Pharmaceutical product for autologous adoptive cellular therapy

Abstract

The present invention concerns a pharmaceutical product for use as an adoptive autologous cellular therapy against a disease in a human or animal subject, comprising two compositions comprising various combinations of autologous immune cells to be administered in three separate doses sequentially to the human or animal subject.

Description

[0001] TITLE

[0002] PHARMACEUTICAL PRODUCT FOR AUTOLOGOUS ADOPTIVE CELLULAR THERAPY

[0003] TECHNICAL FIELD

[0004] The present invention relates to a pharmaceutical product for autologous adoptive cellular therapy in the treatment of a cancer disease in a human or animal subject, comprising two compositions of various combinations of autologous immune cells to be administered sequentially in three doses to the human or animal subject, as well as a method for the manufacture of such a pharmaceutical product.

[0005] PRIOR ART

[0006] According to the WHO, cancer is the second leading cause of death worldwide, accounting annually for about 10 million deaths. Besides surgery, long-established and widely used conventional therapies, such as chemotherapies and radiation therapies, offer rapid results by killing cancer cells, but they have major drawbacks. In most cases, there is no specificity for cancerous cells and as a result, also healthy cells close to target cells are killed. Patients often face severe and long-lasting side effects, and furthermore, tumor cells can develop escape mechanisms and become resistant (Mansoori et al., 2017).

[0007] It is crucial, especially for advanced-stage cancer patients, who have limited treatment options or have developed resistance to front-line therapies, to identify novel and effective therapies (Cheng et al., 2021).

[0008] Immunotherapy triggers a patient's own immune system to fight cancer cells. It is increasingly gaining ground as a potential alternative treatment for cancer patients (Waldman et al., 2020), also due to the reduced side effects it results in. Targeted antibodies, checkpoint inhibitors, vaccines, cytokines, oncolytic viruses and adoptive cell transfer are different forms of established immunotherapies. They can be used alone or in combination with chemotherapies, radiation, surgery or targeted therapies to improve the efficacy of the treatment (Waldman et al., 2020).

[0009] Adoptive cellular therapy is a type of immunotherapy which employs the use of ex vivo activated and expanded immune cells. Clinical trials have shown remarkable results of adoptive cellular therapies against tumors which are highly resistant to conventional therapies (Kirtane et al., 2021). Challenges limiting the efficiency of cellular therapies include immunosuppressive tumor microenvironment, insufficient number of tumor antigens, and poor cell trafficking.

[0010] This is where personalized medicine steps in, by developing ways to determine, following a screening procedure, the potential of each patient to respond to a specific treatment. Each cancer type requires a specific treatment approach. Furthermore, as no two patients are the same, the treatment regimen shall be selected according to the genetic profile and disease history of the specific individual. In oncology, personalized treatment takes into account the inter- and intra-tumor variability of each individual patient (Hoeben et al., 2021). Efficiency of adoptive cellular immunotherapy depends on the immune status of each individual.

[0011] Autologous cell therapies involve the isolation, activation and expansion of each patient's cells outside the body (ex vivo) and reintroduction of cells to the patient (Sarivalasis et al., 2021). Side effects generally are fewer than with other types of therapy.

[0012] Innate immune cells are able to fight cancer cells in an antigen-independent manner, while T cells and B cells, being adoptive immune cells, require pre-activation with antigenic peptides. Activation of adoptive immune cells is performed with dendritic cells (DC), a type of antigen presenting cells (APC). Said immune cells can be generated from patient’s blood itself and then be infused back to the patient, enabling an autologous therapy. Macrophages (MP) and Natural Killer cells (NK) can recognize and kill cancer cells. Macrophages are phagocytic cells and natural killer cells which have the ability to eliminate abnormal cells, i.e. cancer cells in a manner independent of the major histocompatibility complexes class I or II (MHC-independent) (DeNardo et al., 2019; Multhoff et al., 2020; Kundu et al., 2021). DCs are the most efficient APCs and can activate both humoral and cellular immunity. DCs can be differentiated in vitro from their progenitors found in blood. The aim of in vitro manipulation of DCs for immunotherapeutical purposes, especially against cancer, is to obtain loading of both MHC class I and II molecules, for activation of both cellular and humoral immunity. Cytotoxic T-cells (CTLs) are activated against specific antigens by DCs and have the ability to target and kill cancer cells expressing the specific antigen (Hont et al., 2022; Parsonidis et al., 2019). Antibodies produced by plasma cells tag abnormal cells, especially cancer cells, and help their recognition by phagocytes or complement proteins, leading to the subsequent destruction of said targeted abnormal cells (Thomas et al. , 2020).

[0013] Cancer cellular therapies up till now focused on single populations of immune cells, such as T-cells or natural killer cells. Limitations of T-cell therapies include target antigen heterogeneity, which leads to antigen escape. Another barrier is T cell trafficking and infiltration, preventing effector cells from successfully identifying their target cells and effectively eliminating them. Moreover, immunosuppressive tumor microenvironment can have a negative impact on the efficacy of cell therapies (Kirtane et al, 2021).

[0014] It has been suggested that dendritic cells, macrophages and activated B lymphocytes or antibody-producing plasma cells could also be used for the treatment of cancer patients (Parsonidis et al., 2022).

[0015] SUMMARY OF THE INVENTION

[0016] It is an object of the present invention to provide a personalized immunotherapy with combinations of autologous immune cells in the hope that a greater number and more types of effector cells result in a higher efficacy of treatment to fight abnormal cells such as tumor cells. Thereby, the limitations of T-cell therapies shall be overcome.

[0017] The present invention concerns a pharmaceutical product for use in an autologous adoptive cellular therapy for the treatment of a cancer disease of a human or animal subject, wherein the pharmaceutical product according to the present invention comprises

[0018] - a first dose comprising a first composition, comprising autologous macrophages derived from autologous mononuclear cells, and further comprising autologous natural killer cells,

[0019] - a second dose comprising a second composition, and

[0020] - a third dose comprising the same second composition, wherein the second composition comprises ex vivo activated autologous dendritic cells, ex vivo activated autologous CD8+ cytotoxic T-lymphocytes and ex vivo activated autologous plasma cells.

[0021] Dendritic cells, upon recognizing specific antigens, cell surface proteins or genetic material of unwanted cells, such as pathogens or cancer cells with its surface receptors or intracellular receptors, secrete various types of factors and interferons (IFN) and present processed peptides from various antigens on their cell surface. The term "activated" thus can be seen as the dendritic cells having been triggered to display antigenic material to the downstream fighter cells of the adaptive immune response (i.e. T- and B-lymphocytes). The activation / triggering of the DCs encompasses changes in the phenotype and function of the DCs, including the upregulation of costimulatory and adhesion molecules. The DCs are then capable of capturing antigens derived from pathogens, processing and presenting them to naive T lymphocytes or B-lymphocytes as peptides bound to MHC I or II molecules located on the surface of DCs, thereby "priming" or "activating" the respective T- or B-lymphocytes. DCs also secrete various factors which influence the downstream T- and B-cell mediated immune reaction. By this initiation and modulation of the adaptive immune response, T- and B-lymphocytes are triggered or "activated" to fight the cells displaying the unwanted foreign antigens. They not only help activate B lymphocytes to secrete antibodies and macrophages to destroy ingested microbes, but they also help activate cytotoxic T cells to kill infected target cells.

