WO2014019830A1

WO2014019830A1 - Transport of photosensitizing substances using immune cells for tumour treatment

Info

Publication number: WO2014019830A1
Application number: PCT/EP2013/064716
Authority: WO
Inventors: Anja PHILIPPI; André-René BLAUDSZUN; Marc Schneider
Original assignee: Kist Europe Korea Institute Of Science And Technology Europe Forschungsgesellschaft Mbh
Priority date: 2012-08-03
Filing date: 2013-07-11
Publication date: 2014-02-06
Also published as: DE102012107166A1; DE102012107166B4

Abstract

The present invention relates to a modified mammalian immune cell for tumour treatment, and also to its use and preparation. Provision is made for the modified mammalian immune cell to be loaded with a photosensitizing substance, the photosensitizing substance being taken up into the immune cell. The photosensitizing substance is transported from the immune cell to the tumour cell. As a result of the loading of the immune cell with a photosensitizing substance, the immune cell is endowed with a cytotoxic function, or the intrinsic cytotoxic function of the immune cell is boosted.

Description

TRANSPORTING PHOTOSENSIBILIZING SUBSTANCES WITH

HELPING IMMUNOCELLS FOR TUMOR TREATMENT

The present invention relates to a modified mammalian immune cell for tumor treatment. For the treatment of cancer different methods are known. So is u.a. In addition to the surgical removal of the degenerated tissue as well as a pre- and post-treatment with chemo- and radiotherapy, immunotherapy is another approach in cancer therapy. It uses components of the immune system, such as antibodies, cytokines or immune cells, to fight tumor cells.

Immune cells have a natural ability to recognize and target the sites of infection and inflammation, or even tumor cells. This ability is also referred to as "homing." Tumor cells have an altered proteome compared to healthy cells Proteins and peptides on the tumor cell surface act as tumor antigens, which can be specifically recognized by certain immune cells, eg T and B lymphocytes, whereas macrophages, neutrophils and NK cells recognize pathogens and cancer cells independently of tumor antigens.

The approach of cellular immunotherapy is to use immune cells to kill the body's own tumor cells. The immune cells used are able to distinguish tumor cells from healthy cells.

In cellular immunotherapies, donor lymphocyte infusion (DLI), adoptive cell transfer (ACT) and dendritic cell vaccination (DC vaccination) play a major role For example, DLI is used in patients undergoing transplantation of allogeneic hematopoietic stem cells to enhance the graft-versus-tumor response. ACT is the treatment of cancer patients with their own (auto- leukocytes), ex vivo activated and expanded T lymphocytes, whereas in DC vaccination ex vivo manipulated dendritic cells are used to elicit a tumor specific immune response in the patient. While the DLI is already successfully used in the clinic, various ACT and DC vaccination approaches are currently being clinically tested.

A method for transporting certain substances, such as therapeutic or diagnostic substances, in vivo to specific sites of action is described in WO 2010/059253 A2. Nanoparticles, which are provided with the substance to be transported, are bound to the surface of carrier cells. Depending on the cell type and the associated "homing" function, the cell arrives in vivo at its target and thus at the site of action for the corresponding substance.Another approach in cancer therapy is photodynamic therapy (PDT) Treating cancer patients with a photosensitizing substance that preferentially accumulates in malignant tissue Radiation of the tumor with light of a specific wavelength leads to the activation of the light-inducible active substance, which then generates reactive oxygen species, resulting in reaction with neighboring biomolecules Resulting in the destruction of the malignant tissue The photosensitizing substance can be administered systemically or locally Even in the case of local application, the light-inducible substance is absorbed not only by tumor tissue but also by other organs such as the eyes and the skin it comes with light exposure of the patient n also to an unwanted destruction of healthy tissue. Patients treated in this way can only avoid side effects of this kind by strictly avoiding daylight for a long time. In order to reduce or even avoid these limitations of the patients, it is necessary to be able to administer the light-inducible substances in a more targeted manner.

US 2002/0197262 A1 discloses, for the more specific transport of a photosensitizer, a method in which a photosensitizer is conjugated to an antibody. is bound by, which has tumor specificity. In this way, the photosensitizer reaches the tumor region.

The object of the present invention was to find a way to increase the specificity and effectiveness of photodynamic therapy, thereby reducing typical side effects.

The object is achieved by a modified mammalian immune cell which is loaded with a photosensitizing substance, wherein the photosensitizing substance is taken up in the immune cell.

