Combination of GM-CSF and PGE1 to treat hematopoietic insufficiencies and disorders

Abstract

The present invention relates to a drug combination consisting of GM-CSF and PGE1 for use in the parenteral, individual therapy of patients suffering from stem cell-deficiencies and / or hematopoetic insufficiencies.

Description

[0001] COMBINATION OF GM-CSF AND PGE1 TO TREAT HEMATOPOIETIC INSUFFICIENCIES AND DISORDERS

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to a drug combination consisting of GM-CSF and PGE1 for use in the parenteral, individual therapy of patients suffering from stem cell-deficiencies and / or hematopoetic insufficiencies.

[0004] BACKGROUND OF THE INVENTION

[0005] Reference is made to the following documents:

[0006] Amberger, D. C. & Schmetzer, H., Dendritic cells of leukemic origin: specialized antigen-presenting cells as potential treatment tools for patients with myeloid leukemia. REVIEW Transfus Med Hemother 47 (6), 432–443 (2020). DOI: 0.1159 / 000512452

[0007] Amon, L. et al., Harnessing the Complete Repertoire of Conventional Dendritic Cell Functions for Cancer Immunotherapy. REVIEW Pharmaceutics 12 (7), 663 (2020). DOI: 10.3390 /

[0008] Aslan Rejeski, H. et al., Concentration-dependent effects of immunomodulatory cocktails on the generation of leukemia-derived dendritic cells, DCIeu mediated T-cell activation and on-target / off-tumor toxicity. Frontiers in Immunology eCollection 15, 1527961, 1-18 (2025). doi: 0.3389 / fimmu.2024.1527961. PMCID PMC11821930 (published 2025-01-30)

[0009] Atzler M. etal., in vivo Induction of Leukemia Specific Adaptive and Innate Imune Cells By Treatment of AML-Diseased Rats and Therapy-Refractory AML-Patients with Blast Modulating Response Modifiers. Int. J. Mol. Sci. 25 (24), 13469, 1-17 (2024). https: / / doi.org / 10.3390 / xxxx

[0010] Cabeza-Cabrerizo M. etal., Dendritic Cells Revisited. REVIEW, Annual Review of Immunology 39, 131-166 (2021). doi: 10.1146 / annurev-immunol-061020-053707

[0011] Greter M. et al., GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells. Immunity 36 (6), 1031-46 (2012). doi: 10.1016 / j.immuni.2012.03.027.

[0012] Halfter K. et al., Prospective cohort study using the breast cancer spheroid model as a predictor for response to neoadjuvant therapy-the SpheroNEO study. Behalf of the SpheroNEO Study Group. BMC Cancer 15, 519 (2015). doi: 10.1186 / s12885-015-1491-7. PMID: 26169261

[0013] Muzio, M. et al., Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. The Journal of Immunology 164 (11), 5998-6004 (2020). doi 10.4049 / jimmunol.164.11.5998Sallusto F. & Lanzavecchia, A., Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte / macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179 (4), 1109-1118 (1994). doi: 10.1084 / jem.179.4.1109 Schmid, C. et al., Low-dose ARAC, donor cells, and GM-CSF for treatment of recurrent acute myeloid leukemia after allogeneic stem cell transplantation Leukemia 18 (8), 1430-1433 (2004). doi: 10.1038 / sj.leu.2403412

[0014] Schutti O. etal., Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients' Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays. Int. J. Mol. Sci. 25 (13), 6983 1-24 (2024). DOI 10.3390 / ijms25136983

[0015] Unterfrauner M. et al., Granulocyte-Macrophage-Colony-Stimulating-Factor Combined with Prostaglandin El Create Dendritic Cells of Leukemic Origin from AML Patients' Whole Blood and Whole Bone Marrow That Mediate Antileukemic Processes after Mixed Lymphocyte Culture. Int. J. Mol. Sci. 24(24), 17436 (2023). doi 10.3390 / ijms242417436

[0016] Yamanaka R. et al., Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical phase l / ll trial. Clin Cancer Res 11 (11), 4160-4167 (2005). doi 10.1158 / 1078-0432. CCR-05-0120

[0017] Treatment approaches for hematopoietic insufficiencies:

[0018] It is well known that diseases of myelopoiesis such as myeloid leukemias (including acute myeloid leukemia [AML], myelodysplastic syndromes [MDS], myeloproliferative syndromes [MPS], and chronic myeloid leukemia [CML]) as well as diseases of lymphopoiesis such as lymphatic leukemias and lymphomas (including acute lymphatic leukemia [ALL] and chronic lymphatic leukemia [CLL]) are clonal hematopoietic diseases associated with pathologically increased cell proliferation which are due to impaired and insufficient hematopoiesis and impaired hematopoietic regeneration (e.g., stem cells, thrombocytes, erythrocytes, granulocytes, lymphatic cells). Such deficiencies are also found in patients with solid (metastatic) tumor diseases, after chemotherapy, or shortly after stem cell transplantations. These deficiencies lead to an insufficient supply of (regenerative) hematopoietic cells, which (depending on the cell lines affected) is associated with a tendency to bleed, anemia, susceptibility to infection, or reduced antitumor activity. In addition to the acquired deficiencies mentioned above, congenital blood cell formation disorders also result in deficiencies, e.g.: Granulocytopenia (e.g., Chediak-Higashi, Hermansky-Pudlak, and Griscelli syndromes, Cohen and Barth syndromes), erythrocytopenia (e.g., thalassemia, congenital dyserythropoietic anemia) or thrombocytopenia (Fanconi anemia, Wiskott-Aldrich syndrome), immune-mediated thrombocytopenia (idiopathic / anaphylactic) or cytopenia caused by HLA sensitization.

[0019] Depending on the severity of the insufficiency and / or tumor load, affected patients mostly have a poor prognosis: the more tumor cells, deficient stem cells, or autoimmune processes are present in the patient, thefewer functioning platelets, erythrocytes, granulocytes, and immune cells are present. This leads to an increased need for blood transfusions or other therapeutic interventions (e.g., administration of platelet or erythrocyte concentrates), antimicrobial therapies, granulocyte-stimulating therapies [G-CSF]). Allogeneic stem cell transplantation with healthy donor cells is considered the only permanent and curative treatment approach but is severely limited by the availability of matching donors and the fitness of the patients.

[0020] Improvement of antitumor / anti-infective immune competence:

[0021] There is an urgent need for effective (immuno)therapeutic options to stabilize remissions and prevent relapses in patients with acute leukemia (AML, ALL) and patients with MDS / MPS, and to control metastases or prevent metastasis in patients with solid tumors (e.g., colon, breast, bronchial CA, lymphomas). It is equally important to help patients with (chronic) infections and parasitic diseases with new immuno therapies. Antitumor or antimicrobial (e.g., against fungi, viruses, bacteria, parasites) immune responses are mediated in the healthy body by cells of the innate and acquired immune system. In concert with humoral factors such as antibodies, cytokines, chemokines, etc., cellular immune responses are specifically activated and orchestrated by dendritic cells (DC).

[0022] DC phagocytosis-mediated immune activation

[0023] The function of healthy (stem cell- or monocyte-derived) DCs is to enable phagocytosis, processing, and presentation of tumor antigens or pathogen fragments found in or on affected cells and, ultimately, in the context of costimulatory (e.g., MHC) mechanisms, the activation of innate and adaptive (antigen-specific) immune cells which attack infected cells or tumor cells and eliminate them (Cabeza-Cabrerizo 2021).

[0024] It is well known that GM-CSF plays a significant role in activating DC to mature and phagocytose. The activated DC then induce other immune cells to attack infected or abnormal cells (Greter 2012). However, GM-CSF treatment alone does not change the outcome of relapsed AML patients after SCT, pointing to the fact, that additional response modifiers are necessary to create mature and functional DC (Schmid 2004).

[0025] DC / DCieu-mediated immune activation

[0026] It is known that (myeloid) tumor cells can, either spontaneously or triggered by certain molecules, differentiate into 'leukemia-derived DC (in short DCieu). These DCieuexpress the leukemic antigens of the individual patient together with costimulatory MHC antigens to the immune system and activate innate and adaptive immune cells specifically against leukemic cells. This immune activation also works with ex vivo (e.g., from patient monocytes) produced "monocyte-derived DC" that are loaded with leukemic antigens (WT1, PRAME, RHAMM, etc.). In both cases, DCieuor DC loaded with leukemic antigens must be produced ex vivo, purified, and finally adoptively retransfused into patients with AML. It has been shown that these approaches work, with antileukemic effects demonstrated through the induction of leukemia-specific cells in vivo and an improvement in patient survival (Amberger 2020): Patients who had been treated with manipulated DC (presenting leukemia antigens) showed longer survival times. The disadvantage is that these strategies are complex as they involve ex vivo handling of cells under GMP conditions, are often impaired by limited cellyields and are expensive and time-consuming to produce. Therefore, DC-based strategies have struggled to be widely adopted for the immunotherapeutic treatment of leukemia patients. In principle, it is possible that patients with myeloid leukemia (AML) (before or after SCT) produce leukemia-derived DC in vivo when they are given a sufficient dose of the immunomodulatory drug combination GM-CSF+PGE1 (European Patent Specification No: EP 3217975 Bl). These DCieuinduce leukemia-specific immune effector and memory cells, which on the one hand combat the tumor load in the blood or at extramedullary sites and on the other hand prevent relapses by inducing the formation of memory cells which are reactivated when the tumor comes back. Such mechanisms have likely contributed to the successful stabilization of a previously therapyrefractory patient for several months. Specific memory cells can be detected in the blood during the course of treatment by flow cytometric monitoring. The advantage of this in vivo treatment with GM-CSF+PGElcan be repeated indefinitely and without any lead time (since no cell products need to be produced ex vivo, followed by adoptive transfer of the manipulated cells, the yields of which are often not adequate). Ex vivo analyses have shown that this mechanism works with cells from any patients, regardless of age, gender, HLA, mutation, risk, or transplantation status.

[0027] SUMMARY OF THE INVENTION

[0028] The object of the present invention is to provide a pharmaceutical composition comprising a combination of GM-CSF and PGE1 for use in in vivo immunotherapy to stem cell-deficient / hematopoetic insufficient patients, who are suffering from leukemia, solid tumors, infectious diseases, or iatrogenic complications (e.g. after chemotherapies, stem cell transplantation). In preferred embodiments, said combination is for use in the treatment of at least one of:

[0029] a) acute or chronic myeloid / myeloproliferative and lymphatic leukemias (in tumor rich stages, in immunocompetent patients in remission) before and after stem cell transplantation, to induce and produce DC and DCieufrom leukemic cells via the 'DC / DCieu-mediated pathway^thus activating the immune system in a targeted and specific manner against patient-specific leukemia antigens, creating specific adaptive and innate effector cells and memory cells that mediate the killing of leukemic cells, and creating an immunological memory that prevents the progression or recurrence of the diseases;

[0030] b) solid tumors, or lymphomas before and after other therapies, to induce or support a mechanism triggered by the " DC phagocytosis pathway," thereby activating the immune system in a targeted and specific manner against tumor / leukemia / infectious antigens, create specific adaptive and innate effector cells and memory cells that mediate the killing of tumor cells, and create an immunological memory that prevents the progression or recurrence of the diseases;

[0031] c) chronic or acute (microbial / parasitic) infections before and after other therapies, to induce or support a mechanism triggered by the " DC phagocytosis pathway," thereby activating the immune system in atargeted and specific manner against infectious antigens, create specific adaptive and innate effector cells and memory cells that mediate the killing of infected cells, and create an immunological memory that prevents the progression or recurrence of the diseases;

[0032] d) congenital or acquired (disease-related or therapy-related) hematopoietic / stem cell insufficiency or defects, to improve the provision and regeneration of hematopoietic_stem cells and their resulting derivatives (thrombocytes, erythrocytes, granulocytes, immune cells (T, B, NK, iNKT lymphocytes, erythrocytes, thrombocytes, granulocytes, monocytes, and dendritic cells) in patients with congenital or acquired (disease-related or therapy (chemotherapy, stem cell transplantation)-related) hematopoietic insufficiency, thereby counteracting stem cell-triggered regeneration defects, bleeding tendencies, anemia, or infection tendencies.

[0033] The advantage of the combination of GM-CSF and PGE1 is that the treatment of patients with this combination can be carried out for an unlimited period of time or repeated at any time, thus eliminating the need for ex vivo production and purification of (manipulated) DC or DCieuor DC / DCieutriggered immune cells, followed by adoptive transfer of the manipulated and limited cell products.