[0022] For the purpose of the present invention, the activation of the autologous dendritic cells, as well as the activation of the autologous CD8+ cytotoxic T-lymphocytes and the activation of the autologous plasma cells is carried out ex vivo, i.e. outside the body of the human or animal subject to be treated. The ex vivo activation has the purpose of specifically driving the entire immunity against the specific target, i.e. the specific cancer cells. Activated immune cells which already have been activated in vivo, i.e. inside the body of the human or animal subject, are activated against various antigens, i.e. not in a specifically tailored or planned manner. Contrary thereto, the present invention aims at activating naive immune cells ex vivo which can be manipulated, in order to achieve specificity against the specific tumor antigens present in the human or animal subject.

[0023] Preferably, in the first composition the macrophages are M1 -polarized autologous macrophages, which have been differentiated from autologous monocytes. Activated macrophages are usually divided into two categories, M1-like macrophages and M2-like macrophages. Both M1 macrophages and M2 macrophages are closely related to inflammatory responses, however, they adopt different functional programs in response to the signals from their microenvironment. M1-activated macrophages express transcription factors such as Interferon-Regulatory Factor (IRF5), Nuclear Factor of kappa light polypeptide gene enhancer (NF-KB), Activator-Protein (AP-1) and STAT1. This leads to enhanced microbicidal capacity and secretion of high levels of pro-inflammatory cytokines: e.g. IFN-y, IL-1 , IL-6, IL-12, IL-23 and TNF-a. For the purpose of the present invention, it is preferred to keep the macrophages in the M1 -phase. In order to steer autologous monocytes to develop into M1 macrophages, the autologous monocytes isolated from the blood of the human or animal subject are kept in culture conditions in a medium free from GM-CSF and IL-4, which would otherwise trigger them to differentiate into dendritic cells. One day prior to the intended mixture with natural killer cells, the medium is supplemented with IFN-y, in order to signalize the polarization into the M1 state. The classical protocol for M1 polarization is to incubate macrophages in the presence of IFN-y alone or in combination with LPS, in general for 24 h (see Genin et al., 2015).

[0024] In the first composition, the autologous natural killer cells preferably are autologous CD16+ and CD56+ natural killer cells. This means that they express both the surface markers CD16 and CD56 on their cell surface (CD16 and CD56 double positive NK cells).

[0025] According to another preferred embodiment of the present invention, in the second composition, the autologous dendritic cells (DCs) are activated against one or more antigenic peptides.

[0026] It is further preferred that in the second composition, for the second and third dose, the autologous CD8+ cytotoxic T-lymphocytes are, i.e. have been activated against one or more antigenic peptides, such that the activated autologous CD8+ cytotoxic T-lymphocytes (CTL) are capable of recognizing the one or more antigenic peptides. The activation of the autologous CTL preferably has been carried out by ex vivo activation with activated DCs. "Capable of recognizing" in the sense of the invention means that the activated, i.e. primed CTL comprises a T-cell receptor (TCR) which recognizes and interacts with, i.e. is structurally compatible to dock onto MHC I molecules carrying target antigens to recognize either infected cells or tumor cells. Triggering of a CTL requires interaction between its specific T-cell receptor (TCR) and a peptide antigen or tumor antigen presented by an MHC molecule. When the CD8 receptor recognizes an infected cell or tumor cell, it activates the CTL. Structural features are responsible for TCR binding to and activation by the peptide- MHC complex. The CTL activation leads to direct killing of the target cell through induction of apoptotic signals by means of cytotoxic granules and lymphokines.

[0027] It is further preferred, that in the second composition, the autologous plasma cells are activated, i.e. have been activated, against one or more antigenic peptides, such that the autologous plasma cells are capable of producing antibodies against the one or more antigenic peptides. Preferably, the antigenic peptides are isolated from the same human or animal subject.

[0028] Prior to their activation, the plasma cells are actually B-lymphocytes, which after their activation differentiate into antibody secreting plasma cells. Plasma cells are differentiated B-lymphocytes capable of secreting immunoglobulin, or antibody. These cells play a significant role in the adaptive immune response, namely, being the main cells responsible for humoral immunity. For the purpose of the present invention, the B-lymphocytes, prior to their ex vivo activation are termed "autologous plasma cells", and after their ex vivo activation are termed "activated autologous plasma cells".

[0029] According to a further preferred embodiment of the present invention, the ex vivo activated autologous CD8+ cytotoxic T-lymphocytes (CTL) are capable of recognizing at least one of the same antigenic peptides against which the autologous dendritic cells are activated. This means that the activated autologous CTL carry on their cell surfaces receptors which are capable of binding, i.e. structurally compatible to the specific antigens they were activated against, and / or the MHC carrying said antigen(s). This can be a mixture or array of antigens, or a specific single antigen.

[0030] Preferably, the activated autologous plasma cells are capable of producing antibodies against at least one of the same antigenic peptides against which the autologous dendritic cells are activated. This can be antibodies against a mixture or array of antigens, or against a specific single antigen.

[0031] Preferably, the two compositions are adapted for administration to the human or animal subject separately from each other and sequentially at three different points in time, preferably in approximately 2-week intervals, preferably by intravenous injection. Thus, according to a preferred embodiment of the present invention, the first dose comprising the first composition is adapted to be administered to the human or animal subject in week 1 of a treatment schedule, wherein the second dose comprising the second composition is adapted to be administered to the human or animal subject in week 3 of the treatment schedule, and wherein preferably the third dose comprising the same second composition as in the second dose is adapted to be administered to the human or animal subject in week 5 of the treatment schedule. The range of administration times can vary. Preferably, the interval between the dosages within a treatment schedule ranges from 10-20 days and preferably is 14 days.

[0032] The inventive pharmaceutical product is preferably intended for use as a medicament against a cancer disease of a human or animal subject, preferably against a cancer disease selected from a group consisting of the following types of cancer: breast, bronchial, headneck, lung, pancreatic, prostate, colon, mesothelioma, ovarian, skin, thyroid, liver, CUP, bladder, CLL, and myeloma. A use as a medicament against other types of cancer is also conceivable.

[0033] The present invention furthermore concerns a kit-of-parts for use as a medicament against a disease in a human or animal subject, preferably against a cancer disease, comprising the above-described pharmaceutical product.

[0034] The inventive kit-of-parts for use as a medicament against a disease, preferably against a cancer disease in a human or animal subject, comprises:

[0035] - a first dose comprising a first composition, comprising macrophages derived from autologous mononuclear cells, preferably autologous macrophages polarized in a M1- phase, and further comprising autologous natural killer cells, preferably CD16+ / CD56+ natural killer cells,

[0036] - a second dose comprising a second composition, and

[0037] - a third dose comprising the second composition, i.e. the same second composition as is contained in the second dose, wherein the second composition comprises ex vivo activated autologous dendritic cells, ex vivo activated autologous cytotoxic T-lymphocytes and ex vivo activated autologous plasma cells.

[0038] Preferably, the first dose comprising the first composition, the second dose comprising the second composition and the third dose comprising the same second composition each are adapted for administration to the human or animal patient at a different point in time of a treatment schedule, preferably in 2-week intervals, preferably by intravenous injection.

[0039] In said kit-of-parts, the first dose, the second dose and the third dose preferably are provided in separate vials for subsequent administration, preferably by intravenous injection, at three different, separate points in time within a treatment schedule.

