Use Of Immunomodulatory Effective Kits For The Immunotherapeutic Treatment Of Patients Suffering From Myeloid Leukemias

Abstract

The present invention relates to a method for selecting an immunomodulatory kit, selected for an individual patient, for use in the treatment of patients suffering from myeloid leukemias. Different kits are available for selection and ex vivo testing which are composed of substances that have different immunomodulatory effects on leukemia cells. Each kit particularly contains GM-CSF and one (or two) more substances, selected from PICIBANIL, PGE.sub.1, PGE.sub.2, CALCIMYCIN and TNFα, as well as pharmaceutically acceptable adjuvants. The clinical aim is to modify, once the individually selected immunomodulatory kits were administered, blast cells in the body of the patient such that they turn into a “vaccine” which is able to activate the immunoreactive cells (of the patient or of the stem cell donor) in the body against blast cells.

Claims

1-59. (canceled)

60. Kit for use in parenteral, individual therapy and/or prevention of myelogenous leukemia in preferably immunocompetent patients in remission of the disease, containing GM-CSF and at least one further active substance selected from PICIBANIL, PGE1, PGE2, CALCIMYCIN And TNFα as well as pharmaceutically compatible active components comprising water, aqueous solutions, buffers, buffer solutions, binders and/or adjuvants, whereby myeloid leukemia is an acute myeloid leukemia, myelodysplastic disease, myeloproliferative disease or chronic myeloid leukemia.

61. Kit according to claim 60, whereby the treatment is carried out with the following dosage: GM-CSF as i.v. permanent infusion or daily alternating with 15 to 500 μg/day, preferably 75 μg/day, and PICIBANIL i.m., i.d. or s.c. with 20 to 500 μg/day, preferably 50 μg/day, whereby GM-CSF and PICIBANIL are administered in this order in a daily, two-day or three-day interval.

62. Kit according to claim 60, whereby the treatment is carried out with the following dosage: GM-CSF as i.v. permanent infusion or daily alternating with 15 to 500 μg/day, preferably 75 μg/day, and PGE1 i.v. or as a continuous infusion of 0.075 μg/kg/h, up to a maximum of 500 μg/kg/h, whereby GM-CSF and PGE1 are administered in this order in a daily, two-day or three-day interval.

63. Kit according to claim 60, whereby the treatment is carried out with the following dosage: GM-CSF as i.v. permanent infusion or daily alternating with 15 to 500 μg/day, preferably 75 μg/day, and PGE2 with 0.1 to 5 mg/kg/day, whereby GM-CSF and PGE2 are administered in this order at a daily, second-day or three-day interval.

64. Kit according to claim 60, whereby the treatment is carried out with the following dosage: GM-CSF as i.v. permanent infusion or daily alternating with up to three days with 15 to 500 μg/day, preferably 75 μg/day, and PICIBANIL i.m., i.d. or s.c. with 20 to 500 μg/day, preferably 50 μg/week, and PGE2 i.v. with 0.1 to 5 mg/kg/day, with GM-CSF, PICIBANIL and PGE2 being administered in this order in a daily, two-day or three-day interval.

65. Kit according to claim 60, whereby the treatment is carried out with the following dosage: GM-CSF as i.v. permanent infusion or daily alternating with 15 to 500 μg/day, preferably 75 μg/day, and TNFα i.v. 0.04 mg-4 mg/m2/24h, whereby GM-CSF and TNFα are administered in this order in a daily, second-day or three-day interval.

66. Kit according to claim 60, whereby the treatment is carried out with the following dosage: GM-CSF as i.v. permanent infusion or daily alternating with 15 to 500 μg/day, preferably 75 μg/day, and CALCIMYCIN dosage 0.1-10 μg, whereby GM-CSF and CALCIMYCIN are administered in this order in a daily, second-day interval.

67. Kit according to claim 60 for parenteral administration

68. A method for selecting a suitable kit for individual therapy and/or prevention of myelogenous leukemia, comprising the following steps: (A) ex vivo isolation of T cells from a sample of blast-containing or blast-free whole blood from the patient or stem cell donor; (B) subdividing the blast-containing whole blood sample of the patient into several sub-samples; (C) sequentially adding the following individual kits to the individual blast-containing whole blood samples at the beginning and after a first incubation period of the sub-samples, preferably 4 to 6 days, whereby leukemic blasts are converted to leukemic dendritic cells DCleu by the kits in the sub-samples: (i) GM-CSF, PGE.sub.2 and PICIBANIL (ii) GM-CSF and PICIBANIL (iii) GM-CSF and PGE.sub.2 (iv) GM-CSF and PGE.sub.1 (v) GM-CSF and TNFα (vi) GM-CSF and CALCIMYCIN (D) adding the T cells isolated in step (a) to each of the sub-samples obtained in step (c) after incubation and then incubating them during a second incubation period of preferably 7 to 10 days; (E) performing a lysis assay in each of the sub-samples after the incubation period in step (D) for detecting the antileukemic function of the particular kit employed; (F) creating a ranking for the kits used, depending on the quality of the result of the respective lysis tests; and (G) selection of the kit with the best conversion of blasts into DCleu without induction of blast proliferation and the best blast lysis result obtained after antileukemic T cell activation.

