Pharmaceutical composition for treating pancreatic cancer

Abstract

The invention relates to a pharmaceutical composition for treating pancreatic cancer. The present invention relates to a method for treating pancreatic cancer comprising administering to a patient in need thereof a CD40 agonist in combination with dendritic cells loaded with mesothelioma cell lysates. Another aspect of the invention relates to dendritic cells loaded with mesothelioma cell lysates for use in the treatment of pancreatic cancer. A last aspect of the invention relates to a pharmaceutical composition comprising such loaded dendritic cells.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202080011369.6 and the invention name “Pharmaceutical composition for treating pancreatic cancer”. The original application is the PCT international application PCT / NL2020 / 050042 filed on January 28, 2020, which entered the Chinese national phase on July 28, 2021. Technical Field

[0002] The present invention relates to a method for treating pancreatic cancer using dendritic cells loaded with mesothelioma tumor cell lysate in combination with a CD40 agonist. The present invention also relates to loaded dendritic cells and pharmaceutical compositions thereof for treating pancreatic cancer in combination with a CD40 agonist. Background Art

[0003] In the Netherlands, the annual incidence of pancreatic cancer is approximately 3500 patients (1). In 2020, pancreatic cancer is expected to become the second leading cause of cancer-related death (2). The 1-year overall survival (OS) for pancreatic cancer in the Netherlands is 20%; the 5-year OS is only 3% (3). The vast majority of patients present with locally advanced or metastatic disease, which excludes them from curative surgery. Only 15 to 25% of all pancreatic cancer patients are eligible for surgical resection (4). However, ten years after resection, OS is still only 4%, indicating that cure is rare (5). Clearly, based on imaging techniques, the vast majority of patients with (borderline) resectable pancreatic cancer have occult metastatic disease. Compared to chemo-radiotherapy, which has been found to slightly improve pancreatic cancer survival, adjuvant chemotherapy after surgical resection does improve median overall survival to 28 months (6, 7). However, even with new chemotherapy regimens, long-term survival remains exceptional. Because recurrence rates after resection are extremely high, new treatments are needed to inhibit pancreatic cancer progression.

[0004] The potential to harness the power and specificity of the immune system underlies the growing interest in cancer immunotherapy. One approach to activating a patient's immune system uses dendritic cell-based immunotherapy. Dendritic cell-based immunotherapy aims to strengthen a cancer patient's immune system by activating cytotoxic T cells to enhance tumor antigen recognition and, therefore, generate a specific anti-tumor response.

[0005] In this regard, it is well known that dendritic cells are highly mobile and extremely potent antigen-presenting cells located in key locations where the body comes into contact with its environment. In these locations, they "pick up" antigens and transport them to secondary lymphoid organs, where they direct and control the activation of natural killer cells, B lymphocytes, and T lymphocytes, effectively activating these cells to combat the antigens. This characteristic makes them attractive candidates for use in therapeutic strategies targeting cancer. In addition, dendritic cells can be produced in large quantities in vitro.

[0006] Cancer induces a highly immunosuppressive tumor microenvironment (TME), leading to dysfunction of multiple immune effector cells (8, 9). For example, cytokines associated with the anti-inflammatory Th2 phenotype and immunosuppressive regulatory T cells are elevated in the peripheral blood of patients with pancreatic cancer compared with healthy controls (10, 11), while the accumulation of cytotoxic CD8 T cells is delayed (12). This leads to infiltration of tumors by non-cytotoxic T cells and may explain the low response rates of immune checkpoint antibodies (e.g., PD-1 / PD-L1) (13). In pancreatic cancer, early trials have indeed shown disappointing results with these antibodies, suggesting a need for more fundamental activation of the immune system (14 to 16). The induction of robust immune effector cells can enhance CD8 T cell infiltration and shift the balance in favor of anti-cancer responses. One way to activate the patient's immune system and induce tumor-directed cytotoxic T cells is through the use of cancer vaccines. Cancer vaccines have produced promising results in several preclinical and clinical studies (17). In complex immuno-tumors, cell therapy has been shown to be more effective than other types of vaccination (18). Multiple types of cell-based vaccination have been tested in pancreatic cancer in the context of phase I or II trials. Below, we discuss the most promising types of treatment in pancreatic cancer (i.e., tumor cell-based vaccination, adoptive T cell transfer, and dendritic cell vaccination).

[0007] Tumor cell-based vaccines

[0008] In pancreatic cancer, only two types of tumor cell-based vaccines (without adoptive cell transfer) are currently known. Their goal is to sensitize a robust immune response by activating different immune effector cells. Algenpantucel-L consists of two irradiated human pancreatic cancer cell lines (HAPa-1 and HAPa-2) that express the murine enzyme α-1,3-galactosyltransferase (α-GT) (19). Although two Phase III clinical trials with Algenpantucel-L are still ongoing, a recent press release announced that in one of these Phase III trials, Algenpantucel-L failed to improve OS relative to standard of care. The median OS in the intervention group was 27.3 months, while the control group with standard of care showed a median OS of 30.4 months (20).

[0009] The second tumor cell-based vaccine tested in pancreatic cancer patients is GVAX. The GVAX vaccine is based on irradiated tumor cells modified to express granulocyte-macrophage colony-stimulating factor (GM-CSF) (21, 22). It was combined with CRS-207 (Listeria monocytogenes engineered to express mesothelin). Some patients treated with GVAX / CRS-207 and radiochemotherapy developed an immune response against mesothelin and showed improved progression-free survival and OS (21, 23). However, the phase 2b trial ECLIPSE did not meet the primary endpoint of improved OS in patients with pancreatic cancer (24).

[0010] Adoptive T cell transfer

[0011] Tumor-specific effector CD8+ T cells are considered to be the last and most crucial step in immune-mediated cancer eradication. Therefore, adoptive cell transfer (ACT) using effector T cells has been developed, which includes tumor-infiltrating lymphocytes (TIL) therapy and receptor-modified T cell therapy (25). However, due to practical obstacles, the widespread clinical application of TIL in solid tumors is limited. In particular, in pancreatic cancer, due to the presence of significant desmoplastic stroma in pancreatic cancer, the harvesting of tumor cells is extremely challenging (26, 27). To date, there are no clinical trials for the treatment of pancreatic cancer patients with TIL. In addition, lymphocytes can be modified by introducing genes encoding anti-tumor α-β T cell receptors (T cell receptor, TCR) or chimeric antigen receptors (CAR) into mature T cells (28). However, there are some concerns and weaknesses about TCR and CAR T cell therapy. ACT with effector T cells carries the risk of toxicity when the target antigen is shared by tumors and normal tissues or when the target antigen is highly similar to self-antigens (29–31). Unexpected lethal toxicity has been observed in many trials due to previously unknown cross-reactivity (32–34). Furthermore, results in solid tumors have been less encouraging due to the presence of an immunosuppressive microenvironment that can adversely affect the recruitment and activation of adoptive CD8 T cells (35).

[0012] Dendritic cell vaccination

[0013] Dendritic cells (DCs) are the most potent activators of the immune system and play a fundamental role in the effectiveness of cancer vaccines (36). DCs can capture, process, and present tumor-associated antigens (TAAs) in the context of Major Histocompatibility Complex (MHC) class I or II (37). Subsequently, DCs can sensitize naive T cells, memory T cells, and B cells required to induce a robust anti-cancer response (38, 39). DCs pulsed with TAAs have shown beneficial effects in animal models of tumors (40, 41), showing that they are essential for inducing a powerful anti-cancer response. Clinical studies have demonstrated the safety and efficacy of DC immunotherapy (42, 43). The safety of DC-based immunotherapy in patients with pancreatic cancer has been studied in several phase I and phase II studies. To date, approximately 20 clinical DC immunotherapy trials in pancreatic cancer have been conducted worldwide. DCs were pulsed with TAAs such as Wilms' tumor 1 (WT-1), MUC-1, carcino-embryonic antigen (CEA), survivin, human telomerase reverse transcriptase (hTERT), or autologous tumor material (44 to 51), with varying results. Summary of the Invention

[0014] There remains a need for effective curative, palliative, or preventive treatments for pancreatic cancer. This is particularly true for patients who have not undergone or are not suitable for surgery, or for patients with recurrent pancreatic tumors. The present invention provides for such treatments for pancreatic cancer by combining a CD40 agonist with dendritic cells loaded with an allogeneic tumor cell line lysate, or a pharmaceutical composition comprising such dendritic cells loaded with such an allogeneic tumor lysate.

[0015] A first aspect of the present invention relates to a method for treating pancreatic cancer comprising administering to a patient in need thereof a CD40 agonist in combination with dendritic cells loaded with a lysate, wherein the lysate is obtainable by a method comprising the steps of:

[0016] i) providing human mesothelioma tumor cells from at least two different mesothelioma tumor cell lines;

[0017] ii) inducing necrosis in said tumor cells; and

[0018] iii) lysing the necrotic tumor cells to obtain lysates.

[0019] Surprisingly, it was found that a mesothelioma cell lysate (54), previously used successfully in clinical trials for the treatment of mesothelioma, is also very useful in the treatment of pancreatic cancer, particularly when combined with a CD40 agonist.

[0020] A second aspect of the present invention relates to dendritic cells loaded with a lysate for use in treating pancreatic cancer, wherein the dendritic cells are administered to a patient in need thereof in combination with a CD40 agonist, and wherein the lysate is obtainable by a method comprising the steps of:

[0021] i) providing human mesothelioma tumor cells from at least two different mesothelioma tumor cell lines;

[0022] ii) inducing necrosis in said tumor cells; and

[0023] iii) lysing the necrotic tumor cells to obtain a lysate.

[0024] The third aspect of the present invention relates to a pharmaceutical composition for treating pancreatic cancer together with a CD40 agonist, wherein the composition can be obtained by a method comprising the following steps:

[0025] i) providing allogeneic mesothelioma tumor cells from at least two different cell lines, and preparing lysates thereof;

[0026] ii) providing dendritic cells;

[0027] iii) loading dendritic cells with tumor cell lysate and, optionally, providing and adding a pharmaceutically acceptable carrier.

[0028] definition

[0029] The term "antigen" as used herein has its conventional meaning and refers to a molecule capable of inducing an immune response. In the context of the present invention, an antigen may be a protein or a fragment thereof, such as a (poly)peptide representing an epitope of the protein. However, it is also possible that the antigen used is an artificial peptide or peptidomimetic, such as by incorporating rigid non-natural amino acids (e.g., 3-aminobenzoic acid) into a peptide to rigidify the peptide backbone. The antigen used in the present invention is preferably obtained from a tumor cell or derived from a protein or portion thereof of a tumor cell.

[0030] The term "epitope" as used herein has its conventional meaning and refers to the part of an antigen that is recognized by the immune system, particularly by an antibody, B cell or T cell. In the context of the present invention, an antigen is a protein and an epitope is a part thereof (i.e., a (poly)peptide, a fragment or aggregate thereof).

[0031]

[0046] The term "cancer" is used herein with its conventional meaning and refers to a broad class of disorders characterized by hyperproliferative cell growth in the body.

