to induce an effective immune response that can fight infectious diseases or cancer, cells or active molecules in the innate immune system and the adaptive immune system 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 (DC), and neutrophils, as well as active molecules such as complement system in serum. The lymphocytes and antibodies involved in adaptive immunity have the ability to recognize the structure of non-self antigens or amino acid sequences in detail with high accuracy.

[0008] In the complex mechanism of interplay between the inflammatory (activated immune system) and anti-inflammatory (tolerogenic) response, an important role is also played by the microbiota.

[0009] Commensal microorganisms colonize barrier surfaces of all multicellular organisms, including those of humans. For more than 500 million years, commensal microorganisms and their hosts have coevolved and adapted to each other. As a result, the commensal microbiota affects many immune and nonimmune functions of their hosts, and de facto the two together comprise one metaorganism. The commensal microbiota communicates with the host via biologically active molecules. Microbial imbalance may play a critical role in the development of multiple diseases, such as cancer, autoimmune conditions, and increased susceptibility to infection as part of a common mechanisms. The gut microbiota also plays a role in anticancer responses: a dysbiotic microbiota composition lacking immunostimulatory bacteria or containing immunosuppressive species causes treatment failure. The gut microbiome was recently recognized to influence the effectiveness of PD-1 -based anticancer immunotherapy, and healthy gut flora is a determinant of the anticancer response. Indeed, the modulation of immune system on the commensal microbiota is critical to avoid development, progression, and immune evasion of cancer, as well as some modulatory effects on the treatment of cancer.

[0010] An example of gut microbial agents and cancer is represented from cervical cancer, a malignancy caused by persistent human papillomavirus (HPV) infection, develops in more than 500,000 women annually. More than 90% of deaths from cervical cancer occur in low- and middle-income countries. A common epidemiological feature of countries with high cervical cancer incidence is a high burden of intestinal helminth infection. The ability of intestinal helminths to trigger immunoregulation, resulting in a "tolerogenic" systemic immune environment, provides fertile soil for the persistence of oncogenic viruses such as HPV. Animal models have shown that intestinal helminth infection permits the persistence of some viruses, however, HPV-specific and human studies are lacking. Large, well-organized trials evaluating the consequences of intestinal helminth infection on the human immune system and HPV persistence may lead to improved strategies for HPV prevention in helminth-endemic regions of the world. The present invention offers the strategy to counteract the specific ways which also intestinal helminth infection may contributes to immunomodulation and identify a new therapeutic target for a range of diseases, from inflammatory disorders to cancer. EPO negative modulation counteract helminth-induced systemic and local immune dysregulation as possible mechanisms by which chronic intestinal helminth infection facilitate HPV persistence. Multiple common mechanisms of immune evasion-based diseases where EPO pathway is a key target for therapy can be analyzed. One of these cases of immune system evasion driven by the same “tolerogenic” stimuli observed in cancer is represented by Plasmodium falciparum, the pathogen of malaria disease. Malaria is one of the most important human infectious diseases and particularly affects populations living in tropical and subtropical countries. Nowadays, although some antimalarial drugs are available, malaria remains a major public health problem with 241 million cases and 627,000 deaths per year. This infection is caused by the protozoan parasite Plasmodium. Five species are responsible for malaria in human beings: P. vivax, P. malariae, P. ovale, P. knowlesi or P. falciparum. While P. vivax is the most widespread, P. falciparum is responsible for almost the totality of severe and lethal malaria cases. Immune evasion strategy is used to avoid immune response attack. P. falciparum parasites evade mosquito immune response to transmit to a new host. The main and critical P. falciparum gene used for evasion of Anopheles mosquito immune response is Pfs47. It inhibits JNK-mediated apoptosis by preventing activation of several caspases and Jak-STAT pathway is the downstream pathway of EPO / EPO-R signal. The above-described pathway is also important on the modulation of macrophages and lymphocytes in their answer against mycobacterium tuberculosis, HIV and solid tumors, where immune cells can become part of the strategy of resistance to pharmacological therapies. Furthermore, abnormal erythropoiesis has been observed in malaria patients and could potentially be instrumental in anemia. The relation between erythropoietic defects and malarial anemia has been poorly investigated so far; however, recent findings may provide new insights. Tsubata and colleagues in 2005 reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection.

[0011] Cancer is a disease characterized by abnormal, localized cell growth that has the potential to spread throughout the body. In the tumor core, aberrant vascularization, and impaired bloodflow limit oxygen supply, creating a hypoxic environment. Hypoxia attracts Tregs into the TME, which in turn suppresses the functions of effector T-cells and promotes tumor growth. This condition upregulates the expression of Hypoxia-Inducible Factors (HIFs), including HIF-1a and HIF-i p. Hypoxia also enhances glucose uptake and intensifies glycolysis by cancer cells, exacerbating glucose paucity in the TME. Among these factor, EPO and Sphingosine-1- Phosohate (S1 P) play a central role on the conditioning of immunosuppressive tumor microenvironment. S1 P is generated from sphingosine by sphingosine kinase 1 (SphK1) upon apoptosis induction. S1 P exerts its function through a family of receptors, S1 PR1-5, and murine macrophages express only S1 PR1 and S1 PR2. S1 P has been identified as a find-me signal; however, the S1 P concentration in circulation (4 mM) is higher than that in tissues (low nM) and that released from dying cells (400 nM), suggesting that S1 P might work locally or has other functions in the context of apoptotic cell clearance. Apoptotic cell-released find-me signal S1 P has been shown to induce macrophage HIF-1a (hypoxia inducible factor-1a), the oxygen- labile subunit of the HI F complex, which is the master transcription factor for EPO, suggesting that dying cell-derived S1 P might activate macrophage EPO signaling. EPO receptors, EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer (EPORs) has been identified on macrophages, through which, EPO suppresses inflammatory gene expression in macrophages, indicating the involvement in orchestrating tolerogenic responses in macrophages. In addition, EPO has been shown to improve outcomes of autoimmune diseases. However, the involvement of EPO in dying cell clearance and immunotolerance remains unknown. Here we found that dying cell- derived S1 P activated macrophage EPO signaling promoted immune silent clearance of dying cells and immune tolerance in mice.

[0012] This mechanism of immune cell tolerance is common to many types of cancer and infectious diseases refractory to therapy 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, streptococci, SARS, Sars-Cov, MERS prion disease.

[0013] In the past, the most common methods to treat oncological cancer were surgery, radiation therapy or chemotherapy. However, it has recently been demonstrated that cancer immunotherapy has many promises as a treatment for oncology. The most fundamental problem in tumor immunity is how to activate the immune system to recognize and eliminate antigens. In this respect, a novel method of genetically engineering tumor cells to secrete specific cytokines has led to major advances in tumor immunity. The theoretical background of genetically modified tumor vaccines based on immunotherapy is that the host possesses antigens that can recognize tumors as external factors. Human T and B lymphocytes can discriminate almost infinite antigen differences in the form of antigen receptors through the development process. However, to succeed in tumor immunity, the following two criteria must be met. First, tumor cells must express novel antigens (peptides) that are not expressed in normal cells. Second, immune cells must be properly activated to recognize these antigens. Co-administration for the treatment of cancer is becoming more and more common as the benefit of attacking the disease through multiple means is recognized. Co-administration is useful even when resistance to anticancer drugs is shown. In addition, co-administration has the advantage of reducing the amount of the anticancer agent administered by enhancing the efficacy of the anticancer agent. Through this, it is possible to increase the anticancer efficacy while minimizing the toxicity and side effects on each organ of the body. Several cancer immunotherapies that train or stimulate the inherent immunological systems to recognize, attack, and eradicate tumor cells with minimal damage to healthy cells have demonstrated promising clinical responses in recent years. However, most of these immunotherapeutic strategies only benefit a small subset of patients and cause systemic autoimmune side effects in some patients. Immunogenic cell death (ICD)-inducing modalities not only directly kill cancer cells but also induce antitumor immune responses against a broad spectrum of solid tumors. Such strategies for generating vaccine-like functions could be used to stimulate a "cold" tumor microenvironment to become an immunogenic, "hot" tumor microenvironment, working in synergy with immunotherapies to increase patient response rates and lead to successful treatment outcomes.

[0014] Immunotherapies that block immune checkpoints, such as PD-1 or CTLA-4 (e.g. Abatacept) have revolutionized the treatment paradigm for many patients with advanced-stage tumors. Immune checkpoint inhibitors, are already in broad use to treat people with many types of cancer, including melanoma, lung, kidney, bladder, and lymphoma.

[0015] But another form of immunotherapy, called CAR T-cell therapy, and lastly tumor-infiltrating lymphocytes (TILs) therapy, have also generated substantial interest among researchers and oncologists. These therapies have shown the ability to eradicate very advanced leukemias and lymphomas. However, metabolic constraints and soluble factors create an immunosuppressive TME exacerbating the functional exhaustion of tumour-infiltrating T-cells and inducing a poor T cell expansion and short-term T cell persistence.

[0016] Compared to hematological malignancies, in fact, solid tumor CAR-T cell therapy is limited by the ability of CAR-T cells to traffic to and infiltrate solid tumors due to the immunosuppressive tumor microenvironment. One strategy to ameliorate these limitations is through the utilization of delivery routes other than systemic delivery as local administration, to eliminates the need for CAR-T cells to traffic to disease sites and to limit on-target off-tumor toxicities as the CAR- T cells’ on-target activity is directed on tumor cells minimizing interaction with normal tissues. Preclinical models have demonstrated superior therapeutic efficacy of intraventricular injection of CAR-T cells targeting HER2 / IL13Ra2, in breast cancer brain metastases and in glioblastoma (NCT02208362, NCT03389230, NCT03696030).

[0017] Another important aspect to take into consideration is the physical tumor barriers such as the tumor stroma, which limits the penetration and mobility of CAR-T cells. Stroma is mostly composed of extracellular matrix, also known as the matrisome, which helps determine tumor invasion, adhesion, and growth. In matrisome heparin sulfate proteoglycans (HSPG), such as glypican (GPC 1 -6) and syndecan (SDC 1 -4), are the primary component that CAR-T cells must degrade to pass into the tumor. CAR-T cells that have been engineered to express heparanase, an enzyme that degrades HSPG, show enhanced tumor infiltration and antitumor activity. Similarly, fibroblast activation protein (FAP)-targeted CAR-T cells demonstrate increased cytotoxic function through reducing tumor fibroblasts in animal models. In addition, a promising strategy is to guide T cells through tumor-specific chemokines, provided that the matching chemokine receptors are expressed on T cells.

[0018] Here we demonstrate that EPOR is expressed on T cells, as well as monocytes and macrophage -derived form monocytes. We further demonstrate that surprisingly EPO-EPORs pathways is more active on the tolerogenic version of immune system allowing the development and maintenance of cancer and infectious diseases. From this evidence, we decided to negative modulating EPO pathway to modify the pathophysiological mechanism and boost the immune system to answer to the pathogenic stimuli. More in detail for example we treated glioblastoma stem cells, breast cancer cells, melanoma cells, and colon cancer cells with CAR- T cells in co-administration of anti-EPO monoclonal antibody. Surprisingly, results revealed that the combined treatment of anti-EPO monoclonal antibody with CAR-T facilitated migration and penetration of T cells into tumoral tissue, until now little employed in solid malignancies because the poor tumor penetration, surprisingly observing an higher killing activity on cancer cells compared to single administration.

[0019] Therefore, several approaches have been investigated to ameliorate and potentiate CAR-T cell response. Firstly, the combination immunotherapy with CAR-T cells, checkpoint blockade (e.g. nivolumab, relatlimab, Ipilimumab, anti-CD47) which induces the infiltration of CAR-T and provides PD-1 / PD-L1 blockade or SIRPa / CD47, and chemotherapy, as cyclophosphamide. In hematological malignancy, combination PD-1 blockade and CD19 CAR-T cell therapy in children with heavily pretreated B-ALL resulted in improved persistence of CAR-T cells and better outcomes. Secondly, tumors, in particular solid tumors, may have cell-intrinsic resistance mechanisms to CAR-T cell cytotoxicity and combining other forms of immunotherapy strategies may still be necessary to combat the inhibitory signal present in the tumor microenvironment. Indeed, IFNyR signaling loss in tumors has been reported in clinical studies of checkpoint blockade resistance owing to downstream effects on antigen presentation. Indeed, the loss of genes in the interferon-y receptor (IFNyR) signaling pathway (IFNGR1 , JAK1 or JAK2) was demonstrated to render glioblastoma and other solid tumors more resistant to killing by CAR- T cells both in vitro and in vivo. More recently, first and second generation of bispecific antibodies have been introduced in the clinical practice. In cancer immunotherapy, the ability of bispecific antibodies to form ‘immunological synapses’ between tumor cells and immune effector cells such as T cells, affords the possibility to precisely target an immunological response to a particular cancer cell. Nowadays, third-generation T cell engager have been developed. This T cell engager consists of two bispecific antibodies, each of which incorporates a binding module for a tumor antigen as well as half of the domain required to bind a CD3 subunit of the T cell receptor. This strategy of splitting the anti-CD3 paratope ensures that each of the two drug components is completely inactive until both bind simultaneously to target antigens on the same tumor cell.

[0020] In solid tumors, such as brain tumors, the immunosuppressive microenvironment is shaped by mutations in the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2 in glioma cells, which inhibit STAT1 expression and decrease the production of CD8 T cells, type 1 related effector molecules, and chemokines e.g. CXCL10. In line with the findings, T cell infiltration was significantly lower in IDH-mutant gliomas compared to IDH-wildtype gliomas.

[0021] One of the challenges in targeting solid tumor antigens is that solid tumor antigens are often also expressed on normal tissues at varying levels. Therefore, antigen selection is crucial in CAR design to not only ensure therapeutic efficacy but also to limit “on-target off-tumor” toxicity. A potential avenue to overcome the targeting of antigens on solid tumors that are also present on normal tissues is the targeting of tumor-restricted post-translational modifications such as solid tumor overexpressed truncated O-glycans such as Tn (GalNAcal -O-Ser / Thr) and sialyl- Tn (STn) (NeuAca2-6-GalNAca1 -O-Ser / Thr). In line with this consideration, we showed that anti-EPO antibody which binds specifically a splice variant of human EPO, the EV-3 isoform highly expressed on tumor cells, can increase immune cell response, without affecting normal cells. In parallel the inhibitor of EPO and EV-3 are able to induce a cell death mechanism based on ferroptosis. Ferroptosis, like apotosis, cuproptosis, autophagy and pyroptosis is a key celldeath pathway implicated in several human diseases including cancer. Another important characteristic of CAR-T cell products is the ratio of CD4+ / CD8+ subsets. Although CD8+ T cells have long been considered as the primary 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 to provide helper function to evoke intrinsic antitumor immune responses. In particular, the presence of a CD4+ subset correlated with CAR-T persistence in solid tumors. Together, these findings suggest an essential role of CD4-mediated immune responses against tumors, which is also highlighted by some ongoing clinical studies where CD4+ doses are precisely controlled. We demonstrated that the administration of anti-EPO monoclonal antibody is effective to induce CD4+ activation and migration into tumor specimens, inhibiting tolerogenic behavior in immune response.

