Cancer vaccine composition and method of use thereof

Abstract

The present disclosure provides a cancer vaccine composition comprising inactivated cancer cells that are replication-incompetent and an adjuvant. Also provided is a method for making the cancer vaccine composition, the method comprising contacting cancer cells with light (e.g., UV light) in the presence of a photosensitizer (e.g., riboflavin).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 755,741 filed on 5 November 2018; U.S. Provisional Patent Application No. 62 / 688,051 filed on 21 June 2018; and U.S. Provisional Patent Application No. 62 / 645,975 filed on 21 March 2018, each of which is incorporated herein by reference in whole for all purposes.

[0002] This disclosure generally relates to compositions and methods for inhibiting tumor growth and promoting anti-tumor immune responses. More specifically, this disclosure relates to cancer vaccine compositions and methods for activating the immune system's response against tumors. This disclosure also relates to methods for producing cancer cell vaccines. [Background technology]

[0003] Cancer immunotherapy involves the use of compositions and methods for inducing and enhancing an individual's own immune system against cancer cells or cancer-predisposing infections. Cancer vaccines are introduced into the body in a non-carcinogenic form and work by inducing the immune system to initiate a response to an antigen (e.g., typically a protein, peptide, or carbohydrate) that induces the body to confine immunity or acquire a persistent "memory" immune response. Once the immune system response is established, exposure of the immune system to this antigen (e.g., in the form of a cancerous tumor) results in a rapid and robust immune response. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] One challenge in cancer immunotherapy is the significant variation in clinical responses between patients. Some patients may exhibit remarkable and durable responses, while others may not demonstrate clear clinical benefit. Therefore, there is a need in this field for compositions that can reliably and effectively stimulate the immune system as cancer immunotherapy.

Means for Solving the Problem

[0005] The present specification provides a cancer vaccine composition comprising inactivated cancer cells that do not have the ability to replicate. The cancer cells may be isolated or derived from a patient suffering from one or more types of cancer.

[0006] Furthermore, a method for treating cancer in a patient who needs it, the method comprising administering the cancer vaccine of the present disclosure to the patient, is provided.

[0007] Furthermore, a method for producing a cancer vaccine composition, the method comprising treating cancer cells with light (e.g., UV light) in the presence of a photosensitizer (e.g., riboflavin), is provided.

[0008] Furthermore, a cancer vaccine composition for use in a method of treating cancer is provided.

[0009] Furthermore, a cancer vaccine composition for use as a medicament for treating cancer, and use of the cancer vaccine composition in the manufacture of a medicament for treating cancer are provided. These and other aspects are further detailed below.

Brief Description of the Drawings

[0010] [Figure 1] The growth of CAMA cells after treatment with riboflavin and UV light is illustrated at the treatment day (day 0), and at 2, 4, 6, and 8 days after treatment. The cells were treated using a Mirasol® PRT illumination device at illumination intensities of 10%, 20%, 30%, 40%, 50%, or 100%. Cells treated with UV light (live) were included as a control. [Figure 2] Shows the viability of CAMA cells after treatment with riboflavin and UV light. [Figure 3] Shows the expression of the surface marker EpCAM in CAMA cells after treatment with riboflavin and UV light. [Figure 4]Present fluorescence microscopy images comparing surface marker expression on CAMA cells at various time intervals after treatment with riboflavin and UV light (20% illumination intensity). [Figure 5] Show the relative expression of surface markers EpCAM (front row of bars) and CD38 (back row of bars) within the viable population after treatment with various doses of UV light. [Figure 6] Show the caspase-3 concentration within CAMA cells after treatment with riboflavin and UV light. [Figure 7] Show the correlation between surface marker expression and viability of CAMA cells after treatment with riboflavin and UV light. [Figure 8] Show the tumor growth curves in mice with PyMT mammary tumors injected with saline (control, no vaccine) compared to mice injected with inactivated whole cell vaccine and mice injected with lysate vaccine (4T1 spheroid lysate vaccine as described in International Publication No. WO 2016 / 161309, which is incorporated herein by reference in its entirety). Results showed a statistically significant decrease in tumor cell growth in the inactivated whole cell vaccine group compared to the untreated control group starting on day 23 after injection (p = 0.02 on day 23 and p < 0.001 on day 25). [Figure 9] Show overall survival in the vaccinated (inactivated whole cell vaccine) group versus the untreated / saline (control) group of mice with PyMT tumors. Mice injected with the inactivated whole cell vaccine had a significantly extended survival period compared to the saline-treated control group (p = 0.009, Mantel-Cox log-rank test). [Figure 10] Show the size of 4T1 tumors in mice before surgical removal. After surgery, the mice were placed in 3 groups, and each group was shown to have similar mean tumor size (p = 0.9) and dissemination. PBS (“control”), n = 5 mice; adjuvant, n = 8 mice; inactivated whole cell vaccine, n = 8 mice. [Figure 11A]This report shows the results of experiments in which mice were treated weekly with PBS (control), losartan and cationic liposome-DNA complexes (CLDCs) (adjuvant), or inactivated whole-cell vaccine (adjuvant + vaccine) starting 24 hours after surgical removal of the primary tumor. Metastatic disease in the lungs was quantified using IVIS imaging after intraperitoneal injection of 100 μl of luciferin. As shown in Figure 11A, the vaccine-treated mice showed a significant reduction in the measured metastatic burden compared to adjuvant-treated mice (day 14, p=0.0157), and compared to both control and adjuvant-treated mice (day 16, p=0.0119 and p=0.0021, respectively). Figure 11B shows the photon flux data for each mouse in each group over time. [Figure 11B] This report shows the results of experiments in which mice were treated weekly with PBS (control), losartan and cationic liposome-DNA complexes (CLDCs) (adjuvant), or inactivated whole-cell vaccine (adjuvant + vaccine) starting 24 hours after surgical removal of the primary tumor. Metastatic disease in the lungs was quantified using IVIS imaging after intraperitoneal injection of 100 μl of luciferin. As shown in Figure 11A, the vaccine-treated mice showed a significant reduction in the measured metastatic burden compared to adjuvant-treated mice (day 14, p=0.0157), and compared to both control and adjuvant-treated mice (day 16, p=0.0119 and p=0.0021, respectively). Figure 11B shows the photon flux data for each mouse in each group over time. [Figure 12] This shows the frequency of primary tumor regrowth resulting from incomplete removal of the primary tumor in various treatment groups. [Figure 13]This survival curve shows enhanced survival in mice treated with an inactivated whole-cell vaccine. Mice were euthanized when they were near death (i.e., weight loss >10%, seizures, reduced mobility, sloppy appearance, etc.). The median survival time for the control and adjuvant groups was 17.5 days, while it was 24 days for the inactivated whole-cell vaccine-treated group. This difference in survival was not statistically significant (p=0.1), but it is biologically important and demonstrates the invasiveness of 4T1 tumors. [Figure 14] As described in Example 3, this graph shows the doubling time of tumor size. The doubling time was longer in mice treated with the inactivated whole-cell vaccine (p=0.01). [Figure 15] This graph shows the tumor growth area at 3, 5, 7, 10, 13, and 19 days after tumor injection in mice with subcutaneous Lewis lung cancer tumors (LLC) treated with PBS control or inactivated LLC vaccine. At 13 days (p=0.02) and 19 days (p=0.001) after tumor cell injection, tumor growth was significantly reduced in mice vaccinated with inactivated LLC vaccine. [Figure 16] These graphs show the subtypes of T cells in tumors obtained from control and vaccinated mice from the LLC study. Figure 16A shows the percentage of T cells that were either CD4+CD25+ (putative regulatory T cells) or CD8+CD25+. A significant decrease in CD4+CD25+ T cells was observed in vaccinated mice. Figure 16B shows the percentage of CD8+ T cells expressing the immunosuppressive proteins PD-1, Lag3, or Tim3. Figure 16C shows the percentage of CD4+ T cells expressing the immunosuppressive proteins PD-1, Lag3, or Tim3. In each dataset shown in Figures 16A-C, the control is shown on the left and the vaccine is shown on the right. [Figure 17]This shows IFNg production (pg / ml) after isolation of splenocytes from healthy naive B6 mice that were vaccinated and booster-immunized with an inactivated whole-cell vaccine (derived from 4T1 mouse tumor cells) and various immune adjuvants. Next, splenocytes were restimulated in vitro with inactivated 4T1 tumor cells for 72 hours, and IFNg was measured by ELISA. The CLDC adjuvant system produced the best IFNg response. [Figure 18] Figure 17 shows the mean fluorescence intensity (MFI) of serum IgG antibodies derived from mouse blood at a 1:1000 dilution, bound to 4T1 living cells. All vaccine / adjuvant systems demonstrated significantly higher binding affinity than control or inactivated cells alone. [Figure 19] The results of an experiment in which mice were injected with 4T1 mammary tumor cells, then surgically removed and used to produce an inactivated whole-cell vaccine, after which metastatic disease in the lungs was quantified using IVIS imaging are shown. Mice shown in the right panel were treated with the inactivated whole-cell vaccine, while mice shown in the left panel were not administered any vaccine. Scale bars for luminescence are also presented. 62% of vaccinated mice were negative for lung metastases at the same time point, while 80% of untreated mice developed lung tumors 16 days after tumor cell removal. [Figure 20] This paper presents a representative scheme for inactivating cells using UV light and riboflavin, preparing a vaccine composition, and treating patients who require it. [Figure 21] This shows the cell surface staining of mouse LLC cells after UV+Rf (UV light + riboflavin) inactivation. [Figure 22] This shows the surface staining of mouse 4T1 breast cancer cells after UV+Rf inactivation. [Figure 23] This shows GFP expression in mouse melanoma GFP+B16 tumor cells after inactivation by either UV+RF or gamma rays in vitro. [Figure 24] This shows the expression of the mouse tumor-associated antigen gp70 after UV+Rf inactivation of mouse colon cancer, CT26, and tumor cells. [Figure 25]Surface protein staining of inactivated in vitro canine tumor tissue 1 hour and 48 hours after UV+Rf inactivation is shown. Cells were maintained at 4°C for 48 hours after inactivation. [Figure 26] Two additional samples of in vitro canine tumor tissue exhibit UV+RF inactivation. [Figure 27] This chart shows the staining of inactivated human hepatocellular carcinoma (HepG2) cells for the surface marker GLUT1. Inactivated cells are shown in the left panel, and live cells are shown in the right panel. The chart below shows the percentage of antibody-positive and antibody-negative cells when HepG2 cells are stained for the surface markers GLUT1 and HLA1. [Figure 28] This shows the proliferation of T cells derived from the spleen of untreated 4T1 tumor-carrying mice. The T cells proliferated in vitro when cultured with inactivated 4T1 tumor cells. [Figure 29] This shows the lack of proliferation of inactivated 4T1 mouse mammary cancer cells in culture at various time points after inactivation. [Figure 30] The left panel shows the lack of proliferation of inactivated human liver cancer cells HepG2, and the right panel shows data on the lack of proliferation of both HepG2 and human colon cancer cells CRL-2577. [Modes for carrying out the invention]

[0011] A method for inactivating cells and inhibiting their replication using UV light and riboflavin is provided herein. This chemical process is specific to the DNA / RNA present in the cells. Thus, while the DNA and / or RNA of the cells are modified, proteins (including cell surface antigens, enzymes, etc.) remain uncontacted during the process. By inhibiting the replication process while preserving the antigens and phenotype of the cells, the treated cancer cell preparations can be used as vaccine compositions. Due to the fact that the antigens are present in their native state on the cells of the vaccine composition, the immune response may be enhanced to a degree exceeding that observed with single antigen or protein preparations intended to induce the same response. This immunological effect is further enhanced by the combination of inactivated whole cells and an adjuvant.

[0012] This technology can be used autologously or by similar methods, i.e., using tumor cells isolated from the patient, cancer stem cell preparations, or cells grown in a culture system. When administered to a patient, the whole-cell vaccine reduces tumor growth, decreases metastasis, and extends survival.

[0013] Therefore, the techniques described herein provide a rapid method for isolating, preparing, and administering cancer cell vaccines to patients to generate a response in patients competing with the use of standard chemotherapeutic agents.

[0014] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. The terminology used in the forms for carrying out the inventions herein is intended solely to describe specific embodiments and is not intended to limit them.

[0015] definition The following terms are used in the description and the attached claims herein.

[0016] The singular forms "a," "an," and "the" are also intended to include the plural form unless otherwise specified in the context.