[0032] As used herein, the term "mesothelioma cancer cells" or "mesothelioma tumor cells" has its conventional meaning and refers to cells from malignant mesothelioma.

[0033] As used herein, the terms "pancreatic cancer cells" or "pancreatic tumor cells" have their conventional meaning and refer to cells from malignant pancreatic cancer.

[0034] As used herein, the term "for treating pancreatic cancer" has its ordinary meaning and refers to reducing the size of a pancreatic tumor or the number of pancreatic cancer cells, causing pancreatic cancer to go into remission, or preventing or delaying further growth in size or cell number of pancreatic cancer cells.

[0035] As used herein, the term "cold tumor" has its conventional meaning and refers to a tumor in which infiltrating cytotoxic T cells are absent or minimally present.

[0036] As used herein, the term "hot tumor" has its conventional meaning and refers to a tumor in which cytotoxic T cells activated or inactivated by, for example, different immune checkpoints are present in considerable numbers.

[0037] As used herein, the term "progression free survival (PFS)" has its conventional meaning and refers to the time from treatment (or randomization) to first disease progression or death.

[0038] As used herein, the term "overall survival (OS)" has its conventional meaning and refers to patients who are still alive from randomization or from initial diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Experimental Setup Example 3. Immunocompetent C57bl / 6 mice were treated with a DC vaccine consisting of monocyte-derived DCs loaded with either pancreatic cancer lysate (KPC-3) or mesothelioma lysate (AE17). Untreated groups were also included. Subsequently, pancreatic tumors were induced using the KPC-3 tumor cell line and tumor growth was tracked.

[0040] Figure 2Figure 2: Tumor growth after DC vaccination. (A) Tumor size measured over time in untreated and treated mice. (B) Tumor growth curve for each mouse. N = 8 per group. Significance was determined using the nonparametric Mann–Whitney U test. Data are presented as mean ± sem. *P < 0.015. KPC-3 = pancreatic cancer lysate DC treatment; AE17 = mesothelioma lysate DC treatment.

[0041] Figure 3 : Terminal analysis after DC vaccination. (A) CD3 T cells in treated and untreated mice 27 days after DC vaccination as determined by flow cytometry. + 、CD4 + and CD8 + TIL accounts for CD45 + (B) CD44 or Ki67 positive CD4 in treated and untreated mice. + and CD8 + (C) Percentage of TIL in peripheral blood of treated and untreated mice + 、CD4 + and CD8 + T cells account for CD45 + Percentage of surviving subpopulation. (D) CD4 + and CD8 + CD44 on peripheral blood T cells + CD62L - Subpopulation or Ki67 positive. (E) CD8 + PD-1 in TILs + TIM-3 - LAG - (F) Treg (CD4 + CD25 + FoxP3 + ) as a percentage of the CD45 survival subpopulation. All non-Treg CD4 + FoxP3 - N = 8 / group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. *P < 0.05, **P < 0.01, **P < 0.001.

[0042] Figure 4 : Tumor-reactive T cell response after DC treatment. +Fresh spleen cells purified by MACS (assayed on the day of sacrifice (day 27)) were co-cultured with KPC-3 tumor cells. KPC-3 tumor cells were first stimulated with INFγ (40 U / ml) overnight, after which 100,000 cells were co-cultured with CD8 + T cells were seeded together in a 96-well flat-bottom plate at a 1:1 ratio and incubated with 10 μg / ml CD107a-FITC (BD-Bioscience) at 37°C in a humidified atmosphere with 5% CO2 for 5 hours. After one hour, the protein transport inhibitor Golgi stop TM (BD-Bioscience). For the markers granzyme B and TNFα, splenocytes were stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma) for 5 hours. N = 8 per group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. **P < 0.01, ***P < 0.001.

[0043] Figure 5 : Experimental setup Example 4. KPC-3C57B1 / 6 mice were treated with either unloaded (ie in the absence of tumor lysate) but matured DCs (stimulated with CpG) or DCs matured and loaded with mesothelioma AE17 lysate.

[0044] Figure 6 Figure 2: Tumor growth after DC vaccination. Tumor volume measured over time in mice treated with DCs pulsed with and without mesothelioma lysate. N = 7 per group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. *P < 0.05 **P < 0.01.

[0045] Figure 7 : Schematic overview of Example 5. Tumors and spleens from treated and untreated tumor-bearing mice from Example 4 were snap-frozen and stored as single cell suspensions, respectively. Bone marrow was harvested from wild-type non-tumor-bearing mice for mature DC culture.

[0046] Figure 8: Tumor-reactive T cell response after DC vaccination. Thawed splenocytes from pancreatic tumor-bearing mice were co-cultured with GM-CSF-cultured DCs (shown on the x-axis) loaded with 70 ug of autologous pancreatic tumor lysate or control lung lysate. 100,000 DCs were co-cultured with splenocytes from untreated tumor-bearing mice (the first and fourth lanes in each figure), tumor-bearing mice treated with unloaded DCs (the second and fifth lanes in each figure), and tumor-bearing mice treated with AE17-loaded DCs (the third and sixth lanes in each figure) at a ratio of 1:10 in 96-well round-bottom plates and incubated at 37°C in a humidified atmosphere at 5% CO2 for 24 hours. After 20 hours, the protein transport inhibitor Golgi Stop was added. TM (BD Bioscience), and 23 hours later, 10 μg / ml CD107a-FITC (BD Bioscience) was added per well. CD107, granzyme B, IFNγ, and TNFα were determined by flow cytometry. N = 5 to 8 per group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. **P < 0.01, ***P < 0.001.

[0047] Figure 9 : Experimental Setup Example 8. Immunocompetent C57bl / 6 mice were subcutaneously injected with 1×10 5 pancreatic tumor cells and treated with DC vaccine, CD40 agonist monoclonal antibody, or both, as indicated in the figure. 6 DCs were cultured and received CD40 agonistic mAbs or their isotypes on days 6 and 12, as indicated in the figures.

[0048] Figure 10 Tumor growth. (A) Tumor size measured over time in untreated mice and mice treated with mesothelioma lysate DCs, FGK45 (a CD40 agonist monoclonal antibody), or both. (B) Tumor volume on day 18 after tumor injection. N = 8 / group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. *P < 0.05, **P < 0.01.

[0049] Figure 11 : Peripheral blood analysis after DC vaccination and FGK antibody injection. (A) Peripheral blood CD69+ and Ki67+ cells in treated and untreated mice accounted for CD4 + and CD8 + (B) Percentage of CD44 T cells in treated and untreated mice + CD62L - and CD44- CD62L + Subpopulations account for CD4 + and CD8 + Percentage of peripheral blood T cells. N = 8 / group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001.

[0050] Figure 12 : Analysis of terminal tumors. CD3 T cells in treated and untreated mice at the end of the disease phase as determined by flow cytometry + 、CD4 + 、CD8 + and CD4 + CD25 + FoxP3 + TIL accounts for CD45 + Percentage of surviving subpopulations and absolute cell counts / mg tumor. N = 8 per group. Significance was determined using the nonparametric Mann-Whitney U test. Data are expressed as mean ± sem. *P < 0.05, **P < 0.01, **P < 0.001.

[0051] Figure 13 : Study protocol. In this figure, the study protocol is provided. Mice were administered tumor cells on day 0. Thereafter, they received DC vaccination (AE17) followed by administration of the agonistic CD40 antibody FGK45.

[0052] Figure 14 : Tumor growth. In this figure, the growth of tumors in the mice is shown. Clearly, the combination treatment resulted in a significant reduction in the growth of pancreatic tumors.

[0053] Figure 15 : Survival. In this figure, Kaplan Meier curves of the treated mice are shown. It is clear from this that the combined treatment of the present invention significantly improved the overall survival of the mice. DETAILED DESCRIPTION

[0054] A first aspect of the present invention relates to a method for treating pancreatic cancer comprising administering to a patient in need thereof a CD40 agonist in combination with dendritic cells loaded with a lysate, wherein the lysate is obtainable by a method comprising the steps of:

[0055] i) providing human mesothelioma tumor cells from at least two different mesothelioma tumor cell lines;

[0056] ii) inducing necrosis in said tumor cells; and

[0057] iii) lysing the necrotic tumor cells to obtain a lysate.

[0058] A second aspect of the present invention relates to dendritic cells loaded with a lysate for use in treating pancreatic cancer, wherein the dendritic cells are administered to a patient in need thereof in combination with a CD40 agonist, and wherein the lysate is obtainable by a method comprising the steps of:

[0059] i) providing human mesothelioma tumor cells from at least two different mesothelioma tumor cell lines;

[0060] ii) inducing necrosis in said tumor cells; and

[0061] iii) lysing the necrotic tumor cells to obtain a lysate.

[0062] The third aspect of the present invention relates to a pharmaceutical composition for treating pancreatic cancer together with a CD40 agonist, wherein the composition can be obtained by a method comprising the following steps:

[0063] i) providing allogeneic mesothelioma tumor cells from at least two different cell lines, and preparing lysates thereof;

[0064] ii) providing dendritic cells;

[0065] iii) loading dendritic cells with tumor cell lysate and, optionally, providing and adding a pharmaceutically acceptable carrier.

[0066] Surprisingly, it was found that pancreatic cancer can be effectively treated using a CD40 agonist in combination with dendritic cells loaded with a lysate obtainable by the above-mentioned method (or a pharmaceutical composition thereof).

[0067] It is particularly effective in treating patients with unresectable pancreatic tumors.

[0068] In particular, patients with primary pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer, or borderline resectable pancreatic cancer can be treated using the present invention.

[0069] It has been found to be particularly advantageous for treating patients suffering from or at risk of metastatic pancreatic cancer.The present invention and the various options described herein may therefore be used in patients suffering from metastatic pancreatic cancer.

[0070] In the context of the present invention, "unresected" means that the tumor has not been partially or completely removed by surgery. Such a tumor may be a primary or metastatic secondary pancreatic tumor.

[0071] CD40 agonists

[0072] "CD40 agonist" means an agonist of the cell surface receptor CD40, which has potential immunostimulatory and anti-tumor activity. Similar to the endogenous CD40 ligand (CD40L or CD154), the CD40 agonist is preferably capable of binding to CD40 on a variety of immune cell types. Binding of the agonist to the CD40 molecule can trigger cell proliferation and activation of antigen-presenting cells (APCs), as well as activation of B cells and T cells, leading to an enhanced immune response. Particularly preferred are agonistic CD40 monoclonal antibodies (also referred to herein as CD40 agonist monoclonal antibodies), fragments or derivatives thereof, such as single domain antibodies (also referred to as nanobodies), single chain antibodies, single chain variable fragments (scFv), Fab fragments, or F(ab')2 fragments.