[0040] The present invention further concerns a method for the production of a medicament for autologous adoptive cellular therapy in the treatment of a disease, preferably of a cancer disease of a human or animal subject. Said method comprises the following steps to be carried out ex vivo: a.) providing a population of autologous macrophages, preferably autologous macrophages polarized in a M1 -phase; b.) providing a population of autologous natural killer cells, preferably CD16+ / CD56+ natural killer cells; c.) providing a population of ex vivo activated autologous dendritic cells; d.) providing a population of autologous CD8+ cytotoxic T-lymphocytes; e.) providing a population of autologous plasma cells; f.) mixing of the population resulting from step a.) with the population resulting from step b.) for the provision of a first composition for administration to a human or animal subject in a first dose; g.) mixing of a first portion of the population resulting from step c.) with the population from step d.), resulting in a population of ex vivo activated CD8+ cytotoxic T-lymphocytes; h.) mixing of a second portion of the population resulting from step c.) with the population resulting from step e.), resulting in a population of ex vivo activated autologous plasma cells; i.) mixing of the population resulting from step g.) with the population of step h.) for the provision of a second composition; j.) splitting up the second composition resulting from step i.) into two preferably equal portions, resulting in a second dose comprising the second composition and a third dose comprising the same second composition for administration to the human or animal subject.

[0041] According to a further preferred embodiment of the method according to the present invention, in step g.), for the purpose of providing a population of activated autologous CD8+ cytotoxic T-lymphocytes, the population resulting from step c.) is incubated in the presence of p2-microglobulin, preferably for 6-18 hours, preferably for 8-12 hours, and preferably subsequently also incubated with at least one, preferably all of the following group consisting of IL-1 p, TNF-a, IL-6, PGE2 prior to mixing the population resulting from step c.) with the autologous CD8+ cytotoxic T-lymphocytes, preferably for 24-72 hours, more preferably for 48 hours. Preferably, I L-1 b is added at 10-50ng / ml, more preferably at about 25ng / ml, TNF-a is added at 25-100ng / ml, more preferably at about 50ng / ml), and IL-6 is preferably added at 5-25ng / ml more preferably at about 10ng / ml, and PGE2 is added preferably at 0.5-5pM, more preferably at about 1 pM.

[0042] Preferably, in step g.), for the purpose of providing a population of ex vivo activated autologous CD8+ cytotoxic T-lymphocytes, the population resulting from step c.), after mixing with the population resulting from step d.), is further incubated with at least one of the cytokines of the group consisting of IL-2, IL-7, and IL-15, preferably for 8-15 days, more preferably for 10 days.

[0043] Preferably, IL-2 is added at 2-30ng / ml, more preferably at about 10ng / ml, while IL-7 is added preferably at 1-20ng / ml, more preferably at about 5ng / ml), and IL-15 is preferably added at 5-50ng / ml, more preferably about 25ng / ml.

[0044] It is further advantageous, in step g.), for the purpose of providing a population of ex vivo activated autologous plasma cells, to further incubate the population resulting from step c.), after mixing with the population resulting from step e.), with at least one of the cytokines of the group consisting of IL-6, IL-10, TNF-a, and sCD40L, preferably for 8-15 days, more preferably for 10 days. Preferably, IL-6 is added at 50-200ng / ml, more preferably at about 150ng / ml, while IL-10 is preferably added at 50-200ng / ml, more preferably about 100ng / ml, TNF-a is preferably added at 5-25ng / ml, more preferably about 10ng / ml, and sCD40L is preferably added at 0.5-5ng / ml, more preferably about 1ng / ml.

[0045] Preferably, step c.) includes providing an antigen preparation for the purpose of ex vivo activation of the autologous dendritic cells and autologous CD8+ cytotoxic T-lymphocytes in step d.) and for the purpose of ex vivo activation of autologous B lymphocytes in step e.). In case of an intended use of the medicament against a cancer disease of the human or animal patient, said antigen preparation is preferably prepared by isolating antigens present on circulating tumor cells (CTC) and / or cancer stem cells (CSC) of the human or animal subject. For this purpose, the CTC and / or the CSC are preferably isolated using magnetic cell separation, preferably using magnetic beads selective for recognition of at least one tumor antigen selected from a group consisting of CD44, CD133, EpCAM, PanCK.

[0046] Preferably, the activation of dendritic cells in step c.) comprises the followings steps: lysing of a sample of peripheral blood from the human or animal subject to be treated, preferably by NH4CI;

[0047] - washing the peripheral blood, preferably with phosphate-buffered saline (PBS); isolating peripheral blood mononuclear cells (PBMC), preferably by magnetic cell separation; isolating monocytes from the PBMC;

[0048] - washing of the monocytes, preferably with PBS; resuspending of the monocytes in culture medium, preferably supplemented with GM-CSF, IL-4 and penicillin / streptomycin in two flasks, preferably for six days in an incubator, preferably at 37°C and 5% CO2, wherein preferably half way through, GM-CSF and IL-4 are added.

[0049] In an especially preferred embodiment of the abovementioned method, in step a.), for the provision of a population of autologous macrophages which are polarized in a M1 -phase, autologous monocytes are isolated and incubated in a medium free from GM-CSF and free from IL-4, wherein one day prior to step f.), IFN-y is added to the medium in order to trigger a polarization of the macrophages into M1-stage. Preferably, IFNy is added at 5-50ng / ml, more preferably at about 20ng / ml.

[0050] For the purpose of providing the populations of steps a.)-e.), preferably a sample of peripheral blood is collected from the human or animal patient, wherein preferably the populations resulting from steps a.)-e.) are derived from the same sample of peripheral blood. This makes the pharmaceutical product and according to the present invention highly specific to the individual, thus enabling a personalized treatment.

[0051] The present invention further concerns a method of treating or preventing a disease, preferably a cancer disease in a human or animal subject, comprising the following steps:

[0052] - administering a first dose comprising a first composition comprising macrophages derived from autologous mononuclear cells, wherein the macrophages are preferably polarized in a M1-phase, i.e. are M1 -polarized, and further comprising autologous natural killer cells, preferably CD16+ / CD56+ natural killer cells, to the human or animal subject;

[0053] - administering a second dose comprising a second composition, comprising ex vivo activated autologous dendritic cells, ex vivo activated autologous CD8+ cytotoxic T- lymphocytes and ex vivo activated autologous plasma cells, to the human or animal subject;

[0054] - administering a third dose comprising the same second composition as in the second dose, thus also comprising ex vivo activated autologous dendritic cells, activated autologous CD8+ cytotoxic T-lymphocytes and ex vivo activated autologous plasma cells, to the human or animal subject;

[0055] - wherein preferably the first dose, the second dose and the third dose are sequentially administered to the human or animal subject, preferably in intervals of at least one week, more preferably in intervals of 2 weeks.

[0056] Further embodiments of the invention are laid down in the dependent claims.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0059] Fig. 1 shows, with respect to example 1 , the results of total CTOs count and EpCAM positive cells present per 7,5ml blood tested with flow cytometry prior to treatment and 120 days post treatment.

[0060] Fig. 2 shows, with respect to example 1 , the results of cytokine release (I FNy, IL-4, IL- 2, TNFa) with ELISA assay and immune cell markers (CD28+ B cells, CD28+ T cells, CD80+ cells, CD86+ cells) with flow cytometry.

[0061] Fig. 3 shows, with respect to example 2, the results of total CTCs count and EpCAM positive cells present per 7,5ml blood tested with flow cytometry prior to treatment and 120 days post treatment.

[0062] Fig. 4 shows, with respect to example 2, the results of cytokine release (IFNy, IL-4, IL- 2, TNFa) with ELISA assay and immune cell markers (CD28+ B cells, CD28+ T cells, CD80+ cells, CD86+ cells) with flow cytometry.

[0063] Fig. 5. shows, with respect to example 3, the results of total CTCs count and EpCAM positive cells present per ml blood tested with flow cytometry prior to treatment and 120 days post treatment.

