Method for producing BK-riv compounds for programming neutrophil granulocytes to the antigen-presenting phenotype, and medical use thereof

Abstract

The invention relates to a new method for in vitro production of BK-RiV-forte nanoparticles, which enables a higher yield of particulate compositions. The produced BK-RiV-forte nanoparticles differ in their composition from known BK-RiV particles. The BK-RiV-forte nanoparticles produced by the new method increase the diversity of the T cell repertoire and find a wide range of applications in medicine, for example in the treatment of septic diseases as well as wounds, tumours, strokes and inflammatory processes of all kinds.

Description

[0001] Methods for the production of BK-RiV preparations for programming neutrophils to the antigen-presenting phenotype and their medical use

[0002] The invention relates to a new method for producing BK-RiV nanoparticles, which enables a higher yield of particulate compositions. The produced BK-RiV nanoparticles (BK-RiV-forte) differ in their composition from known BK-RiV particles. The BK-RiV-forte nanoparticles produced by the new method increase the diversity of the T-cell repertoire and have numerous applications in medicine, for example, in the treatment of septic diseases.

[0003] Technological background

[0004] Neutrophils have important protective functions, for example, in fighting invading pathogens or in the event of injury. Neutrophils migrate from the peripheral blood to sites of infection or injury and are then activated by contact with pathogens and cellular debris, thereby initiating inflammatory responses. Thus, this protective role of neutrophils is simultaneously linked to tissue-damaging mechanisms such as degranulation, reactive oxygen species (ROS) release, and netosis. Under certain conditions, the induced activation of neutrophils can lead to uncontrolled inflammatory responses, causing severe tissue damage, life-threatening situations such as sepsis, or chronic inflammation, as observed, for example, in various autoimmune diseases.However, recent reports show that neutrophils possess a phenotypic and functional heterogeneity that extends far beyond their pro-inflammatory and tissue-damaging roles.

[0005] For example, neutrophils can also function as antigen-presenting cells. By presenting antigens, they can activate CD4 T cells and thus regulate adaptive immune responses.

[0006] The regulation of adaptive immune responses is of considerable importance. On the one hand, it allows for the highly specific control of infections with pathogens or tumor growth with immunological memory. On the other hand, proper regulation is crucial to prevent adaptive immune responses against self-antigens, as these could otherwise lead to autoimmune diseases. In all these cases, signals from neutrophils, cells of the innate immune system that are the first cells on the scene, could be decisive in influencing the direction and effectiveness of the adaptive immune response.

[0007] Among the most important phenotypic changes that distinguish a pro-inflammatory neutrophil from a regulatory, antigen-presenting neutrophil is the expression of the following surface markers: CD80, CD86, and MHCII. The factors that induce this antigen-presenting and regulatory neutrophil phenotype are not well understood. Given the crucial role of neutrophils in initiating and terminating inflammatory processes, there is an urgent need to establish methods for regulating neutrophil function.

[0008] Significant efforts are being made to specifically inhibit the pro-inflammatory and tissue-damaging effects of neutrophils. However, there are few therapeutic approaches to induce an antigen-presenting phenotype in neutrophils. To date, antigen-presenting neutrophils have been described during induced or naturally occurring inflammatory reactions or infections (e.g., glycogen-induced peritonitis or Fusobacterium nucleatum). It is assumed that the antigen-presenting function of neutrophils is induced by cytokines present in the surrounding microenvironment. The following molecules are used to induce antigen-presenting neutrophils in vitro: GM-CSF, IFNγ, TNFα, IL-3, and IL-4, usually in various combinations.

[0009] The problem is that the published data to date are characterized by high variability. The reasons for this are unknown. Possible causes include the short lifespan of cytokines, the lack of availability of the corresponding receptors, or the inconsistent expression of the surface marker MHCII and the costimulatory molecules CD80 and CD86. The underlying mechanism leading to antigen presentation is unknown. Furthermore, it is unclear which antigen is presented by neutrophils and how.