[0073] The CD40 agonist to be administered in combination with the lysate-loaded dendritic cells or compositions according to the present invention can be a natural CD40 ligand, such as CD40L, or a functional fragment thereof having agonistic properties. The CD40 agonist can also be a monoclonal antibody having agonistic properties, such as CP-870, CP-893 (61), CDX-1140, APX005M, RG7876 / selicrelumab, ADC-1013 / JNJ-64457107, ABBV-428, SEA-CD40, or MEDI5083 (62), or a functional fragment thereof having agonistic properties. The CD40 agonist can also be a small molecule, such as, for example, a small molecule designed to mimic the natural ligand or agonistic antibody, such as, for example, MiniCD40Ls-1 or MiniCD40Ls-2 (63).

[0074] Without wishing to be bound by any theory, the present inventors believe that pancreatic cancer is generally an immunologically cold tumor, in contrast to, for example, melanoma and non-small cell lung cancer. It is believed that the characteristic desmoplastic stroma of established pancreatic cancer promotes this phenotype, acting as a physical as well as an immunosuppressive barrier, leading to the exclusion of T cells (64). The present inventors explored the hypothesis that CD40 agonists could convert pancreatic cancer into an immunologically hot tumor through both T cell-dependent and T cell-independent mechanisms.

[0075] It was also observed that the combined treatment according to the invention was also able to upregulate the expression of VEGFa, adm and Flt1 compared to mice treated with CD40 agonist alone. This indicates that angiogenesis and blood vessel formation are triggered, which promotes the infiltration of immune cells into the tumor.

[0076] The inventors observed that the growth of established tumors was unexpectedly reduced when treated with a combination of dendritic cell therapy and a CD40 agonist. The addition of a CD40 agonist thus enhanced the dendritic cell therapy, resulting in a significant reduction in tumor growth compared to untreated mice or mice treated with either the CD40 agonist or dendritic cell therapy alone.

[0077] The CD40 agonist according to the invention is preferably administered to the patient after administration of the dendritic cells. However, the CD40 agonist can also be administered simultaneously with (ie concomitantly with) the loaded dendritic cells.

[0078] Mesothelioma cell lysate

[0079] Since differential antigen expression occurs in tumors from different patients, it is not sufficient to provide lysates derived from only one cell line to the patient panel.

[0080] In the present invention, this is achieved by preparing mesothelioma tumor cell lysates from at least two different cell lines. By using different cell lines, multiple antigens are present in the lysate, which can be used to load dendritic cells. This approach, by downregulating specific antigens, reduces the chances of pancreatic tumor cells escaping from the patient.

[0081] Furthermore, the use of tumor cell lysates is essential to the present invention. Using these lysates allows for direct application of diverse antigens from diverse tumor cell lines to dendritic antigen-presenting cells. In addition to the extensive antigen library, the advantages of using allogeneic lysates are their ready availability and superior quality compared to autologous lysates.

[0082] A key problem associated with the use of autologous tumor cells is that the amount of tumor cells obtained from the resected tumor material (following surgery or by biopsy) is limited in quantity and quality. Furthermore, the tumor material obtained from the patient is highly heterogeneous in addition to the total tumor mass, which makes standardization difficult, and is "contaminated" with normal cells (e.g., macrophages, lymphocytes). When this tumor material is then used to treat pancreatic cancer, different results in phenotype and stimulatory capacity can be expected, with a potential negative impact on efficacy and complicating the development of a commercial product. For the reasons mentioned above, allogeneic mesothelioma tumor cells are used to prepare lysates.

[0083] In the context of the present invention, the term "allogeneic" has its usual scientific meaning and refers to tumor cells derived from an individual different from the individual to whom the lysate produced by the method according to the present invention will later be administered. The use of tumor cell lysates from cell lines derived from allogeneic mesothelioma tumor cells provides a more standardized and easier approach, bypassing the need for separately prepared autologous tumor lysates. It also creates opportunities for selecting the optimal source, dose, and delivery to dendritic cells or manipulation to improve the immunogenicity of the cells. The use of robust and validated large-scale manufacturing processes also requires fewer product batches for quality control testing, such as identity, purity, quantity, and sterility / safety testing. The main advantages of the allogeneic approach over autologous are the selection and optimization, bulk storage of tumor cell lines, and because the supply of lysates is readily available, the timeliness of manufacturing / quality control does not affect the patient's immediate disease progression.

[0084] According to the present invention, the term "necrosis" has its usual scientific meaning and refers to the morphological changes of cells. Necrosis is characterized, for example, by a "leakiness" (i.e., increased permeability) of the cell membrane, which also leads to the outflow of cell contents and the influx of substances, disturbing the homeostasis and ion balance of the cell, DNA fragmentation, and ultimately leading to the production of granular structures derived from collapsed cells, i.e., cell debris. Generally speaking, necrosis leads to the secretion of proteins into the surrounding tissue, which, when occurring in vivo, can lead to a proinflammatory response.

[0085] Methods for determining whether cells are necrotic are known in the art. It is not important for a person skilled in the art to choose which method to use, as a variety of methods are known. Necrosis can be induced, for example, by freeze-thaw cycles, heat treatment, triton X-100, or H2O2.

[0086] Necrotic cells according to the present invention can be identified, for example, by light microscopy, fluorescence microscopy, or electron microscopy techniques using classical staining, such as trypan blue, wherein the necrotic cells take up the dye and are thereby stained blue, or by morphological changes including loss of membrane integrity, organelle disintegration, and / or chromatin flocculation. Other methods include flow cytometry, for example by staining necrotic cells with propidium iodide.

[0087] According to the present invention, the term "apoptosis" has its usual scientific meaning and means programmed cell death. If a cell undergoes apoptosis, various changes in the cell occur, such as cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosome DNA fragmentation.

[0088] Apoptotic cells can be determined, for example, by flow cytometry, for example with Annexin V-FITC, with fluorochromes: Flura-red, Quin-2, with 7-amino-actinomycin D (7-AAD); reduced accumulation of Rhodamine 123; detection of DNA fragmentation by endonucleases: TUNEL method (X-UTP nick labeling by terminal deoxynucleotidyl transferase); by light microscopy by staining with Hoechst 33258 dye; by Western blot analysis, for example, by detecting caspase 3 activity, by labeling the 89 kDa product with a specific antibody, or by detecting cytochrome C efflux, by labeling with a specific antibody; or by agarose gel DNA analysis, detecting characteristic DNA fragmentation of a specific DNA ladder.

[0089] According to the present invention, the term "lysis" refers to various methods known in the art for opening / destroying cells. In principle, any method that can achieve tumor cell lysis can be used. The person skilled in the art can choose a suitable one, for example, to open / destroy the cells enzymatically, chemically or physically. Some examples of enzymes and enzyme mixtures that can be used to lyse tumor cells are proteases, such as proteinase K, lipase or glycosidase; some non-limiting examples of chemical substances are ionophores, such as nigromycin, detergents, such as sodium lauryl sulfate, acids or bases; and some non-limiting examples of physical means are high pressure, such as French pressing, osmolarity, temperature, such as heat or cold. A preferred way to lyse cells is to subject the cells to a freeze and thaw cycle. In addition, methods using appropriate combinations of enzymes other than proteolytic enzymes, acids, bases, etc. can also be used.

[0090] According to the present invention, the term "lysate" refers to an aqueous solution or suspension containing cellular proteins and factors produced by tumor cell lysis. Such a lysate may contain macromolecules, such as DNA, RNA, proteins, peptides, carbohydrates, lipids, and / or small molecules, such as amino acids, sugars, fatty acids, and the like, or fractions from the lysed cells. Cell fragments present in such a lysate may have a smooth or granular structure. Preferably, the aqueous medium is water, physiological saline, or a buffered solution.

[0091] The lysate according to the present invention is not limited to lysed necrotic cells. For example, due to different sensitivities of the treated cells or due to the conditions applied (e.g., UVB radiation), lysed apoptotic cells may also form the lysate or be part of the lysate. However, preferably, the lysate contains at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 98% lysed necrotic cells. The lysis method can affect the percentage of lysed necrotic cells. Multiple rapid freezing and thawing in liquid nitrogen, for example, results in a relatively high percentage of necrotic cells, while UVB radiation, for example, results in a relatively high percentage of apoptotic cells. The skilled person is aware of methods for obtaining essentially necrotic cells.

[0092] The term lysate used herein also encompasses prepared products or fractions prepared or obtained from above-mentioned lysate.These fractions can be obtained by methods well known to persons skilled in the art, described method such as chromatogram, comprise such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, reversed-phase chromatography and the chromatography carried out with post or batch method with other chromatographic materials, other fractionation methods, such as filtering methods, such as ultrafiltration, dialysis, dialysis and concentration with size exclusion in centrifugation, centrifugation with density gradient or step matrix (step matrice), precipitation, such as affinity precipitation, salt dissolution or salting out (ammonium sulfate precipitation), alcohol precipitation, or other protein chemistry, molecular biology, biochemistry, immunology, chemistry or physical method are separated above component of lysate.In a preferred embodiment, those fractions that are more immunogenic than other are preferred.Those skilled in the art can select suitable method, and by referring to the specific description in the embodiment above general description and herein and suitably modify or change those methods to determine its immunogenicity potential when necessary.

[0093] In order to obtain a good immunogenic response, it is preferred to prepare the lysate using a mixture of allogeneic mesothelioma tumor cells from at least two mesothelioma tumor cell lines, preferably at least three mesothelioma tumor cell lines, more preferably at least four mesothelioma tumor cell lines. It is particularly preferred to prepare the lysate using a mixture of at least five mesothelioma tumor cell lines.

[0094] Preferably, the at least two, at least three, at least four or at least five mesothelioma tumor cell lines are present in substantially equal cell amounts at equal concentrations prior to lysate preparation. The term "substantially equal cell amounts" has its conventional meaning and preferably means that the cell lines are each present relative to each other in a cell ratio of 1:2 to 2:1, more preferably 2:3 to 3:2, more preferably 3:4 to 4:3, more preferably 4:5 to 5:4, most preferably about 1:1.

[0095] As an example of five cell lines, the cells may be present in a cell ratio of 3:4:2:4:3, where cell line 1 has a 3:4 ratio to cell line 2, a 3:2 ratio to cell line 3, a 3:4 ratio to cell line 4, and a 1:1 ratio to cell line 5. Cell line 2 has a 4:3 ratio to cell line 1, a 2:1 ratio to cell line 3, a 1:1 ratio to cell line 4, and a 4:3 ratio to cell line 5. The cell ratios of cell lines 3, 4, and 5 relative to the other cell lines are calculated identically and all fall within the preferred ratios defined above.

[0096] Using such a mixture of cell lines as a source of tumor lysates has the advantage of providing a broader antigen library of tumor-associated antigens (multiple potential tumor antigens), which will improve the ability of the immune response to recognize and destroy tumor cells because the opportunity to escape immune surveillance by regulating antigen expression is more limited.

[0097] The allogeneic mesothelioma tumor cells used in the methods of the present invention are cultured, for example, in culture flasks. These allogeneic cells are suitable for preparing lysates compared to non-cancerous cells due to their ability to divide indefinitely with minimal loss of immunogenicity. The cell lines used to prepare the lysates used in the present invention are of human origin.