[0017] Furthermore, when the term “about” is used herein, for example, when referring to a measurable value such as volume, dosage, time, or temperature, it means encompassing a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of a particular quantity.

[0018] Furthermore, as used herein, “and / or” refers to and encompasses all possible combinations of one or more of the related enumerated items, and, when interpreted in the context of an alternative ("or"), the absence of any combination.

[0019] Unless otherwise indicated by the context, it is specifically intended that the various features described herein may be used in any combination.

[0020] As used herein, the terms “reduce,” “reduces,” “reduction,” and similar terms mean a reduction of at least about 10%, about 15%, about 20%, about 25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or more.

[0021] As used herein, the terms “enhance,” “enhances,” “enhancement,” and similar terms indicate an increase of at least about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more.

[0022] The terms “treat,” “treating,” or “~treatment” (and their grammatical variations) mean that the severity of a patient’s condition is reduced, at least partially improved, or stabilized, and / or that some reduction, mitigation, reduction, or stabilization of at least one clinical symptom is achieved, and / or that the progression of the disease or disability is delayed.

[0023] The terms “prevent,” “preventing,” and “prevention” (and their grammatical variations) refer to the prevention and / or delay of the onset of disease, disability and / or clinical symptoms in a patient, and / or a reduction in the severity of the onset of disease, disability and / or clinical symptoms compared to what would occur without the methods of this disclosure. Prevention may be complete, for example, the complete absence of disease, disability and / or clinical symptoms. Prevention may also be partial, such that the onset and / or severity of disease, disability and / or clinical symptoms in a patient is less than what would occur in the absence of this disclosure.

[0024] When used herein, “therapeutic dose” means the amount sufficient to affect the treatment of a disease or at least one of its clinical manifestations when administered to a patient. “Therapeutic dose” may vary, for example, depending on the disease and / or symptoms of the disease, the severity of the symptoms of the disease and / or disorder, the age, weight, and / or health of the patient being treated, and the judgment of the prescribing physician. The appropriate dose in any given example may be determined by those skilled in the art or measurable by routine experimentation.

[0025] Cancer vaccine composition A cancer vaccine composition is provided herein. The composition optionally contains, essentially consists of, or comprises inactivated cancer cells in combination with an adjuvant. Cancer cells are inactivated by modifying their DNA and / or RNA, thereby causing them to malfunction. Modification of the cells' DNA and / or RNA does not kill the cells; i.e., the cancer vaccine is a live vaccine in which replication is inactivated. Because cell viability is maintained, the vaccine presents a live antigen target to the patient's immune system. Peripheral administration stimulates an immune response against primary tumors and metastases.

[0026] In some embodiments, the cancer vaccine comprises, essentially consists of, or consists of cancer cells that have been inactivated using a photochemical process that inactivates tumor cell DNA and / or RNA replication while preserving protein type and phenotype. In some embodiments, the cancer cell DNA and / or RNA in the cancer cell vaccine contains modified bases. For example, in some embodiments, the cancer cell DNA in the vaccine may contain modified guanine bases, such as oxidized guanine bases.

[0027] In some embodiments, the cancer cells are autologous cancer cells. As used herein, “autologous” refers to cells removed or induced from the same patient to whom the vaccine is administered. In some embodiments, the cancer cells are allogeneic cells. As used herein, “allogeneic” refers to cells removed or induced from a donor who is not the patient to whom the vaccine is administered.

[0028] In some embodiments, cancer cells are derived from patients with one or more types of cancer. For example, cancer cells may be isolated or induced from patients with cancer. The cancer may be a solid tumor or a liquid tumor. Cancer cells may be isolated or induced from a primary tumor or a metastatic tumor. The cancer may be stage I, stage II, stage III, or stage IV. In some embodiments, cancer cells may be induced from patients with breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer. In some embodiments, skin cancer is melanoma. In some embodiments, hematological cancer is leukemia, lymphoma, or myeloma. In some embodiments, leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. In some embodiments, lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. In some embodiments, myeloma is multiple myeloma.

[0029] In some embodiments, cancer cells are derived from immortalized cancer cell lines. As used herein, “cancer cell line” refers to a transformed cell line derived from a cancer sample. Typically, cancer cell lines have the ability to generate tumors when explanted into a suitable host. Cancer cell lines typically undergo essentially unrestricted cell division in vitro, retaining characteristics common to their originating cancer, including, for example, loss of differentiation and loss of contact inhibition. Cancer cell lines may include cell lines that have been genetically modified to express proteins that allow cells to be more readily recognized by antigen-presenting cells.

[0030] In some embodiments, cancer cells are cancer stem cells.

[0031] In some embodiments, the cells are derived from non-cancerous but abnormal growth, i.e., benign tumors or growths.

[0032] In some embodiments, the cancer vaccine includes, essentially consists of, or comprises leukocytes (e.g., tumor-associated macrophages), tumor-associated endothelial cells, tumor-associated fibroblasts, or any other cell types present in the tumor microenvironment.

[0033] In some embodiments, the cancer vaccine composition further comprises an adjuvant. The effect of the adjuvant is to provide an additional immunological response. In some embodiments, the adjuvant modifies monocyte function.

[0034] Examples of suitable adjuvants include saponin preparations, virosomes, virus-like particles, non-toxic derivatives of enteric lipopolysaccharide (LPS), immunostimulatory oligonucleotides (e.g., immunostimulatory oligonucleotides having a CpG motif), mineral-containing compositions, oil emulsions, polymers, micelle-forming adjuvants (e.g., liposomes), immunostimulatory complex matrices (e.g., ISCOMATRIX), particles, squalene, phosphates, cationic liposome-DNA complexes (CLDCs), DDA, and DNA adjuvants. Examples of suitable adjuvants include γ-insulin, ADP-ribosylated toxin, detoxified derivatives of ADP-ribosylated toxin, Freund's complete adjuvant, Freund's incomplete adjuvant, muramyl dipeptide, monophosphoryl lipid A (MPL), polyIC, CpG oligodeoxynucleotide (ODN), imiquimod, adjuvant AS01, adjuvant AS02, adjuvant AS03, MF59®, and aluminum or aluminum salts (e.g., alum, aluminum phosphate, aluminum hydroxide). Other suitable adjuvants include TLR agonists, NOD agonists, and lipid-DNA agonist complexes.

[0035] In some embodiments, the cancer vaccine composition further comprises one or more agonists or antagonists.

[0036] In some embodiments, the agonist includes a Toll-like receptor (TLR) agonist. In some embodiments, the TLR agonist is an agonist of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, or TLR12. In certain embodiments, the agonist is a TLR3 and / or TLR9 agonist.

[0037] In some embodiments, the antagonist is a CC chemokine receptor type 2 (CCR2) antagonist.

[0038] In some embodiments, the antagonist is an angiotensin receptor blocker (ARB), such as losartan, telmisartan, irbesartan, azilsartan, candesartan, eprosartan, olmesartan, or valsartan. In some embodiments, the ARB is administered in doses between about 5 and about 100 mg / kg, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg / kg.

[0039] In some embodiments, the cancer vaccine comprises at least one (i.e., one, two, or all three) of a TLR agonist, a CCR2 antagonist, and an ARB.

[0040] In some embodiments, an agonist or antagonist (e.g., a TLR3 and / or TLR9 agonist) is contained within or conjugated to a liposome. Liposomes are spherical, self-closed vesicles composed of amphiphilic lipids. Liposomes may be monolayers having a single lipid bilayer membrane, or multilayers having two or more concentrically arranged bilayers. Preferred liposomes may have a selected average particle size (diameter) of about 200–500 nm. Various methods for preparing liposomes and encapsulating therapeutic agents within them are well documented (see, for example, U.S. Patent No. 3,932,657, U.S. Patent No. 4,311,712, and U.S. Patent No. 5,013,556, all of which are incorporated herein by reference). Known methods include reverse-phase evaporation, as described in U.S. Patent No. 4,235,871, which is incorporated herein by reference.

[0041] The lipids used for liposome formation described herein include vesicle-forming lipids having two hydrocarbon chains, typically an acyl chain and a polar head group. This class includes phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains typically have carbon atom lengths between approximately 14 and 22 and exhibit varying degrees of unsaturation. The selection of lipids and their ratios may be modified to achieve a desired degree of flexibility or rigidity, to control stability, and / or to control the release rate of the encapsulated agent. When two or more lipid types are used, an appropriate amount of relatively unsaturated lipids (such as PC) may be used to form stable liposomes. In one embodiment, at least 45–50 mol% of the lipids used to form liposomes is PC.

[0042] Liposomes may also contain lipids derivatized with hydrophilic polymers such as polyethylene glycol (PEG). Suitable hydrophilic polymers include polyvinylpyrrolidone, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, and hydrophilic peptide sequences. Methods for preparing lipids derivatized with hydrophilic polymers are known (see, for example, U.S. Patent No. 5,395,619, incorporated herein by reference).

[0043] In some embodiments, the cancer vaccine comprises a cationic liposome-DNA complex (CLDC).

[0044] In some embodiments, the cancer vaccine further includes a photosensitizer such as riboflavin (vitamin B2). In some embodiments, the cancer vaccine is substantially free of a photosensitizer.

[0045] In some embodiments, the cancer vaccine composition further comprises a carrier. In some embodiments, cells and / or a photosensitizer are suspended in the carrier. In some embodiments, the carrier comprises normal physiological saline (e.g., 0.9% sodium chloride), glucose saline (e.g., 5% glucose in 0.9% sodium chloride), or phosphate-buffered saline (e.g., 137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na2HPO4, 2 mmol / L KH2PO4).

[0046] In some embodiments, the cancer vaccine composition further comprises one or more additional pharmaceutically acceptable components well known to those skilled in the art, such as, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners. Suitable carriers, diluents, excipients, etc., can be found in standard pharmaceutical textbooks. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0047] Method for producing cancer cell vaccines The cancer cell vaccines described herein are prepared using harmless chemicals in a selective process that inhibits the cell replication process while preserving the antigen protein structure. More specifically, the cancer cell vaccines are prepared by the combined application of a photosensitizer and light to render cancer cells replication-deficient while preserving other biological functions of the treated cells and proteins. An exemplary scheme for the preparation and use of the cancer cell vaccines is shown in Figure 20. The process for preparing the cancer vaccines of this disclosure is described in detail below.

[0048] First, cancer cells are prepared. The cancer cells may be autologous, i.e., removed or induced from the subject to be vaccinated. In some embodiments, the cancer cells may be homogeneous. The cancer cells may also be induced from a cancer cell line.

[0049] In some embodiments, cancer cells are cancer stem cells. In some embodiments, the cancer vaccine includes, essentially consists of, or comprises leukocytes (e.g., tumor-associated macrophages), tumor-associated endothelial cells, tumor-associated fibroblasts, or any other cell types present in the tumor microenvironment.

[0050] In some embodiments, cancer cells are provided as a single cell suspension during inactivation. In some embodiments, the cells are suspended in a culture medium during inactivation. Exemplary media that may be used include, but are not limited to, RPMI1640, MEM, DMEM, IMDM, DMEM-F12, Opti-MEM, HAM F12, Media 199, or combinations thereof.

[0051] Next, cancer cells are inactivated using photochemical techniques. This is achieved using photosensitizers that can act as electron transfer agents. By applying a photosensitizer that can be excited near guanine bases in a DNA or RNA construct, selective modification of these bases (e.g., oxidation, crosslinking, fragmentation, deamination) becomes possible. Since the electrochemical reactions can occur primarily over short distances, the photosensitizer needs to be bound to or associated with (i.e., intercalated with) the nucleic acid to carry out the desired chemical reaction.

[0052] In some embodiments, the photosensitizer is a flavin, e.g., riboflavin (vitamin B2), flavin mononucleotide, or flavin adenine dinucleotide. In some embodiments, the photosensitizer is a tertiary aliphatic amine (e.g., 1,4-diazabicyclo(2,2,2)octane), piperazine (e.g., N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1,4-dimethylpiperazine), amino acids (e.g., tyrosine, tryptophan, histidine, methionine), enzymes (e.g., superoxide dismutase), or EDTA (ethylenediaminetetraacetic acid). In some embodiments, the photosensitizer is riboflavin.

[0053] The cells are added to a solution containing a photosensitizer (e.g., riboflavin), or the photosensitizer is added to a solution containing the cells (e.g., a single-cell suspension of the cells in a culture medium).