In the context of the present invention, loading of the immune cell means that the photosensitizing substance is taken up in the cell. If particles provided with a photosensitizing substance, such as nano- or microparticulate particles, are used, they too are enveloped by the membrane of the cell and in this way integrated into the cell. The uptake of the photosensitizing substance into the cell takes place via endocytosis and is also referred to as internalization. By loading an immune cell with a photosensitizing substance and thus combination of PDT and cellular immunotherapy, both the selectivity and efficiency of PDT are increased. The photosensitizing substance is brought by the immune cell directly to the tumor cell and transferred to this. The immediate interaction of the immune cell with the target tumor cell allows smaller amounts of the photosensitizing substance to be used more selectively than if the drug is administered to the patient intravenously / systemically or locally. Targeted drug delivery and lower drug levels contribute to fewer side effects than traditional PDT in this approach.

The use of immune cells also increases the efficiency of PDT. The cells of the human defense system are characterized by the fact that they can detect disease foci and actively penetrate into malignant tissue. NEN. This natural homing function of immune cells should be exploited to promote photosensitizing substances targeted to the site of action. In addition, many immune cells have the ability to kill diseased cells either by cytotoxic mechanisms or by phagocytosis. In addition to the homing function of the immune cells used for the targeted transport of the photosensitizing substance, the intrinsic cytotoxic function or phagocytic activity ("knocking" function) and thus cancer-killing effect of immune cells should be used to fight tumor cells as efficiently as possible The intrinsic cytotoxic activity, such as helper T cells, is given a type of cytotoxic function by the photosensitizing substance, which, by loading the immune cell with a photosensitizing substance, gives it a cytotoxic or intrinsic killing function Immune cell reinforced.

For the purposes of the present invention, tumor or tumor cells are cancer cells or a multiplicity of cancer cells in a composite, such as malignant tissue. The term cancer cell includes cells that undergo oncogenic proliferation.

For the purposes of the present invention, photosensitizing substance is to be understood as meaning a chemical component or a precursor of a chemical component which causes a biological effect on the basis of photoactivation.

In this case, it is preferred that the photosensitizing substance is a photosensitizer, preferably a hematoporphyrin, chlorin, bacteriochlorin, phthalocyanine, benzoporphyrin derivative, 5-aminolevulinic acid (ALA) or esters, hypericin, purpurin, porphycene, indocyanine green (ICG ), as well as their derivatives.

In a preferred embodiment, the photosensitizing substance is a water-soluble photosensitizer complex, for example PSS / mTHPP or PSS / mTHPC, or a chemically modified photosensitizer. Furthermore, it is preferred that the photosensitizing substance is a dendrimeric photosensitizer formulation, a photosensitizer-antibody conjugate, a photosensitizing nanoparticle, a liposomal photosensitizer formulation, or a nano- or microparticulate photosensitizer formulation. The nano- or microparticulate photosensitizer formulation is preferably photosensitizer-gold particles, photosensitizer-PLGA particles or particles coated with a photosensitizer.

For the purposes of the present invention, microparticles are particles having diameters of from 400 nanometers to 10 micrometers, and nanoparticles are particles having diameters from 1 to 400 nanometers. It is further preferred that the immune cell is loaded ex vivo by incubation with the photosensitizing substance, the uptake into the cell being by endocytosis.

To obtain mammalian immune cells, they must first be isolated from a tissue sample or blood. Subsequently, the isolated immune cell is preferably activated ex vivo and / or augmented ex vivo and / or generated ex vivo. Generated by ex vivo is understood to mean that this cell is differentiated into a derived immune cell type. Monocytes can e.g. be differentiated ex vivo to macrophages.

It is further preferred that the immune cell is an autologous cell or an allogeneic cell.

A further object of the present invention is that the immune cell comprises a T lymphocyte, in particular an ex vivo activated and expanded polyclonal T cell, an ex vivo propagated tumor infiltrating T cell, an ex vivo expanded monoclonal antigen-specific T cell, a T cell Cell with a genetically engineered T cell receptor (TCR), or a genetically engineered is a chimeric antigen receptor (CAR) expressing T cell. Alternatively, the immune cell is a monocyte, in particular an autologous monocyte, a macrophage, in particular an autologous macrophage, a tumor-associated macrophage (TAM) or an in vitro-generated macrophage, a neutrophilic granulocyte, a natural killer cell (NK cell) and / or a cell an NK cell line, in particular an NK-92 cell and / or a CAR-expressing NK-92 cell.