[0010] The protein complex BK-RiV (BioComplex Reaction Pattern in Vertebrate Cells) is known to represent a biomolecularly measurable response to cellular stress, which can be highly purified and isolated from vertebrate cells. This response can be triggered by various toxic influences on an organism and its cells. These include viral infections and the resulting immune system reactions with hyperimmunization against organs and their cells, as well as cancer and artificially induced stress situations in cell lines in the laboratory.

[0011] BK-RiV was discovered in the 1970s in East Germany during the frantic search for a vaccine against foot-and-mouth disease. It is a protein complex approximately 50 nm in diameter. Its more than 30 individual proteins (including heat shock proteins, annexins, ezrin, etc.) are attributed with various functions in regulating the immune system. Among other things, they support the cellular immune defense in its intensive fight against viral infections and cancer.

[0012] General description of the invention

[0013] A new method for producing BK-RiV nanoparticles has been discovered that does not stress the cells through cell lysis and results in higher protein concentrations. The BK-RiV nanoparticles produced using this method (BK-RiV-forte) also differ in size from previously known BK-RiV nanoparticles.

[0014] It was also found that the BK-RiV nanoparticles obtained using the new method enable new applications in medicine, particularly by increasing the diversity of the T-cell repertoire.

[0015] The invention therefore relates to a process for producing BK-RiV-forte nanoparticles from eukaryotic cell cultures, characterized in that a. cells or cell cultures are subjected to cell lysis by a freeze-thaw process and ultrasonic treatment; b. the cell lysate forms a sediment by ultracentrifugation; c. the sediment formed is resuspended and stabilized with carbohydrates; d. the suspension is purified of impurities; e. the BK-RiV-forte nanoparticles formed are isolated, sterile-filtered, and lyophilized.

[0016] Without committing to this theory, it is hypothesized that the release of cellular components during cell rupture leads to proteins and nucleic acids from previously separated cell compartments and organelles coming together in a now fused environment, and that aggregation and condensation into nanoparticles are induced by the enzymatic activity of proteases, pH changes, and altered ion concentrations. Detailed description of the invention: a) Production of the nanoparticles according to the invention BK-RiV-forte

[0017] The prior art describes the production of BK-RiV formed by the stress response of vertebrate cells (DE 10 2004 036079 A1). This means that, by definition, this BK-RiV is an endogenous cell organelle that arises naturally through a biological process (such as "stress granules"). The present invention, however, relates to nanoparticles that are formed during the lysis of eukaryotic cells in vitro. The product is called "BK-RiV-forte." A crucial difference from the previous BK-RiV is therefore the formation of the nanoparticles as a result of cell lysis. Due to the release of cellular components during cell rupture, proteins and nucleic acids from previously separated cell compartments and organelles come together in a now fused environment. Mechanisms such as the enzymatic activity of proteases, pH changes, and altered ion concentrations (e.g.,Calcium induces aggregation and condensation into nanoparticles. Components of the cytoskeleton form the basic framework for the nanoparticles. Subsequent ultracentrifugation allows the isolation of the nanoparticle-containing fraction. Further purification steps, such as DNA / RNA removal (e.g., by nuclease treatment), lipid removal (e.g., by solvent treatment with chloroform), and filtration, yield a homogeneous particle class with a median size of 100–200 nm, preferably 110–150 nm, and particularly preferably approximately 125 nm, whereas BK-RiV nanoparticles known from the prior art had a size between 9 and 50 nm. In principle, all eukaryotic cell lines, such as HEK293, HELA, and ER-NR1, can be used for the process according to the invention. The AGE1.CRpIX suspension cell line is particularly preferred.