[0022] However, one of the most challenging limitations of CAR-T cell therapy is the development of tumor resistance to single antigen targeting CAR constructs, a phenomenon called antigen escape, which occurs especially in solid tumors. For example, a CAR-T cell therapy case report that targeted IL13Ra2 in glioblastoma suggested that tumor recurrences displayed decreased IL13Ra2 expression. To reduce the relapse rate in CAR-T cell treatment of both hematological malignancies and solid tumors, many strategies are now relying on targeting multiple antigens. These employ the use of either dual CAR constructs or tandem CARs, which is a single CAR construct that contains two scFvs to concomitantly target multiple target tumor antigens in hematological malignancies (ALL and diffuse large B cell lymphoma) such as CD19 / CD20, CD 19 / CD22 or CD19 / BCMA or in solid tumors (glioblastoma, breast, colon cancer) such as HER2 / IL13Ra2, and HER2 / MUC1 , TAG7228, B7- H3, MUC1 , and MUC16, av 6, CXCR1 or CXCR2. The importance of optimizing the selection of target antigens, not only improve antitumor response, but also decrease antigen escape mechanisms to prevent relapse.

[0023] In addition, recently efforts have focused on engineering CARs that are resistant to immunosuppressive factors in the hostile tumor microenvironment such as TGF p, IL-12, IL- 15, IL-4, IL-10, arginase 1 , indoleamine 2,3-dioxygenase (IDO)-mediated inhibitory signals. From these premises, many studies have investigated numerous cytokines to create these “armored CARs”. Therefore, intriguing strategy involves the engineering of CAR-T cells to provide immunostimulatory signals in the form of stimulatory cytokines that increase survival, proliferation, antitumor activity of T cells, and rebalance the tumor microenvironment. In the last years, also the technology RNA CAR-T has been suggested as tumor therapy. Indeed, current CAR-T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Now, ionizable lipid nanoparticles (LNPs) were designed for mRNA delivery to human T cells. Beside the new immunotherapeutic strategies based on CAR-T cells, nowadays studies based on CAR-engineered macrophages (CAR-M) and CAR-engineered natural killer (NK) cells (CAR-NK) or CAR-GAMMA / Delta are establishing themselves in the field of immuno-oncology.

[0024] All in all, the use of negative functional modulators of EPO is important to overcome the host immunotolerance versus microbial agents inducing the shift of inflammatory response activating macrophages and other immune system cells to eradicate the infection also in combination with antimicrobial agents or potentiating the efficacy of vaccines or developing new delivery systems.

[0025] New approaches as trojan horses, are being reported, capable of destroying tumor cells, not only at the injection site, but also of spreading a systemic immune response to destroy circulating and metastatic tumor cells.

[0026] In addition, functionalized microparticles, microcapsules and microspheres based controlled release systems for example can guarantee to achieve long term therapeutic drug levels while minimizing the number of repetitive injections influencing the pathologic microenvironment as in degenerative diseases or solid tumors.

[0027] Furthermore, the new frontier in cancer therapeutical approach is based on innovative nanomedicine and nano delivery systems as tissue gun or probe, viral and no-viral vectors, nanomaterials for the delivery of bioactive drugs for target delivery, nanoparticle-based methods which combined both the treatment and imaging modalities of cancer diagnosis and therapy, lipid systems like liposomes and micelles, gold or magnetic nanoparticles, also in combination with natural products.

[0028] When the target tissue is the central nervous system, strategy to overcome BBB can be employed like physical stimuli (e.g. Focused ultrasound (FUS) and MRI fields) or trojan horse molecules (e.g. IL13 / IL 13R; transferrin; microvescicles natural or synthetic; resveratrol). In addition, Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Also, local administration of immunotherapy using drug-eluting embolic (DEE) microspheres as drug delivery vehicles for direct infusion into tumor-feeding arteries might increase and prolong tumor drug concentrations and reduce systemic drug exposure, potentially improving the risk-to-benefit ratio of these agents.

[0029] Furthermore, negative functional modulators may have an advantage in anti-tumor therapeutic regimen also in combination with other natural or synthetic medicament. Flavonoids, in particular luteolin, an inhibitor of S1 P kinases has been demonstrated to exert a beneficial role in tumors, modulating reactive oxygen species (ROS)-scavenging enzyme activities, participating in arresting the cell cycle, inducing apoptosis, autophagy, and suppressing cancer cell proliferation and invasiveness showing a synergic additional effect to anti EPO treatment. Rapamycin and its derivatives are promising therapeutic agents with both immunosuppressant and anti-tumor properties. Rapamycin actions are mediated through the specific inhibition of the mTOR protein kinase. mTOR serves as part of an evolutionarily conserved signaling pathway that controls the cell cycle in response to changing nutrient levels. Another molecule that controls cell nutrient levels is metformin. Metformin is a widely used drug in today’s prescriptions by physicians due to its specific effects in treating and curing type II diabetes. Metformin inhibits mTOR activity by activating ATM (ataxia telangiectasia mutated) and LKB1 (liver kinase B1 ) and then adenosine monophosphate-activated kinase (AMPK), and thus prevents protein synthesis and cell growth. In addition, one mechanism by which metformin works is by activating AMPK, an enzyme inside cells that lowers blood sugar by promoting energy utilization. Activating AMPK has broad-ranging effects that extend far beyond blood sugar control. Studies show that boosting AMPK activity can prevent — and even reverse — the life-shortening effects of aging, such as cardiovascular disease, diabetes, neurodegenerative diseases, cancer, and more. Furthermore, strong evidence demonstrates that AMPK negatively regulates the mTOR pathway, thus hypothesizing a strictly inter-play between mTOR, AMPK mediated by metformin. We demonstrated a synergic effect of metformin and anti-EPO antibody on viability of cancer cells, and on decreasing gene expression of PD-1 and increasing over-expression of IFNy and IL-1 B genes promoting a pro-inflammatory change in tumor and infection site microenvironment with a beneficial effect to interrupt the physio-pathological mechanisms and eradicate the pathogenic stimuli.

[0030] Glucagon like peptide 1 (GLP1 ) receptor agonists are widely used for the treatment of type 2 diabetes because of their glucose-lowering capacity with low risk of hypoglycemia. GLP1 binds to its specific G-protein coupled receptor, which activates downstream pathways including cAMP / protein kinase A (PKA), cAMP / guanine-nucleotide exchange factor (Epac) or phosphatidylinositol-3 kinase / PKC pathways. It has eben reported that GLP-1 potentially could involve in carcinogenesis due to its trophic effects.

[0031] Glycoprotein non-metastatic melanoma protein B (GPNMB) , with its extracellular domain, shed from the cell surface, interacts with integrins to facilitate in the recruitment of immune- suppressive and pro-angiogenic cells to the tumor microenvironment, thereby enhancing tumor migration and invasion. Tumor intrinsic GPNMB-mediated effects on cellular signaling, coupled with the ability of GPNMB to influence the primary tumor and metastatic microenvironments in a non-cell autonomous fashion, combine to augment malignant cancer phenotypes. In addition, GPNMB is often overexpressed in a variety of cancers, making it an attractive therapeutic target. In this regard, glembatumumab vedotin, an antibody-drug conjugate (ADC) that targets GPNMB, is currently under investigation. Furthermore, GPNMB has been shown to have antiinflammatory effects in a variety of neurological diseases. From these premises, the modulation of GPNMB seems to be promising also in cancer and infection diseases.

[0032] All in all, the present solution plays vaccine-like functions and could be used to stimulate a "cold" site microenvironment to become an immunogenic, "hot" site microenvironment, common feature of cancer and infection and inflammatory diseases, working in synergy with immunotherapies to increase patient response rates and lead to successful treatment outcomes.

[0033] Also, the present invention offers the possibility to counteracts infectious agents (e.g.TBC, Malaria, HIV, SARS, drug resistant bacteria, fungi and viruses) stimulating the inflammatory host response, potentiating the use of classical, recombinant and peptide or lipid or DNA or mRNA-based vaccines, and antimicrobial therapies.

[0034] In addition, the solution herein presented could be a useful tool to counteract negative related mechanism and effects caused by atmospheric flight or by space explorations, orbital and suborbital flights and the inhibition of host immune system caused by extreme environmental condition like microgravity, high quote, hypoxia, cosmic and UV radiation exposure e.g. during space flight missions.Advantageous Effect

[0035] EPO is specifically involved in the modulation of immune system as pathogenetic mechanism in several diseases like cancer, infectious and autoimmune diseases.

[0036] The cancer immunotherapy adjuvant according to the present invention, when administered in combination with a cancer immunotherapy agent or by itself, activates the function of immune factors without causing in vivo side effects, to exhibit the effect of enhancing the kit for anticancer effect of the cancer immunotherapy agent, and thus can be effectively used as a cancer immunotherapy adjuvant or as adjuvant in vaccine-based prophylaxis or therapy.

[0037] The tumor microenvironment (TME) for example imposes a major obstacle to infiltrating T- lymphocytes and suppresses their function. Several immune checkpoint proteins that interfere with ligand / receptor interactions and impede T-cell anti-tumor responses have been identified. Immunotherapies that block immune checkpoints have revolutionized the treatment paradigm for many patients with advanced-stage tumors. However, metabolic constraints and soluble factors that exist within the TME exacerbate the functional exhaustion of tumour-infiltrating T- cells. The use of a negative modulator of EPO offers a strategy to potentiate the immunotherapeutic strategies also enhancing the effect of current therapies. Similar features are shown in some refractory infectious diseases like malaria and HIV, where paradoxically EPO sometimes is used to counteract iatrogenic anemia associated with antimicrobial and antiviral therapies. In that situation, EPO induce immune response deprivation leading to macrophages and lymphocytes to immune tolerance. The present invention is able to potentiate the efficacy of antimicrobial therapies like antibiotics (penicillins, macrolides cephalosporins, fluoroquinolones beta-lactams with increased activity, tetracyclines, trimethoprim-sulfamethoxazole, urinary antiinfectives, lincosamides anti-viral (reverse transcriptase inhibitors, protease inhibitors and antiviral classes)), antimicotics / antifungal (polyenes, azoles, allylamines and echinocandins), anti-malaric therapies, as combined treatment in prion disease.

[0038] SUMMARY OF THE INVENTION

[0039] The inventors have surprisingly found that by blocking EPO, the mechanisms of regulation of innate and adaptive immune response common to tumors, infections, and inflammatory disease can be overcome, developing new therapeutic strategies to inhibit resistance and progression of the pathology and to abolish the “tolerogenic” stimuli of immune system. The present invention relates to negative functional modulators of EPO for use in a method of activating an innate and adaptative immune response of a patient in need thereof. Said compounds were surprisingly seen to be active in inducing migration of immune cells, inducing engraftment into tumor tissue, and potentiating immunostimulatory effect of immune cells, cytotoxic ability of immune cells also in counteracting infectious process and promoting vaccine efficacy.

[0040] EPO is specifically involved in the modulation of immune system as pathogenetic mechanism in several diseases like cancer, infectious and autoimmune diseases.

[0041] In a first aspect, the invention relates to an anti-EPO negative functional modulator or anti-EPO antigen-binding fragment chosen from the group comprising Fab, -F(ab’)2, single chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies, for use in a method of activating an immune response of a patient in need thereof.

[0042] The immunotherapy adjuvant according to the present invention, when administered in combination with a immunotherapy agent or by itself, activates the function of immune factors without causing in vivo side effects, in cancer, infection and inflammatory diseases, and thus can be effectively used as an immunotherapy adjuvant. In a further aspect the invention relates to a method for activating or boosting the immune response of a patient in need thereof, said method comprising the use of ananti-EPO negative functional modulator or anti-EPO antigen-binding fragment alone or in combination with:

[0043] - a check point inhibitor or immunomudulator (e.g. anti-PDL1 antibody; nivolumab; ipilumab, abetacept, glembatumumab vedotin) therapy; and / or

[0044] - a cell-based immunotherapy (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic activated cells against tumor associated antigens, and / or antigen-presenting cells, tumor associated peptide, engineered monocyte-macrophage or polymorphonucleate cell-based therapies and / or a therapy to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs;

[0045] - an anti-microbial therapy (e.g. antibiotics; antivirals, antimycotics, antifungi, anti-prions)

[0046] - a flavonoid molecule;

[0047] - metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonist,

[0048] - a prophylactic or therapeutic DNA and / or RNA and or peptide or carbohydrate or lipid based vaccine (e.g. anti-HPV, anti-EBV or anti-HIV vaccine);

[0049] - Oncolytic virus based immunotherapy;

[0050] - a chemotherapeutic agent;

[0051] - an anti-cancer drug;

[0052] - an enzyme that degrades heparin sulfate proteoglycans (e.g. heparanase);

[0053] - a negative functional modulator of the sphingosine-1 -phosphate (S1 P) signaling pathway, or

[0054] - EPO mimetics that preserve the erythropoietic function; wherein said anti-EPO antigen-binding fragment is chosen from the group comprising Fab, - F(ab’)2, single chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies and said anti-EPO negative functional modulator is chosen from the group consisting of mono- or multi-specific antibody anti-EPO, gene therapy, DNA decoy, an RNA decoy, a ribozyme, an antagomiR, a shRNA, an LNA, a siRNA, an antisense oligonucleotide or an anti-Epo receptor, said anti-Epo receptor chosen from the group consisting of EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs and EPOR / CD131 heterodimer.