In a preferred embodiment, a modified immune cell is co-administered together with a retargeting substance, wherein the retargeting substance is preferably a bispecific antibody, diabody, BiTE antibody, trispecific antibody and / or tetraspecific antibody, and fragments which, acts. In addition to T lymphocytes activated ex vivo, monocytes, macrophages, NK cells and neutrophilic granulocytes can also be co-administered with one of the abovementioned retargeting substances. Preferably, the respective immune cell is pre-incubated with the corresponding retargeting substance immediately before administration. A immune cell functionalized with the retargeting substance is then administered. T cells have receptors (T cell receptors, TCRs) that can recognize a particular peptide antigen bound to an MHC complex. When an activated T cell is directed to a target cell, e.g. a cancer cell, which presents the corresponding peptide antigen MHC-dependent on its surface, binds the T cell with its T cell receptors to the antigen MHC complexes of the target cell. This specific recognition of the target cell triggers the effector function of the T cell.

For example, CD8-positive cytotoxic T cells release perforin and granzyme, which drive the target cell into programmed cell death (cytolytic activity), while CD4-positive T cells secrete cytokines, thereby attracting other immune cells (helper function). Ex vivo activated polyclonal T cells are not specific for a particular tumor antigen. As a result, they can detect the tumor focus, but it is theirs unable to bind to target cells and exert their natural effector functions.

A bispecific antibody, which recognizes both the TCR component CD3 and is specific for a particular tumor antigen (e.g., EpCAM), cross-links MHC-independent nonspecifically activated T cells and cancer cells, triggering the cytolytic function of cytotoxic T cells. Other T-cell subclasses, such as T-helper cells, are also MHC-independently cross-linked to target cells via the bispecific antibody so that they can exert their effector functions. Thus, the retargeting substance serves both the exact targeting and the stimulation of cytolytic activity and other functions of nonspecifically activated polyclonal T cells.

The antigen-specific recognition of target cells applies only to B and T cells. Macrophages and neutrophils recognize pathogens and cancer cells independent of antigen. If monocytes, macrophages, neutrophils or NK cells are used for the purposes of the present invention, it is advantageous to use a retargeting substance which primarily serves to target the immune cell used. Thus, bispecific antibodies can be used which recognize the Fcy receptors CD64 or CD16 expressed on monocytes, macrophages, neutrophilic granulocytes and NK cells and at the same time have a specificity for a tumor antigen.

Further advantages result from the use of at least one modified immune cell for the targeted transport of a photosensitizing substance for the treatment of tumor cells.

The at least one modified immune cell is preferably used for cancer therapy on tumors on the body surface and / or on tumors that can be achieved in a minimally invasive manner, such as skin tumors, for example a basal cell carcinoma, malignant melanoma, head and neck carcinomas, bronchial and lung cancers , Bladder cancer, prostate carcinoma, colon carcinoma, liver carcinoma, Renal cell carcinoma, tumors of the female genital area, brain tumors or malignant ascites used.

Furthermore, a process for producing a modified mammalian immune cell is the subject of the present invention.

To obtain mammalian immune cells, they are first isolated from a mammalian tissue sample or blood. Subsequently, the isolated immune cell is preferably activated ex vivo and / or augmented ex vivo and / or generated ex vivo. Generated ex vivo is to understand that the isolated immune cell is differentiated into a derived immune cell type. Thereafter, the loading of the immune cell is carried out with a photosensitizing substance, wherein the loading is carried out by incubation with the photosensitizing substance. Preferably, the immune cell is subsequently incubated with a retargeting substance, preferably a bispecific antibody.

Furthermore, it is preferred that the photosensitizing substance is a photosensitizer, preferably a hematoporphyrin, chlorin, bacteriochlorin, phthalocyanine, benzoporphyrin derivative, 5-aminolevulinic acid (ALA) or ester, hypericin, purpurin, porphycene, indocyanine green (ICG), and their derivatives, or a water-soluble photosensitizer complex, a chemically modified photosensitizer, a liposomal photosensitizer formulation, a nano- or microparticulate photosensitizer formulation, a dendrimeric photosensitizer formulation, a photosensitizer-antibody conjugate, and / or a photosensitizing nanoparticle ,

In a preferred embodiment of the method according to the invention, a modified immune cell is co-administered together with a retargeting substance, wherein the retargeting substance is preferably a bispecific antibody, diabody, BiTE antibody, trispecific antibody and / or tetraspecific antibody, and Fragments of these, acts.