[0034] Another advantage is, that this personalized strategy is applicable in all patients, independent of mutation, risk, HLA status, sex, age, etc. and shows efficacy not only against leukemia but also against other tumors as well as infectious agents. This 'DC-triggered' mechanism induces antitumor / infectious / leukemia specific (innate and adaptive) effector cells, which contribute to eliminating target cells. In addition, DC-triggered mechanisms are the only immunological mechanisms enabling the provision of memory cells, responsible for a fast re-provision of effector cells that stabilize the disease or remissions.

[0035] A further advantage is, that the combination enables a recovery of (hemopoetic) stem cells without inducing tumor / leukemia cell proliferation, which reestablishes antitumor / infectious / leukemia immune reactions in immunocompromised patients (due to the disease or to therapy) - resulting in reduced need of antiinfectious treatments; These effects also enable 'non immunological' hemopoetic recovery (e.g. recovery of stem cells, provision of thrombocytes, erythropoiesis) - resulting in reduced need of transfusions or stem cell inducing drugs.

[0036] The present invention envisages all suitable administration forms and dosage regimen. It is preferred that the administration is parenteral. Examples of parenteral administration, contemplated by the present invention, include intravenous, intramuscular, subcutaneous, intradermal, intra-arterial, intrathecal, intraosseous, intra-articular or intraperitoneal. In preferred embodiments the administration is an i.v. administration. In alternative preferred embodiments, the administration is a subcutaneous administration. In further embodiments, the administration may be performed with microneedles, as inhalation or as nasal spray. Dosages for the above mentioned administration forms may be determined according to established procedures and based on suitable literature known to the skilled person. It is preferred that herein belowindicated dosage ranges are used as basis for an adaptation to another administration type, preferably to an administration type as mentioned above.

[0037] In further preferred embodiments the invention relates to the pharmaceutical composition for use according to the invention, wherein said pharmaceutical composition is to be administered in the following dosages:

[0038] According to one embodiment GM-CSF is to be administered parenterally with 15 to 500 μg / day, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration; and

[0039] PGE1 is to be administered parenterally with 0.075 μg / kg / h, up to a maximum of 0.5 μg / kg / h, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration.

[0040] In a further, preferred, embodiment GM-CSF is to be administered parenterally with 75 μg / day, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration.

[0041] In yet another preferred embodiment GM-CSF and PGE1 are to be administered parenterally daily or in two-day or three-day intervals, preferably as i.v. administration, or as subcutaneous administration. In another aspect the present invention relates to a method of treating a patient with stem cell deficiency / hematopoietic insufficiency associated with leukemia, a solid tumor, lymphoma, an infection or a hematopoietic disorder, comprising administering a therapeutically effective amount of a combination of GM-CSF and PGE1, along with pharmaceutically acceptable excipients, wherein the combination triggers hematopoetic stem cells as well as resulting T, B, NK, and iNKT lymphocytes, erythrocytes, platelets, granulocytes, monocytes, and dendritic cells and wherein GM-CSF promotes the regeneration and provision of stem cells, and PGE1, as a "danger signaling factor," stimulates and activates hematopoiesis / the immune system. In preferred embodiments, said combination is for the treatment of at least one of the conditions mentioned above.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1: Hematopoiesis under the influence of the drug combination (GM-CSF+PGE1, also named Kit M).

[0044] Figure 2: Generation of DC / DCieuwith 'lymphatic kits' (Kit 1,2) and GM-CSF+PGE1 (Kit M) from ALL (A), AML (B), CLL (C) blood samples (in blast-containing stages of the disease) compared to healthy blood samples (D), and with a special view on AML-derived DC with myeloid blasts (E) or aberrantly lymphoid antigen expressing blasts (F): Samples were cultured for 7 days compared to controls without added kits, and DC / DCieusubtypes were quantified. Furthermore, we recorded the number of cases in which higher DC subtype values could be generated in ALL and CLL compared to AML (Kit 1: IL-4+CD40L; Kit 2: GM-CSF+IL-4+TNFβ (Muzio 2000).Figure 3: Generation of activated and memory T cells after mixed lymphocyte culture (MLC) of patient / healthy T cells with Kit-pretreated whole blood samples compared to healthy blood samples (see Fig. 2). All samples were cultured for 7 days and T cell subtypes were then quantified. In addition, we recorded the number of cases in which higher T cell subtype values were generated for ALL and CLL compared to AML. Figure 4: Generation of leukemia-specific (interferon gamma-producing or degranulating) immune cells after mixed lymphocyte culture (MLC) of patient / healthyT cells with Kit-pretreated whole blood samples compared to healthy blood samples. All samples were cultured for 7 days and T / B / NK / CIK cell subtypes were quantified. Furthermore, we recorded the number of cases in which higher cell subtype values were generated for ALL and CLL compared to AML.

[0045] Figure 5: Antileukemic activity of immune cells after mixed lymphocyte culture (MLC) of patient / healthy T cells with Kit-pretreated whole blood samples compared to healthy blood samples (see Fig. 2):

[0046] All samples were cultured for 7 days and the antileukemic activity was measured in a cytotoxicity assay. Cases with achieved (vs. not achieved) blast lysis and improved blast lysis compared to controls in ALL, CLL compared to AML are shown..

[0047] Figure 6: Relative frequencies of T cells, NK cells and subpopulations in peripheral blood during treatment with GM-CSF and PGE1: The bars in colors represent treatment phases: yellow bars indicate 1 cycle of Azacitidine; orange bars (starting on day 0) represent the ramp-up phase of experimental treatment with Kit M (each bar indicates one five-day cycle with daily infusion of Kit M: GM-CSF+PGE1); brown bars (starting day 50) indicate the final dose phase of Kit M (each bar dl-5 with daily infusions). A. T cells (T3), B. Interferon gamma (IFNy)-producing T cells (gammaT3), C. Degranulating (CD107a+) T cells (degT3), D. IFNy-producing central memory T cells (garnmaTscm), E. T cells expressing PD-1 (T3PDI), F. Regulatory T cells (Treg), G. T helper 1 cells (TH1), and H. T helper 2 cells (TH2), I. Natural killer cells (NK), J. degranulating (CD107a+) NK cells (degNK). The colored lines illustrate the course of relative frequencies (%) of the given cell subtype measured at different time points during the course of treatment.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] The invention described below is equally applicable to patients with stem cell deficiencies (resulting, for example, in erythro / thrombo / neutropenia / lymphopenia) of any cause, as well as for the in vivo generation of 'mono- / stem cell derived DC (which phagocytose, process, and present tumor / infectious (microbial, parasitic) particles in an immature state) and, at the same time, for the generation of leukemia-derived DC from leukemic cells in leukemia patients.

[0050] Figure 1 shows effects of the combination of GM-CSF and PGE1 on hematopoiesis: regeneration of stem cells and their derivatives, provision of phagocytosis-active DC (DC Phag) via the phagocytosis pathway, conversion of leukemic cells to DCieu(leukemia-derived DC).Healthy hematopoietic stem cells are triggered in the presence of the drug combination (GM-CSF+PGEl), see Figure 1. This leads to the provision of hematopoietic cell descendants that eliminate hematopoietic insufficiency. It is theorized that this process depends on the combined action of (stem cell-active) GM-CSF with the 'danger signal' PGE1 (see Aslan 2025).

[0051] Treatment of patients with hematopoietic deficiency (e.g., as a side effect of chemotherapy or due to other illnesses) with a combination of GM-CSF and PGE1 can induce hematopoietic regeneration, shorten neutropenia periods and reduce susceptibility to infections (diminishing the need for antimicrobial therapies), reduce the need for transfusions of erythrocytes and thrombocytes. By stimulating normal hematopoiesis, the above combination could also generally be used as supportive medication for patients undergoing chemotherapy or having stem cell deficiency.

[0052] Through the redifferentiation of leukemic (myeloid and lymphatic) blasts via the 'DC / DCieupathway', leukemia-derived DCs can be created that activate the immune system and establish immunological memory. The redifferentiation of (myeloid) blasts to DCieuis well known (Amberger 2020, Aslan 2025).

[0053] In European Patent EP 3 217975 Bl, we showed that leukemia-derived DC can be generated from myeloid blasts in the presence of the drug combination (GM-CSF+PGEl) from heparinized whole blood (WB or whole bone marrow (WBM) samples. Myeloid blasts are turned into leukemia-derived DC (DCieu) after exposing them to the drug combination (GM-CSF+PGEl). In T cell enriched mixed lymphocyte cultures (MLC), these DCieuaugment leukemia-specific / antileukemic reactions compared to controls without the added drug combination. This was shown with WB and WBM, independent of patient characteristics such as gender, age, HLA, mutation / transplantation status, subtype / risk group, before / after SCT) and also in a wide dose range of added PGE1. (Aslan 2025, Unterfrauner 2023).

[0054] Through phagocytosis of tumor / leukemic particles, antileukemic / antitumor-effective DC mediators can be formed via the " DC phagocytosis pathway" and immunological effector and memory cells can be created.

[0055] These phagocytosis properties are basic properties of all dendritic cells (Amon 2020, Cabeza-Cabrerizo 2021, Greter 2012).

[0056] In line with the phagocytotic / processing and antigen presenting function of DC induced by the combination of GM-CSF and PGE1 (Kit M), the number of tumor cells (in spheroids) from ascites of patients treated with Kit M was found to be reduced in comparison to untreated cells. In spheroids that also contain fibroblasts and immune cells, tumor cell particles have to be enriched with autologous heparinized whole blood samples from the same patient to increase the amount of monocytes / blood stem cells (= sources for DC) and of naive (lateron activated) adaptive and innate immune effector cells. In cultures treated with Kit M, immature (phagocytotically active) DC are expected to convert to mature DC expressing DC markers such as CD80 / 86 / 206 / 40, together with CCR7 and CD83 as maturation markers as well as tumor antigens. Immune cells that have been specifically activated by this process lead to reduced tumor cell frequencies after 7 daysof culture compared to controls (detected by viability assays). This finding hints at the involvement and induction of a DC-triggered, immune-mediated, anti-tumor immune mechanism mediated by DC via the phagocytosis pathway (Halfter 2015). These data also suggest that Kit M can reverse immune escape (which may be caused by silencing immune cells or masking of tumor antigens, etc.) and activate the immune system specifically against tumor cells. It is assumed that this reversion is effected by DC phagocytosing "tumor cell particles" or "particles from infectious agents", respectively.

[0057] Through phagocytosis of infectious particles, anti-infective DC mediators can be formed via the " DC phagocytosis pathway" and immunological effector and memory cells can be created. As the above mentioned phagocytotic properties are common to all dendritic cells, specific immune responses against various pathogens may be induced and / or enhanced by the activation of DC by Kit M.

[0058] Monocyte / stem cell-derived DCs can be generated from healthy WB samples and these DC increase the proportion of antigen-specific (staphylococcal enterotoxin-detecting) cells after MLC.

[0059] We have demonstrated that these WB / WBM samples simulate the in vivo situation in patients or healthy subjects. Using such ex vivo WB / WBM samples, we generated DC / DCieuthat drive (antigen / leukemia-specific) immune responses in vivo. In the presence of the drug combination (GM-CSF+PGE1), leukemic cell derived DC (DCieu) as well as healthy phagocytic DCs are produced in WB / WBM samples (see Figure 1). Thus, leukemia patients can be treated directly with the drug combination (GM-CSF+PGE1) without prior ex vivo production / purification of DCieu, as the patients themselves can generate DC / DCieuin vivo and activate their immune system in an antigen- or leukemia-specific manner. In addition, memory cells are formed, which could prevent tumor progression or recurrence of tumor cells in patients in whom remission has been achieved. In leukemia patients, not all DCs that develop are "leukemia-derived"; DCs also develop from healthy blood samples (from monocytes and / or stem cells). Hence, both normal and healthy DCs are produced, which mature, take up, process, and ultimately present infectious / tumor / leukemia-derived particles, as is known for DCs (Amberger 2020). In this way, in addition to the proven DCieu-triggered anti-leukemic processes, antileukemic (or anti-infective / antitumor) DC processes can also be initiated via the phagocytosis pathway in leukemia patients. In patients with solid tumors or in patients with (chronic) microbial / parasitic infections or insufficient or inadequate hematopoiesis (e.g., due to congenital diseases or acquired conditions in leukemia patients or after chemotherapy), the regeneration of stem cells and the number of stem cell-derived cells (neutrophils, erythrocytes, thrombocytes, lymphocytes) can be improved.