[0055] In a second aspect, herein described is a pharmaceutical kit comprising a negative functional modulator of EPO / EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) and / or their natural or synthetic variants and: - a check point inhibitor or immunomudulator (e.g. anti-PDL1 antibody; nivolumab; ipilumab, abetacept, glembatumumab vedotin) therapy; and / or

[0056] - a cell-based immunotherapy (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic activated cells against tumor associated antigens) engineered monocyte-macrophage or polymorphonucleate cell-based therapies and / or a therapy to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs; and one or more components selected from the group consisting of:

[0057] - a peptide or antibody, diabody, nanobody against the natural and synthetic variants including physiological and pathological splicing variants of erythropoietin and post-translational modification;

[0058] - an anti-microbial therapy (e.g. antibiotics; antivirals, antimycotics, antifungi, anti-prions)

[0059] - a flavonoid molecule;

[0060] - metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonist;

[0061] - a prophylactic or therapeutic DNA and / or RNA and or peptide or carbohydrate or lipid based vaccine (e.g. anti-HPV, anti-EBV or anti-HIV vaccine);

[0062] - Oncolytic virus based immunotherapy;

[0063] - a chemotherapeutic drug

[0064] - an anti-cancer drug

[0065] - an enzyme that degrades heparin sulfate proteoglycans (e.g. heparanase)

[0066] - a negative functional modulator of the sphingosine-1 -phosphate (S1 P) signaling pathway, or

[0067] - EPO mimetics that preserve the erythropoietic function.

[0068] In a third aspect, the present invention relates to a diagnostic or prognostic method for evaluating the expression of EPO and its somatic mutations or its variants, EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, EPOR / CD131 heterodimer) and their somatic mutations and / or their variants and / or C4 mAb ligands that predict the response to target EPO / EPORs negative modulation therapy to personalize therapy in cancer and infectious diseases immunotherapy or prophylaxis, to stratify patients, optimize patient’s response, said method having the step of measuring the amount of detecting the presence of EPO and its somatic mutations or its variants, EPO receptors and its somatic mutations and their variants in tissues, cells, or human fluid (saliva, blood, cerebrospinal fluid, sweat, or derived-extracellular vesicles) as diagnostic or prognostic markers

[0069] In a fourth aspect, the present invention relates to a diagnostic method for evaluating the methylation of the promoter of the genes of EPO and EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) that can predict an increase expression of EPO / EPO-Rs and their negative role in immune system inhibition in eradicating cancer cells or microbial agents to personalize immunotherapy or prophylaxis and for prognostic purposes, said method having the step of detecting the methylation of EPO and EPO receptors genes in tissues, cells, or human fluid (saliva, blood, cerebrospinal fluid, sweat, or derived-extracellular vesicles) as diagnostic or prognostic markers

[0070] In a further aspect the invention relates to an EPO / EPORs negative modulation agent delivery system administered orally, parenterally, intralesional (intratumoral, and intracavity) intraventricular, intrathecal, intranasal or local in various formulations at the time of clinical administration, based on innovative nanomedicine and nano-delivery systems as tissue gun or probe, viral and no-viral vectors, nanomaterials for the delivery of bioactive drugs for target delivery, plant-based vesicles, nanoparticle-based methods which allow both treatment and in vivo imaging modalities for diagnosis and therapy, lipid systems like liposomes and micelles, gold or magnetic nanoparticles, also in combination with natural product, functionalized nanoparticles, microspheres and biomaterials, as PEG, trojan horse approach, as micro pump to release treatment in tissues to enhance the modulating effects on immune system into the local pathological microenvironment or to attract and increase the homing of cell-based immunotherapies or vaccines.

[0071] In a further embodiment, the invention relates to a method based on EPO / EPORs negative modulation for reprogramming tumor associated immune cells to avoid immune system exhaustion and tolerance.

[0072] In a still further embodiment, the invention relates to a method based on EPO / EPORs negative modulation to improve CAR-T and CAR-Gamma / Delta T Cell Therapies Efficacy against Solid Tumors.

[0073] In a still further aspect the invention relates to an EPO / EPO-Rs and their variant inhibitors able to stimulate CTLs infiltration and suppressing the recruitment of immunosuppressive cells in tumor and in infectious diseases, increasing the tissue penetration of inflammatory and immune cells.

[0074] In a still further aspect relates to an EPO / EPO-Rs and their variants inhibitors able to induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferrooptosis, authophagy, cruproptosis and immuno-stimulated cell death enhancing tumor immunogenicity. A still further aspect describes a product selected among EPO and their natural and synthetic variant inhibitors, able to activate the immune response against cancer and infectious agent, reprogram tumor microenvironment and potentiate immunotherapy and immunomodulatory strategies.

[0075] BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Examples given for illustrative and non-limiting purposes, and from the annexed Figures 1 -19, wherein:

[0077] Figure 1. anti-EPO treatment increases migration of Peripheral Blood Mononucleated Cells (PBMC) in presence of GBM tissue. Figure 1 A schematic scheme of the chemotaxis assay, modified-Boyden chamber, used in migration test, in which is showed the GBM tissue on the bottom of the lower compartment. Migration test was performed on PBMC, stained with Hoechst (blue), after 48h in the following condition, GBM tissue in CTR medium (CTR, Figure 1 B), anti-EPO (C4) at 100p.g / mL (Figure 1 C), and recombinant human EPO (rhEPO, Figure 1 D). In Figure 1 E is stated the total number of migrated cells, counted using the Analyze Particle plugin in Imaged. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0078] Figure 2. anti-EPO treatment induces differentiation, activation of PBMCs and expression of Natural Killer markers. The immunophenotypic profile of PBMC was assessed by flowcytometric acquisition. In Figure 2A are reported the ratio of the marker expression in PBMCs after the following treatments: CTR, anti-EPO (C4), and rhEPO. Data are reported compared to CTR condition. In Figure 2B, the data represent the analysis of the used markers in PBMC treated with C4 compared to those treated with rhEPO. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05 versus CTR; **P<0.01 versus CTR for all treatments.

[0079] Figure 3. anti-EPO treatment induces PBMC migration EPOR-dependent.

[0080] Figure 3A illustrates the schematic drawing of the modified-Boyden chamber used in migration test, in which is showed the GBM tissue on the bottom of the lower compartment. The red arrow indicates the lower face in which photographs were captured.

[0081] Figure 3B shows representative photographs of the immunofluorescence analysis of CD8 (green), CD14 (red), and EPOR (gray). Nuclei were counterstained with Hoechst. In Figure 3C is reported total counts of all cells present in stained samples from bottom wells post 48h of trans-migration and specifically total counts for all CD8+ T cells (green bars) and CD14+ monocytes (red bars, Figure 3D), among which the specific sub-populations of double positive EPOR+CD8+ cells (green bars) and EPOR+CD14+ cells (red bars) were calculated (Figure 3E). n=10 Field of views (FOVs) acquired at 63x magnification, over a mean total of n=500 analysed cells / sample.

[0082] Figure 4. anti-EPO treatment induces molecular signature of PBMC activation and blocks exhaustion phenomenon. Gene expression of markers related to inflammation and exhaustion. The analysis was performed by the assessment of gene expression profile on human PBMC by RealTime PCR. The analysis was conducted on L-1 b (Figure 4A), IL-6 (Figure 4B), IFNg (Figure 4C), IL-10 (Figure 4D), TGFb (Figure 4E), IFNa2 (Figure 4F), PD1 (Figure 4G), LAG-3 (Figure 4H), CTLA4 (Figure 4I) genes. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05, versus CTR for all treatments.

[0083] Figure 5. anti-EPO treatment induces monocytes migration and their activation.

[0084] Monocytes were studied to assess migratory capacity followinganti-EPO (C4) treatment in modified Boyden chamber (Figure 5). Migration test was performed on cells stained with Calcein (green) after 6 days and analysis was performed counting the number of migrated cells on the bottom of the well after the following condition: CTR medium (CTR, Figure 5A), C4 at 10p.g / mL (Figure 5B), and recombinant human EPO (rhEPO, Figure 5C). In Figure 5D is reported the total number of migrated cells, counted using the Analyze Particle plugin in Imaged. Immunophenotypic profile was assessed on infiltrated macrophages monocytes derived evaluating the expression of CD86+ / HLA-DR+ following conditions: CTR, TMZ, C4, TMZ+C4, and rhEPO (Figure 5E), to evaluate differentiation towards M1 phenotype. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05, versus CTR for all treatments.

[0085] Figure 6. anti-EPO (treatment induces macrophages infiltration into GBM tissues and their differentiation versus M1 phenotype.

[0086] Immunophenotypic profile of infiltrated macrophages was evaluated through CD86 and HLA- DR expression in following conditions: CTR, TMZ, C4, TMZ+C4, and rhEPO (Figure 6A). Histograms in Figure 6B shows quantification of infiltrated macrophages in GBM tissue. The co-expression of CD86+ / HLA-DR+ (Figure 6C) and CD86+ (Figure 6D) were assessed on macrophages cells after treatments, demonstrating differentiation towards M1 phenotype. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0087] Figure 7. anti-EPO treatment induces differentiation of PBMC-derived monocytes into macrophages by EPOR surface expression. Cells were fixed and immune-labeled to detect (A, B) Phalloidin, as well as EPOR (C, D) in PBMC-derived macrophages. Figure E and DFshowed colocalization of phalloid with EPOR. Nuclei were stained with DAPI (blue) (scale = 100pm). Representative pictures are presented for C4 (Figure A,C,E) and rhEPO (B, D, F) administration.

[0088] Figure 8. anti-EPO administration induces migration of naive T cells

[0089] Naive T cells were analyzed to assess their migratory capacity following anti-EPO treatment. Modified Boyden chamber was used. Chemotaxis assay was performed on cells stained with Calcein (green) after 48h in the following condition: CTR medium (CTR, Figure 8A), C4 at 10pg / mL (Figure 8B), recombinant human EPO (rhEPO, Figure 8C), and C4+rhEPO (Figure 8D). In Figure 8E is reported the total number of migrated cells, counted using the Analyze Particle plugin in Imaged. Migration of CD8+ was performed also in presence of several negative functional modulators of EPO (Figure 8F). For this purpose, we tested commercial antibody B4 (Santa Cruz Biotechnology), and 16FH11 (StemCell Techologies). Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0090] Figure 9. anti-EPO treatment induces T cells activation by EPOR expression in GBM TME.

[0091] Naive CD4+ T cells were cultured with GSC conditioned media treated with the following conditions: CTR (Figure 9A), Temozolomide (TMZ, Figure 9B), rhEPO (Figure 9C), anti-EPO (C4) (Figure 9D), TMZ+C4 (Figure 9E), and rhEPO+C4 (Figure 9F). The immunophenotypic profile was assessed by evaluating the expression of EPOR and CD69+ with the following conditions: CTR, TMZ, C4, TMZ+C4, rhEPO, and rhEPO+C4 (Figure 9G). Data are the mean ± SD of at least 3 experiments in triplicate. **P<0.01 , ***P<0.001 versus CTR for all treatments.

[0092] Figure 10. anti-EPO treatment induces CTL lymphocytes CD4+ migration in TME

[0093] Cytotoxic CD4+ T cells were studied to assess migratory capacity. Modified Boyden chamber was used (Figure 10A). Migration test was performed on cells stained with Calcein (green) after 48h and analysis was performed counting the number of migrated cells on the bottom of the well (Figure 10B) after the treatments with conditioned media collected from GSC treated in the following condition: CTR medium (CTR, Figure 10C), TMZ at 100p (Figure 10D), C4 at 1 Opg / mL (Figure 10E), TMZ+C4 (Figure 10F), and recombinant human EPO (rhEPO, Figure 10G). In Figure 10B is reported the total number of migrated cells, counted using the Analyze Particle plugin in Imaged. Data are the mean ± SD of at least 3 experiments in triplicate. **P<0.01 , ***P<0.001 ; versus CTR for all treatments. Figure 11. anti-EPO treatment induces CTL Lymphocytes deeply penetration into GBM tissue

[0094] CTL T lymphocytes were studied to assess anti-EPO (C4) migratory capacity in presence of GBM tissue. Modified Boyden chamber was used. On the bottom of the well was positioned a chunk of GBM tissue (Figure 1 1 A). Migration test was performed on cells stained with Calcein (green) after 48h and analysis was performed counting the number of migrated cells on the bottom of the well (Figure 11 B) after the treatments with the following condition: CTR medium (CTR, Figure 11 C), TMZ at 10Op. (Figure 11 D), C4 at 10pg / mL (Figure 1 1 E), TMZ+C4 (Figure 11 F), recombinant human EPO (rhEPO, Figure 11 G), and rhEPO+C4 (Figure 1 1 H). In Figure 11 B is reported the total number of migrated cells, counted on the bottom of the well, using the Analyze Particle plugin in Imaged. Data are the mean ± SD of at least 3 experiments in triplicate. **P<0.01 , ***P<0.001 versus CTR for all treatments, and # P<0.05 versus rhEPO.

[0095] Figure 12. anti-EPO treatment induces CTL Lymphocytes migration into GBM tissue

[0096] Migration assay was performed to assess infiltrative ability of CD4+ CTL T cells into GBM tissue. Cells were colored by calcein and migrated for 48h in modified Boyden chamber (Figure 12A), Afterwards, GBM tissue were collected, digested by trypsinization and filtrated. Single cell suspension was analyzed by flow cytometer and infiltrated cells were counted, as number of events. In Figure 12B is reported the total number of infiltrated cells. Data are the mean ± SD of at least 3 experiments in triplicate. ***P<0.01 ; versus CTR for all treatments.

[0097] Figure 13. EPO related target expression on GBM cells

[0098] Expression patterns of EPO-related, transferrin-related, IL-13-related genes by Real-time PCR in CTR and GBM mRNA extracted from cells. A) EGFR, EPOR, EPHB4, CSF2RB, CRLF3 expression; B) IL-13, IL-13R1 , IL-13R2 expression; c) transferrin receptor 1 (TfR1 ), transferrin receptor 2 (TfR2), and Folate receptor expression in control and GBM. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05, versus CTR for all treatments.

[0099] Figure 14. CAR-T cytotoxic effect is potentiated by anti-EPO treatment against GBM cells To test cytotoxic activity of T-cells, WT T cell, CAR-T cells alone or in combination with C4 were co-cultured for 48h with glioblastoma stem cells, Cytotoxic effect was measured co-culturing immune T cells with GSCs at effector / target (E:T) ratios of 1 :1 , 1 :2, 1 :4, 1 :8, 1 :16, 1 :32, and 1 :64 (Figure 14A). Cytokine quantification of TNFa (Figure 14B), and IFNy (Figure 14C) in supernatants was obtained from 48hr co-cultures of CAR-T cells and GSCs. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments. Figure 15. CAR-T cytotoxic effect is potentiated by anti-EPO treatment on breast, pancreatic and melanoma cancer cells

[0100] To test cytotoxic activity of T-cells, WT cells, CAR-T cells alone or in combination with C4 were co-cultured for 48h with breast (MCF7, Figure 15A), melanoma (Figure 15B) and colon cancer cells (DLD1 , Figure 15C). Cytotoxic effect was measured co-culturing immune T cells with breast, melanoma, and colon cancer cells at effector / target (E:T) ratios of 1 :1 , 1 :2, 1 :4, 1 :8, 1 :16, 1 :32, and 1 :64 (Figure 15A-C). Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0101] Figure 16. CAR-T cytotoxic effect is potentiated by anti-EPO and metformin co-treatment on glioblastoma cancer cells in a synergistic fashion.