Since the photosensitizing substance is transported with the help of immune cells targeted to the target site, the tumor focus, can lower amounts of substance as used in conventional photodynamic therapy. This should significantly reduce the side effects of PDT. Due to the additional cellular "killing" mechanisms of the immune cells, there is a significant increase in efficiency.

The present invention will be explained in more detail with reference to the following figures. Show it:

1 shows a fluorescence microscopic detection of photosensitizer uptake into ex vivo activated human T lymphocytes;

Fig. 2 Results of the determination of the viability of PSS / mTHPP-loaded T cells; 3 shows a fluorescence microscopic detection of the photosensitizer transfer of PSS / mTHPP-loaded T lymphocytes to cancer cells; and

Fig. 4 Results of the determination of the cytotoxic effect of PSS / mTHPP-loaded T cells.

FIG. 1 shows ex vivo activated human T lymphocytes which have taken up the photosensitizer complex PSS / mTHPP. Ex vivo stimulated human T cells were incubated on the seventh day post-activation for 2 hours with a PSS / mTHPP amount corresponding to a mTHPP concentration of 10 g / ml. Immediately after loading, the subcellular distribution of the photosensitizer mTHPP was analyzed by means of an inverse IX 71 fluorescence microscope (Olympus). The cell nuclei were detected by DAPI. Figure A shows the individual fluorescent nuclei (N), Figure B the fluorescence of the photosensitizer (P) and Figure C the nuclear staining and photosensitizer fluorescence in one image.

FIG. 2 graphically shows the results of a viability test as performed in Embodiment 5. FIG. On the fourth (A) or third (B) day After activation, T lymphocytes were loaded with the photosensitizer complex PSS / mTHPP, or only Η ₂ ΟΜΝΝ _{ΡΟΓΘ was} added to the control (uncharged T cells). Some of the samples were stored in the dark after loading. On the other hand, the remaining batches were irradiated either immediately (A) or after 72 hours (B) for 2 minutes with a calhalogen light source (h * v). At the times indicated, T-cell viability was determined. Triton Χ-100-treated T lymphocytes each served as a positive control. In each case triple measurements were carried out. (B) C1, C2, C3: Samples 1, 2, 3.

From Figures A and B it can be seen that loaded and unloaded, unirradiated cells have the same viability rates. Thus, the photosensitizer has no influence on the viability of the loaded T-lymphocytes in the dark. By contrast, irradiation of the cells with light, either immediately (A) or three days (B) after loading, leads to cell death.

Figure 3 shows in Figure A a nuclear staining of Skov-3 cells (SN) (human ovarian carcinoma cell line, ATCC, Manassas, USA) and loaded T cells (TN) as described in Example 4. In addition, in Figure B, the fluorescence of the photosensitizer mTHPP is visible in the cells. By means of this fluorescence microscopy, the distribution of the photosensitizer can be documented. In Figure B, it can be seen that the photosensitizer is also located in the tumor cells (P) and thus transferred from the loaded T lymphocytes (TNP) to the co-cultured Skov-3 cancer cells.

Fig. 4 graphically shows the cytotoxic effect of PS-loaded T lymphocytes on Skov-3 cells (human ovarian carcinoma cell line). After activation of the photosensitizer, by a total of three (3, 2.5 and 2 hours) irradiation of all batches before each measurement, a viability test was carried out at the indicated times. The experimental procedure is described in Example 6. This shows in the control samples of unloaded T cells and Skov-3 cells with medium alone Increase in cancer cell viabilitat. In the case of loaded T cells without bispecific antibodies as a retargeting substance, a similar decrease in cancer cell viabilitvity can be seen as in unloaded T cells with retargeting substance. This shows that loaded T cells without retargeting substance have a cytotoxic effect on the cancer cells similar to uncharged cells with retargeting substance. The most potent cytotoxic effect on the Skov-3 cells is a combination of loaded T cells with bispecific antibody as a retargeting substance. The modification of human or animal mammalian immune cells will be explained in more detail below by way of example with reference to the modification of human T cells, without restricting it thereto.

