Methods for enhancing tumor immunogenicity using modified tumor cells and modified dendritic cells, and compositions for autologous cancer immunotherapy products.

Abstract

To provide an improved composition that stimulates an anti-cancer immune response in cancer patients. [Solution] A composition is provided comprising cancer cells and dendritic cells (DCs) from the same cancer patient, wherein the cancer cells or DCs, or both, are modified ex vivo to improve the accumulation or immunogenicity of tumor-associated antigens (TAAs) expressed by the patient's cancer.

Description

Background Art

[0001] The interaction between malignant cells and the immune system involves, in particular, the elimination of cancer cells by the innate and adaptive immune systems by cytotoxic T lymphocytes (CTLs) that recognize specific tumor-associated antigens (TAAs), or the balance between the immune system and resistant cancer cells, or the avoidance of immune control that enables cancer cells to evade and leads to the ultimate clinical detection of cancer. Certain immunotherapies, such as the cytokine interleukin-2, promote existing immune responses, and checkpoint inhibitors such as anti-CTLA-4, PD-1, and anti-PD-L1 can unleash antitumor responses that were suppressed by such inhibitors. However, many cancer patients lack sufficient immune recognition of malignant cells and cannot effectively control or eliminate cancer in such ways. That is, they do not have evidence of CTL tumor infiltration as a sign of an ongoing immune response or an increase in PD-1 expression, or evidence of an increase in PDL-1 expression as a sign of a blunted immune response. Therefore, there remains a need for improved methods of stimulating an anti-cancer immune response in cancer patients.

Summary of the Invention

Means for Solving the Problems

[0002] The disclosed embodiments include autoimmunotherapy products comprising autologous dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) from autologous cancer cells. Such products, and the pDCs they contain, are generated ex vivo and do not include DCs that occur in the body through natural processes or upon exposure of the body to the class of agents disclosed herein. Nevertheless, these products and compositions, after being generated ex vivo, may be administered to the body of a subject, particularly a subject requiring treatment for cancer. In various embodiments, DCs or cancer cells, or both, are manipulated in vitro to enhance their immunogenicity targeting TAAs. Live cancer cells obtained from the tumor of a patient being treated may be exposed to agents that increase the expression and / or accumulation of TAAs in the tumor cells, thereby increasing the amount of TAA present, or decrease the tolerogenicity of the cancer cells. Similarly, cancer cells may be treated to improve their immunogenicity. DCs may be exposed to aminoglycosides that alter intracellular endosomal-lysosome transport, thereby enhancing the cross-presentation of exogenous antigens. DCs are loaded and treated with TAA because they are exposed to lysed tumor cells or whole but non-viable tumor cells. The TAA-loaded DCs can then be administered as an immunotherapy product to the original donor cancer patient. See Figure 1.

[0003] The disclosed embodiments include methods for modifying cancer cells to improve their TAA-specific and general immunogenicity, and methods for modifying DCs to increase their level of cross-presentation. Further embodiments include compositions comprising modified DCs, modified cancer cells or their lysates, or both. The DCs are loaded with antigens by culturing them in the presence of inactivated cancer cells or cell lysates (cancer cell material). In some embodiments, no procedure is taken to remove residual cancer cell material from the DC culture after antigen loading, so compositions comprising antigen-loaded DCs contain residual cancer cell material that has not yet been taken up by the DCs unless otherwise specified. Autoantigen-loaded DCs are the main components of the personalized anti-cancer immunotherapy products described herein. The use of these immunotherapy products in the treatment of cancer, and methods of cancer treatment including the administration of these immunotherapy products are also disclosed. Finally, specific methods for modifying DCs and cancer cells to improve the immunogenicity and efficacy of immunotherapy products in which they are components are disclosed.

[0004] Cross-processing by DCs can be increased by exposure to aminoglycoside antibiotics such as gentamicin or to Toll-like receptor 4 (TLR-4) agonists such as lipopolysaccharide (LPS). In some embodiments, the cross-processing enhancer is added from the beginning of the process of differentiating monocytes into immature DCs. In one aspect of these embodiments, the concentration of the cross-processing enhancer is relatively low. In some embodiments, the cross-processing enhancer is added or its concentration is increased from the start of the DC maturation and antigen loading process. In one aspect of these embodiments, the concentration of the cross-processing enhancer is relatively high. In some embodiments, a TLR-4 agonist such as LPS is used as a maturation agent.

[0005] Cancer cells can be modified by various approaches that depend on different mechanisms to increase TAA expression or accumulation (and thus improve the TAA-specific immunogenicity of cancer cell material) or to increase the overall immunogenicity of cancer cell material. In some embodiments, the method of cancer cell modification uses a single approach. In other embodiments, the method of cancer cell modification uses multiple approaches. In some embodiments, the multiple approaches depend on a single mechanism. In other embodiments, the multiple approaches each depend on a distinct mechanism. In yet another embodiment, some of the multiple approaches have a common mechanism, but at least one depends on a distinct mechanism. Approaches to improve TAA accumulation include increasing protein expression by epigenetic modification, increasing protein expression by activation of the PI3K / AKT / mTOR pathway, increasing protein accumulation by proteasome inhibition, increasing protein accumulation by reducing autophagy, and increasing protein accumulation by inhibiting apoptosis. Approaches to improve TAA immunogenicity include improving TAA accumulation, increasing general immunogenicity by removing tolerogenic molecules, and increasing general immunogenicity by increasing damage-associated molecular patterns (DAMPs).

[0006] In some embodiments, the mechanism of epigenetic modification includes inhibition of DNA demethylation or histone deacetylation. In some embodiments, the mechanism of activating the PI3K / AKT / mTOR pathway includes inhibition of PTEN or growth factors or This includes the addition of lumon. In some embodiments, the mechanism of proteasome inhibition includes inhibition of proteasome protease activity or inhibition of ubiquitin E3 ligase. In some embodiments, the mechanism of reducing autophagy includes inhibition of lysosomal function by treatment with aminoglycoside antibiotics, for example. In some embodiments, the mechanism of inhibiting apoptosis includes caspase inhibition. In some embodiments, the mechanism of removing tolerogenic signals includes depletion of cholesterol and Wnt ligands. In some embodiments, the mechanism of increasing DAMP includes reduction of autophagy by treatment with aminoglycoside antibiotics, for example.

[0007] For each method of cancer cell modification, there is a corresponding composition comprising modified cancer cells, lysates of cancer cells, or antigen-loaded DCs, where the DCs are loaded with modified cancer cell material. In some embodiments, the DCs are modified DCs. In some embodiments, a specific approach, mechanism, or agent is specifically included. In some embodiments, a specific approach, mechanism, or agent is not specifically included.

[0008] In some embodiments, separate cultures of cancer cells are modified by different approaches, mechanisms, or drugs and then combined for DC antigen loading. In some embodiments, separate cultures of cancer cells are modified by different approaches, mechanisms, or drugs and then used for DC antigen loading in separate DC cultures, after which the antigen-loaded DCs are combined into a single immunotherapy product. In some embodiments, separate cultures of cancer cells are modified by different approaches, mechanisms, or drugs and then used for DC antigen loading in separate DC cultures used to create separate immunotherapy products that are administered separately to patients. In some aspects of these embodiments, the separate immunotherapy products are administered almost simultaneously (within minutes to 48 hours), while in other aspects, the separate immunotherapy products are administered at intervals of several weeks or months. [Brief explanation of the drawing]

[0009] [Figure 1] This outlines the process for manufacturing an immunogenic autoimmune therapy antitumor product using cancer cells and dendritic cells derived from the same patient. [Figure 2] This shows the cell survival response to bortezomib concentrations. The linear portion of the response is shown for concentrations of approximately 1 μM to 5 μM. [Figure 3] These are phase-contrast micrographs of ovarian tumor cell line cultures two days after exposure to various concentrations of bortezomib. [Figure 4] This is a phase-contrast micrograph showing survival rescue with morphological changes after sequential administration of 20 μM Z-VAD-fmk and 5 nM bortezomib. [Figure 5] This is a phase-contrast micrograph showing that the combination of bortezomib and Z-VAd-fmk did not cause any apparent changes in culture morphology or viability. [Figure 6] This shows the average fluorescence intensity of two antigens labeled with different fluorescent agents after tumor cells were exposed to various concentrations of bortezomib. [Figure 7] This image shows the sum of pixels from two different fluorescently labeled antigens after tumor cells have been exposed to varying concentrations of bortezomib. [Modes for carrying out the invention]

[0010] The immune system can specifically recognize and eliminate tumor cells. While the potential to leverage this ability in cancer treatment has long been recognized, success in doing so has been limited at best. Using autologous tumor cells as an immunogen offers several advantages compared to using xenoplasmic tumor cells or individual antigens or epitopes. Cancer cells contain mutated proteins and novel antigens that can function as TAAs, and since these are often unique to each patient, only autologous tumors are a reliable source. Because autologous tumors also contain cancer stem cells and early progenitor cells, TAAs representing their subpopulations are included in the immunogenic composition. Furthermore, using autologous tumors eliminates the need to identify and match each individual antigen to target, as is the case with xenoplasmic cells or pre-made immunogens. By using autologous cells as an antigen source, the immunogenic composition contains a complete complement of antigens against the cancer of an individual patient.

[0011] However, the amount of TAA present in tumor cell preparations may be limited by low steady-state levels of the antigen in tumor cells, or by only a subset of tumor cells expressing the antigen, or both. A specific example is an antigen associated only with rare subpopulations, such as cancer stem cells. A limited amount of TAA in tumor cell preparations can adversely affect the TAA-specific immunogenicity of the preparation. Furthermore, it can improve the general immunogenicity of cancer cells. Cancer cells may also contain tolerogenic molecules such as Wnt ligands, and their removal or depletion can improve the general immunogenicity of cancer cells. Cancer cells may contain pro-inflammatory molecules such as injury-associated molecular patterns (DAMPs), and an increase in DAMPs can increase the general immunogenicity of cancer cells. DAMPs may include various nuclear and cytoplasmic proteins such as heat shock proteins, HMGB-1 (high-mobility group box 1 protein), membrane-bound proteins, and proteins derived from the extracellular matrix after cytotoxicity. DAMPs also include non-protein molecules such as DNA, ATP (adenosine 5'-triphosphate), uric acid, and heparin sulfate. Exposure of cancer cells to gamma interferon, many chemotherapeutic agents, irradiation, certain monoclonal antibodies, activated natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and antibody-dependent cell-mediated injury (ADCC) can also increase DAMP signaling. Therefore, in various embodiments, tumor cells are exposed to one or more agents that cause increased protein expression, decreased proteolysis, promote TAA accumulation in tumor cells, deplete tolerogenic molecules from cancer cells, or increase cancer cell production of DAMP. Some embodiments specifically involve exposure to a particular agent of a class of agents. Other embodiments specifically do not involve exposure to a particular agent of a class of agents.

[0012] Enhancing or strengthening protein expression not only increases the TAA expression level in individual cells but also improves the uniformity of antigen expression across the cancer cell population. Increasing the amount of TAA in tumor cell preparations increases the likelihood, by whatever mechanism, that there is enough substance to be immunogenic. In this way, an immune response can be obtained against TAA that is naturally expressed at levels too low to be effective immunogens in the body. However, low levels of antigen expression by cancer cells in the body may be sufficient to be recognized by cytotoxic T lymphocytes (CTLs) and antibodies, and if such an immune response can be triggered in the first place, it can lead to the destruction of cancer cells.