[0060] Treatments of leukemic rats and of therapy-refractory AML patients with the combination of GM-CSF and PGE1 were very well tolerated, did not induce blast proliferation (on the contrary, they reduced or stabilized the amount of blasts). Anti-leukemic cells were produced in vivo. In addition, leukemia-specific effector and memory cells were induced (see Figure 6, Atzler 2024).Preliminary data from WB samples from ALL / CLL patients treated ex vivo with the drug combination (GM-CSF+PGE1) have shown that 1) DCieu(detectable by coexpression of DC with patient-specific leukemia antigens) are produced, and 2) after mixed lymphocyte culture of blood samples pretreated with the drug combination (GM-CSF+PGEl) and enriched with patient T cells (leukemia-specific), adaptive and innate immune effector and memory cells are increased, and 3) blast lysis (detectable by a blast lysis test) is improved by these cells with similar efficiency to that seen in AML compared to controls.

[0061] Implementation of the invention in clinical practice

[0062] The invention described helps patients with hematopoietic or stem cell deficiencies (resulting in erythrocytopenia, thrombocytopenia, neutropenia, or lymphopenia) of any cause, by promoting the in vivo generation of mono / stem cell-derived DCs via the " DC phagocytosis pathway". In their immature state, stem cell-derived / monocyte-derived DC phagocytose tumor / leukemia / infectious (microbial / parasitic) particles, process them, and present them to the immune system. Furthermore, leukemic cells can be converted directly into leukemia-derived DC (DCieu)jn leukemia patients (see Figure 1). This addresses the overall hemopoetic / stem cell deficiency of these patients.

[0063] Benefit for patients with acute or chronic myeloid / myeloproliferative and lymphatic (acute) leukemias, especially immuno-competent patients in remission

[0064] Patients who have undergone successful chemotherapy or stem cell transplantation for acute or chronic leukemia often relapse because leukemic cells remain in the body and ultimately lead to relapses. Repeated chemotherapy is often ineffective because patients become intolerant (requiring a reduction in dose and thus efficacy) or the leukemic cells become resistant to the therapies. Treatments with milder therapies (e.g., hypomethylating therapies) usually no longer achieve remission of the disease but are used to slow disease progression or to provide palliative care.

[0065] In patients with MYELOID LEUKEMIA (AML) before or after SCT, (blast-containing) blood or bone marrow can be collected and used to produce (leukemia-derived) DCs in ex vivo cell cultures by adding Kit M (GM-CSF+PGEl). Mixed lymphocyte cultures (enriched with patients' T cells) can then be supplemented with these DC to give rise to (leukemia-specific) immune effector and memory cells, leading to a significantly improved blast reduction in cell culture (see Figures 2-5). Treatment with Kit M is effective on cells from patients independent of their age, gender, HLA, mutation, risk, or transplant status.

[0066] Patients with therapy-refractory AML after repeated chemotherapy with hypomethylating drugs or after multiple stem cell transplants benefited from in vivo treatment with Kit M (GM-CSF+PGEl) judged by the following parameters: leukemia-specific suppressor cells were reduced, activating and creating leukemia-specific effector and memory immune cells that were detected in the blood (Figure 6). Overall blood formation was at least partially normalized (lower transfusion requirements), and the disease was stabilized for several weeks or months even though the patients were in the end-stage of their disease.

[0067] A big advantage of in vivo immunomodulatory treatment with Kit M (GM-CSF+ PGE1) is that it can be repeated indefinitely or at any time, as no cell products need to be produced ex vivo and re-infused after manipulation. Frequently, the yields of ex vivo manipulated cells are low and lead times high - it is unfortunately not uncommon that patients do not survive long enough to be able to benefit from such therapies. Another problem of ex vivo immune therapies is the high cost of necessary GMP production, which hampers widespread adoption. In contrast, in vivo therapy is readily available, easy-to-use and cheap. Furthermore, in vivo therapy with Kit M improves overall hematopoiesis, induces both DC and DCieuand exploits the migration competence of DC and DCieutriggered immune cells to extramedullary locations.

[0068] Analogous to AML patient blood samples, DCieucan also be generated from blood samples from ALL and CLL patients using the drug combination (GM-CSF+PGE1). After subsequent mixed lymphocyte culture, (leukemiaspecific) immune cells (effector cells, memory cells) arise that enhance blast lysis (see Figure 2-5). These activities are somewhat lower than the effect of 'lymphatic cocktails' (Kits 1 and 2, Muzio 2000, Yamanaka 2005). Kits 1 and 2 are not approved for in vivo use, Kit M (the components of which are clinically approved) may be used to stabilize remissions / disease in patients despite lower activity. Both the DC / DCieu-triggered mechanism and the 'DC phagocytosis pathway' (phagocytosis of leukemic particles) lead to activated effector and memory cells and stimulate hematopoietic regeneration through the stem cell effect. In principle, patients before and after stem cell transplantation and in various stages and subgroups of the disease should benefit from these in vivo treatments.

[0069] Benefits for patients with solid tumors and lymphomas

[0070] Patients with all sorts of solid tumors (of the internal organs, nervous system, lymphomas, melanomas) are mostly treated with targeted (chemo- / immuno)therapy, which usually reduces but does not eliminate the tumor load. As a result of the therapy, residual tumor cells or particles circulate in the blood. In a healthy organism, tumor cells or tumor cell particles would be phagocytosed by dendritic cells via the " DC phagocytosis pathway," processed, and presented to the DC, leading to specific activation of the immune system, followed by elimination of residual tumor cells and the creation of specific immunological memory. In a healthy immuno-competent organism, these DC-triggered processes normally prevent tumor regrowth. Treatment of patients with the above-mentioned solid tumors (after chemotherapy) with the drug combination (GM-CSF+PGE1) could, on the one hand, improve stem cell / blood formation regenerationthrough its hematopoiesis-enhancing effect (see point 1) and, on the other hand, eliminate (residual) tumor cells / particles in the organism, coupled with DC-induced specific effector cell / memory cell immune activation against the tumor cells, increase anti-tumor effector functions and prevent relapses through the induced immunological memory. The combination of activating and modulating substances is expected to compensate for the insufficient DC generation in these patients.

[0071] Benefit for patients with (chronic) infections

[0072] Patients with (microbial / parasitic) infections that cannot be eliminated by conventional antimicrobial / anti-parasitic therapies suffer from recurrent or persistent infections due to a lack of immune defense. Prolonged use of insufficiently effective therapies can cause the development of resistance (and the microbes / parasites survive in the body). In a healthy organism, infected cells or the microbes / parasites themselves or their particles would be phagocytosed by dendritic cells via the 'DC phagocytosis pathway', processed, and their antigens presented on the DC, leading to specific activation of the immune system, followed by elimination of (residual) microbes / parasites or affected cells, as well as the creation of a specific immunological memory. Due to a sufficient immuno-competence in a healthy organism, these DC-triggered processes prevent or eliminate infections.

[0073] Treatment of patients with persistent / recurrent (chronic) infections with a drug combination (GM-CSF+PGE1) may improve stem cell / blood formation regeneration through its hematopoiesis-enhancing effect as well as eliminate residual infected cells / particles in the organism, coupled with DC-induced specific effector cell / memory cell immune activation against the microbially / parasitically affected cells through 'anti-infective' effector functions, and an induced immunological memory is expected to prevent relapses.

[0074] Benefit for patients with stem cell deficiencies

[0075] There are congenital and acquired stem cell defects. In congenital stem cell-related defects, insufficient amounts of blood cells of all differentiation lines may be present. Acquired stem cell defects can be disease-related (e.g., in patients with leukemia (e.g., AML, ALL), healthy hematopoiesis is suppressed by aggressive therapies that impair bone marrow / blood formation (myeloablative) chemotherapy following treatment for solid tumors or in preparation for allogeneic stem cell transplants), or may also be caused by infectious (microbial / parasitic) blood formation deficiencies (e.g., in HIV, CMV infections).

[0076] The consequence of all these acquired or congenital diseases is that the hematopoietic system cannot produce enough blood cells of all types: Depending on the blood cell lines affected, the symptoms experienced by patients range from impaired stem cell regeneration, thrombocyte, erythrocyte, granulocyte formation, to impaired infection / tumor defense (due to missing or insufficiently activated lymphocytes (due to missing ordeficient 'processing and presentation' of infection and tumor antigens by antigen-presenting cells), or due to insufficient formation of soluble components (cytokines, antibodies).

[0077] Depending on the case, symptomatic treatment with anti-infective (antimicrobial: e.g., antibiotics, anti-parasitics) or antitumor / antileukemic therapies (e.g., chemotherapy), stem cell-activating substances (e.g., GM-CSF, G-CSF), or recreation of a functional hematopoietic system via stem cell transplantation may be tried. Treatment of patients with the aforementioned hematopoietic deficiencies with the drug combination (GM-CSF+PGE1) is expected to counteract the above-mentioned clinical symptoms due to its stem cell efficacy and its hematopoietic-enhancing efficacy.

[0078] Dosage, form of application and duration of use for the above mentioned applications

[0079] The substances according to the invention may, in certain embodiments, be administered in very low, immunomodulatory doses based on the dosages specified by the manufacturer.

[0080] In certain embodiments, the optimal dosage may be determined in animals (tolerability, adverse events, generation of hematopoietic stem cells / derivatives, provision of DC (DCieuin leukemia patients), generation of (leukemia-specific / tumor- / infection-specific antigen-specific) immune effector and memory cells. In cell culture trials (with AML blood samples), it was shown that sequential addition of the substances to whole blood cultures produces comparable amounts of DC subtypes compared to simultaneous addition (data not shown). The order of administration of more than one substance to patients should - initially - be sequential in order to better assess any possible (adverse) effects or side effects of the individual substances (starting with GM-CSF, e.g. parenterally, preferably as i.v. administration or subcutaneous administration, for cytokinebased stimulation of DC differentiation. The second substance should be administered as a "danger signal" on days 2-3, and the third or first substance again on days 3-5. (In the treatment of AML patients in a therapeutic trial, it was shown that the infusion of GM-CSF over 4 hours, with PGE1 infused after 1 hour, was well tolerated and showed an immunostimulatory effect in vivo without inducing proliferation of leukemic cells.

[0081] In animal experiments on rats, it was shown that the simultaneous infusion of GM-CSF and PGE 1 was well tolerated and that immune activation occurred.

[0082] The form of administration (e.g: intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.), intradermal (i.d. ), subdermal, intranasal) may be selected in accordance with the instructions and recommendations of the drug manufacturer. In the case of i.v. administration, a direct influence of the substances on all cells in the blood must be ensured. Alternatively, the drugs may be administered s.c. - this would allow patients to administer the medication themselves (after instruction).The duration of use in patients can range from at least 3 months to 2 years and must also be decided in the context of clinical use in patients: The treatment duration may be upto 180 days, preferably as recommended by the manufacturers of the substances listed here.

[0083] The advantage of using the drug combination (GM-CSF+PGEl) in all of the patient groups mentioned is that the effects on the blood count and on the generation of (specific) immune effector and memory cells can be monitored. If all blood parameters normalize, treatment could then be tapered off; if hematopoietic insufficiency persists or (leukemic / tumor cell markers) become detectable again, treatment could be resumed. How maintenance therapy or regeneration therapy can be designed must be determined in clinical studies:

[0084] In addition to examining blood samples before / after their ex vivo processing, regular examinations of patients' blood samples may be carried out during in vivo treatment with the drug combination (GM-CSF+PGEl) (monitoring) in order to record their efficacy and side effect profiles. These tests may preferably be performed BEFORE the start of application (control values) and at several points during the course of treatment. These points in time should be based on those recommended for other immunotherapeutic treatments: The initial focus is on quantifying leukemic / tumor / infected cells (or their markers) using flow cytometric / hemato-logical / serum measurements and by recording molecular cell profiles (overexpression of tumor / infection antigens, PCR). The present invention further envisages the provision of a monitoring panel that records the cells involved in antileukemic / antitumor / anti-infective responses (e.g., T cell subpopulations (regulatory, CD4 / CD8, memory / effector T cells, T cells with defined vp profile, functional properties of involved T cells, effector and memory cells, as well as NK and iNKT cells). Humoral factors (inhibitory (e.g., IL10, TGF, CXCR4) / stimulatory soluble factors, chemokines, cytokines (e.g., IL-2, IL-12, IL-17, IFNγ), antibodies should also be recorded in order to obtain as accurate a picture as possible of the infection / tumor / leukemia defense. By monitoring soluble and cellular factors at various points in the course of the disease and correlating the data with clinical events (e.g., recurrence, persistence, progression), the clinical relevance, treatment success, and prognostic value of our approach can ultimately be determined.

