WO2012069067A1

WO2012069067A1 - Method for producing an immunotherapeutic agent

Info

Publication number: WO2012069067A1
Application number: PCT/EP2010/007182
Authority: WO
Inventors: Leander Grode; Henning Weigt
Original assignee: Vakzine Projekt Management Gmbh
Priority date: 2010-11-26
Filing date: 2010-11-26
Publication date: 2012-05-31

Abstract

The invention relates to a method for producing a safe and effective immunotherapeutic agent, characterized in that allogeneic tumor cells are cooled to at least 10°C, preferably to below 0°C, and are irradiated in the cooled state with gamma rays in a dose of less than 500 Gy. The invention further relates to the use of said method to produce a medication for immunotherapy and to tumor cells that can be produced by means of said method.

Description

"Method of Making an Immunotherapeutic"

The present invention relates to a method for producing an immunotherapeutic by gamma irradiation of cooled tumor cells and to the use of the method for producing a medicament for immunotherapy. Furthermore, the invention relates to tumor cells that can be produced by this method and compositions containing the tumor cells.

Cancer is one of the leading causes of death worldwide. According to a World Health Organization estimate, approximately 13% of all deaths worldwide (totaling 7.4 million deaths) in 2004 were due to cancer. A tumor disease is characterized by uncontrolled growth of cells, their invasion into adjacent tissues and also the formation of metastases.

The classic treatments for cancer include surgical resection, chemotherapy and radiotherapy. Often, two or even all three forms of therapy are applied to a patient. The latter two methods have significant cytotoxic side effects. The therapeutic range is very low in chemotherapy, so that a high dosage - which would be beneficial for an increase in the effect - is usually excluded. However, if not all cells of the tumor and its metastases are destroyed in the therapy, the further treatment by resistance formation is much more difficult. For years, therefore, research on new therapeutic methods.

CONFIRMATION COPY In the past, among others, the therapeutic approach of active specific immunization (ASI) was considered promising. Such a method is known from WO 2003/072032 and US 5,484,596. Tumor tissue is taken from a patient by biopsy, tumor cells are harvested therefrom, and these tumor cells are irradiated so that they are sterile and non-tumorigenic, and then used as an immunotherapeutic agent in the same patient. However, it has been shown that this therapeutic approach does not allow effective tumor control. Thus, all previous, pivotal studies in this field have failed despite widespread use of different ASI technology platforms.

One of the reasons for this failure is a low immunogenicity of the tumor cells, which is insufficient to induce an effective tumor-specific immune response even when an adjuvant is given. In addition, the problem arose that the gamma irradiation of the tumor cells used for sterilization and proliferation inhibition on the basis of the often bacterially contaminated and very heterogeneous biopsy material provides no reproducible and thus standardizable results. This is contrary to a safe use of the material as a therapy.

A safe inhibition of proliferation is namely a condition for an application of an immunotherapeutic agent in humans. An immunotherapeutic agent with an inadequately reduced ability to proliferate could in the patient itself lead to tumor formation and thus worsen the patient's state of health.

None of the initially hopefully debated experiments have therefore led to an approved drug.

It is therefore an object of the invention to provide a method by means of which a safe and effective cell-based immunotherapeutic can be produced.

Summary of the invention

The object is achieved by the method according to claim 1. Advantageous developments are the subject of corresponding subclaims.

The method according to the invention consists essentially in not using primary cells from tumors of the respective patient for the preparation of the therapeutic agent, as is customary in the prior art, but cells from cell lines. Thus, according to the invention, secondary cells are used. These are first cooled and then irradiated in a cooled state. Cool down to at least 10 ° C. Irradiation does not exceed a maximum dose of 500 Gy. By gamma irradiation of the cells in conjunction with the previous cooling of the cells, it becomes possible to limit the harmful effects of gamma radiation on the cells in such a way that, on the one hand, the proliferation of the cells is reliably inhibited, but, on the other hand, the vitality of the cells, and therefore also the

Expression of genes relevant for tumor therapy remains possible.

The method according to the invention makes it possible to provide a safe immunotherapeutic agent by using cells of an established - and therefore standardisable - cell line in conjunction with a standardized irradiation method. The difficulties of standardization of individual biopsy material (as in WO 2003/072032 A1) are thus overcome. The cells thus obtained are reliably inhibited in their proliferation - and thus tumorigenicity - but at the same time they are still sufficiently vital to retain their immunogenicity. The use of cell lines also means that, according to the invention, allogeneic cells can also be used to produce the immunotherapeutic agent.