[0102] The combined treatment of anti-EPO(C4) and cytotoxic activity of T-cells was tested also in coadministration of metformin. WT cells, CAR-T cells alone or in combination with C4 and / or metformin were co-cultured for 48h with GSCs (Figure 16A). Cytotoxic effect was measured co-culturing immune T cells with glioblastoma cancer cells at effector / target (E:T) ratios of 1 :1 , 1 :2, 1 :4, 1 :8, 1 :16, and 1 :32 (Figure 16A). IL-i p (Figure 16B), IFNy (Figure 16C), and PD-1 (Figure 16D) gene expression was assessed after co-administration of anti-EPO (C4) with metformin (MET), Figure 16B. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0103] Figure 17 anti-EPO treatment stimulates Immune cells infiltration into GBM subcutaneous PDX mouse model as example of solid tumor penetration and efficacy. Representative pictures of intratumoral inflammatory cell infiltration by hematoxylin and eosin staining. Low density of inflammatory cell infiltration in PDX tumor treated with placebo (a: x 100; b x 400, Figure 17A,B). High density of inflammatory cell infiltration in the PDX tumor treated with anti-EPO antibody at 10mg / Kg (c: x 100, d: x 400, Figure 17C,D).

[0104] Figure 18. anti-EPO treatments decreases parasitemia in blood cells

[0105] Parasitemia curves of infected erythrocytes after exposure of placebo (WT) or anti-EPO antibody. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0106] Figure 19. anti-EPO treatment increases migration of PBMC in infection diseases. Figure 19A schematic scheme of the chemotaxis assay, modified-Boyden chamber, used in migration test, in which is showed the migratory capacity of PBMC after anti-EPO (C4) in presence of Lipopolysaccharide (LPS). LPS was administered at 10ug / mL for 48h (Figure 19B), as well as anti-EPO (C4) (Figure 19c) and rhEPO (Figure 19D). PBMC migration was evaluated by modified Boyden Chamber and the number of migrating cells was counted using the Analyze Particle plugin in Imaged Figure 19E). Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05; versus CTR for all treatments.

[0107] Figure 20. In an in vivo orthotopic rodent brain cancer model, compared to the healthy contralateral hemisphere (Figure 20A), the administration of anti-EPO significantly induced the activation of immune system with migration of T lymphocytes (Figure 20B). Surprisingly, the treatment with anti-EPO induced ferroptosis in cancer cells, as markers of cell death (Figure 20C).

[0108] DETAILED DESCRIPTION OF THE INVENTION

[0109] The invention herein provides a method based on the negative modulation of EPO and EPO receptors and their natural splicing and synthetic variants to modulate immune system and uses thereof. Pharmaceutical compositions as well as methods of treatment are also provided. In another form the invention herein provides a method based on the negative modulation of EPO canonical receptor and alternative receptors (EPO-R; EPBH4; CSF2RB; CD131 ; CRLF3, EPO-R soluble, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) or their splicing variants or the somatic mutations or their variants, to modulate immune system uses thereof.

[0110] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized hybridoma methodologies and phage display techniques.

[0111] The present invention describes a method to activate immune system against tumor, infectious agent alone or in combination with standard therapy.

[0112] In a first aspect, the invention relates to an anti-EPO negative functional modulator or anti-EPO antigen-binding fragment chosen from the group comprising Fab, -F(ab’)2, single chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies, for use in a method of activating an immune response of a patient in need thereof.

[0113] In a further aspect the invention relates to a method for activating or boosting the immune response of a patient in need thereof, said method comprising the use of an anti-EPO negative functional modulator or anti-EPO antigen-binding fragment alone or in combination with:

[0114] - a check point inhibitor or immunomudulator (e.g. anti-PDL1 antibody; nivolumab; ipilumab, abetacept, glembatumumab vedotin) therapy; and / or - a cell-based immunotherapy (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic activated cells against tumor associated antigens, and / or antigen-presenting cells, tumor associated peptide, engineered monocyte-macrophage or polymorphonucleate cell-based therapies and / or a therapy to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs;

[0115] - an anti-microbial therapy (e.g. antibiotics; antivirals, antimycotics, antifungi, anti-prions)

[0116] - a flavonoid molecule;

[0117] - metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonist,

[0118] - a prophylactic or therapeutic DNA and / or RNA and or peptide or carbohydrate or lipid based vaccine (e.g. anti-HPV, anti-EBV or anti-HIV vaccine);

[0119] - Oncolytic virus based immunotherapy;

[0120] - a chemotherapeutic agent;

[0121] - an anti-cancer drug;

[0122] - an enzyme that degrades heparin sulfate proteoglycans (e.g. heparanase);

[0123] - a negative functional modulator of the sphingosine-1 -phosphate (S1 P) signaling pathway, or

[0124] - EPO mimetics that preserve the erythropoietic function; wherein said anti-EPO antigen-binding fragment is chosen from the group comprising Fab, - F(ab’)2, single chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies and said anti-EPO negative functional modulator is chosen from the group consisting of mono- or multi-specific antibody anti-EPO, gene therapy, DNA decoy, an RNA decoy, a ribozyme, an antagomiR, a shRNA, an LNA, a siRNA, an antisense oligonucleotide or an anti-Epo receptor, said anti-Epo receptor chosen from the group consisting of EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs and EPOR / CD131 heterodimer.

[0125] In a preferred aspect, in the anti-EPO negative functional modulator or anti-EPO antigenbinding fragment for use according to the invention, the anti-EPO negative functional modulator or anti-EPO antigen-binding fragment bind to EPO, EPO receptors chosen from the group consisting of EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, EPOR / CD131 heterodimer and their variants or the product of their somatic mutations.

[0126] Preferably, said modulator or anti-EPO antigen-binding fragment is an immunotherapy adjuvant and / or elicits an immune cell response (T helper and / or T Cytotoxic and / or B and / or NK Lymphocyte and / or Macrophages and / or Neutrophils and / or Dendritic cell activation antigen presenting cells), wherein said cell response is an anti-tumoral T or anti-microbial cell response of a patient affected with a cancer or infectious disease. In a further preferred aspect, in the use of the negative functional modulator or anti-EPO antigen-binding fragment or in the method for activating or boosting the immune response of a patient in need thereof according to invention, said anti-tumoral T helper and / or T Cytotoxic and / or B and / or NK Lymphocytes and / or Macrophages and / or Neutrophils and / or Dendritic cell are PBMC derived immune cells.

[0127] Preferably, in the use of the negative functional modulator or anti-EPO antigen-binding fragment or in the method for activating or boosting the immune response of a patient in need thereof, said anti-tumoral T cell response is a CD8+ and CD4+ T cell response.

[0128] Preferably, in the use of the negative functional modulator or anti-EPO antigen-binding fragment or in the method for activating or boosting the immune response of a patient in need thereof, said anti-tumoral T cell response is due to CD14, CD69, and EPOR expression in migrated PBMCs.

[0129] In a preferred aspect, in the use of the negative functional modulator or anti-EPO antigenbinding fragment or in the method for activating or boosting the immune response of a patient in need thereof, said anti-EPO antigen-binding fragment is a neutralizing antibody which binds to EPO, EPO variants or EPO receptors and restores T helper and / or T Cytotoxic and / or B and / or NK Lymphocyte and / or Macrophages and / or Neutrophils and / or Dendritic cell activation. In a still preferred aspect, in the use of the negative functional modulator or anti-EPO antigenbinding fragment or in the method for activating or boosting the immune response of a patient in need thereof, said anti-EPO antigen-binding fragment is a neutralizing antibody is chosen from the group consisting of C4, B4 and 16F1 H1 1 .

[0130] An example described in the present invention concerns a purified anti-Erythropoietin (EPO) antibody, also identified as “C4 antibody” or “C4”, wherein said antibody comprises: a. a variable domain of a light chain (VL) having the amino acid sequence of SEQ ID NO:6; and b. a variable domain of a heavy chain (VH) having the amino acid sequence of SEQ ID NO:14. The hybridoma which produces the C4 antibody according to the present invention, said antibody comprising a variable domain of a light chain (VL) having the amino acid sequence of SEQ ID NO:6; and a variable domain of a heavy chain (VH) having the amino acid sequence of SEQ ID NO:14, was deposited at the Leibniz-lnstitute DSMZ with the accession number DSM ACC 3370 on 09.09.2021 .

[0131] In a preferred aspect, the antibody of the present invention is an isolated anti-EPO antibody, wherein said antibody comprises 6 CDR regions, said CDR regions being: a. a VL-CDR1 having the amino acid sequence of SEQ ID NO:4; b. a VL-CDR2 having the amino acid sequence of GAS (Gly-Ala-Ser); c. a VL-CDR3 having the amino acid sequence of SEQ ID NO:5 d. a VH-CDR1 having the amino acid sequence of SEQ ID NO:11 ; e. a VH-CDR2 having the amino acid sequence of SEQ ID NO:12; and f. a VH-CDR3 having the amino acid sequence of SEQ ID NO:13.

[0132] For the purposes of the present disclosure, each sequence has a corresponding SEQ ID NO. as follows:

[0133] SEQ ID NO:1 corresponds to the DNA sequence of the CDR1 region of the variable light chain of the anti-EPO antibody (VL-CDR1 ):

[0134] GAAAGTGTTGACTATTATGGCACAGGTTTA

[0135] GGTGCATCC corresponds to the DNA sequence of the CDR2 region of the variable light chain of the anti-EPO antibody (VL-CDR2)

[0136] SEQ ID NO:2 corresponds to the DNA sequence of the CDR3 region of the variable light chain of the anti-EPO antibody (VL-CDR3):

[0137] CAGCAAACTAGGAAGGTTCCTTCGACG

[0138] SEQ ID NO:3 variable light chain DNA sequence (333bp, CDRs in bold: FR1 -CDR1 -FR2-

[0139] CDR2-FR3-CDR3-FR4):

[0140] GATATCGTTCTCACTCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACC

[0141] ATCTCCTGCAGAGCCAGTGAAAGTGTTGACTATTATGGCACAGGTTTAATGCAGTGGTA

[0142] CCAACAGAGACCAGGACAGCCACCCAAACTCCTCATCTATGGTGCATCCAACGTAGGAT

[0143] CTGGGGTCCCTGCCAGGTTTAGCGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACAT

[0144] CCATCCTGTGGAGGGGGATGATATTGCAATGTATTTCTGTCAGCAAACTAGGAAGGTTC

[0145] CTTCGACGTTCGGTGGAGGCACCAAGTTGGAAATCAAA

[0146] SEQ ID NO:4 corresponds to the amino acid sequence of the CDR1 region of the variable light chain of the anti-EPO antibody (VL-CDR1 ): ESVDYYGTGL

[0147] GAS (Gly-Ala-Ser) corresponds to the amino acid sequence of the CDR2 region of the variable light chain of the anti-EPO antibody (VL-CDR2)

[0148] SEQ ID NO:5 corresponds to the amino acid sequence of the CDR3 region of the variable light chain of the anti-EPO antibody (VL-CDR3): QQTRKVPST

[0149] SEQ ID NO: 6 variable light chain amino acid sequence (1 11 aa, CDRs in yellow: FR1 -CDR1 - FR2-CDR2-FR3-CDR3-FR4): DIVLTQSPASLAVSLGQRATISCRASESVDYYGTGLMQWYQQRPGQPPKLLIYGASNVGSG VPARFSGSGSGTDFSLNIHPVEGDDIAMYFCQQTRKVPSTFGGGTKLEIK

[0150] SEQ ID N0:7 corresponds to the DNA sequence of the CDR1 region of the variable heavy chain of the anti-EPO antibody (VH-CDR1 ):

[0151] GGATTCACTTTCAGTACCTATACC

[0152] SEQ ID NO:8 corresponds to the DNA sequence of the CDR2 region of the variable heavy chain of the anti-EPO antibody (VH-CDR2):

[0153] ATTAGTAATGGTGGTGATAGAACC

[0154] SEQ ID NO:9 corresponds to the DNA sequence of the CDR3 region of the variable heavy chain of the anti-EPO antibody (VH-CDR3):

[0155] GCAAGACATAATATTACTACGGTTCCCTTTACTATGGACTAC

[0156] SEQ ID NQ:10 variable heavy chain DNA sequence (363bp, CDRs in bold: FR1 -CDR1-FR2- CDR2-FR3-CDR3-FR4):

[0157] GAGGTGAAGCTGCAGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTGAAAC TCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATACCATGTCTTGGGTTCGCCAG ACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGATAGAACCT ACTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACGATGCCAAGAACACC

[0158] CTGTTCCTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAG

[0159] ACATAATATTACTACGGTTCCCTTTACTATGGACTACTGGGGTCAAGGAACCTCAGTCA

[0160] CCGTCTCCTCA

[0161] SEQ ID NO:11 corresponds to the amino acid sequence of the CDR1 region of the variable heavy chain of the anti-EPO antibody (VH-CDR1): GFTFSTYT

[0162] SEQ ID NO:12 corresponds to the amino acid sequence of the CDR2 region of the variable heavy chain of the anti-EPO antibody (VH-CDR2): ISNGGDRT

[0163] SEQ ID NO:13 corresponds to the amino acid sequence of the CDR3 region of the variable heavy chain of the anti-EPO antibody (VH-CDR3): ARHNITTVPFTMDY

[0164] SEQ ID NO:14 variable heavy chain amino acid sequence (VH) (121 aa, CDRs in bold: FR1 - CDR1-FR2-CDR2-FR3-CDR3-FR4):

[0165] EVKLQESGGGLVQPGGSLKLSCAASGFTFSTYTMSWVRQTPEKRLEWVAYISNGGDRTYY PDTVKGRFTISRDDAKNTLFLQMSSLKSEDTAMYYCARHNITTVPFTMDYWGQGTSVTVSS SEQ ID NO:15: EPO amino acid sequence (N-terminal signal peptide + protein chain) aa 1 - 193 MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLN ENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVD KAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEA CRTGDR

[0166] SEQ ID N0:16 EPO mature peptide amino acid sequence (aa 28-193)

[0167] APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVE VWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDA ASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

[0168] SEQ ID NO: 17 EPO gene sequence.

[0169] For the purposes of the present invention, the phrase “negative functional modulator of EPO”, “EPO / EPO-Rs negative modulation agent” or “negative functional modulator anti-EPO” intends to relate to an agent that inhibits the function of EPO.