[0098] Five human mesothelioma cell lines have been developed that provide particularly good results. These cell lines are deposited with the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikro-organismen und Zellkulturen) in Germany, hereinafter referred to as DSMZ. The cell lines were initially assigned the following codes and accession numbers: Thorr 01 (Deposit No. DSMACC3191), Thorr 02 (Deposit No. DSMACC3192), Thorr 03 (Deposit No. DSMACC3193), Thorr 04 (Deposit No. DSMACC3194), Thorr 05 (Deposit No. DSMACC3195). Deposits were made under the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure. After the initial deposit, the cell lines were renamed as follows: Thorr 01 was renamed Thorr 03, Thorr 02 was renamed Thorr 01, Thorr 03 was renamed Thorr 02, Thorr 04 was renamed Thorr 05, and Thorr 05 was renamed Thorr 06. Throughout this patent application, the renamed names are used, namely: Thorr 01 (Deposit No. DSMACC3192), Thorr 02 (Deposit No. DSMACC3193), Thorr 03 (Deposit No. DSMACC3191), Thorr 05 (Deposit No. DSMACC3194), Thorr 06 (Deposit No. DSMACC3195).

[0099] Therefore, in a preferred embodiment, a lysate for use according to the present invention is provided, wherein the allogeneic mesothelioma tumor cells used are selected from two or more of the following cell lines: Thorr 01 (Deposit No. DSMACC3192), Thorr 02 (Deposit No. DSMACC3193), Thorr 03 (Deposit No. DSMACC3191), Thorr 05 (Deposit No. DSMACC3194), Thorr 06 (Deposit No. DSMACC3195).

[0100] Necrosis of allogeneic mesothelioma tumor cells can be achieved by methods known in the art. However, freezing and thawing the cells is particularly preferred. Preferably, the cells are necrotized and lysed by freezing at a temperature below -75 degrees Celsius and thawing at room temperature. In particular, rapid freezing in liquid nitrogen at a temperature below -170 degrees Celsius and thawing at room temperature or higher (e.g., at about 37 degrees Celsius in a water bath) is most preferred. Also preferably, the freezing / thawing is repeated at least once, more preferably at least twice, even more preferably at least three times, particularly preferably at least four times, and most preferably at least five times.

[0101] Preferably, the tumor cells are irradiated with at least 50 Gy, preferably at least 100 Gy. This prevents any tumor cells from remaining viable. Irradiation can be performed before or after freezing and thawing the tumor cells.

[0102] In a preferred embodiment of the method according to the present invention, the lysate comprises at least three mesothelioma cancer cell-associated antigens. Preferably, the lysate comprises at least three, more preferably at least five, more preferably at least ten mesothelioma cancer cell-associated antigens. In this regard, it should also be noted that the antigens can be derived from the same protein, that is, the antigens can be different epitopes from the same protein. However, it is preferred to use antigens that are (or are based on) different tumor cell-associated proteins. Preferably, at least three, more preferably at least five, more preferably at least ten mesothelioma cancer cell-associated antigens are also expressed on pancreatic cancer cells, that is, these antigens are shared between mesothelioma cancer cells and pancreatic cancer cells, at least the majority of pancreatic cancer cells to be treated in patients in need thereof.

[0103] It is particularly beneficial for the lysate to contain multiple antigens, ideally covering all tumor cells in the tumor. After all, if a particular tumor cell does not possess a specific antigen, an immune response against it will not be triggered. If other cells are attacked while this cell remains unattacked, it will have an advantage and will be able to grow further, leading to further growth of the tumor. The inventors have now been able to establish the most important antigens that can be used to load dendritic cells and target essentially all tumor cells in pancreatic cancer. This method allowed the inventors to formulate a lysate that is particularly useful for loading dendritic cells and inducing an immune response against pancreatic cancer cells.

[0104] Preferably, at least three, more preferably at least five, and even more preferably at least six mesothelioma cancer cell-associated antigens are selected from the group consisting of RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, and MUC1. Other antigens of importance in the context of the present invention are RAB38 / NY-MEL-1, BING4, MAGE A12, HER-2 / Neu, glypican, and LMP2. When used according to the present invention, a mixture of at least three, preferably at least five, more preferably at least six, and most preferably at least ten of the aforementioned mesothelioma-associated antigens is particularly effective against pancreatic cancer.

[0105] In a preferred embodiment, a lysate for use according to the present invention is provided, wherein the at least three, preferably at least five, more preferably at least six mesothelioma cancer cell-associated antigens are selected from the following group: RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1.

[0106] In another preferred embodiment, a lysate for use according to the present invention is provided, wherein at least three, preferably at least five, more preferably at least seven, more preferably at least nine, and more preferably at least ten mesothelioma cancer cell-associated antigens are selected from the following group: RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1, RAB38 / NY-MEL-1, BING4, MAGEA12, HER-2 / Neu, phosphatidylinositol proteoglycan, and LMP2.

[0107] Surprisingly, it was found that many of the antigens present in the mesothelioma cells used to prepare the lysates of the present invention are shared with pancreatic cancer cells (Table 1). For example, the tumor-associated antigen mesothelin, which is present in large quantities in the lysates of the present invention (also called "PheraLys"), is also present in pancreatic cancer. The presence of mesothelin in pancreatic cancer has led to the initiation of clinical trials targeting mesothelin in this type of cancer worldwide. Combining Listeria monocytogenes expressing mesothelin with the allogeneic pancreatic cancer vaccine GVAX extended the median survival of patients with advanced pancreatic cancer from 3.9 months to 6.1 months (22). However, due to the single antigen approach, the duration of the response is limited.

[0108] Table 1: Target antigens for pancreatic cancer in PheraLys

[0109] <![CDATA[Sorting a > antigen Gene ID <![CDATA[FPKM score b > PheraLys <![CDATA[Pancreatic cancer c > 3 Mesothelin 10232 84.25 ++ ++ 9 Survivin 332 38.71 ++ ++ 18 HER-2 / neu 2064 16.89 + + 21 MUC1 4582 13.13 ++ ++ 29 WT1 7490 10.28 + + 30 KRAS 3845 9.26 + + 36 LY6K 54742 6.84 + / - + / -

[0110] a. A large list of overexpressed, differentiation, and cancer germline antigens (195) was examined for their frequency in each of the five malignant mesothelioma cell lines used to create PheraLys by RNA-seq analysis and ranked according to their mean FPKM score.

[0111] b FPKM = fragments per kilobase million mapped

[0112] c according to www.proteinatlas.org Antigen expression

[0113] ++ = strong expression, + = moderate expression, + / - = expression status varies between samples

[0114] In addition to a large number of antigens with relatively high expression, antigens with relatively low expression can also induce highly specific T cell responses in patients. For example, both dominant and subdominant neoantigens significantly increase the TCR-β repertoire after DC vaccination (55). Therefore, all antigens may be valuable in patients, while others have tried single antigens, or combinations of antigens for dendritic cell loading, the size of the number of antigens in PheraLys is clearly an advantage of the current approach.

[0115] For example, it has been shown that the efficacy of single antigen therapy is often short-lived in solid tumors (56). The tumor is able to downregulate the specific antigen relatively quickly, after which the treatment becomes ineffective. In contrast, immunotherapy with a large number of tumor-associated antigens reduces the likelihood of tumor escape by eliciting a broad immune response, and the clinical response will be more sustained. In one embodiment, the lysate is in the form of a pharmaceutical composition that also includes a pharmaceutically acceptable excipient or carrier for the treatment of pancreatic cancer.

[0116] The lysate can also be loaded onto dendritic cells ex vivo and formulated into a pharmaceutical composition, as will be described in more detail below.

[0117] dendritic cells

[0118] The term "dendritic cell" as used herein has its conventional meaning and refers to the antigen-presenting cells (also referred to as accessory cells) of the mammalian immune system, which capture antigens and have the ability to migrate to the lymph nodes and spleen, where they are particularly active in presenting processed antigens to T cells. The term dendritic cell also encompasses cells with activities and functions similar to dendritic cells. Dendritic cells can be derived from lymphoid or mononuclear phagocyte lineages. Such dendritic cells can be found in lymphoid and non-lymphoid tissues. The latter have been shown to induce T cell responses only when activated and migrate to lymphoid tissues.

[0119] Dendritic cells are known to be one of the most potent activators and regulators of the immune response. An important feature is that they are the only antigen-presenting cells currently known to stimulate naive T cells. Immature dendritic cells are characterized by their ability to absorb and process antigens (a function significantly reduced in mature dendritic cells), which in turn exhibit enhanced presentation of processed antigens on their surface, primarily antigens bound to MHC class I and class II molecules. Maturation is also associated with the upregulation of co-stimulatory molecules (such as CD40, CD80, and CD86) and certain other cell surface proteins (such as CD83 and DC-Sign). Dendritic cell maturation is also typically associated with enhanced migration capacity, resulting in (in vivo) dendritic cells migrating to local lymph nodes, where dendritic cells encounter T and B lymphocytes. In a preferred embodiment, the dendritic cells are immature when loaded with a lysate, but mature and activated when administered to a patient in need thereof.

[0120] Dendritic cells can be obtained from humans using methods known to those skilled in the art (57 to 59). After obtaining monocytes, these cells are differentiated ex vivo into immature dendritic cells, which are further matured and activated.

[0121] Preferably, the cultured dendritic cells are autologous dendritic cells. The advantage of using autologous dendritic cells is that an immune response of the patient against these dendritic cells is avoided and an immune response is triggered against antigens from the mesothelioma tumor cells present in the lysate.

[0122] In a preferred embodiment, the dendritic cells are autologous to the subject with pancreatic cancer. While the use of autologous dendritic cells offers many advantages, the use of allogeneic dendritic cells can also be advantageous. One of the main advantages of using allogeneic dendritic cells is that they provide patients with a ready-to-use medication. In other words, instead of having to differentiate, load, and activate dendritic cells from an individual, loaded allogeneic dendritic cells can be administered immediately. This saves the patient valuable time. Therefore, in a preferred embodiment, the dendritic cells are allogeneic to the subject with pancreatic cancer.

[0123] Loading of dendritic cells with mesothelioma cell lysate

[0124] Dendritic cells or their precursors are differentiated using suitable growth factors and / or cytokines (e.g., GM-CSF and IL-4), and the resulting immature dendritic cells are loaded with a lysate used according to the present invention. The immature dendritic cells loaded with the lysate used according to the present invention are further matured into mature dendritic cells. In special cases, mature dendritic cells can also be loaded (pulsed) with an antigen or immunogen from the lysate.

[0125] Preferably, the dendritic cells are loaded with 1 tumor cell equivalent per 100 dendritic cells to 10 tumor cell equivalents per dendritic cell, more preferably 1 tumor cell per 10 dendritic cells to 1 tumor cell equivalent per dendritic cell, and particularly preferably about 1 tumor cell equivalent per 3 dendritic cells.

[0126] Preferably, the dose administered to the patient comprises 1×10 6 to 1×10 9 loaded dendritic cells, preferably 2×10 6 to 5×10 8 loaded dendritic cells, more preferably 1×10 7 to 1×10 8 loaded dendritic cells, most preferably about 2.5×10 7 Most preferably, the dose comprises about 2.5×10 dendritic cells loaded with about 1 tumor cell equivalent / 3 dendritic cells. 7 dendritic cells.