[0054] In some embodiments, the concentration of the photosensitizer used during inactivation is approximately 10 μM to approximately 100 μM, for example, approximately 10 μM, approximately 15 μM, approximately 20 μM, approximately 25 μM, approximately 30 μM, approximately 35 μM, approximately 40 μM, approximately 45 μM, approximately 50 μM, approximately 55 μM, approximately 60 μM, approximately 65 μM, approximately 70 μM, approximately 75 μM, approximately 80 μM, approximately 85 μM, approximately 90 μM, approximately 95 μM, or approximately 100 μM. In some embodiments, the solution contains the photosensitizer at a concentration of about 1 μM to about 50 μM, for example, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM. In some embodiments, the photosensitizer concentration is less than about 10 μM, for example, less than about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM, about 4 μM, about 3 μM, about 2 μM, or about 1 μM.

[0055] Next, a solution containing the photosensitizer and cells (optionally in the culture medium) is subjected to phototreatment. Phototreatment may include treatment with visible light, ultraviolet light, and / or infrared light. Phototreatment inactivates DNA and / or RNA in cancer cells by modifying the bases of these nucleic acids. In some embodiments, guanine bases are selectively modified. In some embodiments, guanine bases are selectively oxidized. Oxidized guanine bases cannot be repaired by natural enzymes and cell repair mechanisms. Thus, there is no possibility of reversing the induced modification to a form that would restore the cell's ability to replicate.

[0056] In some embodiments, the phototreatment includes, essentially consists of, or comprises treatment with ultraviolet (UV) light. The UV light may be UV-A, UV-B, or UV-C light. The UV light may have wavelengths of 170–400 nm, for example, the entire range and partial range between them. For example, in some embodiments, the UV light has wavelengths of 315–400 nm, 310–320 nm, 280–360 nm, 280–315 nm, or 180–280 nm. The UV light may be provided by a UV light source known in the art, for example, a Mirasol® PRT illumination device (TerumoBCT, Lakewood, Colorado). In some embodiments, cells may be treated simultaneously with light of multiple wavelengths.

[0057] In certain embodiments, when riboflavin is used as a photosensitizer, UV light having a wavelength of 310–320 nm is used. The inventors have determined that this wavelength prevents riboflavin from reacting in free solution, thereby preventing the generation of undesirable oxygen free radicals. At these wavelengths, riboflavin reacts selectively when intercalated with nucleic acids.

[0058] The UV light dose may vary depending on the volume of the solution being treated. For example, the UV light dose may be between 200 and 400 joules (e.g., 300 joules) for a volume of solution of approximately 170 to 370 ml. As will be understood by those skilled in the art, if the volume of material being treated is above or below this range, the dose may be adjusted upward or downward.

[0059] In some embodiments, the dose of UV light may be about 200 joules to about 600 joules, for example, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 joules. In some embodiments, the volume of the cancer cell preparation for luminescence may be about 200 ml to about 600 ml, for example, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 ml. In some embodiments, the UV light dose may be about 0.5 J / ml to about 3.0 J / ml. For example, the UV light dose may be about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0 joules / ml.

[0060] The cells may be treated with UV light for approximately 1 to 60 minutes, for example, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the cells are treated with UV light for approximately 1 to 10 minutes, 1 to 5 minutes, or 1 to 3 minutes.

[0061] In some embodiments, cancer cells are pre-incubated for a predetermined period of time in a solution containing a photosensitizer (e.g., riboflavin) before being phototreated.

[0062] In some embodiments, the cells do not undergo any additional purification or modification steps after phototreatment. In other embodiments, the cancer cells are isolated and / or washed after phototreatment. For example, the cells may be pelletized and optionally washed after phototreatment. By pelletizing and / or washing the cells, the photosensitizer (e.g., riboflavin) may be substantially removed from the composition. In some embodiments, the cancer cells are concentrated after phototreatment.

[0063] In some embodiments, cancer cells are resuspended or combined with one or more additional pharmaceutically acceptable components as described above after phototreatment. In some embodiments, cancer cells are resuspended in a solution containing the adjuvant after phototreatment.

[0064] In some embodiments, the cells remain viable for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after phototreatment. In some embodiments, the cells die (e.g., by apoptosis) on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after treatment.

[0065] Cells produced using this method lack the ability to replicate, but substantially maintain and preserve the antigenic and epitope properties of the original native cells or antigens during processing. In some embodiments, the inactivation process does not substantially alter the metabolic processes, phenotype, or structure of cancer cells. For example, in some embodiments, the inactivation process does not substantially alter the expression of cell surface markers in cancer cells. In some embodiments, the inactivation process does not substantially alter the expression levels of cell surface markers such as EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC class I, PDL-L1, CD45, gp70, GFP, and / or CD90 in cells. In some embodiments, the inactivation process does not impair the integrity of the cell membrane and nuclear membrane of the cells.

[0066] The fact that cells are non-replicating protects the body from modified cell compositions involved in the formation of the native form of disease (cancer) or neoplastic lesions. Therefore, since the specificity of the chemicals preserves antigenic properties and cellular integrity and maintains the protein structure in its native state, the inactivated cells produced by this process provide an improved source for antigen presentation.

[0067] Treatment method The cancer cell vaccine compositions described herein can be used as vaccines or stimulants for priming and recognizing the immune system to cultivate an immune response in cancer patients. This targeted therapy results in reduced side effects compared to traditional treatments such as chemotherapy or radiation. In particular, because the vaccine's cancer cells maintain a normal phenotype, the likelihood of them inducing unwanted side effects is extremely low or nonexistent.

[0068] In some embodiments, a cancer cell vaccine may be administered to a patient to treat or prevent cancer in the patient. The cancer to be treated or prevented may be a solid tumor or a liquid tumor. For example, the cancer to be treated or prevented may be breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer. In some embodiments, skin cancer is melanoma. In some embodiments, hematological cancer is leukemia, lymphoma, or myeloma. In some embodiments, leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. In some embodiments, lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. In some embodiments, myeloma is multiple myeloma.

[0069] In some embodiments, the vaccine may be administered to a patient to treat or prevent non-cancerous but abnormal growths in the patient, i.e., benign tumors or growths. While most benign tumors / growths are treatable by surgery, some are located in positions where surgery may be impossible and / or radiation may not be appropriate. Examples of non-cancerous growths that may be treated include, but are not limited to, adenoma, fibroma, neuroma, hemangioma, seborrheic keratosis, dermatosis papulosa nigra, and sebaceous hyperplasia.

[0070] In some embodiments, the patient is evaluated for immune function and immune status prior to administration of the cancer vaccine. Such evaluation may include, but is not limited to, DTH skin test, blood test, lymph node aspiration test, tumor tissue test, and / or determination of whether the patient is allergic, B cell responsiveness, etc. In some embodiments, the patient is not evaluated for immune function and immune status prior to administration of the cancer vaccine.

[0071] In some embodiments, the patient may be immunocompetent. In other embodiments, the patient may be immunocompromised. Optionally, the vaccine may be used in combination with a genetic test to quantify the degree of immune responder or non-responder.

[0072] It will be understood by those skilled in the art that the appropriate number of cells in the cancer vaccine composition may vary among patients. In some embodiments, the cancer vaccine comprises about 1×10 3 about 1×10 4 about 1×10 5 about 1×10 6 about 1×10 7 about 1×10 8 about 1×10 9 or about 1×10 10 cells, or about 1×10 5 to about 1×10 8 cells.

[0073] In some embodiments, per administration, about 1×10 5 to about 1×108 Individual cells are administered to the patient. For example, approximately 1 x 10⁶ cells per dose. 5 , about 5×10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , or approximately 1 x 10 8 Individual cells may be administered to the patient. In some embodiments, the dose is a divided dose, where the total number of cells for administration is divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 partial doses. One or more partial doses may be administered peripherally to the patient at different locations on the patient's body. Each partial dose may be administered at approximately the same time, or the administration of partial doses may be staggered. For example, partial doses may be administered at intervals of 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 3 hours.

[0074] In some embodiments, the cancer vaccine is administered to the patient once or more times. In some embodiments, the cancer vaccine is administered to the patient once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.

[0075] The cancer vaccine may be administered to the patient daily, approximately every 3 days, approximately every 7 days, approximately every 14 days, approximately once a month, or approximately once a year. In some embodiments, the cancer vaccine is administered at least once a week, at least every 2 weeks, or at least once every 6 months. In some embodiments, the cancer vaccine is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, twelve times, fifteen times, twenty times, or twenty-five times per year.

[0076] In some embodiments, a first cancer vaccine and a second cancer vaccine are administered to the patient. In some embodiments, the second cancer vaccine is administered after the first cancer vaccine to enhance the immune response. In some embodiments, the immune response and / or tumor growth in the patient is monitored between the administration of the first vaccine and the administration of the second vaccine. In some embodiments, the second cancer vaccine is administered when it is determined that the patient has not shown an adequate immune response after the administration of the first vaccine, or when it is determined that the tumor has continued to grow or metastasize after the administration of the first vaccine. In some embodiments, the first and second cancer vaccines include cells isolated or derived from a first tumor extract. For example, tumors removed from a patient may be used to prepare the first and second vaccines, and after the administration of the first vaccine, the second vaccine is stored for later use. In some embodiments, the first and second vaccines include cells isolated or derived from separate tumor extracts. For example, a tumor removed from a patient may be used to produce the first vaccine, and after tumor recurrence or metastasis, a recurrent or metastatic tumor may be removed and used to produce the second vaccine.

[0077] Cancer vaccines may be delivered to patients intramuscularly, intramucosally, intranasally, subcutaneously, intratumorally, intradermally, percutaneously, intravaginally, intraperitoneally, intrarectally, intra-articularly, or intralymphally, or orally, or intravenously. In some embodiments, administration may be via sublingual, buccal, intra-organ (e.g., intraspleen), or inhalation routes. For intravenous, cutaneous, or subcutaneous injection, or injection into tumor sites, cancer cell vaccines may be in the form of parenterally acceptable aqueous solutions having a suitable pH, isotonicity, and stability. Those skilled in the art can adequately prepare suitable solutions using isotonic media such as sodium chloride injection, Ringer's injection, or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0078] In some embodiments, the vaccine is administered peripherally to the patient. In some embodiments, multiple aliquots of the cancer vaccine are administered peripherally to the patient at different sites.

[0079] In some embodiments, the cancer vaccine is administered simultaneously with or consecutively (either before or after) the vaccine enhancer. In some embodiments, the vaccine enhancer is an angiotensin receptor blocker (ARB) or a beta-blocker (BB). Exemplary vaccine enhancers include losartan, telmisartan, irbesartan, azilsartan, candesartan, eprosartan, olmesartan, valsartan, propranolol, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, and timolol. In some embodiments, the vaccine enhancer is selected from the group consisting of losartan and propranolol. In some embodiments, the vaccine enhancer is losartan. In some embodiments, the vaccine enhancer is propranolol.

[0080] In some embodiments, the vaccination protocol described herein comprises administering a cancer cell vaccine composition comprising inactivated live cancer cells and a potent adjuvant comprising liposome-attached TLR3 and / or TLR9 agonists, and further comprising sequential or concurrent administration of a vaccine enhancer (e.g., losartan) administered at or before / after vaccination to reduce the recruitment of immunosuppressive myeloid cells.

[0081] In some embodiments, the vaccination protocol described herein includes administering a cancer cell vaccine composition containing inactivated live cancer cells to a patient in need. An adjuvant may optionally be administered at the time of vaccination. In some embodiments, the adjuvant is administered after vaccination, for example, about 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after vaccination, to enhance the immune response. In some embodiments, the adjuvant comprises liposomes, such as CLDCs. In some embodiments, a vaccine enhancer, such as losartan, may be administered at the time of vaccination or before or after. In some embodiments, the vaccine enhancer, such as losartan, may be administered after vaccination, for example, about 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after vaccination. In some embodiments, the vaccine enhancer, such as losartan, may optionally be administered to the patient daily for a number of therapeutically effective days from the day the vaccine is administered. In some embodiments, the vaccine enhancer (e.g., losartan) is administered in doses between approximately 5 and approximately 100 mg / kg, for example, approximately 5, approximately 10, approximately 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, or approximately 100 mg / kg.

[0082] In some embodiments, the treatment reduces tumor growth or regrowth by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to tumor growth in unvaccinated patients. In some embodiments, the treatment extends patient survival by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to unvaccinated patients. In some embodiments, the treatment reduces the incidence of metastasis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to unvaccinated patients.

[0083] Cancer cell vaccines may induce an immune response in patients. In some embodiments, the immune response may include one or more of the following: (i) upregulation of immunoglobulins (e.g., IgG, IgM), (ii) T cell activation (e.g., production of multiple T cells that match multiple cancer neoantigens), (iii) regulation of innate immune cells (e.g., myeloid cells), and (iv) restoration of “exhausted” T cell populations.