Description

IV. DESCRIPTION OF ILLUSTRATIONS AND TABLES

[0019] FIG. 1: DC/, leukemia-derived DC (DCleu)-mediated antileukemic T cells

[0020] a) DC are produced from stem cells or monocytes in the healthy organism; DCleu arise from ‘redifferentiated’ AML blasts

[0021] b) Antileukemically active, DC/DC-stimulated and activated T cells (ex vivo/in vivo)

[0022] FIG. 2: DC/DC.sub.leu can be generated from AML blast-containing WB samples with standard DC differentiation methods (eg Pici) as well as with kits (patient-specific)

[0023] a) DC/DC.sub.leu generation from WB using standard procedures (eg Pici, left) and with kits (right side)

[0024] b) Comparable average amounts of DC subtypes with all kits

[0025] FIG. 3: Selection of the best WB kit method for generating DC/DC.sub.leu subtypes in individual AML patients: the percentage frequencies of a successful DC/DC.sub.leu generation with individual kits are shown

[0026] a) RANKING: ‘best or second best DC method’ compared to at least 3 other kits

[0027] b) RANKING: DC method with ‘excellent or high quality’ (criteria see 3c.)

[0028] c) Criteria for successful DC generation

[0029] d) Selection of the 6 best kits according to RANKING criteria

[0030] FIG. 4: Antileukemic-blast-lytic activity of T cells stimulated with KIT-TREATED (HS-D, F, I, M, K, A) compared to untreated AML WB samples

[0031] a) Selection according to the patient's individual best lytic value

[0032] b) Selection according to the patient's individual greatest difference to the WB control lysis value

[0033] c) Selection of HS-D, I, K, NI treated WB samples compared to the WB control

[0034] FIG. 5: Treatment of BNML rats with kits leads to reduction in blast proportions and changes in Tcell compositions in blood and spleen compared to placebo control

[0035] a) Experimental design of rats' treatment

[0036] b) Blast reduction in blood (left) and spleen (right)

[0037] c) Increase of CD62L+(Tmem): CD4+(left) and CD8+(right side) in blood and spleen

[0038] d) Reduction of CD4+CD25+FoxP3+Treg in the spleen

[0039] Table 1: Generation of DC/DCLeu from AML blast-containing mononuclear cells (MNC) or whole blood (WB) cells with different DC differentiation methods or specific kits

[0040] a) DC differentiation methods or kits for the generation of DC/DC cells from blast-containing MNC (a1-a4) or WB (a1-a5)

[0041] b) Advantages and value of DC generation from whole blood samples

[0042] Table 2: Ingredients in the kits for the immunomodulation of AML blasts

[0043] Table 3: Composition of the tested kits

V. DETAILED DESCRIPTION OF THE INVENTION

[0044] The invention presented below in the example of the AMLqualifies likewise in patients with other myeloid leukemia. According to the present invention, in vitro, a minimized combination of clinically approved “immunomodulators” was developed and tested. This was done with a test model with “heparinized whole blood (WB) samples” (taken in heparin tubes with standardized heparin addition): WB contains all soluble and cellular components of patients with leukemic disease and also all cellular and soluble influencing variables on the DC generation.

[0045] It is also possible to determine in vitro at the same time whether DC differentiation in vivo is possible and differentiation resistances of the cells can be overcome and thus a functioning humoral and cellular immunity can be (re-)established in the patient. Very cell-rich WB samples were diluted with ca 30-50% serum-free medium (X-vivo) in order to guarantee the comparability of the individual cases. Table 1b shows advantages of generating DC/DCleu from WB: all cell types and soluble factors present in the body can be detected in their “natural environment”. Functionality tests are tested close to the clinical reality and can yield finally conclusions about biological correlations and reaction profiles compared to results with mononuclear cells, the normally used leukocyte fraction for examinations.

[0046] Similar proportions of DC (subtypes, determined by flow cytometry) were obtained both from WB and from MNC of patients with leukemic diseases with the standard methods (CA, PICI, MCM or INTR, Table 1a) what means that a WB model is in general suitable for inducing the modulation of blasts ex vivo, to evaluate the convertibility of blasts to DCleu. Moreover it is a good physiological model to test how successful immunomodulation is. The next step it was shown that the generation of DC (subtypes) from WB with the standard methods was similar to that with minimized Kits consisting of 1-3 clinically approved immunomodulators, from WB while culture controls without the addition of kits, did not give rise to DCs from the blasts (the composition of the kits is given below).

[0047] FIG. 2a shows results of the DC generation from WB using standard method, Pici ‘(left side) compared to WB with kits (right side): many of the 11 kits tested yield similar amounts of DC subtypes from WB of leukemic patients as with standard methods. Average (see bar graphs FIGS. 2a and b) shows similar levels of DC (subtypes (see table):% DC (, Q′), mature/migratable DC (, Z′), DC leu converted blasts, DC leu in the entire cell fraction (, X′) related to all DC (, Y′)) from WB: With at least one method a good DC generation was possible for each patient. Culture controls without the addition of kits are usually unsuccessful. Furthermore, it is also apparent that not every kit is equally suitable for individual patients to generate DC (subtypes) (see table and FIG. 2a).

[0048] Thus, a WB method is a good physiological model for DC/DCleu generations, whereby patient-specific differences are recognizable: individual kits are better than others suitable to generate DC (subtypes). Several kits were (comparatively) tested within the scope of the present invention. All substances are approved for the application in humans and are regularly used in patients with other indications than listed below. A number of individual substances as well as their combinations were used for this purpose regarding their abilities to generate DC (subtypes), to stimulate leukemic cells to proliferate, or the resulting DC products were finally tested for the ability to activate antileukemic T cells.

[0049] Table 2 shows the used individual ingredients, their biological/clinical effect and previous areas of clinical applications: In summary, GM-CSF, IFNα, TNFα and PICIBANIL are used as immunomodulators and immunostimulants, for hematopoietic regeneration and systemic application for the treatment of various diseases (PICIBANIL is also applied locally in tumors). PGE 2 is used in high concentrations to initiate the birth and is administered vaginally locally or systemically. Due to its vasodilatory properties, PGE 1 is systematically used for example for treatment of VOD disease or of ischemias on extremities. TNFα can be administered systemically but also locally in soft tissue tumors in high concentrations. CALCIMYCIN is not yet used systematically in human beings (although experience from animal experiments are available), but is used ex vivo for the treatment of oocytes before in vitro fertilization.