[0018] Two crucial steps for increasing the particle count compared to prior art processes are the elimination of the initial clarification centrifugation and the separate removal of DNA and RNA. DNA represents a process contaminant that must be removed. In the process according to DE102004036079A1, the clarification centrifugation of the cell homogenate was intended to separate a large proportion of the cell nuclei and thus the majority of the DNA. However, this simultaneously reduced the number of nanoparticles in the final product. Furthermore, since the removal of the cell nuclei was incomplete, variable amounts of BK-RiV particle aggregates formed in the ultracentrifuge sediment due to DNA-histone binding. Because of their higher density, these aggregates migrated into the separation layer between the two phases during centrifugation for the two-phase separation (chloroform treatment), leading to further particle losses.

[0019] To increase the overall particle yield, the clarification centrifugation for removing cell nuclei is now omitted. Instead, in the new process, cell nuclei are intentionally destroyed and DNA is selectively released through more intense cell lysis (increased ultrasound intensity). This leads to an increased DNA concentration in the further course of the process, causing DNA to bind more strongly to proteins in the form of loose associations and inducing the formation of nanoparticles. Before solvent treatment, however, the DNA is almost completely removed by enzymatic treatment with a nuclease. This significantly reduces the particle losses that originally occurred during solvent treatment in the process of the present invention. The increase in protein content and particle number due to both changes exceeds 100%. b) Medical use of the BK-RiV nanoparticles according to the invention

[0020] Severe infections, poor vaccination outcomes, cancer, and autoimmune diseases can be signs of a weakened immune system. The cells that largely define a person's immune status are T lymphocytes, as they must be able to distinguish between foreign and self antigens. T cells should react very strongly to foreign antigens, or in the case of cancer, to neoantigens, while they should show only weak reactions to self antigens. A T cell recognizes foreign or self antigens via T-cell receptors (antigen receptors). Each T cell has its own specific antigen receptor. The number of different antigen receptors for the entire T-lymphocyte population of a person is called the T-cell repertoire (TCR). Recent research shows that the diversity of the TCR plays a crucial role in the aforementioned immune system disorders.The more diverse the TCR is, i.e., the more different antigen receptors are present in a person's repertoire, the better the immune status of a person, since this allows the reactions to foreign and self-antigens to be adjusted correctly as needed.

[0021] The diversity of the triglyceride core (TCR) changes throughout life. The TCR is primarily formed in the thymus during childhood. This results in the highest TCR diversity in young adults. As the thymus generates fewer new T cells with age, and DNA damage and cellular senescence occur, TCR diversity continuously declines. The resulting gaps in the TCR are filled by homeostatic cell division. This process primarily proliferates T cells that are already abundant. All T cells derived from a single T cell are called T-cell clones. Homeostatic cell division leads to a shift in the clonal distribution of T cells from youth to old age. In youth, a person has many clones that occur at a low frequency, while in old age, there are fewer clones, but they occur at a high frequency.The diversity of the triticum (TCR) decreases particularly sharply during severe acute (e.g., sepsis) and chronic (e.g., autoimmune diseases and chronic inflammation) events. In these cases, it is therapeutically important to increase TCR diversity in order to compete with and suppress pathogenic and excessive T-cell clones. To counteract a decrease in TCR diversity and thus immune system exhaustion, it is crucial to stimulate high TCR diversity.

[0022] Injections of foreign antigens with and without enhancing adjuvants, open mRNA vaccines, and vaccinations modulate the triatomine reserve (TCR). The T-cell responses with which T cells react to foreign antigens / vaccines are always very intense. The increase in TCR diversity is usually only temporary, occurring until the foreign antigen or vaccine is eliminated. To alter TCR diversity long-term, the antigens / vaccines would have to be injected repeatedly. However, repeated injections are problematic because, after a strong T- and B-cell response, a few specific memory cells (T- and B-cell clones) persist. Upon re-injection, these cells produce specific, usually high-affinity antibodies that cause inflammatory side effects and immediately eliminate the re-injected antigen (drug-adverse effect, allergy). Consequently, these antigens no longer influence TCR diversity.

[0023] Therefore, there is a need for new medical treatment options to increase or maintain the diversity of the TZR in order to overcome the disadvantages of the known options.