Compared to the radiation dose of 1000 to 2000 Gy, which is generally proposed in WO 2003/072032 A1 for reducing the proliferation capacity of the cells removed from the patient, the radiation dose according to the invention is

Procedure surprisingly low. In fact, it is known that cells of a cell line (ie secondary cells) are characterized by an increased radiation resistance or a lower radiation sensitivity compared to primary cells. According to Mcllwrath et al. (Cancer Research 54: 3718-3722.) (1994: Cell Cycle Arrest and Radiosensitivity of Human Tumor Cell Lines: Dependence on Wild-type P53 for Radiosensitivity), this commonly observed increased radiation resistance is due to the fact that the cell lines grow during growth accumulate mutations in genes responsible for cell cycle control in cell culture. These mutations prevent the cells from entering apoptosis or cell cycle arrest in the case of radiation-induced DNA damage. As a result, larger doses of radiation are needed to restrict cells in their proliferation and vitality compared to primary cells. From this finding, in conjunction with the teaching of WO 2003/072032 A1, the radiation dose used for the proliferation inhibition of the secondary cells would therefore have to be more than 2000 Gy. According to the invention, however, the maximum radiation dose is only 500 Gy. In particular embodiments of the invention, the radiation dose used may even amount to as little as 300 Gy, more preferably up to 200 Gy. The area between 30 and 200 Gy, in particular between 40 or 50 and 150 Gy, is particularly preferred.

According to the invention, the cells are cooled to below 10 ° C. before irradiation and irradiated in this cooled state. Preferably, the cells are frozen, i. put into a solid aggregate state. The frozen state is usually reached in an aqueous medium at 0 ° C, so that a cooling to at least 0 ° C is preferred. However, depending on the medium used and the environmental conditions (especially pressure), lower temperatures may be required to reach the frozen state. In a preferred embodiment of the invention, the cells are cooled down to below -20 ° C or below -40 ° C, more preferably down to below -70 ° C, or most preferably below -80 ° C. The cooled and / or frozen state of the cells is substantially preserved during the subsequent irradiation. When the cells are frozen, they are in any case irradiated without being thawed in between.

In a particularly preferred embodiment of the invention, the tumor cells are genetically modified. Preferably, they express recombinant proteins. Preferred are cytokines, more preferably interleukin-2 and interferon-gamma.

The expression of cytokines can enhance the immune response against the tumor antigens present on the tumor cells and thus improve the immunological control of the primary tumor in the patient.

In a very particularly preferred embodiment, the tumor cells are prostate carcinoma cells (for example, LNCaP cells). definitions

For the purposes of the invention, a "tumor cell line" is to be understood as meaning a cell population which is culturable outside of an organism and / or a cell grouping, possibly without further genetic manipulation, such as transfection with a virus, and is due to cells of a tumor a cell line is characterized by clonality, ie the cell population is based on only one tumor origin cell, in particular secondary, immortalized or permanent / established tumor cell lines, preference being given to immortalized or permanent cell lines having unlimited reproductive ability in culture Used cells from cell lines that have already been passaged several times. The term "immunotherapeutic agent" in the sense of the invention means any therapeutic agent which exerts its therapeutic effect at least also via influencing the immune system, in particular via activation of immune cells (eg cytotoxic T cells).

For the purposes of the invention, "gamma irradiation" is to be understood as meaning electromagnetic radiation which has quantum energies above 200 keV, in particular the electromagnetic radiation which results from the decomposition of radioactive nuclides.

For the purposes of the invention, "allogeneic tumor cells" are to be understood as meaning any cells in which the tumor cells used in the patient originate not from the patient himself but from a donor of the same species, which includes secondary or stable cell lines.

According to the invention, "tumor antigens" or "tumor antigens" are substances produced by tumor cells which are capable of triggering an immune response in the affected organism. The tumor antigens can be located freely in the extracellular space in the cytoplasm or on the cell membrane or by antigen shedding. While the tumor-specific antigens are novel foreign proteins that are only produced by cancer cells, the tumor-associated antigens are gene products that are also expressed by healthy cells. "Cytokines" within the meaning of the invention are proteins, in particular glycoproteins, which exert regulatory functions on the growth and differentiation of cells and are preferably a group of peptides which initiate or regulate the proliferation and differentiation of target cells Cytokines may belong to one of the following five major groups: interferons, interleukins, colony stimulating factors, tumor necrosis factors and chemokines, with particular preference for interferons and interleukins.

According to the invention, a "recombinant protein" is a protein produced by means of a genetically engineered cell, most conveniently the genetic information for the protein is cloned into a vector (e.g.

Plasmid), which is then transformed or transfected into the host organism.