[0050] DC/DCleu-Generation Ex Vivo-Selection of the Best Kits (Ranking):

[0051] It was shown that not every Kit is equally well suited for DC (subtype) generation in individual patients (FIG. 2a). Therefore, we have created a “ranking list” for DC generation kits where the percentual values of DC, DCleu as well as mature migratory DC (DC mig) were used for the evaluation: FIG. 3a shows the frequencies of individual kits (composition see Table 3) being BEST OR SECOND best DC generation method compared to at least 3 other kits: HS-F, D, M, I, A, K ranked among all as best or second best methods, while the remaining kits ranked worse (FIGS. 3a and d). Alternatively, the frequencies are shown where DC-generation with kits was ranked as “EXCELLENT OR HIGH” (only oriented on DC subtype qualities), (FIG. 3b, criteria for the classification are shown in FIG. 3c). Again the kits HS-K, F, M, I, D, A were ranked as ‘EXCELLENT OR HIGH’ DC generation if fractions of mature DCmig were included in the ranking (FIG. 3d, middle column) or excluded (right column). All in all, our results showed that the first 6 kits mentioned are in any case capable to produce good DC/DCleu proportions, while the remaining kits or individual substances or controls only rarely produced sufficient amounts of DC at all.

[0052] Induction of Blast Proliferation Ex Vivo:

[0053] It must be excluded that administered kits to patients induce blasts' proliferation (in vivo). Blast proliferation-tests were performed in a WB-model on 51 samples: flow cytometry showed expression of the proliferation marker CD71 (transferrin receptor) on the blasts before (proportions of double CD71+ and blast-markers+blasts: CD71+Bla+/Bla+′) and after influence of the kits (here the proportions of DCleu (expressing the blast markers) were excluded: CD71+Bla+DC−/Bla+)). Only cases with an expression of more than 10% of proliferation markers before culture were included in the analyses. CD71 is expressed regularly but not by all AML cases on the (unmodulated) blasts. Apart from a few exceptions (approximately 14% of the cases), no increase in CD71+, proliferating blasts was found after Kit-culture. Thus, with the exception of a few cases, it can be assumed that the proliferation of blasts under influence of kits will not be induced, however, the proliferation of blasts under the Influence of specific, patient-individually selected kits (in particular with regard to an in vivo application) should be studied. In the future, further methods to study proliferation (CFDA assay, Flow cytometric measurement of intracellular proliferation markers) should be applied in order to obtain a proliferation state in all patients before WB cultivation.

[0054] The used kits stimulate only in exceptionally cases (or only individual kits in individual patients) the blast proliferation in the WB-system. Therefore it is advisable to test the influence of different Kits on blasts in WB samples in all individual patients with leukemia and to chose the Kit for in vivo application which does not induce any blast proliferation and produces at the same time high DCleu proportions.

[0055] Preparation of Antileukemic T-Cells after Kit-Stimulation:

[0056] Ultimately, the antileukemic function of the T cells in the body is to maintain a long-lasting remission. Therefore, according to the invention, an optimization of the antileukemic T cell function and generation/reactivation of T-memory cells would be a great clinical benefit to fight (residual) leukemic cells in the patients' body. According to the invention it was therefore examined whether the Kit treatment of WB blasts leads to a better and more efficient antileukemic activity of T cells. FIG. 1b shows schematically how the ex vivo detection of the T cell function is performed: First, leukemic blasts are converted with a Kit to DCleu (b1). To the DC-containing (=Kit-modulated blasts) as well as to blasts without Kit treatment (control) in WB cultures isolated T cells from the patient were added and cultured together in mixed lymphocyte cultures for 7-10 days (b2). Finally, the antileukemic function of the cells is determined by the addition of (labeled) leukemic cells in a lysis-assay (flow cytometry): after incubation of these target cells with T-effector cells the decrease as well as the increase of blasts can be quantified (b3). Thereby it should be worked out whether the Kit treatment of WB blasts ends up in a better and more efficient antileukemic activity of T cells. FIG. 4 shows results obtained with Kit-treated “Modulated WB blasts” in comparison to WB controls: due to the ranking criteria and the fact that the T cells are limited our trials functional tests were performed with the HS-A, F, D, I, K, M Kits. It can be seen that not always antileukemically effective Tcells are provided after Kit treatment of WB (T cells without blastlytic activity can also arise) and that the lytic efficiency of the resulting T-cells can be variable or individual best kits can be selected (FIG. 4a). In addition only WB (“blast-stimulated”) Tcells can also be capable to give rise to antileukemically stimulated and cytotoxic Tcells (ex vivo), although less frequently and less efficiently. FIG. 4b shows that patient-specific Kits could be selected that improve the antileukemic function of the T cells most compared to the WB control. In almost all cases (at least) one Kit could be found, which leads to an improvement of the antileukemic Tcell function: selecting the best achieved LYSIS value after Kit influence and compared it with the WB control without addition of Kits at least one Kit could be always found (with the exception of some cases) in that the antileukemic function and at the same time an improvement over the control (without addition of Kit) could be shown. FIG. 4c shows (parallelly obtained) results with HS-D, I, K, M-treated kits. These 4 kits were selected preferentially and the kits HS-A and F were put on hold since the systemic application of TNFα or CALCIMYCIN-containing kits to patients is currently not allowed. It can be seen that kit-treated WB samples show differences in the provision of efficient antileukemic T cells. Therefore, a ranking of the antileukemic function was performed by us. This revealed that kits HS-I, K, F, M, followed by HS-A, D were most antileukemically effective ex vivo compared to whole blood control (data not shown). In addition to the results listed, we also compared the antileukemic efficacy WB-in comparison to kit-stimulated WB samples or also to unstimulated T cells. These studies have also shown that not all kits are able to provide comparable and patient individually variable and antileukemically (more) effective T-cells, so that, in principle, detailed functional analyzes are needed compared to different controls in order to find the patient individual-best kit which provides the best antileukemic T cell function.