[0024] It has been shown that the nanoparticles according to the invention (BK-RiV-forte) increase the diversity of the triglyceride core (TCR) without inducing a complete T- and B-cell response. While not committing to this theory, it is hypothesized that BK-RiV-forte, as a particulate structure consisting primarily of intracellular proteins, is preferentially taken up by antigen-presenting cells (APCs) and presented to T cells without costimulation. This type of "silent" presentation occurs continuously in homeostasis. Because BK-RiV consists of a mixture of intracellular proteins, activation with allergy development or autoimmunity does not occur despite ongoing T-cell reactions, as only low-affinity T-cell clones can be stimulated (clonal selection in childhood). Homeostatic proliferation is thus expanded to include all these epitopes, thereby increasing the diversity of the TCR.The nanoparticles according to the invention (BK-RiV-forte) can be injected preventively in a healthy state, concurrently with infections, or during chronic inflammation and autoimmune diseases, thus diversifying and expanding the TCR (transient inflammatory response). In addition to the treatment of sepsis, their potential applications in the treatment of wounds (including diabetic foot ulcers), tumors, strokes, and inflammatory processes of all kinds are particularly noteworthy.

[0025] The advantages of using BK-RiV-forte nanoparticles are:

[0026] 1. BK-RiV-forte nanoparticles are produced in vitro from intracellular contents of eukaryotes.

[0027] 2. BK-RiV-forte nanoparticles contain many intracellular proteins and therefore many antigens with countless epitopes.

[0028] 3. Intracellular proteins are only subliminally recognized by T cells because they bind to the TCR with low affinity and therefore do not induce T and B cell activation. Due to the high homology of intracellular proteins across many species, BK-RiV-forte nanoparticles can be produced from autologous or exogenous cells.

[0029] 4. BK-RiV-forte nanoparticles are particulate and are recognized and absorbed very quickly by APCs, thus leading to the presentation of many different antigens.

[0030] 5. BK-RiV-forte nanoparticles can be injected during infections or autoimmune diseases and are therapeutically effective.

[0031] 6. BK-RiV-forte nanoparticles can be used advantageously for the treatment of sepsis.

[0032] 7. BK-RiV-forte nanoparticles can be used in a healthy state to expand the TZR (anti-aging).

[0033] Brief description of the characters

[0034] The invention is explained in more detail with reference to the following exemplary embodiments and accompanying figures. The figures show:

[0035] Fig. 1 Schematic sequence of the manufacturing process according to the invention in comparison to the prior art process (DE 10 2004 036079 A1) and comparison of the end products

[0036] Fig. 2 Demonstrate the increase in T-tell repertoire diversity through comparative treatment of mice with autoimmune skin pathology. Two groups of mice are shown, one treated with BK-RiV-forte and the other without. The T-cell receptor repertoire (TCR repertoire) was determined and compared between animals within each group. White fields indicate similar, black fields dissimilar TCR sequences. The numbers indicate the number of matching TCR sequences.

[0037] (Note: The samples M1_BK-RiV to M6_BK-RiV shown in the figure are preparations that have been treated with the BK-RiV-forte according to the invention.)

[0038] Fig. 3: Exemplary result of the particle size analysis of a batch of BK-RiV-forte nanoparticles, produced according to the manufacturing example described below. The particle suspension was analyzed using the NanoSight LM20 instrument via nanoparticle tracking analysis (NTA). In this process, the samples are irradiated with a laser, and the scattered light is detected using a microscope. The particle size is calculated based on Brownian motion, and the particle count is automatically recorded.