A "cryopreservation medium" in the sense of the invention is a medium which contains at least one substance (a so-called cryoprotectant) which protects biological objects (in particular cells) from freeze-thaw damage and preferably in the

Metabolism does not occur or occurs only in cryoprotective ineffective concentrations. you distinguishes low molecular weight cell membrane permeating and predominantly intracellular acting (eg glycerol, dimethylsulfoxide, dimethylacetamide) and high molecular weight, extracellular cryoprotectants (eg polyvinylpyrrolidone, dextran, hydroxyethyl starch). Cryoprotectants usually act by colligative, antioxidant and / or water-binding properties.

According to the invention, "vitality" is defined as the proportion of living cells in a population of cells One of the key features for distinguishing living from dead cells is the physical integrity of the plasma membrane.Some calorimetric dyes such as trypan blue can enter the cell only in the event of a defective cell membrane An incubation of the cells with trypan blue and subsequent counting of the stained cells (so-called trypan blue labeling) in relation to the total cell number can therefore preferably be used to determine the vitality.

For the purposes of the invention, a "primary packaging material" is a container that is in direct contact with the preparation containing the immunotherapeutic agent, the primary packaging material being suitably designed so that it does not enter into physical or chemical interactions with the contents as much as possible. Bottles, syringes, syringes or

Bag.

For the purposes of the invention, a "finished medicinal product" is a medicinal product which has been finished in a dosage form and packaged in predetermined pack sizes and is intended to be sold to the consumer in this dosage form.

Within the meaning of the invention, "recultivation" is to be understood as a further treatment of the tumor cells, which comprises a culturing step, which in particular includes thawing of the cells with subsequent conversion into a

Cell culture medium and cultivation under suitable cell culture conditions with possible passage of the cells.

A "clean room" in the sense of the invention is defined as a space in which the number of particles contained in the room air is limited, In such rooms usually smooth surfaces are used to avoid the deposition of dust or other particles Separators are used to limit the amount of dust particles, lint, etc. to a set minimum. Detailed description of the invention

The invention provides a method of making an immunotherapeutic agent comprising treated cells of a tumor cell line. The patient to be treated usually has a tumor or should be protected from a tumor, which in its antigenic endowment at least in an antigen with the cells of the

Immunotherapeutic agent, i. in that the tumor cells of the patient and the tumor cells of the therapeutic agent have at least one common antigen. In a particular embodiment of the invention, the patient's tumor is of the same type as that from which the tumor cell of the therapeutic agent is derived. Methods for determining the tumor type are known to those skilled in the art. The patient's immune response is usually based on tumor-specific antigens presented in the MHC complexes of the tumor cell used for immunotherapy.

The tumor cells carry tumor antigens (or tumor antigens substances), which are present either as tumor-specific antigens (TSA) or as tumor-associated antigens (TAA) In contrast to healthy cells, tumor-associated antigens are overexpressed in tumor cells (cancer cells) and correspondingly frequently over the

Main histocompatibility complex presented on the cell membrane. Since they are ubiquitous proteins, an immune response often stops. However, in an immunogenic context, the cancer cells can be recognized and destroyed by specific T cells.

In one embodiment of the invention, tumor cells are used which express the prostate-specific antigen (PSA), the prostate-specific membrane antigen PSMA and / or the epithelial cell adhesion molecule EpCAM. With such a therapeutic agent prepared according to the invention, therefore, patients can be treated with prostate carcinoma or men are at risk of developing a prostate cancer.

Carcinoma be protected.

The tumor cells preferably used according to the invention are genetically modified and preferably express at least one recombinant protein. In a particular embodiment, they express at least two recombinant proteins.

The recombinant proteins are preferably selected from the group of cytokines. Especially preferred are interleukin-2 and / or interferon-gamma.

For recombinant expression, the tumor cells can be transiently or stably transfected with the appropriate gene constructs. Preference is given here

Tumor cells that have been stably transfected. The stable transfection allows one permanent and stable expression of the cytokines, so that a stronger immune response is triggered.

According to a preferred embodiment, the tumor cell line used according to the invention comprises at least one vector which effects the expression of one or more of the polypeptides defined above. The vector may be episomal or integrated into the genome of the cells. Methods for the cloning of vectors are known to the person skilled in the art, for example from Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor: Cold Spring Harbor Laboratory.

In a very particularly preferred embodiment of the invention, the tumor cells are cells of a prostate cancer cell line and preferably cells of the LnCaP cell line. These may be transfected to express both interleukin 2 and interferon-gamma. The cell line is also a carrier of known tumor-associated antigens (TAA), such as the prostate-specific antigen

(PSA), the prostate-specific membrane antigen PSMA or the epithelial cell adhesion molecule EpCAM.

