Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

• the invention relates to a method for the in vitro investigation of the action of substances in in vivo processes as well as an in vitro detection method for the identification of immunomodulating compounds and / or the detection of the effect of immunomodulating compounds and the identification of apoptosis and / or necrosis inducing compounds by means of Immune system in in vivo processes.
• Chemotherapy which has been the only treatment option for advanced and widespread cancer since the 1950s, has one goal in mind: to develop therapies that have fewer side effects for the patient but are highly effective in achieving the therapeutic goal.
• immunotherapy which aims to enhance the natural immune response against the tumor disease by genetic engineering modifications, ie the "attention" of the immune system to cancer cells and thus to influence the defense reaction so that the tumor is controlled by the body itself.
• immunomodulatory substances are under development, which should cause the immune system to fight tumor cells.
• These immunomodulatory agents aim to induce or "program" the immune system to specifically attack and ultimately destroy tumor cells, thus immunomodulatory agents acting indirectly through the immune system on the tumor or underlying tumor cell type.
• a method that allows in vitro to investigate the effect of new substances on in vivo processes, such as the destruction of tumor cells, would avoid on the one hand with great ethical reservations in vivo experiments and on the other hand allow, in a short time a variety of substances in a variety of different tumor cells to test.
• it would be possible by such a method to represent the progress of a therapy regarding the induced in vivo effects in a so-called "therapy monitoring".
• Effector cells of the mixture of immune cells e.g. PBMC [Peripheral monoclonal immune system of blood (human or higher mammalian) cells, spleen cells (animal models), etc.] or sub-populations sorted by FACS or MACS, e.g. B, T and NK cells, monocytes, dendritic cells, etc.
• PBMC Peripheral monoclonal immune system of blood (human or higher mammalian) cells, spleen cells (animal models), etc.
• FACS or MACS e.g. B, T and NK cells, monocytes, dendritic cells, etc.
• immunomodulatory compounds are to be understood as meaning substances which are capable of influencing the reaction of the immune system or even of only individual cells thereof, in particular of the effector cells. These include not only chemical compounds but also DNA Constructs, proteins, antibodies, sugar molecules or other substances that have properties that cause the immune system or cells of the immune system to react. This relates in particular to the cells of the immune system designated as effector cells in the present invention, which are capable of effecting or transmitting reactions of the immune system. This transfer takes place by means of the distribution of specific messengers.
• an in vitro detection method for the identification of immunomodulating compounds and / or the detection of the effect of immunomodulating compounds as well as the identification of apoptosis and / or necrosis inducing compounds by the immune system in in vivo processes comprising the following sequence of Process steps comprise a) primary incubation of effector cells of the immune system with an immunomodulatory effect or apoptosis or necrosis inducing substance, followed by b) recovery of the supernatant or mixture of cells and supernatant of the primary incubation followed by c) secondary incubation of target cells with the supernatant or mixture of cells and supernatant of the primary incubation, and finally d) the immunomodulating and / or apoptosis and / or necrosis-inducing effect is analyzed by means of suitable detection methods.
• the effect of already performed / performed therapy can be tracked (by analyzing relevant parameters).
• this use of the method according to the invention is also referred to as "therapy monitoring.” This merely refers to the in vitro tracking of the in vivo therapy effects
• the methods according to the invention are not associated with the therapy itself, except that the therapeutic success is controlled or can be tracked.
• the isolated cells are in a preferred embodiment of the method according to the invention to effector cells of the immune system according to the definition given above.
• the methods of the invention are particularly useful to study effects of substances on cells mediated by the immune system.
• target cells human cells or cells of higher mammals are preferably provided.
• isolated cells are used for the primary incubation, in particular cells of the immune system, and as target cells for the secondary incubation.
• target cells for the secondary incubation.
• tumor cells of different origin can be considered tumor cells.
• the aim of a "functional in vitro immunoassay" is to identify or investigate substances which are suitable for inducing apoptosis or necrosis by means of the immune system in tumor cells.