[0170] For the purposes of the present invention, the “antibody” or “monoclonal antibody” is a “negative functional modulator of EPO” against human EPO. In particular, the antibody is against the mature form of EPO which corresponds to amino acids (AA) 28-193 of the whole 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. The anti-EPO antibody is a molecule capable of recognizing and binding an amino acid sequence included in Erythropoietin, capable of direct or indirect interaction with EPO, and / or direct or indirect interaction with the biosynthetic pathway of EPO, wherein said interactions have resulted in a decrease in the levels of EPO, rather than a decrease in the stimulation of the signal transduction cascade in which EPO is involved. In a further embodiment, said negative functional modulators of EPO act on EPO which has undergone post-translational modifications, such as EV-3.

[0171] In a more preferred aspect, the isolated anti-EPO antibody of the invention is a monoclonal antibody, a chimeric antibody and / or is humanized or human, is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or (Fab')2fragment and more preferably further comprises a framework sequence and at least a portion of the framework sequence is a human consensus framework sequence.

[0172] The main objective of humanization process is to reduce antibodies immunogenicity in order to improve tolerance in humans and improve their biophysical properties. Briefly, variable regions sequences information is generated by Reverse Transcription of total RNA extraction obtained from hybridoma cell line. Variable regions of the heavy (VH) and light chains (VL) are amplified by PCR and cloned into shuttle vector for sequencing. A total of 5 independent clones are sequenced for each variable chain. Sequences of the hybridoma are determined from the sequencing results of the VH and VL. A chimeric construct is designed and expressed by combination of mouse VH and VL variable regions with human IgG 1 constant regions in order to confirm affinity / binding and biological function related to the parental mouse hybridoma. Antibody sequences are humanized by grafting the three CDRs from the light chain variable region (VL) into human VL germlines which are as homologous as possible to the mouse antibody VL. Similarly, the three CDRs from the heavy chain variable region (VH) are grafted into human VH germlines which are as homologous as possible to the mouse antibody VH. In addition, because different framework context might bring added value to the resulting antibody, CDRs are grafted into human VH and VL germlines which are well-known to exhibit good overall biophysical properties even if they are less homologous. A total of 9-18 VH / VL combinations are generated between the CDR-grafted VH, the CDR-grafted VL, and the chimeric versions of both VH and VL. Proof of concept quantities of each recombinant humanized antibody are transiently produced through XtenCHOTM platform and evaluated for binding / biological activity / biophysical properties compared to the chimeric version of parental mouse hybridoma. Comprehensive antibody affinity maturation services can be done via phage display using custom libraries generated by random or target mutagenesis. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In addition, antibodies may be prepared by different techniques. For example, monoclonal antibodies may be purified from cells that naturally express them, such as hybridoma cells, or produced in recombinant expression system both from mammalian system or prokaryotes (e.g. Escherichia Coli). More recently, fragment antibodies have been introduced in clinical practice. Indeed, fragment antibodies are emerging as great tools in imaging and diagnostics because they are capable of detecting cellular proteins with high affinity and specificity. Antibody fragments include, but not limited to: Fab, F(ab’)2, single chain antibodies, nanobodies, diabodies, triabodies, tetrabodies, and domain antibodies. They can be easily linked to radioisotopes, fluorescent molecules or enzymes that tag specific biomarkers in patients. They also have a shorter half-life in the body which results in faster clearance and may result in fewer risks of side effects from potentially invasive diagnostic agents. Where desired the affinity of the monoclonal antibody or fragment antibody according to the invention, containing one or more of CDRs above-mentioned, can be improved by affinity maturation procedures.

[0173] Preferably the antibody herein described is a full-length monoclonal antibody and / or can be a bispecific anti-EPO antibody. The anti-EPO antibody has an amino acid sequence identical to or comprising 0, 1 , 2, or 3 amino acid residue substitutions relative to the VL of SEQ ID NO:6 and to the VH of SEQ ID NO:14.

[0174] The advantageous properties of the EPO negative modulation of the present invention will be apparent in the experimental section.

[0175] In a still preferred aspect, in the use of the negative functional modulator or anti-EPO antigenbinding fragment or in the method for activating or boosting the immune response of a patient in need thereof, the negative functional modulator or anti-EPO antigen-binding fragment elicits a T helper and / or T Cytotoxic and / or B and / or NK Lymphocyte and / or Macrophages and / or Neutrophils and / or Dendritic cell, and / or antigen-presenting cells, said cell response is further enhanced by a CAR-T, CAR-M, CAR-NK, engineered monocyte-macrophage or polymorphonucleate cell based therapies or to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs.

[0176] In a still preferred aspect, in the use of the negative functional modulator or anti-EPO antigenbinding fragment or in the method for activating or boosting the immune response of a patient in need thereof, said patient in need thereof is suffering from cancer, proliferative pathologies, chronic inflammatory diseases on an autoimmune and non-autoimmune basis, neurodegenerative diseases, Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1 , endometriosis, Crohn's disease, ulcerative colitis, neuro- inflammatory and infectious diseases or a patient undergoing organ or tissue transplantation, and wherein said cancer is selected from the group consisting of cerebral astrocytoma, cerebellar astrocytoma, astrocytoma of the pineal gland, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, glioblastoma multiforme, glioblastoma grade II fibrillary astrocytoma, protoplasmic, grade III gemistocytic, anaplastic astrocytoma, including gliomatosis cerebri, pituitary adenoma, ependymoma, medulloblastoma, neural ectoderm tumor, neuroblastoma, hypothalamic glioma, breast cancer, lung cancer, colon cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, esophageal cancer, basal cell carcinoma, cholangiocarcinoma, cancer of the spleen, osteosarcoma, intraocular melanoma, retinoblastoma, stomach cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, cancer of the oral cavity, nasal and paranasal cancer, cancer of the salivary glands, nasopharyngeal cancer, throat cancer, thyroid cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, gastric and hepatic carcinoma, colorectal cancer, rectal cancer, testicular cancer, renal cell cancer, melanoma, sarcoma, mesothelioma, pheochromocytoma, hematological cancers or chronic myeloid leukemia, diffuse midline glioma (DMG), diffuse intrinsic pontine glioma (DIPG), embryonal tumors, brain stem glioma, pineoblastoma, choriod plexus carcinoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, haemangioblastoma.

[0177] In a more preferred aspect, said cancer is glioblastoma multiforme.

[0178] In a further aspect said negative functional modulator or anti-EPO antigen-binding fragment promotes an immune system response in a patient affected by a refractory or persistent infectious diseases, wherein said persistent infectious diseases include tuberculosis, malaria, HIV, EBV, or HPV induced pre-cancerous alterations and their prevention.

[0179] Preferably the negative functional modulator or anti-EPO antigen-binding fragment promotes immune system response in a patient affected by streptococci, staphylococci, fungi, viruses; Sars-Cov2, SARS, MERS, prion agents resistant to antimicrobial therapies and / or in presence of immune system tolerance.

[0180] More preferably the negative functional modulator or anti-EPO antigen-binding fragment is for use in promoting immune system response and boosting the efficacy of prophylactic and therapeutic DNA and or RNA or peptides and or lipidic based vaccines in infectious diseases and / or cancer immunotherapy. In a second aspect, herein described is a pharmaceutical kit comprising a negative functional modulator of EPO / EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) and / or their natural or synthetic variants and:

[0181] - a check point inhibitor or immunomudulator (e.g. anti-PDL1 antibody; nivolumab; ipilumab, abetacept, glembatumumab vedotin) therapy; and / or

[0182] - a cell-based immunotherapy (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic activated cells against tumor associated antigens and / or antigen-presenting cells) engineered monocyte-macrophage or polymorphonucleate cell-based therapies and / or a therapy to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs; and one or more components selected from the group consisting of:

[0183] - a peptide or antibody, diabody, nanobody against the natural and synthetic variants including physiological and pathological splicing variants of erythropoietin and post- translational modification;

[0184] - an anti-microbial therapy (e.g. antibiotics; antivirals, antimycotics, antifungi, anti-prions)

[0185] - a flavonoid molecule;

[0186] - metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonist;

[0187] - a prophylactic or therapeutic DNA and / or RNA and or peptide or carbohydrate or lipid based vaccine (e.g. anti-HPV, anti-EBV or anti-HIV vaccine);

[0188] - Oncolytic virus based immunotherapy;

[0189] - a chemotherapeutic drug;

[0190] - an anti-cancer drug;

[0191] - an enzyme that degrades heparin sulfate proteoglycans (e.g. heparanase);

[0192] - a negative functional modulator of the sphingosine-1 -phosphate (S1 P) signaling pathway, or

[0193] - EPO mimetics that preserve the erythropoietic function.

[0194] In a third aspect, the present invention relates to a diagnostic or prognostic method for evaluating the expression of EPO and its somatic mutations or its variants, EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, EPOR / CD131 heterodimer) and somatic mutations and / or their variants, C4 mAb ligands that predict the response to target EPO / EPO-Rs negative modulation therapy to personalize therapy in cancer and infectious diseases immunotherapy or prophylaxis, to stratify patients, optimize patient’s response, said method having the step of measuring the amount of / detecting the presence of EPO and its somatic mutations or its variants, EPO receptors and its somatic mutations and their variants in tissues, cells, or human fluid (saliva, blood, cerebrospinal fluid, sweat, or derived- extracellular vesicles) as diagnostic or prognostic markers

[0195] In a fourth aspect, the present invention relates to a diagnostic method for evaluating the methylation of the promoter of the genes of EPO and EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) that can predict an increase expression of EPO / EPO-Rs, C4 ligands and their negative role in immune system inhibition in eradicating cancer cells or microbial agents to personalize immunotherapy or prophylaxis and for prognostic purposes, said method having the step of detecting the methylation of EPO and EPO receptors genes in tissues, cells, or human fluid (saliva, blood, cerebrospinal fluid, sweat, or derived-extracellular vesicles) as diagnostic or prognostic markers.

[0196] As immunotherapeutic or immunotherapy adjuvant protocol, according to the present invention, the compound represented by a negative modulator of EPO and its variants and or EPO receptors and their variants, as an example reported on formula 1 or the pharmaceutically acceptable salt thereof can be administered orally, parenterally, intralesional (intratumoral, and intracavity) intraventricular, intrathecal, intranasal or local in various formulations at the time of clinical administration. More preferably, they can be parenteral formulations. The method described in the present invention can also include the compound represented by formula 1 or the pharmaceutically acceptable salt thereof can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant. Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing the compound represented by formula 1 or the pharmaceutically acceptable salt thereof of the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, and emulsions. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. The cancer immunotherapy adjuvant described here can include the compound represented by formula 1 or the pharmaceutically acceptable salt thereof as an active ingredient can be administered by parenterally and the parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0197] To prepare the compound represented by formula 1 or the pharmaceutically acceptable salt thereof as a formulation for parenteral administration, the compound represented by formula 1 or the pharmaceutically acceptable salt thereof is mixed with a stabilizer or a buffering agent in water to produce solution or suspension, which is then formulated as ampoules or vials. The composition herein can be sterilized and additionally contains preservatives, stabilizers, wettable powders or emulsifiers, salts and / or buffers for the regulation of osmotic pressure, and other therapeutically useful materials, and the composition can be formulated by the conventional mixing, granulating or coating method.

[0198] The formulations for oral administration are exemplified by tablets, pills, hard / soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, and troches, etc. These formulations can include diluents (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine) and lubricants (for example, silica, talc, stearate and its magnesium or calcium salt, and / or polyethylene glycol) in addition to the active ingredient. Tablets can include binding agents such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrolidone, and if necessary, disintegrating agents such as starch, agarose, alginic acid or its sodium salt or azeotropic mixtures and / or absorbents, coloring agents, flavors, and sweeteners can be additionally included thereto.

[0199] The cancer immunotherapy adjuvant can enhance the efficacy of the cancer immunotherapy agent, and more specifically, it can enhance the efficacy of the cancer immunotherapy agent by activating immune factors to assist the anticancer activity of the cancer immunotherapy agent. The immune factor 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 Lymphocyte (TILs) and cytokines.

[0200] The cancer immunotherapy adjuvant can be administered simultaneously or sequentially with the cancer immunotherapy agent, and when administered sequentially, the cancer immunotherapy adjuvant can be administered after the cancer immunotherapy agent is administered, or the cancer immunotherapy adjuvant can be administered after the cancer immunotherapy agent is administered. However, the administration method is only an example, and the administration method may be changed to enhance the anticancer immune effect. In an embodiment of the present invention, the cancer immunotherapy adjuvant was administered by intravenous injection every day, and the cancer immunotherapy agent was administered by intraperitoneal injection 3 times a week, but not always limited thereto.

[0201] The cancer immunotherapy adjuvant can activate one or more immune factors selected from the group consisting of helper T cells, cytotoxic T cells, natural killer cells (NK cells) and cytokines. The cancer immunotherapy adjuvant exhibits the effect of enhancing the anticancer effect of the cancer immunotherapy agent by activating the immune factors.

[0202] At this time, the cancer immunotherapy adjuvant can prevent or treat cancer by being administered in combination with the cancer immunotherapy agent.

[0203] The cancer can be at least one selected from the group consisting of cancer is selected from the group consisting of brain cancer, metastatic brain cancer, brain stem glioma cerebral astrocytoma, cerebellar astrocytoma, astrocytoma of the pineal gland, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, uterine sarcoma, rhabdomyosarcoma, Kaposi's sarcoma, glioma, glioblastoma multiforme, glioblastoma grade II fibrillary astrocytoma, protoplasmic, grade III gemistocytic, anaplastic astrocytoma, including gliomatosis cerebri, ependymoma, medulloblastoma, neural ectoderm tumor, neuroblastoma, hypothalamic glioma, breast cancer, triple-negative breast cancer, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, colon cancer, colorectal cancer, ovarian cancer, ovarian epithelial cancer, gestational villous disease, cervical cancer, endometrial cancer, uterine cancer, ovarian germ cell cancer, esophageal cancer, basal cell carcinoma, cholangiocarcinoma, choroidal melanoma, choriod plexus carcinoma, cancer of the spleen, osteosarcoma, intraocular melanoma, malignant melanoma, retinoblastoma, stomach cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, cancer of the oral cavity, nasal and paranasal cavity cancer, cancer of the salivary glands, nasopharyngeal cancer, throat cancer, thyroid cancer, parathyroid cancer, thymus cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, gastric and hepatic carcinoma, gastric lymphoma, colorectal cancer, rectal cancer, rectal carcinoma, small intestine cancer, gastrointestinal stromal cancer, testicular cancer, renal cell cancer, adrenal cancer, renal pelvic cancer, malignant mesothelioma, mesothelioma, pheochromocytoma, hematological cancers or chronic myeloid leukemia lip cancer, tonsil cancer, squamous cell carcinoma, ampullar of vater 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, hduodenal cancer, malignant soft tissue cancer, malignant lymphoma, chronic myelogenous leukemia, gallbladder cancer, biliary tract cancer, chronic lymphocytic leukemia , malignant bone cancer, metastatic bone cancer, eye cancer, vulvar cancer, ureter cancer, mediastinal cancer, urethral cancer, cancer of unknown primary site, vaginal cancer, spinal cord cancer, vestibular schwannoma, diffuse midline glioma (DMG), diffuse intrinsic pontine glioma (DIPG), embryonal tumors, pineoblastoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, and haemangioblastoma, and proliferative pathologies, chronic inflammatory diseases on an autoimmune and non-autoimmune basis, neurodegenerative diseases, Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1 , endometriosis, Crohn's disease, ulcerative colitis, neuro-inflammatory and infectious diseases, mycosis fungoides, and infectious diseases as malaria, tuberculosis, HIV1 and 2, sickle cells, SARS, SARS-COV, MERS.