The production of stably transfected cells expressing interleukin 2 and interferon gamma is described in European Patent EP 0 684 831 B1. This is fully incorporated in the disclosure of the present application.

As stated above, the tumor cells according to the invention are first cooled and / or frozen and then irradiated. For this purpose, the cells are preferably present in a cryopreservation medium. In one embodiment of the invention, the cryopreservation medium used is a serum-free medium, which is preferably free of animal proteins. The medium Cryostor CS10 is particularly preferably used. The culture medium preferably contains at least one cryoprotectant. preferred

Cryopreservatives are DMSO or glycerol. The use of a cryopreservation medium allows the cells to cool and freeze without being damaged or destroyed by single crystals. The cooling of the cells can be carried out at a rate of 0.5 to 2 ° C / min and preferably at 1 ° C / min. Such chosen cooling rate reduces freezing damage to the cells.

The radiation source for the gamma radiation is preferably a Cs-137 source. In a further embodiment of the invention, the tumor cells used for the irradiation are present in a cell density of 1.10 × 10 ⁵ to 1.0 × 10 ⁹ , in particular in a cell density of 1.5 × 10 ⁷ cells / ml. In one embodiment, after irradiation, the cells are stored further in the cooled or frozen state. The storage temperature is preferably below - 10 ° C, more preferably below 0 ° C, more preferably below -70 ° C and especially in the temperature range at -80 to -196 ° C. The cells can be stored in liquid nitrogen.

In one embodiment, the tumor cells after irradiation express at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 80% or 90% of the recombinantly expressed cytokines compared to the tumor cells prior to irradiation.

In another embodiment, the tumor cells after irradiation express comparable amounts of tumor antigens as compared to the tumor cells prior to irradiation, but at least 25%, more preferably at least 50%, most preferably at least 80% or 90%.

In an additional embodiment of the invention, after irradiation, the tumor cells have a vitality of at least 25%, preferably at least 50%, most preferably at least 75%, based on the cells taken in culture, for example as determined by trypan blue labeling.

The proliferation of the tumor cells, for example as determined by the incorporation of ³ H-thymidine in the cell culture, is preferably reduced to a dose of 1 million cells by the irradiation to a value of less than 300, preferably less than 200, in particular less than 100, vital cells ,

In one possible embodiment of the invention, the process according to the invention fulfills one or more, preferably all, of the following conditions:

i) The provision of the tumor cells for freezing represents the last step in the production of the immunotherapeutic agent;

ii) the tumor cells irradiated in the step after cooling are in

Primary packaging before;

iii) the tumor cells obtained after irradiation represent the finished medicinal product;

iv) the steps from the provision of the tumor cells up to and including the closing of the primary packaging material are carried out in a clean room; v) the tumor cells obtained after irradiation are applied to the patient without recultivation of the cells.

In one embodiment of the invention, the tumor cells provided for irradiation are present in the final formulation buffer, so that the irradiation preferably represents the last production step on the otherwise finished immunotherapeutic agent. Thus, according to the invention, the entire production process of the therapeutic can be controlled and centralized. In view of the required standardization and safety of the therapeutic agent, this is of great advantage compared to the prior art, according to which cells for cancer therapy are usually localized, ie, finalized on site for the individual treatment on the patient.

Preferably, the tumor cells used for the irradiation are already present in the primary packaging. This has the advantage that after the irradiation no renewed filling or aliquoting must take place, but the cells remain in the primary packaging. This reduces labor and costs and reduces the risk of contamination.

In a particularly preferred embodiment, the tumor cells obtained after the irradiation are applied to the patient without further recultivation of the cells.

One aspect of the invention relates to the use of the method according to the invention for the production of a medicament for the immunotherapy of a tumor.

In a further aspect, the invention relates to tumor cells characterized by having prostate cancer associated tumor antigens, preferably from the group consisting of PSA, PSMA, EpCAM, and at least one gene construct for recombinant expression of a cytokine, said cells being characterized by inventive method can be produced.

One aspect of the invention relates to a medicament for immunotherapy which can be produced by the method according to the invention. Example 1: Method for irradiation of tumor cells ("VPM4001 cells")

1.1 Preparation of VPM 4001 cells

The VPM 4001 cells are based on the LNCaP cell line, which has been stably transfected to co-express the human interferon-gamma and the human interleukin-2.