[0013] Antigen processing of protein antigens proceeds via two paradigm pathways. In one, exogenous antigens are phagocytosed by antigen-presenting cells (APCs), including DCs, and partially degraded in the endosomal compartment to produce peptides (epitopes) associated with class II MHC. These peptide-MHC II complexes are displayed on the surface of APCs, where, among other possibilities, they are recognized by CD4+ T cells and authorized to provide T cell assistance to B cells, supporting the induction and maturation of an antibody response against the antigen. In the other paradigm pathway, endogenous protein antigens are typically degraded by proteasomes, and the resulting peptides (epitopes) are associated with class I MHC in the endosomal compartment. These peptide-MHC I complexes are displayed on the surface of APCs and recognized by CD8+ T cells that mature into CTLs.

[0014] Antibodies can be a crucial part of the antitumor response to the extent that TAAs are expressed on the surface of tumor cells. However, many TAAs are intracellular proteins and therefore generally do not access antibodies, but rather need to be targeted by CTLs. There are examples of paradigm-shifting antigen processing pathways in which exogenously encountered antigens are bypassed to endogenous antigen processing pathways, leading to antigen presentation and induction of CTL responses associated with class I MHCs. Such cross-presentation can be increased by altering endosomal transport, particularly by delaying phagosome-lysosome fusion, allowing more phagosome material to be released into the cytosol. Such delays in phagosome-lysosome fusion are achieved by exposing DCs to aminoglycoside antibiotics or by activating TLR4 by exposing DCs to TLR4 agonists such as LPS (lipopolysaccharide), glucuronoxylomannan, and morphine-3-glucuronide. In this way, DCs can more efficiently stimulate CTL responses to a wider range of TAAs. Since the treatment does not eliminate presentation in relation to class II MHC, humoral and cellular immunity is stimulated.

[0015] In some embodiments, both live cancer cells and DCs are manipulated to enhance TAA expression and cross-presentation, respectively. In other embodiments, cancer cells with enhanced TAA expression are used to deliver antigens to unmanipulated DCs. In yet another embodiment, live cancer cells that have not been manipulated to enhance TAA expression are used to deliver antigens to manipulated DCs to enhance cross-presentation. However, the combined effect of improved antigen availability for uptake by DCs and enhanced cross-presentation by DCs is expected to synergistically improve the immunogenicity of manipulated antigen-loaded DCs as a cellular immunotherapy product. Therefore, in various embodiments, the immunotherapy product is prepared using modified cancer cells (or their lysates), modified DCs, or both.

[0016] In some embodiments, the patient is human. In other embodiments, the patient is a non-human mammal, such as a dog, cat, or horse. In some embodiments, the non-human mammal is not a rodent.

[0017] Isolation of live cancer cells from tumors During tumor removal or bulking surgery, live tumor cells are removed from the cancer patient's body. In some cases, the surgery involves the removal of the entire tumor(s). In other cases, it is necessary to remove an entire organ or a substantial portion of it. If both diseased and normal tissue are present in the removed tissue, the tumor tissue is separated from the normal tissue. In other cases, the surgery involves biopsies, including but not limited to punch or needle biopsies. In the case of solid tumors, the tissue is fragmented and dissociated by enzymatic digestion. In the case of leukemia, live cancer cells can be recovered from the blood, for example, by density gradient sedimentation of whole blood or leukocytapheresis. In the case of ascites tumors, live tumor cells can be recovered by draining and then sedimenting the ascites fluid. The number of live cancer cells recovered varies depending on the size of the tumor and the specific recovery method, but generally, a larger number of cells is preferred, especially in the case of tumors that do not grow well in culture. Therefore, in various embodiments, 10 5 from 10 7 from 10 9 Or an order of magnitude more than one order of live cancer cells are recovered. After separation from digested extracellular matrix and other debris, the live cells are transferred to a cell culture medium rich in nutrients for proliferation and / or exposed to one or more agents to enhance TAA expression and accumulation.

[0018] In some embodiments, the tumor or cancer cells are derived from any malignant neoplasm. Some embodiments specifically include a particular class or type of cancer, while others specifically exclude it. They may be classified as solid tumor formation or as cells suspended in bodily fluids. They may be classified according to the tissue of origin, such as the brain, head and neck, esophagus, lungs, liver, pancreas, kidneys, stomach, colon, prostate, breast, uterus, cervix, ovaries, skin, bone, blood, eye, or retina. They may be classified into specific types, such as melanoma, non-small cell lung cancer, glioblastoma, and renal cell carcinoma. They may be further subdivided according to the expression of biomarkers, such as triple-negative breast cancer, hormone-resistant prostate cancer, and PD-L1-positive (or negative) lung cancer. They may also be subdivided according to the progression of the disease: non-invasive, invasive, metastatic; stage 0, 1, 2, 3, or 4; and various scales associated with a particular cancer. Cancers can also be classified according to the phenotypically significant mutations they carry, such as mutations in p53 or B-Raf.

[0019] Enhancement and strengthening of antigen expression and immunogenicity in isolated cancer cells. A variety of agents are available that can increase the accumulation or exposure of TAAs or reduce the tolerogenicity of tumor cells by any of a number of mechanisms. These include increased protein expression, reduced proteolysis, inhibition of apoptosis, and depletion of cholesterol and Wnt ligands from the cell membrane. In some embodiments, the isolated cancer cells are cultured for from 24 hours to 4 weeks. In other embodiments, the culture period extends from 4 to 6 weeks, from 4 to 8 weeks, or longer. In still other embodiments, the culture period is short, lasting for a number of hours, such as 2, 3, 4, 5, 6 hours or more, but not exceeding 24 hours. In some embodiments, the culture period is divided into a growth phase to increase the number of available cancer cells, followed by an enhancement phase in which the cancer cells are modified to improve their overall immunogenicity. In other embodiments, the growth phase and the enhancement phase coincide or there is no growth phase. Depending on the enhancer and its mechanism of action, in some embodiments, the enhancer will be present throughout the enhancement phase. In other embodiments, the enhancer is present only during the last few hours of the culture or only on the last day of the enhancement phase, or is present throughout the enhancement phase but the concentration of the agent will increase during this final period. In still other embodiments, the enhancer is present only during the first few hours of the culture or only on the first day of the enhancement phase, or the concentration of the agent decreases after this initial period but is maintained at a lower concentration throughout the remainder of the enhancement phase. In some embodiments, the cancer cells will be exposed to the agent(s) during exactly one of the enhancement procedures described hereinbelow. In other embodiments, the cancer cells are exposed to multiple enhancement procedures, such as 2, 3, 4, or more of the enhancement procedures described hereinbelow. In some embodiments, these modified cancer cells will be exposed to autologous DCs immediately after the enhancement phase. In other embodiments, these modified cancer cells are cryopreserved for later exposure to autologous DCs.

[0020] Epigenetic modification Protein expression levels can be increased by epigenetic modifications in cancer cells. Compared to physiologically normal cells, cancer cells have epigenetic modifications that reduce the expression of various proteins. Therefore, by reversing the acquired epigenetic modifications in cancer cells, the expression of downregulated TAAs can be restored.

[0021] Transcriptional activation is associated with acetylation of lysine residues in the histone tail. One epigenetic mechanism that can downregulate protein expression is deacetylation of lysine residues in the histone tail. Exposure to histone deacetylase inhibitors (HDIs) can be used to increase mRNA transcription, which is accompanied by an increase in protein expression and the antigen content of cancer cells. Examples of HDIs include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (such as trapoxin B), and depsipeptides, benzamides, electrophilic ketones, and fatty acid compounds such as phenylbutyrate and valproic acid. Second-generation HDIs include the hydroxamic acid vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589); and benzamides: entinostat (MS-275), CI994, and mocetinostat (MGCD0103). Furthermore, class III histone deacetylases are NAD + -dependent and are thus inhibited by nicotinamide and derivatives of NAD such as dihydrocoumarin, naphthopyranone, and 2-hydroxynaphthaldehyde. Therefore, these agents can be used to increase the transcriptional activation and expression levels of TAAs.

[0022] The promoters of protein-coding genes typically exhibit an increased frequency of CgG dinucleotides, known as CpG islands. These CgG dinucleotides can be methylated, and hypermethylation of these dinucleotides in CpG islands can lead to transcriptional silencing. Such hypermethylation is a stable epigenetic change that can be inherited by daughter cells after mitosis. While such silencing plays a role in normal physiological functions, such as regulating gene quantity, abnormal hypermethylation is frequently observed in cancer. Demethylating agents can be used to reverse the expression of silenced genes, including germline or tumor-specific genes that may be expressed by cancer cells.

[0023] Demethylating agents such as 5-azacitidine (azacitidine, 5-aza-CR; Vidaza®, Celgene Corp., Summit, NJ, USA) and 5-aza-2'-deoxycytidine (decitabine, 5-aza-CdR (Dacogen®, SuperGen, Inc., Dublin, CA, USA)) were previously used as anticancer agents, but they operate through different mechanisms. At high concentrations, these drugs disrupt normal polynucleotide physiological functions to a degree of cytotoxicity. Azacitidine, which is preferentially incorporated into RNA, disrupts protein synthesis, while decitabine is incorporated only into DNA and, at low concentrations, inactivates DNA methyltransferase and disrupts the heritability of CpG methylation patterns. Therefore, these demethylating agents can be used to increase TAA expression, and a preferred embodiment uses decitabine as the demethylating agent.

[0024] Inhibition of proteomic degradation Proteasome inhibitors are well-known therapeutic agents used to disrupt the metabolic turnover of tumor cell proteins that trigger caspase-activated cell death. In normal cells, proteasomes regulate protein expression and function by degrading ubiquitinated proteins, and further cleanse cells of abnormal or misfolded proteins.

[0025] The proteasome is the cell's primary neutral protein degradation machinery, playing a major role in normal protein turnover, as well as in the degradation of cytosolic and nuclear damage, misfolds, and abnormal proteins, particularly ubiquitinated proteins. While prolonged blockage of protein degradation leads to cell death, it should initially cause protein accumulation, otherwise degradation. Proteasome inhibitors and enzymes in the non-victination pathway, especially E3 ligases, can be used to block protein degradation.

[0026] Misfolded proteins are generated in all compartments of a cell due to a variety of stress conditions, including transcriptional and translational failures, genomic mutations, or oxidation or heat. These misfolded proteins target proteolytic pathways, most notably the ubiquitin-proteasome system and the autophagy vacuole (lysosome) system. Therefore, inhibiting ubiquitin, a component of the proteasome system, leads to the accumulation of misfolded or mutant (nascent antigen) proteins that may have antigenic value. As a secondary consequence, increased lysosomal processing can lead to increased MHC presentation to the immune system.

[0027] While multiple mechanisms are likely involved, proteasome inhibition is thought to prevent the degradation of pro-apoptotic factors, thereby inducing programmed cell death in newly formed cells. Proteasome inhibition has also been shown to alter the balance of intracellular peptides after short-term administration at relatively high doses (50-500 nM).

[0028] Various non-peptide and peptide, reversible and irreversible inhibitors of the 20S proteasome have been identified that can enter mammalian cells and inhibit protein degradation via the ubiquitin-proteasome pathway. The first non-peptide proteasome inhibitor discovered was the natural product lactacystin. Other proteasome inhibitors include disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770), the natural selective inhibitor of epixomycin, beta-hydroxybeta-methylbutyrate (HMB), bortezomib, carfilzomib, and ixazomib. E3 ligase inhibitors include nutlin-3, JNJ-26854165 (cell demethan), NVP-CGM097, NSC207895, N-(4-butyl-2-methylphenyl)acetamide (SKP2E3 ligase inhibitor II), 5-(3-dimethylaminopropylamino)-3,10-dimethyl-10H-pyrimido[4,5-b]quinoline-2,4-dione (Hdm2E3 ligase inhibitor II), and 9H-indeno[1,2-e][1,2,5]oxadiazolo[3,4-b]pyrazine-9-one (SMER3). Therefore, these drugs can be used to induce the accumulation of TAA in cancer cells.