[0057] T cells (functional profiles) as well as cytokine profiles before and after stimulation of T cells with whole blood samples (with or without kit treatment) are investigated comparatively to determine whether regulatory or antileukemic, effective T cells as well as memory cells or inhibitory/activating cytokine profiles are generated. For this purpose, flow cytometric analyzes for the determination of cellular profiles as well as analyzes for the determination of cytokine profiles as well as functional analyzes are carried out.

[0058] Thus, the presented results suggest that the immuno-modulation of blasts with the tested substance mixtures is basically possible and can be achieved on a regular basis, although patient-specific differences occur. These kits can modulate AML blast-containing WB samples, resulting in a good antileukemic efficacy of the T cells stimulated therewith, although this does not work equally well for each kit or individual patient sample. Ultimately, however, the best combination of kits in WB can be determined and ultimately optimized to select the patient's individual best kit for an in vivo application.

[0059] Kit Selection:

[0060] Table 3 shows the kits prepared according to the invention as well as the most important findings and clinical considerations before use in the patient (details below): The patient-individual best from the first 4 primarily selected kits (HSD, I, K, M) is to be determined and finally administered to the patient. HS-C, F and A are first presented as subordinate kits. The reason for this is that CALCIMYCIN has not yet been used as well as TNFα can no longer be used systemically and TNFα is to be administered in a triple combination. The combination HS-E as well as the single substances HS-G, H and L were tested, are basically possible for use on the patient, but not as efficient in the provision of DC (subtypes) compared to the other combination preparations; HS-E, G, H and L are therefore also subordinated. The preferential kit selection according to the invention shown here is therefore: HS-D, HS-I, HS-K, HS-M. Conceptionally, the selection of these kits means that they should develop different immunomodulatory/stimulatory effects (Table 1, 2): in all combination kits GM-CSF is contained, a cytokine that induces and stimulates myeloid cell proliferation and differentiation, the provision and regeneration of stem cells, immunocompetent cells, granulocytes and also of DC generally works without promoting blast proliferation (Table 3).

[0061] In the case of kit HS-I (GM-CSF and PICIBANIL), the additional application of the streptococcal lysate is intended to activate immunoreactive cells and the conversion of the myeloid blasts to DCleu, as is already known for the ex vivo generation of DC. Possibly AML patients treated in this way could also benefit from the influence of PICIBANIL on the vascular system (antiangiogenetic effect, increased permeability of the endothelium for immunoreactive cells or humoral factors).

[0062] In the case of HS-M (GM-CSF and PGE1), PGE1 is to be administered in addition to GM-CSF. In analogy to PGE2, it was shown that PGE1 also promotes the differentiation of DC; PGE1 is also used (in higher concentrations) for the treatment of VOD disease of patients after SCT. Thus, HS-M might provide a desired additional vasodilatory effect for patients after SCT.

[0063] HS-K (GM-CSF and PGE2) and HS-D (GM-CSF and PGE2 and PICIBANIL) are kits containing PGE2. PGE2 increases the maturity or the ability to migrate. HS-D also contains PICIBANIL, possibly enhancing the immunomodulating function of the kit (followed by improved T cell activity).

[0064] Subordinate Kits:

[0065] TNFα-containing kits HS-A (GM-CSF and TNFα) as well as HS-C (GM-CSF and TNFα and PGE2) contain TNFα in two- or triple combinations: TNFα is involved as an acute phase protein in inflammatory processes, as a regulator cytokine of cellular processes as well as for activating the immune system—for example, as an inductor of DC maturation. TNFα has so far been used locally for the treatment of soft tissue tumors (in high concentration) and systemically (low concentration) for the treatment of patients with advanced neoplasia. Initially HS-A and HS-C should be used in leukemia patients (in low doses of TNFα) as soon as experience with the first-mentioned kits in the treatment of patients are available. HS-F (GM-CSF and CALCIMYCIN) are also mentioned: CALCIMYCIN has antibiotic action as well as effect on the calcium ion exchange, acts as a decoupler and ATPase inhibitor. In the animal experiment, it has been shown that (i.v. administered) it triggers (in extremely high dosage) inflammations or anaphylaxis. CALCIMYCIN has been used extracorporally to fertilize oocytes in man, but has not yet been applied systematically to humans. It is planned to use HS-F in leukemia patients (in low CALCIMYCIN doses), as soon as experiences with the first-mentioned kits in the patient treatment are available. In the case of kit HS-E (GM-CSF and INTRON) additional activation of the cytokine IFNα2b (=INTRON) could lead to an activation of the immune system as well as to the conversion of the myeloid blasts to DCleu as is known from chronic myelogenous leukemia; HS-E (GM-CSF), HS-H (IFNα2b) and HS-L (PICIBANIL) were basically possible for use on the patient but were shown less efficient in comparison to the combination preparations in providing DC (subtypes); HS-E, G, H and L are therefore to be subordinated. In the last step, CORRELATION ANALYSIS must be performed to investigate the role or amount of subpopulations of DC, T cells and cytokine concentrations in the context of the antileukemic lytic function. The data are not yet available for evaluation.

