CZ29538U1 - Kit for the preparation of antitumor vaccine, antitumor vaccine per se inclusive of efficiency intensification - Google Patents

Description

Technical field

The present invention relates to enhancing the anti-tumor effect of an anti-tumor vaccine containing dendritic cells (DB) using immunomodulatory agents and a kit for preparing such an anti-tumor vaccine.

BACKGROUND OF THE INVENTION

Dendritic cells (DB) are cells of the immune system that form the link between native and adaptive immune mechanisms. They are found primarily in the skin, mucous membranes and, to a small extent, in other tissues and blood. Their main function is the processing and presentation of antigens (foreign material) so that a targeted and effective immune response is triggered if the antigen is recognized as dangerous by DB. DB develops from hematopoietic precursors in the bone marrow into myeloid or lymphoid DB. Under physiological circumstances, the body is mainly the so-called "immature" DB, maintaining the state of tolerance of the immune system, especially to its own proteins, molecules and cells. Immature DBs have a very good ability of tagocytosis to absorb solid particles, including foreign antigens. If such an antigen is recognized as dangerous, the DB will gradually mature and travel from the tissue to the lymph node. DB maturation consists in inhibition of phagocytosis of new particles and the expression of HLA (human leukocyte antigen) molecules I. and II. class, which are necessary for targeted and specific activation of T lymphocytes in the lymph node. T lymphocytes are a powerful component of the immune system, which is able to produce the necessary cytokines (helper Th lymphocytes) and cytotoxic potential against target antigens (cytotoxic Tc lymphocytes) based on information transmitted from mature DBs. Helper Th lymphocytes can be divided into Th1 and Th2. Th1 lymphocytes have the ability to produce a spectrum of cytokines (particularly interferon gamma) that activate Tc lymphocytes and further increase their cytotoxic potential, which can then advantageously be used in anti-tumor immunotherapy. Th2 lymphocytes are responsible for stimulation of B lymphocytes and production of antibodies; they are not significantly involved in anti-tumor immunity. For activation of Th1 lymphocytes, IL-12 interleukin family cytokines that produce some mature DBs are important. DB1, which produce IL-12 and polarize the immune response by activating Th1 lymphocytes, is currently considered important for anti-tumor immunotherapy.

Thus, DBs play a central role in the initiation of an immune response that is mediated by T cells. Thus, DBs function as professional antigen-presenting cells, which are capable of targeted presentation of antigens (including tumor cells) and effectively stimulate an immune response directed against a given antigen. It is possible to use these cells to treat cancer. After in vitro vaccination of a DB patient, activation of the patient's own immune system, particularly helper and cytotoxic T lymphocytes of the patient in vivo, can be utilized. With increasing knowledge in the field of DB immunobiology, procedures for its preparation are being developed to achieve optimal properties, which are currently considered particularly immunophenotype, IL-12 cytokine production and the ability to stimulate both intrinsic and allogeneic T lymphocytes.

DB precursors are normally obtained from peripheral blood by leukapheresis. Leukapheresis is the aspiration of a white blood cell fraction from whole blood, with the red blood cells and blood plasma being returned to the patient's bloodstream. Monocytes that have the ability to adhere to surfaces are then isolated from these white blood cells. Monocytes represent the starting material - precursor DB.

Clinical studies are currently underway that show the benefit of differently prepared DBs in some selected cancers (prostate cancer, melanoma, glioblastoma and others).