[0084] Suitable patients include both birds and mammals. The term "birds," as used herein, includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, parrots, and parakeets. The term "mammals," as used herein, includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, horses, cats, dogs, and rabbits. Human subjects include newborns, infants, young adults, adults, and the elderly. The terms "subjects" and "patients" are interchangeable within this specification.

[0085] Cancer cell vaccines may be administered to patients who have a pre-existing condition that interferes with treatment with other therapies such as radiation, chemotherapy, or surgical resection.

[0086] Combination therapy Cancer cell vaccines may be administered alone or in combination with other treatments / therapies, simultaneously or sequentially, depending on the condition of the patient being treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (e.g., administration of drugs, e.g., active agents including chemotherapeutic agents); surgery; and radiotherapy. Further examples of treatments and therapies include immunotherapy, e.g., antibody therapy, adoptive cell therapy (ACT), and vaccine-based therapy. In some embodiments, the cancer cell vaccines described herein may be administered after another treatment / therapy to eliminate any remaining tumor cells.

[0087] In some embodiments, the cancer vaccine may be administered in combination with one or more of the following therapies: checkpoint inhibitors (e.g., PD-1 or PD-L-1 inhibitors), antibody therapies, genetically modified dendritic cells, or genetically modified T cells (e.g., CAR-T cells).

[0088] In some embodiments, cancer vaccines may be administered alone or in combination with chemotherapeutic agents. “Chemotherapeutic agents” are compounds useful in the treatment of cancer, regardless of their mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, spindle toxin plant alkaloids, cytotoxic / antitemogenic antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy.

[0089] Examples of suitable chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), and paclitaxel (TAXOL®, Bristol-Myers Squibb). Examples include Oncology (Princeton, NJ), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentozabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethaneamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1 / 2, HPPD, and rapamycin.

[0090] Further examples of chemotherapeutic agents include oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, International Publication No. 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), and SF-1126 (PI3K inhibitor, Semafore (Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), ronafarnib (SARASAR®, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA®, Johnson & Johnson), ABRAXANE® (cremofol-free), albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chlorambucil, AG1478, AG1571 (SU5271;Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), camphospamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and pigosulfan; aziridines such as benzodopa, carbocone, metsuredopa and uredopa; etilenimine and methylamelanamine, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelanamine; acetogenins (especially bratacin and bratacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (its adzelesin, calzelcin) and including biceresin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongstatin; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembitin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, e.g., engine antibiotics (e.g., calicheamicin, calicheamicin γ1I, calicheamicin ωI1 (Angew) Chem.Intl.Ed.Engl.(1994)33:183-186); Dinemycin, Dinemycin A; Bisphosphonate, Clodronate, etc.; Esperamicin;(and neocartinostatin chromophores and related pigment proteins enediin antibiotic chromophores), acrasinomycin, actinomycin, authramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcelomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, keramycin, rhodo Rubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; frolinic acid Folic acid supplements such as acid; acegraton; aldofsphamide glycosides; aminolevulinic acid; enyluracil; amsacrin; bestrabutyl; bisanthren; edatraxate; defofamine; demecoltin; diazicon; elfornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; mytansinoids such as mytansin and ansamitosin; mitogluazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid;2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, OR); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecin (especially T-2 toxin, Beraclin A, Loridine A and Anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; 6-Thiogunine; Mercaptopurine; Methotrexate; Cisplatin and Carboplatin Examples include platinum analogs such as tin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and any pharmaceutically acceptable salts, acids, and derivatives of the above.

[0091] Furthermore, the definition of "chemotherapeutic agents" includes (i) anti-hormone drugs that act to regulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modifiers (SERMs), such as tamoxifen (NOLVADEX®; including tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), and formestanier (for (i) mestanie), phadrozol, RIVISOR® (borozol), FEMARA® (letrozole; Novartis), and ARIMIIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (International Publication No. 2007 / 044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, e.g., PKC-α, Raf, and H-Ras, e.g., oblimersen (GENASENSE®, Genta Inc.)(vii) Ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) Vaccines, such as gene therapy vaccines, such as ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) Anti-angiogenic drugs such as bevacizumab (Avastin®, Genentech); and any pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0092] Furthermore, the definition of "chemotherapeutic agents" includes therapeutic antibodies, such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen); rituximab (RITUXAN®, Genentech / Biogen Idec); ofatumumab (ARZERRA®, GSK); pertuzumab (PERJETA®, OMNITARG®, 2C4, Genentech); trastuzumab (HERCEPTIN®, Genentech); tositumomab (Bexxar, Corixia); and the antibody drug conjugate gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

[0093] Humanized monoclonal antibodies that have therapeutic potential as chemotherapeutic agents in combination with the vaccines disclosed herein include alemtuzumab, apolizumab, aselizumab, atlizumab, bapineozumab, bevacizumab, vivacuzumab meltansine, cantuzumab meltansine, sedelizumab, certolizumab pegol, cidofcituzumab, cidotuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erulizumab, felbizumab, fontrizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, rabetuzumab, lintuzumab, matsuzumab, mepolizumab, and motabizumab. Motobizumab, natalizumab, nimotuzumab, norovizumab, numabizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecufcituzumab, pectuzumab, pertuzumab, paxerizumab, larivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, roberizumab This includes bruplizumab, cibrotuzumab, ciprizumab, sontuzumab, tacutuzumab tetraxetan, tadocizumab, talizumab, tefivazumab, tocilizumab, tralizumab, trastuzumab, tucotsuzumab cermoloykin, tuccituzumab, umabizumab, urtoxazumab, and vizilizumab.

[0094] Numbered Embodiments of the Invention 1. A cancer vaccine composition containing inactivated cancer cells that lack the ability to replicate. 2. The cancer vaccine composition of Embodiment 1, wherein the cancer cells are derived from a patient suffering from one or more types of cancer. 3. The cancer vaccine composition of Embodiment 2, wherein the patient has one or more of the following cancers: breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, and hematological cancer. 4. The cancer vaccine composition of Embodiment 3, wherein the skin cancer is melanoma. 5. The cancer vaccine composition of Embodiment 3, wherein the blood cancer is leukemia, lymphoma, or myeloma. 6. The cancer vaccine composition of Embodiment 5, wherein the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. 7. The cancer vaccine composition of Embodiment 5, wherein the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. 8. The cancer vaccine composition of Embodiment 5, wherein the myeloma is multiple myeloma. 9. The cancer vaccine composition of Embodiment 2, wherein the patient has a benign tumor. 10. The cancer vaccine composition of Embodiment 2, wherein the cancer is metastatic cancer. 11. A cancer vaccine composition from any one of Embodiments 1 to 10, wherein the cancer cells are derived from an immortalized cell line. 12. A cancer vaccine composition from any one of Embodiments 1 to 11, wherein the cancer cells are autologous. 13. A cancer vaccine composition from any one of Embodiments 1 to 11, wherein the cells are of the same species. 14. The composition contains about 1 × 10 5 ~Approx. 1×10 8 A cancer vaccine composition comprising any one of embodiments 1 to 13, wherein each cancer cell is present. 15. A cancer vaccine composition in any one of Embodiments 1 to 14, wherein the DNA of cancer cells contains modified guanine bases. 16. A cancer vaccine composition which further comprises an adjuvant, any one of Embodiments 1 to 15. 17. The cancer vaccine composition of Embodiment 16, wherein the adjuvant modifies monocyte function. 18. The cancer vaccine composition of Embodiment 16, wherein the adjuvant contains aluminum hydroxide. 19. The cancer vaccine composition of Embodiment 16, wherein the adjuvant comprises CLDC. 20. The cancer vaccine composition of Embodiment 16, wherein the adjuvant comprises polyIC, CpG oligodeoxynucleotide (ODN), or imiquimod. 21. The cancer vaccine composition of Embodiment 16, wherein the adjuvant comprises liposomes. 22. A cancer vaccine composition of Embodiment 21, wherein liposomes are conjugated to an agonist. 23. The cancer vaccine composition of Embodiment 22, wherein the agonist is at least one agonist of TLR3 and TLR9. 24. Any one of Embodiments 1 to 23, further comprising a pharmaceutically acceptable carrier. 25. The cancer vaccine composition of Embodiment 24, wherein the pharmaceutically acceptable carrier is normal physiological saline, glucose saline, or phosphate-buffered saline. 26. A cancer vaccine composition in which cancer cells are inactivated using phototherapy, any one of Embodiments 1 to 25. 27. The cancer vaccine composition of Embodiment 26, wherein the phototreatment is performed for approximately 1 to 3 minutes. 28. A cancer vaccine composition of Embodiment 26 or 27, wherein the structure of the antigen protein on cancer cells is not substantially altered by phototreatment. 29. A cancer vaccine composition from any one of embodiments 26 to 28, wherein the DNA of cancer cells is modified by phototreatment. 30. The cancer vaccine composition of Embodiment 29, wherein guanine bases in the DNA of cancer cells are selectively oxidized by phototreatment. 31. Any one of the cancer vaccine compositions of Embodiments 26 to 30, wherein phototreatment does not substantially alter the metabolic processes, phenotype, or structure of cancer cells. 32. Any one of the cancer vaccine compositions of Embodiments 26 to 31, wherein phototreatment does not substantially alter the expression or activity of surface markers in cancer cells. 33. The cancer vaccine composition of Embodiment 32, wherein the expression levels of EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC class I, PDL-L1, CD45, gp70, GFP, or CD90 in cells do not change upon phototreatment. 34. A cancer vaccine composition from any one of Embodiments 26 to 33, wherein the integrity of the cell membrane or nuclear membrane of a cell is not impaired by phototreatment. 35. A cancer vaccine composition from any one of Embodiments 26 to 34, wherein the phototreatment includes treatment with UV light. 36. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 170 to 400 nm. 37. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 315 to 400 nm. 38. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 310-320 nm. 39. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 280 to 360 nm. 40. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 280 to 315 nm. 41. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 180 to 280 nm. 42. The cancer vaccine composition of Embodiment 35, wherein the UV light has a wavelength of 170-200 nm. 43. A cancer vaccine composition from any one of embodiments 35 to 42, wherein the UV light dose is approximately 200 joules to approximately 600 joules. 44. The cancer vaccine composition of Embodiment 43, wherein the UV light dose is approximately 200 joules to 400 joules. 45. The cancer vaccine composition of Embodiment 44, wherein the UV light dose is approximately 300 joules. 46. ​​A cancer vaccine composition in any one of embodiments 26 to 45, wherein the phototreatment is carried out by exposing cancer cells to light in the presence of a photosensitizer. 47. The cancer vaccine composition of Embodiment 46, wherein the concentration of the photosensitizer is approximately 1 μM to approximately 50 μM. 48. The cancer vaccine composition of Embodiment 46 or 47, wherein the concentration of the photosensitizer is less than approximately 10 μM. 49. A cancer vaccine composition from any one of embodiments 46 to 48, wherein the photosensitizer is riboflavin. 50. A method for treating cancer in a patient in need thereof, comprising administering to the patient one of the cancer vaccine compositions of Embodiments 1 to 49. 51. The method of Embodiment 50, wherein the cancer vaccine composition is administered simultaneously with or consecutively with the vaccine enhancer. 52. The method of Embodiment 51, wherein the vaccine enhancer is an angiotensin receptor blocker (ARB) or a beta-blocker (BB). 53. The method of Embodiment 51 or 52, wherein the vaccine enhancer is losartan. 54. The method of Embodiment 53, wherein the dose of losartan is between approximately 5 and approximately 100 mg / kg. 55. The method of Embodiment 54, wherein the dose of losartan is approximately 60 mg / kg. 56. The method of Embodiment 51 or 52, wherein the vaccine enhancer is propranolol. 57. Any one of embodiments 50 to 56, wherein the cancer vaccine composition is administered to a patient once. 58. Any one of embodiments 50 to 56, wherein the cancer vaccine composition is administered to a patient two or more times. 59. The method of Embodiment 58, wherein the cancer vaccine composition is administered to the patient 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. 60. The method of Embodiment 58 or 59, wherein the cancer vaccine composition is administered to a patient at least once every 7 days. 61. The method of Embodiment 58 or 59, wherein the cancer vaccine composition is administered to the patient at least once every 14 days. 62. The method of Embodiment 58 or 59, wherein the cancer vaccine composition is administered to a patient at least once every six months. 63. Any one of Embodiments 50 to 62, wherein the cancer vaccine composition is administered by a route selected from subcutaneous, intramuscular, intravenous, intranasal, sublingual, buccal, inhalation, intradermal, intratumoral, intraorganic, oral, and intraperitoneal. 64. The method of Embodiment 63, wherein the cancer vaccine composition is administered by subcutaneous injection. 65. The method of Embodiment 63, wherein the cancer vaccine composition is administered by intravenous injection. 66. The method of Embodiment 63, wherein the cancer vaccine composition is administered by intramuscular injection. 67. Any one of embodiments 50 to 62, wherein the patient is immunocompetent. 68. Any one of embodiments 50 to 62, wherein the patient is immunocompromised. 69. Any one of embodiments 50 to 68, wherein the treatment reduces tumor growth by at least 10% compared to tumor growth in unvaccinated patients. 70. The method of Embodiment 69, wherein the treatment reduces tumor growth by at least 20% compared to tumor growth in unvaccinated patients. 71. The method of Embodiment 70, wherein the treatment reduces tumor growth by at least 50% compared to tumor growth in unvaccinated patients. 72. Any one of embodiments 50 to 71, wherein the treatment extends the patient's survival by at least 10% compared to unvaccinated patients. 73. The method of Embodiment 72, wherein the treatment extends the patient's survival by at least 20% compared to unvaccinated patients. 74. The method of Embodiment 73, wherein the treatment extends the patient's survival by at least 50% compared to unvaccinated patients. 75. Any one of embodiments 50 to 74, wherein the treatment upregulates IgG and / or IgM in the patient. 76. Any one of embodiments 50 to 75, wherein the treatment activates T cells in the patient. 77. Any one of Embodiments 50 to 76, wherein the cancer is breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer. 78. The method of Embodiment 77, wherein the skin cancer is melanoma. 79. The method of Embodiment 77, wherein the blood cancer is leukemia, lymphoma, or myeloma. 80. The method of Embodiment 79, wherein the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. 81. The method of Embodiment 79, wherein the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. 82. The method of Embodiment 79, wherein the myeloma is multiple myeloma. 83. Any one of embodiments 50 to 82, wherein the cancer is metastatic cancer. 84. Any one of embodiments 50 to 83, wherein a cancer vaccine composition is administered to a patient in combination with one or more additional therapeutic agents. 85. The method of Embodiment 84, wherein one or more additional therapeutic agents are selected from the group consisting of checkpoint inhibitors, antibody therapeutics, genetically modified dendritic cells, genetically modified T cells, and chemotherapeutic agents. 86. A method for producing a cancer vaccine, comprising contacting cancer cells with UV light in the presence of riboflavin. 87. The method of Embodiment 86, wherein the DNA of cancer cells is modified by UV light. 88. The method of Embodiment 87, wherein guanine bases in the DNA of cancer cells are selectively oxidized by UV light. 89. The method of Embodiment 86, wherein the structure of the antigen protein on cancer cells is not substantially altered by phototreatment. 90. Any one of the embodiments 86 to 89, wherein UV light does not substantially alter the metabolic processes, phenotype, or structure of cancer cells. 91. Any one of Embodiments 86 to 90, wherein UV light does not substantially alter the expression or activity of surface markers in cancer cells. 92. The method of Embodiment 91, wherein UV light substantially alters the expression levels of EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC class I, PDL-L1, CD45, gp70, GFP and / or CD90 in cells. 93. Any one of embodiments 86 to 92, wherein inactivation does not impair the integrity of the cell membrane and nuclear membrane of the cell. 94. Any one of embodiments 86 to 93, wherein cancer cells are exposed to UV light for about 1 to 3 minutes in the presence of riboflavin. 95. Any one of the embodiments 86 to 94, wherein the UV light has a wavelength of 170 to 400 nm. 96. Any one of embodiments 86 to 94, wherein the UV light has a wavelength of 315 to 400 nm. 97. Any one of embodiments 86 to 94, wherein the UV light has a wavelength of 310 to 320 nm. 98. Any one of embodiments 86 to 94, wherein the UV light has a wavelength of 280 to 360 nm. 99. Any one of the embodiments 86 to 94, wherein the UV light has a wavelength of 280 to 315 nm. 100. Any one of the embodiments 86 to 94, wherein the UV light has a wavelength of 180 to 280 nm. 101. Any one of the methods of Embodiments 86 to 94, wherein the UV light has a wavelength of 170 to 200 nm. 102. Any one of embodiments 86 to 101, wherein the dose of UV light is approximately 200 joules to approximately 600 joules. 103. The method of Embodiment 102, wherein the dose of UV light is approximately 200 joules to approximately 400 joules. 104. The method of Embodiment 103, wherein the dose of UV light is approximately 300 joules. 105. Any one of embodiments 86 to 104, wherein cancer cells are present in a single-cell suspension. 106. The method of Embodiment 105, wherein riboflavin is added to a single-cell suspension. 107. Any one of embodiments 86 to 106, wherein cancer cells are pre-incubated in a riboflavin-containing solution before being exposed to UV light. 108. The method of Embodiment 107, wherein the solution contains 10 to 100 μM of riboflavin. 109. The method of Embodiment 107, wherein the solution contains about 1 μM to about 50 μM of riboflavin. 110. The method of Embodiment 107, wherein the solution contains less than approximately 10 μM of riboflavin. 111. A cancer vaccine composition, one of Embodiments 1 to 49, for use as a pharmaceutical agent. 112. Any one of Embodiments 1 to 49 of a cancer vaccine composition for use as a drug for treating cancer. 113. Any one of the cancer vaccine compositions from Embodiments 1 to 49 for use in a method of treating cancer. 114. Use of any one of the cancer vaccine compositions of Embodiments 1 to 49 in the manufacture of a drug for treating cancer. 115. Use of any one of the cancer vaccine compositions of Embodiments 111 to 113, or Embodiment 114, wherein the cancer is breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer. 116. The cancer vaccine composition or use of Embodiment 115, wherein the skin cancer is melanoma. 117. The cancer vaccine composition or use of Embodiment 115, wherein the blood cancer is leukemia, lymphoma, or myeloma. 118. The cancer vaccine composition or use of Embodiment 117, wherein the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. 119. The cancer vaccine composition or use of Embodiment 117, wherein the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. 120. The cancer vaccine composition or use of Embodiment 117, wherein the myeloma is multiple myeloma. 121. Use of any one of the cancer vaccine compositions of Embodiments 111 to 113, or Embodiment 114, wherein the cancer is metastatic cancer. [Examples]