[0204] The cancer immunotherapy adjuvant can be co-administered together with the conventionally known and well-known cancer immunotherapy agent to those skilled in the art without limitation. For example, the cancer immunotherapy adjuvant can be administered in combination with one or more cancer immunotherapy agents selected from the group consisting of anti-PD1 , anti-PDL1 , anti-CTLA4, anti-LAG3, anti-VISTA, anti-BTLA, anti-TIM3, anti-HVEM, anti-CD27, anti-CD137, anti-OX40, anti-CD28, anti-PDL2, anti-GITR, anti-ICOS, anti-SIRPa, anti-ILT2, anti-ILT3, anti-ILT4, anti-ILT5, anti-EGFR, anti-CD19 and anti-TIGIT, but not always limited thereto.

[0205] In another aspect of the present invention, the present invention provides a combination drug for cancer immunotherapy.

[0206] Particularly, the present invention provides a combination drug for cancer immunotherapy comprising a cancer immunotherapy agent and a cancer immunotherapy adjuvant.

[0207] The specific description of the cancer immunotherapy agent, the cancer immunotherapy adjuvant and the combination drug is the same as the specific description of the cancer immunotherapy adjuvant. In another aspect of the present invention, the present invention provides a pharmaceutical composition for use in enhancing the efficacy of a cancer immunotherapy agent.

[0208] Particularly, the present invention provides a pharmaceutical composition for use in enhancing the efficacy of a cancer immunotherapy agent comprising a compound represented by sequence 1 , an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0209] In addition, the specific description of the pharmaceutical composition for use in enhancing the efficacy of a cancer immunotherapy agent is the same as the specific description of the cancer immunotherapy adjuvant.

[0210] In another aspect of the present invention, the present invention provides a pharmaceutical composition for use in enhancing immunity.

[0211] Particularly, the present invention provides a pharmaceutical composition for use in enhancing immunity comprising a compound represented by sequence 1 , an isomer thereof, a solvate thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. In addition, the specific description of the pharmaceutical composition for use in enhancing immunity is the same as the specific description of the cancer immunotherapy adjuvant.

[0212] In another aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising a step of administering a cancer immunotherapy agent and a cancer immunotherapy adjuvant to a subject in need thereof.

[0213] The cancer immunotherapy adjuvant and the cancer immunotherapy agent can be administered in combination or at different times.

[0214] In another aspect of the present invention, the present invention provides a use of a cancer immunotherapy adjuvant and an immunotherapy agent in the prevention or treatment of cancer.

[0215] In another aspect of the present invention, the present invention provides a combination therapy for the treatment of cancer comprising a step of administering a cancer immunotherapy adjuvant and a cancer immunotherapy agent to a subject in need thereof.

[0216] In another aspect of the present invention, the present invention provides a kit for preventing or treating cancer comprising a cancer immunotherapy agent and a cancer immunotherapy adjuvant as an active ingredient. Also, the present invention includes the use of negative modulators of EPO- EPO receptors pathways in the treatment of autoimmune pathology and infectious diseases where said diseases can be HIV, Tuberculosis, Malaria, streptococci, staphylococci, fungi, viruses like Sars-Cov2, MERS, prionic disease, parasites infections, in single administration or in combination with antimicrobial agents or vaccines and recombinant cytokines. Furthermore, the present invention describes a method for boosting the efficacy of prophylactic and therapeutic vaccines in infectious diseases and in cancer immunotherapy stimulating the immune system inflammatory response.

[0217] In a further aspect the invention relates to an EPO / EPO-Rs negative modulation agent delivery system administered orally, parenterally, intralesional (intratumoral, and intracavity) intraventricular, intrathecal, intranasal or local in various formulations at the time of clinical administration, based on innovative nanomedicine and nano-delivery systems as tissue gun or probe, viral and no-viral vectors, nanomaterials for the delivery of bioactive drugs for target delivery, plant-based vesicles, nanoparticle-based methods which allow both treatment and in vivo imaging modalities for diagnosis and therapy, lipid systems like liposomes and micelles, gold or magnetic nanoparticles, also in combination with natural product, functionalized nanoparticles, microspheres and biomaterials, as PEG, trojan horse approach, as micro pump to release treatment in tissues to enhance the modulating effects on immune system into the local pathological microenvironment or to attract and increase the homing of cell-based immunotherapies or vaccines.

[0218] In a further embodiment, the invention relates to a method based on EPO / EPO-Rs negative modulation for reprogramming tumor associated immune cells to avoid immune system exhaustion and tolerance.

[0219] In a still further embodiment, the invention relates to a method based on EPO / EPO-Rs negative modulation to improve CAR T and CAR-Gamma / Delta T Cell Therapies Efficacy against Solid Tumors.

[0220] In a still further aspect the invention relates to an EPO / EPO-Rs and their variant inhibitors able to stimulate CTLs infiltration and suppressing the recruitment of immunosuppressive cells in tumor and in infectious diseases, increasing the tissue penetration of inflammatory and immune cells.

[0221] In a still further aspect relates to an EPO / EPO-Rs and their variants inhibitors able to induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferrooptosis, authophagy, cruproptosis and immuno-stimulated cell death enhancing tumor immunogenicity. A still further aspect describes a product selected among EPO and their natural and synthetic variant inhibitors, able to activate the immune response against cancer and infectious agent, reprogram tumor microenvironment and potentiate immunotherapy and immunomodulatory strategies. Hereinafter, the present invention will be described in detail by the following examples and experimental examples.

[0222] EXAMPLES

[0223] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.

[0224] Example 1. anti-EPO) increases migration of Peripheral Blood Mononucleated Cells (PBMC) in presence of GBM tissue

[0225] Figures 1 shows the effects of treatment with anti-EPO antibody and rhEPO on PBMC migration added on trasnwell membrane with GBM chunk in bottom chamber (Figure 1 A). On the day of the surgery, after excision, GBM tissues were cut in sterile condition and placed on the bottom of 24 well plate with the following treatments: CTR (Figure 1 B), anti-EPO antibody at 10p.g / mL (Figure 1 C) and rhEPO at 100 ng / mL (Figure 1 D). Migration capacity of PBMCs was evaluated at TO, T24, and T48h. 2x10A4 PBMC (for each well) were resuspended in cell medium for 10OuL for each well and PBMC suspension was placed in the top chamber. In the bottom chamber, of transwell plate received treatment, C4 or rhEPO, in 500 pL of medium with GBM tissue. After 48h of incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and PBMCs were coloured with Hoechst (1 :1000 in PBS for 15 mins at 37°C), and the number of PBMC on the bottom chamber (Figure 1 E) were counted under the microscope and analyzed by Imagej with analyze particle plug in. Immunolabeling acquisition was performed by Nikon Crest (NikonTi+Andor Du888+ Zyla 4.6 + 16-led CoolLed + 4 lasercubes LDI + Crest Optics Spinnig Disk and VCS-sim XlightV2 / VCS) with Nis-Elements V.5.3.2 software module for acquisition and GA3 module for analysis.

[0226] The analysis revealed that in presence of anti-EPO antibody a significant higher number of migrated PBMCs was recorded. (Figure 1 E). Interestingly, the analysis showed an inhibitory effect of rhEPO on PBMC migration.

[0227] Example 2. anti-EPO induces differentiation, activation of PBMC and expression of Natural Killer markers.

[0228] Flow Cytometry analysis was performed on PBMCs following 48h of migration to assess CD56, CD86, and CD69 expression. To perform the analysis, first an indirect conjugation with primary and secondary antibodies was performed, and then a mix of conjugated antibodies was added. In detail, EPOR as AB-I was added to PBMCs and incubated in blocking solution with PBS + 5% BSA+2% donkey serum for 1 h at RT. AB-I were removed and, after three washes with PBS + 0.1 % BSA, AB-II was added for 1 h at RT. Then PBMCs were washed with PBS and the following mix with conjugated antibody: CD56-APC, CD86-PE-Cy7, and CD69-APC-Cy7 was administered. The analysis was conducted using FACS Canto II flow cytometer and FACSDiva software (BD Bioscience, version 5.0). Forward- versus side-scatter (FSC-A vs. SSC-A) gating was used to identify intact PBMC based on size and granularity in CTR. Results on Figure 2 demonstrated that anti-EPO administration induced a significant over-expression of CD56, CD86, CD69 and EPOR compared to CTR condition, PBMCs without any treatments (Figure 2A). Surprisingly, when the analysis was performed compared to rhEPO, anti-EPO administration induced a potent expression of CD56, CD86, CD69 and EPOR in migrated PBMCs (Figure 2B).

[0229] Example 3. anti-EPO treatment induces PBMC migration EPOR-dependent

[0230] PBMCs collected from n=3 GBM patients were used for migration analysis in Boyden Chamber for 48h (Figure 3A). 2x10A4 PBMC (for each well) were resuspended in cell medium for 100uL for each well and PBMC suspension was placed in the top chamber. Bottom chamber of Transwell plate received treatment, anti-EPO or rhEPO, in 500 pL of medium with GBM tissue. After 48h of incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and the insert was fixed with PFA 4% for 10 mins in ice. Following, the insert was removed and flipped on a glass slide to perform immunolabeling for CD14, CD8, and EPOR targets. PBMCs were blocked in PBS + 5% BSA+2% donkey serum for 30 min at RT. Incubation with primary antibodies (AB-I) diluted in blocking buffer was performed overnight at 4 °C. The following AB- I were used: anti-CD14 (ThermoFisher Scientific), anti-CD8 (Abeam, Cambridge, UK), anti- EPOR (SantaCruz Technology). The following day, the AB-I were removed and, after three washes with PBS + 0.1% BSA, AB-II was added for 1 h at RT. PBMCs were treated with PBS + 0.5% Triton X-100 to permeabilize the cell membranes and incubated directly with DAPI. The Immunolabeling was acquired using a high-resolution SP5 confocal microscope. Interestingly, CD8+ cells presented a larger and rounded morphology respect to both CTR and rhEPO treatments (Figure 3B).

[0231] In addition, the analysis revealed that anti-EPO treatment induced a significant migration for both CD14+ and CD8+ PBMC-derived cells compared to CTR and rhEPO administration (Figure 3C). Surprisingly, photomicrographs, and relative quantification (Figure 3D-E) revealed that migrating CD8+ and CD14+ cells showed a higher expression of EPOR when anti-EPO was added in the medium. These data demonstrate that anti-EPO antibody is a potent immunomodulatory stimulus triggering effects on GBM patient-PBMCs. Surprisingly, stimulation effects are mediated by an over-expression of EPOR on migrated PBMCs. Example 4. anti-EPO induces molecular signatures of activation and blocks exhaustion phenomenon.

[0232] Gene expression analysis was performed on PBMCs cultured for 48h in CTR, anti-EPO, and rhEPO condition by RealTime PCR. PBMC were collected as describe above and were assessed for migration by Boyden Chamber assay. Following 48h of migration, PBMC were collected from the bottom of the well, centrifuge 300g x 10 mins and the pellet was resuspended in Tri-Reagent for RNA extraction, following manufacture’s’ instruction. RNA was quantified with NanoDrop 1000 Spectro-photometer (Thermo Fisher Scientific). Reverse transcriptase reaction was executed using TranScriba Kit (A&A Biotechnology), loading 1 pg of RNA (A260 / A280 > 1.8), according to manufacturer’s instructions. qRT-PCR was performed using StepOnePlus™ (Thermo Fisher Scientific), 1 pg of cDNA, forward and reverse primers (250 nM each) Titan HotTaq EvaGreen® qPCR Mix (Bioatlas). Data were normalized to TBP expression, used as endogenous control. Relative gene expression was determined using the 2-AACt method (Figure 4). Results demonstrated that the treatment with anti-EPO induced PBMC activation and importantly, blocked exhaustion phenomenon, modulating the expression of IL-1 b (Figure 4A), IL-6 (Figure 4B), IFNg (Figure 4C), IL-10 (Figure 4D), TGFb (Figure 4E), IFNa2 (Figure 4F), PD1 (Figure 4G), LAG-3 (Figure 4H), CTLA4 (Figure 4I) genes. Data are the mean ± SD of at least 3 experiments in triplicate. *P<0.05, versus CTR for all treatments.

[0233] Example 5. anti-EPO induces monocytes migration and their activation

[0234] Monocytes migration was evaluated using a 24-well, Transwell plate (8.0pm pore size; Corning, Corning, NY). In brief, monocytes were washed once with RPMI1640 medium, cell count readjusted (5x10A5 cells / mL) in RPM 11640+10%FBS. Monocytes were colored with Calcein (1 :1000 in RPMI medium for 15 mins), washed with PBS and an aliquot (100 pL) of cell suspension was placed in the top chamber. Bottom chamber of Transwell plate received specific treatment in 500pL of medium. After 48h of incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and the number of cells on the bottom of the chamber were counted under the microscope and analyzed by Imagej with analyze particle plug in in the following condition: CTR (Figure 5A), anti-EPO (Figure 5B), rhEPO (Figure 5C). Interestingly, after 6days of migration, anti-EPO treatment increased monocytes migration compared to CTR condition. Remarkably, migration was decrease in presence of rhEPO and above all rhEPO (Figure 5D). Surprisingly, phenotypic analysis on migrated monocytes revealed that C4 treatment can significantly induce CD86 and HLA-DR over-expression (Figure 5E). When monocytes migration was performed in presence of GBM fragment on the bottom of the transwell, after 6 days of experimental conditions, tissue was collected from the bottom of the well, digested with Trypsin 0,25% for 30mins, filtered by 70p.m pore-size mash and analyzed by flowcytometer to count green positive infiltrated cells. Results demonstrated that the administration of anti-EPO antibody significantly increased the number of infiltrated macrophage-derived monocytes (Figure 6A,B). Fascinatingly, the immunophenotypic profile of infiltrated macrophage reveled a significant higher expression for CD86+ / HLA-DR+, indicating a M1 macrophage polarization (Figure 6C), and remarkably, infiltrated macrophages overexpressed EPOR (Figure 6D).