[0127] Preferably, the dendritic cells are loaded with more than one mesothelioma cancer cell-associated antigen. Thus, preferably, the composition used to load the dendritic cells comprises at least three, preferably at least five, and more preferably at least ten mesothelioma cancer cell-associated antigens. In this regard, it is also noted that the antigens can be derived from the same protein, i.e., the antigens can be different epitopes from the same protein. However, it is preferred to use antigens that are (or are based on) different tumor cell-associated proteins.

[0128] In order for T cells to attack all tumor cells, it is important to ensure that the dendritic cells are loaded with antigens that ideally cover all tumor cells in the tumor. After all, if a particular tumor cell does not have a specific antigen, an immune response against that cell will not be triggered. If other cells are attacked while this cell remains unattacked, it will have an advantage and will be able to grow further, leading to further growth of the tumor. The inventors have now been able to create a lysate containing the most important antigens that can be used to load dendritic cells and target pancreatic cancer. This method allowed the inventors to formulate an antigen composition that is particularly useful for loading dendritic cells and inducing an immune response against pancreatic tumor cells.

[0129] Mesothelioma cancer cell-associated antigens are preferably selected from the group consisting of RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, and MUC1. These antigens have been shown for the first time to be capable of inducing a strong immune response against pancreatic tumor cells via dendritic cell immunotherapy. Other antigens of interest in the context of the present invention include RAB38 / NY-MEL-1, BING4, MAGE A12, HER-2 / Neu, glypican, and LMP2.

[0130] In addition, for these tumor cell associated proteins, it should be noted that a portion of these proteins (i.e., their epitopes) can also be used as antigens for loading dendritic cells. In this respect, it is also noted that polypeptides or peptide mimetics of such epitopes can also be used for loading dendritic cells. In one embodiment, the antigen composition comprises an antigen that is only selected from the antigen group shown in Table 1. This is advantageous from an adjustment perspective.

[0131] In another embodiment, the mesothelioma cancer cell-associated antigens are obtained from lysates of allogeneic mesothelioma tumor cells from at least two different mesothelioma tumor cell lines, preferably at least three tumor cell lines, more preferably at least four tumor cell lines, and most preferably at least five tumor cell lines. The advantage of using such lysates is that many tumor-associated antigens will be present in the lysates, and dendritic cells will be loaded with a considerable number of antigens, thereby reducing the chance that tumor cells will not be recognized and escape the immune response.

[0132] The mesothelioma tumor cell line used for preparing such lysates is preferably selected from Thorr 01 (Deposit No. DSMACC3192), Thorr 02 (Deposit No. DSMACC3193), Thorr 03 (Deposit No. DSMACC3191), Thorr 05 (Deposit No. DSMACC3194), Thorr 06 (Deposit No. DSMACC3195).

[0133] The lysate was prepared as follows: 10 × 10 6 to 200×10 6 tumor cells / ml, preferably 20×10 6 to 100×10 6 , more preferably 30×10 6 to 75×10 6 , more preferably 40×10 6 to 60×10 6 , most preferably about 50×10 6 Therefore, the lysate according to the present invention contains 10×10 6 to 200×10 6 Equivalent, preferably 20×10 6 to 100×10 6 , more preferably 30×10 6 to 75×10 6 , more preferably 40×10 6 to 60×10 6 , most preferably about 50×10 6 Tumor cell equivalents / ml. In the present context, by equivalent is meant the amount of tumor cells present in the solution before lysis, since after lysis only cell fragments are present.

[0134] Also find that the total protein content of the lysate used according to the present invention has dependency, because this is directly related to the number of the tumor cells that are used to prepare compositions.If the amount of protein (i.e. antigen) is too low, then the load of dendritic cell will be very poor, and the immunne response of induction will be restricted.If protein concentration is too high, then the interaction between the different proteins will take place, making antigen more difficult for being absorbed by dendritic cell, and causing stability problem.Therefore, the total amount of protein is preferably 5 to 25mg protein / ml, more preferably 6 to 20mg protein / ml, more preferably 7 to 15mg, most preferably 7.9 to 11.8mg protein / ml in the antigen composition.

[0135] It is also preferred that only fragmented DNA is present in the lysate. First, the lysate is preferably subjected to a freeze-thaw cycle (reducing DNA size) and preferably irradiated with a very high dose of 50Gy, preferably 100Gy irradiation, which causes irreparable double-strand breaks and therefore causes distortion (distort) and difficult-to-read information (reducing the risk of carcinogenesis and infection of the residual DNA). In addition, dendritic cells are preferably purified from the lysate component that is not incorporated by density gradient centrifugation, thereby removing residual small DNA fragments. After removing the lysate from the dendritic cells, the dendritic cells are preferably incubated in vitro for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours before purification, thereby allowing free-floating nucleic acids (RNA / DNA) to be degraded by natural nucleases. These measures result in almost no complications (no viscosity or complex formation, showing that there is no sizable DNA fragmentation) in the downstream processing of the pharmaceutical composition and lysate comprising dendritic cells. Although DNA present in lysates and / or pharmaceutical compositions is considered a cellular contaminant rather than a risk factor by the WHO Expert Committee on Biological Standardization, a dose limit of 10 ng / dose has been set for it.

[0136] Therefore, the pharmaceutical composition according to the invention preferably comprises less than 10 ng free DNA per dose, preferably less than 100 pg, more preferably less than 1 pg, most preferably less than 0,01 pg free DNA per dose.

[0137] In a preferred embodiment, a lysate for use according to the present invention is provided, wherein the lysate is loaded onto autologous dendritic cells prior to administration to a patient. Preferably, the dendritic cells are loaded with 1 tumor cell equivalent per 100 dendritic cells to 10 tumor cell equivalents per dendritic cell, more preferably 1 tumor cell equivalent per 100 dendritic cells to 1 tumor cell equivalent per dendritic cell, and most preferably about 3 dendritic cells per 1 tumor cell equivalent.

[0138] To induce a sufficiently large immune response, 1×10 6 to 1×10 9 Loaded dendritic cells / administration, preferably 2×10 6 to 5×10 8 loaded dendritic cells, more preferably 1×10 7 to 1×10 8 loaded dendritic cells, most preferably about 2.5×10 7 It is advantageous to administer a plurality of dendritic cells / day.

[0139] The dendritic cells used may be autologous or allogeneic. However, the use of autologous dendritic cells is particularly preferred. MHC class II molecules expressed on these autologous dendritic cells present peptides to TCRs expressed on T cells present in the treated patient. The ability of the TCR to distinguish foreign peptides from self peptides presented by "self" MHC molecules is a prerequisite for an effective adaptive immune response. The use of intratumorally injected allogeneic dendritic cells has also been described, but such allogeneic dendritic cells are unlikely to present tumor antigens directly to the patient's T cells (60). Without being bound by theory, it is believed that such allogeneic dendritic cells, when injected at the tumor site, can effectively recruit other immune cells to the site, such as NK cells, which ultimately kill the allogeneic dendritic cells, thereby providing both tumor antigens and "danger signals" to the autologous dendritic cells within the tumor, which then induce a specific (T cell) immune response against the tumor antigen. Therefore, in a preferred embodiment, the dendritic cells of the present invention are allogeneic to the patient receiving them, wherein preferably, the dendritic cells are administered intratumorally. Preferably, the lysate is provided as a ready-to-use product that can be used to load dendritic cells obtained from a patient suffering from pancreatic cancer.After loading and appropriate formulation for intravenous and / or intradermal administration, the loaded dendritic cells are administered to the patient.

[0140] Pharmaceutical composition

[0141] The lysate itself and the loaded dendritic cells can be formulated as a pharmaceutical composition or kit. A skilled person will be able to prepare suitable pharmaceutical compositions based on his or her general knowledge.

[0142] The pharmaceutical composition according to the present invention may comprise a physiologically acceptable carrier or may be administered to a patient together with a physiologically acceptable carrier, as described herein. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle that is administered together with the therapeutic agent. Such a pharmaceutical carrier may be a sterile liquid, such as water and a buffer.

[0143] Some examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions may comprise a therapeutically effective amount of cell lysate or loaded dendritic cells (preferably in purified form) and a suitable amount of carrier to provide a form for proper administration to the patient. The formulation should be suitable for the mode of administration.

[0144] In one embodiment, the composition is in a water-soluble form, such as a pharmaceutically acceptable salt, which is meant to include both acid addition salts and base addition salts.

[0145] The composition can be prepared in various forms, such as injection solutions, tablets, pills, suppositories, capsules, suspensions and the like.

[0146] Pharmaceutical grade organic or inorganic carriers and / or diluents suitable for oral and topical use can be used to construct compositions containing therapeutically active compounds. Diluents known in the art include aqueous media, plant and animal oils and fats. Stabilizers, wetting agents and emulsifiers, salts for modifying osmotic pressure or buffers for ensuring an appropriate pH value, and skin penetration enhancers can be used as adjuvants. The composition may also contain one or more of the following: a carrier protein, such as serum albumin; a buffer; a filler, such as microcrystalline cellulose, lactose, corn and other starches; a binder; sweeteners and other flavorings; a colorant; and polyethylene glycol. Additives are well known in the art and are used in a variety of formulations.

[0147] The pharmaceutical composition can be formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous and / or intradermal administration to humans. Typically, compositions for intravenous and / or intradermal administration are solutions in sterile isotonic aqueous buffer.

[0148] The present invention will be further illustrated by the following non-limiting examples.

[0149] Example

[0150] Example 1

[0151] Description of the PheraLys Manufacturing Process

[0152] PheraLys is considered a highly heterogeneous source of tumor-associated antigens (TAAs) because it contains five highly heterogeneous MPM tumor cell lines.

[0153] The cell lines named Thorr 01, Thorr 02, Thorr 03, Thorr05 and Thorr 06 (Thorr is the abbreviation of Thoracic Oncology Research Rotterdam) from 5 different patients with MM were selected for PheraLys preparation. These cell lines were deposited at the Leibniz Institute DSMC-Collection of Microorganisms and Cell Cultures (DSMZ) for patent purposes according to the Budapest Treaty: Thorr 01 (preservation No.DSMACC3192), Thorr 02 (preservation No.DSMACC3193), Thorr 03 (preservation No.DSMACC3191), Thorr 05 (preservation No.DSMACC3194), Thorr06 (preservation No.DSMACC3195).

[0154] Individual Thorr cell lines were incorporated into the culture and incubated overnight at 37°C in a humidified atmosphere of 5% CO2, 95% air, followed by a medium change and PBS wash the next day. The cells were washed and expanded in fresh medium until a sufficient number of cells of each individual Thorr cell line was obtained. The cells were washed thoroughly with PBS, counted, and plated at a concentration of 50 × 10 in PBS at <-70°C in a controlled environment. 6 cells / ml and stored until further use.