In the treatment of prostate cancer, the efficacy of a single tumor antigen-loaded DB vaccine has been demonstrated in a Phase III clinical study, and the Sipuleucel-T vaccine has been authorized in the US for the treatment of hormone refractory prostate cancer (Kantoff, et al. , 363: 479-81. 512 patients participated in the study, of which 341 patients received the vaccine and 171 patients received placebo. The study demonstrated a median survival of 4.1 months (from approximately 22 to 26 months) using Sipuleucel-T over placebo. Acts

So far, this is the only clinical study that has led a DB-based product to its marketing authorization by the US FDA. The disadvantage of such a procedure, however, may be the use of only a single tumor antigen as opposed to a tumor tissue lysate that contains the complete spectrum of tumor antigens, and thus the anti-tumor response may be substantially broader and more effective. An example of this is a clinical trial using DB vaccination in glioblastoma patients with standard median survival of 12-14 months, with autologous DB vaccine loaded with autologous glioblastoma tumor cell lysate in combination with standard treatment, up to 24 months median survival (Ardon H, et al. J Neurooncol 2010, 99: 261-72). The disadvantage of this study is the fact that the tumor lysate was obtained only from the patient himself and thus only patients able to undergo neurosurgical surgery could participate in this treatment. Patients who were unable to undergo surgery to obtain tumor antigens were thus excluded from this treatment. Another disadvantage was the use of DB without demonstrable IL-12 production, which could affect vaccination efficiency. Finally, in the treatment of metastatic melanoma, very good results were obtained in a phase II clinical study using a DB-based vaccine loaded with tumor antigens from an autologous tumor lysate (Dillman RO, et al. Cancer Biother Radiopharmaceutical 2009, 24: 311-9). When the vaccination was performed only by tumor line, the median survival was 31 months, while using a DB-based lysate vaccine from the autologous tumor line, the median survival was significantly prolonged to 64 months with a five-year survival of 54% of the patients treated. The disadvantage of this study is again the autologous approach, since not all patients can obtain and grow their own tumor line for the use of their own antigens, as well as administration of DB without demonstrable IL-12 production. A DB-based vaccine that produces a sufficient amount of IL-12 has also been described (Dohnal A, et al. Cytotherapy. 2007, 9: 755-70). This vaccine utilizes standard culture of immature DB with GM-CSF and IL-4 in AIM-V medium and maturation with LPS and IFNγ. However, the vaccine has been used in combination with an autologous tumor lysate, which greatly limits its spread and cannot be used in patients whose own tumor tissue is not available. The results of this Phase I study in pediatric cancer patients have shown good tolerability and induction of an immune response in severely pretreated patients.

The effect of an anti-tumor vaccine can be enhanced by the use of immunomodulatory agents that suppress the effect of tolerogenic T lymphocytes, which attenuate the anti-tumor response, and in turn stimulate the effect of anti-tumor cytotoxic T lymphocytes that are stimulated by DB. These include cyclotosphamide at a low, so-called 50 mg, metronomic dose, taken daily for 2-4 weeks prior to the start of DB vaccination and for 3-12 months during vaccination (Ferrandina G, et al. BMC Cancer. 2014, 14: 947, Perroud HA, et al., Future Oncol. 2013, 9: 451-62). To boost non-specific immunity, including NK cells, it is also possible to combine metronomic doses of cyclophosphamide with betaglucan 300 mg daily for 14 days prior to DB vaccination followed by 100 mg daily for DB vaccination (Hamack U, et al. Anticancer Res. 2011 , 31: 1169-72; Masuda Y, et al. J Leukoc Biol. 2015, 98: 1015-25; Tian J, et al. Eur J Immunol. 2013, 43: 1220-30).

The present invention aims to provide a kit for the preparation of an anti-tumor vaccine and a resultant anti-tumor vaccine combined with immunomodulation resulting in preferential activation of cytotoxic T lymphocytes, suppression of tolerogenic T lymphocytes and wider use, i.e. a autologous tumor lysate independent vaccine.

The essence of the technical solution

The present invention provides a kit for the preparation of an anti-tumor vaccine based on dendritic cells from blood monocytes comprising the cytokines recombinant human interleukin IL-4 and recombinant human granulocytes and colony monocytes stimulating GM-CSF, lipopolysaccharide (LPS) and interferon gamma (IFN-) γ), medium, and at least two different tumor lysates from the same tumor type.

