WO2024242545A1 - Cancer vaccine composition comprising calcium lactate as active ingredient and use thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising, as active ingredients: a) calcium lactate; and b) at least one substance selected from the group consisting of a phosphate, stem cell, cytokine, cancer cell line, antigen derived from the cancer cell line, polynucleotide encoding the antigen, antigen gene construct in which the polynucleotide is operably linked to a promoter, and expression vector comprising the gene construct. The composition of the present invention has an anti-tumor immunological memory effect, induces apoptosis of cancer cells, and thus can be used as an effective immunotherapy candidate for solid cancers, a vaccine candidate for in-situ treatment of solid cancers, and a cancer-treating agent administered into tumors.

Description

Cancer vaccine composition comprising calcium lactate as an active ingredient and its use

The present invention relates to a pharmaceutical composition comprising calcium lactate as an active ingredient and its use, and more specifically, to a cancer vaccine composition comprising calcium lactate as an active ingredient and its use.

Current cancer treatments typically involve systemic chemotherapy with non-targeted small molecules or antibody-specific cytotoxic agents that preferentially enter or bind to cancer cells (in the case of antibody-specific agents) by a variety of mechanisms and kill them.

Systemic chemotherapy can reach many distant metastatic sites, except brain metastases, but in too many patients, responses are usually short-lived (months to years) and ultimately result in tumor recurrence.

Immunological approaches are rapidly gaining ground in the cancer treatment paradigm, as the body's natural immune system can destroy cancer cells after recognition. However, some cancer cells, and even cancer stem cells, are initially able to evade immune surveillance, and in fact acquire the ability to evolve, ultimately surviving by remaining relatively immune-invisible [Gaipi et al, Immunotherapy 6:597-610, 2014].

One possible mechanism by which traditional vaccine adjuvants, such as alum, enhance immune responses is through the release of DAMPs. Adjuvants, such as alum, can promote Th2 responses and induce the release of host cell DNA that can induce T cell responses and the production of IgG1 and IgE. Ideally, adjuvants should be molecularly defined and enhance the magnitude and duration of a specific immune response to an antigen that provides protection against intracellular pathogens and/or reduces tumor burden.

Activation of the STING (Stimulator of Interferon Genes) protein can produce an activated or primed immune system similar to that produced by an adjuvant. This can produce a protective or preventive state when challenged or re-challenged by intracellular pathogens or viruses or tumors that inhibit the growth or proliferation of intracellular pathogens or tumors.

It may also be recognized that when a STING activator is therapeutically administered to a system in which a tumor/pathogen is present, it may act beneficially in two different but related ways. First, by direct suppression of tumor/pathogen eradication via upregulation of type-I interferons and cytokines, as described above, to act directly against the tumor/pathogen. Second, a STING activator will also induce a persistent immune response such that re-challenge or re-inoculation with the pathogen or tumor can be resisted through both a general activation of the immune system and a potential antigen-specific response to the pathogen or tumor.

Meanwhile, necroptosis is a programmed form of necrosis or inflammatory cell death mechanism. Since it was first named in 2005, key factors related to it, such as RIPK3 (receptor-interacting protein kinase 3), have been identified since 2009.

Necroptosis causes rupture of the cell membrane, and has the characteristic of accompanying a stronger immune response than immunogenic cell death induced by conventional chemotherapy or recently emerging photodynamic therapy. This is because it does not involve protein degradation or oxidation within the cell, so damage-associated molecular patterns (DAMPs) and tumor antigens that lose immunogenicity during the above process are released in their original immunogenic state, which effectively induces an anticancer immune response. Due to the above characteristics, it was expected that necroptosis could maximize the therapeutic efficacy by amplifying the responsiveness to conventional anticancer treatments.

However, although tumor necrosis factor cytokines including TRAIL, pan-caspase inhibitors including zVAD, and MLKL interfering RNA were developed, their efficacy was limited.

Therefore, since the current clinical application of anticancer treatment through necroptosis is limited, the development of necroptosis-inducing agents that selectively act on cancer cells is necessary.

The present invention has been made to solve the above problems and meet the above needs, and the purpose of the present invention is to provide a novel pharmaceutical composition.

Another object of the present invention is to provide a novel cancer treatment or cancer vaccine composition.

Another object of the present invention is to provide a novel method for treating or preventing cancer.