[0029] In vivo use of bortezomib has been shown to dramatically impair the ability of innate human blood dendritic cells (DCs) to modulate innate and adaptive antitumor immunity, which influences the design of therapeutic strategies combining DC vaccination and bortezomib treatment. Proteasome inhibitors lead to the accumulation of caspase cascade components that are normally degraded by proteasomes, resulting in apoptosis-induced cell death.

[0030] In some embodiments, ex vivo treatment of cancer cells with proteasome inhibitors such as bortezomib is used to avoid dendritic cell (DC) damage and death resulting from in vivo exposure. By utilizing ex vivo treatment, DC exposure to proteasome inhibitors can be avoided or reduced. Furthermore, caspase blockade with pancaspase inhibitors (i.e., Z-VAD-fmk) can prevent the initiation of apoptosis in either tumor cells or DCs, which can be counterproductive.

[0031] The simultaneous or sequential use of caspase inhibitors (such as Z-VAD-fmk, a caspase with a broad therapeutic range) and proteasome inhibitors (such as bortezomib) is expected to lead to the accumulation of various peptides and proteins, including novel tumor antigens, thereby increasing antigen loading and triggering a better immune response. Furthermore, in vitro use of proteasome inhibitors with tumor cells avoids exposure of dendritic cells (DCs) to the proteasome inhibitor, thus preventing interference with the antigen presentation and antitumor immune activation functions of DCs.

[0032] Reduce autophagy Aminoglycoside antibiotics are toxic to mammalian cells through selective accumulation in lysosomes and inhibition of lysosomal enzymes. In vitro treatment of tumor cells with gentamicin in the absence of iron ions reduces lysosomal processing and increases TAA accumulation along with enhanced DAMP signaling favorable to phagocytosis by DCs. This effect becomes more pronounced following stress that increases autophagy, such as irradiation or starvation. Therefore, aminoglycoside antibiotics such as gentamicin can be used to reduce or delay autophagy, thereby increasing intracellular protein "junk" and increasing DAMP signaling. In some embodiments, cancer cells are stressed and then exposed to 50-150 μg / ml of gentamicin. Increased DAMP signaling, in addition to the accumulation of TAA-containing proteins, enhances the general immunogenicity of cancer cells.

[0033] Activation of the PI3K / AKT / mTOR pathway The PI3K / Akt / mTOR pathway is a complex signaling pathway involved in many cellular processes, including cell proliferation and survival, cell growth and differentiation, insulin action, protein synthesis and autophagy regulation, and plays a central role in maintaining malignancy in many cancers. Mechanisms of pathway activation include inhibition of tumor suppressor PTEN function, amplification of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), amplification or mutation of Akt, and amplification of growth factor receptors. One of the downstream effects of pathway activation is the activation of mTOR, the main regulator of protein translation. mTOR exists in two complexes, the TORC1 and TORC2 complexes. In the TORC1 complex, mTOR signals its downstream effectors S6 kinase / ribosomal protein S6 and 4EBP-1 / eIF-4E, regulating protein translation. Although mTOR is generally considered a downstream substrate of Akt, mTOR can provide positive feedback to the pathway via the TORC2 complex.

[0034] Both mTORC1 and mTORC2 respond to hormones and growth factors. In particular, mTORC1 appears to be rapidly regulated by nutrients such as amino acids and glucose. The presence of large amounts of leucine and arginine in cell culture media can increase cell growth through increased ribosome and protein biosynthesis and suppression of autophagy. The proliferation of human hepatocellular carcinoma cell lines has been shown to be leucine concentration-dependent in vitro, with growth rates significantly reduced in 0.05 mM leucine-containing media compared to 0.2 mM. In various embodiments, the molar ratio of leucine or arginine to alanine is at least 10:1 or greater, e.g., 25:1, 50:1, or 100:1. Therefore, higher than standard levels of leucine and / or arginine in culture media can increase the proliferation and protein expression (including TAA expression) of cultured cancer cells.

[0035] PTEN (with deletion of the phosphatase and tensin homologs on chromosome 10) is a phosphatidylinositol-3,4,5-triphosphate 3-phosphatase that converts phosphatidylinositol-3,4,5-triphosphate 3-phosphatase to phosphatidylinositol-4,5-diphosphate, counteracting PI3K-mediated PKB / Akt activation. PTEN is a 50kD cytosolic enzyme that transiently interacts with the cell membrane to metabolize its lipid substrates. Loss of function, due to several different mechanisms, is frequently observed in many tumor types. Inhibition of PTEN activity has potent effects on cell proliferation, growth, survival, and associated metabolic changes in many cell lineages. Vanadium compounds, such as sodium orthovanadate, have long been recognized as phosphatase inhibitors. Peroxovanadium compounds such as bisperoxovanadium 1,10-phenanthroline (bpV(phen)) and bisperoxovanadium 5-hydroxypyridine-2-carboxyl (bpV(HOpic)) exhibit increased biological potency and higher target selectivity than simple vanadate compounds. N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide (SF1670) is also a potent and specific inhibitor of PTEN. In some embodiments, PTEN inhibitors are used in combination with agents that act via the PI3K pathway. Such factors include known growth factors (e.g., fibroblast growth factor (FGF), epidermal growth factor (EGF), ventricle growth factor-inducible (VGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), etc.), hormones, integrins (laminin), and other receptor-mediated signaling factors. Therefore, these factors can be used to increase the level of cell activation, including protein synthesis, and to increase the level of TAA expression in cultured cancer cells. Preferred combinations of factors to be added to the cell culture medium are insulin, thyroid hormones, basic FGF, and EGF. Thus, PTEN inhibitors, especially when used in combination with growth factors, can not only increase protein synthesis but also accelerate the growth and proliferation of cancer cells.

[0036] Inhibition of apoptosis Apoptosis is a controlled process of programmed cell death initiated by various stresses and biochemical signals. This is in contrast to necrosis caused by cellular trauma. This process is regulated by a family of enzymes called caspases, which are cytosolic aspartate-specific cysteine ​​proteases. They are involved in the initiation and execution of the apoptotic program. Caspases are expressed as latent zymogens and are activated by autoproteolytic mechanisms or by treatment with other proteases (often other caspases). Human caspases can be subdivided into three functional groups: cytokine activation (caspase-1, -4, -5, and -13), apoptosis initiation (caspase-2, -8, -9, and -10), and apoptosis execution (caspase-3, -6, and -7). Caspases respond to a variety of stimuli, including APAF1, CFLAR / FLIP, NOL3 / ARC, and members of the apoptosis inhibitor (IAP) family such as BIRC1 / NAIP, BIRC2 / cIAP-1, BIRC3 / cIAP-2, BIRC4 / XIAP, BIRC5 / Survivin, and BIRC7 / Livin. IAP activity is regulated by DIABLO / SMAC or PRSS25 / HTRA2 / Omi. In vitro exposure to caspase inhibitors with a broad therapeutic range prevents cell death and enables rapid proliferation of tumor cells through TAA accumulation.

[0037] Table 1 shows non-limiting examples of cell-permeable, reversible or irreversible peptide caspase inhibitors or non-peptide caspase inhibitors of natural or synthetic origin. These agents may be used to increase the number and percentage of viable cancer cells in culture and to enable TAA accumulation.

[0038] [Table 1]

[0039] Depletion of DC inhibitory signaling molecules A common mode of intercellular communication is the secretion of signaling molecules, which are received by neighboring cells. One such signaling pathway is mediated by Wnt ligands, a large family of lipid-modified hydrophobic glycoproteins. In fact, it is the lipid modification that provides hydrophobicity, as the primary sequence is relatively hydrophilic. Reception of Wnt ligands on the cell surface initiates an intracellular signaling cascade that leads to changes in gene transcription. In dendritic cells (DCs), Wnt signaling leads to the activation of β-catenin, a crucial process that promotes tolerance and limits inflammation.

[0040] Abnormal regulation of Wnt signaling is common to many tumor types. Epigenetic and genetic changes associated with malignant status lead to increased Wnt pathway activity. More recently, it has been revealed that Wnt signaling levels identify stem-like tumor cells involved in promoting tumor growth. In tumors, the accumulation of Wnt signaling is partially involved in immune evasion through the regulation of antigen-presenting cells accompanied by excessive Wnt signaling. Furthermore, cancer stem cells, one of the most desirable targets for cancer immunotherapy, appear to be particularly tolerogenic.

[0041] Wnt ligands are preferentially found in the surfactant-resistant portions of cell membranes of cells rich in lipid rafts, cholesterol, gangliosides, and sphingolipids. Depletion of membrane cholesterol disrupts the integrity of lipid rafts and simultaneously depletes Wnt. Among the various cholesterol-depleting agents available, methyl-β-cyclodextrin (MCD), a highly water-soluble cyclic heptasaccharide consisting of β-glucopyranose units, is the most effective agent for cholesterol depletion, along with other lipid-modifying membrane components containing Wnt ligands from cells. In Wnt-enriched tumors, the immunogenicity of cancer cell therapies is improved because capturing and removing Wnt ligands from tumor cells before exposure to DCs prevents the programming of DCs into a tolerogenic state. Therefore, treatment with cholesterol-depleting agents such as MCD also depletes Wnt ligands, resulting in improved immunogenicity of tumor cells, particularly cancer stem cells.

[0042] Enhancement of antigen treatment Cytolytic immune responses are primarily mediated by CD8+ T cells. To stimulate such a response, DCs need to present antigens in association with Class I MHC cells loaded with endogenously expressed antigens. Phagotics are primarily presented in association with Class II MHC cells, but there is a process called cross-presentation that leads to the presentation of phagocytic antigens in association with Class I MHC cells. After ingestion, phagosomes undergo a process called "phagosome maturation," which involves the degradation of phagosome contents through a series of fusion and division events, first in the endosomal compartment and then in the lysosomal compartment. DCs have developed specialized phagocytic pathways that enable optimal conditions for cross-presentation. These specialties include a mildly degradable phagosome environment, transport of antigens to the cytosol for proteasome-mediated degradation, and effective loading of synthesized peptides in the endoplasmic reticulum (ER) or phagosome. The normal fusion of phagosomes and lysosomes leads to the degradation of phagosome contents. Delays in the fusion process enhance the export of phagosome contents to the cytoplasm. Therefore, intentionally delaying phagosome processing of tumor cells may lead to increased cross-presentation of TAAs and enhanced cytotoxic immune responses.

[0043] Aminoglycoside antibiotics accumulate in lysosomes, inhibiting lysosomal enzymes and leading to the construction of autophagy substances. This accumulation triggers a cellular stress response, including the generation of reactive oxygen species (ROS). In the presence of ROS and lysosomal iron, these substances penetrate the lysosomal membrane and are released into the cytosol, inducing apoptosis and cell death. However, the toxicity of DCs can be reduced by the absence of iron in the culture medium. Therefore, low concentrations of aminoglycoside antibiotics, such as gentamicin, increase the level of cross-presentation. TLR4 agonists also mediate the delay of phagosome-lysosome fusion. Therefore, in some embodiments, aminoglycoside antibiotics are supplemented with TLR4 agonists such as LPS, glucuronoxyromannan, or morphine-3-glucuronide.