• a further aim of the method according to the invention is also the detection of tumor cells by the immune system, caused by the increased expression of MHC-I (eg HLA-ABC) and adhesion (eg ICAM-1) molecules on the surface of the tumor cells, to investigate.
• MHC-I eg HLA-ABC
• adhesion eg ICAM-1
• kits according to the invention For the application of the method according to the invention for the investigation of changes of the expression of surface molecules due to an immune reaction induced by the immunomodulating substance a kit according to the invention is provided.
• the kit contains storage-ready aliquots of cells, preferably immune system effector cells, for primary incubation with the substances to be tested, means for performing primary and secondary incubation, and suitable means for analyzing the expression pattern of the surface molecules of the cells of the secondary incubation.
• the kit according to the invention contains means for carrying out an RT-PCR for analyzing the expression pattern of surface antigens of the target cells of the secondary incubation, for which the kit contains suitable primers for amplification of the mRNA of surface molecules, enzymes for the duplication and the required buffers and / or means for a FACS Analysis for which the kit contains suitable fluorescently labeled antibodies directed against surface antigens and apoptosis / necrosis markers, and also means for processing the target cells, such as buffers and chemicals.
• the methods according to the invention are also suitable for monitoring therapy, in which, in the primary incubation, whole blood, blood cells, blood serum or the blood plasma of a patient before, during and / or after a treatment (eg immunotherapy or therapy containing the Immune system changed or influenced) is used.
• a treatment eg immunotherapy or therapy containing the Immune system changed or influenced
• the treatments in which the methods according to the invention are intended as therapy monitoring for the effectiveness of the therapeutics used in each case are preferably diseases such as cancer, infections, allergies and autoimmune diseases.
• CpG motif-containing oligodeoxynucleotides and dSLIM double stem-loop immunomodulating oligodeoxyribonucleotides, see EP 1 196 178 BI
• dSLIM double stem-loop immunomodulating oligodeoxyribonucleotides
• biomolecules such as natural or genetically engineered antibodies, DNA or RNA-based substances (antisense oligodeoxynucleotides, si-RNA, etc.), amino acid compounds, messengers or other immune modulators (such as aluminum salts, Imidazoquinolines, lipopolysaccharides, saponin derivatives, phospholipids, squalene, etc.).
• compounds which induce apoptosis and / or necrosis are, in particular, those compounds which are suitable for sustainably disrupting the processes necessary for obtaining the cell.
• DNA- or RNA-based substances antisense oligodeoxynucleotides, si-RNA, etc.
• antibodies or chemotherapeutic agents come into consideration.
• the methods of the invention may be further used to identify messengers that are secreted by the cells as a result of incubation of the isolated cells in the primary incubation with immunomodulatory or apoptosis and / or necrosis inducing substances.
• the supernatant of the primary incubation is preincubated with antibodies which specifically recognize potential messengers prior to addition to the target cells of the secondary incubation. Due to the interaction between the antibody and the epitope of the messenger, it is no longer able to mediate signals to the target cells and is thus blocked in its function.
• This embodiment of the method according to the invention is important for detecting which specific messengers are responsible for an induced effect, e.g. Apoptosis, are responsible.
• perforated plates having 24-96 holes are preferably used, the area of each hole of a plate being coated with an antibody which is resistant to an epitope of a messenger (eg IFN).
• a messenger eg IFN
• kits for applying the methods of the invention for identifying messenger substances which are released as a reaction of incubation of the cells of the primary incubation with a substance to be examined also subject of the present invention.
• a kit contains storable prepared aliquots of cells, preferably effector cells of the immune system, for the primary incubation with the substances to be examined, means for performing primary and Sekund sourcetububation, and further multi-well plates with a number of holes between 24 - 96 holes in which the surfaces of the Holes are coated with an antibody, wherein the surfaces of different holes are coated with different antibodies, but preferably at least two holes are each provided with an identical antibody.
• the incubation steps necessary in the method according to the invention preferably take place in an incubator with 5% CO 2 content.