[0066] Conclusions and Implementation of the Invention in the Clinic:

[0067] Even in successfully chemotherapy or stem cell transplanted AML patients often relapse because leukemic cells remain in the body and are ultimately responsible for the relapse. The present invention addresses these residual blasts: they are to be modulated to DCleu with the aid of individually selected and applied kits (consisting of clinically approved substances, growth factors and immune modulators) in such a way that, as a leukemia-antigen presenting DC vaccine′ activate antileukemic T cells, create an immunological T-cell memory, additionally help to overcome other cellular and humoral immunological barriers and thus protect the patients from relapses.

[0068] It could be principally demonstrated that DC/DCleu can be generated from WHOLE BLOOD (WB) of patients with myeloid leukemia (AML) using “standard procedures” as well as minimalized combinations of immunomodulators (kits) and that antileukemic T cells after stimulation arise without inducing blast proliferation. Patient-specific differences were observed: not every kit was successful in every patient, and the antileukemic T-cell activation was not successful in every patient. Thus, in a clinical context, it should be possible-after pre-testing in WB samples- and application of (optimally selected) optimal combinations of immunomodulators to induce the in vivo production of ‘DC/DCleu’, inducing in vivo antileukemic T cells and thus allow the maintenance of remissions. This TESTING should be performed in cell culture with blast-rich WB samples (taken in acute, blast-rich disease stages), the TREATMENT of the patients should be done in stages, in which the patients are not too much (eg disease- or chemotherapie-induced) immunocompromised, to enable a regeneration of immune-competent cells. The present invention is potentially useful for the treatment of all patients of all ages with myeloid leukemia (chronic or acute myelogenous leukemia or myelodysplasia) before or after SCT. The approach is not HLA-restricted and possible without knowledge of leukemic antigens involved. In principle, patients in acute as well as in chronic disease stages/remissions can be treated with the presented concept. The clinical efficacy, tolerability and safety of the individually selected (very low-dose) kits is to be tested in about 10-15 patients at relapse after SCT.

[0069] Ex vivo, we have shown that different kits have different effects on individual patient samples or are differently efficient, so that a personalized treatment protocol is to be derived. In every given patient-initially with AML at relapse—the following kits are to be tested on WB samples (HS-I, K, M and possibly D) and the most efficient kit ex vivo for each patient should be selected: a kit that achieves the best blast modulation (Best DC/DCleu subtypes) without inducing a blast proliferation, that induces the most effective antileukemic T-cell response with patient T cells (without causing overshooting T cell responses, eg, in GvH) and produces the best micromilieu/cytokine profile. By treating the patient with this kit, an overcoming of immunological barriers could be achieved and a functioning cellular and humoral immunity in the patient could be restored. Primarily, 3-4 kits are to be used in selected relapsed patients after SCT, whose individual substances are well tolerated or even minimize clinically/therapeutically known complications in the mentioned patient group.

[0070] In principle, kits with potentially risky components (eg TNFα or CALCIMYCIN) could also be used, although severe side effects are not to be expected because of the very low dosage. In principle, the application of single substances (GM-CSF, INTRON, PICIBANIL) would also be possible, but possibly not so effectively in the provision of antileukemic T cells.

[0071] The application form (intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.), intradermal (i.d.)) is to be carried out according to instructions and recommendations of the pharmaceutical manufacturer (Table 3, column 4). I.v. applications are generally preferred (if possible) in order to ensure as direct a direct influence of the substances on leukemic cells in the blood.

[0072] In some cell culture preliminary experiments, it was shown that the sequential (compared to the simultaneous addition) of the substances to the WB cultures gave rise to comparable amounts of DC subtypes (data not shown). It must also be clarified whether GM-CSF i.v. should be administered as a permanent infusion or in frequencies as recommended by the manufacturer.

[0073] The duration of the application to the patient can last from at least 3 months to 2 years and must also be decided in the context of clinical application to the patient: The manufacturers of the substances listed here recommend a treatment period of up to 180 days (Table 3, column 7). It is expected that the application will be limited to 3 to 4 months if patients are treated for relapse after SCT. The advantage of using the kits in this patient group is that the effects on (numerous blood) blasts can be directly examined. If these treatments were to accompany DLI treatments, a further advantage would be the administration of defined amounts of T cells, and thus the mainly addressed effector cells. Treatment of patients in remission of the disease in the course of a preservation therapy should be carried out over a period of up to 2 years (in analogy to other maintenance therapies). Depending on the results of a monitoring (see above), an extension or shortening of the recommended treatment time could result.

[0074] The dosage of the substances is to be carried out with very low, immunomodulating doses and is based on the concentrations used in the WB cultures; it could in principle also be increased to higher doses as recommended by the manufacturer (Table 3, columns 4 and 5). Probably the optimal dosage will have to be worked out after preliminary experiments in the animal model: First, the selected kits are to be administered in some animal preliminary tests to immunocompromised rats or mice (eg NOD-SCID) (to which human AML blasts and T cells were administered): The 2-3 best kits have to be tested in different dosages in parallel (compared to blood samples without kit treatment) in 2-3 immunocompromised mice/rat groups in order to clarify whether there is a blast reduction, an antileukemic T cell activation, T cell subtypes (eg effector cells, memory cells) are provided and an activating cytokine milieu is installed.