[0085] The present invention further envisages the development of an "in vivo protocol" that enables the application of minimized combinations of immunomodulators (GM-CSF+PGEl) in patients. This eliminates the need for a clean room (" GM P facility") for the manufacture of cell products.

[0086] The detailed design of a personalized treatment protocol for patients with hematopoietic insufficiencies / leukemia / tumors / lymphomas / infections with the drug combination (GM-CSF+PGEl) together with a detailed concept for immune monitoring may be carried out in consultation with the attending physicians before the start of treatment and may additionally be based on further input, e.g. other immunotherapeutic treatment protocols. A diagnostic companion program for the examination of routinely collected blood samples and clinical findings on patient progress may also be provided and used.

[0087] This means that existing experience, expertise, and synergies can be used for the clinical implementation of the invention to help patients with the aforementioned diseases.EXAMPLES

[0088] Example 1

[0089] Functional and Immunological Monitoring of Immune-Reactive and AntiLeukemic Cells (T, NK, CIK, B-Cells) arising in WB from AML, ALL, or CLL Patients under Treatment with Several Immunomodulatory Approaches

[0090] Materials and Methods

[0091] Sample Collection

[0092] Sample acquisition was conducted after obtaining informed consent of the blood donor and in accordance with the Helsinki protocol and the ethics committee of the Ludwig-Maximilians-University-Hospital Munich (Vote-No 339-05). Heparinised peripheral whole blood (WB) samples were provided by the University Hospitals of Munich, Augsburg, Oldenburg, Tubingen, Frankfurt, the Rotkreuzklinikum in Munich, the Diakonieklinikum in Stuttgart, and the St.-Josefs-Hospital in Hagen between 2016 and 2023.

[0093] Flow Cytometry to characterize and quantify cell subsets and their functionality

[0094] To evaluate and quantify phenotypes of DC / DCieu, leukemic blasts, monocytes, and immune-reactive cell subsets of the adaptive and innate immunity, analyses were conducted via flow cytometry, using a fluorescenceactivating cell-sorting flow cytometer (FACS CaliburTM). Using a refined gating technique and the analysis software Cell Quest-Pro (Becton Dickinson, Heidelberg, Germany), functionalities of cells (proliferation, cytokine production, degranulation, and cytotoxicity) could be investigated, as shown before

[0012] , Evaluation and quantification of stained cells was obtained with the fluorescence-activated cell sorting flow cytometer FACS Calibur (Becton Dickinson) and the analysis software Cell Quest-Pro 6.1 (Becton Dickinson), applying a refined gating strategy, as shown before

[0035] ,

[0095] Dendritic Cell Culture (DCC)

[0096] The generation of DC / DCieufrom healthy and leukemic WB was performed using 3 different DC-generating methods: " Kit-1" containing 20pg / mL IL-4 (ThermoFischer Scientific, Darmstadt, Germany), 3pg / mL CD40L(ThermoFischer Scientific, Darmstadt, Germany); " Kit-2" containing 800U / ml GM-CSF; Sanofi-Aventis, Frankfurt, Germany), 20pg / mL IL-4 (ThermoFischer Scientific, Darmstadt, Germany) and 10ng / mL Tumor Necrosis Factor Alpha (TNFa; ThermoFischer Scientific, Darmstadt, Germany) and " Kit-M" containing 800 U / ml GM-CSF; Sanofi-Aventis, Frankfurt, Germany) and 1 µg / ml Prostaglandin-E1 (PGE1; Santa Cruz Biotechnology) [21-35],T Cell-Enriched Mixed Lymphocyte Culture (MLC)

[0097] To generate T cell-enriched immune-reactive cells, thawed autologous T cells were stimulated with DC / DCieucontaining Kit-pretreated WB. Flow cytometric analyses of T-cell subsets were quantified using a refined gating strategy, as shown before

[0035] ,

[0098] Intracellular Cytokine Assay (INTCYT) and De-granulation Assay (DEG)

[0099] INTCYT and DEG cultures were set up as described

[0035] to detect intracellular IFNy producing leukemiaspecific cells and leukemia-specific cells, as given in Table 1. Flow cytometric analyses of the INTCYT- and the DEG-assays were quantified using a refined gating strategy.

[0100] Table 1: Cells and cell subsets as evaluated by flow cytometry

[0101] Name of Abbreviati Surface Marker Referre Abbreviation Reference Subgroups on of d to

[0102] Subgroups

[0103] Blast cells Blasts BLA BLA e.g. WB BLA / WB (Schmetzer CD19+, CD20+, (whole et al., 2007) CD34+, CD117+ blood)

[0104] Proliferating BLAprol-CD71 BLA+DC-CD71+ BLA BLAprOi-cD7i / BLA (Plett et al., blasts 2017) Proliferating BLAprol-IPO38 BLA+DC-IPO38+ BLA BLAproi-ipo38 / BLA (Ansprenger blasts et al., 2020) Dendritic cells Dendritic DC DC+ e.g. CD80+, WB DC / WB (Klauer et cells CD206+ al., 2025) Leukemia- DC|eu DC+BLA+ WB or DCieu / WB (Klauer et derived DC DCieu / BLA al., 2025) DC or BLA

[0105] Mature DCmal DC+CD197+ WB DCmai / WB (Klauer et migratory al., 2025) DC

[0106] Mature DC|eu-mat DC+BLA+CD197+ BLA DCieu-mai / BLA (Klauer et migratory al., 2025) DC|eu

[0107] T lymphocytes CD3+ pan T CD3+ CD3+ lympho T3+ / cells (Klauer et cells cytes al., 2025) Non-naive T Tnon-naive CD3+CD45RO+ CD3+ TnOn-naive / T3+ (Klauer et cells al., 2021) Central Tcm CD3+CD45RO+ CD3+ Tcm / T3+(Klauer et memory CD197+ al., 2021) T cells

[0108] Effector Tem-eff CD3+CD45RO+ CD3+ Tem / err / T3+ (Klauer et memory CD197- al., 2021) T cells

[0109] Proliferating Tprol-early CD3+CD69+ CD3+ Tproi-eariy / T3+ (Klauer et T cells al., 2021)

[0110]

[0111] - earlyName of Abbreviati Surface Marker Referre Abbreviation Reference Subgroups on of d to

[0112] Subgroups

[0113] Leukemia-specific cells

[0114] B lymphocytes CD19+ B B cell107a+CD19+CD107a+ B cell deg+B / B

[0115] cells

[0116] T lymphocytes deg+ / IFNy+ deg+T3+CD3+CD107a+ T3+deg+T3+ / T3+ (Aktas et CD3+ pan IFNy+T3+CD3+IFNy+ T3+IFNy+T3+ / T3+ al., 2009) T cells (Klauer et al., 2021) deg+ / IFNy+ deg+TnOn- CD3+CD45RO+ Tnon-naive deg+Tnon-naive / (AktaS et Non-naive T naive CD107a+ Tnon-naive Tnon-naive al., 2009) cells IFNy+TNOn- CD3+CD45RO+IFNy+ IFNy+ Tnon-naive / (Klauer et naive Tnon-naive al., 2021) deg+ / IFNy+ deg+TcmCD3+CD45RO+ Tcmdeg+Tcm / Tcm (Aktas et Central IFNy+TcmCD197+CD107a+ Tcm IFNy+Tcm / Tcm al., 2009) memory T CD3+CD45RO+ (Klauer et cells CD197+IFNy+ al., 2021) deg+ / IFNy+ deg+Tem / err CD3+CD45RO+ Tem / err deg+ Tem / err / (Aktas et Effector 1 FNy+Tem / err CD197+CD107a+ Tem / eff Tem / eff IFNy+ al., 2009) memory T deg+T4+reg CD3+CD45RO+ T4+reg+ Tem / eff / Tem / eff (Klauer et cells CD197+IFNy+ deg+ T4+reg+ / al., 2021) deg+Regulat CD4+CD25+ T4+reg+ (Klauer et oryT cells CD127lowCD107a+ al., 2021) Cytoki deg+ / IFNy+ deg+CIK CD3+CD56+ CIK cell deg+CIK / CIK (Aktas et ne- CD3+ CD56+ IFNy+CIK CD107a+ CIK cell IFNy+ CIK / CIK al., 2009) Innat indue CIK cells CD3+CD56+IFNy+ (Klauer et e ed al., 2021) immu killer

[0117] ne cells

[0118] syste Natur CD3-CD56+ deg+NK CD3+CD56+ NK cell deg+NK / NK (Aktas et m al NK cells IFNy+NK CD107a+ NK cell IFNy+ NK / NK al., 2009) killer CD3+CD56+IFNy+ (Klauer et

[0119]

[0120] cells al., 2021)

[0121] Table 1 (continued): Cells and cell subsets as evaluated by flow cytometryCytotoxicity Fluorolysis Assay (CTX)

[0122] The Cytotoxicity Fluorolysis Assay was performed to analyse the blast lytic activity of T cell-enriched immune-reactive cells in MLCWB DC(Kit-1, Kit-2, Kit-M)and MLCWB DC(Control). Flow cytometric analyses were performed using a refined gating strategy. Achieved blast lytic activity was defined as the percentage difference of viable target cells (blasts) between the effector-target-cell culture and the control, as shown before

[0035] ,

[0123] Statistical Methods

[0124] Statistical analyses and figures were implemented with Excel 2022 (Microsoft, Redmond, WA, USA) and Prism 9 (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation. In some comparisons / correlations, relatively changed frequencies of cell subsets (deltas) between Kit-treated and untreated settings are given. Statistical analyses were conducted using t-tests: Differences were considered as "highly significant" in cases with p-values ≤ 0.005, as "significant" with p-values <0.05, and as "borderline significant" with p-values between 0.05 and 0.10.

[0125] Results

[0126] In the samples, between 2% and 94% of blasts and varying proportions of the remaining hematopoietic cells were detectable. Details of the cellular composition of AML / ALL / CLL samples are shown in Table 2. In this study, uncultured immune reactive cells from AML, ALL, CLL, and healthy donors' WB samples and samples after the influence of several Kits for DC generation were analyzed. In two patients with low blasts (<5%), DC / DCieu could not be quantified. Furthermore, (leukemia-specific) immune cells (IFNy-producing / degranu-lating T, NK, CIK, B-cells) following DC / MLC culture were quantified. Results were correlated with (ex vivo) functional as well as patients' clinical and prognostic data.

[0127] Stimulation of uncultured (AML, ALL, CLL, and Healthy) WB with LAA / SEB increases frequencies of (antigenspecific) intracellularly IFNY-producing and degranulating immune cells

[0128] In AML patients' uncultured WB, frequencies of leukemia-specific intracellularly IFNγ-producing or degranulating cells were comparable thereby confirming data already demonstrated before (e.g., Unterfrauner et al., 2023

[0037] ). The frequencies of intracellular IFNy-producing and degranulating adaptive and innate immune cells (e.g, Tnon-naive, Tcm, Tem / eff, CIK, NK cells) are increased (or decreased: T4+reg) after the addition of leukemic (WT1, PRAME) antigens. The results of AML-Myo and AML-Ly were comparable (data not shown). In ALL patients' uncultured WB, non-significantly higher frequencies of intracellular IFNy-producing cells with LAA (STIM) vs. without LAA stimulation (UNSTIM) (e.g., IFNy+Tcm / Tcm, IFNy+T3+i?7+ / T3+i?7+) were found, significantly or borderline higher frequencies (e.g., % IFNy+Tcm / Tcm: UNSTIM: 28.93±20.88, STIM: 36.72±18.56, p= 0.01; IFNγ+T3+β7+ / T3+β7+: UNSTIM: 31.05±15.93, STIM: 37.49±16.30, p= 0.08) compared to unstimulated WB were observed. For degranulating T cells or B cells with / without LAA stimulation,differences were pronounced. In CLL patients' uncultured WB, no significant differences in intracellular I FNy-producing or in degranulating cells with vs. without LAA stimulation were observed. In healthy samples exposed to Staphylococcal Enterotoxin B (SEB) stimulation, (non)significantly higher frequencies of intracellular IFNy-producing and degranulating cells with vs. without SEB stimulation were observed, thereby confirming data shown before