In order to achieve the above purpose, the present invention comprises: a) calcium lactate and

b) A pharmaceutical composition comprising, as an active ingredient, at least one substance selected from the group consisting of a phosphate, a stem cell, a cytokine, a cancer cell line, an antigen derived from the cancer cell line, a polynucleotide encoding the antigen, an antigen gene construct in which the polynucleotide is operably linked to a promoter, and an expression vector comprising the gene construct is provided.

In one embodiment of the present invention, the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA), the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, and GM-CSF, and the phosphate is preferably, but not limited to, monosodium phosphate.

In another embodiment of the present invention, the composition preferably further comprises a carbonate, but is not limited thereto.

In another embodiment of the present invention, in the composition, the calcium lactate is preferably 2.5 mM to 54 mM, and the phosphate is preferably 5 mM or more, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for treating cancer, comprising as an active ingredient at least one substance selected from the group consisting of a) calcium lactate and b) phosphate, stem cells, cytokines, cancer cell lines, antigens derived from the cancer cell lines, polynucleotides encoding the antigens, antigen gene constructs in which the polynucleotides are operably linked to a promoter, and expression vectors including the gene constructs.

In one embodiment of the present invention, the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA), the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, and GM-CSF, and the phosphate is preferably, but not limited to, monosodium phosphate.

In another embodiment of the present invention, the composition preferably further comprises a carbonate, but is not limited thereto.

In another embodiment of the present invention, in the composition, the calcium lactate is preferably 2.5 mM to 54 mM and the phosphate is preferably 5 mM or more, but is not limited thereto.

In another embodiment of the present invention, the cancer is preferably, but not limited to, a cancer selected from the group consisting of colon cancer, lung cancer, stomach cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel cell carcinoma, prostate cancer, blood cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof.

In addition, the present invention provides a cancer vaccine composition comprising, as an active ingredient, at least one substance selected from the group consisting of a) calcium lactate and b) phosphate, stem cells, cytokines, cancer cell lines, antigens derived from the cancer cell lines, polynucleotides encoding the antigens, antigen gene constructs in which the polynucleotides are operably linked to a promoter, and expression vectors comprising the gene constructs.

In one embodiment of the present invention, the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA), the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, and GM-CSF, and the phosphate is preferably, but not limited to, monosodium phosphate.

In another embodiment of the present invention, the composition preferably further comprises a carbonate, but is not limited thereto.

In another embodiment of the present invention, in the composition, the calcium lactate is preferably 2.5 mM to 54 mM and the phosphate is preferably 5 mM or more, but is not limited thereto.

In another embodiment of the present invention, the composition is preferably administered intratumoral, but is not limited thereto.

In another embodiment of the present invention, the cancer is preferably, but not limited to, a cancer selected from the group consisting of colon cancer, lung cancer, stomach cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel cell carcinoma, prostate cancer, blood cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof.

In addition, the present invention provides a method for treating or preventing cancer, comprising administering to a subject having a cancer a composition comprising, as an active ingredient, at least one substance selected from the group consisting of a) calcium lactate and b) phosphate, stem cells, cytokines, cancer cell lines, antigens derived from the cancer cell lines, polynucleotides encoding the antigens, antigen gene constructs in which the polynucleotides are operably linked to a promoter, and expression vectors comprising the gene constructs.

In one embodiment of the present invention, the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA), the cytokine is at least one cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, and GM-CSF, and the phosphate is preferably, but not limited to, monosodium phosphate.

In another embodiment of the present invention, the composition preferably further comprises a carbonate, but is not limited thereto.

In another embodiment of the present invention, in the composition, the calcium lactate is preferably 2.5 mM to 54 mM and the phosphate is preferably 5 mM or more, but is not limited thereto.

In another embodiment of the present invention, the composition is preferably administered intratumoral, but is not limited thereto.

In another embodiment of the present invention, the cancer is preferably, but not limited to, a cancer selected from the group consisting of colon cancer, lung cancer, stomach cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel cell carcinoma, prostate cancer, blood cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof.

The present invention is further described below.

The composition of the present invention is a pharmaceutical composition, and comprises, in addition to the calcium lactate described above, a pharmaceutically acceptable excipient or carrier.

The term "pharmaceutically acceptable" as used herein refers to properties and/or materials that are acceptable to the patient from a pharmacological/toxicological point of view with respect to composition, formulation, safety, patient acceptability and bioavailability, and acceptable to the compounding pharmacist from a physical/chemical point of view. A "pharmaceutically acceptable carrier" refers to a medium that does not interfere with the biological activity of the active ingredient(s) and is nontoxic to the host upon administration.

The subject of application of the pharmaceutical composition of the present invention may be any animal, and specifically, mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs, etc. The most preferred subject is humans.