• other incubation conditions are also conceivable, each of which depends on the requirements of the cells to be incubated.
• the recovery of the supernatants or the mixture of supernatant and cells from the primary incubation is carried out according to the invention by centrifugation.
• all other methods are also conceivable which are suitable for separating the cells from the supernatants, for example filtration of the cells at a mesh width which allows passage only to the supernatant, but not to the cells or existing cell debris.
• cell separations or sortings by means of specific antibodies and subsequent magnetic (MACS) or fluorescence-based (FACS) selection are provided.
• FACS measurements Fluorescent Activated Cell Sorting
• Western blots gel filtrations or cytospins are possible.
• RNAse protection assays for example, RNAse protection assays and Northern and Southern blots.
• apoptosis assays are provided, such as the staining of the cells with annexin V or the so-called tunel assay or cell cycle analyzes, for example by Propidiumjodid-dyeings.
• peripheral blood mononuclear cells were obtained from whole blood or so-called "buffy coat.” This is a by-product which is produced from whole blood during the production of erythrocyte concentrates.
• Ficoll is an uncharged sucrose polymer whose density is adjusted so that when layered with whole blood or buffy coat followed by centrifugation, the lower density portions pass through the ficoll layer and collect at the bottom, while lymphocytes and monocytes intercalate in the interphase Collect plasma (top) and ficoll (bottom).
• the interphase containing the cells after centrifugation was isolated and washed several times with PBS. Subsequently, the isolated cells in cell culture absorbed and adjusted to a concentration of 1 - 4 x 10 6 cells per milliliter.
• Double-stranded immunomodulatory oligodeoxynucleotides dSLIM
• Double-stranded immunomodulatory oligodeoxynucleotides are molecules with CpG sequences.
• linear oligodeoxynucleotides ODN
• ODN linear oligodeoxynucleotides
• dumbbell-shaped molecules dumbbell-shaped molecules, called the dSLIM, "double stem-loop immunomodulator.”
• the immunomodulatory effect is based on non-specific activation of the immune system by the unmethylated CpG sequences that bind to Toll-like receptors, and especially the special Structure of the dSLIM molecules
• Each loop of the dSLIM contains three unmethylated CpG motifs.
• Double stranded immunomodulators dSLIM of the ISS30 type (e.g., dSLIM-30L1) were prepared after SOP and final quality control in the class B laboratory.
• dSLIM-30L1 Double stranded immunomodulators dSLIM of the ISS30 type (e.g., dSLIM-30L1) were prepared after SOP and final quality control in the class B laboratory.
• ODN Oligode- soxyribonukleotide
• T4 DNA ligase T4 DNA ligase.
• the resulting dSLIM molecules were concentrated by ethanol and sodium magnesium acetate precipitation and dissolved in PBS. The exact method is shown in WO 01/07055.
• the isolated cells were seeded in multi-well-plates.
• the size of the batches and, accordingly, the size of the wells was chosen so that the culture supernatant harvested later had exactly the volume needed for the secondary incubation with the target cells.
• Target cells e.g., HT-29
• the optimum concentration and the volume had to be determined beforehand in which they were sown.
• the aim was that after the secondary incubation at least 5 x 10 5 target cells per well are present for the analysis. Care was taken to ensure that the cells had optimal growth conditions for 3 days, were as dense as necessary and seeded as thinly as possible, so that after 3 days they are almost confluent. Non-optimal growth conditions also lead to necrosis or apoptosis, which would lead to a falsification of the test result.
• HT-29 co-ion carcinoma cells were used as target cells.
• the cells were seeded in the previously determined optimal density in correspondingly large batches and incubated overnight at 37 degrees Celsius in a CO 2 incubator (eg 2.4 ⁇ 10 5 cells in 700 ⁇ l per well in 24 well-plate).
• the approaches - direct stimulation and indirect stimulation - were again incubated for 48 hours at 37 degrees Celsius in the CO 2 incubator. Subsequently, the respective desired analysis could be carried out with the cells.