[0095] The following examples included herein are for illustrative purposes only and are not intended to be limiting.

[0096] Example 1. Inactivation of tumor cells derived from tumor cell lines CAMA cells (human breast tumor cell line) were inactivated using UV light treatment in the presence of riboflavin. Cells were treated with a Mirasol® PRT illumination system at illumination intensities of 10% (190 joules), 20% (380 joules), 30% (570 joules), 40% (760 joules), 50% (950 joules), or 100% (1896 joules). Cells not treated with UV light (live) were included as a control. Proliferation (Figure 1), viability (Figure 2), cell surface marker expression (Figures 3, 4, and 5), caspase activity (Figure 6), and cell membrane and nuclear membrane integrity were tested (day 0) and on days 2, 4, 6, and 8 after treatment.

[0097] As shown in Figure 1, treatment with riboflavin / UV light rendered cells unable to replicate in culture. This effect was immediately observed on the day of treatment, even at low doses of approximately 190-380 joules (10-20% illumination intensity). Even though they were inactivated, the cells remained substantially viable after treatment (Figure 3). Specifically, the cells maintained an intact and metabolically and functionally sound state (Figure 4). After 4 days, caspase-3 concentration significantly increased, consistent with apoptotic cell death (Figure 6). Therefore, after treatment, the cells no longer proliferated and slowly died over time.

[0098] Cell surface marker expression (EpCAM and CD38) was maintained at relatively consistent levels after treatment, from low doses of approximately 190–380 joules (10–20% illumination intensity) to high doses of approximately 1896 joules (100% illumination intensity) (Figures 3 and 5). Cell viability was positively correlated with surface marker expression (Figure 7). This indicates that, even with a wide range of UV doses, cells maintained the cell surface antigens required to generate an immune response after UV treatment.

[0099] This data establishes a dynamic therapeutic range for preparing inactivated cells without damaging the cell surface marker proteins required to stimulate antibody production.

[0100] Example 2. Vaccine safety: Inactivation of autologous tumor cells and injection into healthy test mice. PyMT tumor cells were injected into wild-type C57Bl6 mice. After the tumors grew, tumor tissue was collected and a cancer cell vaccine was prepared. A total of 2 × 10⁶ cells were collected from 7 C57Bl6 mice. 8 PyMT in vitro tumor cells were resuspended in a solution containing (i) 265 ml of DMEM medium supplemented with 20% fetal bovine serum and glutamine (without antibiotics) and (ii) 35 ml of riboflavin. The cells were then treated with UV light at a total dose of 300 joules.

[0101] 1 × 10⁶ of treated cells6 The cells were placed in culture medium and incubated under optimal conditions. After one month in culture, no evidence of growth or proliferation was observed, which corresponds to 100% inactivation of the replication capacity of the treated cells.

[0102] In addition, a total of 10 C57 / Bl6 mice were given 1x10 6 Inactivated cells were injected subcutaneously. Additional doses were administered after 1, 2, and 3 weeks (a total of 4 doses, 1 × 10¹⁶ doses each). 6 (Individual cells). Animals were monitored for 160 days after the initial injection. During the 160-day monitoring period, no tumors were observed in any of the study subjects, and no side effects from the injection were observed, which corresponds to complete inactivation of cells.

[0103] Finally, 1 × 10¹⁶ septa were placed on the flanks of eight immunodeficient NOD / SCID mice. 6 Inactivated PyMT cells were injected into the animals. The animals were monitored for 5 months after injection. No tumor growth was observed during this monitoring period.

[0104] In summary, these data suggest that there are no safety concerns regarding the injected cell preparations.

[0105] Example 3. Vaccine efficacy: Injection of inactivated tumor cells into test mice with breast cancer inhibits tumor growth. In the mammary fat pad of C57Bl / 6 mice, 2.5 × 10⁶ 5 10 viable PyMT cells were injected. Three days later, the mice were treated with either saline (control, n=10), a vaccine containing inactivated tumor cells (n=10), or a pre-tested lysate vaccine (positive control, n=6). The vaccine was co-mixed with a CLDC adjuvant system (lipid + TLR agonist) at a dose of 1 × 10⁶. 6The mice were given a dose of inactivated cells / mouse subcutaneously into both forelimbs under anesthesia (typically 100-110 μl / limb). The mice were then given losartan (60 mg / kg) by intraperitoneal injection once daily for 3 days, in a total of 3 doses, starting from the day of vaccination. Finally, 24 hours after vaccination, the mice received a booster immunization with CLDC adjuvant (100 μl, intraperitoneal). Vaccination was repeated weekly for a total of 5 vaccine / losartan / CLDC booster immunization cycles. Tumor growth was measured over time using calipers (length × width). The degree of mortality observed over 2 months post-injection was also monitored.

[0106] Figure 8 shows tumor growth curves for saline injection (control, no vaccine) compared to those administered with inactivated whole-cell vaccine and those administered with lysate vaccine (4T1 Spheroid Lysate Vax). A statistically significant reduction in tumor cell growth was observed in the inactivated whole-cell vaccine group, which began treatment 23 days after injection, compared to the untreated control group (p=0.02 on day 23 and p<0.0001 on day 25).

[0107] Figure 9 shows overall survival. In this experiment, mice were euthanized when the longest tumor diameter exceeded 15 mm. Mice administered with the inactivated whole-cell vaccine had a significantly longer survival period compared to the saline-treated control group (p=0.0038). The median overall survival time for mice administered with the inactivated whole-cell vaccine was 34 days compared to 26 days for the control group (an increase of approximately 30%).

[0108] Figure 14 shows the doubling time. In mice treated with the inactivated whole-cell vaccine, the doubling time of tumor growth was significantly longer compared to control mice (p=0.01).