Preferably, the DB medium is CellGro, which is also suitable for clinical use. This medium is commercially available and has a standardized composition.

In a preferred embodiment, the tumor lysates are a combination of at least one tumor lysate obtained from the patient and at least one tumor lysate from the tumor line.

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In another preferred embodiment, the tumor lysates are a combination of lysates of at least two tumor lines of the same tumor type.

Preferably, DB vaccination is supplemented by the administration of 2 immunomodulatory agents cyclophosphamide and beta-glucans which enhance the anti-tumor effect of vaccination.

The preparation of the dendritic cell vaccine using the kit according to the present invention is carried out by culturing blood monocytes under standard conditions (in an incubator in a 5% CO ₂ atmosphere at 37 ° C) for 6 days in the presence of recombinant human IL interleukin cytokines -4 and recombinant human granulocytes and colony stimulating factor (GM-CSF) monocytes on immature dendritic cells that are further cultured in the presence of the recombinant human IL-4 and recombinant human GM-CSF cytokines and in the presence of at least two different tumor lysates of the same type tumor, followed by addition of lipopolysaccharide (LPS) and interferon γ (IFN-gamma) to mature dendritic cells. GM-CSF and IL-4 are standardly used for in vitro preparation of DB from peripheral blood monocytes. LPS and IFN-γ molecules will allow full maturation of DB and IL-12 production. Obtaining sufficient DB is ensured by leukapheresis, a standard procedure used to isolate white blood cells from peripheral blood. By using a combination of tumor antigens, which is not known in the art, the anti-tumor effect of the vaccine thus prepared is intensified. In addition, in a situation where an autologous tumor is not available, tumor lines or a combination of multiple tumor lines with the same tumor type as the patient's tumor can be utilized.

The tumor lysate is preferably prepared from tumor tissue by grinding and passing through a suitable sieve (e.g., 100 µm). Preferably, the tumor donor is subjected to tests for the presence of blood-borne diseases such as HIV, hepatitis and syphilis, and in the event of a positive test result, the tissue is not used.

Another object of the present invention is a dendritic cell-based anti-tumor vaccine comprising dendritic cells producing interleukin 12 (IL-12) and presenting antigens of at least two tumor lines of the same tumor type or of tumor cell antigens obtained from a patient and at least one tumor line from the same tumor type.

A dendritic cell vaccine can be used as adjunctive therapy for patients with glioblastoma, renal carcinoma, ovarian carcinoma, and melanoma when loaded with the appropriate tumor lysate from individual tumor types and tumor lines. It is intended for individual administration in cases indicated by the responsible physician or for testing in clinical studies in accordance with EC No. 1394/2007, Directive 2001/83 / EC and the Drug Act 378/2007.

Especially in the case of strictly indicated patients, the therapeutic effect is best achieved in a situation where partial or complete remission is induced by standard therapy, or when it is a minimal residual disease where the patient's immune system is not weakened enough to prevent its activation by a vaccine. The weakened immune system of the patient can be enhanced by administering immunomodulatory agents cyclophosphamide and beta-glucans. In these situations, the maximum effect of the vaccine can be expected.

Example of technical solution

Preparation of tumor lysates:

Reagents

- DPBS (Dulbecco's Phosphate Buffered Saline, 10x PBs), (Lonza. No .: BE17-515F)

- HBSS (Hanks Balanced Salt Solution) (Lonza, No .: BE10-547F or BE10-527F)

- Aqua injectable water for injection of ardeaphanna (Ardeapharma, No .: 76/926/95-C), sterile water for injection of Fresenius (Fresenius Kabi, No .: 87/405/97-C) or Aqua for injection (B. Braun) , No .: 87/02/98-C

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- KLH: Hemocyanin, Keyhole Limpet, Megathura crenulata, Glycerol (Calbiochem, No .: 374817)

- NaCl 0,9% Sodium chloride in water for injection (Fresenius Kabi, No.:76/365/96-C)