The pharmaceutical composition of the present invention may be freeze-dried or formulated as a liquid preparation by any suitable means in the art. Non-limiting examples of preparations in liquid form include solutions, suspensions, syrups, slurries and emulsions. Suitable liquid carriers include any suitable organic or inorganic solvent, such as water, alcohol, saline, buffered saline, physiological saline, dextrose solution, propylene glycol solution, and the like, preferably in sterile form.

The pharmaceutical compositions of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the active polypeptide), which are formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or with organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. In addition, salts formed from the free carboxyl groups may be derived from inorganic bases, such as sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases, such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The pharmaceutical composition of the present invention is preferably formulated for inoculation or injection into a subject. For injection, the composition of the present invention may be formulated in an aqueous solution, for example, water or alcohol, or in a physiologically compatible buffer, for example, Hanks' solution, Ringer's solution or physiological saline buffer. The solution may contain formulatory agents, for example, suspending agents, preservatives, stabilizers and/or dispersing agents. In addition, the injection formulation may be prepared as a solid form preparation which is converted to a liquid form preparation suitable for injection just prior to use, for example, by reconstitution with a suitable vehicle, for example, sterile water, saline or alcohol, prior to use.

Additionally, the compositions of the present invention may be formulated as sustained release vehicles or depot preparations. Such long-acting preparations may be administered by inoculation or implantation (e.g., subcutaneously or intramuscularly) or by injection.

The compositions of the present invention may be administered by infusion or injection (e.g., intravenously, intramuscularly, intradermally, subcutaneously, intrathecally, intraduodenal, intraperitoneally, etc.). In addition, the compositions of the present invention may be administered intranasally, vaginally, rectally, orally, or transdermally. Additionally, the compositions of the present invention may be administered by a "needle-less" delivery system. Preferably, the compositions are administered by intradermal injection. Administration may be as directed by a physician or medical assistant.

The injection may be divided into several injections, and these divided injections are preferably administered substantially simultaneously. When administered as divided injections, the dose of the immunogen is preferably, but not necessarily, equally distributed in each separate injection. When an adjuvant is present in the vaccine composition, the dose of the adjuvant is preferably, but not necessarily, equally distributed in each separate injection. The separated injections for divided injections are in some aspects administered substantially adjacent to each other in the patient's body.

A variety of alternative pharmaceutical delivery systems may be utilized. Non-limiting examples of such systems include liposomes and emulsions. Certain organic solvents, such as dimethyl sulfoxide, may also be used. Additionally, the compositions of the present invention may be delivered using delayed release systems, such as semipermeable matrices of solid polymers containing the therapeutic agent. A variety of available delayed release materials are well known to those skilled in the art. Delayed release capsules, depending on their chemical properties, may release the vaccine composition over a period ranging from several days to several weeks to several months.

To prevent recurrence of cancer in a patient in remission, a therapeutically effective amount of a composition of the present invention is administered to the subject. The therapeutically effective amount will provide a clinically significant increase in the number of tumor-associated antigen-specific cytotoxic T-lymphocytes (CD8+) and a clinically significant increase in the cytotoxic T-lymphocyte response to the antigen in the patient, as measured by any means suitable in the art. Typically, in the patient, the therapeutically effective amount of the vaccine composition will destroy any remaining microscopic disease, thereby significantly reducing or eliminating the risk of recurrence of the cancer in the patient.

The effective amount of the composition of the present invention may depend on many variables, including but not limited to, race, breed, size, height, weight, age, the overall health of the patient, the type of formulation, the mode or method of administration, or the presence or absence of risk factors that significantly increase the likelihood that the cancer will recur in the patient. Such risk factors include but are not limited to, the type of surgery, the status and number of positive lymph nodes, the size of the tumor, the histologic grade of the tumor, the presence/absence of hormone receptors (estrogen and progesterone receptors), HER2/neu expression, lymphovascular invasion, and genetic predisposition (such as BRCA 1 and 2). In some preferred embodiments, the effective amount depends on whether the patient is lymph node positive or lymph node negative, and if the patient is lymph node positive, the number and extent of positive lymph nodes. In all cases, a suitable effective amount can generally be determined by one skilled in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors apparent to one of ordinary skill in the art. Preferably, a therapeutically effective amount of a vaccine composition described herein will provide a therapeutic prophylactic benefit without causing substantial toxicity to the subject.

The toxicity and therapeutic efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Compositions that exhibit a large therapeutic index are preferred. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in patients. The dosage of such compositions preferably falls within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized.