1 . Cell isolation, activation and extension

Human T lymphocytes are isolated from fresh buffy coat blood using the RosetteSep® Human T Cell Enrichment Cocktail (StemCell Technologies, Grenoble, France) according to the manufacturer's instructions. In each case 20 ml of blood are mixed with 0.5 ml of RosetteSep® antibody cocktail and incubated with gentle shaking for 20 min at room temperature. The mixture is then mixed with 10 ml of PBS (phosphate-buffered saline), carefully pipetted onto 15 ml Pancoll human (PAN Biotech, Aidenbach, Germany) and centrifuged for 20 min at 1200 * g with the brake off. The then enriched in the boundary layer between Pancoll and serum T cells are transferred to PBS and sedimented. After removal of residual erythrocytes with the aid of BD Pharm Lysis Lysing buffer (BD Biosciences, Heidelberg, Germany), the remaining T cells are washed twice more with PBS and finally in culture medium (RPMI 1640, PAN Biotech + 10% (v / v ) heat-inactivated FBS, PAN Biotech + 1% (v / v) penicillin / streptomycin, Sigma-Aldrich, Steinheim, Germany).

After determining the cell number using a Casy® Cell Counter + Analyzer system (Model TT, Schärfe System GmbH, Reutlingen, Germany), the isolated T lymphocytes using the T-Cell Activation / Expansion Kit human (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. For this purpose, first the cell concentration in culture medium is adjusted to 2.5 × 10 ⁶ T cells / ml. Subsequently, the cells are added in a ratio of 2: 1 with anti-CD3, anti-CD28 and anti-CD2 coupled microbeads and incubated for 72 hours in a water vapor-saturated 5% CO2 / air mixture at a temperature of 37 ° C.

Subsequently, the culture medium is removed and the cell density in fresh medium again adjusted to 2.5 × 10 ⁶ T cells / ml. To expand the activated T-lymphocytes, the cultures are mixed with 20 U / ml of human Interleukin-2 (PAN Biotech) and cultured for one day at a temperature of 37 ° C in the CO2 incubator.

In order to remove the "activation beads" and dead cells from the batches, the T lymphocytes are subjected to another Pancoll density gradient centrifugation on the fourth day after activation by layering the cells resuspended in 20 ml of culture medium over 20 ml of Pancoll, and 20 After transferring the T cells enriched in the intermediate layer into PBS, they are sedimented again and taken up in culture medium.After cell count determination, the T lymphocytes are either used immediately for further experiments or at a cell density of 2.5 × 10 ⁶ cells / ml in turn with 20 U / ml interleukin-2 and cultured for a further 1 -3 days at 37 ° C in a CO2 incubator.

2. Loading ex vivo stimulated human T lymphocytes with a water-soluble photosensitizer

By way of example, the water-soluble photosensitizer PSS / mTHPP is used in this embodiment. The water-soluble photosensitizer complex PSS / mTHPP which is present in lyophilised form is prepared with sterile H2OMIII _P ore at a concentration of 5 mg / ml. The stock solution is kept protected from light for a maximum of two weeks at 4 ° C. For loading ex vivo stimulated human T cells with PSS / mTHPP, the cell density in culture medium (RPMI 1640 + 10% FBS and 1% Pen / Strep) is adjusted to 2.5 × 10 ⁶ cells / ml 4-7 days after activation. Then the cells are mixed with an amount of PSS / mTHPP stock solution corresponding to the respectively indicated mTHPP concentration and incubated for 2 hours at 37 ° C. in the CO ₂ incubator. To remove unaccumulated complex, the T lymphocytes are then washed three times with ice-cold culture medium. Thereafter, the cells are either immediately used for further use or taken up in IL-2-containing culture medium and further cultivated until their later use. Only T cells incubated with Η ₂ ΟΜΝΝ _d each served as a control (uncharged cells).

As background light, a Dukalux SL lamp (Kindermann GmbH, Ochsenfurt, Germany, emission _max ~ 61 1 nm) is used in all work steps. To avoid premature activation of the light-inducible drug, the incubations are done in the dark.

If a non-water-soluble photosensitizer is used, it is first dissolved in ethanol. The photosensitizer dissolved in ethanol is then added to the cell suspension and incubated with it.