[0155] Equal cell amounts of different cell lines were mixed and stored at <-70°C. For the preparation of lysates, the intermediate product was thawed and aliquoted in 50 ml tubes, containing 30 ml of cell suspension. These 50 ml tubes were frozen and thawed 5 times by rapid freezing with liquid nitrogen. Thereafter, the 50 ml tubes were irradiated with 100 Gy by gamma irradiation with a radioactive 137 cesium irradiation source (Cis Bio International). At this point, there were no more tumor cells present in the final lysate, so the concentration was described in terms of tumor cell equivalents (TCE). 50 × 10 6 TCE is equal to 50×10 6 The content of tumor cells.

[0156] Example 2

[0157] Expression of tumor-associated antigens in Thorr cell lines

[0158] Use Affymetrix expression array to characterize five kinds of tumor cell lines by RNA sequencing.The expression spectrum of cell line is evaluated for the list of 195 kinds of known antigens.These 195 kinds of antigen lists have covered all differentiation / overexpressed antigens as target or prognosis markers published in the literature.In addition, it comprises all cancer germline antigens that are currently listed as cancer specific targets in the cancer / testis antigen database (www.cta.lncc.br).Cancer germline antigens are particularly interesting because these have greater chance to trigger a strong immune response because they are only expressed by cancer cells (and not by healthy tissue).

[0159] FPKM (fragments per kilobase per million) approximates the relative abundance of transcripts based on the fragments observed from RNA-seq experiments. If transcript expression is identical, longer genes will have more fragments than shorter genes. This is adjusted by dividing FPM by gene length to produce the metric fragments per kilobase of transcript per million mapped reads (FPKM).

[0160] The results showed that the TAAs of interest were heterogeneously expressed by different Thorr cell lines (Table 2). This illustrates the potential of the five selected Thorr cell lines to serve as a source of a wide range of clinically relevant TAAs.

[0161] Table 2: Most relevant antigens present in model cell lines (RNA sequencing results)

[0162]

[0163] *FPKM value (fragments per kilobase of exon per million mapped fragments).

[0164] Example 3

[0165] Immune responses against pancreatic tumors by treatment with DCs loaded with autologous pancreatic or allogeneic mesothelioma lysates

[0166] Immunocompetent C57bl / 6 mice were treated with a DC vaccine consisting of monocyte-derived DCs loaded with pancreatic cancer lysate (KPC-3) or mesothelioma lysate (AE17). The loading was comparable to that in humans, i.e., 1 tumor cell equivalent per 3 DCs. An untreated group was also included. Subsequently, pancreatic tumors were induced by subcutaneous injection of 100,000 cells of the pancreatic cancer KPC-3 cell line, and tumor growth was followed (see schematic setup: Figure 1). This experimental setting corresponds to the situation of pancreatic cancer patients with only micrometastases remaining after surgery.

[0167] In this preclinical setting, 2 × 10 6 DCs, and 1×10 6 DCs were administered after surgery, with neither clinical signs of established tumors nor the presence of the unique desmoplastic stroma of established pancreatic cancer. Vaccination in our mouse model was performed before tumor establishment, closely resembling the clinical setting. By treating mice before macroscopic tumor formation and desmoplasia were established, we simulated resected patients with potentially micrometastatic disease. DCs were stimulated with CpG overnight and loaded with mesothelioma lysate (AE17 cell line; Professor Nelsons, Curtin University, Perth, Australia) or pancreatic cancer lysate (KPC-3). DCs were generated as previously described (54).

[0168] Systemic immune responses were monitored 4 and 11 days after DC vaccination (interim analysis). At the end of the disease (27 days after DC vaccination), T cell phenotypes (including activation, proliferation, and exhaustion status) were analyzed in tumors, spleen, and peripheral blood (terminal analysis).

[0169] Tumor growth was significantly delayed in treated animals compared to untreated animals. The relative delays in tumor growth and tumor size at different time points were comparable in treated animals regardless of the type of DC loading, indicating that treatment with DCs derived from mesothelioma cells was as effective as treatment with DCs derived from autologous pancreatic cell lysates ( Figure 2 ).

[0170] In both groups of DC-treated mice, tumor growth was delayed and the frequency of tumor-infiltrating lymphocytes (TILs) was increased compared with that in untreated mice ( Figure 3 A). In addition, CD44 expression was higher on both CD4+ and CD8+ TILs in treated mice, indicating a more pronounced effector memory T cell phenotype. The proliferation marker Ki67 was also higher on CD8+ TILs in treated mice compared to untreated mice ( Figure 3 B). In addition, a higher frequency of PD-1+LAG-3-TIM-3-CD8+TILs was observed in treated mice, although with significant changes. This phenotype is associated with truly activated, non-exhausted T cells required for a robust anti-tumor response ( Figure 3 E).

[0171] After DC treatment, suppressive intratumoral CD4+FoxP3+Tregs were not increased ( Figure 3 F), which further confirmed the potent anti-tumor CD8+ T cell response.

[0172] In peripheral blood, increased frequencies of T cell subsets were observed as early as four days after DC treatment. 27 days after treatment, increased frequencies of T cells in peripheral blood and spleen (not shown) were still present, whereas the earlier observed enhanced values ​​of CD44+CD62L- subsets and Ki67 markers returned to the untreated baseline ( Figure 3 C, D).

[0173] To demonstrate the induction of tumor-specific T cell responses, splenocytes were isolated on the day mice were sacrificed in Experiment 1. + MACS-purified spleen cells were stimulated with pancreatic tumor cells (KPC-3) in vitro.

[0174] After stimulation with pancreatic tumor cells, CD8 + T cells expressed elevated frequencies of multiple activation and degranulation markers.

[0175] The production of interferon-γ (IFNγ) and tumor necrosis factor α (TNFα) was assessed by intracellular cytokine staining, and the expression of CD107a, CD69, and granzyme B was also assessed by flow cytometry. Notably, IFNγ expression was significantly increased in all treated mice after stimulation with tumor cells compared to untreated mice. + and CD107a + CD8 expression + In the case of CD69, granzyme B, and TNFα, higher frequencies were observed only in mice treated with mesothelioma-pulsed DCs ( Figure 4 ).

[0176] Example 4

[0177] Loading DCs with (shared) tumor-associated antigens that are essential for an effective antitumor response

[0178] It was investigated whether delayed tumor growth depends on the induction of a tumor-specific immune response induced by DCs loaded with tumor-associated antigens shared between mesothelioma and pancreatic cancer cell lysates or by the administration of mature DCs themselves. To this end, KPC-3C57B1 / 6 mice were treated with unloaded (i.e. in the absence of tumor lysates) but matured DCs (stimulated with CpG) or DCs matured and loaded with mesothelioma AE17 lysates (see schematic setup: Figure 5 ).

[0179] Unloaded but mature DCs (referred to as unloaded DCs or DCs only) are not intentionally loaded with tumor-specific antigens. However, mature DCs will present peptides that come into contact with them, and without bound peptides in the MHC groove, DCs will never express MHC molecules. In this experiment, DCs will absorb peptides during the culture process. These peptides / antigens will likely not overlap with tumor-associated antigens.

[0180] Mice treated with mesothelioma lysate-loaded DCs had a significant tumor growth delay, indicating that loading DCs with mesothelioma lysate induced a tumor-specific immune response against pancreatic tumors ( Figure 6 ).

[0181] Example 5

[0182] Induction of pancreatic tumor-specific immune responses

[0183] To monitor whether DCs loaded with mesothelioma lysate induced pancreatic tumor-specific immune responses, splenocytes and tumors were isolated on the day of sacrifice from treated and untreated tumor-bearing mice of Example 4. Bone marrow was harvested from wild-type non-tumor-bearing mice for mature DC culture.

[0184] DCs were cultured from mouse bone marrow with GM-CSF and loaded with autologous pancreatic tumor lysate or healthy lung lysate as a control. Autologous pancreatic lysate and healthy lung lysate were prepared by bead-mediated homogenization of rapidly frozen terminal tumor or lung tissue, respectively. DCs loaded with autologous pancreatic tumor or control lung lysate were co-cultured with thawed splenocytes for 24 hours. A schematic overview of this (potency) assay is shown in Figure 7 middle.

[0185] After co-culturing DCs loaded with autologous pancreatic tumor lysate with splenocytes from mice treated with mesothelioma-loaded DCs, we found increased expression of the cytotoxic markers CD107 and granzyme B, as well as the proinflammatory cytokines IFNγ and TNFα, in CD8+ T cells compared with splenocytes from untreated mice or from mice treated with unloaded DCs ( Figure 8 ).

[0186] No increase in these cytotoxic markers and proinflammatory cytokines was observed when DCs loaded with control lung lysate were co-cultured with splenocytes from treated or untreated mice.

[0187] Example 6

[0188] Description of the manufacturing process for the MesoPher drug substance (dendritic cells loaded with tumor lysate) for clinical application.

[0189] The apheresis product is the cell starting material produced by a standard 9L leukapheresis procedure to collect mononuclear cells using an apheresis unit according to hospital procedures. After the procedure, the product is transferred to a clean room and prepared for the CliniMACS procedure by labeling it with CD14+ microbeads. The CD14+ mononuclear cell product is transferred to a 200 ml conical tube, centrifuged, and resuspended in X-VIVO 15 medium supplemented with 2% human serum / HS (= culture medium) to a final concentration of 100×10 6 / 30ml. The cell suspension was inoculated into 225cm 2 30 ml / bottle of culture medium was added. The flask was incubated overnight in a 37°C, 5% CO2 incubator. The remaining cells were cryopreserved in 10% DMSO.

[0190] On day 2, 15 ml of culture medium was replaced per flask with 15 ml of fresh culture medium supplemented with the cytokines GM-CSF and IL-4. The final cytokine concentrations were 800 IU / ml GM-CSF and 500 IU / ml IL-4. The monocytes were cultured at 37°C, 5% CO2 for 4 days.

[0191] On day 5, cells were harvested from the flasks into 200 ml tubes and centrifuged. The final volume was a maximum of 840 ml (420 × 10 6 DC) and minimum 200ml (100×10 6 DC) Cell products were diluted to 0.5×10 6 / ml. The suspension was supplemented with 800 IU / ml GM-CSF, 500 IU / ml IL-4, 1:3 TCE PheraLys product / DC (TCE: tumor cell equivalent), and 10 μg / ml endotoxin-free keyhole limpet hemocyanin (KLH). The cell suspension was plated into 6-well plates. The 6-well tissue culture plates were incubated in a 37°C, 5% CO2 incubator for an additional 2 days.

[0192] On day 8, DCs were matured by adding fresh culture medium supplemented with maturation factors to a final concentration of 5 ng / ml IL-1β, 15 ng / ml IL-6, 20 ng / ml TNF-α, and 10 μg / ml PGE2. The 6-well tissue culture plates were incubated for an additional 2 days at 37°C in a 5% CO2 incubator.

[0193] On day 10, mature DCs were harvested and centrifuged. After centrifugation, the culture supernatants were collected separately. The cells were resuspended and combined in 50 ml of PBS. This suspension was subjected to a density gradient centrifugation (Lymphoprep) step in 2 x 50 ml tubes to remove excess PheraLys. Cells (DCs) were collected from the gradient interface and washed in PBS by centrifugation. The final volume of the suspension was 50 ml in a 50 ml tube. The total cell number was determined by cell counting.