[0075] Table 3 shows the kits according to the invention presented here and gives an overview of their composition, the concentrations of individual substances used ex vivo, as well as clinically relevant considerations on the dosage, application form and duration of the treatment with these. The primary selection of kits according to the invention is: HS-D, HS-M, HS-K, HS-I. Moreover some data of the DC generation efficiency and antileukemic activity of kit-treated T cells are presented. Here, too, it can be seen that the above mentioned kits show good DC generation or antileukemic activity of stimulated T cells. Overall, it should be mentioned that no specific treatment protocol for patients is available at the moment. Therefore, dosages, application suggestions, etc. indicated in the text or in the illustrations are intended only as a guide.

[0076] Besides examinations of the WB samples before/after their ex vivo processing, regular studies of blood samples of the patients have to be carried out during the in vivo kit treatment (monitoring) in order to record their efficacy and side effects profiles. These examinations must be carried out BEFORE beginning the application (control values) as well as at several points in the course of the treatment. These times should be based on the time points recommended for other immunotherapeutic treatments: First, the quantification of leukemic cells by means of flow cytometry (blast profile) and molecular profiles (overexpression of leukemia-associated antigens, PCR); possibly also DC/DCleu, if they are detectable in the blood. In addition, a characterization of the role involved in the leukemia defense of cellular and soluble factors has to be performed: T cell subpopulations (regulatory, CD4/CD8, memory/effector T cells, T cells with defined Vβ profile, functional characteristics of involved T cells (antileukemic activity) possibly detection of LAA-specific T-cells (Tetramer-analyses), humoral factors (inhibitory (e.g. IL-10, TGFβ, CXCR4)/stimulatory soluble factors, chemokines, cytokines (eg, IL-2, IL-12, IL-17, IFNγ) measured in cytokine detection assays).

[0077] By monitoring of soluble and cellular factors at different timepoints in the course of the disease and by correlating the data with clinical events (for example, relapse), the clinical relevance, treatment success and the prognostic value of our approach can be developed.

[0078] It is within the scope of the present disclosure that comments on the various aspects of the invention, as set forth in the appended independent claims, as specifically set forth in connection with one aspect, also provide an explanation of all other aspects of the invention. To this extent, embodiments of an aspect of the invention are also embodiments of the other aspects of the invention.

[0079] Test Results from the Use of Kits in Rats with Leukemia:

[0080] In the meantime, the kits were tested in a leukemia rat model, which should also be shown in vivo as an example of the effectiveness of kits:

[0081] BN—(, brown norway) rats as well as PVG.1N rats are rats with the same MHC profile. In BN rats an AML-M3 can be induced by injection of BNML leukemic cells. The leukemia manifests itself within 14-17 days in the spleen, later also in the peripheral blood and then leads to death within 7 days. Blood samples can be taken from the tail vein for cell analyses. After the sacrificing of the animals (after 23 days, shortly before their AML-related death), cell analyses can also be carried out on spleen and blood cells after heart punctions.

[0082] Ex Vivo DC/DCleu Generating from WB of Leukemically Diseased Rat:

[0083] In analogy to human blast-containing blood, we performed cultivation of AML rat WB with kits HS-D, I, K, M as compared to WB controls. (The same substances as in humans were used, except for rat GM-CSF). First evaluations showed that DCleu can be generated from rat blood from 5 animals as well as from human leukemic WB (on average 10-35% within the entire cell fraction with a convertibility of the blasts to DCleu of 30-50%). The most successful kit was HS-I, followed by HS-M and HS-K. An induction of blast proliferation (co-expression of Ki67) was seen after treatment with none of the kits. In addition, after co-culture of DCleu-containing kit-treated WB samples with autologous rat T cells, increased blast lytic activity against BNML blasts could be detected in all kits. The highest antileukemic activity had HS-I-WB-stimulated T cells. More detailed analyzes are still pending. Since basically blasts in rat blood were convertible to DCleu now in vivo treatments should be carried out.

[0084] In Vivo Treatment of PVG.1N Rats with Picibanil, PGE1 and PGE2 (Safety Data):

[0085] Safety data were performed on a total of 4 rats of the healthy strain PVG.1N: they were given the clinically intended final concentrations of the single substances iv in the following doses: Picibanil (0.35 μg/rat), PGE2 (1 μg/rat), PGE1 (1 μg/rat) and placebo (PBS). The rats showed no (severe) side effects (S) AE: breathing, sleep, food intake, mobility, skin, weight were completely normal or as in control animals (data not shown). Flow cytometric analyzes on blood cells of the PVG.1N rats showed no difference compared to the placebo controls (Teff, Treg, CD4, CD8, NK, NKT cells, data not shown).

[0086] In Vivo Treatment of Leukemic Diseases BN Rats with Kits (Efficacy Data):

[0087] Since the tolerability of the substances we use is very good, now BN rats were treated after induction of leukemia (day 0) on days 14 and 17 with 4 kits compared to the placebo controls (iv administration in penile vein, see FIG. 5 a): 3 rats each received:

[0088] HS-I.sub.low: GM-CSF (1 μg/rat)+Picibanil (0.175 μg/rat, corresponding to 0.5 KE in humans)

[0089] HS-I.sub.high: GM-CSF (1 μg/rat)+Picibanil (0.35 μg/rat, corresponding to 1 KE in humans)

[0090] HS-K: GM-CSF (1 μg/rat)+PGE2 (1 μg/rat)

[0091] HS-M: GM-CSF (1 μg/rat)+PGE1 (1 μg/rat)

[0092] Placebo: PBS

[0093] The efficacy of the kits was determined on day 23 as a comparison of the blast proportions in blood and spleen after kit treatment compared to placebo control: While HS-I.sub.10 (with low-dose picibanil) and HS-K (PGE2-containing) did not show differences in blast proportions compared to controls the blast portions after treatment with HS-I.sub.high were reduced by 4.4-6.3% in blood or spleen, after HS-M treatment the blast portions were even significantly reduced by 10.7-15.8%.