[0235] Intriguingly, immunofluorescence analysis performed on infiltrated macrophages, showed a higher expression for EPOR, as highlighted by cells colored by red (Figure 7A-F).

[0236] All in all, these data illustrated that anti-EPO shows a potent chemotactic ability to induce monocytes migration. Surprisingly, anti-EPO antibody can increase macrophage-derived monocytes infiltration into tumoral tissue, and importantly, polarized macrophages towards a M1 phenotype. Intriguing, this mechanism is mediated by EPOR expression on macrophages. Example 6. anti-EPO is a potent migratory stimulus for T cells

[0237] The migration testing or "chemotaxis assay" was carried out to highlight the migratory capacity of naive T cells, a phenomenon that is observed in response to an immune response. For this purpose, transwell multiwells were used and equipped with inserts with a polycarbonate membrane. The holes in the membrane of a diameter of 8pM can retain the cells and the culture medium but allow the active transmigration of cells through the membrane to reach the lower well. 2x10A4 naive CD4+ T cells (for each well) were coloured with Calcein (1 :1000 in PBS) for 10 mins and then resuspended in cell medium for 100uL for each well and cells were resuspended in the presence of the following treatments: Bottom chamber of Transwell plate received treatment, CTR (Figure 8A) C4 (10 j g / mL, Figure 8B), rhEPO (100 ng / mL, Figure 8C), or rhEPO+anti-EPO Ab in combination (Figure 8D) in 500pL of medium. After 48h of incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and migrated T cells on the bottom of the chamber were counted under the microscope and analyzed by Imagej with analyze particle plug in (Figure 8E). Immunolabeling was acquired using a Leica Time-Lapse microscope. High resolution imaging was performed on all stained wells using the whole-well approach though large-mosaic rendering during high-magnification acquisition to obtain a complete field of view (FOV) of the whole well for each instance of IF labelling. Results show that T cells are chemoattracted by anti-EPO antibody and that this effect is significantly reduced in presence of rhEPO. Interestingly, when anti-EPO is co-administrated with rhEPO, a significant higher number of migrated T cells was recorded. Therefore, it can be concluded that the anti-EPO treatment a is a potent stimulus and significantly increased T cell migration. Treatment with rhEPO, on the contrary, blocks T cell migration, indicating an immunosuppressive role for erythropoietin. *P<0.05 versus CTR treatment.

[0238] Immunophenotypic profile was assessed on T cells after C4 administration by evaluating the expression of EPOR in CD69+ subpopulation in the following conditions: CTR (Figure 9), Temozolomide (TMZ, Figure 9B), rhEPO (Figure 9C), C4 (Figure 9D), TMZ+C4 (Figure 9E), and rhEPO+C4 (Figure 9F). Results demonstrated that in activated CD69+ positive cells, EPOR expression was significantly increased in all the conditions in which anti-EPO antibody was added. Indeed, migrated T cells showed higher expression for CD69 and EPOR compared to CTR and rhEPO conditions (Figure 9G). Data are the mean ± SD of at least 3 experiments in triplicate. **P<0.01 , ***P<0.001 versus CTR for all treatments.

[0239] Example 7. anti-EPO induces CTL lymphocytes CD4+ migration in TME and deeply penetration into GBM tissues

[0240] Lymphocyte migration was evaluated using a 24-well, Transwell plate (8.0pm pore size; Corning, Corning, NY). In brief, T cells were washed once with RPMI1640 medium, cell count re-adjusted (5x10A5 cells / mL) in T cell medium (RPMI1640+10%FBS). T cells were colored with Calcein (1 :1000 in RPMI medium for 15 mins), washed with PBS and an aliquot (100 pL) of T-cell suspension was placed in the top chamber. Bottom chamber of Transwell plate received chemokine in 500pL of conditioned medium (CM), Figure 10A. After 48h of incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and the number of T cells on the bottom of the chamber were counted under the microscope and analyzed by Imagej with analyze particle plug in. Figure 10B shows the number of migrated T cells on the bottom of the well. Data demonstrated that anti-EPO can induce a higher migration of T cells when administered alone or in combination with TMZ (Figure 10B), as represented in photomicrographs of the well in the following conditions: CTR (Figure 10C), TMZ (Figure 10D), anti-EPO (Figure 10E), TMZ+C4 (Figure 10F), and rhEPO (Figure 10G).

[0241] Surprisingly, in presence of a fragment of GBM tissue (Figure 1 1 A), the number of migrated T cells was significantly higher after C4 administration, also in combination with TMZ (Figure 11 B). The photomicrographs in Figure 1 1 C-H represented the whole bottom of the wells in the following conditions: CTR (Figure 11 C), TMZ (Figure 11 D), C4 (Figure 1 1 E), TMZ+C4 (Figure 11 F), rhEPO (Figure 11 G), and rhEPO+C4 (Figure 11 H). At the end of the migration analysis, the fragment of GBM tissue (Figure 12A) was collected from the bottom of the well, digested with T rypsin 0,25% for 30mins, filtered by 70pm pore-size mash and analyzed by flowcytometer to count green positive infiltrated cells. Results demonstrated that the administration of anti- EPO antibody significantly increased the number of infiltrated T cell, alone and in presence of TMZ, or rhEPO (Figure 12B). All in all, these data illustrated that anti-EPO shows a potent chemotactic ability to induce T cell migration. Surprisingly, anti-EPO antibody was able to induce T cell infiltration into GBM tissue, revealing a powerful capacity as chemotactic molecule.

[0242] Example 8. Expression on GBM cells of EPO related target

[0243] Expression patterns of EPO-related, transferrin-related, IL-13-related genes by Real-time PCR in CTR and GBM mRNA was performed to identify specific GBM signature to develop CAR construct to harness the immune system to attack the tumor.

[0244] To obtain information about expression of specific genes, Real-Time PCR was performed. In detail: gene expression analysis was performed on GBM cells and CTR mRNA (Takara) Cells were collected, centrifuge 300g x 10 mins and the pellet was resuspended in Tri-Reagent for RNA extraction, following manufacture’s’ instruction. RNA was quantified with NanoDrop 1000 Spectro-photometer (Thermo Fisher Scientific). Reverse transcriptase reaction was executed using TranScriba Kit (A&A Biotechnology), loading 1 pg of RNA (A260 / A280 > 1.8), according to manufacturer’s instructions. qRT-PCR was performed using StepOnePlus™ (Thermo Fisher Scientific), 1 pg of cDNA, forward and reverse primers (250 nM each) Titan HotTaq EvaGreen® qPCR Mix (Bioatlas). Data were normalized to 18S gene expression, used as endogenous control. Relative gene expression was determined using the 2-AACt method (Figure 13). Figure 13 shows the results of gene expression of EGFR, EPOR, EPHB4, CSF2RB, CRLF3 in tumor cells and commercial CTR mRNA. (Figure 13A). In addition, gene expression for IL-13, IL-13R1 , IL-13R2 (Figure13B), and TfR1 , TfR2, and folate receptor (Figure 13C) are shown. The results revealed that there are high expression levels of EGFR, EPHB4, CSF2RB and CRLF3 in GBM cells, compared to healthy cells, as well as for IL-13, IL-13R1 , IL-13R2, TfR1 , TfR2, and folate receptors, indicating a possible target to develop CAR construct.

[0245] Example 9. CAR-T efficacy of anti-EPO treatment with cancer cells

[0246] Since EGFR amplification, overexpression, or mutation is present in approximately half of glioblastomas and other malignant CNS tumors, including ependymoma and medulloblastoma, in both children and adults, EGFR CAR-T (EGFR scFv-4-1 BB-CD3 CAR T-cells, Promab) were used. To examine the effect of T cells (WT) and EGFR-CAR-T cells in presence of absence of anti-EPO, a killing test on glioblastoma stem cells (Figure 14), and cancer cells from colon, breast, and melanoma was performed (Figure 14). Target cells were seeded in triplicate in 96-well plate at a concentration of 1x10A4 cells per well in DMEM: F12 (ThermoFisher Scientific) media supplied with 10% FBS. The target cells were cultured with T cells, or CAR-T alone or in presence of anti-EPO at a serial of E:T (effector to target) ratio of 1 :1 , 1 :2, 1 :4, 1 :8, 1 :16, 1 :32, and 1 :64 for 24h at 37°C and 5%CO2 (Figure 14A). Each well was stained by crystal violet staining solution (ThermoFisher Scientific). The images were observed under an inverted microscope (TS100, NIKON Instruments Inc) and photographed after staining. Data were reported as % of viable cells (Figure 14A). Under such conditions, significant differences were observed when T-cells were cultured in presence of anti-EPO treatment. Indeed, T-cells mediated a better control of target cell number. Thus, the cytotoxic activity of CAR-T cells+anti- EPO, was highly efficient at lower effector abundances.

[0247] In addition, to determine if the observed enhanced cytolytic activity was accompanied by a similar significant increase in TNF-a and IFN-y secretion, GSCs were co-cultured with EGFR- CAR-T cells alone or in combination with C4 antibody for 24 h and cytokines were measured by ELISA kit (R&D System) follow the instructions. As shown in Figure 14, both EGFR-CAR-T cells alone spontaneously produced detectable levels of TNF-a (Figure 14B) and IFN-y (Figure 14C). Culturing these cells with CAR-T in combination with anti-EPO antibody, induced TNF-a and IFN-y, with significantly higher levels of cytokines produced. These results agree with the aforementioned cytotoxicity data, and together indicate that co-administration of anti-EPO antibody with EGFR-CAR-T can significantly enhance T cell effector functions in response to EGFR+ glioma cells.

[0248] Furthermore, cytotoxic effect of EGFR-CAR-T cells was assessed on the following cellular models: i) Breast cancer cell line (MCF-7); breast cancer cells. This cell line has been shown to be reactive to human erythropoietin (rHuEPO) treatment in terms of increased cell proliferation. In addition, it has been reported that MCF7 cell line express EGFR (Figure 15A). ii) prostate cancer prostate cancer cell lines (LNCAP); human prostate cancer cells used in cancer research and drug development. Human hepatocellular receptors (Ephs) producing erythropoietin have been reported to be overexpressed and associated with poor prognosis and reduced survival in prostate cancer patients and are considered predictive markers of aggressive prostate cancer behavior. Furthermore, it has recently been shown that in LNCAP, the simultaneous overexpression of EPO and EPOR in resistant prostate cancer plays an important role in progression and is responsible for the development of a neuroendocrine phenotype; Interestingly, it has been reported that EGFR is expressed on LNCAP cell line (Figure 15B). ill) Melanoma cell lines, derived from a primary amelanotic tumor A375. Elevated levels of EGFR, expression was detected. From these premises we decided to use A375 cell lines as model for cytotoxic activity of EGFR-CAR-T cells (Figure 15C). Under such conditions, significant differences were observed when T-cells were cultured in presence of anti-EPO treatment, in MCF7 breast cancer cell line (Figure 15A), in LNCAP prostate cell line (Figure 15B), and in A375 melanoma cell line (Figure 15C). Indeed, T-cells mediated a better control of target cell number. Thus, the cytotoxic activity of CAR-T cells+anti-EPO, was significant highly efficient at lower effector abundances.

[0249] Furthermore, results showed that CAR-T cytotoxic effect was potentiated by anti-EPO and metformin (MET) co-treatment on glioblastoma cancer cells (Figure 16A). In addition gene expression analysis was performed on PBMC treated in the following conditions: CTR, anti- EPO, MET, and anti-EPO+MET. Results revealed that co-administration of anti-EPO-i- MET induces on PBMCs an overexpression of IL-i p (Figure 16B) and IFNy (Figure 16C) with a downregulation of PD-1 (Figure 16D).

[0250] Example 10. Immune cells infiltration into GBM subcutaneous PDX mouse model after anti-EPO treatments

[0251] SCID mice (n=10) were inoculated subcutaneously with GBM fragment and monitored until tumor masses reached 50-100 mm3. Then, mice were randomized in 2 groups as follows: CTRL treated with PBS, and anti-EPO treated with intravenous (IV) injections of 15 mg / Kg for 3 doses / week. Tumor mass weights were measured with a caliber before each dose. Mice body weight was monitored for the entire duration of the experiment. The day of scarify, tumor mass was excised and processed for hematoxylin and eosin staining. An immunohistochemical evaluation was carried out by two independent pathologists who were blinded to the clinical information. Following anti-EPO treatment (Figure 17 A,B), increased polymorphonuclear leukocyte infiltration was observed in GBM mass at 21 days post injection, compared to CTR condition (Figure 17C,D).

[0252] Example 11. anti-EPO treatment ameliorates malaria in infected erythrocytes.

[0253] Leukocyte-depleted human erythrocytes were processed. Erythrocytes were washed upon arrival with filtered RPM1 1640 medium (containing 25 mM HEPES and 50 pg / ml hypoxanthine). Plasmodium parasites were grown in complete culture medium (cRPMI) containing 1% AlbuMax II (ThermoFisher Scientific), 0.21% sodium bicarbonate (Gibco), and 20 pg / ml gentamicin (ThermoFisher Scientific) in RPM1 1640 (ThermoFisher Scientific) at 5% hematocrit, 37°C, and a gas mixture containing 90% N2, 5% CO2, and 5% 02. To monitor parasitemia, erythrocytes were prepared on glass slides, fixed with 100% methanol, and stained for 15 min with 20% Giemsa staining (Sigma-Aldrich, St. Louis, MO). Using bright-field microscopy erythrocytes were counted, and the number of parasitized cells was used to estimate percentage parasitemia in CTR condition or following anti-EPO antibody. When anti-EPO antibody was administered into erythrocytes infected all cells showed decreased parasitemia and recovered from the infection (Figure 18A). When monocytes were cultured with infected cells, immunophenotypic profile showed a higher expression of CD86+ / HLA-DR+ positivity, indicating a M1 polarization (Figure 18B). These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria (Figure 18).