[0039] Process flow for BK-RiV-forte nanoparticles (overview):

[0040] The starting material for the active ingredient is, for example, a (eukaryotic) AGEl .CRpIX suspension cell line. The cell bulk is produced via cell culture and processed further without the culture supernatant. A freeze-thaw process and ultrasound rupture the cells and form the particles. A nanoparticulate fraction is then obtained by ultracentrifugation. The resulting material is resuspended in a phosphate-buffered saline solution with Ca / Mg. DNA and RNA impurities are removed by incubation with a nucleic acid-digesting enzyme (e.g., benzonase, DNase I, micrococcal nuclease (MNase), S1 nuclease, preferably benzonase). Subsequent heat treatment at 60°C for 10 h serves as an inactivation step for non-enveloped viruses.The subsequent treatment with an organic solvent (10% v / v chloroform, dichloromethane, acetone, diethyl ether, preferably chloroform) by shaking is an inactivation step for enveloped viruses and simultaneously serves to remove lipids. After centrifugation, the upper aqueous phase (bulk product) is isolated. After sterile filtration, the bulk product is lyophilized.

[0041] The resulting nanoparticles have a median size of, for example, 130 nm. They consist of approximately 20 main proteins, which make up 90% of the protein content. The nanoparticles are associated with traces of RNA and DNA. The final product has a concentration of at least 10 11 Particles per ml.

[0042] Exemplary manufacturing process for BK-RiV-forte nanoparticles

[0043] The starting material for the active ingredient is the AGEl.CRpIX suspension cell line (developed by ProBioGen for pharmaceutical manufacturing). Incubation takes place at 37°C (+ / - 0.5°C) and an 8% (+ / - 0.5%) CO2 atmosphere. The culture medium (specifically developed by ProBioGen for this cell) is CDU7 medium with the addition of IGF1 and glutamine in standardized concentrations. The fill volumes and shaking frequencies for cell culture in Erlenmeyer flasks are as follows: 125 ml culture flask with 50 ml medium at 135 rpm, 500 ml culture flask with 160 ml medium at 120 rpm, 2000 ml culture flask with 600 ml medium at 105 rpm. Cell culture is carried out until a cell density of 5-6 x 10⁻⁶ is reached. 6 Cells / ml. 1.8 liters of cell bulk are prepared per batch. The cells are centrifuged in a swing-out rotor at 300 g for 8 minutes. The culture supernatant is discarded, and the cell sediments are transferred to a total of 600 ml of PBS with Ca 2+ / Mg 2+(Potassium chloride 0.22 g / l, sodium phosphate 2.39 g / l, potassium phosphate 0.22 g / l, sodium chloride 8.88 g / l, magnesium chloride 0.10 g / l, calcium chloride 0.13 g / l) absorbed.

[0044] The cells are ruptured by a freeze-thaw process (-25°C to room temperature) and ultrasound (device: Sonopuls HD5050, sonotrode: TS104, vessel: 150 ml Corning® centrifuge beaker, volume: 100 ml, power: 80%, time: 45 seconds). Following ultracentrifugation (3 x 200 ml, 54,000 g, 180 min, 4°C), the three resulting sediments are then mixed in 200 ml of PBS (1:1 mixture with / without calcium). 2+ / Mg 2+The sample is resuspended, then 10% v / v D-sucrose is added and homogenized by ultrasound (as above, but time: 15 seconds). DNA and RNA impurities are removed by incubation with the nucleic acid-digesting enzyme Benzonase® (8500 units, 8 h, 20°C). Subsequent heat treatment at 60°C for 10 h serves as an inactivation step for non-enveloped viruses. The following treatment with an organic solvent (10% v / v chloroform) by shaking (250 rpm, 5 min) is an inactivation step for enveloped viruses and simultaneously serves to remove lipids. After clarification centrifugation in a swing-out rotor (7500 g, 15 min, 4°C), the upper aqueous phase (approx. 200 ml bulk product) is isolated. The bulk product is then lyophilized into 40 x 5 ml vials in 10 ml vials until a residual moisture content of < 5% is reached. After resuspension of the lyophilisate in WFI, a final sterile filtration (0.22 pm PES filter) is performed. Store at +2 to +8°C. Stability: 12 months.The final product has a concentration of at least 10. 11 Particles per ml.