[0109] Example 4. Vaccine efficacy: Injection of inactivated tumor cells into test mice with invasive breast cancer reduced lung metastases, limited tumor regrowth, and enhanced survival. 4T1 breast cancer is a highly neoplastic, invasive, transplantable tumor cell line that, unlike most tumor models, can spontaneously metastasize from its primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lungs, brain, and bone. 1 × 10⁶ cells were transplanted into the mammary fat pad of Balb / c mice. 6 Individual 4T1 luciferase tumor cells were injected. Eleven days after injection (average tumor area was 52 mm²). 2 (When this was the case), the primary tumor was measured and then surgically removed. One mouse died during surgery. The remaining 21 mice were divided into groups according to their preoperative tumor size, with equal mean tumor size (Figure 10). The following groups were determined: PBS ("control"), n=5 mice; adjuvant, n=8 mice; inactivated whole-cell vaccine, n=8 mice.

[0110] Next, the surgically removed tumor tissue was placed in culture medium overnight at 4°C. The following day, the tumor tissue was divided and then treated with collagenase. The cells were filtered to remove tissue fragments and then quantified. A portion of the cells was used to create the first vaccine. Inactivated 4T1 tumor cells (1.7 × 10⁶) were administered to mice. 6 Patients were vaccinated with a single cell dose plus an adjuvant once a week, followed by a booster immunization with the adjuvant 24 hours after vaccination. From the day of vaccination, losartan was administered at a daily dose of 60 mg / kg for three doses. This cycle was repeated weekly.

[0111] Mice were periodically imaged using an IVIS device to detect the development of metastatic disease. Mice were injected with 100 μl of luciferin, followed by IVIS 10 minutes later. Photon flux numbers were calculated using IVIS-based software and compared between groups (Figures 11A-B).

[0112] Starting 24 hours after surgical removal of the primary tumor, mice were treated weekly with PBS alone (control), cationic liposome-DNA complex (CLDC) and losartan (adjuvant), or inactivated whole-cell vaccine (adjuvant + vaccine). After intraperitoneal injection of 100 μl of luciferin, metastatic disease in the lungs was quantified using IVIS imaging (Figure 19). As shown in Figure 11A, a significant reduction in the measured metastatic burden was observed in vaccine-treated mice compared to adjuvant-treated mice (day 14, p=0.0157), and also compared to both control mice and adjuvant-treated mice (day 16, p=0.0119 and p=0.0021, respectively). Figure 11B shows the time-course photon flux data for each mouse in each group.

[0113] In some mice, regrowth of the primary tumor was observed due to incomplete resection. The number of mice with primary tumor regrowth was recorded 17 days post-surgery (Figure 12). Interestingly, fewer mice treated with the inactivated whole-cell vaccine exhibited primary tumor regrowth compared to other treatment groups. 60% of the control group, 63% of the adjuvant-only group, and only 38% of the inactivated whole-cell vaccine group experienced primary tumor regrowth.

[0114] The vaccine's effect on survival was also tested. Mice were euthanized when they showed signs of disease (greater than 10% weight loss, low activity, epileptic seizures, etc.), and their postoperative days were evaluated (Figure 13). The median survival time for mice treated with the inactivated whole-cell vaccine was 24 days, compared to 18 days for control mice and 17.5 days after surgical removal of the primary tumor in adjuvant-only mice. Therefore, vaccine treatment increased median survival by approximately 6 days. All mice in all groups eventually succumbed to metastatic disease. Any observed effects are noteworthy given that the 4T1 metastatic model is highly invasive and typically unresponsive to many conventional treatments.

[0115] Example 5. Test efficacy of inactivated whole-cell vaccine in different mouse tumor models (LLCs). To test the efficacy of inactivated whole-cell vaccines in different mouse tumor models, healthy B6 mice were injected with Lewis lung cancer (LLC) cells. When primary tumors grew, they were excised, and tumor cells were inactivated with 300 J. 5 × 10⁶ cells were used in 19 other B6 mice. 5 Each LLC cell was subcutaneously injected into the flank. Three days after the tumor cell injection, the mice were given 1.7 × 10⁶ cells as their first vaccine. 6 Individual inactivated cells / mice / vaccines were administered. Furthermore, the mice were given a CLDC adjuvant, e.g., losartan, and a CLDC booster immunization 24 hours after vaccination. The mice received these vaccines weekly for a total of three doses. On day 19, all mice were euthanized, tumor tissue was collected, and immunocells were stained.

[0116] As shown in Figure 15, tumor growth was significantly reduced / delayed in mice treated with inactivated whole-cell vaccine (p=0.02 on day 13 and p=0.001 on day 19). Two mice in the vaccine group were tumor-free on day 19 (complete remission = 20%). Final tumor weight in mice with tumors decreased in vaccine-treated mice, but not statistically (p=0.0547). Tumor doubling time also did not decrease statistically, but was shorter in vaccine-treated mice (p=0.0570). In tumors, a significant decrease in CD4+CD25+ T cells was observed in vaccinated mice (p=0.004, putative immunosuppressive regulatory T cells), along with a decrease in CD4+ T cells expressing GITR as another marker of regulatory T cells (p=0.02) (Figure 16A). In relation to this, a significant decrease in CD8+ T cells expressing the immunosuppressive proteins Lag3 (p=0.01) and Tim3 (p=0.05) was observed in vaccinated mouse tumors (Figure 16B), as well as a significant decrease in CD4+ T cells expressing the proteins Lag3 (p=0.005) and Tim3 (p=0.02) (Figure 16C).

[0117] Example 6. Determining whether or not in vitro canine tumor tissue contains tumor cells that maintain protein surface expression. Two tumor tissues were surgically obtained from dogs with spontaneous cancer undergoing treatment. One tissue was obtained from anal gland carcinoma (ASA) and the other from thyroid cancer (TC). The tissues were digested with collagenase, and the resulting single-cell suspensions were frozen at -80°C in multiple vials. The tumor cells were then thawed, and one cell bullet was used as a control cell, while the other was used as an inactivated cell. The canine cells were inactivated using 300 J. The number of cells used was smaller than in previous cases used to inactivate tumor cells. It took 1 minute and 38 seconds to inactivate each group of cells. Next, the tumor cells were stained for the expression of canine CD44, CD90, PD-L1, and CD45. All hematopoietic cells were gated out using CD45 (Figures 25A-D). "Tumor" cells were analyzed against CD45-negative cells (including tumor cells, fibroblasts, endothelial cells, and other cell types).

[0118] In ASA, 1 hour after inactivation, 15% of cells expressed CD44 (compared to 20% in the control), 1% expressed CD90 (compared to 0.3%), and 7.7% expressed PD-L1 (compared to 3.1%). In TC, after inactivation, 7.1% expressed CD44 (compared to 6.8% in control cells), 0.6% expressed CD90 (compared to 0.4%), and 0% expressed PD-L1 (compared to 0.2%). The remaining cells were placed at 4°C for 48 hours and then stained again. After 48 hours, in ASA, CD44=10% (compared to 11% in the control), CD90=2% (compared to 3.2% in the control), and PD-L1=9.5% (compared to 3% in the control). In the TC group, CD44 was 5.8% (compared to 8.9% of the control group), CD90 was 3.1% (compared to 3.2% of the control group), and PD-L1 was 3% (compared to 2.9% of the control group).

[0119] Further tumor tissue samples were obtained from two different dogs undergoing surgery; one from the GI population and the other from the lung population. The tumor tissue was digested with collagenase, washed, and then stained to prepare single-cell suspensions. All cells were then frozen in cell freezing medium. Later, the cells were thawed, and half of the cells were inactivated using the UV+RF (UV light + riboflavin) protocol. The other half were maintained on ice. The cells were then stained for surface expression of canine MHC class I, CD44, CD90, and PD-L1 (Figure 26).

[0120] In summary, these data indicate that surface markers are maintained on in vitro canine tumor cells after inactivation.

[0121] Example 7. Testing of different adjuvants to generate significant cellular and humoral immune responses against inactivated tumor cells. Healthy Balb / c mice were either vaccinated with PBS or inactivated cells alone, or with an inactivated 4T1 vaccine (used in the metastatic mouse tumor test described in Example 4) mixed with a CLDC adjuvant, topical imiquimod (2 mg / kg) applied to the skin before vaccination, or CpG ODN (50 μg / vaccine). The vaccine was administered subcutaneously in equal doses to both the left and right forelimbs near the feet of the mice. 1.7 × 10⁶ mice 6 Individual cells / mice / vaccine were administered. The vaccine was administered on day 1 and then again on day 14. Mice were euthanized on day 8 after administration of the booster vaccine, and their spleens and blood were collected. Splenocytes were cultured with inactivated 4T1 cells (to inhibit replication), and IFNg production was measured after 72 hours (Figure 17). Splenocytes were cultured in a ratio of 25 splenocytes to 1 inactivated 4T1 cell.

[0122] As shown in Figure 17, the CLDC adjuvant system exhibited the best recall response in terms of IFNg production, followed by CpG ODN. The vaccine alone also produced some IFNg in 3 out of 4 mice, but the difference was not significant compared to the control.

[0123] Example 8. Further testing of adjuvants to generate significant cellular and humoral immune responses against inactivated tumor cells. Healthy Balb / c mice were either vaccinated with PBS or inactivated cells alone, or with an inactivated 4T1 vaccine (used in the metastatic mouse tumor test described in Example 4) mixed with a CLDC adjuvant, topical imiquimod (2 mg / kg) applied to the skin before vaccination, or CpG ODN (50 μg / vaccine). The vaccine was administered subcutaneously in equal doses to both the left and right forelimbs near the feet of the mice. 1.7 × 10⁶ mice 6 Individual cells / mice / vaccine were administered. The vaccine was administered on day 1 and then again on day 14. Mice were euthanized on day 8 after administration of the booster vaccine, and their spleens and blood were collected. Serum from these mice was screened for IgG antibody binding against 4T1 live tumor cells by incubating the serum with the cells at either a 1:500 or 1:1000 dilution, staining with a fluorescently labeled donkey anti-mouse secondary antibody, and then subtracting background staining (normal mouse serum at 1:500 and 1:1000).

[0124] As shown in Figure 18, unvaccinated mice and those treated with inactivated whole-cell vaccine alone did not produce any IgG specific to 4T1 tumor cells. Imiquimod yielded variable levels in 3 out of 5 mice with very high staining. CpG ODN showed a reliably significant increase in staining at a 1:1000 dilution. Stains not significantly higher than background were detected at a 1:500 dilution.

[0125] Example 9. Preservation of surface antigens The mouse lung cancer cell line LLC maintained surface antigen expression after inactivation (Figures 21A-21B). LLC cells were treated with riboflavin (RF, 50 μM) and UV light (300 J), and stained for surface expression of CD34, CD117, CD44, and CD90. All four antigens were maintained on the cell surface. In addition, the surface marker expression of CD44, Sca1, and EpCAM in the mouse mammary cancer cell line 4T1 was evaluated before and after UV+RF inactivation (Figure 22).

[0126] Mouse melanoma tumor cell line B16, transfected with green fluorescent protein, was injected into C57Bl6 mice, removed from the mice, and converted into a single-cell suspension. GFP+ tumor cell expression was then analyzed before and after UV+RF inactivation and after gamma irradiation (100 Gy). None of the cell inactivation methods negatively affected GFP expression by tumor cells in in vitro tumor tissue (Figure 23).

[0127] Finally, mouse colon cancer cell line CT26 was inactivated with UV+RF, and then the surface expression of the known tumor-associated antigen gp70 was analyzed (Figures 24A-24B). UV+RF inactivation enhanced gp70 expression on CT26 cells.

[0128] Furthermore, human hepatocellular carcinoma cell line HepG2 was inactivated, and after UV+RF inactivation (300J, Figure 27), it was stained for human HLA and GLUT1 expression and imaged on an adherent cell cytometer.

[0129] The results from all of these tests indicate that inactivation can be performed without significant modification of cell-specific antigens on the cell surface. Furthermore, these markers were maintained in morphologically intact cells over long storage periods after treatment, and these observations were observed in cells induced from all three test species (mouse, dog, and human).

[0130] Example 10. Induction of the immune system by inactivated cells Splenocytes were removed from mice with 4T1 tumor growth but no treatment, placed in culture, and their T cell immune response to either cells derived from a UV+RF-inactivated 4T1 cell line or cells derived from a live 4T1 cell line was tested (Figure 28). Splenocytes were cultured in 1 × 10⁶ cells. 6 Seed cells / well, then 4×10 5 Cells / well were co-cultured with (live or inactivated) tumor cells for 72 hours. After 48 hours of culture, the growth dye EdU was added. Next, splenocytes were collected and stained for CD4+ T cell and CD8+ T cell proliferation. A significant increase in the proliferation of both CD4+ T cells and CD8+ T cells was observed when these cells were co-cultured with UV+RF inactivated 4T1 tumor cells.