[0064] Fig. 6 shows, with respect to example 3, the results of cytokine release (IFNy, IL-4, IL- 2, TNFa) with ELISA assay and immune cell markers (CD28+ B cells, CD28+ T cells, CD80+ cells, CD86+ cells) with flow cytometry.

[0065] Fig. 7 shows, with respect to example 4, the results of total CTCs count and EpCAM positive cells present per ml blood tested with flow cytometry prior to treatment and 120 days post treatment.

[0066] Fig. 8. shows, with respect to example 4, the results of cytokine release (IFNy, IL-4, IL- 2, TNFa) with ELISA assay and immune cell markers (CD28+ B cells, CD28+ T cells, CD80+ cells, CD86+ cells) with flow cytometry.

[0067] Fig. 9. Shows, with respect to example 5, the results of total CTCs count and EpCAM positive cells present per ml blood tested with flow cytometry prior to treatment and 120 days post treatment.

[0068] Fig. 10. shows, with respect to example 5, the results of cytokine release (IFNy, IL-4, IL- 2, TNFa) with ELISA assay and immune cell markers (CD28+ B cells, CD28+ T cells, CD80+ cells, CD86+ cells) with flow cytometry.

[0069] Fig. 11 . shows, with respect to example 6, the results of total CTCs count and EpCAM positive cells present per ml blood tested with flow cytometry prior to treatment and 120 days post treatment.

[0070] Fig. 12. shows, with respect to example 6, the results of cytokine release (IFNy, IL-4, IL- 2, TNFa) with ELISA assay and immune cell markers (CD28+ B cells, CD28+ T cells, CD80+ cells, CD86+ cells) with flow cytometry.

[0071] Fig. 13 shows the patient distribution according to cancer type (Fig. 13A) and stage (Fig. 13B) for CTC and EpCam positive cell determination, prior to treatment compared to 120 days and 210 days after treatment.

[0072] Fig. 14 shows the patient distribution according to cancer type (Fig. 14A) and stage (Fig. 14B) for immune status determination, prior to treatment compared to 120 days and 210 days after treatment.

[0073] Fig. 15 shows a circulating tumor cell (CTC) count prior to, 120 days and 210 days after treatment.

[0074] Fig. 16 shows an EpCam positive cell count prior to, 120 days and 210 days after treatment.

[0075] Fig. 17 shows a determination of immune status prior to and 120 days after treatment, with respect to immunity markers CD19, CD28 B cells, CD62L, IFN-y, IL-4, IL- 2, CD28 T cells, CD80 and CD86.

[0076] Fig. 18 shows a determination of immune status prior to and 210 days after treatment, with respect to immunity markers CD19, CD28 B cells, CD62L, IFN-y, IL-4, IL- 2, CD28 T cells, CD80 and CD86.

[0077] DESCRIPTION OF PREFERRED EMBODIMENTS

[0078] The studies performed indicate that combination adoptive cellular therapy involving macrophages, NK cells, dendritic cells, cytotoxic T lymphocytes and antibody-producing plasma cells is feasible in patients with advanced solid tumors and is well tolerated. According to a first preferred embodiment of the pharmaceutical product according to the present invention, the first dose comprises autologous M1 -polarized macrophages and autologous cytotoxic CD16+ / CD56+Natural Killer cells (NK cells). The second and the third dose each comprise autologous dendritic cells (DC) activated against tumor peptides, and further comprise autologous activated CD8+ cytotoxic T lymphocytes and activated autologous plasma cells producing monoclonal antibodies against specific tumor antigens.

[0079] Said first preferred embodiment was prepared according to the following method:

[0080] For the preparation of the pharmaceutical product according to example 1 , an initial sample of approximately 120 ml of whole peripheral blood was collected, i.e. peripheral blood without any prior processing or separation steps. Of the 120 ml blood sample collected, 10 ml were used for serum isolation by centrifugation at 3'500 rpm for 15 minutes. The serum was used for cytokine release determination. Next, the pellet was lysed with NH4CI and the RNA was isolated using Trizol (TRI Reagent; Sigma Aldrich Cat. No: T9424). An additional 30 ml of the blood sample were used for DC preparation, 15 ml were used for macrophage preparation, 15 ml were used for NK cell preparation, 15 ml were used for T cell preparation, 15 ml were used for B cell / plasma cell preparation and 15 ml were used for isolation of circulating tumor cells (CTCs) and CSCs (cancer stem cells). The remaining blood volume was retained for quality control and assays to compare samples in the follow-up process.

[0081] For the macrophage manufacturing, whole peripheral blood was lysed with NH4CI and washed with phosphate-buffered saline (PBS). Next, mononuclear cells were isolated from the blood sample using negative isolation magnetic beads (BD Biosciences, Cat. No: 558454). The negative fraction that contained the isolated monocytes was washed with PBS and resuspended in 10 ml Roswell Park Memorial Institute (RPMI) medium (PAN BIOTECH, Cat. No: P04-17500) supplemented with penicillin / streptomycin (Sigma-Aldrich, Cat. No: P0781) in T75 flask (Biologix, Cat. No: 07-8075 100) for eight to ten days in an incubator (37°C, 5% CO2). On the tenth day, IFNy (20ng / ml) (Immuno-Tools, Cat. No: 11343536) was added to the culture overnight. The next day, cells were mixed with NK cells (prepared as described below) and cryopreserved.

[0082] For the NK manufacturing, whole peripheral blood was lysed with NH4CI, washed with phosphate-buffered saline (PBS) and resuspended in 10 ml Roswell Park Memorial Institute (RPMI) medium supplemented with penicillin / streptomycin in T75 flask in an incubator (37°C, 5% CO2). On the third day, the culture was supplemented with IL-2 (20ng / ml) (ImmunoTools, Cat. No: 11340025), IL-15 (20ng / ml) (ImmunoTools, Cat. No: 11340155) and IL-21 (20ng / ml) (ImmunoTools, Cat. No: 11340215). Next, NK cells were isolated using negative isolation magnetic beads (Thermo Fischer Scientific, Cat. No: 11349D). The negative fraction that contained the isolated monocytes was mixed with the macrophages, washed two times with PBS and cryopreserved in a first vial (Dose 1) (CryoKI NG cryogenic vial 1.5ml, Biologix Corp., Cat. No: 04201011)) in serum-free cell freezing medium (Sigma- Aldrich, Cat. No: C2639) until shipment. Cryovials were stored for 24 h at -80°C in a freezing container (Nalgene Mr Frosty, Thermo Fischer Scientific, Cat. No: 5100-0001) and then transferred to liquid nitrogen until requested. When ready to be shipped, cells were thawed, washed using PBS, counted with erythrosin B and checked for bacterial and yeast contamination. Then they were resuspended in 6 ml of isotonic solution and added in an aseptic vial that seals tightly.

[0083] For the preparation of DCs, whole peripheral blood was lysed with NH4CI and washed with phosphate-buffered saline (PBS). Next, mononuclear cells were isolated using negative isolation magnetic beads. The negative fraction containing the isolated monocytes was washed with PBS and resuspended in 10 ml of Roswell Park Memorial Institute (RPMI) medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 ng / mL) (ImmunoTools, Cat. No: 11343127), IL-4 (60 ng / ml) (ImmunoTools, Cat. No: 11340047), and penicillin / streptomycin in two T75 flasks for six days in an incubator (37°C, 5% CO2). Halfway, fresh GM-CSF and IL-4 was added.