The size of the dose administered in the prevention of recurrent cancer will vary depending on, among other factors, the severity of the patient's condition, the relative risk of recurrence, or the route of administration. The severity of the patient's condition can be assessed, for example, in part by standard prognostic assessment methods.

The vaccine composition of the present invention can be administered to a patient on any suitable schedule to induce and/or support a cytotoxic T lymphocyte response to induce and/or support protective immunity against recurrence of cancer. For example, after administering the vaccine composition to a patient as described and exemplified herein as a primary immunization, a booster can be administered to support and/or maintain protective immunity. In some aspects, the vaccine composition can be administered to the patient once, twice or more times per month.

The vaccine administration schedule, including primary and booster shots, may continue for as long as the patient requires, for example over the course of several years, to prolong the patient's life. In some aspects, the vaccine schedule includes more frequent administrations at the beginning of the vaccine regimen, and less frequent administrations (e.g., boosters) for a period of time to maintain protective immunity.

The vaccine composition may be administered at a lower dose at the beginning of the vaccine regimen and at higher doses over time. Additionally, the vaccine may be administered at a higher dose at the beginning of the vaccine regimen and at lower doses over time. The number of primary vaccine and booster administrations and the dose of antigen administered may be tailored and/or adjusted to meet the specific needs of the individual patient, as determined by the attending physician using any means suitable in the art.

In one embodiment of the present invention, the vaccine composition is for treating and preventing cancer.

As used herein, the term “prevention” refers to any success or sign of success in preventing the recurrence/relapse of cancer in a patient in clinical remission, as measured by any objective or subjective variable, including the results of radiological or physical examinations.

The term 'METI-101' used in the specification and/or drawings of the present invention means calcium lactate.

The calcium lactate of the present invention can act as an agonist of STING by increasing the expression of STING, an immune activation inducer inside tumor cells, and at the same time inducing the activation of STING, and can induce an anti-tumor immune response through the activation of the immune system, and can also have an effect on secondary immunity. It was confirmed that it has an anti-tumor immune memory effect as a result of a re-challenge experiment.

The composition of the present invention can be used as an effective solid cancer immunotherapy candidate substance, an in situ cancer treatment vaccine candidate substance, and an intratumoral treatment substance by inducing apoptosis necrosis of cancer cells.

Figure 1 is a drawing showing the comparison of cell death patterns and cell-specific effects of human cancer cell lines by METI-101 (#p<0.0001 vs Control, N.S. Not Significant).

do 2 is a graph showing the ratio of the cell death effect of human cancer cell lines by METI-101 (***p<0.001 vs Control, #p<0.0001 vs Control, NS Not Significant),

do Figure 3 shows the confirmation of necroptosis factor expression by Meti-101.

do 4 is Figure showing the quantification of STING and activated STING induced by Meti-101 (***p<0.001 vs Control, #p<0.0001 vs Control),

Fig. 5 is A graph showing the mRNA expression levels of Interferon α and β induced by METI-101 in human cancer cell lines (**p<0.005 vs Control, ***p<0.001 vs Control, #p<0.0001 vs Control),

Figure 6 is a drawing showing the stimulation of Interferon receptor synthesis in human cancer cell lines by Meti-101 (**p<0.005 vs Control, ***p<0.001 vs Control, #p<0.0001 vs Control).

do 7 is Figure showing the change of IRF7 and increase of MHC1, which transmits type I interferon signal in human cancer cell lines (*p<0.01 vs Control, ***p<0.001 vs Control, #p<0.0001 vs Control),

do 8 is A diagram showing the change in the expression of chemokines, immune response inducers, by Meti-101 treatment in human cancer cells (**p<0.005 vs Control, ***p<0.001 vs Control, #p<0.0001 vs Control),

do 9 shows the decrease in CD24 and CD47 of human cancer cell lines by Meti-101 treatment (**p<0.005 vs Control, ***p<0.001 vs Control),

do Figure 10 shows the inhibition of angiogenesis in HUVEC cells by inhibition of VEGF production.

do 11 is Figure showing the differentiation of fibroblasts by TGF-β treatment and the differentiation inhibitory effect of Meti-101.

do 12 is Figure showing the reduction in TGF-β secretion in human cancer cell lines by Meti-101.

Figure 13 shows the changes in intracellular lactate concentration and pH concentration (*<p<0.01 vs Control, **p<0.005 vs Control).