[0131] Example 11: Pharmacokinetics: In vitro and in vivo proliferation and persistence of tumor cells To test the safety and effectiveness of the inactivation process, PyMT tumor cells were removed from solid tumors on B6 mice and inactivated using UV light (300J). 1.5 × 10 6 10¹¹ "viable" inactivated PyMT cells were subcutaneously injected into five healthy B6 mice. This was repeated over four doses on days 7, 14, and 21. Approximately 160 days after the initial injection of inactivated tumor cells, the mice were euthanized and excised. No evidence of tumor growth was observed. To test the safety of inactivated cells in immunodeficient mice, 1 × 10¹¹ cells were administered to eight NOD / SCID mice. 6 One inactivated PyMT cell was subcutaneously injected into the right flank. The mice were monitored thereafter. During the experiment, three mice died from what was likely an unrelated cause (possibly a viral or bacterial infection). A necropsy was performed on one mouse, but no tumor was found. Skin analysis was performed on the other two mice, but no tumors were found. 252 days after tumor cell injection, the mice were euthanized, and no tumors were found in the remaining five mice.

[0132] Cell proliferation in culture after inactivation was also evaluated (Figure 29). 4T1 cells were injected into the fat body of Balb / c mice, and tumor growth was monitored until the tumor diameter reached approximately 10 mm. The tissue was removed, digested with collagenase, washed, inactivated with UV+Rf, and then cultured at 37°C for 24 and 72 hours. No proliferation of 4T1 cells was observed 24 and 72 hours after inactivation. This was also compared to uninactivated in vitro tumor cells (partial proliferation) and 4T1 tumor cell lines (proliferation). Similar tests were performed using the human liver cancer cell line HepG2 and the human colon cancer cell line CRL-2577 (Figure 30). Proliferation tests were performed using Click-iT EdU, which labels newly formed DNA. The fluorescence of the labeled novel DNA was compared with whole DNA staining using an adherent cell cytometer. This study identified as many as 20–35% high-EdU cells in live controls of CRL-2577, and thousands of replicated cells, representing approximately 75% of the longer-labeled HepG2 population.

[0133] While the teachings of this disclosure describe various applications, methods, and compositions, it will be understood that various modifications and alterations can be made without departing from the teachings herein and the following claims. The aforementioned examples are provided to better illustrate the teachings of this disclosure and are not intended to limit the scope of the teachings presented herein. While the teachings are described in terms of these exemplary embodiments, those skilled in the art will readily understand that a great many variations and alterations of these exemplary embodiments are possible without excessive experimentation. All such variations and alterations are within the scope of the teachings herein.

[0134] All references cited herein, such as patents, patent applications, articles, textbooks, GenBank(trademark) or acceptance numbers, and any references cited therein, are hereby invoked by reference in their entirety unless they have not yet been invoked. If one or more of the invoked documents and similar materials differ from or contradict this application, including, but not limited to, defined terms, usage of terms, or described technology, this application shall prevail.

[0135] The prior description and examples detail specific embodiments of the present invention and describe the best mode considered by the inventors. However, regardless of how the prior description and examples are detailed in the text, it will be understood that the present invention may be carried out in many ways and that the present invention should be interpreted in accordance with the affixed claims and any equivalents thereof. In certain embodiments, for example, the following items are provided: (Item 1) A cancer vaccine composition containing inactivated cancer cells that lack the ability to replicate. (Item 2) The cancer vaccine composition according to item 1, wherein the cancer cells are derived from a patient suffering from one or more types of cancer. (Item 3) The cancer vaccine composition described in item 2, wherein the patient has one or more of the following cancers: breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, and hematological cancer. (Item 4) The cancer vaccine composition according to item 3, wherein the skin cancer is melanoma. (Item 5) The cancer vaccine composition according to item 3, wherein the blood cancer is leukemia, lymphoma, or myeloma. (Item 6) The cancer vaccine composition according to item 5, wherein the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. (Item 7) The cancer vaccine composition according to item 5, wherein the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. (Item 8) The cancer vaccine composition according to item 5, wherein the myeloma is multiple myeloma. (Item 9) The cancer vaccine composition described in item 2, wherein the patient has a benign tumor. (Item 10) The cancer vaccine composition described in item 2, wherein the cancer is metastatic cancer. (Item 11) The cancer vaccine composition according to any one of items 1 to 10, wherein the cancer cells are derived from an immortalized cell line. (Item 12) The cancer vaccine composition according to any one of items 1 to 11, wherein the cancer cells are autologous. (Item 13) The cancer vaccine composition according to any one of items 1 to 11, wherein the cells are of the same species. (Item 14) The aforementioned composition is about 1 × 10 5 ~Approx. 1×10 8 A cancer vaccine composition according to any one of items 1 to 13, comprising a single cancer cell. (Item 15) The cancer vaccine composition according to any one of items 1 to 14, wherein the DNA of the cancer cells contains modified guanine bases. (Item 16) The cancer vaccine composition according to any one of items 1 to 15, wherein the composition further comprises an adjuvant. (Item 17) The cancer vaccine composition according to item 16, wherein the adjuvant modifies monocyte function. (Item 18) The cancer vaccine composition according to item 16, wherein the adjuvant comprises aluminum hydroxide. (Item 19) The cancer vaccine composition according to item 16, wherein the adjuvant comprises CLDC. (Item 20) The cancer vaccine composition according to item 16, wherein the adjuvant comprises polyIC, CpG oligodeoxynucleotide (ODN), or imiquimod. (Item 21) The cancer vaccine composition according to item 16, wherein the adjuvant comprises liposomes. (Item 22) The cancer vaccine composition according to item 21, wherein the liposome is conjugated with an agonist. (Item 23) The cancer vaccine composition according to item 22, wherein the agonist is at least one agonist of TLR3 and TLR9. (Item 24) A cancer vaccine composition according to any one of items 1 to 23, further comprising a pharmaceutically acceptable carrier. (Item 25) The cancer vaccine composition according to item 24, wherein the pharmaceutically acceptable carrier is normal physiological saline, glucose saline, or phosphate-buffered saline. (Item 26) The cancer vaccine composition according to any one of items 1 to 25, wherein the cancer cells are inactivated by phototreatment. (Item 27) The cancer vaccine composition according to item 26, wherein the phototreatment is performed for approximately 1 to 3 minutes. (Item 28) The cancer vaccine composition according to item 26 or 27, wherein the structure of the antigen protein on the cancer cells is not substantially altered by the phototreatment. (Item 29) The cancer vaccine composition according to any one of items 26 to 28, wherein the DNA of the cancer cells is not altered by the phototreatment. (Item 30) The cancer vaccine composition according to item 29, wherein the guanine bases in the DNA of the cancer cells are selectively oxidized by the phototreatment. (Item 31) A cancer vaccine composition according to any one of items 26 to 30, wherein the phototreatment does not substantially alter the metabolic process, phenotype, or structure of the cancer cells. (Item 32) The cancer vaccine composition according to any one of items 26 to 31, wherein the expression or activity of surface markers in cancer cells is not substantially altered by the aforementioned phototreatment. (Item 33) The cancer vaccine composition according to item 32, wherein the phototreatment does not alter the expression levels of EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC class I, PDL-L1, CD45, gp70, GFP, or CD90 in the cells. (Item 34) The cancer vaccine composition according to any one of items 26 to 33, wherein the integrity of the cell membrane or nuclear membrane of the cell is not impaired by the phototreatment. (Item 35) The aforementioned phototreatment includes treatment with UV light, the cancer vaccine composition according to any one of items 26 to 34. (Item 36) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 170 to 400 nm. (Item 37) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 315 to 400 nm. (Item 38) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 310 to 320 nm. (Item 39) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 280 to 360 nm. (Item 40) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 280 to 315 nm. (Item 41) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 180 to 280 nm. (Item 42) The cancer vaccine composition according to item 35, wherein the UV light has a wavelength of 170 to 200 nm. (Item 43) A cancer vaccine composition according to any one of items 35 to 42, wherein the UV light dose is approximately 200 joules to approximately 600 joules. (Item 44) A cancer vaccine composition as described in item 43, wherein the UV light dose is approximately 200 joules to 400 joules. (Item 45) A cancer vaccine composition as described in item 44, wherein the UV light dose is approximately 300 joules. (Item 46) The cancer vaccine composition according to any one of items 26 to 45, wherein the phototreatment is carried out by exposing the cancer cells to light in the presence of a photosensitizer. (Item 47) The cancer vaccine composition according to item 46, wherein the concentration of the photosensitizer is approximately 1 μM to approximately 50 μM. (Item 48) The cancer vaccine composition according to item 46 or 47, wherein the concentration of the photosensitizer is less than approximately 10 μM. (Item 49) The cancer vaccine composition according to any one of items 46 to 48, wherein the photosensitizer is riboflavin. (Item 50) A method for treating cancer in a patient in need thereof, comprising administering to the patient a cancer vaccine composition described in any one of items 1 to 49. (Item 51) The method according to item 50, wherein the cancer vaccine composition is administered simultaneously with or consecutively with the vaccine enhancer. (Item 52) The method according to item 51, wherein the vaccine enhancer is an angiotensin receptor blocker (ARB) or a beta-blocker (BB). (Item 53) The method according to item 51 or 52, wherein the vaccine enhancer is losartan. (Item 54) The method described in item 53, wherein the dose of losartan is between approximately 5 and approximately 100 mg / kg. (Item 55) The method described in item 54, wherein the dose of losartan is approximately 60 mg / kg. (Item 56) The method according to item 51 or 52, wherein the vaccine enhancer is propranolol. (Item 57) The method according to any one of items 50 to 56, wherein the cancer vaccine composition is administered to the patient once. (Item 58) The method according to any one of items 50 to 56, wherein the cancer vaccine composition is administered to the patient two or more times. (Item 59) The method according to item 58, wherein the cancer vaccine composition is administered to the patient two, three, four, five, six, seven, eight, nine, or ten times. (Item 60) The method according to item 58 or 59, wherein the cancer vaccine composition is administered to the patient at least once every seven days. (Item 61) The method according to item 58 or 59, wherein the cancer vaccine composition is administered to the patient at least once every 14 days. (Item 62) The method according to item 58 or 59, wherein the cancer vaccine composition is administered to the patient at least once every six months. (Item 63) The method according to any one of items 50 to 62, wherein the cancer vaccine composition is administered by a route selected from subcutaneous, intramuscular, intravenous, intranasal, sublingual, buccal, inhalation, intradermal, intratumoral, intraorganic, oral, and intraperitoneal. (Item 64) The method according to item 63, wherein the cancer vaccine composition is administered by subcutaneous injection. (Item 65) The method according to item 63, wherein the cancer vaccine composition is administered by intravenous injection. (Item 66) The method according to item 63, wherein the cancer vaccine composition is administered by intramuscular injection. (Item 67) The method according to any one of items 50 to 62, wherein the patient is immunocompetent. (Item 68) The method according to any one of items 50 to 62, wherein the patient is immunocompromised. (Item 69) The method according to any one of items 50 to 68, wherein the treatment reduces tumor growth by at least 10% compared to tumor growth in unvaccinated patients. (Item 70) The method according to item 69, wherein the treatment reduces tumor growth by at least 20% compared to tumor growth in unvaccinated patients. (Item 71) The method according to item 70, wherein the treatment reduces tumor growth by at least 50% compared to tumor growth in unvaccinated patients. (Item 72) The method according to any one of items 50 to 71, wherein the treatment extends the survival of the patient by at least 10% compared to an unvaccinated patient. (Item 73) The method according to item 72, wherein the treatment extends the survival of the patient by at least 20% compared to an unvaccinated patient. (Item 74) The method according to item 73, wherein the treatment extends the survival of the patient by at least 50% compared to an unvaccinated patient. (Item 75) The method according to any one of items 50 to 74, wherein the treatment upregulates IgG and / or IgM in the patient. (Item 76) The method according to any one of items 50 to 75, wherein the treatment activates T cells in the patient. (Item 77) The method according to any one of items 50 to 76, wherein the cancer is breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer. (Item 78) The method according to item 77, wherein the skin cancer is melanoma. (Item 79) The method according to item 77, wherein the blood cancer is leukemia, lymphoma, or myeloma. (Item 80) The method according to item 79, wherein the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. (Item 81) The method according to item 79, wherein the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. (Item 82) The method according to item 79, wherein the myeloma is multiple myeloma. (Item 83) The method according to any one of items 50 to 82, wherein the cancer is metastatic cancer. (Item 84) The method according to any one of items 50 to 83, wherein the cancer vaccine composition is administered to the patient in combination with one or more additional therapeutic agents. (Item 85) The method according to item 84, wherein the one or more additional therapeutic agents are selected from the group consisting of checkpoint inhibitors, antibody therapeutics, genetically modified dendritic cells, genetically modified T cells, and chemotherapeutic agents. (Item 86) A method for producing a cancer vaccine, comprising exposing cancer cells to UV light in the presence of riboflavin. (Item 87) The method according to item 86, wherein the DNA of the cancer cells is modified by the UV light. (Item 88) The method according to item 87, wherein the guanine bases in the DNA of the cancer cells are selectively oxidized by the UV light. (Item 89) The method according to item 86, wherein the phototreatment does not substantially alter the structure of the antigen protein on the cancer cells. (Item 90) The method according to any one of items 86 to 89, wherein the metabolic processes, phenotype, or structure of the cancer cells are not substantially altered by the UV light. (Item 91) The method according to any one of items 86 to 90, wherein the expression or activity of the surface marker in the cancer cells is not substantially altered by the UV light. (Item 92) The method according to item 91, wherein the UV light does not substantially alter the expression levels of EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC class I, PDL-L1, CD45, gp70, GFP and / or CD90 in the cells. (Item 93) The method according to any one of items 86 to 92, wherein the inactivation does not impair the integrity of the cell membrane and nuclear membrane of the cell. (Item 94) The method according to any one of items 86 to 93, wherein the cancer cells are exposed to UV light for about 1 to 3 minutes in the presence of riboflavin. (Item 95) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 170 to 400 nm. (Item 96) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 315 to 400 nm. (Item 97) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 310 to 320 nm. (Item 98) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 280 to 360 nm. (Item 99) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 280 to 315 nm. (Item 100) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 180 to 280 nm. (Item 101) The method according to any one of items 86 to 94, wherein the UV light has a wavelength of 170 to 200 nm. (Item 102) The method according to any one of items 86 to 101, wherein the dose of UV light is approximately 200 joules to approximately 600 joules. (Item 103) The method described in item 102, wherein the UV light dose is approximately 200 joules to approximately 400 joules. (Item 104) The method according to item 103, wherein the dose of UV light is approximately 300 joules. (Item 105) The method according to any one of items 86 to 104, wherein the cancer cells are present in a single-cell suspension. (Item 106) The method according to item 105, wherein the riboflavin is added to the single-cell suspension. (Item 107) The method according to any one of items 86 to 106, wherein the cancer cells are pre-incubated in a solution containing riboflavin before the cells are exposed to the UV light. (Item 108) The method according to item 107, wherein the solution contains 10 to 100 μM of riboflavin. (Item 109) The method according to item 107, wherein the solution contains approximately 1 μM to approximately 50 μM of riboflavin. (Item 110) The method according to item 107, wherein the solution contains less than approximately 10 μM of riboflavin. (Item 111) A cancer vaccine composition described in any one of items 1 to 49, for use as a pharmaceutical agent. (Item 112) A cancer vaccine composition as described in any one of items 1 to 49, for use as a drug for treating cancer. (Item 113) A cancer vaccine composition according to any one of items 1 to 49, for use in methods of treating cancer. (Item 114) Use of any one of the cancer vaccine compositions described in item 1 to 49 in the manufacture of a drug for treating cancer. (Item 115) The cancer vaccine composition described in any one of items 111 to 113, or the use described in item 114, wherein the cancer is breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer. (Item 116) The cancer vaccine composition or use described in item 115, wherein the skin cancer is melanoma. (Item 117) The cancer vaccine composition or use described in item 115, wherein the blood cancer is leukemia, lymphoma, or myeloma. (Item 118) The cancer vaccine composition or use described in item 117, wherein the leukemia is acute lymphoblastic leukemia or acute myeloid leukemia. (Item 119) The cancer vaccine composition or use described in item 117, wherein the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma. (Item 120) The cancer vaccine composition or use described in item 117, wherein the myeloma is multiple myeloma. (Item 121) The cancer is metastatic cancer, and the cancer vaccine composition described in any one of items 111 to 113, or the use described in item 114.