3. Fluorescence microscopic analysis of photosensitizer uptake into activated T cells

In order to detect the uptake of the photosensitizer complex into activated human T lymphocytes, T cells are incubated on the seventh day after activation with a PSS / mTHPP amount dissolved in H ₂ O, which has a mTHPP concentration of 10 g / ml. ml corresponds. Immediately after loading, the cells are subjected to fluorescence microscopic analysis. For this purpose, 3 × 10 ⁶ loaded T lymphocytes are first fixed for 10 min in 4% (w / v) paraformaldehyde (Sigma-Aldrich) solution. Following two PBS washes, the cells are embedded in FluorSave mounting medium (Calbiochem) which has been previously probed with 1 g / ml DAPI (Sig. ma-Aldrich) has been added. For curing, the samples are stored overnight protected from light at room temperature. Subsequently, the preparations are stored darkened at 4 ° C until microscopy. With the help of an inverse IX 71 fluorescence microscope from Olympus, equipped with a digital camera (F-View II, Olympus) and the Cell P software (version 2.8, Olympus), the internalized photosensitizer is visualized. For viewing the cells, preferably a 60x / 1 .42 oil immersion objective is chosen. While a NU filter (excitation wavelength: 360-370 nm, emission> 420 nm) is preferably used to detect the DAPI signals, the subcellular distribution of the photosensitiser is preferably determined by means of a WG filter (excitation: 510-550 nm , Emission> 590 nm). The exposure times varied depending on the signal intensity.

4. Fluorescence microscopic analysis of photosensitizer transfer of T lymphocytes to cancer cells

To test whether the photosensitizer is transferred from the loaded T lymphocytes in vitro to cocultivated cancer cells, live cell fluorescence microscopy can be used. On the fourth day after the ex vivo activation, T lymphocytes are loaded for three hours with a quantity of photosensitizer complex dissolved in Η2ΟΜΝΝ _ΡΟΓΘ , which corresponds to a mTHPP concentration of 45 g / ml. 3 cells (human ovarian carcinoma cell line, ATCC, Manassas, USA) seeded in a Clear black 96-well plate (Greiner Bio-one). Following two medium washes, the loaded T cells are added to the cancer cells in a 4: 1 ratio. The batches are then cultured at 37 ° C in the CO2 incubator. After a co-incubation time of 28 hours, the samples are washed three times with medium, removing most of the T cells from the batches. Thereafter, the cells are visualized for 20 min with 5 g / ml Hoechst dye (Invitrogen) to visualize the cell nuclei. Subsequently, the distribution of the photosensitizer is analyzed by means of an inverse Olympus IX 71 fluorescence microscope. The photo sensitizer complex mTHPP is autofluorescent. The procedure is carried out as described under 3..

5. Determination of the viability of photosensitizer-loaded T cells

In order to determine the viability of modified immune cells for control purposes, the procedure is preferably as follows. On the fourth or third day after the / nw ^{'fro-stimulating} T-cells are incubated in culture medium having a dissolved in _Η2ΟΜΝΝ ΡΟΓΘ complex quantity corresponding to a mTHPP concentration of 10 g / ml, or only with H ₂ O staggered (unloaded T cells). After transfer of the loaded and unloaded T lymphocytes in 96-well plates (2.5x10 ⁵ T cells / well), half of the samples are stored in the dark. The remaining batches are illuminated either immediately or after a 72-hour incubation in the CO2 incubator. For each 2-minute irradiation of the cells, a Kalthalogenlichtquelle (KL 200, Schott, Mainz, Germany) is used. T-cells treated with 1% (w / v) Triton X-100 can serve as positive controls.

With the help of the Cell Proliferation Reagent WST-1 (Roche Applied Science), the viability of the T lymphocytes is determined at different times (see FIG. 1). The WST-1 assay measures the mitochondrial activity of cells, indicating their viability. The test is based on the photometric detection of a formazan dye resulting from the reduction of a tetrazolium salt by living cells. When carrying out the viability tests, proceed according to the manufacturer's instructions. In each case 100 μΙ cell suspension with 10 μΙ WST-1 reagent are added. After a 1-hour incubation in a CO2 incubator (37 ° C), the absorption measurement is carried out in a Microplate reader (Spectra ELISA Reader, Tecan, Crailsheim, Germany) at a wavelength of 450 nm (reference wavelength: 690 nm). Triple measurements are carried out in each case.