[0044] Isolation of dendritic cells DCs for use in the embodiments described herein may be obtained by differentiation of monocytes isolated from the blood of the same patient from whom tumor cells are isolated. Techniques for differentiating DCs from monocytes are well established in the art. Briefly, in a typical protocol, peripheral blood mononuclear cells (PBMCs) are isolated from whole blood by density gradient centrifugation. The PBMCs are cultured. Monocytes are adherent, and non-adherent cells are washed away after 1–24 hours. Alternatively, monocytes can be isolated from PBMCs using immunomagnetic beads. Monocytes are cultured for 5–8 days in the presence of GM-CSF and IL-4 to differentiate into immature DCs. At this point, the immature DCs are loosely adherent and can be harvested by gentle pipetting. Next, the immature DCs are cultured for a further 2 days in the presence of maturation factors, typically TLR-4 agonists such as LPS. Alternatively, a monocyte maturation cocktail containing, for example, TNFα, IL-6, IL-1β, and PGE2 can be used. Typically, maturation and antigen loading are performed simultaneously. The procedure can be performed on newly isolated or cryopreserved PBMCs.

[0045] harvesting and inactivation of cancer cells after TAA enhancement. Cancer cell cultures are harvested by enzymatic digestion (e.g., by trypsin TrypLE, collagenase, or dispase) or mechanical scraping after undergoing one or more procedures to enhance TAA expression or accumulation, or to enhance immunogenicity, including but not limited to those described herein. The collected cells are washed with repeated precipitation cycles in a neutral buffer (e.g., phosphate buffer, saline, Hanks equilibrium salt solution, Ringer's solution, etc.) until the medium and enzyme solution are depleted. Total protein can be determined by the biuret method or spectrophotometric method using a dye (Bradford, 3',3'',5',5''-tetrabromophenolphthalein ethyl ester-TBPEE, or erythrosine-B).

[0046] Since these cancer cells are used to prepare immunotherapy products administered to patients / donors, it is important to inactivate them (prevent them from dividing) to ensure that viable malignant cells are not re-administered to patients. One method of inactivation is gamma irradiation by exposure to a radioactive source (such as Cs-137 or Co-60) up to a total cumulative dose of 10–100 Gy (1,000–10,000 Rad). Alternatively, exposure to X-ray or UV irradiation can be used for the same purpose. All irradiated cells can be combined with DCs for antigen loading.

[0047] Cell lysis can be used for inactivation, either in place of or in addition to irradiation. Lysis is achieved by repeated freeze-thaw cycles in isotonic or hypotonic solutions without the addition of cryoprotectants. Mechanical lysis can also be produced by exposure to high-intensity ultrasound using an ultrasonic device. Both bath-type and probe-type ultrasonic devices can be used, but special care must be taken with the latter to avoid cross-contamination of samples. Osmotic lysis is achieved by exposing cells to hypotonic buffer. Other lysis methods consistent with current Good Manufacturing Practices may also be used. Lysates can be combined with DCs for antigen loading. Cancer cell lysates and inactivated whole cancer cells are collectively called cancer cell material.

[0048] Various quality control methods are available to assess whether inactivation is complete. One method is dye exclusion, in which living cells exclude the dye, while inactivated cells are stained. Suitable dyes include trypan blue, which can be used for evaluation by light microscopy or automated methods using cell counters (Vi-CELL®; Beckman Coulter, etc.), and propidium iodide or 7-aminoactinomycin D (7-AAD), which can be used for evaluation by fluorescence microscopy or flow cytometry. Viability can be assessed according to particle size analysis using a cell counter or flow cytometer. Finally, there are various proliferation assays that can be used to detect living cells. These include proliferation assays that rely on the incorporation of radiolabeled nucleotides into DNA, as well as assays that rely on chromogenic products such as formazan dyes formed by the reduction of corresponding tetrazolium salts, as in the MTT and MTS assays.

[0049] In some cases, patients may not be ready to receive immunotherapy products. For example, an immunotherapy regimen may require multiple doses on a specific schedule, but the time for the next dose has not yet arrived. In such cases, inactivated cells or cell lysates may be frozen for future use. In the case of whole inactivated cells, cryoprotectants such as DMSO, glycerol, trehalose, and sucrose are used, and the cells are stored at approximately -135°C to approximately -196°C in an LN2 freezer or similar. Lysates may be stored at temperatures below -20°C. In some embodiments, cancer cells are maintained in culture throughout the treatment period or a significant portion thereof, allowing for multiple cycles of enhanced culture, with fresh harvesting after each scheduled dose. In other embodiments, a single enhanced period and harvest provides cancer cell material for multiple, or even all, doses.

[0050] DC antigen loading Inactivated and enhanced cancer cells or cell lysates (cancer cell material) are added to a culture of immature DCs along with maturation factors such as LPS. The DCs are cultured for a further period to allow antigen uptake and antigen processing to occur. In various embodiments, this antigen treatment period lasts from 4 to 36 hours. In preferred embodiments, the DCs are treated with an aminoglycoside antibiotic, in which case such treatment is continued throughout the treatment period. At the end of the antigen treatment period, aliquots of the DCs are frozen in liquid nitrogen in the presence of an antifreeze until needed. In some embodiments, the antigen-loaded DCs are purified from the cancer cells or cell lysates before administration to the patient. In other embodiments, the mixture is administered to the patient. Single precipitation and resuspension washing may be used to purify the DCs from the lysates. Immunoaffinity methods, such as using immunomagnetic beads, may be used to remove all cancer cells remaining after the antigen treatment period. In other embodiments, the antigen preparation and DCs are not separated. The mixture is used in the immunogenic composition.

[0051] immunity TAA-loaded DCs (DCs combined with cancer cell material) are administered to the patient / donor. Administration is by injection or infusion via a medically appropriate route of injection, including, for example, intravenous, subcutaneous, intramuscular, intradermal, intralymphatic (i.e., into afferent lymphatic vessels), and intranodal (e.g., into inguinal or axial lymph nodes). TAA-loaded DCs may be used as monotherapy or in combination with immune checkpoint inhibitors such as antibodies against CTLA4 (e.g., ipilimumab), PD-1 (e.g., pembrolizumab or nivolumab), and / or PD-L1 (e.g., atezolizumab).

[0052] In some embodiments, the dose is administered weekly for the first month, and then monthly for a total of eight doses. Other embodiments involve only a single dose. Other embodiments continue regular administration until the disease is no longer detectable or there is clear progression of the disease. In yet another embodiment, the immunotherapy product is administered as a series of infusions over several hours, days, weeks, or months. In some embodiments, a new batch of the product is generated from new metastases if and when new metastases appear. The number of DCs administered can vary greatly, from about 1 to about 20 million DCs per dose, for example, 10 million DCs. However, the number of cells used can also be increased or decreased. For example, in some embodiments using multiple bolus injections, the number of DCs per injection may be at or below the lower end of the above range. For example, in some embodiments using infusions over a long period, the total number of DCs administered may approach or exceed the upper end of the above range. In some embodiments, the number of DCs administered is limited by the amount of tumor tissue or DCs that can be obtained from the patient.

[0053] logistics The expertise and infrastructure required to perform the procedures described herein may not be available at all hospitals, clinics, outpatient surgery centers, etc. (collectively, patient treatment facilities) where cancer patients receive treatment. In some embodiments, a central facility equipped with the necessary infrastructure and trained personnel receives the patient's tumor tissue and blood from the treatment site. The central facility may perform procedures to modify cancer cells and / or DCs as described herein to produce a personalized anti-cancer immunotherapy product containing autologous DCs loaded with autologous TAAs, and deliver the immunotherapy product to a patient treatment facility where it can be administered to the patient. In some embodiments, the central facility performs specific modifications of cancer cells and / or DCs in accordance with instructions from the patient's physician that are particularly beneficial to the patient in the physician's judgment. In some embodiments, the immunotherapy product is administered to the patient in accordance with guidance provided by the central facility (such as the time frame in which administration is performed, the dosage, the frequency of administration, the rate of infusion in the case of infusion, or how the immunotherapy product is stored from receipt to administration) and the resulting benefits to the patient (as well as the physician and / or patient treatment facility receiving the benefit of being able to treat the patient). The patient treatment facility where tumor tissue is removed from the patient and the patient treatment facility where the immunotherapy product is administered may be the same or different. “Central facility” means a facility that provides services to multiple patient treatment facilities. The central facility may be located in the same building or on the same campus as one of the patient treatment facilities, or it may be located separately from all of the patient treatment facilities.

[0054] In some cases, the patient benefits in the form of an improvement in cancer (i.e., the progression of the cancer stops, regresses, goes into remission, secondary symptoms improve, or adverse side effects of other treatments are avoided). In aspects of these embodiments, physicians, other healthcare professionals, healthcare facilities (such as patient treatment facilities), health maintenance organizations, and / or central facilities are acting at the patient's request. In other embodiments, the patient's benefit is conditional on agreeing to donate to the organization and receiving the administration of immunotherapy products in accordance with the orders and directions of physicians, other healthcare professionals, healthcare facilities (such as patient treatment facilities), health maintenance organizations, and / or central facilities, or arranging payment for various necessary steps in the method of implementation. In other cases, physicians, other healthcare professionals, healthcare facilities (such as patient treatment facilities), health maintenance organizations, and / or central facilities may obtain reputational or business benefits by being paid to obtain positive outcomes for patients or to perform one or more required steps of a method, and the patients or other stakeholders of the rest of the physician's chain, other healthcare professionals, healthcare facilities (such as patient treatment facilities), health maintenance organizations, and / or central facilities may act at their request. In other forms, the benefits of physicians, other healthcare professionals, healthcare facilities (such as patient treatment facilities), health maintenance organizations, and / or central facilities may be subject to the orders or endorsements of the patients or other stakeholders of the rest of the physician's chain, other healthcare professionals, healthcare facilities (such as patient treatment facilities), health maintenance organizations, and / or central facilities.

[0055] Frozen immunotherapy products (i.e., frozen antigen-loaded DCs) are stored at a central facility until the patient is ready for administration. The single dose is then shipped to the patient's treatment facility, where it is thawed and administered to the patient without further processing or manipulation. If the patient's treatment facility and the central facility are in the same location and the patient is ready to receive the immunotherapy product when the antigen processing phase is complete, the immunotherapy product does not need to be frozen and can instead be administered to the patient rapidly.