Tumor tissue collected from the donor or obtained from the culture dish (if it is a tumor line) is placed in a tube containing NaCl. The tube should be stored at 4 ° C and processed within 3 days of tumor surgery. A 120 Gy tube should be irradiated before processing the tumor sample. Furthermore, it is advisable to subject the donor (if the patient's tumor tissue) to blood-borne diseases (HIV, hepatitis B, hepatitis C, syphilis) prior to tumor processing;

Tumor tissue (if patient tumor) is transferred to a sterile Petri dish with 5-10 ml HBSS. Necrotic and connective tissue are removed with the help of scalpel and tweezers. Using a scalpel, cut the tumor into particles of about 0.5 mm in size and then crush the back end of the syringe. The obtained tumor cell suspension is repeatedly pushed through a syringe with a needle. The cell suspension is then passed through a nylon screen (100 µm) into a clean 50 ml tube. This gives a single cell suspension. The petri dish with the suspension residue is rinsed with HBSS and passed through a nylon screen again. The strainer is then flushed with the remaining HBSS. The resulting suspension is centrifuged at 1600 rpm, 7 min at 4 ° C. The supernatant is aspirated by pipette, and the cell pellet is resuspended in 2 ml of injection water. The suspension is dispensed into microtubes of approximately 1 ml. The tubes with the tumor cell suspension are frozen in liquid nitrogen (-196 ° C) until it is frozen - about 3 minutes - and thawed in a water bath (37 ° C) until liquid. This process is repeated 5 times to achieve lysis of tumor cells. The suspension is then centrifuged at 1600 rpm, 7 min at 4 ° C.

The supernatant from all microtubes is aspirated with a needle into one syringe so that the pellet of cells is not aspirated and the supernatant does not remain in the needle. The supernatant is syringe filtered through a 0.2 µm membrane filter into a sterile 50 ml tube.

DPBS (10xPBS) was added to the tumor lysate in a volume equivalent to 1/10 of the lysate volume. Protein concentration is determined spectrophotometrically.

Monocyte culture into vaccine:

Reagents • CellGro DC Medium (CellGenix, No .: 2007) • IL-4: Recombinant Human Interleukin 4 (CellGenix, GMP-grade, No .: 1003-050) • GM-CSF: Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor ( CellGenix, GMP-grade No .: 1012-050) • KLH: Hemocyanin, Keyhole Limpet, Megathura crenulata, Glycerol (Calbiochem, No .: 374817) • LPS: Lipopolysaccharide Endotoxin (US Pharmacopeia, No .: 1235503) • IFN- γ: interferon-γ, Imukin (Boehringer Ingelheim, No: 1-19480) • Accutase, 100 ml (PAA, No .: LI 1-007) • CryoStor CS2 / DLite cryopreservation solution (BioLife Solutions, No .: 650211 - 50 ml; 650212 100 ml) or CryoStor CS5 (BioLife Solutions, No .: 610202 - 100 ml) • HBSS, 500 ml (Lonza, No .: BE10-547F or BE10-527F).

Monocytes are separated from donor leukopheresis by elutriation (Elutra Cell Separation System, Gambro BCT) or magnetic separation (CliniMACS).

IL-4 was dissolved in CellGro to a final concentration of 32,000 U / ml. Fill 750 microns and 170 microns in microtubes. GM-CSF is dissolved in CellGro medium to a final concentration of 100,000 U / ml. Fill 750 microns and 170 microns in microtubes. KLH (supplied dissolved

-4389538 Ul in glycerol, concentration 22.7 mg / ml) - To 200 μΐ of the KLH solution in glycerol add 45.2 ml of HBSS. The final concentration is 100 pg / ml. Fill 500 μΐ into microtubes. CryoStor CS2 / DLite or CryoStor CS5 - with a sterile syringe with a needle, withdraw 30 ml and dispense about 1.5 ml into 20 microtubes.