6. Determination of the cytotoxic effect of photosensitizer-loaded T lymphocytes in vitro To determine the cytotoxic effect, the "killing effect", of PS-loaded T lymphocytes, the WST-1 assay can also be used, as described under 5. First, T cells are incubated on the fourth day after activation loaded in Η ₂ ΟΜΝΝ _ΡΟΓΘ, corresponding to a mTHPP concentration of 30 g / ml, or only incubated with H ₂ O (uncharged T cells). After two medium washes, 80,000 T lymphocytes (loaded and unloaded) are added to the cancer cells in triplicates, half of the batches are additionally supplemented with a retargeting substance, (human ovarian carcinoma cell line). In this case 0.1 g / ml bispecific antibody HEA125xOKT3 (Gerhard Moldenhauer, DKFZ Heidelberg, Germany) was added to Skov-3 cells treated with 1% (w / v) Triton X-100 as well as untreated cancer cells . Negative control serve. All preparations are incubated in the dark at 37 ° C and 5% CO2.

In order to determine the survival of the tumor cells in the different cultures, WST-1 assays (Roche Applied Science) are performed at the indicated times. The photosensitizer is activated by irradiating all of the batches three times (3, 2.5, and 2 hours) under standard conditions with halogen light (Haloline Eco, 400 W, 9000 Im, OS RAM) for 2 minutes before each measurement. The viability tests are carried out according to the manufacturer's instructions, as described under 5.. In order to record only the mitochondrial activity of the Skov-3 cells, the absorption of 80,000 T-lymphocytes is subtracted from the respective values of the cocultures.

7. Further use in vivo

After recovery of mammalian immune cells and subsequent activation and / or propagation and loading of the immune cell with a photosensitizing substance, the modified immune cell is preferably applied to a patient. Preferably, the modified immune cell is administered to the patient intravenously or locally. In addition, a retargeting substance can be applied to photosensitizer-loaded To crosslink immune cells at the site of action with the target cells and thereby possibly trigger the intrinsic cytotoxic activity of the carrier cells. It is preferred that the modified immune cells, once they have reached the target site, first perform their natural "killing" function before, at a defined time, the photosensitizing substance is activated by light irradiation and thus additionally acts on the tumor cells tumors can be used in a single radiation session, but there is a possibility of repeated radiation treatment, as well as treatment with loaded immune cells and retargeting substance once or several times.

LIST OF REFERENCE NUMBERS

N nucleus

P photosensitizer

TN T-cell nucleus

SN nucleus Skov-3

TNP T-cell nucleus + photosensitizer

SNP nucleus Skov-3 + photosensitizer

Claims

claims

1 . Use of at least one modified mammalian immune cell for the targeted transport of a photosensitizing substance for the treatment of tumor cells, characterized in that it is loaded with a photosensitizing substance, wherein the photosensitizing substance is taken up in the immune cell.

2. Use according to claim 1, characterized in that the photosensitizing substance is a photosensitizer, preferably a hematoporphyrin, chlorin, bacteriochlorin, phthalocyanine, benzoporphyrin derivative, 5-aminolevulinic acid (ALA) or ester, hypericin, purpurin, porphycene, indocyanine green ( ICG), as well as their derivatives.

3. Use according to claims 1 or 2, characterized in that the photosensitizing substance is a water-soluble photosensitizer complex, a chemically modified photosensitizer, a liposomal photosensitizer formulation, a nano- or microparticulate photosensitizer formulation, a dendrimeric photosensitizer Formulation, a photosensitizer-antibody conjugate and / or a photosensitizing nanoparticle.

4. Use according to one of the preceding claims 1 to 3, characterized in that the immune cell is loaded ex vivo by incubation with the photosensitizing substance.

5. Use according to one of the preceding claims 1 to 4, character- ized in that the immune cell activated ex vivo and / or propagated ex vivo and / or ex vivo generated.

6. Use according to one of the preceding claims 1 to 5, characterized in that the immune cell is an autologous cell or an allogeneic cell.

7. Use according to one of the preceding claims 1 to 6, characterized in that the immune cell is a T lymphocyte, in particular an ex vivo activated and expanded polyclonal T cell, an ex vivo propagated tumor infiltrating T cell, an ex vivo expanded monoclonal antigenspezifi T cell, a T cell with engineered T cell receptor (TCR), or a genetically engineered CAR (chimeric antigen receptor) expressing T cell;

or a monocyte, in particular an autologous monocyte, a macrophage, in particular an autologous macrophage, a tumor-associated macrophage (TAM) and / or an in vitro-generated macrophage, a neutrophilic granulocyte, a natural killer cell (NK cell) and / or a cell of one NK cell line, in particular an NK-92 cell and / or a CAR-expressing NK-92 cell.