[0056] List of specific embodiments The following list of embodiments is illustrative of various embodiments relating to the width, combinations and subcombinations, classes of invention, etc., described herein, but is not intended to be an exhaustive list of all embodiments for which support is found herein. Embodiment 1. A composition comprising cancer cells and dendritic cells (DCs) from the same cancer patient, A composition wherein the cancer cells or DCs, or both, are modified ex vivo to improve the accumulation or immunogenicity of tumor-associated antigens (TAAs) expressed by the patient's cancer. Embodiment 2. A composition comprising DCs from a cancer patient, wherein the DCs are loaded with antigenic material from cancer cells isolated from a tumor removed from the cancer patient, and the cancer cells or the DCs, or both, are modified to improve the accumulation or immunogenicity of TAA expressed by the patient's cancer. Embodiment 3. The composition according to Embodiment 1 or 2, wherein the modification for improving TAA accumulation includes increased protein expression by epigenetic modification. Embodiment 4. The composition according to any one of Embodiments 1 to 3, wherein the modification for improving TAA accumulation includes increasing protein expression by activating the PI3K / AKT / mTOR pathway. Embodiment 5. The modification for improving TAA accumulation is the composition according to any one of Embodiments 1 to 4, comprising increased protein accumulation by proteasome inhibition. Embodiment 6. The composition according to any one of Embodiments 1 to 5, wherein the modification for improving TAA accumulation includes increasing protein accumulation by reducing autophagy. Embodiment 7. The composition according to any one of Embodiments 1 to 6, wherein the modification for improving TAA accumulation includes increasing protein accumulation by inhibiting apoptosis. Embodiment 8. The composition according to any one of Embodiments 1 to 7, wherein the modification for improving the immunogenicity of TAA includes the removal of a tolerogenic molecule. Embodiment 9. The composition according to any one of Embodiments 1 to 8, wherein the modification for improving the immunogenicity of TAA includes increasing general immunogenicity by increasing damage-associated molecular patterns (DAMPs). Embodiment 10. The composition according to any one of Embodiments 1 to 9, wherein the DC is modified to have an increased level of cross-presentation. Embodiment 11. The composition according to Embodiment 10, wherein the DC is modified by exposure to an aminoglycoside antibiotic. Embodiment 12. The composition according to Embodiment 11, wherein the aminoglycoside antibiotic comprises gentamicin. Embodiment 13. The composition according to any one of Embodiments 10 to 12, wherein the DC is modified by exposure to a Toll-like receptor 4 agonist. Embodiment 14. The composition according to any one of Embodiments 1 to 13, wherein the cancer cells are modified to express or accumulate an increased amount of TAA. Embodiment 15. The composition according to Embodiment 14, wherein the cancer cells are modified by exposure to a genome demethylating agent. Embodiment 16. The genome demethylating agent is the composition of Embodiment 15, comprising decitabine. Embodiment 17. The composition according to Embodiment 14, wherein the cancer cells are modified by exposure to a histone acetylation promoter. Embodiment 18. The composition according to Embodiment 17, wherein the histone acetylation promoter comprises a histone deacetylase inhibitor. Embodiment 19. The composition according to claim 18, wherein the histone deacetylase inhibitor comprises valproic acid. Embodiment 20. The composition according to Embodiment 14, wherein the cancer cells are modified by exposure to a proteasome inhibitor. Embodiment 21. The composition according to Embodiment 20, wherein the proteasome inhibitor comprises lactacystin, epixomycin, beta-hydroxy-beta-methylbutyrate, or any combination thereof. Embodiment 22. The composition according to Embodiment 14, wherein the cancer cells are modified by exposure to an E3 ligase inhibitor. Embodiment 23. The composition according to Embodiment 14, wherein the cancer cells are modified by exposure to a PI3K / AKT / mTOR pathway activator. Embodiment 24. The composition according to Embodiment 23, wherein the PI3K / AKT / mTOR pathway activator comprises ultrastandard concentrations of leucine or arginine, or both, in the cell culture medium. Embodiment 25. The composition according to Embodiment 23 or 24, wherein the PI3K / AKT / mTOR pathway activator comprises a PTEN inhibitor. Embodiment 26. The PTEN inhibitor is the composition according to Embodiment 25, comprising bisperoxovanadium-1,10-phenanthroline (bpV(phen)), bisperoxovanadium-5-hydroxypyridine-2-carboxyl (bpV(HOpic), bisperoxo-(bipyridine)-oxovanadate bpV(bipy), or any combination thereof. Embodiment 27. The composition according to Embodiment 25 or 26, wherein the PTEN inhibitor is used in combination with one or more hormones or growth factors. Embodiment 28. The composition according to Embodiment 27, wherein the one or more hormones or growth factors include insulin, thyroid hormone, basic FGF, EGF, or a combination thereof. Embodiment 29. The composition according to Embodiment 14, wherein the cancer cells are modified by exposure to an apoptosis inhibitor. Embodiment 30. The composition according to Embodiment 29, wherein the apoptosis inhibitor is a caspase inhibitor. Embodiment 31. The caspase inhibitor is the composition according to Embodiment 30, comprising Z-VAD-fmk. Embodiment 32. The composition according to any one of Embodiments 1 to 13, wherein the cancer cells are modified by depletion of tolerogenic compounds. Embodiment 33. The composition according to Embodiment 32, wherein the tolerogenic compound comprises a Wnt ligand. Embodiment 34. The composition according to Embodiment 32 or 33, wherein the cancer cells are modified by depletion of tolerogenic compounds due to exposure to beta-methylcyclodextrin. Embodiment 35. The composition according to any one of Embodiments 1 to 13, wherein the cancer cells are modified to increase the production of damage-associated molecular patterns (DAMPs). Embodiment 36. The composition according to Embodiment 35, wherein the cancer cells are modified by exposure to gentamicin. Embodiment 37. The composition according to any one of Embodiments 1 to 36, wherein the composition does not contain living cancer cells. Embodiment 38. The composition according to Embodiment 37 for use in the treatment of cancer in a patient. Embodiment 39. A personal immunotherapy product comprising the composition described in Embodiment 37. Embodiment 40. Use of the composition described in Embodiment 37 or the personalized immunotherapy product described in Embodiment 39 in the treatment of the patient's cancer. Embodiment 41. A method for treating cancer, comprising administering to the patient the composition described in Embodiment 37 or the personalized immunotherapy product described in Embodiment 39. Embodiment 42. A method for manufacturing a personalized immunotherapy product for cancer for individual cancer patients, To collect tumor tissue from the aforementioned patient, To collect blood from the aforementioned patient, The process includes manipulating the tumor tissue and the blood in order to produce a personalized immunotherapy product. The manipulation involves ex vivo modification of cancer cells or DCs, or both, obtained from the patient in order to improve the accumulation or immunogenicity of TAA expressed by the patient's cancer. method. Embodiment 43. A method for manufacturing a personalized immunotherapy product for cancer for individual cancer patients, To collect tumor tissue from the aforementioned patient, To collect blood from the aforementioned patient, Manipulating the tumor tissue to increase the accumulation of TAA, The blood is manipulated to separate monocytes and differentiate them into dendritic cells, and, if necessary, to enhance the antigen-presenting ability of the dendritic cells. Includes, To prepare a personalized immunotherapy product containing the dendritic cells and cancer cell material from the manipulated tumor tissue, method. Embodiment 44. A modification to improve the accumulation or immunogenicity of TAA is a method of Embodiment 42 or 43, comprising carrying out the modification described in any one of Embodiments 1 to 36. Embodiment 45. The method according to any one of Embodiments 42 to 44, wherein the operation includes inactivating cancer cells. Embodiment 46. The method according to Embodiment 45, wherein inactivation includes exposure to gamma-ray irradiation. Embodiment 47. The method of Embodiment 45, wherein inactivation includes exposure to UV irradiation. Embodiment 48. The method according to Embodiment 45, wherein inactivation includes exposure to X-ray irradiation. Embodiment 49. The method according to any one of Embodiments 45 to 48, wherein inactivation includes cell lysis. Embodiment 50. The method according to any one of Embodiments 42 to 49, wherein the sampling includes physically removing the tumor tissue and the blood from the patient. Embodiment 51. The method according to claim 50, further comprising sending the tumor tissue and the blood to a central facility having the ability to separate cancer cells from the tumor tissue and distinguish DCs from monocytes in the blood, wherein the central facility further has the ability to 1) modify the DCs to increase cross-processing, or 2) modify the cancer cells to increase their TAA content or enhance their immunogenicity, or 3) both. Embodiment 52. The method according to any one of Embodiments 42 to 49, wherein the collection includes receiving the tumor tissue and the blood sample. Embodiment 53. The method according to any one of Embodiments 42 to 52, further comprising isolating cancer cells from the tumor tissue. Embodiment 54. The method according to any one of Embodiments 42 to 53, further comprising obtaining DCs from the blood by differentiating monocytes. Embodiment 55. The method according to any one of Embodiments 42 to 54, further comprising combining the cancer cell material, which includes the cancer cells or their lysates, with the DCs in the presence of a DC maturation factor. Embodiment 56. The method according to Embodiment 55, wherein the DC is an immature DC when combined. Embodiment 57. The method according to Embodiment 55 or 56, wherein the DC maturation factor is lipopolysaccharide (LPS). Embodiment 58. The method according to any one of Embodiments 42 to 57, further comprising modifying the DC to have an increased level of cross-presentation. Embodiment 59. The method according to any one of claims 42 to 58, further comprising modifying the cancer cells to increase DAMP production. Embodiment 60. The method according to Embodiment 58 or 59, which is modified to include exposure of the DC or cancer cells to an aminoglycoside antibiotic. Embodiment 61. The method of Embodiment 60, wherein the aminoglycoside antibiotic comprises gentamicin. Embodiment 62. A modification of any one of Embodiments 42 to 62, comprising exposure of the DC to a Toll-like receptor 4 agonist. Embodiment 63. The method according to any one of Embodiments 42 to 62, further comprising modifying the cancer cells to express or accumulate an increased amount of TAA. Embodiment 64. The method according to Embodiment 63, wherein the cancer cells are modified by exposure to a genome demethylating agent. Embodiment 65. The method according to Embodiment 64, wherein the genome demethylating agent comprises decitabine. Embodiment 66. The method according to any one of Embodiments 42 to 65, wherein the cancer cells are modified by exposure to a histone acetylation promoter. Embodiment 67. The method according to Embodiment 66, wherein the histone acetylation promoter comprises a histone deacetylase inhibitor. Embodiment 68. The method according to Embodiment 67, wherein the histone deacetylase inhibitor comprises valproic acid. Embodiment 69. The method according to any one of Embodiments 42 to 68, wherein the cancer cells are modified by exposure to a proteasome inhibitor. Embodiment 70. The method according to Embodiment 69, wherein the proteasome inhibitor comprises lactacystin, epixomycin, beta-hydroxy-beta-methylbutyrate, or any combination thereof. Embodiment 71. The method according to any one of Embodiments 42 to 70, wherein the cancer cells are modified by exposure to an E3 ligase inhibitor. Embodiment 72. Any one of Embodiments 42 to 71, wherein the cancer cells are modified by exposure to a PI3K / AKT / mTOR pathway activator. Embodiment 73. The method according to Embodiment 72, wherein the PI3K / AKT / mTOR pathway activator comprises leucine or arginine, or both, in ultrastandard concentrations in the cell culture medium. Embodiment 74. The method according to Embodiment 72 or 73, wherein the PI3K / AKT / mTOR pathway activator includes a PTEN inhibitor. Embodiment 75. The method according to Embodiment 74, wherein the PTEN inhibitor comprises bisperoxovanadium-1,10-phenanthroline (bpV(phen)), bisperoxovanadium-5-hydroxypyridine-2-carboxyl (bpV(HOpic), bisperoxo-(bipyridine)-oxovanadate bpV(bipy), or any combination thereof. Embodiment 76. The method according to Embodiment 74 or 75, wherein the PTEN inhibitor is used in combination with one or more hormones or growth factors. Embodiment 77. The method according to Embodiment 76, wherein the one or more hormones or growth factors include insulin, thyroid hormone, basic FGF, EGF, or a combination thereof. Embodiment 78. The method according to any one of Embodiments 42 to 77, wherein the cancer cells are modified by exposure to an apoptosis inhibitor. Embodiment 79. The method according to Embodiment 78, wherein the apoptosis inhibitor is a caspase inhibitor. Embodiment 80. The method according to Embodiment 79, wherein the caspase inhibitor includes zVAD.fmk. Embodiment 81. The method according to any one of Embodiments 42 to 80, wherein the cancer cells are modified by depletion of tolerogenic compounds. Embodiment 82. The method according to Embodiment 81, wherein the tolerogenic compound comprises a Wnt ligand. Embodiment 83. The method according to Embodiment 81 or 82, wherein the cancer cells are modified by depletion of tolerogenic compounds by exposure to beta-methylcyclodextrin. Embodiment 84. The method according to any one of Embodiments 42 to 83, further comprising adding a cryoprotectant to the combination of DC and cancer cell material 24 to 48 hours after the combination of DC and cancer cell material, and cryopreserving the combined material. Embodiment 85. A method for treating cancer, comprising administering the personalized immunotherapy product, manufactured by any one of Embodiments 42 to 84, to the individual cancer patient. Embodiment 86. Use of the personal immunotherapy product manufactured by any one of Embodiments 42 to 84 in the treatment of cancer. Embodiment 87. Use of any one of Embodiments 1 to 37 in the manufacture of a drug for the treatment of cancer in the patient. Embodiment 88. The personal immunotherapy product according to Embodiment 39, wherein the patient is a human. Embodiment 89. 1-20x10 per dose 6 A personalized immunotherapy product according to Embodiment 88, including DC. Embodiment 90. A therapeutic method according to Embodiment 41 or 85, including administration by injection, or a use according to Embodiment 39 or 86. Embodiment 91. A therapeutic method according to Embodiment 41 or 85, or a use according to Embodiment 39 or 86, including administration by injection. Embodiment 92. The therapeutic method according to Embodiment 41 or 85, or the use according to Embodiment 39 or 86, further comprising the administration of an immune checkpoint inhibitor. Embodiment 93. The method according to claim 92, wherein the immune checkpoint inhibitor is an antibody specific to CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, B7-H3, B7-H4, BTLA, ICOS, or OX40. Embodiment 94. A treatment method according to Embodiment 41 or 85, or a use according to Embodiment 39 or 86, including administration of a single dose. Embodiment 95. A treatment method according to Embodiment 41 or 85, or a use according to Embodiment 39 or 86, including administration at weekly intervals. Embodiment 96. A treatment method according to Embodiment 41 or 85, or a use according to Embodiment 39 or 86, including administration at monthly intervals. Embodiment 97. The treatment method according to Embodiment 41 or 85, or the use according to Embodiment 39 or 86, wherein the cancer is a carcinoma. Embodiment 98. The treatment method according to Embodiment 41 or 85, or the use according to Embodiment 39 or 86, wherein the cancer is a sarcoma. Embodiment 99. The treatment method according to Embodiment 41 or 85, or the use according to Embodiment 39 or 86, wherein the cancer is leukemia or lymphoma. Embodiment 100. The treatment method according to Embodiment 41 or 85, or the use according to Embodiment 39 or 86, wherein the cancer is a cancer of the brain, head and neck, esophagus, lung, liver, pancreas, kidney, stomach, colon, prostate, breast, uterus, cervix, ovary, skin, bone, blood, eye, or retina. Embodiment 101. The treatment method according to Embodiment 41 or 85, or the use according to Embodiment 39 or 86, wherein the cancer is melanoma, non-small cell lung cancer, glioblastoma, renal cell carcinoma, or colorectal cancer.