Here, the IFN-gamma gene is under the control of an immediate early promoter of the cytomegalovirus and the IL-2 gene is under the control of the thymidine kinase promoter of the herpes simplex virus These expression cassettes were introduced into the retroviral N2 vector and became stable transfected into the LNCaP cells, this vector derived from the Moloney leukemia virus carries a gene

Neomycin resistance. After neomycin selection, an IL-2 and IFN-gamma expressing clone was obtained from which a master cell bank was prepared. The working cell bank derived from this master cell bank was used to prepare the immunotherapeutic agent of the present invention.

1.2 Pretreatment of the tumor cells

The tumor cells obtained after expansion and cultivation of the working cell bank are suspended in a 0.9% NaCl solution and the vitality and the number of cells are determined. Thereafter, the suspension was adjusted to a cell count of 1.5 × 10 7 cells / ml by adding a 0.9% NaCl solution. The number of cells thus set was verified by a further cell count determination.

With the help of a bottling plant, 1 ml of cell suspension is filled into 2 ml screw-capped cryogenic vessels. Then 1 ml Kryomedium Cryostor CS100 are added to each vessel, the vessels are then closed and placed on ice.

1.3 Freezing the cells

The cryo-vessels obtained from step 1.2 are transferred to a -80 ° C refrigerator for cryoprotection.

1.4 Radiation of the cells

After the cells are frozen, they are packed on dry ice and placed in the irradiation apparatus. During the irradiation, the cryogenic vessels are surrounded by dry ice to prevent heating of the cells or even thawing. The total dose is 100 Gy.

The irradiation is carried out with the aid of the irradiation device GSR-D1 of the company "Gamma- Service Medical GmbH" and is equipped with a Cs-137 source which has an activity of 180 terabecquerels The irradiation chamber has a chamber volume of about 64 L. 1.5 Storage of the irradiated cells

After irradiation, the cells are stored in a liquid nitrogen tank with the vessels in the gas phase above the liquid nitrogen. 1.6 Characterization of irradiated cells

For use as an immunotherapeutic, the irradiated tumor cells were examined for the following parameters:

Expression of the cytokines IFN-gamma and IL-2

Expression of the tumor markers PSA, PSMA and EpCAM

· Formation of the complement complex

In vitro activation of effector cells by VPM4001

• Phagocytosis of VPM4001 cells by antigen-presenting cells

• cellular integrity

• vitality

· Proliferation

1.6.1 Expression of the cytokines IFN-gamma and IL-2

The expression of the cytokines IFN-gamma and IL-2 was determined at the protein level by ELISA assay. In this case, 10 to 150 Gy irradiated cells were seeded on 48-well plates in a cell density of 1 x 10 ⁵ cells / cm ² . cell-free

Culture supernatants were removed 24, 48, 72 and 96 hours after seeding the culture, stored at -80 ° C and subjected to the ELISA test. For this purpose, the samples were diluted with DMEM medium. DME medium was used as the negative control, and a representative sample of the VPM4001 cells as a positive control. A dilution series of IFN-g and IL-2 standard solutions was used

Standard curve measured. The cytokine concentration was measured from this standard curve in pg / ml and converted to IU / ml (for IL-2) or U / ml (IFN-g) using the following equations: IL-2 [IU / ml] = 0.021 x Quantikine IL-2 value [pg / ml]

IFN-g [lU / ml] = 0.010 x quantikine IFN-g value [pg / ml]

The ELISA quantification of the cytokine IFN-gamma is shown in Figure 1A: The amount of interferon-gamma released was higher in the unirradiated control group and nearly unchanged in the irradiated samples over the entire dose range. For all samples, a decrease in IFN-gamma release was generally observed with increasing culture duration.

The ELISA quantification of the cytokine IL-2 is shown in FIG. 1B: IL2 secretion increased from the first to the fourth day regardless of the radiation dose.

As with the cytokine IFN-gamma, IL-2 release was irradiated cells higher than in the irradiated cells. As the radiation dose increased, IL-2 release decreased only slightly.

1.6.2 Expression of Tumor Markers PSA, PSMA and EpCAM

The marker PSA is quantified using an ELISA assay based on the ELISA method described in 1.6.1. The concentration of the PSA was determined using a standard curve and converted into ng / ml using the following equation: PSA [ng / ml] = 0.505 x Quantikine PSA value [ng / ml]

The expression of the markers PSMA and EpCAM is determined at the protein level by means of flow cytometry. For EpCAM, a fluorochrome-conjugated EpCAM-specific antibody was used. The negative control used was a fluorochrome-coupled isotype control. For PSMA, a murine antibody directed against an extracellular domain of PSMA was used. Detection was done by a second anti-mouse IgG antibody coupled to a fluorochrome. As the PSMA negative control, an isotype control was used. In the flow cytometry both the proportion of PSMA or PSA positive cells was detected as well as the mean fluorescence intensity (MFI) of the positive cells, the

Represents the amount of each expressed PMSA or PSA.