Approximately 167x10 ⁶ monocytes are seeded into 70 ml medium into each culture flask. For about 167x10 ⁶ monocytes (1 culture flask) we will need 150 pg of tumor lysate. The following is a procedure for culturing 500x10 ⁶ monocytes. The monocyte suspension in the culture flasks is centrifuged at 1500/10 min / 22 ° C. Remove supernatant. The pellet is resuspended in a small amount of CellGro medium and made up to 35 ml with CellGro medium. Pour into flasks.

The remaining medium (35 ml per bottle) with cytokines is added to the culture flasks:

For 167x10 ⁶ monocytes in 70 ml medium (1 culture flask):

IL-4 (32,000 U / ml) 320 U / ml 700 μΐ

GM-CSF (100,000 U / ml) 1000 U / ml 700 μΐ

Mix by gently turning the bottle from side to side. The cells are cultured in a thermostat at

37 ° C, 5% CO ₂ for 3 days. The same amount of medium (70 ml) and cytokines (700 μΐ IL-4 and 700 μΐ GM-CSF) were then added to the cells as at the beginning of the culture. Cells are cultured in a thermostat at 37 ° C, 5% CO ₂ for an additional 3 days.

The resulting cell suspension of immature DB with medium is poured from each culture flask into three 50 ml tubes. 5 ml of fresh CellGro without cy20 tokin was added to each culture flask to survive the cells remaining adhered to the bottle surface. The cell suspension in the tubes is centrifuged at 1500/10 min / 22 ° C. Remove supernatant. The pellet is resuspended in about 2 ml CellGro medium. Transfer all cells originating from one culture flask to one 50 ml tube. Make up to 10 ml of each tube with CellGro. Mix gently by inverting the tube.

The required amount of maturation cytosines, KLH and tumor lysate obtained from the patient's tumor and from the tumor line or from two tumor lines are added to each of the three 50 ml cell tubes as follows:

For immature DB in 15 ml medium (1 culture flask):

IL-4 (32,000 U / ml) 320 U / ml 150 μΐ GM-CSF (100,000 U / ml) 1000 U / ml 150 μΐ KLH (100 pg / ml) 1 pg / ml 150 μΐ tumor lysate (or line) 10 pg / ml 150 pg tumor cell lysate 10 pg / ml 150 pg

The cell suspension with the medium and reagents is transferred back to the appropriate culture flasks, which are mixed by gently turning the bottle from side to side. Cells are incubated in a thermostat at 37 ° C, 5% CO _{2 for} 1.5 to 2 hours.

LPS (10x10 ⁴ U / vial activity) is dissolved in 0.5 ml HBSS (preferably the same HBSS as previously used to prepare the tumor lysate of the donor). The final concentration would be 20,000 U / ml.

Maturation of cytokines for DB is added to immature DB according to the following schedule:

For immature DB in 15 ml medium (1 culture flask):

LPs (20,000 U / ml) 200 U / ml 150 μΐ

IFN-γ (200,000 ng / ml) 50 ng / ml 37.5 μΐ

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Mix by gently turning the bottle from side to side. Cells are incubated in a thermostat at 37 ° C, 5% CO _{2 for} 6 hours.

Filling and freezing the vaccine:

The MiniCooler can be cooled in the fridge. The cell culture flask is agitated by shaking to release the adherent cells from the bottle walls. Pour the volume of each culture flask into a labeled 50 ml tube. Each culture flask is rinsed twice with approximately 10 ml of HBSS (preferably the same HBSS as previously used to prepare the tumor lysate of the donor). Mix by shaking and pour the entire volume of HBSS into the appropriate cell tube. Approximately 5 ml of HBSS is poured into each culture flask and placed in a box.