[0011] ,Table 2: Patients' characteristics

[0129] “lmtAge. Sex Stage Blast phenotype {CD1 Experiments Conducted « ith WB NO UtaSSlilCatlOn JtLNibWiLUNCvNl WB 1419 65, f dgn. pAML _y toeT-e 19. 54. •, T13 7 94 DC MLC. CTX 1461 Mm dgn. pAML (lyj Adverse 19, 34, 22, 1&.65, 33 611 DC, MLC, CTX 1568 29, m dp. p*ML(LyJ llnternwiate 19, 34, 20, 117, 13 60 DC, MIC, D< UG,q>, lnCyt(UC, G), CTX 1574 ». ■ dpi sAMJLyj nd 19, 34, 117, 15 33 DC, MLC, Defl(LIC, CJ, InCyffUC. C), CTX 1® 62,, f dgn. sAML (Mp) Interned ate 34, 117, 13, 14 34 CL r.’u. 3eg(UC, CJ, lnCyt(iJC, C|, GTX 1567 9fl,f dgn., 34, 117, 1565 16 DC, MILC, De< JC|, InCyl(UC), CTX A>dVvf-38 1570 37, f dgn. pAML (Mjo) Fawatte 34, 11765,13,33 8 DC, MILC, Deg(UtC): MytjUC. CJ, CTX 1572 63, f dgn. pAML Pyojl Ad«e 34, 11766, 33, 13 12 DC, MIC, DeglC), IhCytlJC], CTX 1573 61, ■ dgn pAML (Bjoj Miwse 34. 117, 65, 13 13 DC, MIC, Deg(UC, Q, toCytfQ, CTX 1594 70, f dp- pAMLJMyo) Favorable 34, 1176633, 56 2 DC,, MIC, DegUC, Qi, lnCji:(UC, CJ, CTX 1641 65, f ReteSCT IAML Pye) ltd. 34. 117,33,65 6 DC, MIC, DegflJC. GK lnCyt(UC. G), CTX 14B 6D;f ReteSCT sAML(Ly) nJ. 19, 34, 2, 3, 13, » 6 DC, MLC, CTX 1587 57, ■ dp CALL (Bl) High risk 19, 34, 1115 33 DC, MIC, Deg(UC, Q, hCj1fUC. C]i. CTX 1666 56. f dp. rtLLIBB), 19, 34, 10, 20.22 30 DC MIC, DegfUGC). W(UC,q, CTX High n» 1626 28, in dgn. rtLLpil):. 19, 34, 10, 20, 22 20 DC, MLC, DegfUGA hCytfUC. CJ, CTX 1646 26, ■ dgn. tALLpB) M^trisk 19, 34, 10, 20,22 40 DC, MIC, DeB(WC)4lnCyt(UC, C) 1653 77, in dgn poBALL(BI) Hflh ris* 19, 34, 22 55 DC, MIC, Deg(UC, C), hCji(UC, Q, CTX 1662 46, ■ dgn. tALLflll) Staw& Kt 19, 10,22,65 « DC, MLC, DegUCfl, InCytfUC. C), CTX 1670 23, m dgn. pro^ALL(BI) StarnM 19, 34,3, 33 20 DC, MIC, DegfUCfl, hCyt(UC, C| CTX 1676 55,1 dp. cALLpll) Hgh ns* 19, 34, 10, 73a 61 t. L.. “■ 1677 73, f dgn. prfLALL(BI] St'darC 19, 34, 22, 65, 79a 26 DC, MLC, DegflJCft lnCyi(UC, Q, CTX W 61, tn ReL CALL (01) StentM 19, 34, 10,20,23 2 L.. 1 2.“ ■ 1657 35, ■ ReL Pte-TJ«1(BIO H^irisk 5, 34, 2,4, 7 30 DC, MLC, D< UC, Cj, ln& KUC, CJi, CTX 1589 61,1 Pers, BCLL A 19, 20,5, 15,23 45 DC, MLC. CTX 1591 82, ■ ta. ELCLL A 19. 5, 15- 2D 52 DC, MLC, CTX 1639 75, 1 Pte. 84X1 nJ. ia, M 23 57 DC, MLC, Deg(UC, G}, W(UG,qCTX 1678 50, ■ Pera. 8CLL A 19, 5,20, 22,23,79b 80 DC, MLC, DegflJ& C), InCjt (UC, Q, CTX 1681 66, ■ Pera. B4JLL A 19, 5 83 DC, MLC, DegfC), InCyUCJi, CTX 1687 37, « Pera. 8431 A 19, 5,20 17 DC, MLC, DegflJQ, hCjtfUC) 1658 66, m Pera. 801 A 19, 5, 20, 79b 65 DC, MLC, DegfUQ, InCWUQ 1563 26, f Heafcy DC, MLC 1566 54, f Heafcy DC, MLC, DegUQ, InCyl (UC) 1652: 43, ■> Hedfty DC, MLC 1661 34, f Healthy CL MLC De:, JC Ci 1666 30, « Hedlhy DC, MLC, Deg(UC, CJ, bCy»(UC, Q 1667 25, f Healthy L.l'_: L J. t: CT t

[0130]

[0131] ■-C8 60 f Healthy DC MLC Dej. JC Ci -CyiLK C; Legend: t female: irc mate; WHO classifcat»i: World Health Organization classMcafion: acute myeloid teutemia; pAML: preliminary AML; sAML secondary AML: IAML: thsHipyeretatetf AML; AML(Ly): AML Masts with lymphoid matter expression; AML(Myp): AML blasts without lymphoid marker expression; ELN: European Leukemia Network; NCCN: National Qomprefensiw Cancer Network; GMALL: German MulticeniBr Study Group for Adult Acute LyenphotfasBc Leukemia; dgn: first diagnosis; Pers: persistence; Ret: retapse; ReL a. SGT: relapse after SCT; WB: whole btood; CD: Ouster of dMerertjation; told: antibody wed for expression analyses,; n.d„: no data; DC: dendritic cell culture; MILC: mired lymphocyte culture; Deg: degranulation assay; InCyt intracelular cytokine assay; CTX: cytotoxicity assay; UC: measurements in uncultured samples; C: measure merits after culture:

[0132] (Leukemia) derived DC / DC / DCleuare increased in AML, ALL, CLL patients, or healthy donors' WB, compared to Control

[0133] In AML patients' samples, it has been shown before (e.g., Unterfrauner et al., 2023

[0037] ), that the frequencies of (mature / leukemia-derived) DC can be significantly increased under the influence of Kit-M (vs control) without induction of AML blasts' proliferation (data not shown) were confirmed. Additionally, only non-significantly higher frequencies of DC / DCieuin Kit-1 and Kit-2 pretreated WB compared to the control were found. In a detailed analysis, it is confirmed that more cases with higher DC / DCieuvalues compared to control were found in AML using Kit-M compared to Kit-1 or Kit-2-pretreatment (e.g., DC / WB: Kit-1: 1 / 8 (1 of 8 cases); Kit-2: 4 / 8; Kit-M: 8 / 11; DCieu / BLA: Kit-1: 3 / 8; Kit-2: 4 / 8; Kit-M: 8 / 11) (see Fig.2B). The results of DC / DCieuin AML-Myo and in AML-Ly' WB under the influence of Kit-M were comparable (see Fig.2E, F) (in one case, with <5% blasts in PB, DC could not be quantified). In ALL patients' samples, higher average frequencies of leukemia derived DC / DCieusubtypes following treatment with Kit-1 (DCWB (Kit-i>), Kit-2 (DCWB (Kit-2>) and Kit-M (DCWB (Kit-M)) compared to control group (DCWB(controi)) (e.g., %DC / WB: Kit-l:13.59±7.85; Kit-2: 12.27±3.58; Kit-M: 13.33±4.08; Control: 9.37±2.18; p= 0.12, p= 0.003, p= 0.008;o / oDCieu / BLA: Kit-1: 35.59±20.39; Kit-2: 33.83±13.7; Kit-M: 32.28±16.54; Control: 22.60±9.32; p= 0.01, p= 0.02; p= 0.01, (see Fig. 2 A)) (in one case with <5% blasts in PB DCieucould not be quantified) were generated. Proliferation of blasts (as detected by the co-expression of CD71 or IPO-38) from leukemic WB was not induced by Kit-M, Kit-1, and Kit-2 treatment (data not shown).

[0134] In summary, higher frequencies of DCs and their subtypes in Kit-l-treated ALL-WB, followed by Kit-M and Kit-2, compared to the control group were observed. Compared to AML, in general frequencies of DC / DCieusubtypes were (non-significantly) lower. In a detailed analysis, "cut-off" values with respect to frequencies of DC subtypes obtained in > two-thirds of AML cases using Kit-M were defined. In ALL cases, more cases with DC / DCieu were seen using Kit-1 followed by Kit-2 and Kit-M-pretreatment (e.g., DC / WB: Kit-1: 5 / 8 (5 of 8 cases); Kit-2: 4 / 9; Kit-M: 5 / 10; DCieu / BLA: Kit-1: 3 / 8; Kit-2: 3 / 9; Kit-M: 3 / 10) (see Fig. 2A). Comparing the results between ALL and AML patients' samples, it was found that AML samples, treated with Kit-M, generated more DCieu / BLA (p= 0.05) and DC ieu-mat / BLA (p= 0.08) (see Fig. 2A and B). In CLL patients' samples, slightly increased frequencies of DC / DCieu / DCieu-mat were found in the blasts' fractions using Kit-M or Kit-1 vs. Control (e.g., %DCieu-mat / BLA: Kit-1: 15.61±11.89; Control: 12.64±12.64, p= 0.03). Proliferation of blasts was not induced by Kit-M, Kit-1, and Kit-2 treatment (data not shown).

[0135] Compared to ALL and AML patients' samples, fewer cases with DC / DCieuvalues higher than the control values were found (see Fig. 2C). Due to the low number of CLL cases, a comparison with AML patients' samples was not performed. In healthy samples, we found non-significantly higher frequencies of DC within WB when using Kit-M (DCwB(Kit-M)), Kit-1 (DCwB(Kit-i)), Kit-2 (DCWB(Kit-2>) compared to control (DCWB(controi)) (see Fig. 2D).

[0136] Activated and memory T cells are increased after MLC with Kit-1, Kit-2, and Kit-M- pretreatment of patients' or healthy donors' WB

[0137] In AML patients' samples, significantly higher frequencies of Tcmsubsets and non-naive T cell subsets in M LCwB-DC(Kit- ) compared to M LCWB-Dc(controi) (%Tcm / T3+: Kit-M: 22.53±15.55; Control: 14.92±15.23; p= 0.05 and %TnOn-naive / T3+: Kit-M: 55.96±19.95; Control: 48.63±17.64; p= 0.04) were found. In a detailed analysis, we confirmed that there were more cases with higher frequencies of TnOn-naive and Tcmin AML using Kit-M thanKit-1 or Kit-2-pretreatment (e.g., Tproi-eariy / T3+: Kit-1: 5 / 9; Kit-2: 4 / 8; Kit-M: 8 / 12; Tem / eff / T3+: Kit-1: 5 / 9 (5 of 9 cases); Kit-2: 5 / 8; Kit-M: 8 / 12) (Fig. 3B). Comparable results were obtained comparing AML-Myo and AML-Ly of T cell subsets after MLC with Kit-M-pretreated WB (MLCWB-DC(Kit -M; (see Fig. 3E, F).

[0138] In ALL patients' samples, (significantly) higher frequencies of early proliferating T cells, TnOn-naive or Tcmin

[0139]

[0140] and MLCwB-DC(Kit-M) were found compared to (e.g.: Kit-1: 42.06117.17, Kit-2: 36.64±12.42, Kit-M: 34.08±13.07, Control: 26.53±13.19; p= 0.000, p= 0.02, p= 0.02), with the highest values obtained with Kit-1. Moreover, "cut-off" values with respect to frequencies of cell subsets obtained in > two-thirds of AML cases using Kit-M were defined. It was found that, in ALL using Kit-1- pretreatment, more cases with higher activated and memory T cells than with Kit-2- or Kit-M-pretreatment were found (e.g., Tnon-naive / T3+: Kit-1: 6 / 8; Kit-2: 5 / 9; Kit-M: 5 / 11; Tcm / T3+: Kit-1: 4 / 8 (4 of 8 cases); Kit-2:4 / 9; Kit-M: 4 / 11) (see Fig. 3A). In CLL patients' samples, no higher frequencies of T cell subsets after MLC with Kit-1, Kit-2, and Kit-M pretreated WB compared to control (see Fig. 3C) were found. In healthy samples, borderline significantly higher frequencies of Tcmand Tproi-eariy in MLCWB-DC(Kit -M) compared to M LCWB-Dc(controij (%Tcm / T3+: Kit-M: 8.62±6.16; Control: 4.46±2.55; p= 0.06 and %TprOi-eariy / T3+: Kit-M: 42.29±14.57; Control: 35.37±14.74; p= 0.08) were found. (Non-significantly) increased frequencies of TnOn-naive / T3+ in MLCWB-DC(Kit-ij and MLCWB-DC(Kit-2) compared to M LCWB-Dc(controij (see Fig. 3D) were also found..