The proportion of PSMA and EpCAM positive cells after irradiation is shown in FIG. 2A. Regardless of the irradiation dose, 100% of the cells expressed both PSA and PSMA.

When determining the MFI (see FIG. 2B), it was found that the mean fluorescence intensity for both PSMA and EpCAM actually increased with increasing radiation dose.

1.6.3 Formation of the complement complex

The adhesion of proteins of the complement system to the cell surface was determined by flow cytometry. For this, the irradiated VPM4001 cells were incubated after thawing and determination of vitality and cell number for 60 minutes at 37 ° C, 5% C0 ₂ in the presence of 20% native human serum (nhs). Heat inactivated human serum (ihs, inactivation by incubation at 56 ° C for 30 minutes) was used as a negative control to determine the background level. As a positive control VPM4001 cells were used, which were taken from the current culture incubated with antibodies that induce complementation. After incubation, cells were washed and purified by flow cytometry using biotin-coupled anti-iC3b and anti-C5b-9 antibodies analyzed. Antibody binding was detected by fluorochrome-conjugated streptavidin.

The deposition of the complement proteins iC3b and C5b-9 on the nhs-treated cells was independent of the radiation dose (16-21% for iC3b and 23-34% for C5b-9, see Figure 3). The ihs-treated tumor cells showed only a marginal deposition of iC3b (<3%) and a C5b-9 deposition of a maximum of 15% of the cells. The positive control showed a deposition of both complement proteins on almost all tumor cells.

1.6.4 In ^' iro-Activation of Effector Cells by VPM4001

Here, the dose-dependent killing of VPM4001 cells by multinuclear cells and natural killer cells is measured in a cytotoxicity assay. Furthermore, the cytolytic activity of Z cells on VPM4001 is also analyzed.

For this purpose, mononuclear cells (MNCs) were isolated from donor blood by density centrifugation. Both T cells and NK cells were subsequently separated by magnetic depletion of non-T cells and non-NK cells, respectively. The successful separation and the purity of the cell population were confirmed by

Flow cytometry verified. MNC, T and NK cells were co-cultured with VPM4001 cell in 96-well plates at various effector: target (E: T) ratios at 37 ° C, 5% C0 ₂ in a humidified atmosphere. NK cell-mediated cytotoxicity was determined after 24 hours by quantification of the remaining VPM4001 target cells using the XTT Proliferation Kit II. The T cell and NK cell mediated

Cytotoxicity was determined after 3, 4 or 5 days of culture using the XTT proliferation kit II (Roche olecular Biochemicals, Penzberg, FRG).

After three days of culture and an E: T ratio of 10: 1, complete MNC-mediated killing of the tumor cells was observed (Figure 4A). After 4 or 5 days of culture, complete kill was achieved at an E: T ratio of 5: 1.

Figure 4B depicts NK cell-mediated killing of VPM4001 cells. The VPM4001 cells were completely killed at an E: T ratio of 10: 1.

With T cells from the same donor, only partial killing of VPM4001 cells was observed at all E: T ratios (Figure 4C). At an E: T ratio of 10: 1, a maximum kill of 30% of the cells was observed after 3, 4 or 5 days of coculture. In summary, VPM4001 can effectively activate the immune cell types studied

1.6.5 Phagocytosis of VPM4001 cells by antigen-presenting cells

The phagocytosis of VPM4001 cells by macrophages was measured by

Macrophages were incubated with calcein-labeled VPM4001 cells and the macrophages were subsequently analyzed by cell flow cytometry with calcein detection. For this purpose, the cryopreserved VPM4001 cells irradiated with a dose of 10 to 50 Gy were thawed and labeled with calcein-AM after determination of the survival rate and the cell number. From peripheral donor blood, macrophages were isolated by adhesion to a plastic surface followed by GM-CSF induced differentiation for 11-15 days. The coculture of macrophages and VPM4001 cells was incubated in 48-well plates with low binding capacity for 4 and 24 hours at 37 ° C and 5% C0 ₂ . Antigen uptake was assessed by flow cytometry (FIG. 5A) and FIG

Fluorescence microscopy examined (Fig. 5B). Calcein-labeled VPM4001 cells were differentiated from macrophages by CD14 labeling. As a positive control, unirradiated VPM4001 cells from the on-going culture were incubated with antibodies (ATG, anti-thymocyte globulin) which induce antibody-mediated cellular phagocytosis (so-called ADCP).