[0194] The cell suspension produced in step 10 is defined as the MesoPher drug substance (DS).

[0195] Example 7

[0196] The lysate or pharmaceutical composition according to the present invention is used for clinical use in treating pancreatic cancer.

[0197] A Phase II study of MesoPher in patients with pancreatic cancer is currently recruiting. The study overview is as follows:

[0198] Objective: To investigate the feasibility, safety, and toxicity of, and the induced immune responses following vaccination with allogeneic tumor cell lysate loaded onto autologous dendritic cells in patients with resected pancreatic cancer receiving standard of care therapy.

[0199] Study Design: Open-label, single-center phase II study

[0200] Study Population: Patients 18 years and older with surgically resected pancreatic cancer receiving standard of care

[0201] Sample size: 10 patients

[0202] Investigational treatments:

[0203] preparation :MesoPher:Autologous monocyte-derived dendritic cells loaded with PheraLys

[0204] dose : 25 million DC loads

[0205] Route of administration : 1 / 3 injected intradermally in the forearm, and 2 / 3 injected intravenously

[0206] Number of doses : A total of 5 vaccinations.

[0207] Dosage regimen : 3 biweekly doses and 2 additional gifts (3 and 6 months after the last dose)

[0208] Inclusion criteria :

[0209] Pancreatic cancer that has been surgically removed.

[0210] Complete postoperative standard of care. Standard of care treatment includes the option of adjuvant chemotherapy. Patients who do not complete adjuvant chemotherapy due to toxicity or who cannot start standard of care for specific reasons will be allowed to participate in the study after approval by the coordinating investigator.

[0211] No disease activity as assessed by radiographic imaging.

[0212] Patients must be at least 18 years of age and must be able to give written informed consent.

[0213] • Patients must be ambulatory (WHO-ECOG performance status 0, 1, or 2) and medically stable.

[0214] Patients must have normal organ function and adequate bone marrow reserve: absolute neutrophil count >1.0×109 / l, platelet count >100×109 / l, and Hb >6.0 mmol / l (as determined during screening).

[0215] • Positive DTH skin test (induration > 2 mm after 48 hours) to at least one positive control antigen, tetanus toxoid (see section 8.3 for DTH skin test procedure).

[0216] Females of childbearing potential must have a negative serum pregnancy test at Screening and a negative urine pregnancy test on Day 1, prior to the first study drug administration, and must be willing to use an effective method of contraception (intrauterine device, hormonal contraceptives, birth control pills, implants, transdermal patches, hormonal vaginal devices, extended-release infusions) or true abstinence (when consistent with preferred and usual lifestyle)* during the study and for at least 12 months after the last study drug administration.

[0217] *True abstinence is acceptable when it is consistent with the subject's preferences and usual lifestyle. Cyclic abstinence (e.g., calendar, ovulation, symptothermal, postovulation) and withdrawal are unacceptable methods of contraception.

[0218] Men must be willing to use effective contraception (eg, condoms, vasectomy) during the study and for at least 12 months after the last study drug administration.

[0219] ·Ability to return to the hospital for adequate follow-up as required by the protocol.

[0220] Written informed consent according to ICH-GCP.

[0221] Exclusion criteria:

[0222] Medical or psychological barriers to adherence to the protocol.

[0223] Current or previous treatment with immunotherapeutic agents.

[0224] Current use of steroids (or other immunosuppressants). Patients must have been off steroids for 6 weeks and must have stopped any such treatment during the study period. Prophylactic use of dexamethasone during chemotherapy was excluded from the 6-week interval.

[0225] Previous malignancy, except for adequately treated basal cell or squamous cell skin cancer, superficial bladder cancer or carcinoma in situ, or other cancers in which the patient has been disease-free for five years.

[0226] Serious concomitant illness or active infection.

[0227] History of autoimmune disease or organ transplant (or active acute or chronic infection, including HIV and viral hepatitis).

[0228] Serious concurrent chronic or acute illness, such as pulmonary disease (asthma or COPD), cardiac disease (NYHA class III or IV), liver disease, or other illness that the study coordinator determines constitutes an unwarranted high risk for investigational DC treatment.

[0229] Known allergic reaction to shellfish (may contain keyhole limpet hemocyanin (KLH)).

[0230] Pregnant or breastfeeding women.

[0231] Inadequate peripheral venous access for leukocyte apheresis.

[0232] Concomitant participation in another clinical intervention trial (except for participation in a biobank study).

[0233] Organic brain syndrome or other significant psychiatric disorder that would impair the ability to give informed consent and prevent participation in the full protocol and follow-up.

[0234] There was no guarantee of adherence to the protocol. There was no availability of follow-up assessments.

[0235] Example 8

[0236] CD40 agonist enhances mesothelioma lysate-pulsed DC immunotherapy

[0237] Tumor growth delay was observed in mice treated with CD40 and DC vaccination

[0238] Immunocompetent C57bl / 6 mice were injected subcutaneously in the right flank with 1×10 5 Pancreatic tumor cells were injected subcutaneously on day 5 after tumor injection. Mice were treated with a DC vaccine consisting of monocyte-derived DC loaded with mesothelioma lysate (AE17), a CD40 agonist monoclonal antibody (FGK45, Bio X Cell), or both. 6 DCs and 1 × 10 6 In addition, 100 μg of CD40 agonistic monoclonal antibody or its isotype (clone 2A3, Bio X Cell) was injected on days 6 and 12. Mice were monitored by measuring tumor size 2 to 3 times per week until the tumor reached 1000 mm. 3 ( Figure 9 ).

[0239] Tumor growth was significantly delayed in mice vaccinated with DCs and treated with CD40 agonistic monoclonal antibodies compared to untreated mice. This delay in tumor growth was not observed in mice treated with DC monotherapy or CD40 agonistic monotherapy alone ( Figure 10 ).

[0240] DC vaccination has a significant effect on the CD4+ T cell compartment

[0241] FACS analysis was performed on blood samples from all mice on day 9 after tumor injection. Compared to untreated mice, the frequencies of activation markers CD69 and proliferation marker Ki67 on both CD4 and CD8 T cells were increased in mice treated with both DC vaccination and CD40 agonist treatment. + There were significant differences in T cell activation and proliferation ( Figure 11 A) Characterization of CD44 + CD62L - Similar effects were observed in the T cell effector memory compartment ( Figure 11 B).

[0242] Tumors were isolated and analyzed by flow cytometry at the end of the disease. An increase in the frequency of tumor-infiltrating lymphocytes was observed in all treated groups compared to untreated mice. In the group receiving DC vaccination, CD4 + The increase in T cell numbers was even more significant. Compared to untreated mice, the frequency and number of T regulatory cells did not increase after CD40 agonist treatment, DC treatment, or combined treatment ( Figure 12 ).

[0243] Treatment of larger tumors (10 days)

[0244] To determine whether the positive effects observed in 5-day-old tumors were also seen in larger tumors (10-day-old), a more intensive treatment regimen ( Figure 13 In this experimental setting, tumor growth and survival of mice treated with either monotherapy DC vaccination (i.e., AE17) or CD40 agonistic monoclonal antibody (i.e., FGK45) alone were not significantly different from those of untreated tumor-bearing mice. However, combination therapy significantly delayed tumor growth, as Figure 14 It also resulted in improved survival, as shown in Figure 15 Interim peripheral blood analysis showed that both DC vaccination and CD40 agonistic monoclonal antibody treatment induced higher frequencies of CD69+, Ki-67+, and PD-1+ T cells. Combination therapy induced higher frequencies of CD69+, Ki-67+, and PD-1+ for both CD4+ and CD8+ T cells.

[0245] in conclusion

[0246] Reduced growth of established tumors was observed in mice treated with DC therapy in combination with a CD40 agonist. DC therapy induced unique properties of immune cells in the circulation as well as in the tumor. This was primarily present in the CD4+ T cell compartment. Addition of a CD40 agonistic monoclonal antibody enhanced DC vaccination, resulting in a significant reduction in tumor growth compared to untreated mice. This was not seen in mice treated with agonistic CD40 monotherapy or DC treatment alone. This may be the result of modulation of the desmoplastic stroma characteristic of pancreatic cancer, leading to an influx of tumor-specific T cells.

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[0313] The embodiments corresponding to the original claims of the parent case of this application are hereby incorporated into this specification:

[0314] 1. A method for treating pancreatic cancer comprising administering to a patient in need thereof a CD40 agonist in combination with dendritic cells loaded with a lysate, wherein the lysate is obtainable by a method comprising the steps of:

[0315] i) providing human mesothelioma tumor cells from at least two different mesothelioma tumor cell lines;

[0316] ii) inducing necrosis in said tumor cells; and

[0317] iii) lysing the necrotic tumor cells to obtain lysates.

[0318] 2. The method of claim 1 , wherein the pancreatic cancer is unresected pancreatic cancer.

[0319] 3. The method according to any one of the preceding items, wherein the pancreatic cancer is primary pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer or borderline resectable pancreatic cancer.

[0320] 4. The method according to any one of the preceding items, wherein the pancreatic cancer is recurrent pancreatic cancer.

[0321] 5. The method according to any one of the preceding items, comprising a pharmaceutical composition comprising the dendritic cells loaded with the lysate and a pharmaceutically acceptable carrier.

[0322] 6. The method according to any of the preceding items, wherein the CD40 agonist is administered to the patient after administration of the dendritic cells.

[0323] 7. The method according to any of the preceding items, wherein the CD40 agonist is selected from CP-870, CP-893, CDX-1140, APX005M, RG7876 / serilumab, ADC-1013 / JNJ-64457107, ABBV-428, SEA-CD40, or MEDI5083.

[0324] 8. The method according to any of the preceding items, wherein the induction of necrosis of the mesothelioma tumor cells is achieved by subjecting the cells to freeze-thaw cycles.

[0325] 9. The method according to any of the preceding items, wherein after inducing necrosis and lysis of the tumor cells, the lysate obtained is irradiated with at least 50 Gy, preferably at least 100 Gy.

[0326] 10. The method according to any of the preceding items, wherein the mesothelioma tumor cells provided comprise tumor cells from at least three, preferably at least four, most preferably at least five mesothelioma tumor cell lines.

[0327] 11. The method according to any of the preceding items, wherein the mesothelioma tumor cells from the at least two, at least three, at least four or at least five mesothelioma tumor cell lines are provided in essentially equal amounts.

[0328] 12. The method according to any one of the preceding items, wherein the allogeneic mesothelioma tumor cells used are selected from two or more of the following cell lines: Thorr 01 (Deposit No. DSM ACC3192), Thorr 02 (Deposit No. DSM ACC3193), Thorr 03 (Deposit No. DSM ACC3191), Thorr 05 (Deposit No. DSM ACC3194), Thorr 06 (Deposit No. DSM ACC3195).

[0329] 13. The method according to any one of the preceding items, wherein the lysate comprises at least three, preferably at least five, more preferably at least ten mesothelioma cancer cell-associated antigens.