[0057] (Examples) The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the representative embodiments currently being considered. These examples should not be construed as limiting any of the embodiments described herein, but they may support certain limitations found in the claims.

[0058] (Example 1) Isolation of live cancer cells from tumors In a typical preparation, tumor tissue obtained by surgical excision is incised from normal tissue, mechanically chopped into fragments 2-3 mm in diameter, and dissociated with enzymes in cell culture medium. The chopped tumor tissue is continuously stirred at 37°C for 0.5-3 hours in the presence of collagenase trypsin. Alternatively, dipase is used at low concentrations at 37°C, refrigerated overnight or up to 72 hours (approximately 4°C), room temperature (approximately 25°C), or 37°C. Digested extracellular matrix and other debris are removed by repeated cycles of centrifugation and resuspension. Live cancer cells are transferred to cell culture vessels and grown in nutrient-rich medium.

[0059] (Example 2) Increased protein expression due to inhibition of histone deacetylases The isolated live cancer cells are placed in tissue culture. During the enhancement phase, the tissue culture medium is supplemented with valproic acid, phenyl butyrate, or both at concentrations of 0.01 mM to 10 mM, respectively. Levels of histone acetylation, mRNA transcription, and protein expression, including TAA expression, all increase.

[0060] (Example 3) Increased protein expression due to inhibited DNA methyltransferase The isolated live cancer cells are placed in tissue culture. The tissue culture medium is supplemented with decitabine, depending on the concentration, for at least one hour from the start of the potentiation phase until the end of the potentiation phase. Concentrations of 100–500 nM may be used throughout the potentiation phase of the culture. Alternatively, a higher concentration of 1 μM–10 μM may be used at the start of the potentiation phase and then removed or reduced to a lower concentration. Hypermethylation is reversed, and the expression of silenced germline and tumor-specific antigens increases, resulting in a more homogeneous overall cancer cell population.

[0061] (Example 4) Increased protein content due to proteasome inhibition The isolated live cancer cells are placed in tissue culture. The tissue culture medium is supplemented with lactacystin at a concentration of 0.1–1 μM, epixomycin at a concentration of 1–2 μM, and / or HMB at a concentration of 10–150 μg / mL. The inhibitor may be present throughout or in part of the culture's enhancement phase, but at least in the last 24 hours before harvest. The lower limit of the listed dose range is suitable for long-term exposure. The upper limit of the listed dose range may be used for the remaining 24 hours of culture. The protein content (including normal proteins, mutant proteins, and misfolded proteins) in cancer cells increases.

[0062] (Example 5) Increased cell proliferation and protein production due to PTEN inhibition Isolated live cancer cells are placed in tissue culture medium supplemented with leucine and arginine at a 70:1 arginine:alanine ratio and a 25:1 leucine:alanine ratio. The tissue culture medium is further supplemented with bpV(phen), bpV(HOpic), or bpV(bipy) at concentrations of 5–20 μM. This is applied for at least 24 hours at the start of cell culture and, if applicable, continued at lower concentrations throughout the cell culture period. Throughout the period of PTEN inhibition, the medium is further supplemented with insulin, thyroid hormones, basic FGF, and EGF.

[0063] (Example 6) Rapid proliferation of cancer cells and accumulation of TAA due to suppression of apoptosis. The isolated live cancer cells are placed in tissue culture. The tissue culture medium is supplemented with the broad-spectrum caspase inhibitor Z-VAD-fmk at a concentration of 5–50 μM. The caspase inhibitor is added at the start of cell culture and maintained throughout the growth and enhancement phases of the culture. The cultured cancer cells proliferate and maintain a high viability, while proteins including TAA accumulate at higher levels than in untreated cultures.

[0064] (Example 7) Increased immunogenicity due to depletion of Wnt signaling ligands The isolated live cancer cells are placed in tissue culture. 30 minutes to 3 hours before the end of the potentiation phase and DC loading preparation, the tissue culture medium is supplemented with beta-methylcyclodextrin at a concentration of 0.5–20 mM. Cholesterol and Wnt ligands are depleted from the plasma membrane of the cancer cells.

[0065] (Example 8) Harvesting and inactivation of cancer cells after enhanced culture. Once the culture enhancement stage is complete, cancer cells are isolated by trypsin treatment. The harvested cells are washed with 3 cycles of centrifugation in phosphate-buffered saline until the culture medium and trypsin solution are depleted. The cells are then irradiated with a total dose of 100 Gy and lysed in cryoprotectant-free medium using 3-5 freeze / thaw cycles. Total protein is determined by the Biuret method.

[0066] (Example 9) Promotion of cross-processing in DCs by treatment with aminoglycoside antibiotics. PBMCs are seeded in culture medium, monocytes are allowed to adhere, and then non-adherent cells are washed away. Fresh medium supplemented with GM-CSF, IL-4, and 5-10 μg / ml of gentamicin is added, and the cells are incubated for 3-5 days. Inactivated cancer cells or cell lysates and LPS are added, the gentamicin concentration is increased to 50-150 μg / ml, and the DCs are incubated for a further 24-48 hours.

[0067] (Example 10) In vitro application of proteasome inhibitors and caspase inhibitors to increase protein accumulation in tumor cells While the increased antigen levels obtained by the use of proteasome inhibitors are beneficial, proteasome inhibitors may be used in combination with caspase inhibitors to avoid inducing apoptosis. Initially, to find the concentration of proteasome inhibitors that induced minimal cell death, various bortezomib dilutions were tested in vitro in established ovarian tumor lines after reaching 70–90% confluence. Cultures were maintained in standard DMEM:F12 medium containing 5% FBS. Bortezomib was reconstituted with DMSO and added directly to the cultures at concentrations of 0.1–100 nM.

[0068] At concentrations above 5 nM bortezomib, all cells died within 24 hours. Between 5 nM and 1 nM bortezomib, cell survival increased proportionally with decreasing concentration. Below 1 nM concentration, bortezomib had no effect on survival (Figures 2 and 3).

[0069] Next, the caspase inhibitor Z-VAD-fmk was tested in the same cell culture system at concentrations ranging from 10 to 100 μM. It was found that the caspase inhibitor did not affect cell survival without apoptosis exposure (challenge). For further experiments, a concentration of 20 μM was selected.

[0070] Next, the sequential application of caspase inhibitors and proteasome inhibitors was tested. Tumor cells were cultured for 24 hours in the presence of the caspase inhibitor Z-VAD-fmk, then the medium was replaced with a medium containing bortezomib, and the cells were cultured for a further 48 hours. Bortezomib was used at 5 nM, the lowest concentration that induced cell death. A cell viability of approximately 75% was observed, with a characteristic cell morphology consisting of multinucleated and enlarged cell bodies, as illustrated in Figure 4.

[0071] Concomitant administration of bortezomib and Z-VAD-fmk was also tested using 1 nM bortezomib with 1 or 20 μM Z-VAD-fmk. These treatments did not cause any apparent changes in cell morphology or survival (see Figure 5).

[0072] To assess protein content, we searched for two targets commonly found in cancer: CA125, typically specific to ovarian cancer, and MUC1, common in many types of cancer (e.g., colon, breast, ovarian, lung, and pancreatic). Proteins were labeled using antibodies conjugated to Alexa Fluor 488 (green; anti-MUC1) or 594 (red; anti-CA125). Cells were imaged with an epifluorescence microscope (Nikon) with pre-set parameters for exposure and magnification. Using Nikon NIS-Elements software, each channel was analyzed for maximum pixel intensity (indicating similar imaging parameters), average intensity indicating an increase or decrease in the amount of target protein, and the total number of labeled pixels proportional to the labeled target protein content. As seen in Figures 6 and 7, bortezomib at concentrations of 0.1 nM to 1.0 nM (without caspase inhibitor) increased the number and content of these antigens in treated tumor cells.

[0073] Furthermore, Ki67 labeling was used to analyze differences in tumor cell proliferation under various conditions. Ki67 labeling did not show any difference in proliferation.

[0074] These data demonstrate that the protein content of tumor cells can be increased in vitro by exposing cells to low concentrations of proteasome inhibitors, optionally combined with caspase inhibitors, without affecting cell viability or proliferative capacity. This treatment increases protein content, subsequently making new antigens available to antigen-presenting cells. Simultaneously, in vitro treatment of tumor cells before exposure to dendritic cells avoids harmful in vivo exposure of antigen-presenting cells to proteasome inhibitors. These findings are significant for the immediate applicability of dendritic cell-based immunotherapy.

[0075] Finally, while aspects of this specification are emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily grasp that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methodologies, protocols, and / or reagents, etc., described herein. Accordingly, various modifications, changes, or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of this specification. Finally, the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention as defined solely by the claims. Therefore, the invention is not limited to what has been shown and described.

[0076] Specific embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Of course, variations of these described embodiments will be obvious to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend to carry out the Invention in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the appended claims, as permitted by applicable law. Furthermore, unless otherwise indicated herein or clearly rejected by the context, any combination of the above embodiments in all possible modifications is incorporated into the Invention.