[0094] Analysis of the immunoreactive cells in the blood at the time of the sacrifice of the rats revealed the following picture: CD62L+CD4+bw CD8+T (memory) cells were increased by 10.4% (CD4+) or 21.1% (CD8+) in rats' blood (FIG. 5c). The percentual reduction of CD4+FoxP3+CD25+ regulatory T cells after treatment of the rats with all kits (by −24.3% (HS-I.sub.low) to −55.6% (HS-M)) in the spleen was also noticeable in comparison to control (FIG. 5d). Further data on immunoreactive cells are not yet available.

[0095] In summary, our results obtained with rat blood show that the ex vivo generation of DC/DCleu is similar to that achieved with human WB without increasing the proliferation of the blasts. The treatment of healthy rats with the single substances showed a very good compatibility of the substances; The treatment of AML-M3 leukemia-mediated BN rats with different kits HS-K, M, I showed that a blast reduction of up to 15.8% could be achieved in only 10 days of treatment with our immunomodulatory treatment concept Well in this leukemia form, but not every kit works equally well in this leukemia form.

CONCLUSIONS

[0096] Based on the present invention, it will be possible at an early stage to develop an “in vivo protocol” which allows the application of minimalized combinations of immunomodulators (without need for a GMP facility) for patients with myeloid leukemia. The detailed design of a personalized treatment protocol for patients with myeloid leukemias with different or differently combined immunomodulatory single substances as well as the detailed concept of an immune monitoring must be carried out after consultation with treating physicians before the start of the treatments and will be based on other immunotherapeutic treatment protocols. Expertise in immunotherapeutic treatments is given to the institutions for the treatment of patients. A routinely collected diagnostic support program for the examination of the blood samples as well as clinical findings on patient profiles is available. Thus, experience, expertise and synergisms available for the clinical implementation of the invention can be used to treat—initially—AML patients at relapse after allogeneic SCT.

TABLE-US-00001 TABLE 1 Generation of DC/DCleu from AML-blast-containing Mononuclear cells (MNC) or Whole Blood (WB)-cells with various DC-differentiation methods or specific kits a) DC-differentiation methods or specific kits for the generation of DC/DCleu from blast-containing MNC (a1-a4) or WB (a1-a5) DC- ,Kits': HS-A, C, D, E, generating ‘CA’ ‘MCM’ ‘PICI’ ‘INTR’ F, G, H, I, K, L, M* methods a.1 a.2 a.3 a.4 a.5 Culture time 3-4 days 10-14 days 9-11 days 10-14 days 8-10 days [d] DC- IL-4, GM-CSF, IL- GM-CSF, GM-CSF, Various combinations generating CALCIMYCIN 4, TNFα, IL- TNFα, PGE.sub.2, INTRON, of 1-3 clinically methods 1β, IL-6, PICIBANIL TNFα approved single PGE.sub.2, substances/immune modulators Mechanism of Bypass of the Cytokine- Bacterial Cytokine- DC differentiation by Action cytokine induced DC- lysate and induced DC- combination of induced DC differentiation; PGE.sub.2 differentiation immune modulators/ differentiation PGE.sub.2 stimulate DC- danger-factors/ increases differentiation cytokines CCR7- expression and improves migration b): Advantages and value of DC generation from whole blood samples Comments on the clinical relevance or Advantages implementation in a clinical application Simulation of a physiological system Transferability to the clinic Capability to quantify and qualitatively characterize cells Conclusions on biological mechanisms/reaction profiles involved in responses possible increase in blasts can be quantified Proportions of blasts interesting immunereactive cells (e.g.: regulatory, T cell (sub)populations, NK, NKTcells effectormemory, integrin-positive Tcells, NK, NKT-cells Success of a blast conversion to DCleu or In vivo, DC in the blood are difficult to quantify because DCsubtypes well quantifiable of their migration into the tissue Analysis of the microenvironment on immunostimulating Detection of, escape'- or antileukemic mechanisms and and -inhibiting influences possible possible interactions; Development of appropriate therapeutic strategies Functional tests possible in whole blood Antileukemic responses quantifiable (decrease/increase of blasts), effector cell profiles measurable (proliferation, subtypes) PGE.sub.2: Prostaglandin E2; IL-4, -1β, -6: interleukin 4, -1β, -6; PICIBANIL: OK432 (Bacterial lysate from stretococcus pyogenes); INTRON: Interferon alpha 2b; FL FLT3 Ligand TNFα: tumor necrosis factor alpha; GM-CSF: granulocyte macrophage colony stimulating factor; *Details see Table 2