[0330] 14. The method according to the preceding item, wherein the at least three, preferably at least five, more preferably at least ten mesothelioma cancer cell-associated antigens are selected from the following group: RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1, RAB38 / NY-MEL-1, BING4, MAGE A12, HER-2 / Neu, glypican, LMP2.

[0331] 15. The method according to any of the preceding items, wherein the lysate is loaded onto the patient's autologous dendritic cells.

[0332] 16. The method according to any one of the preceding items, wherein the dendritic cells are loaded with 1 tumor cell equivalent / 100 dendritic cells to 10 tumor cell equivalents / 1 dendritic cell.

[0333] 17. The method according to any one of the preceding items, wherein 1×10 6 to 1×10 9 loaded dendritic cells, preferably 1×10 7 to 1×10 8 loaded dendritic cells, most preferably about 2.5×10 7 Loaded dendritic cells / dose.

[0334] 18. Dendritic cells loaded with a lysate for use in treating pancreatic cancer, wherein the dendritic cells are administered to a patient in need thereof in combination with a CD40 agonist, and wherein the lysate is obtainable by a method comprising the steps of:

[0335] i) providing human mesothelioma tumor cells from at least two different mesothelioma tumor cell lines;

[0336] ii) inducing necrosis in said tumor cells; and

[0337] iii) lysing the necrotic tumor cells to obtain a lysate.

[0338] 19. The dendritic cell for use according to item 1, wherein the pancreatic cancer is unresected pancreatic cancer.

[0339] 20. Dendritic cells for use according to any one of the preceding items, wherein the pancreatic cancer is primary pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer or borderline resectable pancreatic cancer.

[0340] 21. Dendritic cells for use according to any of the preceding items, wherein the pancreatic cancer is recurrent pancreatic cancer.

[0341] 22. The dendritic cell for use according to any one of the preceding items, comprising a pharmaceutical composition comprising the dendritic cell loaded with the lysate and a pharmaceutically acceptable carrier.

[0342] 23. Dendritic cells for use according to any of the preceding items, wherein the CD40 agonist is administered to the patient after administration of the dendritic cells.

[0343] 24. The dendritic cell for use according to any one of the preceding items, wherein the CD40 agonist is selected from CP-870, CP-893, CDX-1140, APX005M, RG7876 / selumab, ADC-1013 / JNJ-64457107, ABBV-428, SEA-CD40 or MEDI5083.

[0344] 25. Dendritic cells for use according to any of the preceding items, wherein the induction of necrosis of the mesothelioma tumor cells is achieved by subjecting the cells to freeze-thaw cycles.

[0345] 26. Dendritic cells for use according to any of the preceding items, wherein after inducing necrosis and lysis of the tumor cells, the lysate obtained is irradiated with at least 50 Gy, preferably at least 100 Gy.

[0346] 27. Dendritic cells for use according to any of the preceding items, wherein the mesothelioma tumor cells provided comprise tumor cells from at least three, preferably at least four, most preferably at least five mesothelioma tumor cell lines.

[0347] 28. Dendritic cells for use according to any of the preceding items, wherein said mesothelioma tumor cells from said at least two, at least three, at least four or at least five mesothelioma tumor cell lines are provided in essentially equal amounts.

[0348] 29. The dendritic cell for use according to any of the preceding items, wherein the allogeneic mesothelioma tumor cells used are selected from two or more of the following cell lines: Thorr 01 (Deposit No. DSMACC3192), Thorr 02 (Deposit No. DSMACC3193), Thorr 03 (Deposit No. DSMACC3191), Thorr 05 (Deposit No. DSMACC3194), Thorr 06 (Deposit No. DSMACC3195).

[0349] 30. Dendritic cell for use according to any of the preceding items, wherein the lysate comprises at least three, preferably at least five, more preferably at least ten mesothelioma cancer cell-associated antigens.

[0350] 31. The dendritic cell for use according to the preceding item, wherein the at least three, preferably at least five, more preferably at least ten mesothelioma cancer cell-associated antigens are selected from the following group: RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1, RAB38 / NY-MEL-1, BING4, MAGE A12, HER-2 / Neu, glypican, and LMP2.

[0351] 32. Dendritic cells for use according to any of the preceding items, wherein the lysate is loaded onto autologous dendritic cells of the patient.

[0352] 33. Dendritic cells for use according to any of the preceding items, wherein the dendritic cells are loaded with 1 tumor cell equivalent / 100 dendritic cells to 10 tumor cell equivalents / 1 dendritic cell.

[0353] 34. Dendritic cells for use according to any one of the preceding items, wherein 1×10 6 to 1×10 9 loaded dendritic cells, preferably 1×10 7 to 1×10 8 loaded dendritic cells, most preferably about 2.5×10 7 Loaded dendritic cells / dose.

[0354] 35. A pharmaceutical composition for use in treating pancreatic cancer together with a CD40 agonist, wherein the composition is obtainable by a method comprising the following steps:

[0355] i) providing allogeneic mesothelioma tumor cells from at least two different cell lines, and preparing lysates thereof;

[0356] ii) providing dendritic cells;

[0357] iii) loading said dendritic cells with said lysate of tumor cells and, optionally, providing and adding a pharmaceutically acceptable carrier.

[0358] 36. The pharmaceutical composition for use according to the preceding item, wherein the pancreatic cancer is unresected pancreatic cancer.

[0359] 37. The pharmaceutical composition for use according to any one of the preceding items, wherein the pancreatic cancer is primary pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer or borderline resectable pancreatic cancer.

[0360] 38. The pharmaceutical composition for use according to any one of the preceding items, wherein the pancreatic cancer is recurrent pancreatic cancer.

[0361] 39. A pharmaceutical composition for use according to any one of the preceding items, wherein the dose of the composition administered to a patient comprises 1×10 6 to 1×10 9 loaded dendritic cells, preferably 1×10 7 to 1×10 8 loaded dendritic cells, most preferably about 2.5×10 7 loaded dendritic cells.

[0362] 40. The pharmaceutical composition for use according to any one of the preceding items, wherein the dendritic cells are loaded with 1 tumor cell equivalent / 100 dendritic cells to 10 tumor cell equivalents / 1 dendritic cell.

[0363] 41. Pharmaceutical composition for use according to any of the preceding items, wherein the composition comprises an adjuvant.

Claims

1. Use of a combination of a CD40 agonist and lysate-loaded dendritic cells for the manufacture of a medicament for treating pancreatic cancer in a patient in need thereof, wherein the dendritic cells are autologous to the patient, The lysate can be obtained by a method comprising the following steps: i) providing human mesothelioma tumor cells that are allogeneic to said patient from at least two different mesothelioma tumor cell lines; ii) inducing necrosis in said tumor cells; and iii) lysing the necrotic tumor cells to obtain a lysate, The mesothelioma tumor cells used therein are selected from two or more of the following cell lines: Thorr 01 deposited under Deposit No. DSMACC3192, Thorr 02 deposited under Deposit No. DSMACC3193, Thorr 03 deposited under Deposit No. DSMACC3191, Thorr 05 deposited under Deposit No. DSMACC3194, and Thorr 06 deposited under Deposit No. DSMACC3195. The use according to claim 1 , wherein the pancreatic cancer is unresected pancreatic cancer.

3. The use according to claim 1, wherein the pancreatic cancer is primary pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer or borderline resectable pancreatic cancer. The use according to claim 1 , wherein the pancreatic cancer is recurrent pancreatic cancer. The use according to claim 1 , comprising a pharmaceutical composition comprising the dendritic cells loaded with the lysate and a pharmaceutically acceptable carrier. The use according to claim 1 , wherein the CD40 agonist is a monoclonal antibody.

7. The use according to claim 1, wherein the CD40 agonist is selected from CP-870, CP-893, CDX-1140, APX005M, RG7876 / selukumab, ADC-1013 / JNJ-64457107, ABBV-428, SEA-CD40 or MEDI5083.

8. The use according to claim 1, wherein the induction of necrosis of the mesothelioma tumor cells is achieved by subjecting the cells to freeze-thaw cycles. 9 . The method according to claim 1 , wherein after inducing necrosis and lysis of the tumor cells, the obtained lysate is irradiated with at least 50 Gy. 10 . The use according to claim 9 , wherein after inducing necrosis and lysis of the tumor cells, the obtained lysate is irradiated with at least 100 Gy.

11. The use according to claim 1, wherein the mesothelioma tumor cells provided comprise tumor cells from at least three mesothelioma tumor cell lines.

12. The use according to claim 11, wherein the mesothelioma tumor cells provided comprise tumor cells from at least four mesothelioma tumor cell lines.

13. The use according to claim 11, wherein the mesothelioma tumor cells provided comprise tumor cells from at least five mesothelioma tumor cell lines.

14. The use according to claim 1, wherein the mesothelioma tumor cells from the at least two mesothelioma tumor cell lines are provided in substantially equal amounts.

15. The use according to claim 11, wherein the mesothelioma tumor cells from the at least three mesothelioma tumor cell lines are provided in substantially equal amounts.

16. The use according to claim 12, wherein the mesothelioma tumor cells from the at least four mesothelioma tumor cell lines are provided in substantially equal amounts.

17. The use according to claim 13, wherein the mesothelioma tumor cells from the at least five mesothelioma tumor cell lines are provided in substantially equal amounts.

18. The use according to claim 1, wherein the lysate comprises at least three mesothelioma cancer cell-associated antigens.

19. The use according to claim 18, wherein the lysate comprises at least five mesothelioma cancer cell-associated antigens.

20. The use according to claim 18, wherein the lysate comprises at least ten mesothelioma cancer cell-associated antigens.

21. The use according to claim 18, wherein the at least three mesothelioma cancer cell-associated antigens are selected from the group consisting of RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1, RAB38 / NY-MEL-1, BING4, MAGEA12, HER-2 / Neu, glypican, and LMP2.

22. The use according to claim 19, wherein the at least five mesothelioma cancer cell-associated antigens are selected from the group consisting of RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1, RAB38 / NY-MEL-1, BING4, MAGEA12, HER-2 / Neu, glypican, and LMP2.

23. The use according to claim 20, wherein the at least ten mesothelioma cancer cell-associated antigens are selected from the group consisting of RAGE1 / MOK, mesothelin, EphA2, survivin, WT1, MUC1, RAB38 / NY-MEL-1, BING4, MAGEA12, HER-2 / Neu, glypican, and LMP2.

24. The use according to claim 1, wherein the lysate is loaded onto the patient's autologous dendritic cells.

25. The use according to claim 1, wherein the dendritic cells are loaded with 1 lysed mesothelioma tumor cell / 100 dendritic cells to 10 lysed mesothelioma tumor cells / 1 dendritic cell.

26. The use according to claim 1, wherein the dose of the drug to be administered to a patient in need thereof for said treatment comprises 1×10 6 to 1×10 9 loaded dendritic cells.

27. The use according to claim 26, wherein the dose comprises 1×10 7 to 1×10 8 loaded dendritic cells.

28. The use according to claim 26, wherein the dose comprises 2.5×10 7 loaded dendritic cells.

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