[0045] 60% of the nanoparticles consist of 50 proteins, each contributing more than 0.5% to the total protein content. Qualitative analysis of a batch:

[0046] Protein content: 75 pg / ml

[0047] Number of nanoparticles & median size: 2.4 x 10 11 Particles / ml 125 nm (Figure 1)

[0048] Protein and mass spectrometry data: (Appendix)

[0049] Protein percentage

[0050] Heat shock protein family A (Hsp70) member 2 5.84200553

[0051] Peptidyl-prolyl cis-trans isomerase 5.00003554

[0052] Beta-actin 3,13090321

[0053] ATP synthase subunit alpha 3.05977862

[0054] Annexin A11 2,69918243

[0055] RAB7A, member RAS oncogene family 2.25980451

[0056] Ferritin 2.09395236

[0057] Nucleolin 1 ,95186105

[0058] Single stranded DNA binding protein 1 1 ,43180683

[0059] Annexin A2 1 ,42036059

[0060] Pyruvate kinase 1 ,36202421

[0061] Helix-destabilizing protein 1 ,35839298

[0062] H(+)-transporting two-sector ATPase 1 ,32500152

[0063] Heterogeneous nuclear ribonucleoprotein A2 / B1 1 ,30945042

[0064] Small nuclear ribonucleoprotein Sm D3 1 ,28363716 ATP synthase subunit gamma 1 ,10026043

[0065] Heterogeneous nuclear ribonucleoprotein A3 1 ,06426396 L-lactate dehydrogenase 1 ,05700151

[0066] Small RNA binding exonuclease protection factor La 1 ,05384393 60S ribosomal protein L12 1 ,04366072 phosphopyruvate hydratase 0,96795762

[0067] Annexin A7 0,94427574 Heat shock protein family A (Hsp70) member 8 0,92114643 60S ribosomal protein L10a 0,91648899

[0068] Small nuclear ribonucleoprotein E 0,90662154 Polyadenylate-binding protein 0,86959886 Calreticulin 0,83226042

[0069] Heterogeneous nuclear ribonucleoprotein A / B 0,83154996

[0070] Ubiquitin-40S ribosomal protein S27a 0,78986984

[0071] Peroxi redoxin 1 0,77055331

[0072] Malate dehydrogenase 0,735141

[0073] Peptidase M24 domain-containing protein 0,73476209

[0074] RAB1 A, member RAS oncogene family 0,73432003

[0075] Peroxi redoxin 3 0,68497487

[0076] Peptidyl-prolyl cis-trans isomerase 0,6803648

[0077] Proteasome subunit alpha type 0,67456274

[0078] 40S ribosomal protein SA RPSA 0,66167979

[0079] 60S acidic ribosomal protein PO 0,62704108

[0080] Major vault protein 0,59800709

[0081] Heterogeneous nuclear ribonucleoprotein R 0,58224285

[0082] Splicing factor, arginine / serine-rich 1 0.57939313

[0083] Moesin 0.5730227

[0084] RAP1 B, member of RAS oncogene family 0.5628316

[0085] VRK serine / threonine kinase 1 0.56011607

[0086] Proteasome subunit alpha type 0.55464556

[0087] GTP-binding nuclear protein Ran 0.55221422

[0088] Adenylyl cyclase-associated protein 0.54318353

[0089] Stomatin 0.53636314

[0090] Heterogeneous nuclear ribonucleoprotein D like 0.5263536 Proteasome subunit alpha type 0.50356373 Table 1 Comparison of the manufacturing process according to the invention with the prior art process (DE 10 2004 036079 A1) and comparison of the end products

[0091] In-vitro studies

[0092] A culture of mouse neutrophils isolated from bone marrow, treated with nanoparticles according to the invention (BK-RiV-forte), showed the following findings: BK-RiV-forte was found to be taken up by 20% of the neutrophils.

[0093] 1. Neutrophils that have ingested BK-RiV-forte develop an antigen-presenting phenotype: a. The expression of CD80 is accelerated and increased.