[0084] The DCs were pulsed with the mixture of antigenic peptides from the patient. For this purpose, circulating tumor cells were isolated from the blood of patient, and then underwent a step of cell membrane disruption, with at least 4 cycles of freezing and thawing and centrifuging at least at 20'000 g, in order to isolate the cell surface antigenic peptides.

[0085] For activation of the DCs, on the sixth day, antigens which have been prepared were resuspended in 1ml RPMI and equally split (500pl) into the two T75 flasks. The first flask was also supplemented with p2-microglobulin (3pg / ml) (Promocell, Cat. No: C-69303). Both flasks were left overnight and the next day the first flask was additionally supplemented with IL-1 p (25 ng / ml) (ImmunoTools, Cat. No: 11340015), TNF-a (50 ng / ml) (ImmunoTools, Cat. No: 11343015), IL-6 (10 ng / ml) (ImmunoTools, Cat. No: 11340066), and PGE2 (10“6M) (Sigma-Aldrich, Cat. No: P5640-1 MG) and incubated for 48 hours.

[0086] For the preparation of CTLs, whole peripheral blood was lysed with NH4CI, washed with PBS and cryopreserved until further use. Cells were thawed when DCs were ready for coculture. For T cell activation, DCs from the first flask (with p2-microglobulin) were used and the culture was supplemented with IL-2 (10ng / ml), IL-7 (5ng / ml) (ImmunoTools, Cat. No: 11340075) and IL-15 (25ng / ml) for 10 days. Halfway, fresh cytokines were added.

[0087] For the preparation of B cells / plasma cells, whole peripheral blood was lysed with NH4CI, washed with PBS and cryopreserved until further use. Cells were thawed when DCs were ready for co-culture. For B cell / plasma cell activation, DCs from the second flask (without p2-microglobulin) were used and the culture was supplemented with IL-6 (150ng / ml), IL-10 (100ng / ml) (Cat. No: 11340105), TNF-a (10ng / ml), sCD40L (1ng / ml) (ImmunoTools, Cat. No: 11343345) for 10 days. Halfway, fresh cytokines were added.

[0088] Cells from the two flasks were mixed, washed two times with PBS and cryopreserved in serum-free cell freezing medium in two vials (comprising Dose 2 and Dose 3, respectively) (CryoKING cryogenic vials 1.5ml, Biologix Corp., Cat. No: 04201011) until shipment. Cryovials were stored for 24 hours at -80°C in a freezing container (Nalgene Mr Frosty) and then transferred to liquid nitrogen until requested. When ready to be shipped, cells were thawed, washed using PBS, counted with erythrosin B and checked for bacterial and yeast contamination. Then the cells were resuspended in 6 ml of isotonic solution and added in a tight-sealing aseptic vial.

[0089] Patient trials:

[0090] Example 1 :

[0091] Female patient (age 70) with breast cancer stage IV. Received first dose on 29 November 2021 , second dose on 13 December 2021 and third dose on 27 December 2021 , wherein the compositions of each of the doses 1 -3 were prepared pursuant to the method described above according to the first preferred embodiment.

[0092] The immunogenicity evaluation was performed on day 0 (prior to the production of the pharmaceutical product according to the present invention) and on day 120 (after administration of the first dose) and the potential efficacy of the pharmaceutical product according to the present invention (decrease of Circulating Tumor Cells (CTCs), decrease of EpCAM positive cells and change in their immunophenotype) was assessed.

[0093] Results:

[0094] Figure 1 shows the decrease of total number of circulating tumor cells (CTCs) and EpCAM positive cells in patient’s blood 120 days after the administration of first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment (in each case, for both prior to treatment and 120d post treatment, the left bar represents the CTC count and the right bar represents the EpCAM positive cell count, respectively, likewise in figures 3, 5, 7, 9 and 11).

[0095] Figure 2 shows an increase in the percentage of cytokines present in the serum from patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment (for each cytokine or immune cell marker, respectively, the left bar represents the respective percentage prior to treatment, and the right bar represents the respective percentage 120d post treatment, likewise in figures 4, 6, 8, 10 and 12). Cytokines related to the activation of immunity (I FNy, IL-4, IL-2) appeared to be detectable at elevated levels compared to control reference serum, while TNFa, which is related to immune suppression, showed a decreased levels. Regarding the immune cell phenotyping, CD28 positive B cells and T cells (memory cells), and CD80 and CD86 positive cells, which are expressed as cell surface molecules by antigen presenting cells (APCs), appeared to have increased 120 days after the administration of first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment.

[0096] Example 2

[0097] Female patient (age 47) with colorectal cancer stage III. Received first dose on 22 August 2022, second dose on 5 September 2022 and third dose on 19 September 2022 (same doses and compositions as in example 1).

[0098] The immunogenicity evaluation was performed on day 0 (prior to the production of the pharmaceutical product according to the present invention) and on day 120 (after the administration of the first dose) and the potential efficacy of the pharmaceutical product according to the present invention (decrease of Circulating Tumor Cells (CTCs), decrease of EpCAM positive cells and change in their immunophenotype) was assessed.

[0099] Results:

[0100] Figure 3 shows a decrease of total number of CTCs and EpCAM positive cells in patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0101] Figure 4 shows an increase in the percentage of cytokines present in the serum from patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment. Cytokines related to the activation of immunity (I FNy, IL-4, IL-2) appeared to show elevated percentages, while TNFa, which is related to immune suppression, showed a decreased percentage. Regarding the immune cell phenotyping, CD28 positive B cells and T cells (memory cells), and CD80 and CD86 positive cells, which are expressed as cell surface molecules by APCs, appeared to have increased 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment.

[0102] Example 3

[0103] Female patient (age 43) with breast cancer stage IV. Received first dose on 4 October 2021 , second dose on 18 October 2021 and third dose on 1 November 2021 (same doses and compositions as in example 1).

[0104] The immunogenicity evaluation was performed on day 0 (prior to the production of the pharmaceutical product according to the present invention) and on day 120 (after the administration of the first dose) and the potential efficacy of the pharmaceutical product according to the present invention (decrease of CTCs, decrease of EpCAM positive cells and change in their immunophenotype) was assessed.

[0105] Results:

[0106] Figure 5 shows a decrease of total number of CTCs and EpCAM positive cells in patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0107] Figure 6 shows an increase in the percentage of cytokines present in the serum from patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment. Cytokines related to the activation of immunity (I FNy, IL-4, IL-2) appeared to be with elevated percentages, while TNFa, which is related to immune suppression, showed a decreased percentage. Regarding the immune cell phenotyping, CD28 positive B cells and T cells (memory cells), and CD80 and CD86 positive cells, which are expressed as cell surface molecules by APCs, appeared to have increased 120 days after the administration of first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0108] Example 4

[0109] Male patient (age 70) with prostate cancer stage IV. Received first dose on 11 April 2022, second dose on 25 April 2022 and third dose on 9 May 2022 (same doses and compositions as in example 1).

[0110] The immunogenicity evaluation was performed on day 0 (prior to the production of the pharmaceutical product according to the present invention) and on day 120 (after the administration of the first dose) and the potential efficacy of the pharmaceutical product according to the present invention (decrease of CTCs, decrease of EpCAM positive cells and change in their immunophenotype) was assessed.