[0141] Increased frequencies of intracellular IFNY-producing and degranulating immune cells after MLC, depending on Kit-1, Kit-2, and Kit-M-pre-treatment of AML, ALL, CLL, and healthy donors' WB

[0142] In AML, compared to Kit-1 and Kit-2, frequencies of intracellular I FNv+ producing or degranulating cells were significantly upregulated in samples pretreated (vs not pretreated) with Kit-M-pretreatment (e.g., % deg+Tem / eff / Tem / eff: M LCwB-DCfKit-Mj: 37.16114.23%, MLCwB-DC(controi): 30.53117.08%, p= 0.00; deg+NK / NK: MLC wB-DC(Kit-M): 7.27111.37%, MLCwB-DQControij: 5.5115.72%, p= 0.03).

[0143] Moreover, it was also confirmed that there were more cases with increased intracellular IFNy-producing and degranulating immune cells after MLC in AML using Kit-M than Kit-l-pretreatment (e.g., IFNY+Tcm+ / Tcm: Kit-1: 3 / 6 (3 of 6 cases); Kit-2: 4 / 5; Kit-M: 6 / 8; deg+Tnon-naive / Tnon-naive: Kit-1: 3 / 7; Kit-2: 5 / 7; Kit-M: 5 / 7; deg+Tcm / Tcm: Kit-1: 3 / 7; Kit-2: 5 / 7; Kit-M: 5 / 7) (see Fig. 4B). Frequencies of intracellular I F Nv+ producing or degranulating cells of AML-Myo and AML-Ly were comparable. However, due to the low number of cases, studied data are not presented in detail.

[0144] In ALL patients' samples, (non-significantly) increased intracellular IFNy-producing immune cells were observed using Kit-pretreatment after MLC compared to control (e.g., %IFNY+Tcm / T

[0145]

[0146] M

[0147]

[0148] 63.36±16.44, MLCWB 55.04123.04, MLCWB 58.42117.18, MLCWB 51.44118.15; p= 0.24, p= 0.34, p= 0.07) (see Fig. 4(1)A). Additionally, significantly higher frequencies of deg+T3+ / T3+ and deg+B / B in compared to M LCWB-Dc(controij were found, (e.g., % deg+T3+ / T3+: MLCwB-DCfKit-ij: 32.51116.49, M 24.43114.92, p= 0.03; deg+B / B: MLCWB 28.0716.25, MLCWB 18.5415.67, p=0.04). A significant decrease in frequencies of deg+T4+reg / T4+reg in Kit-M treated samples compared to the control group (Fig. 4(2)A) was demonstrated. Moreover, "cut-off" values with respect to cell frequencies obtained in > two-thirds of AML cases were defined using Kit-M. It was found that, in ALL using Kit-1-pretreatment, there were more cases with increased intracellularly IFNy producing and degranulating immune cells after MLC than with Kit-2 or Kit-M-pretreatment (e.g., IFNy+TnOn-naive / Tnon-naive: Kit-1: 4 / 6 (4 of 6 cases); Kit-2: 2 / 6; Kit-M: 3 / 7; IFNy+Tcm+ / Tcm: Kit-1: 5 / 6; Kit-2: 3 / 6; Kit-M: 3 / 7; deg+Tnon-naive / Tnon-naive: Kit-l: 5 / 7; Kit-2: 3 / 7; Kit-M: 2 / 7; deg+Tcm / Tcm: Kit-1: 7 / 7; Kit-2: 5 / 7; Kit-M: 3 / 7) (see Fig. 4(1)A and (2)A). It is concluded that Kit-l-pretreatment, followed by Kit-M (vs. no treatment), led to the most effective increase in production of intracellular I FNy and degranulation activity in ALL samples' adaptive immune cells.

[0149] In three CLL samples, we found (non-significantly) upregulated frequencies of intracellular I FNv+ producing or degranulating cells in samples with Kit-l-pretreatment (vs. no treatment) (see Fig. 4(1)C). In healthy samples, we found significantly higher frequencies of antigen-specific deg+T3+ / T 3+, deg+TnOn-naive / Tnon-naive, deg+Tcm / Tcm, deg+CIK / CIK, comparing MLCWB-DC(Kit -M) with M LCWB-Dc(controij (e.g., % deg+Tcm / T cm* M LCyvB-DC(KiL-M)' 49.00±22.78%, MLCWB -Dc(controi): 28.26+16.55%, p- 0.03). And (non-significantly) decreased frequencies of deg+T 4+reg compared to MLCwB-DC(Kit- ), M LCwB-DQKit-ij, and MLCwB-DC(Kit-2j with M LCwB-DC(controi) (see Fig. 4(2)D) were also observed.

[0150] Increased antileukemic cytotoxicity after MLC of Kit-1, Kit-2, and Kit-M-pretreated (vs. untreated) AML, ALL, and CLL patients' WB

[0151] After co-culture of 'effector cells' (T cell-enriched MLC with Kit-l / Kit-2 / Kit-M-pretreated (vs. not pretreated WB) with 'target cells' (thawed blast-containing MNC), blast lysis of M LCWB DC(Klt l)('Kit-1'), M LCWB DC(Klt-2)('Kit-2'), and M LCWB DC(Klt lvl)('Kit-M') vs. M LCWB DC(Control)('Control') were compared, using a cytotoxicity fluorolysis assay.

[0152] Analyses were conducted after 3 or 24 h of incubation of the target with effector cells, and finally, the best antileukemic effectivity after either 3 or 24 h was selected as the 'best' achieved lysis value. The lytic activity was calculated and defined as the frequency of (non-)viable target cells compared to a control [35,37],

[0153] AML

[0154] After 3 hours of coincubation of effector with target cells, blast lysis was observed in 44.44% of cases (4 / 9) after Kit-l-pretreatment (MLCWB-DC(Kit-i;), 66.67% of cases (6 / 9) after Kit-2 pretreatment (MLCWB-DC(Kit-2)), and 58.33% of cases (7 / 12) after Kit-M-pretreatment (MLCWB-DC

[0155]

[0156] VS. 50% of cases (6 / 12) in the Control (MLCwB-DQcontroij). After 24 hours, blast lysis was observed in 66.67% of cases (6 / 9) after Kit 1-pretreatment (MLCWB-DC(Kit-l)), 77.76% of cases (7 / 9) after Kit-2 pretreatment (MLCWB-DC(Kit-2)), and 75% of cases (9 / 12) after Kit-M-pretreatment (MLCWB DC vs. 58.33% of cases (7 / 12) in the Control (MLCSelecting the 'best' achieved lysis value after 3 h and 24 h of incubation time, more cases with lysis after

[0157]

[0158] M LCWB-DC vs. M ( M 83.33% vs. 56.67% cases with lysis) were found Frequencies of lysed blasts in MLCWB-DC(Kit -M), MLCWB-DCIKIW) were (borderline / significantly) lower compared to M LCWB-Dc(controij (%lysed blasts: MLCWB-DC(Kit -M>: 29.16±41.13% VS. MLCWB-Dc(controij: 6.9±38.79%; p= 0.02; M

[0159]

[0160] LCWB 25.41±30.93% vs. M

[0161]

[0162] LCWB 6.9±38.79%, p= 0.06) (see Fig. 5B(1)). After 24 hours of incubation of effector with target cells, all cases showed improved blast lysis after

[0163]

[0164] -M; compared to M LCwB-DC(controi), which led to improved lysis of 33.05±25.99% (see Fig. 5B(2)). In summary, compared to the effects seen after Kit-1 and Kit-2-pretreatment, the most effective and improved blast killing was achieved after pretreatment vs. no pretreatment of AML samples with Kit-M, thereby confirming data shown before (e.g, Unterfrauner 2023

[0037] ).

[0165] ALL

[0166] After 3 hours of coincubation of effector with target cells, blast lysis was observed in 87.5% of cases (7 / 8) after Kit-l-pretreated

[0167]

[0168] (M LCWB-DC 62.5% of cases (5 / 8) after Kit-2 pretreated (MLCWB-DC(Kit-2)), and 80% of cases (8 / 10) after Kit-M-pretreated (M LCWB-DC -M;), VS. 50% of cases (5 / 10) in the Control

[0169]

[0170] (MLC After 24 hours, blast lysis was observed in 100% of cases (8 / 8) after Kit-l-pretreated (MLCWB-DC(Kit-i)), 87.5% of cases (7 / 8) after Kit-2 pretreated (MLCWB-DC(Kit-2)), and 90% of cases (9 / 10) after Kit-M-pretreated vs. 80% of cases (8 / 10) in the Control (MLCWB-DC(controij). Selecting the 'best' achieved lysis value after 3 h and 24 h of incubation time, more cases with lysis after M LCWB-DC vs. M LCWB-DC(controi;

[0171] 100% vs. MLCWB-DC(controij: 80% cases with lysis were found. Frequencies of lysed blasts in MLCwB-DC(Kit-i), MLCWB-DC(Kit-2) and MLCwB-DC(Kit-M) were (borderline / significantly) lower compared to M LCwBDC(controi) (%lysed blasts: MLCwB-DC(Kit-i): 58.19+21.60% vs. MLCwB-DC(controi): 15.57+18.08%, p= 0.004;

[0172] 43.68120.24% vs. MLCWB 15.57118.08%, p= 0.005; MLCWB 37.34116.75% vs.

[0173] 15.57118.08%, p=0.002) (see Fig. 5A(1)). Cases with improved blast lysis were found in all

[0174]

[0175] cases (100%) after MLCWB M LCwB-DC(Kit-2), and MLCwB-DC(Kit-M) compared to M which led to improved blast lysis of 63.36117.43% in M LCwB-DC(Kit-i), 38.29115.70% in MLCWB-DC(Kit-2), and 33.44117.43% in M LCwB-DC(Kit-M) (see Fig.5A(2)). In summary, ALL patients' samples pretreated with different Kits (Kit-lfollowed by Kit-2 / Kit-M) achieved significantly improved blast lysis after MLC.

[0176] CLL

[0177] After 3 hours of coincubation of effector with target cells, blast lysis was observed in 75% of cases (3 / 4) after Kit-l-pretreatment ( M LCWB-DC (KH-I)), 50% of cases (2 / 4) after Kit-2 pretreatment (MLCWB-DC(Kit-2)), and 40% of cases (2 / 5) after Kit-M-pretreatment (M LCWB-DC vs. 20% of cases (1 / 5) in the Control (MLC

[0178]

[0179] After 24 hours, blast lysis was observed in 75% of cases (3 / 4) after Kit-l-pretreatment (MLCWB-DC(Kit-i)), 75% of cases (3 / 4) after Kit-2-pretreatment (MLCWB-DC(Kit-2)), 60% of cases (3 / 5) after Kit-M-pretreatmentvs. 60% of cases (3 / 5) in the Control (MLCwB-Dqcontroij). Selecting the 'best' achieved lysis value after 3 h and 24 h of incubation time, more cases with lysis after M LCwB-ocfKit-i) vs. M LCWB-Dc(controi)

[0180]

[0181] 100% vs. MLCWB-Dc(controij: 60% cases of lysis were found. Frequencies of lysed blasts in M LCwB-DC(Kit-i), M LCwB-DQKit-2), M LCwB-DC(Kit-M) were (non-significantly) lower compared to MLC wB-DC(controi) (see Fig. 5C(1)). Improved blast lysis was found in 75% of cases (100%) after M LCwB-ocfKit-i) and M LCWB-DC(Kit-2j, in 80% of cases in MLCWB-DC(Kit -M) compared to M LCWB-Dc(controi), which led to improved lysis of 31.91±5.51% in M

[0182]

[0183] 10.81±8.22 % in M LCWB and 22.23+19.79% in M LCwB-DC(Kit-M) (see Fig- 5C(2)). In summary, CLL patients' samples treated with different Kits (Kit-1 followed Kit-2 / Kit-M) showed less improved blast lysis after MLC compared to ALL or AML. Stimulatory effect of Kit-1, Kit-2, or Kit-M-pretreated (vs. untreated) WB on the cytotoxic activity after 3 h and 24 h of co-culture of immunoreactivity cells ('effector cells') and blasts ('target cells') of ALL, AML, and CLL. Given are the proportions of cases with blast lysis and the frequencies ± standard deviation of increased or lysed blasts after MLCWB-DC(Kiti) (Kit-1) M LCWB-DCIKM) (Kit-2) and (Kit-M) compared to M LCWB-Dc(controi) (Control) after 3h and 24h and the 'best' achieved blast lysis after 3h or 24h (see Fig. 5(1)).