Figure 14 shows the analysis of PD-L1 expression in A549 and H1975 lung cancer cell lines.

do Figure 15 shows the histological analysis of VEGF changes and CD31 in the tumor (**p<0.005 vs Control).

do Figure 16 shows the evaluation of the cancer growth inhibition effect of combined administration of MSC and Meti-101 in a xenograft mouse model using the mouse colon cancer cell line MC-38.

do 17 is a comparative diagram showing the confirmation of immune memory effect using MC-38 cell line (*p<0.01 vs Control).

do Figure 18 shows changes in white pulp in the mouse spleen.

do 19 is A diagram showing the immune distribution according to tumor size and white pulp size.

do 20 is Figure showing the confirmation of co-administration of peptide vaccine and Meti-101 in a lung cancer cell animal model using TC-1 cell line.

Figure 21 is a drawing showing the confirmation of antitumor evaluation in the MC-38 animal model by combined administration of IL-7 and Meti-101.

Figures 22 to 24 are photographs of cultures treated with calcium lactate in RPMI1640 medium containing 10% FBS (Figure 22), serum-free RPMI1640 medium (Figure 23), and serum-free RPMI1640 medium (Figure 24).

Figure 25 is a photograph showing the effect of calcium lactate on inducing apoptosis and necrosis in HCT-116 colon cancer cell lines cultured in serum-free RPMI1640 medium.

Figure 26 is a graph showing the cell viability of HCT-116 cells cultured in serum-free RPMI1640 medium and cells cultured in serum-free RPMI1640 medium treated with calcium lactate.

Figure 27 is a photograph of HCT-116 colon cancer cells cultured in serum-free DMEM, IMEM, and RPMI1640 media with calcium lactate added.

Figure 28 is a graph comparing the survival rates of colon cancer cells in serum-free DMEM, IMEM, and RPMI1640 media treated with calcium lactate.

Figure 29 is a graph confirming protein factors related to immune induction in cancer cells when apoptosis necrosis of cancer cells is induced with METI-101, Figure 30 is a photograph confirming immune markers and signaling core proteins of cancer cells undergoing apoptosis necrosis by immunoblotting, and Figure 31 is a graph confirming the expression level of interferon at the mRNA level.

Figure 32 is a graph confirming the TSPAN8 protein, a type of tumor-associated antigen, through enzyme-linked immunosorbent assay, and Figure 33 is a graph confirming the CA9 protein, a type of tumor marker, through enzyme-linked immunosorbent assay.

Figure 34 shows the results of measuring the amount of nucleic acids leaked out of cells when cells are damaged and die, and Figure 35 shows the results of measuring the amount of representative cell damage proteins leaked out of cells when cells are damaged and die.

Figure 36 is a photograph showing that METI-101 induced apoptosis and necrosis in a tumor grown by xenografting the human cancer cell line HCT116 into mice through direct intratumoral injection, and that the marker, active MLKL, was confirmed by immunohistochemical staining.

Figure 37 is a photograph showing the tumor grown by xenografting human cancer cell line HCT116 into mice, in which METI-101 was directly injected into the tumor to induce apoptosis and necrosis, and the distribution of lymphocytes within the tumor was confirmed using hematoxylin-eosin staining.

Figure 38 is a schematic diagram of an animal experiment to confirm that cancer vaccination is induced by standard treatment, radiation therapy, and intratumoral injection of METI-101.

Figure 39 is a graph showing the frequency of tumor formation when cancer cells were re-administered with radiation therapy and intratumoral injection of METI-101.

Figure 40 is a photograph of cells in which apoptosis was induced by METI-101 in a NaH ₂ PO ₄ + NaHCO ₃ solution, which is a condition showing the same phenomenon as apoptosis using Welgene RPMI1640 medium, and Figure 41 is the result of measuring apoptosis and turbidity by METI-101 in a NaH ₂ PO ₄ + NaHCO ₃ solution compared to Welgene RPMI1640 medium.

The present invention will be described in more detail through the following non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1. Apoptosis-induced cell death in various cancer cells

Quantification of immune-induced cytotoxic peptides (DAMPs)

The main component, calcium lactate pentahydrate, was quantified to produce a level of DAMP, an immune-inducing substance. DAMP was activated and selective immune-induced cell death was confirmed in human cancer cell lines. For this purpose, human cancer cells, mouse cancer cells, and human normal cells, fibroblast cells, were compared.