[0111] Results:

[0112] Figure 7 shows a decrease of total number of CTCs and EpCAM positive cells in patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0113] Figure 8 shows an increase in the percentage of cytokines present in the serum from patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment. Cytokines related to the activation of immunity (I FNy, IL-4, IL-2) appeared to show elevated percentages, while TNFa, which is related to immune suppression, showed a decreased percentage. Regarding the immune cell phenotyping, CD28 positive B cells and T cells (memory cells), and CD80 and CD86 positive cells, which are expressed as cell surface molecules by APCs, appeared to have increased 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0114] Example 5

[0115] Female patient (age 77) with liver cancer stage IV. Received first dose on 25 July 2022, second dose on 8 August 2022 and third dose on 22 August 2022 (same doses and compositions as in example 1).

[0116] The immunogenicity evaluation was performed on day 0 (prior to the production of the pharmaceutical product according to the present invention) and on day 120 (after the administration of the first dose) and the potential efficacy of the pharmaceutical product according to the present invention (decrease of CTCs, decrease of EpCAM positive cells and change in their immunophenotype) was assessed.

[0117] Results:

[0118] Figure 9 shows the decrease of total number of CTCs and EpCAM positive cells in patient’s blood 120 days after the administration of first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment. Figure 10 shows increase in the percentage of cytokines present in the serum from patient’s blood 120 days after the administration of first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment. Cytokines related to the activation of immunity (I FNy, IL-4, IL-2) appeared to show elevated percentages, while TNFa, which is related to immune suppression, showed a decreased percentage. Regarding the immune cell phenotyping, CD28 positive B cells and T cells (memory cells), and CD80 and CD86 positive cells, which are expressed as cell surface molecules by APCs, appeared to have increased 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0119] Example 6

[0120] Male patient (age 80) with prostate cancer stage IV. Received first dose on 18 July 2022, second dose on 1 August 2022 and third dose on 15 August 2022 (same doses and compositions as in example 1).

[0121] The immunogenicity evaluation was performed on day 0 (prior to the production of the pharmaceutical product according to the present invention) and on day 120 (after the administration of the first dose) and the potential efficacy of the pharmaceutical product according to the present invention (decrease of Circulating Tumor Cells (CTCs), decrease of EpCAM positive cells and change in their immunophenotype) was assessed.

[0122] Results:

[0123] Figure 11 shows the decrease of total number of CTCs and EpCAM positive cells in patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment.

[0124] Figure 12 shows an increase in the percentage of cytokines present in the serum from patient’s blood 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior treatment. Cytokines related to the activation of immunity (I FNy, IL-4, IL-2) appeared to show elevated percentages, while TNFa, which is related to immune suppression, showed a decreased percentage. Regarding the immune cell phenotyping, CD28 positive B cells and T cells (memory cells), and CD80 and CD86 positive cells, which are expressed as cell surface molecules by APCs, appeared to have increased 120 days after the administration of the first dose of the pharmaceutical product according to the present invention compared to the baseline set prior to treatment. In summary, with respect to the results of the six examples, autologous adoptive cellular therapy has been seen to have positive results regarding the efficiency of therapy. The results on day 120 of the treatment, including 3 doses, i.e. 120 days after the administration of the first dose of the pharmaceutical product to 6 patients according to examples 1-6, revealed that 6 of 6 patients (100%) showed a decrease in total numbers of CTCs and in the numbers of EpCAM positive cells.

[0125] Regarding the immune response, 4 of 6 patients (66.7%) had over 15% positive CD80 and CD86 cells. Furthermore, 3 of 6 patients (50%) had elevated numbers of CD28+ B cells and CD28+ T cells after treatment with the inventive pharmaceutical product. B cells and T cells expressing the CD28 marker are related with the establishment of humoral and cellular immunological memory, respectively. Higher percentages of secreted cytokines, which is related with immune activation (I FNy, IL-4 and IL-2), and a decrease of secreted TNFa, which is related with immune suppression, were detected in all 6 cases.

[0126] Example 7:

[0127] For the longer term tests, the results of which are described below and shown in Fig. 13- 18, circulating tumor cells and EpCam positive cells of 49 patients with various types and stages of cancer were determined by flow cytometry for 120 days (D120) and 210 days (D210) post-treatment, respectively, with the same doses and compositions as in example 1. First, CTCs were identified as the cell population that is CD45 negative, CD31 negative and pan-cytokeratin-positive (according to Ntanovasilis et al., 2019) (Fig. 15). In addition, EpCam positive cancer cells were identified by adding EpCam antibody (Fig. 16). Furthermore, the immune activation was determined using enzyme-linked immunoassay (ELISA) against a number of markers representing different arms of immunity, for 120 days and 210 days post-treatment, respectively (Fig. 17-18). The markers which were determined and the arm of immunity they are indicative of are the following: CD19 for plasma cells; CD28-B for memory cells of the B-cell lineage; CD62L for CTLs; IFN-y for CD4 cell-mediated immunity; IL-2 and IL-4 each for CD4 humoral immunity; CD28-T for memory cells of the T-cell lineage; and CD80 and CD86 each for dendritic cells.

[0128] Fig. 13 shows the patient distribution according to stage and cancer type for CTC and EpCam positive cell determination in longer term tests, in which samples from 49 patients from different types and stages of cancer were analyzed over 120 days and 210 days, respectively. Fig. 13A shows percentages, starting with breast cancer (27%), followed in clockwise direction by bronchial (2%), head-neck (8%), lung (6%), pancreatic (8%), prostate (19%), colon (12%), mesothelioma (2%), ovarian (2%), prostate (4%), skin (2%), thyroid (2%), followed by liver (2%), CUP (cancer of unknown primary) (2%), as well as bladder cancer (2%). The significant percentages include 27% (14 patients) suffering from breast cancer, 12% (6) from colon cancer, 19% (10) from prostate cancer, and 8% each from pancreatic cancer and head-neck cancer, respectively. Of the 49 patients, 27% (14) suffered from stage I, 3% from stage II, 20% from stage III, and 48% from stage IV of the respective cancer type, as illustrated in Fig. 13B.

[0129] Fig. 14 shows the patient distribution according to stage and cancer type for immune status determination in longer term tests, in which samples from 42 patients from different types and stages of cancer were analyzed over 120 days and 210 days, respectively. Fig. 14A shows percentages, starting with breast cancer (34%), followed in clockwise direction by CLL (3%), head-neck (3%), lung (10%), N-H lymphoma (3%), pancreatic (2%), prostate (20%), skin (2%), mesothelioma (2%), myeloma (2%), thyroid (2%), bladder (2%), and colon (15%). The significant percentages include 34% (14 patients) suffering from breast cancer, 20% (6) from prostate cancer, 15% (7) from colon cancer, and 10% (4) from lung cancer. Of the 42 patients, 38% (16) suffered from stage I, 7% (3) from stage II, 19% (8) from stage III, and 36% (15) from stage IV of the respective cancer type, as illustrated in Fig. 14B.

[0130] In Fig. 15, the count of CTCs prior to treatment (represented by the left bar in each comparison), as well as 120 days and 210 days after treatment (represented by the right bar, respectively, in each comparison) is illustrated. By day 120 (D120) and for 40 subjects, CTCs were found to have decreased in 100% of the subjects (40 / 40) (i.e. 40 subjects of the abovementioned 49 treated subjects were tested, and a decrease was detected in all 40 subjects). Particularly, the average of CTC counts prior to treatment was found to be 3.28, while for D120 it was 2.87. This reduction of CTCs was found to be statistically significant (p = 3.75e-16< 0.05, paired Student’s t-test). Furthermore, for D210 and 25 subjects, CTCs were found to have decreased in 96% of the subjects (24 / 25) by D210. Particularly, the average of CTCs’ counts prior to treatment was found to be 3.27, while for D240 it was 2.68. This reduction in CTC counts was found to be statistically significant (p = 6.93e'10< 0.05, paired Student’s t-test).