[0184] Given are the proportions of cases with an improvement in blast lysis and the frequencies ± standard deviation of improved blast lysis after M LCwB-DC(Kit-i), M LCWB-DCIKM) and MLCWB-DC(Kit -M) in relation to M LCwB-D controi) after 3h and 24h and the 'best' achieved improvement in blast lysis after 3h or 24h (see Fig.

[0185] 5(2)). Statistical analyses were conducted using a t-test: Differences were considered as highly significantly different with p values < 0.005, as significantly different with p values <0.05, and as borderline significantly different with p values between 0.05 to 0.10. Abbreviations of cell types are given in Table 1.

[0186] Summary and Conclusion

[0187] Composition and function of uncultured (leukemia- specific) immune cells in AML, ALL, CLL

[0188] Compared to immunoreactive cells without LAA (UNSTIM) stimulation, frequencies of many uncultured and antigen leukemia-specific (degranulating or IFNy- producing) immunoreactive cells with (STIM) vs. without LAA stimulation (UNSTIM) were higher in AML, ALL, CLL patients' and healthy donors' samples. These data confirm that LAA stimulation increases the provision of leukemia-specific immunoreactive (uncultured) cells in AML. [11,12,37], but as we show here also in ALL and CLL. It is also confirmed that the addition of SEB in WB of healthy volunteers significantly increased the degranulation activity in healthy T cells, T3+, TnOn-naive, and Tem / eff, as shown before [11,37],

[0189] DC-Based Immunotherapy

[0190] Ex vivo, DC / DCieu subtypes can be generated from AML or ALL patients' monocytes using combinations of response modifiers (GM-CSF, PGE1, IL-4, CD40L, TN Fa) and loaded with leukemic antigens. [21-35] And DCieucan migrate to tissues in the whole body, present the patients' entire leukemic antigen repertoire in a costimulatory manner to immune cells, and induce leukemia-specific cytotoxic lymphocytes, thereby providing an antileukemic strategy in the whole body [6,11,35,53], An elegant way to improve DC-based immunotherapies (using DC / DCieuproduced under GMP, followed by adoptive transfer to patients [6,10]) is to directly produce DC and DCieuin vivo (from monocytes or blasts) in the patients, resulting in activation of (leukemia- specific / antileukemic) immune cells. Ex vivo, this strategy can be simulated using (heparinized) patients' WB containing the patients' complete cellular or soluble (potentially inhibitory or immune activating composition- as shown for AML samples treated with Kit-M or other Kits before (e.g., Schwepcke et al., 2021 [5], Unterfrauner et al., 2023

[0037] ).

[0191] Here, it is confirmed that this Kit-M-mediated antileukemic approach for AML samples: Kit-M-pretreated leukemic WB produces leukemia-derived DC, which later increases leukemia-specific / antileukemic cells after MLC (see Fig.2B; Fig.3B). It is also noted that Kit-M pretreatment of AML samples with versus without aberrant lymphoid marker expression yields comparable frequencies of DC subtypes, as well as immune cell subtypes, after MLC (see Fig.2E, F; Fig. 3). Recently, it was shown that generation of DC and DCieufrom AML-WB using Kit-M is independent of patients' ELN risk, mutation, HLA status, age, or sex. Treating leukemia-diseased rats and therapy-refractory AML patients with these clinically approved drugs (GM-CSF and PGE1) in Kit-M, it was demonstrated that the treatment is safe, improves anti-leukemic responses in vivo, and improves hematopoietic recovery without increasing blast counts

[0039] , Previous findings show that DC and DCieu subtypes can be produced from AML-WB with Kits containing GM- CSF, combined, e.g. with PGE1 (Kit-M), with Picibanil (Kit-I), or with IFNa (Kit-E) -with Kit-M being the most effective Kit to induce antileukemic reactions against AML [5,12], It is further added that 'lymphoid' Kits (Kit-1 containing CD40L and IL-4 or Kit-2 containing GM- CSF, TN Fa, and IL-2) are partially able to produce DC / DCieusubtypes-followed by induction of leukemia-specific or antileukemic immune cells from AML-WB. Studying the influence of response modifiers in various combinations on the generation of DC / DCieuand DC / DCieumediated leukemia-specific immune reactions in ALL-WB, we confirm data demonstrated before [29,32], that DC / DCieucan be generated with combined "lymphoid" Kit-1 and Kit- 2 without inducing blast / monocyte proliferation. Especially, with Kit-l-pretreatment, we found the highest frequencies of DCieu / BLA (Fig.2A). In addition, Kit-1 and Kit-2 are effective in generating DC / DCieufrom ALL blasts, followed by antileukemic immunoactivation after T cell-enriched MLC [29,30,32],

[0192] Moreover, it was shown that Kit-M is also able to produce DC and DCieufrom ALL-WB, followed by antileukemic immunoactivation after T cell-enriched MLC, although with less efficiency compared to Kit-1 or Kit-2 (see Fig.3A). These data show that ALL is a stem cell disease with comparable cellular differentiation capabilities to AML [54,55], In CLL patients' WB samples, DC / DCieucould be generated under Kit-1-, Kit-2, and Kit-M-pretreatment with less success and in fewer cases (see Fig.2C). This could indicate that CLL (characterized by mature monoclonal B cells) may be less influenced by Kits' treatment, whereas thoseaffecting stem cell diseases may respond more effectively. Treating healthy donors' WB samples with various Kits, we confirm previous findings that DC / DCieucan be generated with Kit-M- pretreated (vs. not pretreated) healthy patients' WB samples without inducing monocyte proliferation (see Fig.2D) [11,35],

[0193] Kit-M is composed of approved drugs that can be used for clinical applications- a phase 1 trial for AML patients is in preparation.

[0039] ,

[0194] Enhanced provision and activation of leukemia-specific immune cells after Kit-1 / 2 and Kit-M pretreatment of AML / ALL / CLL patients' WB

[0195] The present study confirms preliminary data that Kit-M-treated (vs. untreated) AML-WB leads to the provision of activated and Tcmcells (see Fig.3B) [10,12], In addition, a generally higher activation status of immune cells after MLC in ALL patients' Kit-l-pretreated (vs. untreated) WB was shown, leading to increased frequencies of proliferating TnOn-naive and Tcm(see Fig.3A). Memory T cells are critical for immune surveillance and the enhancement of anti-leukemic immunity, playing a key role in detecting and eliminating residual leukemia cells post-treatment, thereby improving treatment outcomes in ALL [52,56], In CLL patients' samples, Kit-l-pretreatment showed (non-significantly) increased frequencies of activated or Tcmcells (see Fig.3C).

[0196] Enhanced Intracellular Cytokine Production and Degranulation of Immune Cells after MLC in Kit- 1-treated ALL patients' WB and in Kit M-treated AML patients' WB

[0197] In AML samples, previous studies indicating that Kit-M treatment of WB enhances anti- leukemic response after MLC were confirmed. This includes an increase in IFNy production by various T cell subtypes (e.g., IFNy+Tnon-naive, IFNy+ Tem / eff, and IFNy+Tcm). Additionally, increased degranulation activity (CD107a expression) in these T cell subtypes (e.g., deg+TnOn-naive, deg+Tem / eff ) and of NK cells (deg+NK) was observed. Furthermore, Kit-M treatment resulted in decreased frequencies of degranulating Tregin MLC wB-oc(Kit-M) compared to M LCwB-D controi), consistent with previous findings, (see Fig. 4(1)B and 4(2)B) [35,37], Through stimulation of immunoreactive cells using DC / DCieu-Kit-l in ALL samples, an increase in IFNy-producing and degranulating adaptive and innate immune cells (e.g., IFNy-producing TnOn-naive, Tcm, NK, and CIK cells) in MLCWB-DC(Kit i) compared to M LCWB-Dc(controi) was observed, as well as an increase in degranulating T- or B-cell (e.g., deg+T3+ and deg+B) subtypes (see Fig. 4(1)A and 4(2)A) These findings support the notion that Kit-1 treatment enhances the functional activity of adaptive immunity. Notably, previous studies have indicated that ALL-derived DCs might serve as a vaccine platform capable of eliciting LAA-specific T-cell responses.

[0030] , The herein provided results further substantiate this concept by demonstrating that Kit-l-treated ALL samples exhibit increased IFNy production upon DC / DCieustimulation, highlighting the activation of functionally competent leukemia-specific immune cells. Importantly, in both ALL and AML samples, the production ofIFNy and other immunological markers was independent of patients' age, sex, or blast frequency. This underscores the broad applicability of DC / DCieu-based / inducing strategies in inducing leukemia-specific immune responses across diverse patient subgroups. In CLL samples, similarly increased IFNy production of T cell subtypes (e.g., IFNy+TnOn-naive, IFNy+Tem / eff and IFNy+Tcm) was observed, as well as enhanced degranulation activity (CD107a expression) of T cells (e.g., deg+T3+, deg+Tcm), and (non-significantly) reduced frequencies of degranulating Treg(see Fig. 4(1)C and 4(2)C)). Overall, the herein provided data demonstrate increased antileukemic immunological activity in innate and adaptive immune compartments across all tested leukemia subtypes, further supporting the potential of Kit-based treatments to induce functionally active leukemia-specific immune cells.

[0198] Improved Blastolytic Activity after MLC in Kit-M- treated AML patients' WB and Kit-l-treated ALL patients' WB

[0199] In AML samples, previous studies indicating that a higher number of cases with improved blast lysis was achieved after MLC with Kit-M-pretreated vs. untreated WB, following 3 or 24 hours of co-incubation of blast targets with effector cells (vs. Control), and also compared to Kit- 1 or Kit-2 pretreated (Fig.5B(l))

[0200] [11,13,34,35] were confirmed. Blast lysis was superior in some cases after 3 hours of incubation of targets with effector cells, and in other cases after 24 hours in Kit-M-pretreated WB (see Fig.5B(2)). These effects may be attributed to distinct, independent blastolytic mechanisms: the faster perforin / granzyme pathways, which lead to blast lysis predominantly after 3 hours of co-incubation, and the slower Fas / FasL pathways, resulting in blast lysis predominantly after 24 hours of co-incubation

[0035] , In ALL samples, a significantly improved lytic activity against leukemic blasts in WB through Kit- 1, followed by Kit-2 and Kit-M mediated pretreatment (see Fig.5A(l)) was demonstrated. Kit-1 pretreatment of blasts in WB has previously been demonstrated to enhance the antileukemic activation of immunoreactive cells, as evidenced by the induction of apoptosis in leukemic cells. [29,30], Blast lysis was superior in some cases after 3 hours of incubation of the target with effector cells and in other cases after 24 hours in Kit-l-pretreated WB (see Fig.5A(2)). In CLL samples, also a higher number of cases with improved blast lysis achieved after MLC with Kit-l-(followed by Kit-M and Kit- 2) pretreated WB compared to untreated WB was observed, following 3 or 24 hours of incubation of blast targets with effector cells (vs. Control) (Fig.5C(l)), furthermore, blast lysis was superior in some cases after 3 hours of incubation of targets with effector cells, and in other cases after 24 hours in Kit-l-pretreated WB (Fig.5C(2)), the sample size was too small to draw definitive conclusions about the effectiveness of Kits treatments.

[0201] AML, ALL, CLL patients and healthy donors' WB were treated ex vivo with Kit-1 (IL-4 and CD40L), Kit-2 (GM-CSF, IL-4 and TNFa or Kit-M (GM-CSF and PGE-1). It was shown that DCieu-generation was best in AML patients 'WB with Kit-M (followed by Kit-1 and Kit-2) and in ALL patients' WB with Kit-l(followed by Kit-Mand Kit-2). However, both Kits were effective in AML and ALL and can induce leukemia-specific antileukemic immune effector and memory responses. Kit-pretreatment of leukemic WB not only activates antileukemic T cells, but also B, NK, and CIK cells, thereby improving the overall antileukemic response.