The phagocytosis of the tumor cells was already observed after 4 hours of incubation and showed an increase after 24 hours of incubation (see Fig. 5A). This phagocytosis rate was independent of the radiation dose. The positive control showed a strong phagocytosis of> 80%, which was due to the sufficient activity of the

Macrophages indicated.

1.6.6 Cellular integrity (survivability)

The cellular integrity was determined by staining the cells with trypan blue and subsequent light microscopic evaluation. For this, the cells were thawed after irradiation with 0, 10, 50, 100 or 150 Gy and stained using trypan blue. Living cells can re-donate the dye while staining dead cells. The proportion of living and dead cells can be evaluated in a Neubauer chamber under a microscope.

After direct thawing, a radiation dose of up to 150 Gy causes no cell death or cell destruction (Figure 6A). When the irradiated VPM4001 cells are cultured, cell viability decreases regardless of the radiation dose with increasing culture time (Figure 6B). 1.6.7 Proliferation

For proliferation determination, cells were incubated in a ³ H-thymidine containing medium for up to 7 days and then analyzed microscopically.

For this purpose, the VPM4001 cells irradiated with a dose between 0 and 150 Gy were thawed after thawing in fibronectin-coated 96-well plates with a cell density of 1 x 10 ⁵ cells / cm ² and 37 ° C and 5% C0 ₂ in one incubated in a humid atmosphere. Proliferation was measured on day 3 and day 7 by quantification of ³ H-thymidine incorporation with the ³ H-thymidine containing medium added 20 hours before. The quantification was carried out with the aid of a beta counter (decay events per minute, cpm).

The results are shown in FIG. The proliferation of non-irradiated cells increased from day 3 to day 5. The decrease on days 6 and 7 is probably due to critical consumption of nutrients. Irradiation of the cells resulted in a dose-dependent decrease in proliferation. Here, the proliferation rate of the 20, 40 and 50 Gy doses on day 3 is lower than that of the non-irradiated and 10 Gy samples.

A similar result was shown by the microscopic analysis of the samples (Figure 8). For this purpose, the VPM4001 cells irradiated with a dose between 0 and 150 Gy were thawed, the cell count and the survivability were determined and seeded in T25 cell culture bottles. The cells were cultured at 37 ° C and 5% C0 ₂ in a humid atmosphere. There was a change of media at least once a week. A confluent growth was considered a significant proliferation and the culture was stopped for that particular radiation dose.

For the unirradiated samples, the cells were confluent after 15 days, but at 10 and 20Gy it took 38 days. The cells irradiated at 40 and 50 Gy were not confluent even after 38 days, but showed a marked cell death on microscopic analysis.

2. Effect of gamma irradiation on the cellular parameters

The following table summarizes the influence of irradiation on the cellular parameters described above. The cells were irradiated with a radiation dose of 10-150 Gy.

Parameters results

IFN-gamma reduction of IFN-gamma expression by about 50% over an expression dose range of 10 to 150 Gy over unirradiated

cell IL-2 expression Reduction of IFN-gamma expression by> 50% compared to unirradiated cells. Expression decreases slightly with increasing gamma radiation over a dose range of 10 to 150 Gy.

PSMA and over a dose range of 10 to 150 Gy was none

EpCAM change in PSMA and EpCAM expression was observed.

expression

Complement In about 30% of VPM4001 cells, a complement complex formation complex is deposited on the cell surface. The deposition was unchanged over a dose range of 10 to 150 Gy.

Cellular integrity of cells integrity measured immediately after thawing was unchanged over a dose range of 10 to 150 Gy.

Vitality When cultivating the irradiated cells increased with vitality with the

Duration of culture. For radiation doses above 50 Gy, it was independent of the radiation dose.

Proliferation Radiation of the cells resulted in a decrease in proliferation.

The proliferation levels of the 20, 30 and 50 Gy doses were significantly lower than the proliferation levels of unirradiated and 10 Gy irradiated cells. Irradiation with 40 Gy to 150 Gy prevented confluent growth of VPM 4001 cells in culture.

3. Conclusion

An irradiation dose of 40 to 150 Gy resulted in VPM4001 in a marked inhibition of proliferation, cell characteristics such as vitality, expression of the

Tumor antigens and the recombinant cytokines were preserved. It is precisely these cell properties that, coupled with reduced proliferation, allow application of VPM4001 as an immunotherapeutic agent.