[0131] In Fig. 16, the count of EpCam positive cells prior to treatment (represented by the left bar in each comparison), as well as 120 days and 210 days after treatment (represented by the right bar, respectively, in each comparison) is illustrated. By D120 and for 40 subjects, the EpCam positive cell count was found to have decreased in 100% of the subjects (40 / 40) (i.e. 40 subjects of the abovementioned 49 treated subjects were tested, and a decrease was detected in all 40 subjects). Particularly, the average EpCam positive cell count prior to treatment was found to be 1.67, while for D120 it was 1.45. This reduction of EpCam positive cells was found to be statistically significant (p = 4.94e-15< 0.05, paired Student’s t-test). Furthermore, by D210 and for 25 subjects, the EpCam positive cell count was found to have decreased in 96% of the subjects (24 / 25). Particularly, the average EpCam positive cell count prior to treatment was found to be 1 .84, while for D240 it was 1.51 . This reduction of EpCam positive cells was found to be statistically significant (p = 1.67e-09< 0.05, paired Student’s t-test).

[0132] In Fig. 17, the immune status determination prior to treatment (left bar), as well as 120 days after treatment (right bar) is illustrated. For marker CD19, an increase was detected in 88.57% of the subjects (31 / 35) (i.e. 35 subjects of the abovementioned 42 treated subjects were tested, and of those 35, an increase was detected in 31 of the 35 subjects). Particularly, the average percentage for the baseline was found to be 0.31 , while for D120 it was 1.52. This increase was found to be statistically significant (p=0.00098 < 0.05, paired Student’s t-test). For marker CD28 for B cells an increase was detected in 73.68% of the subjects (28 / 38). Particularly, the average percentage for the baseline was found to be 0.51 , while for D120 it was 1 .50. This increase was found to be statistically significant (p=0.0037 < 0.05, paired Student’s t-test). For marker CD62L, an increase was detected in 77.14% of the subjects (27 / 35). Particularly, the average percentage for the baseline was found to be 1.24, while for D120 it was 3.27. This increase was found to be statistically significant (p=0.00098 < 0.05, paired Student’s t-test). For marker IFN-y, an increase was detected in 95.00% of the subjects (38 / 40). Particularly, the average percentage for the baseline was found to be 2.08, while for D120 it was 5.30. This increase was found to be statistically significant (p=2.80e'°9< 0.05, paired Student’s t-test). For marker IL4, there was found an increase in a percentage of 95.00% of the subjects (38 / 40). Particularly, the average percentage for the baseline was found to be 1 .86, while for D120 it was 4.47. This increase was found statistically significant (p=5.21e'°8< 0.05, paired Student’s t-test). For marker IL2, an increase was detected in 95.00% of the subjects (38 / 40). Particularly, the average percentage for the baseline was found to be 1 .99, while for D120 it was 4.03. This increase was found to be statistically significant (p=1.99e'1° < 0.05, paired Student’s t-test). For marker CD28 for T cells, an increase was detected in 89.19% of the subjects (33 / 37). Particularly, the average percentage for the baseline was found to be 0.72, while for D120 it was 2.93. This increase was found to be statistically significant (p=4.85e'°5< 0.05, paired Student’s t-test). For marker CD80, an increase was detected in 97.50% of the subjects (39 / 40). Particularly, the average percentage for the baseline was found to be 10.67, while for D120 it was 20.50. This increase was found to be statistically significant (p=3.69e'09< 0.05, paired Student’s t-test). For marker CD86, an increase was detected in 97.44% of the subjects (38 / 39). Particularly, the average percentage for the baseline was found to be 7.33, while for D120 it was 18.65. This increase was found to be statistically significant (p=8.99e_11< 0.05, paired Student’s t-test).

[0133] Fig. 18 illustrates an immune status determination prior to treatment (represented by the left bar in each comparison), as well as 210 days after treatment (represented by the right bar in each comparison). For marker CD19, an increase was detected in 92.86% of the subjects (13 / 14) (i.e. 14 subjects of the abovementioned 42 treated subjects were tested, and an increase was detected in 13 of those 14 subjects). Particularly, the average percentage for the baseline was found to be 0.44, while for D120 it was 3.09. This increase was found to be statistically significant (p=0.0083 < 0.05, paired Student’s t-test). For marker CD28 for B cells, an increase was detected in 81.25% of the subjects (13 / 16). Particularly, the average percentage for the baseline was found to be 0.87, while for D120 it was 2.21. This increase was found to be statistically significant (p=0.0044 < 0.05, paired Student’s t-test). For marker CD62L, an increase was detected in 85.71% of the subjects (12 / 14). Particularly, the average percentage for the baseline was found to be 1.34, while for D120 it was 4.17. This increase was found to be statistically significant (p=0.0013 < 0.05, paired Student’s t-test). For marker IFN-y, an increase was detected in 87.50% of the subjects (14 / 16). Particularly, the average percentage for the baseline was found to be 3.35, while for D120 it was 5.87. This increase was found to be statistically significant (p=0.0026

[0134] < 0.05, paired Student’s t-test). For marker IL4, an increase was detected in 87.50% of the subjects (14 / 16). Particularly, the average percentage for the baseline was found to be 2.86, while for D120 it was 4.75. This increase was found to be statistically significant (p=0.014 < 0.05, paired Student’s t-test). For marker IL2, an increase was detected in 87.50% of the subjects (14 / 16). Particularly, the average percentage for the baseline was found to be 3.18, while for D120 it was 4.92. This increase was found to be statistically significant (p=0.0022

[0135] < 0.05, paired Student’s t-test). For marker CD28 for T cells, an increase was detected in 93.75% of the subjects (15 / 16). Particularly, the average percentage for the baseline was found to be 1.01 , while for D120 it was 3.44. This increase was found to be statistically significant (p=0.00016 < 0.05, paired Student’s t-test). For marker CD80, an increase was detected in 100.00% of the subjects (16 / 16). Particularly, the average percentage for the baseline was found to be 15.30, while for D120 it was 26.15. This increase was found to be statistically significant (p=8.9e'°5< 0.05, paired Student’s t-test). For marker CD86, an increase was detected in 100.00% of the subjects (16 / 16). Particularly, the average percentage for the baseline was found to be 10.78, while for D120 it was 23.86. This increase was found to be statistically significant (p=2.39e'05< 0.05, paired Student’s t-test).

[0136] The results of these tests among patients suffering from various types of cancer across various stages (Example 7) again show that administration of the present autologous cell composition activates the immune system. This activation on the one hand leads to a statistically significant decrease in circulating tumor cells (CTCs) and EpCam positive cells (EpCam). The activation of the immune system also is evident in that various immune markers were increased in a statistically significant manner. The long-lasting positive effect of the inventive treatment was evident across various different cancer types and stages at least 7 months after administration.

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Claims

CLAIMS1. Pharmaceutical product for use in an autologous adoptive cellular therapy for the treatment of a cancer disease of a human or animal subject, comprising- a first dose comprising a first composition, comprising autologous macrophages derived from autologous mononuclear cells, and further comprising autologous natural killer cells,- a second dose comprising a second composition, and- a third dose comprising the second composition, wherein the second composition comprises ex vivo activated autologous dendritic cells, ex vivo activated autologous CD8+ cytotoxic T-lymphocytes and ex vivo activated autologous plasma cells.

2. Pharmaceutical product according to claim 1 , wherein in the first composition the macrophages are M1 -polarized autologous macrophages.