[0077] Grouping of alternative embodiments, elements, or processes of the present invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of such inclusion or removal, the specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0078] Unless otherwise indicated, all figures used herein and in the claims to represent features, items, quantities, parameters, characteristics, terms, etc., should be understood to be modified in all cases by the term “approximately.” As used herein, “approximately” means that the thus limited characteristic, item, quantity, parameter, feature, or term encompasses a range of up to 10 percent above and below the value of the specified characteristic, item, quantity, parameter, feature, or term. Therefore, unless otherwise indicated, the numerical parameters described in the specification and the appended claims are variable approximations. Each numerical representation should be interpreted, at least, by applying common rounding techniques, taking into account the number of significant figures reported, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims. Although numerical ranges and values ​​describing the broad scope of the invention are approximations, the numerical ranges and values ​​described in specific embodiments are reported as accurately as possible. However, numerical ranges or values ​​inherently contain certain errors that inevitably arise from the standard deviation found in each test measurement. The enumeration of numerical ranges of values ​​herein is intended solely as a simple way to refer individually to the individual numerical values ​​that fall within that range. Unless otherwise specified herein, individual values ​​within a numerical range are incorporated herein as if they were individually listed herein.

[0079] Unless otherwise indicated herein or unless clearly contradicted by the context, the terms “a,” “an,” “the,” and similar references used in the context describing the present invention (particularly in the context of the appended claims) should be construed to encompass both singular and plural forms. All methods described herein may be carried out in any suitable order unless otherwise indicated herein or unless clearly contradicted by the context. The use of any examples or illustrative words provided herein (e.g., “etc.”) is intended merely to better illuminate the present invention and not to limit the scope of the claimed invention. Language herein should not be construed to indicate non-claimed elements essential to the practice of the present invention.

[0080] The specific embodiments disclosed herein may be further limited in the claims using the transitional term "consisting of" or "essentially consisting of." Where used in the claims, whether submitted or added in each amendment, the transitional term "consisting of" excludes any element, process, or component not specified in the claims. The transitional term "essentially consisting of" limits the scope of the claims to a specific substance or process and not substantially affect any basic and novel properties. Embodiments of the invention as claimed in this manner are essentially or expressly described and enabled herein.

[0081] All patents, patent publications, and other publications referenced and identified herein are expressly incorporated herein by whole by reference for the purpose of describing and disclosing, for example, compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for disclosure prior to the filing date of this application. In this regard, the inventors should not be construed as acknowledging that they have no prior rights to such disclosures, either because of prior inventions or for any other reason. All statements regarding dates or expressions regarding the contents of these documents are based on information available to the applicant and do not constitute an endorsement of the accuracy of the dates or contents of these documents.

[0082] (Note) (Note 1) A composition comprising cancer cells and dendritic cells (DCs) from the same cancer patient, The cancer cells or DCs, or both, are modified ex vivo to improve the accumulation or immunogenicity of tumor-associated antigens (TAAs) expressed by the patient's cancer. composition.

[0083] (Note 2) A composition containing DCs from cancer patients, The DCs are loaded with antigenic substances from cancer cells isolated from tumors removed from the cancer patient, and the cancer cells, the DCs, or both are modified to improve the accumulation or immunogenicity of TAA expressed by the patient's cancer. composition.

[0084] (Note 3) The composition according to Appendix 1 or 2, wherein the DC is modified to have an increased level of cross-presentation.

[0085] (Note 4) The composition described in Appendix 3, wherein the DC is modified by exposure to an aminoglycoside antibiotic.

[0086] (Note 5) The aminoglycoside antibiotic is the composition described in Appendix 4, comprising gentamicin.

[0087] (Note 6) The composition according to any one of the appendices 3 to 5, wherein the DC is modified by exposure to a Toll-like receptor 4 agonist.

[0088] (Note 7) The composition according to any one of Appendix 1 to 6, wherein the cancer cells are modified to express or accumulate an increased amount of TAA.

[0089] (Note 8) The composition according to Appendix 7, wherein the cancer cells are modified by exposure to a genome demethylating agent.

[0090] (Note 9) The genome demethylating agent is the composition described in Appendix 8, comprising decitabine.

[0091] (Note 10) The composition according to Appendix 7, wherein the cancer cells are modified by exposure to a histone acetylation promoter.

[0092] (Note 11) The composition according to Appendix 10, wherein the histone acetylation promoter comprises a histone deacetylase inhibitor.

[0093] (Note 12) The histone deacetylase inhibitor is the composition described in Appendix 11, comprising valproic acid.

[0094] (Note 13) The composition according to Appendix 7, wherein the cancer cells are modified by exposure to a proteasome inhibitor.

[0095] (Note 14) The proteasome inhibitor is the composition according to Appendix 13, comprising lactacystin, epixomycin, beta-hydroxy-beta-methylbutyrate, or any combination thereof.

[0096] (Note 15) The composition according to Appendix 7, wherein the cancer cells are modified by exposure to an E3 ligase inhibitor.

[0097] (Note 16) The composition described in Appendix 7, wherein the cancer cells are modified by exposure to a PI3K / AKT / mTOR pathway activator.

[0098] (Note 17) The PI3K / AKT / mTOR pathway activator is the composition described in Appendix 16, comprising leucine or arginine, or both, in ultrastandard concentrations in cell culture medium.

[0099] (Note 18) The PI3K / AKT / mTOR pathway activator is a composition according to Appendix 16 or 17, comprising a PTEN inhibitor.

[0100] (Note 19) The PTEN inhibitor is the composition described in Appendix 18, comprising bisperoxovanadium-1,10-phenanthroline (bpV(phen)), bisperoxovanadium-5-hydroxypyridine-2-carboxyl (bpV(HOpic), bisperoxo-(bipyridine)-oxovanadate bpV(bipy), or any combination thereof.

[0101] (Note 20) The PTEN inhibitor is used in combination with one or more hormones or growth factors, as described in Appendix 18 or 19.

[0102] (Note 21) The composition according to Appendix 20, wherein the one or more hormones or growth factors include insulin, thyroid hormone, basic FGF, EGF, or a combination thereof.

[0103] (Note 22) The composition according to Appendix 7, wherein the cancer cells are modified by exposure to an apoptosis inhibitor.

[0104] (Note 23) The composition according to Appendix 22, wherein the apoptosis inhibitor is a caspase inhibitor.

[0105] (Note 24) The caspase inhibitor is the composition described in Appendix 23, comprising Z-VAD-fmk.

[0106] (Note 25) The composition according to any one of the appendices 1 to 6, wherein the cancer cells are modified by depletion of tolerogenic compounds.

[0107] (Note 26) The composition according to Appendix 25, wherein the tolerogenic compound comprises a Wnt ligand.

[0108] (Note 27) The composition according to Appendix 25 or 26, wherein the cancer cells are modified by depletion of tolerogenic compounds due to exposure to beta-methylcyclodextrin.

[0109] (Note 28) The composition according to any one of the appendices 1 to 6, wherein the cancer cells are modified to increase the production of damage-associated molecular patterns (DAMPs).

[0110] (Note 29) The composition described in Appendix 28, wherein the cancer cells are modified by exposure to gentamicin.

[0111] (Note 30) The composition described above is the composition according to any one of Appendix 2 to 29, which does not contain living cancer cells.

[0112] (Note 31) Personal immunotherapy products containing the composition described in Appendix 30.

[0113] (Note 32) Use of personal immunotherapy products as described in Appendix 31 in the treatment of cancer.

[0114] (Note 33) A method for treating cancer, comprising administering the personal immunotherapy product described in Appendix 31 to the patient.

[0115] (Note 34) A method for manufacturing personalized immunotherapy products for cancer for individual cancer patients, To collect tumor tissue from the aforementioned patient, To collect blood from the aforementioned patient, Manipulating the tumor tissue to increase the accumulation of TAA, The blood is manipulated to separate monocytes and differentiate them into dendritic cells, and, if necessary, to enhance the antigen-presenting ability of the dendritic cells. Includes, To prepare a personalized immunotherapy product containing the dendritic cells and cancer cell material from the manipulated tumor tissue, method.

[0116] (Note 35) The method according to Appendix 34, wherein the collection includes physically removing the tumor tissue and the blood from the patient.

[0117] (Note 36) The method according to Appendix 35, further comprising sending the tumor tissue and the blood to a central facility having the ability to separate cancer cells from the tumor tissue and distinguish DCs from monocytes in the blood, wherein the central facility further has the ability to 1) modify the DCs to increase cross-processing, or 2) modify the cancer cells to increase their TAA content or enhance their immunogenicity, or 3) both.

[0118] (Note 37) The method according to Appendix 34, wherein the collection includes receiving the tumor tissue and the blood sample.

[0119] (Note 38) The isolation of cancer cells from the aforementioned tumor tissue, By differentiating monocytes, DCs can be obtained from the blood, Modifying the aforementioned cancer cells, the aforementioned DCs, or both, The cancer cell material, including the cancer cells or their lysates, is combined with the DCs in the presence of DC maturation factors. The method described in Appendix 35 or 37, further including the method described in Appendix 35 or 37.

[0120] (Note 39) The method according to Appendix 35 or 37, further comprising combining cancer cell material, including cancer cells or lysates derived from the tumor tissue, with immature DCs derived from the blood, in the presence of DC maturation factors, wherein the cancer cells, the DCs, or both are modified.

[0121] (Note 40) The DC maturation factor is lipopolysaccharide (LPS) as described in Appendix 38 or 39.

[0122] (Note 41) The method according to any one of the appendices 38 to 40, further comprising modifying the DC to have an increased level of cross-presentation.

[0123] (Note 42) The method according to any one of the appendices 38 to 41, further comprising modifying the cancer cells to increase DAMP production.

[0124] (Note 43) The modification is the method described in Appendix 41 or 42, which includes exposure of the DC or cancer cells to an aminoglycoside antibiotic.

[0125] (Note 44) The method described in Appendix 43, wherein the aminoglycoside antibiotic includes gentamicin.

[0126] (Note 45) The modification is the method described in any one of the appendices 38 to 44, which includes exposure of the DC to a Toll-like receptor 4 agonist.

[0127] (Note 46) The method according to any one of the appendices 38 to 45, further comprising modifying the cancer cells to express or accumulate an increased amount of TAA.

[0128] (Note 47) The method described in Appendix 46, wherein the cancer cells are modified by exposure to a genome demethylating agent.

[0129] (Note 48) The genome demethylating agent described above is the method described in Appendix 47, comprising decitabine.

[0130] (Note 49) The method according to any one of the appendices 38 to 48, wherein the cancer cells are modified by exposure to a histone acetylation promoter.

[0131] (Note 50) The method according to Appendix 49, wherein the histone acetylation promoter includes a histone deacetylase inhibitor.

[0132] (Note 51) The histone deacetylase inhibitor is the method according to Appendix 50, comprising valproic acid.

[0133] (Note 52) The method according to any one of the appendices 38 to 51, wherein the cancer cells are modified by exposure to a proteasome inhibitor.

[0134] (Note 53) The method according to Appendix 52, wherein the proteasome inhibitor comprises lactacystin, epixomycin, beta-hydroxy-beta-methylbutyrate, or any combination thereof.

[0135] (Note 54) The method according to any one of the appendices 38 to 53, wherein the cancer cells are modified by exposure to an E3 ligase inhibitor.

[0136] (Note 55) The method according to any one of the appendices 38 to 54, wherein the cancer cells are modified by exposure to a PI3K / AKT / mTOR pathway activator.

[0137] (Note 56) The method according to Appendix 55, wherein the PI3K / AKT / mTOR pathway activator comprises leucine or arginine, or both, in ultrastandard concentrations in the cell culture medium.