• the supernatants were removed from the cells and the cells were washed with PBS. The cells were removed from the wells by means of trypsin / EDTA and, after a further washing step, transferred to a centrifugation tube, in order subsequently to determine the cell number. Staining of surface antigens
• the cells from the stimulation batches were centrifuged off and washed with a special staining buffer. Subsequently, the cell suspension was adjusted to a concentration of 1 ⁇ 10 6 cells per milliliter. 500 ⁇ i (0.5 ⁇ 10 6 cells) of this cell suspension was centrifuged off in a FACS tube and, after uptake in 50 ⁇ l of staining buffer, the antibodies (eg ICAM-1 (CD54) conjugated with FITC and HLA-ABC conjugated with PE) were added. For each antibody, an appropriate isotype control was included as well as a single stained positive sample for device adjustment and compensation. After an incubation step, the cells were washed twice with PBS and resuspended for measurement in 500-1000 ⁇ l of PBS. For the discrimination of dead cells, 7-AAD was added and incubated for a further 10 minutes. Subsequently, the FACS measurement was carried out.
• the antibodies eg ICAM-1 (CD54) conjugated with FITC and HLA
• Apoptotic cells were stained with annexin V-PE, which detects apoptotic processes in the cell. To distinguish from necrotic cells, a counterstaining with 7-AAD was performed.
• the cells from the stimulation batches were centrifuged off and washed twice with PBS. Subsequently, the cells were diluted in a special annexin binding buffer and a cell concentration of 1 ⁇ 10 6 cells per milliliter was set. Per 100 .mu.l (1 ⁇ 10 5 cells) of this cell suspension were mixed with 5 .mu.l of annexin V-PE and 7-AAD and incubated after good mixing for 15 min at room temperature. Subsequently, 400 .mu.l binding buffer were added and the measurement was carried out immediately in the FACS.
• Fluorescence 2 (annexin V-PE) and fluorescence 3 (7-AAD) were measured.
• the device setting was done with unstimulated cells (direct approaches) or untreated cells (indirect approaches).
• the cell population was set to be in the middle. Subsequently, the PMT settings and compensation for fluorescence 2 and 3 were performed. Thereafter, all samples were measured (5000 cells).
• the device setting was carried out with lin-30L1 stimulated cells with appropriate isotype controls (in double staining) for the adjustment of non-specific binding and with the fluorescence-labeled antibodies (in single stains).
• the cell population was set to be in the middle. Subsequently, PMT settings were performed for fluorescence 1, 2 and 3 with the isotype controls, as well as the compensation with single staining. Thereafter, all samples were measured (10000 cells). The dead cells (7 AAD-positive cells) were excluded (fluorescence 3 versus FSC in the dot blot).
• a dot blot 7-AAD versus annexin V is formed. Then quadrants are set based on untreated cells. Depending on their position in the respective quadrant, the cells belong to the apoptotic or necrotic fraction.
• Living cells are annexin negative and 7AAD negative
• Apoptotic cells are annexin-positive and 7AAD-negative
• Necrotic cells are annexin-positive and 7AAD-positive
• FIG. 1 Schematic representation of the method according to the invention
• Fig. 6 Analysis of the mechanism of action of dSLIM by detecting apoptosis and necrosis in HT-29 tumor cells using the method of the invention.
• Fig. 7 Analysis of the mechanism of action of dSLIM by detecting the expression of HLA-ABC surface markers in HT-29 tumor cells using the method of the invention.
• Fig. 12 In vitro tracking of viable tumor cells during therapy of a cancer patient
• FIG. 13 In vitro tracking of apoptotic / necrotic tumor cells during the therapy of a cancer patient
• Figure 14 In vitro follow-up of surface markers of tumor cells during therapy of a cancer patient
• FIG. 1 shows a schematic representation of the sequence of the method steps in the method according to the invention.
• FIG. 2 shows the results of an analysis of the in vitro action of dSLIM immunomodulator using the method according to the invention.