3. Pharmaceutical product according to claim 1 or 2, wherein in the first composition the autologous natural killer cells are autologous CD16+ and CD56+ natural killer cells.

4. Pharmaceutical product according to one of the preceding claims, wherein in the second composition contained in the second dose and in the third dose, the autologous dendritic cells are activated against one or more antigenic peptides, and wherein the autologous CD8+ cytotoxic T-lymphocytes are activated against one or more antigenic peptides, such that the activated autologous CD8+ cytotoxic T- lymphocytes are capable of recognizing the one or more antigenic peptides, and wherein the autologous plasma cells are activated against one or more antigenic peptides, such that the autologous plasma cells are capable of producing antibodies against the one or more antigenic peptides, wherein the antigenic peptides are isolated from the same human or animal subject.

5. Pharmaceutical product according to claim 4, wherein the activated autologous CD8+ cytotoxic T-lymphocytes are capable of recognizing at least one of the same antigenic peptides against which the autologous dendritic cells are activated, and / or wherein the activated autologous plasma cells are capable of producing antibodies against at least one of the same antigenic peptides against which the autologousdendritic cells are activated.

6. Pharmaceutical product according to one of the preceding claims, wherein the three doses are adapted for administration to the human or animal subject separately from each other and sequentially at three different points in time, wherein preferably the first dose comprising the first composition is adapted to be administered to the human or animal subject in week 1 of a treatment schedule, preferably by injection, wherein preferably the second dose comprising the second composition is adapted to be administered to the human or animal subject in week 3 of the treatment schedule, preferably by injection, and wherein preferably the third dose comprising the second composition is adapted to be administered to the human or animal subject in week 5 of the treatment schedule, preferably by injection.

7. Pharmaceutical product according to one of the preceding claims, wherein each of the first, second and third dose is adapted to be administered to the human or animal subject by intravenous injection.

8. Pharmaceutical product according to one of the preceding claims, for use as a medicament against a cancer disease of a human or animal subject.

9. Kit-of-parts for use as a medicament against a disease in a human or animal subject, preferably against a cancer disease, comprising the pharmaceutical product according to one of claims 1-7.

10. Kit-of-parts for use as a medicament against a cancer disease in a human or animal subject, comprising:- a first dose comprising a first composition, comprising macrophages derived from autologous mononuclear cells, preferably autologous macrophages polarized in a M1 -phase, and further comprising autologous natural killer cells, preferably CD16+ / CD56+ natural killer cells,- a second dose comprising a second composition, and- a third dose comprising the second composition, wherein the second composition comprises ex vivo activated autologous dendritic cells, ex vivo activated autologous cytotoxic T-lymphocytes and ex vivo activated autologous plasma cells; and wherein preferably the first dose, the second dose and the third dose each are adapted for administration to the human or animal patient ata different point in time of a treatment schedule, preferably in 2-week intervals.

11. Method for the production of a medicament for autologous adoptive cellular therapy in the treatment of a cancer disease of a human or animal subject, comprising the following steps ex vivo: a.) providing a population of autologous macrophages, preferably autologous macrophages polarized in a M1 -phase; b.) providing a population of autologous natural killer cells, preferably CD16+ / CD56+ natural killer cells; c.) providing a population of ex vivo activated autologous dendritic cells; d.) providing a population of autologous CD8+ cytotoxic T-lymphocytes; e.) providing a population of autologous plasma cells; f.) mixing of the population resulting from step a.) with the population resulting from step b.) for the provision of a first composition for administration in a first dose to a human or animal subject.; g.) mixing of a first portion of the population resulting from step c.) with the population from step d.), resulting in a population of ex vivo activated CD8+ cytotoxic T- lymphocytes; h.) mixing of a second portion of the population resulting from step c.) with the population resulting from step e.), resulting in a population of ex vivo activated autologous plasma cells; i.) mixing of the population resulting from step g.) with the population of step h.) for the provision of a second composition; j.) splitting up the second composition resulting from step i.) into two preferably equal portions, resulting in a second dose of the second composition and a third dose of the second composition for administration to the human or animal subject.

12. Method according to claim 11 , wherein in step g.), for the purpose of providing a population of activated autologous CD8+ cytotoxic T-lymphocytes, the population resulting from step c.) is incubated in the presence of p2-microglobulin, preferably for 6-18 hours, preferably for 8-12 hours, and preferably also incubated with at least one, preferably all of the following group consisting of IL-1 p, TNF-a, IL-6, PGE2 prior to mixing the population resulting from step c.) with the autologous CD8+ cytotoxic T-lymphocytes, preferably for 24-72 hours, more preferably for 48 hours.

13. Method according to claim 11 or 12, wherein in step g.), for the purpose of providinga population of ex vivo activated autologous CD8+ cytotoxic T-lymphocytes, the population resulting from step c.), after mixing with the population resulting from step d.), is further incubated with at least one of the cytokines of the group consisting of IL-2, IL-7, and IL-15, preferably for 8-15 days, more preferably for 10 days.

14. Method according to claim 11 , wherein in step g.), for the purpose of providing a population of ex vivo activated autologous plasma cells, the population resulting from step c.), after mixing with the population resulting from step e.), is further incubated with at least one of the cytokines of the group consisting of IL-6, IL-10, TNF-a, and sCD40L, preferably for 8-15 days, more preferably for 10 days.

15. Method according to one of claims 11-14, wherein step c.) includes providing an antigen preparation for the purpose of ex vivo activation of the autologous dendritic cells and autologous CD8+ cytotoxic T-lymphocytes in step d.) and for the purpose of ex vivo activation of autologous B lymphocytes in step e.).

16. Method according to claim 15, wherein, in case of an intended use of the medicament against a cancer disease of the human or animal patient, the antigen preparation is prepared by isolating antigens present on circulating tumor cells (CTCs) and / or cancer stem cells (CSCs) of the human or animal subject, wherein preferably, the circulating tumor cells and / or the cancer stem cells are isolated using magnetic cell separation, preferably using magnetic beads selective for recognition of at least one tumor antigen selected from a group consisting of CD44, CD133, EpCAM, PanCK.

17. Method according to one of claims 11-16, wherein in step a.), for the provision of a population of autologous macrophages which are polarized in a M1-phase, autologous monocytes are isolated and incubated in a medium free from GM-CSF and free from IL-4, wherein one day prior to step f.), IFN-y is added to the medium in order to trigger a polarization of the macrophages into M1 -stage.

18. Method according to one of claims 11-17, wherein for the purpose of providing the populations of steps a.)-e.), a sample of whole peripheral blood is collected from the human or animal patient, wherein preferably the populations resulting from steps a.)- e.) are derived from the same sample of whole peripheral blood.

9. Method of treating or preventing a disease in a human or animal subject, comprising the following steps:- administering a first dose comprising a first composition, said first composition comprising macrophages derived from autologous mononuclear cells, wherein the macrophages are preferably polarized in a M1 -phase, and further comprising autologous natural killer cells, preferably CD16+ / CD56+ natural killer cells, to the human or animal subject;- administering a second dose comprising a second composition, said second composition comprising ex vivo activated autologous dendritic cells, ex vivo activated autologous CD8+ cytotoxic T-lymphocytes and ex vivo activated autologous plasma cells, to the human or animal subject;- administering a third dose comprising said second composition, to the human or animal subject;- wherein preferably the first dose, the second dose and the third dose are sequentially administered to the human or animal subject in intervals, preferably in intervals of at least one week, more preferably in intervals of 2 weeks.

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