Figure legends

Fig. 1A: Interferon-gamma release of irradiated VPM4001 cells after 24, 48, 72 and 96 h of culture

Figure 1 B: lnterleukin-2 release of irradiated VPM4001 cells after 24, 48, 72 and

96 h culture time

FIG. 2A: Percentage of EpCAM or PSMA-positive VPM4001 cells as a function of the radiation dose. FIG

FIG. 2B: Mean fluorescence intensity of the EpCAM or PSMA-positive VPM4001 cells as a function of the radiation dose FIG. 3: The deposition of the complement proteins iC3b and C5b-9 on irradiated. FIG

(10, 50, 100, 150 Gy) and unirradiated (0 Gy) VPM4001 cells in response to native human serum (nhs) and heat-inactivated human serum (ihs). The functionality of the serum was detected by antibody opsonization (positive control).

Fig. 4A: Result of MNC-mediated cytotoxicity to VPM4001 cells after 3 days (diamond), 4 days (square) or 5 days (triangle) versus E: T ratio. Fig. 4B: NK cell-mediated cytotoxicity to VPM4001 cells after 24

Hours of coculture depending on the E: T ratio.

Figure 4C: Result of T-cell mediated cytotoxicity to VPM4001 cells after 3 days (diamond), 4 days (square) or 5 days (triangle) versus E: T ratio.

Fig. 5A: Recording of irradiated (10, 20, 40, 50 Gy) and unirradiated (0 Gy)

VPM4001 cells by macrophages. Cells from the running culture were used as control (ctr). The positive control was incubation with ATG antibodies (ctr-ATG).

Fig. 5B: Photographic documentation of colocalization and recording

(Arrows) of irradiated (50 Gy) VPM 4001 cells by macrophages (stained cells in the right panel). Figure 6A: Survival and cell counts of irradiated and unirradiated VPM4001 cells after thawing

Fig. 6B: survivability and cell number of the irradiated and unirradiated

VPM4001 cells after 50 and 70 days in culture

Fig. 7: Proliferation of irradiated (10, 20, 40, 50 Gy) and unirradiated (0 Gy)

VPM4001 cells 3 to 5 days after sowing. Fig. 8: Photomicrographs of the VPM4001 cells after irradiation with 0 (control), 10, 20, 40 or 50 Gy. The cells were photographed by light microscopy 2, 8, 15 and 38 days after seeding the previously cryo-irradiated cells. The medium was changed once a week.

Claims

claims

A method of making an immunotherapeutic comprising the steps of:

a) providing cells of a tumor cell line;

b) cooling the tumor cells to at least 10 ° C; preferably at below 0 ° C; c) Gamma irradiation of the tumor cells with a dose of up to 500 Gy.

2. The method according to claim 1, characterized in that the maximum radiation dose is less than 200 Gy.

3. The method according to any one of the preceding claims, characterized in that the cells in step b) and in step c) are cooled to a temperature of below -20 ° C, preferably to a temperature of below -70 ° C.

4. The method according to claim 1, characterized in that the tumor cells express at least one recombinant protein, preferably from the group of cytokines, more preferably interleukin-2 and / or interferon-gamma.

5. The method according to claim 1 to 3, characterized in that they are tumor cells of a prostate cancer cell line, preferably cells of the LnCaP cell line.

6. The method according to any one of the preceding claims, characterized in that the tumor cells in the irradiation in step b) are present in a Kryokonservierungs- medium, which preferably contains a cryoprotectant, which is particularly preferably DMSO or glycerol and is especially Cryostor CS10.

7. The method according to any one of the preceding claims, characterized in that the cells express after irradiation compared to the tumor cells before irradiation at least 10%, preferably at least 25% of the recombinantly expressed cytokines.

8. The method according to any one of the preceding claims, characterized in that the cells express after irradiation in comparison to the tumor cells before irradiation equal amounts of tumor-associated antigens.

9. The method according to any one of the preceding claims, characterized in that the cells have a determined by trypan blue marker vitality of at least 50%, preferably 70% and particularly preferably 85% based on the cells taken in culture after irradiation.

10. Method according to one of the preceding claims, characterized in that it fulfills one or more and preferably all of the following conditions:

i) the addition of the medium in step a) represents the last step in the preparation of an immunotherapeutic agent;

ii) the tumor cells irradiated in step b) are in the primary packaging; iii) the irradiated tumor cells obtained after step c) represent the finished medicinal product;

iv) steps a) and b) are carried out in a clean room;

v) the tumor cells obtained after step c) are applied to the patient without recultivation of the cells.

11. Application of the method according to one of the preceding claims for the production of a medicament for immunotherapy.

12. Tumor cells, producible by a method according to the previous claims.

13. Tumor cells according to claim 12, characterized in that the cells after irradiation have a survivability of at least 20% and an expression of the recombinant cytokines of at least 25% compared to the cells before irradiation.

14. Medicament for immunotherapy containing tumor cells produced by a method according to one of the preceding claims.