• Use of the supernatant from dSLIM-incubated PBMCs induces apoptosis and necrosis in HT-29 tumor cells (colon carcinoma), as in the right part of the figure You can see.
• HT-29 tumor cells colon carcinoma
• FIG. 4 shows that dSLIM induces apoptosis (annexin V) and necrosis (7-AAD).
• apoptosis annexin V
• necrosis 7-AAD
• FIG. 5 shows the enhanced induction of the HLA-ABC surface markers by the incubation of the target cells (HT-29) with the dSLIM supernatant of the PBMC.
• Figure 6 shows the results of an analysis of the mechanism of action of dSLIM in HT-29 cells using the method of the invention and the detection of apoptosis and necrosis.
• an antibody anti-IFN- ⁇ , green frame
• experiments were performed with antibodies (anti-IFN- ⁇ , anti-TNF ⁇ ) to prove the specificity. It is easy to see (green frame) that the number of apoptotic as well as necrotic cells is minimized by the anti-IFN- ⁇ antibody.
• FIG. 7 the application of the method according to the invention corresponds to that of FIG. 6, but the expression of the surface marker ICAM-1 (CD54) was analyzed in the target cells (HT-29). In the lower part of the figure is shown comparatively the shift of the cell population.
• Figures 8 and 9 show results of experiments using the method of the invention in Renca tumor cells, comparing the effect of dSLIM with linear ODNs.
• the linear CpG-containing oligodeoxynucleotides also have a different sequence than the dSLIM and are protected against degradation by means of phosphorothioate.
• Figure 8 shows that treatment of the target cells with dSLIM results in enhanced expression of the surface marker HLA-ABC (upper part), whereas a linear CpG ODN has no effect.
• the table in the right part of the figure compares the numerical differences.
• dSLIM is significantly more potent than linear CpG ODN.
• the lower part shows the difference in induction of apoptosis in percent.
• Figures 10 and 11 respectively compare dSLIM with linear CpG ODN using the method of the invention in HT-29 cells as target cells.
• the results of this experiment correspond to the results obtained with the Renca tumor cells and are shown in FIGS. 8 and 9.
• the structure of the figures is also according to the figures 8 and 9.
• Figures 12, 13 and 14 illustrate the use of the inventive method for in vitro tracking of the number of viable tumor cells ( Figure 12) and apoptotic / necrotic cells (Figure 13) as well as the change in expression of the surface markers ICAM-1 / HLA-ABC ( Figure 14) in the course of therapy of a cancer patient.
• the plasma was isolated and incubated with cells of the tumor cell line HT-29. Subsequently, the number of viable (FIG. 12) and apoptotic / necrotic cells was determined and the expression of the surface markers ICAM-1 / HLA-ABC was investigated.
• Figure 12 shows the results of incubation of HT-29 cells with plasma from eight blood samples. There is a clear decrease in viable HT-29 cells as early as the second day of administration of dSLIM. The number of viable cells decreases on the second day to less than half of the cells of the first day, which is comparable to the number of viable cells of the controls.
• FIG. 13 shows the tracking of apoptotic / necrotic tumor cells during the treatment of the cancer patient on days 1, 2, 5 and 20. It can be seen in this evaluation of the tracking of the in vivo effects that the number of apoptotic / necrotic cells increases significantly already one day after the administration of dSLIM.
• Figure 14 illustrates results of the studies on changing the expression of the surface markers ICAM-1 / HLA-ABC during the therapy of the cancer patient with reference to the plasma of the blood of samples 1, 2, 3 and 8.
• sample 1 is used as a reference value to show changes the expression of the two surface markers used.
• ICAM-1 is significantly more expressed on the second day of therapy, as shown in the lower part of the figure by the shift in the position of the fluorescence intensity, which shows that ICAM-1 is more strongly expressed.
• target tissue e.g. tumor
• test substance e.g. dSLIM
• immune cells e.g. PBMC
• test substance e.g. dSLIM
• target cells e.g. tumor cells

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• 2006
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