[0254] Example 12. Migration of PBMCs: model of chemotaxis in infection disease stimulated by anti-EPO

[0255] Figure 19 shows the effects of lipopolysaccharide (LPS) treatment with anti-EPO antibody and rhEPO on PBMC migration. LPS is a molecule present on the membrane of the Gram-negative bacteria. The migration testing or "chemotaxis assay" was carried out to highlight the migratory capacity of PBMC, a phenomenon that is observed in response to an immune response. For this purpose, transwell multiwell plates (24-well) were used and equipped with inserts with a polycarbonate membrane. The holes in the membrane of a diameter of 8pM can retain the cells and the culture medium but allow the active transmigration of cells through the membrane to reach the lower well. 2x10A4 PBMC (for each well) were resuspended in cell medium for 100uL for each well and PBMC suspension was placed in the top chamber (Figure 19A). Bottom chamber of Transwell plate received treatment, CTR+LPS (Figure 19B), anti-EPO+LPS at 10p.g / mL (Figure 19C) or rhEPO+LPS at 100 ng / mL (Figure 19D), in 500 pL of medium. After 48h of incubation at 37°C in a 5% CO2 atmosphere, the top chamber was removed, and PBMC were coloured with Hoechst (1 :1000 in PBS for 15 mins at 37°C), washed with PBS, and the number of PBMC on the bottom chamber were counted under the microscope and analyzed by Imagej with analyze particle plug in (Figure 19E). Immunolabeling was acquired using a high-resolution Nikon Ti spinning disk microscope (Nikon Instruments, Florence, Italy) equipped with a CREST-optics spinning disk head, a VCS structure illumination module for super-resolution (CREST-Optics, Rome, Italy), and Andor cameras for resolution (Andor Zyla, Andor Technology, Oxford Instruments, Oxford, UK) and quantum efficiency (Andor Technology, Oxford Instruments, Oxford, UK). The NIS-Elements V.5.3.2 was used to deconvolve the spinning disk confocal images, whereas specific VCS-Studio algorithms (CREST Optics, Rome, Italy) were employed for correct structure illumination reconstruction. High resolution imaging was performed on all stained wells using the whole-well approach though large-mosaic rendering during high-magnification acquisition to obtain a complete field of view (FOV) of the whole well for each instance of IF labelling. Intriguingly, PBMC were chemoattracted by anti-EPO antibody and this effect was significantly reduced in presence of recombinant human EPO (rhEPO). Therefore, it can be concluded that in presence of LPS, condition mimicking an infection disease, anti-EPO treatment significantly increase PBMC migration, because of a potent stimulus. Treatment with rhEPO, also in presence of LPS, on the contrary, blocks PBMC migration, indicating an immunosuppressive role for EPO. *P<0.05 versus CTR. treatment.

[0256] Example 13. Administration of anti-EPO in a rodent orthotopic model of GBM induces elevated immune cell migration, with tumor penetration of T lymphocytes.

[0257] In an in vivo orthotopic GBM model, the administration of anti-EPO, compared to the healthy contralateral hemisphere (Figure 20A), surprisingly induced the activation of immune system with migration of T lymphocytes (Figure 20B, white arrows). Interestingly, following anti-EPO administration, ferroptosis was evaluated by histological analysis that showed iron deposits, marker of cellular death.

Claims

CLAIMS1. A method for activating or boosting the immune response of a patient in need thereof, said method comprising the use of an anti-EPO negative functional modulator or anti-EPO antigenbinding fragment alone or in combination with:- a check point inhibitor or immunomudulator (e.g. anti-PDL1 antibody; nivolumab; ipilumab, abetacept, glembatumumab vedotin) therapy; and / or- a cell-based immunotherapy (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic activated cells against tumor associated antigens, and / or antigen-presenting cells, tumor associated peptide, engineered monocyte-macrophage or polymorphonucleate cell-based therapies and / or a therapy to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs;- an anti-microbial therapy (e.g. antibiotics; antivirals, antimycotics, antifungi, anti-prions)- a flavonoid molecule;- metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonist,- a prophylactic or therapeutic DNA and / or RNA and or peptide or carbohydrate or lipid based vaccine (e.g. anti-HPV, anti-EBV or anti-HIV vaccine);- Oncolytic virus based immunotherapy;- a chemotherapeutic agent;- an anti-cancer drug;- an enzyme that degrades heparin sulfate proteoglycans (e.g. heparanase);- a negative functional modulator of the sphingosine-1 -phosphate (S1 P) signaling pathway, or- EPO mimetics that preserve the erythropoietic function; wherein said anti-EPO antigen-binding fragment is chosen from the group comprising Fab, - F(ab’)2, single chain antibodies, diabodies, triabodies, tetrabodies, repebodies, or domain antibodies and said anti-EPO negative functional modulator is chosen from the group consisting of mono- or multi-specific antibody anti-EPO, gene therapy, DNA decoy, an RNA decoy, a ribozyme, an antagomiR, a shRNA, an LNA, a siRNA, an antisense oligonucleotide or an anti-Epo receptor, said anti-Epo receptor chosen from the group consisting of EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs and EPOR / CD131 heterodimer.

2. The method according to claim 1 , wherein the negative functional modulator or anti-EPO antigen-binding fragment elicits a immune cell response (T helper and / or T Cytotoxic and / or B and / or NK Lymphocyte and / or Macrophages and / or Neutrophils and / or Dendritic cell activation,antigen presenting cells), wherein said cell response is an anti-tumoral T or anti-microbial cell response of a patient affected with a cancer or infectious disease.

3. The method according to claim 2, wherein said anti-tumoral T helper and / or T Cytotoxic and / or B and / or NK Lymphocytes and / or Macrophages and / or Neutrophils and / or Dendritic cell are PBMC derived immune cells.

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

5. The method according to any one of claims 2 to 4, wherein said anti-tumoral T cell response is due to CD69, and EPOR expression in migrated PBMCs.

6. The method according to any one of claims 1 to 5, wherein said anti-EPO antigen-binding fragment is a neutralizing antibody which binds to EPO, EPO variants or EPO receptors and restores T helper and / or T Cytotoxic and / or B and / or NK Lymphocyte and / or Macrophages and / or Neutrophils and / or Dendritic cell activation.

7. The method according to any one of claims 1 to 6, wherein said anti-EPO antigen-binding fragment is a neutralizing antibody is chosen from the group consisting of C4, B4 and 16F1 H11 .

8. The method according to any one of claims 1 to 7, wherein the negative functional modulator or anti-EPO antigen-binding fragment elicits a T helper and / or T Cytotoxic and / or B and / or NK Lymphocyte and / or Macrophages and / or Neutrophils and / or Dendritic cell, and / or antigen- presenting cells, wherein said cell response is further enhanced by a CAR-T, CAR-M, CAR- NK, engineered monocyte-macrophage or polymorphonucleate cell based therapies or to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs.

9. The method according to any one of claims 1 to 8, wherein said patient in need thereof is suffering from cancer, proliferative pathologies, chronic inflammatory diseases on an autoimmune and non-autoimmune basis, neurodegenerative diseases, Hippel-Lindau disease(VHL), multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1 , endometriosis, Crohn's disease, ulcerative colitis, neuro-inflammatory and infectious diseases, mycosis fungoides, and infectious diseases as malaria, tuberculosis, HIV1 and 2, sickle cells, SARS, Sars-Cov, MERS or a patient undergoing organ or tissue transplantation, and wherein said cancer is selected from the group consisting of brain cancer, metastatic brain cancer, brain stem glioma cerebral astrocytoma, cerebellar astrocytoma, astrocytoma of the pineal gland, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, uterine sarcoma, rhabdomyosarcoma, Kaposi's sarcoma, glioma, glioblastoma multiforme, glioblastoma grade II fibrillary astrocytoma, protoplasmic, grade III gemistocytic, anaplastic astrocytoma, including gliomatosis cerebri, ependymoma, medulloblastoma, neural ectoderm tumor, neuroblastoma, hypothalamic glioma, breast cancer, triple-negative breast cancer, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, colon cancer, colorectal cancer, ovarian cancer, ovarian epithelial cancer, gestational villous disease, cervical cancer, endometrial cancer, uterine cancer, ovarian germ cell cancer, esophageal cancer, basal cell carcinoma, cholangiocarcinoma, choroidal melanoma, choriod plexus carcinoma, cancer of the spleen, osteosarcoma, intraocular melanoma, malignant melanoma, retinoblastoma, stomach cancer, heart cancer, liver cancer, hypopharyngeal cancer, laryngeal cancer, cancer of the oral cavity, nasal and paranasal cavity cancer, cancer of the salivary glands, nasopharyngeal cancer, throat cancer, thyroid cancer, parathyroid cancer, thymus cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, gastric and hepatic carcinoma, gastric lymphoma, colorectal cancer, rectal cancer, rectal carcinoma, small intestine cancer, gastrointestinal stromal cancer, testicular cancer, renal cell cancer, adrenal cancer, renal pelvic cancer, malignant mesothelioma, mesothelioma, pheochromocytoma, hematological cancers or chronic myeloid leukemia lip cancer, tonsil cancer, squamous cell carcinoma, ampullar of vater 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 myelogenous leukemia, gallbladder cancer, biliary tract cancer, chronic lymphocytic leukemia .malignant bone cancer, metastatic bone cancer, eye cancer, vulvar cancer, ureter cancer, mediastinal cancer, urethral cancer, cancer of unknown primary site, vaginal cancer, spinal cord cancer, vestibular schwannoma, diffuse midline glioma (DMG), diffuse intrinsic pontineglioma (DI PG), embryonal tumors, pineoblastoma, germ cell tumors, acoustic neuroma, schwannoma, meningioma, and haemangioblastoma.

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

11. The method according to any one of claims 1 to 10, wherein said negative functional modulator or anti-EPO antigen-binding fragment promotes an immune system response in a patient affected by a refractory or persistent infectious diseases, wherein said persistent infectious diseases include tuberculosis, malaria, HIV, EBV, or HPV induced pre-cancerous alterations and their prevention.

12. The method according to any one of claims 1 to 1 1 , wherein said negative functional modulator or anti-EPO antigen-binding fragment promotes immune system response is in a patient affected by streptococci, staphylococci, fungi, viruses; Sars-Cov2, SARS, MERS, prion agents resistant to antimicrobial therapies and / or in presence of immune system tolerance.

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

14. A pharmaceutical kit comprising a negative functional modulator of EPO / EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) and / or their natural or synthetic variants and:- a check point inhibitor or immunomudulator (e.g. anti-PDL1 antibody; nivolumab; ipilumab, abetacept, glembatumumab vedotin) therapy; and / or- a cell-based immunotherapy (CAR-T, CAR-M, CAR-GAMMA / DELTA, CAR-NK, dendritic activated cells against tumor associated antigens) engineered monocyte-macrophage or polymorphonucleate cell-based therapies and / or a therapy to enhance and reprogramming the answer of tumor associated lymphocytes TILs or Tumor associated macrophages TAMs; and one or more components selected from the group consisting of:- a peptide or antibody, diabody, nanobody against the natural and synthetic variants including physiological and pathological splicing variants of erythropoietin and post-translational modification;- an anti-microbial therapy (e.g. antibiotics; antivirals, antimycotics, antifungi, antiprions)- a flavonoid molecule;- metformin and / or rapamycin and / or GLP-1 receptor agonists, GPNMB antagonist;- a prophylactic or therapeutic DNA and / or RNA and or peptide or carbohydrate or lipid based vaccine (e.g. anti-HPV, anti-EBV or anti-HIV vaccine);- Oncolytic virus based immunotherapeitic drug;- a chemotherapy- an anti-cancer drug- an enzyme that degrades heparin sulfate proteoglycans (e.g. heparanase)- a negative functional modulator of the sphingosine-1 -phosphate (S1 P) signaling pathway, or- EPO mimetics that preserve the erythropoietic function.

15. A diagnostic or prognostic method for evaluating the expression of EPO and its somatic mutations or its variants, EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, EPOR / CD131 heterodimer) and their somatic mutations and / or their variants and / or C4 mAb ligands that predict the response to target EPO / EPORs negative modulation therapy to personalize therapy in cancer and infectious diseases immunotherapy or prophylaxis, to stratify patients, optimize patient’s response, said method having the step of measuring the amount of / detecting the presence of EPO and its somatic mutations or its variants, EPO receptors and its somatic mutations and their variants in tissues, cells, or human fluid (saliva, blood, cerebrospinal fluid, sweat, or derived-extracellular vesicles) as diagnostic or prognostic markers.

16. A diagnostic method for evaluating the methylation of the promoter of the genes of EPO and EPO receptors (EPOR EPHB4, CSF2RB, CRLF3, tissue protection factors, TPRs, e.g. EPOR / CD131 heterodimer) that can predict an increase expression of EPO / EPO-Rs and their negative role in immune system inhibition in eradicating cancer cells or microbial agents to personalize immunotherapy or prophylaxis and for prognostic purposes, said method having the step of detecting the methylation of EPO and EPO receptors genes in tissues, cells, orhuman fluid (saliva, blood, cerebrospinal fluid, sweat, or derived-extracellular vesicles) as diagnostic or prognostic markers.

17. An EPO / EPO-Rs negative modulation agent delivery system administered orally, parenterally, intralesional (intratumoral, and intracavity) intraventricular, intrathecal, intranasal or local in various formulations at the time of clinical administration, based on innovative nanomedicine and nano-delivery systems as tissue gun or probe, viral and no-viral vectors, nanomaterials for the delivery of bioactive drugs for target delivery, plant-based vesicles, nanoparticle-based methods which allow both treatment and in vivo imaging modalities for diagnosis and therapy, lipid systems like liposomes and micelles, gold or magnetic nanoparticles, also in combination with natural product, functionalized nanoparticles, microspheres and biomaterials, as PEG, trojan horse approach, as micro pump to release treatment in tissues to enhance the modulating effects on immune system into the local pathological microenvironment or to attract and increase the homing of cell-based immunotherapies or vaccines.

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

19. A method based on EPO / EPO-Rs negative modulation to improve CAR T and CAR- Gamma / Delta T Cell Therapies Efficacy against Solid Tumors.

20. An EPO / EPO-Rs and their variant inhibitors able to stimulate CTLs infiltration and suppressing the recruitment of immunosuppressive cells in tumor and in infectious diseases, increasing the tissue penetration of inflammatory and immune cells.

21. An EPO / EPO-Rs and their variants inhibitors able to induce inflammation, associated pyroptosis, immunogenic cell death, necroptosis, ferrooptosis, authophagy, cruproptosis and immuno-stimulated cell death enhancing tumor immunogenicity.

22. A product selected among EPO and their natural and syntetic variant inhibitors, able to activate the immune response against cancer and infectious agent, reprogram tumor microenvironment and potentiate immunotherapy and immunomodulatory strategies.

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