8. Use according to one of the preceding claims 1 to 7, characterized in that the immune cell is co-applied together with a retargeting substance, wherein the retargeting substance is preferably a bispecific antibody, diabody, BiTE antibody, trispecific Antibodies and / or tetraspecific antibodies, as well as fragments thereof.

9. Use of at least one modified immune cell according to one of the preceding claims 1 to 8, characterized in that they are preferably used for cancer therapy on tumors on the body surface and / or on tumors that can be achieved minimally invasive, such as skin tumors, basal cell carcinoma, malignant melanoma, head and neck cancer, bronchial and lung cancer, bladder cancer, prostate carcinoma, colon carcinoma, hepatic carcinoma, renal cell carcinoma, tumors of the female genital area, brain tumors or malignant ascites.

10. Modified mammalian immune cell, characterized in that it is loaded with a photosensitizing substance, wherein the photosensitizing substance is taken up in the immune cell.

1 1. Modified immune cell according to claim 10, characterized in that the photosensitizing substance is a photosensitizer, preferably a hematoporphyrin, chlorin, bacteriochlorin, phthalocyanine, benzoporphyrin derivative, 5-aminolevulinic acid (ALA) or esters, hypericin, purpurin, porphycene, indocyanine green (ICG ), as well as their derivatives.

12. Modified immune cell according to claims 10 or 11, characterized in that the photosensitizing substance is a water-soluble photosensitizer complex, a chemically modified photosensitizer, a liposomal photosensitizer formulation, a nano- or microparticulate photosensitizer formulation, a dendrimeric photosensitizer toric formulation, a photosensitizer-antibody conjugate and / or a photosensitizing nanoparticle.

13. Modified immune cell according to one of the preceding claims 10 to 12, characterized in that the immune cell is loaded ex vivo by incubation with the photosensitizing substance.

14. Modified immune cell according to one of the preceding claims 10 to 13, characterized in that the immune cell activated ex vivo and / or propagated ex vivo and / or generated ex vivo.

15. Modified immune cell according to one of the preceding claims 10 to 14, characterized in that the immune cell is an autologous cell or an allogeneic cell.

16. Modified immune cell according to one of the preceding claims 10 to 15, characterized in that the immune cell comprises a T lymphocyte, in particular an ex vivo activated and expanded polyclonal T cell, an ex vivo multiple tumor infiltrating T cell, an ex vivo expanded monoclonal anti-gene specific T cell, a T cell with genetically engineered T cell receptor (TCR), or a genetically engineered CAR (chimeric antigen receptor) expressing T cell is;

17. Modified immune cell according to one of the preceding claims 10 to 16, characterized in that it is co-applied together with a retargeting substance, wherein the retargeting substance is preferably a bispecific antibody, diabody, BiTE antibody, trispecific Antibodies and / or tetraspecific antibodies, as well as fragments thereof.

18. A method for producing a modified mammalian immune cell for use according to one of the preceding claims, comprising at least the following steps:

Isolation of a mammalian immune cell from a mammalian tissue sample or blood,

ex vivo activation and / or ex vivo propagation and / or ex vivo generation of the isolated immune cell,

- Loading the immune cell with a photosensitizing substance, wherein the loading takes place via incubation with the photosensitizing substance.

19. A method for producing a modified mammalian immune cell according to claim 18, characterized in that the photosensitizing substance is a photosensitizer, preferably a hematoporphyrin, chlorin, bacteriochlorin, phthalocyanine, benzoporphyrin derivative, 5-aminolevulinic acid (ALA) or ester, Hypericin, purpurin, porphycene, indocyanine green (ICG), as well as their derivatives, or a water-soluble photosensitizer complex, a chemically modified photosensitizer, a liposomal photosensitizer formulation, a na- or microparticulate photosensitizer formulation, a dendrimeric photosensitizer formulation, a photosensitizer-antibody conjugate and / or a photosensitizing nanoparticle is.

20. A method for producing a modified mammalian immune cell according to claims 18 or 19, characterized in that a modified immune cell is co-administered together with a retargeting substance, wherein the retargeting substance is preferably a bispecific antibody, diabody, BiTE antibodies, trispecific antibodies and / or tetraspecific antibodies, as well as fragments thereof.