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[0257] Clinical stabilization of a highly refractory acute myeloid leukemia under individualized treatment with immune response modifying drugs by in vivo generation of dendritic cells of leukemic origin (DCieu) and modulation of effector cells and immune escape mechanisms

[0258] Allogeneic stem cell transplantation (alloSCT) remains the most effective curative approach for high-risk acute myeloid leukemia (AML). Nevertheless, 30–60% of patients relapse post-transplant, frequently based on defined immune escape mechanisms, including mis-matched HLA loss, regulatory T- / B-cell expansion, or upregulation of immune checkpoints (PD-1 / CTLA-4 / TIM-3) on effector cells [1-3], Dendritic cell (DC)-based immunotherapy may circumvent immune escape [4], It was previously demonstrated that leukemia derived DCs (DCieu) can be generated ex-vivo using the immune response modifiers GM-CSF and PGE1 (termed " Kit-M") and induce leukemia-specific B-, T-, and NK cell responses in preclinical models [5, 6],

[0259] Here the clinical translation of this approach in a patient with refractory AML is reported. A 65-year-old male with secondary AML (adverse genetics: BCR:: ABL1, ASXL1, KRAS, RUNX1, FLT3-ITD, IKZF1), initially treated with standard induction and alloSCT in MRD-positive first complete remission, presented with hematological and extramedullary (malignant pleural effusions requiring paracentesis 2-3x / week) relapse after multiple lines of therapies, including a second haplo-identical alloSCT six months earlier. Donor lymphocyte infusions were precluded due to genomic HLA loss in leukemic blasts [1], and the leukemia was refractory to Azacitidine salvage therapy. In the absence of approved treatment options, an individualized immunotherapeutic approach using Kit-M was discussed, thereby re-purposing two compounds approved for clinical use in other indications [7], Previously, successful generation of DCieusubsets had been demonstrated after stimulation of the patient's blood with Kit-M. Beyond, mixed lymphocyte cultures had confirmed activation of effector and memory T- and NK cells, downregulation of regulatory T cells (Treg), and induction of blast-directed cytotoxicity. Based on this observation, it was hypothesized that systemic administration of the compounds of Kit-M to the patient would promote in-vivo antileukemic immune reactions and potentially induce clinical response. The patient was extensively informed about the experimental treatment nature. Following written consent for both treatment and sequential collection of blood samples for immune monitoring; and following approval by the patient's health care provider and the local ethics committee (LMU #33905), Kit-M treatment was administered in eight 5-day cycles over a total 105 days, with dose escalation during the initial four cycles to ensure safety (see Table 3).Table 3: Experimental treatment protocol of Kit M (GM-CSF + PGE1). PGE1: Prostaglandin El, GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor, i.v.: intravenous

[0260] Phase Day Drug Dosage (i.v.) Schedule Ramp up phase 0-2 GM-CSF 75 μg / m24 h (9 am - 1 pm)

[0261] PGE1 20 pg 2 h (1 pm – 3 pm) 3-4 GM-CSF 75 μg / m24 h (9 am - 1 pm)

[0262] PGE1 40 pg 2 h (1 pm – 3 pm) 7-11 GM-CSF 75 μg / m24 h (9 am – 1 pm)

[0263] PGE1 40 pg 2 h (1 pm – 3 pm) 15-19 GM-CSF 75 μg / m24 h (9 am - 1 pm)

[0264] PGE1 40 pg 2 h (1 pm – 3 pm) 22-26 PGE1 40 pg 2 h (9 am - 11 arn)

[0265] GM-CSF 75 μg / m24 h (11 am – 3 pm) PGE1 40 pg 2 h (3 pm - 5 pm) Treatment break* Final dose" 51-55 PGE1 40 pg 2 h (9 am – 11 am)

[0266] GM-CSF 75 μg / m24 h (11 am – 3 pm) PGE1 40 pg 2 h (3 pm - 5 pm) • prolonged by one week due to minor surgery

[0267]

[0268] *• repeated in five-day cycles from d71-75, d87-91, and d101-105, slight differences in the intervals due to patient's preference

[0269] Treatment was well tolerated; no serious adverse events or graft-versus-host disease were observed. The patient's clinical status remained stable (Karnofsky performance score 90). Red cell transfusion requirements decreased by 25% compared to the prior treatment phase, and frequency of pleural paracentesis could be decreased to < lx / week. Peripheral leucocyte (WBC) and blast counts remained stable (median: WBC: 2.28 / nl; blasts: 9%), indicating controlled leukemia burden. Hence, Kit-M seemed to support granulopoiesis without promoting blast proliferation. After four months, AML progressed (peripheral blood [PB] blasts: >40%), prompting Kit-M discontinuation. Palliative chemotherapy was initiated, and the patient died four weeks later from disease progression. Comprehensive immune monitoring during treatment revealed sustained increases in mature DCs and DCieuin PB. Concurrently, activation of both innate and adaptive immune compartments was observed, including expansion of IFN-γ + memory γδ T cells, de-granulating cytotoxic T- / NK cells, and invariant NKT cells. TH1 polarization increased, while TH2 and Treg frequencies declined. Notably, frequencies of effector T-cells expressing inhibitory checkpoint receptors PD-1, CTLA-4, and KLRG-1 were markedly decreased (see Fig.6). Regulatory B cells were downregulated, while memory B cells expanded.

[0270] These changes imply a systemic immunologic reprogramming toward a more activated state. Importantly, while stable during Kit-M administration, PB blast counts increased during treatment breaks, suggesting that continuous immune activation contributed to temporary leukemic containment. Despite pre-existing HLA loss on malignant blasts, leukemia-directed immune responses were observed, potentially either due to targeting of HLA-retaining subclones or involved (HLA-independent) NK-mediated mechanisms. Beyond generation of DCieu, extensive immune monitoring demonstrated a broader immune stimulation by Kit-M, involving NK cell stimulation, reversal of T cell exhaustion and inhibition via immune checkpoints, wherebythe relative role of the different mechanisms to clinical effects remains to be defined. Nonetheless, final disease progression occurred. Decrease of mature DC (corresponding to inefficient antileukemic functionality) and increase of immune checkpoints on T cells and blasts (2B4, TIM-3, data not shown) suggested the emergence of various immune escape mechanisms.

[0271] This case illustrates the potential of Kit-M to elicit leukemia-reactive immune responses, even in heavily pretreated patients, that were able to stabilize both hematologic and extramedullary disease. Unlike traditional DC-based strategies such as vaccines, requiring intensive ex-vivo manipulation, Kit-M induces DCieuin-vivo, thereby avoiding the need of complex cell manufacturing [8],

[0272] References

[0273] 1. Vago L, Perna SK, Zanussi M, Mazzi B, Barlassina C, Stanghellini MTL, et al. N Engl J Med. 2009;361(5):478–88.

[0274] 2. Toffalori C, Zito L, Gambacorta V, Riba M, Oliveira G, Bucci G, et al. Nat Med. 2019;25(4):603–11.

[0275] 3. Sauerer T, Velázquez GF, Schmid C. Mol Cancer. 2023;22(1):180.

[0276] 4. Van Acker HH, Versteven M, Lichtenegger FS, Roex G, Campillo-Davo D, Lion E et al. J Clin Med. 2019;8(5).

[0277] 5. Atzler M, Baudrexler T, Amberger DC, Rogers N, Rabe A, Schmohl J et al. Int J Mol Sci. 2024;25(24).

[0278] 6. Unterfrauner M, Rejeski HA, Hartz A, Bohlscheid S, Baudrexler T, Feng X et al. Int J Mol Sci. 2023;24(24).

[0279] 7. Amberger DC, Doraneh-Gard F, Gunsilius C, Weinmann M, Mobius S, Kugler C et al. Int J Mol Sci.

[0280] 2019;20(18).

[0281] 8. Palomares F, Pina A, Dakhaoui H, Leiva-Castro C, Munera-Rodriguez AM, Cejudo-Guillen M et al. Vaccines (Basel). 2024;12(2).

Claims

CLAIMS1. A pharmaceutical composition comprising a combination of GM-CSF and PGE1, along with pharmaceutically acceptable excipients, wherein the combination triggers hematopoetic stem cells as well as resulting T, B, NK, and iNKT lymphocytes, erythrocytes, platelets, granulocytes, monocytes, and dendritic cells and wherein GM-CSF promotes the regeneration and provision of stem cells, and PGE1, as a "danger signaling factor," stimulates and activates hematopoiesis / the immune system, for use in the treatment of patients with stem cell deficiency / hematopoietic insufficiency associated with leukemia, a solid tumor, lymphoma, an infection or a hematopoietic disorder.

2. The pharmaceutical composition for use of claim 1, wherein said leukemia is an acute or chronic myeloid / myeloproliferative or lymphatic leukemia, before and after stem cell transplantation.

3. The pharmaceutical composition for use of claim 1, wherein said solid tumor or lymphoma is a solid tumor or lymphoma before and after various therapies, such as chemotherapy, antihormonal therapy, targeted therapy, immunotherapy or stem cell transplantation.

4. The pharmaceutical composition for use of claim 1, wherein said infection is a chronic or microbial or parasitic infection due to hematopoietic disorders or insufficiencies, before and after various therapies, such as antimicrobial / antiparasitic therapies, including various therapies for cancer.

5. The pharmaceutical composition for use of claim 1, wherein said hematopoietic disorder is a disorder or insufficiency due to (i) congenital or acquired diseases or therapies, such as acquired stem cell defects following chemotherapy, stem cell transplantation, or other therapies that impair hematopoiesis, or (ii) congenital stem cell defects that lead to thrombocytopenia, erythrocytopenia, neutropenia, or lymphocytopenia.

6. The pharmaceutical composition for use of any one of claims 1 to 5 for parenteral administration.

7. The pharmaceutical composition for use of any one of claims 1 to 6, wherein said pharmaceutical composition is to be administered according to the following dosage regime:GM-CSF is to be administered with 15 to 500 μg / day, preferably as i.v. administration with continuous infusion or daily batch administration or as subcutaneous administration; andPGE1 IV is to be administered with 0.075 μg / kg / h, up to a maximum of 0.5 μg / kg / h, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration.

8. The pharmaceutical composition for use of claim 7, wherein GM-CSF is to be administered parenterally with 75 μg / day, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration.

9. The pharmaceutical composition for use of claim 7 or 8, wherein GM-CSF and PGE1 are to be administered daily or in two-day or three-day intervals.

10. A method of treating a patient with stem cell deficiency / hematopoietic insufficiency associated with leukemia, a solid tumor, lymphoma, an infection or a hematopoietic disorder, comprising administering a therapeutically effective amount of a combination of GM-CSF and PGE1, along with pharmaceutically acceptable excipients, wherein the combination triggers hematopoetic stem cells as well as resulting T, B, NK, and iNKT lymphocytes, erythrocytes, platelets, granulocytes, monocytes, and dendritic cells and wherein GM-CSF promotes the regeneration and provision of stem cells, and PGE1, as a "danger signaling factor," stimulates and activates hematopoiesis / the immune system.

11. The method of claim 10, wherein said leukemia is an acute or chronic myeloid / myeloproliferative or lymphatic leukemia, before and after stem cell transplantation.

12. The method of claim 10, wherein said solid tumor or lymphoma is a solid tumor or lymphoma before and after various therapies, such as chemotherapy, antihormonal therapy, targeted therapy, immunotherapy or stem cell transplantation.

13. The method of claim 10, wherein said infection is a chronic or microbial or parasitic infection due to hematopoietic disorders or insufficiencies, before and after various therapies, such as antimicrobial / antiparasitic therapies, including various therapies for cancer.

14. The method of claim 10, wherein said hematopoietic disorder is a disorder or insufficiency due to (i) congenital or acquired diseases or therapies, such as acquired stem cell defects following chemotherapy, stem cell transplantation, or other therapies that impair hematopoiesis, or (ii) congenital stem cell defects that lead to thrombocytopenia, erythrocytopenia, neutropenia, or lymphocytopenia.

15. The method of any one of claims 10 to 14, wherein for said administration is a parenteral administration.

16. The method of any one of claims 10 to 15, wherein said administration is performed according to the following dosage regime:GM-CSF is to be administered parenterally with 15 to 500 μg / day, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration; andPGE1 is to be administered parenterally with 0.075 μg / kg / h, up to a maximum of 0.5 μg / kg / h, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration.

17. The method of claim 16, wherein GM-CSF is to be administered parenterally with 75 μg / day, preferably as i.v. administration with continuous infusion or daily batch administration, or as subcutaneous administration.

18. The method of claim 16 or 17, wherein GM-CSF and PGE1 are to be administered parenterally daily or in two-day or three-day intervals, preferably as i.v. administration, or as subcutaneous administration.

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