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), and breast cancer (MDA-MB-231) cell lines and mouse colon cancer (MC-38, CT-26) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 × 10 ⁵ cells/well, respectively. As a control, a cell line derived from fetal lung tissue (MRC-5) was used and seeded under the same conditions as the cancer cells. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS to remove FBS and then cultured for 22 h under normoxic conditions (37°C, 5% CO ₂ ) and hypoxic conditions (37°C, 5% CO ₂ , 1% O ₂ ) in medium without FBS. The cultured cells were washed with PBS and then treated with Meti-101 at a concentration of 2.5 mM in medium without FBS for 2 h. Afterwards, cell images were obtained through a microscope, and a certain amount of cell culture medium was taken and the absorbance at a wavelength of 660 nm was measured using a spectrophotometer.

As a result of cell observation, human cancer cells treated with 2.5 mM Meti-101 did not show normal cell morphology, and it was confirmed that cell byproducts flowed into the medium and the absorbance of the medium increased at a wavelength of 660 nm (p<0.0001). In the case of mouse cancer cells, the effect of Meti-101 was relatively smaller than that of human cancer cells, and it was confirmed that cell death due to Meti-101 hardly occurred in the case of normal cells other than cancer cells. These results confirmed that Meti-101 is an effective anticancer agent that minimizes damage to normal cells and significantly induces death in human cancer cells (Fig. 1).

Quantification of immune-induced apoptosis

To confirm that the main ingredient, calcium lactate pentahydrate, selectively induces immune-induced apoptosis in human cancer cell lines, colon cancer, pancreatic cancer, breast cancer, and lung cancer were selected, and mouse colon cancer was used as a comparison group.

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), non-small cell lung cancer (H1299), and breast cancer (MDA-MB-231) cell lines and mouse colon cancer (MC-38, CT-26) cell lines were seeded at a cell number of 3.5 × 10 ⁵ cells/well in 60 mm cell culture dishes, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ for cell stabilization. The stabilized cells were washed with PBS to remove FBS and then cultured in FBS-free medium under normoxic conditions (37°C and 5% CO ₂ ) and hypoxic conditions (37°C and 5% CO ₂ , 1% O ₂ ) for 22 h. The cultured cells were washed with PBS and treated with Meti-101 at a concentration of 2.5 mM in FBS-free medium for 2 h. After incubation, the cells were detached with trypsin, collected in a conical tube, and centrifuged to sediment the cells. After discarding the supernatant, the solution was resuspended in a medium, and 10 μl was taken to count the number of living cells using a hemocytometer.

In the case of human cancer cell lines, Meti-101 treatment induced cell death and no living cells could be found. However, in the case of mouse cancer cell lines, Meti-101 significantly induced cancer cell death, although it did not induce cell death like human cancer cell lines. Based on this, it was confirmed that Meti-101 acts specifically on cancer cells and can specifically induce immune-induced cell death in human cancer cell lines (Fig. 2).

Identification of apoptosis-related factors in immune-induced necroptosis

To confirm necroptosis, one of the types of cell death, changes in RIP1, p-RIP1, RIP3, p-RIP3, MLKL, and p-MLKL were observed. Human colon cancer, pancreatic cancer, lung cancer, and breast cancer cell lines were used to confirm intracellular proteins by immunomagnetic banding (WB).

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), lung cancer (H1299), and breast cancer (MDA-MB-231) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 x 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS and cultured in RPMI-1640 medium containing 0% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 1 h. The cultured cells were collected with RIPA buffer and proteins were extracted. The extracted proteins were quantified using a protein assay, separated by size, and then RIP1, p-RIP1, RIP3, p-RIP3, MLKL, and p-MLKL-specific antibodies were attached to the proteins to determine the level of fluorescence, thereby comparing the expression levels of activated proteins of necroptosis-related factors induced by Meti-101 in cancer cells.

The experimental results showed that Meti-101 decreased the expression of RIP1, RIP3, and MLKL among necroptosis-related factors, and simultaneously decreased the activation of p-RIP1, p-RIP3, and p-MLKL. This suggests that Meti-101 induces necroptosis, a type of cell death (Fig. 3).

Example 2. Confirmation of induction of antitumor immune activity

Identification of tumor-intrinsic immune activation inducer STING and signaling

To confirm the induction of antitumor immune response by calcium lactate pentahydrate, we observed the change in STING, an immune activation inducer inside tumor cells. Using human colon cancer and pancreatic cancer cell lines, we confirmed the induction of STING, a response factor to dsDNA, an intracellular DAMP, and active STING by immunomagnetic blotting (WB).