Claims

1. A cancer vaccine composition comprising inactivated cancer cells and an adjuvant, Cancer cells are inactivated by treatment with UV light in the presence of riboflavin, wherein the UV light has a wavelength of 310-320 nm. The structure of one or more antigen proteins on the inactivated cancer cells is substantially the same as that on the cancer cells before treatment with UV light. The DNA of the inactivated cancer cells contains oxidized guanine bases, A cancer vaccine composition wherein the adjuvant comprises a TLR3 agonist, a TLR9 agonist, a TLR4 agonist, or a TLR7 agonist.

2. The following markers on the aforementioned inactivated cancer cells: EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC Class I, PD-L1, CD45, gp70, GFP, and CD90 The cancer vaccine composition according to claim 1, wherein the expression level of one or more of the substances is substantially the same as that on the cancer cells before treatment with UV light.

3. The cancer vaccine composition according to claim 1 or 2, wherein the integrity of the cell membrane and nuclear membrane of the inactivated cancer cells is not impaired, and the inactivated cancer cells are non-replicating and intact.

4. The cancer vaccine composition according to any one of claims 1 to 3, wherein the treatment with UV light continues for approximately 1 to 3 minutes.

5. The cancer vaccine composition according to any one of claims 1 to 4, wherein the UV light dose is approximately 0.5 joules / ml to approximately 3 joules / ml.

6. The cancer vaccine composition according to any one of claims 1 to 4, wherein the UV light dose is in the range of about 200 joules to about 600 joules, or about 0.6 joules / ml to 2 joules / ml.

7. The cancer vaccine composition according to claim 6, wherein the UV light dose is approximately 300 joules, or 190 to 380 joules, or approximately 1 joule / ml.

8. The cancer vaccine composition according to any one of claims 1 to 7, wherein the riboflavin is concentrated at a concentration of about 10 to 100 μM, or about 1 μM to about 50 μM.

9. The cancer vaccine composition according to any one of claims 1 to 8, wherein the cancer cells are breast cancer cells, lung cancer cells, liver cancer cells, bladder cancer cells, gynecological cancer cells, brain cancer cells, gastric cancer cells, prostate cancer cells, skin cancer cells, thyroid cancer cells, pancreatic cancer cells, colon cancer cells, or hematological cancer cells.

10. The cancer vaccine composition according to claims 1 to 9, wherein the cancer cells are isolated from a subject having cancer.

11. The cancer vaccine composition according to any one of claims 1 to 9, wherein the cancer cells are derived from an immortalized cell line.

12. The cancer vaccine composition according to any one of claims 1 to 9, wherein the cancer cells are isolated from a benign tumor.

13. The composition is approximately 1 × 10 ５ ~Approx. 1×10 ８ A cancer vaccine composition according to any one of claims 1 to 12, comprising a single cancer cell.

14. The composition is approximately 1 × 10 ４ ~Approx. 1×10 ７ A cancer vaccine composition according to any one of claims 1 to 12, comprising a single cancer cell.

15. The cancer vaccine composition according to any one of claims 1 to 14, wherein the adjuvant modifies monocyte function.

16. The cancer vaccine composition according to any one of claims 1 to 15, wherein the adjuvant comprises a cationic liposome-DNA complex (CLDC), poly-IC, CpG oligodeoxynucleotide (ODN), imiquimod, or liposome.

17. The cancer vaccine composition according to claim 16, wherein the adjuvant is CLDC.

18. The cancer vaccine composition according to claim 16, wherein the adjuvant is a CpG oligodeoxynucleotide (ODN).

19. The cancer vaccine composition according to any one of claims 1 to 18, wherein the composition comprises a pharmaceutically acceptable carrier.

20. The cancer vaccine composition according to claim 19, wherein the pharmaceutically acceptable carrier is normal physiological saline, glucose saline, or phosphate-buffered saline.

21. A cancer vaccine composition according to any one of claims 1 to 20, for use as a drug for treating cancer in subjects requiring cancer treatment.

22. A cancer vaccine composition for use according to claim 21, wherein the cancer is breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or hematological cancer.

23. The cancer vaccine composition for use according to claim 21 or 22, wherein the cancer is metastatic cancer.

24. The cancer vaccine composition for use according to any one of claims 21 to 23, wherein the inactivated cancer cells are autologous.

25. The cancer vaccine composition for use according to any one of claims 21 to 24, wherein the cancer vaccine composition is formulated for subcutaneous injection, intravenous injection or intramuscular injection.

26. The cancer vaccine composition for use according to any one of claims 21 to 25, wherein the cancer vaccine composition is for administration to the subject once.

27. The cancer vaccine composition for use according to any one of claims 21 to 25, wherein the cancer vaccine composition is to be administered to the subject two, three, four, five, six, seven, eight, nine, or ten times.

28. The cancer vaccine composition for use according to claim 27, wherein the cancer vaccine composition is to be administered at least once every 7 days, at least once every 14 days, or at least once every 6 months.

29. The cancer vaccine composition for use according to any one of claims 21 to 28, wherein the cancer vaccine composition is to be administered simultaneously with or consecutively with a vaccine enhancer.

30. The cancer vaccine composition for use according to claim 29, wherein the vaccine enhancer is an angiotensin receptor blocker (ARB) or a beta-blocker (BB).

31. The cancer vaccine composition for use according to claim 29 or 30, wherein the vaccine enhancer is losartan.

32. The cancer vaccine composition for use according to claim 31, wherein the dose of losartan is about 5 to about 100 mg / kg.

33. The cancer vaccine composition for use according to claim 32, wherein the dose of losartan is approximately 60 mg / kg.

34. The cancer vaccine composition for use according to claim 29 or 30, wherein the vaccine enhancer is propranolol.

35. A cancer vaccine composition for use according to any one of claims 21 to 34, wherein the adjuvant is administered after administration of the cancer vaccine composition, and the adjuvant modifies monocyte function.

36. The cancer vaccine composition for use according to any one of claims 21 to 35, wherein the subject is a human.

37. An in vitro method for producing cancer vaccines, (1) Pre-incubating cancer cells in a solution containing riboflavin, wherein the solution contains 10 to 100 μM of riboflavin, (2) Inactivating the cancer cells by exposing them to UV light in the presence of riboflavin. A method comprising UV light having a wavelength of 310 to 320 nm, the UV light having a dose of about 0.5 joules / ml to about 3 joules / ml or about 200 joules to about 600 joules, and the UV light selectively oxidizing the guanine bases in the DNA of the cancer cells.

38. The method according to claim 37, wherein the cancer cells are breast cancer cells, lung cancer cells, liver cancer cells, bladder cancer cells, gynecological cancer cells, brain cancer cells, gastric cancer cells, prostate cancer cells, skin cancer cells, thyroid cancer cells, pancreatic cancer cells, colon cancer cells, or hematological cancer cells.

39. The method according to claim 37 or 38, wherein the inactivation results in no substantial change in the expression levels of EpCAM, CD38, CD34, CD117, CD44, CD24, Sca1, HLA, Glut1, MHC class I, PD-L1, CD45, gp70, GFP and / or CD90 on the cancer cells, and / or the inactivation results in no substantial change in the structure of one or more antigen proteins on the cancer cells.

40. The method according to any one of claims 37 to 39, wherein the inactivation does not impair the integrity of the cell membrane and nuclear membrane of the cells, and the inactivated cancer cells are non-replicating and intact.

41. The method according to any one of claims 37 to 40, wherein the cancer cells are exposed to UV light for about 1 to 3 minutes in the presence of riboflavin.

42. The method according to any one of claims 37 to 41, wherein the dose of the UV light is approximately 200 joules to approximately 400 joules, or approximately 200 joules to approximately 380 joules, or approximately 0.6 joules / ml to approximately 2 joules / ml.

43. The method according to claim 42, wherein the UV light has a dose of about 300 joules or about 1 joule / ml.

44. The method according to any one of claims 37 to 43, wherein the cancer cells are present in a single-cell suspension, and the riboflavin is added to the single-cell suspension for pre-incubation.

45. The method according to any one of claims 37 to 44, wherein the solution contains about 1 μM to about 50 μM of riboflavin.

46. The method according to claim 45, wherein the solution contains about 50 μM of riboflavin.

47. Cancer cells are approximately 1 x 10 ５ ~Approx. 1×10 ８ The method according to any one of claims 37 to 46, wherein the concentration is [number].

48. The aforementioned cancer cells are approximately 1 × 10 ４ ~Approx. 1×10 ７ The method according to any one of claims 37 to 46, wherein the concentration is [number].

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