TABLE-US-00002 TABLE 2 Active ingredients in the kits for the immunomodulation of AML blasts Active substance Functions/effects Previous clinical applications GM-CSF induces proliferation and (1) AML patients >55 years: after chemotherapy to Leukine differentiation of reduce neutrophil recovery, to reduce the incidence Sargramostin) hematopoietic cells of infections (precursor cells, (2) mobilization of stem cells into the PB (for the neutrophils, monocytes, collection of stem cells) macrophages, myeloid (3) Myeloid cell reconstitution after autologous/ DC) allogenic SCT of ALL, NHL, HD reduces duration of (4) Treatment of transplant failure after autologous/ neutropenia allogeneic SCT reduces incidence of (5) Monocyte/DC activation in melanoma patients infections in vivo reduces administrations (6) Immune modulation of blasts (s.c or iv) of antimicrobial substances TNFα Cytokine, which activates (1) Local treatment of sarcoma, non-resectable Kachexin acute phase proteins tumors, melanomas of the extremities Induction of apoptosis, (2) Systemic (i.v.) treatment of patients with cell proliferation, - advanced neoplasia differentiation, cytokine release triggering of inflammation, necrosis, cachexia, fever Autoimmune-reactions, INTRON Cytokine with (1) Treatment of hepatitis B and C IFNα2b immunomodulating effect (2) Treatment of CML important in the defense (3) treatment of myelomas, of virus infected cells, (4) Treatment of follicular lymphomas important in the (5) Treatment of carcinoid tumors activation of the immune (6) treatment of melanomas, system (7) Treatment of hairy cell leukemia regulators of cell (Administration i.v, i.m., s.c.: in healthy volunteers i.v proliferation and s.c/i.m. applications comparable) PGE.sub.1 vasodilatorial properties (1) Treatment of Venoocclusive Disease (VOD) (Alprostadil) (increase in blood flow) after SCT anti platelet activity (2) treatment of erectile dysfunction (3) Preservation of the Ductus Arteriosus Botalli (4) Treatment of ischemia of the extremities (I.v administration) PGE.sub.2 is formed by (activated) (1 induction of labour, birth promotion, introduction (ProstinE2) blood platelets, possibly of abortion produced by erythrocytes (2) Application iv, sc, im (by activation of Ca transport in Erys): involved in coagulation! contracting PICIBANIL Streptococcus pyogenes (1) intralasional treatment of benign lymphatic OK432 (low virulent, H2O2 and tumors (lymphangiomas, cystic hygromae), head (Chugai Pharma) penicillin-treated) and neck tumors induces activation of (2) intrauterine treatment of cystic hygroma immunoreactive cells, (3) Intratumoral injection of immature DC and cytokine release, OK432 in or near tumors of pancreas carcinoma increases permeability of (4) Induction of pleurodesis by pleural infusion of endothelium, OK432 antiangiogenetic effect (5) Immunomodulation in squamous cell carcinomas (Hitayama 2013) (Id or near the tumor) I.m., i.d or s.c according to approval CALCIMYCIN, Antibiotic activity against (1) Ex vivo: Treatment of oocytes before in vitro CalciumIonophor, gram-positive bacteria fertilization A23187 increases intracellular (2) Pig: i.v. administration (high dose 5 mg/kg) (Sigma) Ca + 2 ion concentrations indicates anaphylactic shock Product of decoupler of oxidative (3) Rat: intrapleural injection produces pleurisy Streptomyces phosphorylation (4) Guinea pigs: Inhalation produces pneumonia chartreusensis inhibitor of mitochondrial ATPase

TABLE-US-00003 TABLE 3 Composition of the tested kits Comments on 1, 2, 3, Clinical considerations: PLANNED in vivo method of administration (AA), dosage (Do): indicated as range (Do.sup.R) as well as in a immunemodulatory context/related to 6 L blood (Do.sup.I), frequency of administration (VF) and -duration (VD) Do.sup.R Do.sup.I/[6I] .sup.(1)μg/m.sup.2 .sup.(2)mio I .Math. E/m.sup.2 .sup.(3)μg/day (2)anti- .sup.(4)mg/kg/day VD (1)Efficiency leukemic AA .sup.(5)μg/m.sup.2/day (overall: 3 of DC- T-cell- in CELL (bold: .sup.(6)μg/week (=we) months up generation function Kits CULTURE Planned) .sup.(7)mg/m.sup.2/day VF to 2 years) N/NN (%) N/NN (%) HS-D: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 38/43 (88%) 10/14 (71) PICIBANIL  10 μg/ml, im, sc, id 20-500.sup.(3) 50.sup.(6) ca3-7x/we  -21 days PGE.sub.2  1 μg/ml, iv, sc, im 0.1-5.sup.(4)   ca3-7x/we  7-42 days? HS-E: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 15/25 (60%) 6/9 (67) INTRON 500 U/ml iv/sc/im 2-5.sup.(2)  3-3.5 mio ca 3-7x/we -180 days HS-I: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 30/30 (100%) 14/19 (74) PICIBANIL  10 μg/ml im, sc, id 20-500.sup.(3) 50.sup.(6) ca3-7x/we  -21 days HS-K: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 13/13 (100%) 7/9 (78) PGE.sub.2  1 μg/ml, iv, sc, im 0.1-5.sup.(4)   ca3-7x/we  7-42 days? HS-M: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 3/4 (75%) 3/3 (100) PGE.sub.1  1 μg/ml, iv 0.075-500.sup.(3)   ca7x/we  -30 days HS-A: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 36/44 (82%) 3/3 (100) TNFα  10 ng/ml iv 0.04-4.sup.(7)    HS-C: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 30/37 (81%) 5/9 (56) TNFα  10 ng/ml, iv 0.04-4.sup.(7)    ca3-7x/we PGE.sub.2  1 μg/ml, iv, sc, im 0.1-5.sup.(4)   ?  7-42 days? HS-F: GM-CSF 800 U/ml, ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 42/45 (93%) 11/14 (79) CALCIMYCIN 375 ng/ml ex vivo 5-10 μM once once HS-G: GM-CSF 800U ic/sc/iv 15-500.sup.(1) 75.sup.(5) ca3-7x/we 7-42 days 18/27 (66%) HS-H: INTRON 500 U/ml iv/sc/im 2-5.sup.(2)  3-3.5 mio ca 3-7x/we -180 days 4/12 (33%) HS-L: PICIBANIL  10 μg/ml im, sc, id 20-500.sup.(3) 50.sup.(6) ca3-7x/we  -21 days 6/9 (67%) w/o 6/41 (15%) 16/22 (73) * Based on 6 liters of blood, according to the drug concentrations used in vitro W/o WB control without kits