Human colon cancer (HCT-116) and pancreatic cancer (AsPC-1) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 x 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 hours at 37°C under 5% CO ₂ conditions to stabilize them. The stabilized cells were washed with PBS and then cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 24 hours. The cultured cells were collected with RIPA buffer and proteins were extracted. The extracted proteins were quantified using a protein assay, separated by size, and then STING-specific antibodies were attached to confirm the degree of protein fluorescence, thereby comparing the protein expression levels of STING (Stimulator of Interferon Genes) and activated STING induced by Meti-101 in cancer cells.

The experimental results showed that Meti-101 increased the expression of STING, an immune activation inducer inside tumor cells, and simultaneously caused STING activation. This suggests that Meti-101 can act as an agonist of STING, and can induce anti-tumor immune responses through immune system activation, as well as exert effects on secondary immunity (Fig. 4).

Determination of tumor-intrinsic immune activity Type I interferon production

To confirm the induction of antitumor immune response by calcium lactate pentahydrate, the production of type I interferons IFN-α and IFN-β produced by tumor cells was quantified. The amount of type I interferon mRNA induced by activated STING was quantified using RT-PCR for human colon cancer, breast cancer, and pancreatic cancer cell lines.

Human colon cancer (HCT-116), breast cancer (MDA-MB-231), and pancreatic cancer (AsPC-1) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 x 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS and cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 24 h. mRNA was extracted from the cultured cells using trizol. The mRNA was quantified to synthesize the same amount of cDNA, and qRT-PCR was performed based on Syber green and the target primer to quantitatively compare the expression level of type I interferon.

IFN-α and IFN-β, which belong to type I interferon, were confirmed to have an approximately 2.5 to 3-fold increase in mRNA expression by Meti-101. The increased interferon activates natural killer cells (NK cells) and macrophages to increase the innate immune response and promotes the synthesis of major histocompatibility complex (MHC) to express antigens on the cell surface. Therefore, the increase in mRNA expression of type I interferon by Meti-101 presents immune cells to recognize cancer cells as antigens and helps them to attack cancer cells smoothly (Fig. 5).

Confirmation of expression of type I interferon receptor on the surface of tumor cells

In order to confirm the induction of antitumor immune response by calcium lactate pentahydrate, the mRNA and protein expression levels of IFNAR1 and IFNAR2, which are Type I interferon receptors produced by tumor cells, were quantified. This was confirmed by RT-PCR and immunomagnetic spectroscopy (WB) for human colon cancer, breast cancer, and pancreatic cancer cell lines. Through this, the reactivity of cancer cells to Type I interferon was confirmed.

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), and breast cancer (MDA-MB-231) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 x 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS and cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 24 h. RIPA buffer was used to obtain proteins from the cultured cells, and mRNA was extracted using Trizol. The extracted proteins were quantified using a protein assay, separated by size, and then the fluorescence level of the proteins was confirmed by attaching an Interferon receptor-specific antibody, thereby comparing the protein expression levels of Interferon receptor1 and Interferon receptor2 by Meti-101 in cancer cells. Similarly, mRNA was quantified to synthesize the same amount of cDNA, and qRT-PCR was performed based on Syber green and the target primer to quantitatively compare the expression levels of Interferon receptors.

The experimental results showed that the expression of Interferon receptor, a cell surface membrane protein, increased in human cancer cell lines treated with METI-101. The mRNA expression of IFNAR1 was significantly increased in human cancer cell lines, and the mRNA of IFNAR2 was also confirmed to increase by about 1.5 to 3 times. The protein expression of IFNAR1 and IFNAR2 was also found to increase by Meti-101 in three types of human cancer cells. The increase in interferon receptor strengthens the signaling intensity of Type I interferon, leading to the activation of STING, which can promote the antitumor immune response through the feedback action (Fig. 6).

Confirmation of Type I interferon signaling on the tumor cell surface

To confirm the immune response in tumor cells by calcium lactate pentahydrate, Type I interferon signaling (IRF7, MHC I) was confirmed. It was confirmed by RT-PCR and immunomagnetic blotting (WB) for human colon cancer, breast cancer, and pancreatic cancer cell lines. Through this, the increase in Type I interferon signaling in cancer cells was confirmed.

Human colon cancer (HCT-116 and HT29), pancreatic cancer (AsPC-1), and breast cancer (MDA-MB-231) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 × 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS and cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 24 h. RIPA buffer was used to obtain proteins from the cultured cells, and mRNA was extracted using Trizol. The extracted proteins were quantified using a protein assay, separated by size, and then an antibody specific for Type I interferon signaling protein was attached to check the level of protein fluorescence, thereby comparing the protein expression levels of IRF-7 and MHC I by METI-101 in cancer cells. Similarly, mRNA was quantified to synthesize the same amount of cDNA, and qRT-PCR was performed based on Syber green and the target primer to quantitatively compare the level of mRNA expression of IRF-7.