[0138] (Note 57) The PI3K / AKT / mTOR pathway activator according to the method described in Appendix 55 or 56, comprising a PTEN inhibitor.

[0139] (Note 58) The PTEN inhibitor is the method according to Appendix 57, comprising bisperoxovanadium-1,10-phenanthroline (bpV(phen)), bisperoxovanadium-5-hydroxypyridine-2-carboxyl (bpV(HOpic), bisperoxo-(bipyridine)-oxovanadate bpV(bipy), or any combination thereof.

[0140] (Note 59) The PTEN inhibitor is used in combination with one or more hormones or growth factors, as described in Appendix 57 or 58.

[0141] (Note 60) The method according to Appendix 59, wherein the one or more hormones or growth factors include insulin, thyroid hormone, basic FGF, EGF, or a combination thereof.

[0142] (Note 61) The method according to any one of the appendices 38 to 60, wherein the cancer cells are modified by exposure to an apoptosis inhibitor.

[0143] (Note 62) The method according to Appendix 61, wherein the inhibitor of apoptosis is a caspase inhibitor.

[0144] (Note 63) The caspase inhibitor is the method described in Appendix 62, comprising zVAD.fmk.

[0145] (Note 64) The method according to any one of the appendices 38 to 63, wherein the cancer cells are modified by depletion of tolerogenic compounds.

[0146] (Note 65) The method according to Appendix 64, wherein the tolerogenic compound contains a Wnt ligand.

[0147] (Note 66) The method according to Appendix 64 or 65, wherein the cancer cells are modified by depletion of tolerogenic compounds due to exposure to beta-methylcyclodextrin.

[0148] (Note 67) The method according to any one of the appendices 38 to 66, further comprising adding a cryoprotectant to the combination of DC and cancer cell material 24 to 48 hours after the combination of DC and cancer cell material, and cryopreserving the combined material.

[0149] (Note 68) A method for treating cancer, comprising administering the personal immunotherapy product manufactured by the method described in any one of Appendix 34 to 67 to the individual cancer patient.

[0150] (Note 69) Use of the aforementioned personal immunotherapy product manufactured by any one of the methods described in Appendix 34 to 67 in the treatment of cancer.

Claims

1. A composition comprising cancer cells and dendritic cells (DCs) from the same cancer patient, The cancer cells or DCs, or both, are modified in ex vivo to improve the accumulation or immunogenicity of tumor-associated antigens (TAAs) expressed by the patient's cancer. composition.

2. A composition containing DCs from cancer patients, The DCs are loaded with antigenic substances from cancer cells isolated from tumors removed from the cancer patient, and the cancer cells, the DCs, or both are modified to improve the accumulation or immunogenicity of TAA expressed by the patient's cancer. composition.

3. The composition according to claim 1 or 2, wherein the DC is modified to have an increased level of cross-presentation.

4. The composition according to claim 3, wherein the DC is modified by exposure to an aminoglycoside antibiotic.

5. The composition according to claim 4, wherein the aminoglycoside antibiotic comprises gentamicin.

6. The composition according to any one of claims 3 to 5, wherein the DC is modified by exposure to a Toll-like receptor 4 agonist.

7. The composition according to any one of claims 1 to 6, wherein the cancer cells are modified to express or accumulate an increased amount of TAA.

8. The composition according to claim 7, wherein the cancer cells are modified by exposure to a genome demethylating agent.

9. The composition according to claim 8, wherein the genome demethylating agent comprises decitabine.

10. The composition according to claim 7, wherein the cancer cells are modified by exposure to a histone acetylation promoter.

11. The composition according to claim 10, wherein the histone acetylation promoter comprises a histone deacetylase inhibitor.

12. The composition according to claim 11, wherein the histone deacetylase inhibitor comprises valproic acid.

13. The composition according to claim 7, wherein the cancer cells are modified by exposure to a proteasome inhibitor.

14. The composition according to claim 13, wherein the proteasome inhibitor comprises lactacystin, epixomycin, beta-hydroxy-beta-methylbutyrate, or any combination thereof.

15. The composition according to claim 7, wherein the cancer cells are modified by exposure to an E3 ligase inhibitor.

16. The composition according to claim 7, wherein the cancer cells are modified by exposure to a PI3K / AKT / mTOR pathway activator.

17. The composition according to claim 16, wherein the PI3K / AKT / mTOR pathway activator comprises leucine or arginine, or both, in ultrastandard concentrations in the cell culture medium.

18. The composition according to claim 16 or 17, wherein the PI3K / AKT / mTOR pathway activator comprises a PTEN inhibitor.

19. The composition according to claim 18, wherein the PTEN inhibitor comprises bisperoxovanadium-1,10-phenanthroline (bpV(phen), bisperoxovanadium-5-hydroxypyridine-2-carboxyl (bpV(HOpic), bisperoxo-(bipyridine)-oxovanadate bpV(bipy), or any combination thereof.

20. The composition according to claim 18 or 19, wherein the PTEN inhibitor is used in combination with one or more hormones or growth factors.

21. The composition according to claim 20, wherein the one or more hormones or growth factors include insulin, thyroid hormone, basic FGF, EGF, or a combination thereof.

22. The composition according to claim 7, wherein the cancer cells are modified by exposure to an apoptosis inhibitor.

23. The composition according to claim 22, wherein the apoptosis inhibitor is a caspase inhibitor.

24. The composition according to claim 23, wherein the caspase inhibitor comprises Z-VAD-fmk.

25. The composition according to any one of claims 1 to 6, wherein the cancer cells are modified by depletion of tolerogenic compounds.

26. The composition according to claim 25, wherein the tolerogenic compound comprises a Wnt ligand.

27. The composition according to claim 25 or 26, wherein the cancer cells are modified by depletion of tolerogenic compounds due to exposure to beta-methylcyclodextrin.

28. The composition according to any one of claims 1 to 6, wherein the cancer cells are modified to increase the production of damage-associated molecular patterns (DAMPs).

29. The composition according to claim 28, wherein the cancer cells are modified by exposure to gentamicin.

30. The composition according to any one of claims 2 to 29, wherein the composition does not contain living cancer cells.

31. A personal immunotherapy product comprising the composition described in claim 30.

32. Use of the personalized immunotherapy product according to claim 31 in the treatment of cancer.

33. A method for treating cancer, comprising administering the personalized immunotherapy product described in claim 31 to the patient.

34. A method for manufacturing personalized immunotherapy products for cancer for individual cancer patients, To collect tumor tissue from the aforementioned patient, To collect blood from the aforementioned patient, Manipulating the tumor tissue to increase the accumulation of TAA, The blood is manipulated to separate monocytes and differentiate them into dendritic cells, and, if necessary, to enhance the antigen-presenting ability of the dendritic cells. Includes, To prepare a personalized immunotherapy product containing the dendritic cells and cancer cell material from the manipulated tumor tissue, method.

35. The method according to claim 34, wherein the collection includes physically removing the tumor tissue and the blood from the patient.

36. The method according to claim 35, further comprising sending the tumor tissue and the blood to a central facility having the ability to separate cancer cells from the tumor tissue and to distinguish DCs from monocytes in the blood, wherein the central facility further has the ability to 1) modify the DCs to increase cross-processing, or 2) modify the cancer cells to increase their TAA content or enhance their immunogenicity, or 3) both.

37. The method according to claim 34, wherein the collection includes receiving the tumor tissue and the blood sample.

38. The isolation of cancer cells from the aforementioned tumor tissue, By differentiating monocytes, DCs can be obtained from the blood, Modifying the aforementioned cancer cells, the aforementioned DCs, or both, The cancer cell material, including the cancer cells or their lysates, is combined with the DCs in the presence of DC maturation factors. The method according to claim 35 or 37, further comprising:

39. The method according to claim 35 or 37, further comprising combining a cancer cell substance, comprising cancer cells or lysates derived from the tumor tissue, with immature DCs derived from the blood, in the presence of a DC maturation factor, wherein the cancer cells, the DCs, or both are modified.

40. The method according to claim 38 or 39, wherein the DC maturation factor is lipopolysaccharide (LPS).

41. The method according to any one of claims 38 to 40, further comprising modifying the DC to have an increased level of cross-presentation.

42. The method according to any one of claims 38 to 41, further comprising modifying the cancer cells to increase the production of DAMP.

43. The method according to claim 41 or 42, wherein the modification includes exposure of the DC or cancer cells to an aminoglycoside antibiotic.

44. The method according to claim 43, wherein the aminoglycoside antibiotic comprises gentamicin.

45. The modification comprises exposure of the DC to a Toll-like receptor 4 agonist, according to any one of claims 38 to 44.

46. The method according to any one of claims 38 to 45, further comprising modifying the cancer cells to express or accumulate an increased amount of TAA.

47. The method according to claim 46, wherein the cancer cells are modified by exposure to a genome demethylating agent.

48. The method according to claim 47, wherein the genome demethylating agent comprises decitabine.

49. The method according to any one of claims 38 to 48, wherein the cancer cells are modified by exposure to a histone acetylation promoter.

50. The method according to claim 49, wherein the histone acetylation promoter comprises a histone deacetylase inhibitor.

51. The method according to claim 50, wherein the histone deacetylase inhibitor comprises valproic acid.

52. The method according to any one of claims 38 to 51, wherein the cancer cells are modified by exposure to a proteasome inhibitor.

53. The method according to claim 52, wherein the proteasome inhibitor comprises lactacystin, epixomycin, beta-hydroxy-beta-methylbutyrate, or any combination thereof.

54. The method according to any one of claims 38 to 53, wherein the cancer cells are modified by exposure to an E3 ligase inhibitor.

55. The method according to any one of claims 38 to 54, wherein the cancer cells are modified by exposure to a PI3K / AKT / mTOR pathway activator.

56. The method according to claim 55, wherein the PI3K / AKT / mTOR pathway activator comprises leucine or arginine, or both, in ultrastandard concentrations in the cell culture medium.

57. The method according to claim 55 or 56, wherein the PI3K / AKT / mTOR pathway activator comprises a PTEN inhibitor.

58. The method according to claim 57, wherein the PTEN inhibitor comprises bisperoxovanadium-1,10-phenanthroline (bpV(phen), bisperoxovanadium-5-hydroxypyridine-2-carboxyl (bpV(HOpic), bisperoxo-(bipyridine)-oxovanadate bpV(bipy), or any combination thereof.

59. The method according to claim 57 or 58, wherein the PTEN inhibitor is used in combination with one or more hormones or growth factors.

60. The method according to claim 59, wherein the one or more hormones or growth factors include insulin, thyroid hormone, basic FGF, EGF, or a combination thereof.

61. The method according to any one of claims 38 to 60, wherein the cancer cells are modified by exposure to an apoptosis inhibitor.

62. The method according to claim 61, wherein the apoptosis inhibitor is a caspase inhibitor.

63. The method according to claim 62, wherein the caspase inhibitor comprises zVAD. fmk.

64. The method according to any one of claims 38 to 63, wherein the cancer cells are modified by depletion of tolerogenic compounds.

65. The method according to claim 64, wherein the tolerogenic compound comprises a Wnt ligand.

66. The method according to claim 64 or 65, wherein the cancer cells are modified by depletion of tolerogenic compounds due to exposure to beta-methylcyclodextrin.

67. The method according to any one of claims 38 to 66, further comprising adding an antifreeze to the combination of DC and cancer cell material and freezing the combined material 24 to 48 hours after the DC and cancer cell material have been combined.

68. A method for treating cancer, comprising administering the personalized immunotherapy product manufactured by the method described in any one of claims 34 to 67 to the individual cancer patient.

69. Use of the personal immunotherapy product manufactured by the method of any one of claims 34 to 67 in the treatment of cancer.

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