[0094] 2. Neutrophils that have taken up BK-RiV-forte alter the expression of cell adhesion molecules and their migration behavior: a. Reduction in the expression of lipopolysaccharide (LPS)-induced CD11 β (integrin) b. Reduction in the expression of CD54 (ICAM) after stimulation with LPS c. Neutrophils are not induced to migrate by CXCL12

[0095] 3. The following additional parameters were found to characterize the altered neutrophil phenotype after BK-RiV-forte treatment: a. BK-RiV-forte activates neutrophils very rapidly but only temporarily (expression of CDHβ, 45', 24 h) b. BK-RiV-forte has no effect on the survival rate of neutrophils c. BK-RiV-forte slightly reduces activation by LPS (24 h) d. BK-RiV-forte does not induce tissue-damaging effector functions in neutrophils (phagocytosis, ROS release, netosis) 45', 24 h e. BK-RiV-forte has no effect on LPS-stimulated phagocytosis (24 h). BK-RiV-forte accelerates and increases the expression of CD80 (costimulatory molecule for antigen presentation) (45', 24 h). BK-RiV-forte increases the proportion of apoptotic neutrophils after induction of cell death by phorbol-12-myristate-13-acetate (PMA).

[0096] Exemplary increase in the diversity of the T-cell repertoire

[0097] In C57BI6 mice, autoantibodies were injected that induce infiltration of inflammatory cells by binding to type 7 collagen in the skin. The resulting skin wounds become visible after approximately 5 days and cover 15% of the total skin surface after about 2 weeks. The wounds then heal over a period of 2-3 weeks. BK-RiV-forte was injected 3 times per week from day 16 until the end of the experiment (5 weeks). Lymph nodes were removed and the T-cell repertoire (TCR) was determined. The results of the analysis are shown in Figure 2. It demonstrates that the application of BK-RiV-forte nanoparticles resulted in a significantly higher diversity of the T-cell repertoire (TCR) than comparable treatment with PBS (phosphate-buffered saline). The numbers in the boxes represent the number of identical (overlapping) T-cell clones between the mice. The lighter the color, the more overlaps; the darker the color, the fewer overlaps.

Claims

Patent claims 1. A method for the in vitro production of BK-RiV-forte (BioComplex reaction pattern in vertebrate cells) nanoparticles from eukaryotic cell cultures, characterized in that a. cells or cell cultures are subjected to cell lysis by a freeze-thaw process and ultrasonic treatment; b. the cell lysate forms a sediment by ultracentrifugation; c. the formed sediment is resuspended and stabilized with carbohydrates; d. the suspension is purified of impurities; e. the formed BK-RiV-forte nanoparticles are isolated, sterile filtered, and lyophilized.

2. Method according to claim 1, characterized in that the AGE1 .CRpIX suspension cell line is used as the eukaryotic cell line.

3. Method according to claim 1, characterized in that sucrose is used as the carbohydrate for stabilization.

4. Method according to claim 1, characterized in that a nucleic acid digesting enzyme is used to remove DNA and RNA impurities.

5. The method according to claim 4, characterized in that benzoase is used to remove DNA and RNA impurities.

6. Method according to claim 1, characterized in that heat treatment and / or treatment with an organic solvent is carried out to remove viral contaminants.

7. Method according to claim 1, characterized in that the BK-RiV-forte nanoparticles have a median particle size of 100-200 nm, preferably 110-150 nm, particularly preferably approximately 125 nm.

8. Use of a preparation containing BK-RiV-forte nanoparticles in purified form, according to any one of claims 1 to 7, for the manufacture of a medicinal product in in human or veterinary medicine for diagnosis, therapy, metaphylaxis and / or prophylaxis 9. Use of a preparation according to claim 8 for the manufacture of a medicament for increasing the diversity of the T-tell repertoire.

10. Use of a preparation according to claim 8 for the manufacture of a Drug for the treatment of sepsis.

11. Use of a preparation according to claim 8 for the manufacture of a medicament for the treatment of wounds, tumors, strokes and inflammatory processes of any kind.

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