The cell suspension in each tube is made up to 50 ml with HBSS and mixed by inverting the tube. The tubes were centrifuged at 1500/10 min / 4 ° C. The supernatant is decanted and approximately 5 ml HBSS is added to the cell pellet. Cells are resuspended in HBSS and pooled from all tubes into one 50 mL tube. Make up to 50 ml with HBSS. Mix by inverting the tube. Take 10 μΐ of cell suspension in the Burker counting chamber. If cellularity greater than l, 4x 10 ⁶ / ml, skip the next paragraph. If cellularity was lower than l, 4x 10 ⁶ / ml used to detach the cells from flasks Accutase.

Freeze aliquots of 5x10 ⁶ DB. At least 6 aliquots should be frozen for patient administration, one quality control cryovial, immunomonitoring, itest, mycoplasma tests and three arbitration cryotubes.

Centrifuge the cells at 1500/10 min / 4 ° C. The cryovials are transferred to a cooled MiniCooler. The supernatant is decanted from the centrifuged cells and CryoStore (freezing medium) is added to the centrifuged cell sediment. Pipette the product into cryotubes.

Immediately after filling, transfer all cryovials of the medicinal product to an ethanol box and freeze to -80 ° C. As soon as possible after a minimum of 24 hours, transfer all cryotubes from -80 ° C to a Dewar liquid nitrogen (-196 ° C) vessel designed for CP only.

Table 1 gives an example of three manufactured DB-based vaccines with the respective test parameters. There were three different donors. All three vaccines met the acceptance criteria.

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Table 1:

Type of examination Sample 1 Sample 2 Sample 3 Criteria acceptability Sterility of the finished product sterile sterile sterile sterile Mycoplasma test negative negative negative negative Number of cells (x10 ^{3 4 * 6} ) 3.6 4.8 3.9 1-5 x10 ⁶ Viability (%) 93.8 91.4 87.8 70-100% Purity (%) 98.0 98.1 98.2 70-100% Impurities CD3 (%) 0.7 0.2 1.1 total 0-30% CD19 (%) 1.3 1.7 0.7 Phenotype * CD80 (%) 98.4 98.1 95.2 60-100% CD86 (%) 99.3 99.2 99.0 60-100% MHC II 86.2 99.3 74.6 60-100% CD83 (%) 97.6 86.5 86.8 60-100% CD14 (%) 1,8 26.2 0.3 0-40% IL-12 production (pg / ml) 9639,3 11874,6 836.63 ≫ 100 pg / ml alioMLR activated T lymphocytes (%) ** DC.PBMC ratio 1: 5 87.1 81.1 90.6 ž 30% DC: PBMC in ratio 1:10 77.2 75.1 80.6 > 30% DC: PBMC ratio 1:20 62.7 68.4 46.2 ž15% Conclusion compliant compliant compliant -

* Of the 5 phenotypic traits listed, at least 3 must meet acceptance criteria ** Of the 3 alioMLR listed, at least 2 must meet acceptance criteria

PROTECTION REQUIREMENTS

Claims (5)

An anti-tumor dendritic cell vaccine comprising dendritic cells producing interleukin IL-12 and presenting antigens of at least two 5 tumor lines of the same tumor type or tumor cell antigens obtained from a patient and at least one tumor line from the same tumor type. The anti-tumor vaccine of claim 1, further comprising cyclophosphamide and betaglucan in a metronomic dose. A kit for the preparation of an anti-tumor vaccine based on dendritic cells from blood monocytes, according to claim 1, characterized in that it comprises recombinant human interleukin IL4. recombinant human granulocytes and colony monocytes stimulating GM-CSF, lipopolysaccharide and interferon gamma, medium, and at least two different tumor lysates from the same tumor type. The kit for preparing an anti-tumor dendritic cell vaccine from blood monocytes 15 according to claim 1, wherein the tumor lysates are a combination of at least one tumor lysate obtained from the patient and at least one tumor lysate from the tumor line. A kit for the preparation of an anti-tumor vaccine based on dendritic cells from blood monocytes according to claim 1, characterized in that the tumor lysates are a combination of lysates of at least 5 two tumor lines of the same tumor type.