Type I interferon signal transduction is mediated by IRF-7, and it was confirmed that the mRNA and protein expression of IRF-7 in human cancer cell lines increased when Meti-101 was treated. As a result of signal transduction by IRF-7, it was also confirmed that the protein expression of MHC1, a cell surface antigen presentation factor, increased (Fig. 7).

Confirmation of immune-induced chemokine expression by type I interferon signaling on the tumor cell surface

In order to confirm the immune response in tumor cells by calcium lactate pentahydrate, the expression of immune cell-induced chemokines was confirmed. The mRNA and protein production of CXCL-9 and CXCL10 were confirmed by RT-PCR and immunomagnetic blotting (WB) for human colon cancer, breast cancer, and pancreatic cancer cell lines. Through this, the increase in immune-induced chemokines in cancer cells was confirmed.

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), and breast cancer (MDA-MB-231) cell lines were seeded in 60 mm cell culture dishes at a density of 3.5 x 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS and cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 24 h. RIPA buffer was used to obtain proteins from the cultured cells, and mRNA was extracted using Trizol. The extracted proteins were quantified using a protein assay, separated by size, and then an antibody specific for Type I interferon signaling protein was attached to check the level of protein fluorescence, thereby comparing the protein expression levels of CXCL-9 and CXCL-10 induced by Meti-101 in cancer cells. Similarly, mRNA was quantified to synthesize the same amount of cDNA, and qRT-PCR was performed based on Syber green and the target primer to quantitatively compare the level of mRNA expression of the chemokine.

The experimental results confirmed that Meti-101 increases the secretion of Chemokine, a type of immune activation protein. The mRNA increase of CXCL-9 and CXCL-10 in human cancer cell lines increased from about 1.5 to 4.5 times. It was confirmed that the protein amount of CXCL-10 increased along with the mRNA of Chemokine. As a result, human cancer cells treated with METI-101 induce an immune response, which can make the immune system sensitive to cancer cells (Fig. 8).

Confirmation of tumor cell immune evasion factor expression

The immune response induction ability was confirmed by measuring the decrease in the immune evasion factor of tumor cells by calcium lactate pentahydrate. The mRNA and protein production of CD24 and CD47 were confirmed by RT-PCR and immunomagnetic fluorescence (WB) for human colon cancer, breast cancer, and pancreatic cancer cell lines. Through this, the immune response of cancer cells was confirmed to increase.

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), and non-small cell lung cancer (H1299) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 x 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ₂ to stabilize them. The stabilized cells were washed with PBS and then cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ) for 24 h. RIPA buffer was used to obtain proteins from the cultured cells, and mRNA was extracted using Trizol. The extracted proteins were quantified using a protein assay, separated by size, and then specific antibodies were attached to the immune evasion factor proteins to check the level of protein fluorescence, thereby comparing the protein expression levels of CD24 and CD47 by Meti-101 in cancer cells. Similarly, mRNA was quantified to synthesize the same amount of cDNA, and qRT-PCR was performed based on Syber green and target primers to quantitatively compare the levels of mRNA expression of immune evasion factors.

CD24 and CD47 are membrane proteins that enable immune evasion, and we confirmed that CD24 and CD47 were significantly reduced when Meti-101 was treated in human cancer cell lines. All human cancer cell lines used in the experiment showed a decrease in CD24 and CD47 in mRNA and protein. Human cancer cell lines treated with Meti-101 showed a decrease in CD24 and CD47, which eliminated anti-immune responses and increased immune sensitivity to cancer cells (Fig. 9).

Example 3. Confirmation of reduction of immunosuppressive factors and accessibility of immune cells in the tumor microenvironment

Expression of angiogenic factor VEGF and inhibition of angiogenesis

The inhibitory effect of calcium lactate pentahydrate on the expression of the immunosuppressive angiogenic factor VEGF in tumor cells (Fig. 12) and the inhibitory effect of angiogenesis in vascular cells (Fig. 10) were confirmed. This is an immunosuppressive factor that inhibits the entry of immune cells into tumors. For confirmation, the expression level of the angiogenic factor VEGF mRNA was measured using RT-PCR for two types of human colon cancer cells (Fig. 12). In addition, the degree of angiogenesis was confirmed using human human umbilical vein endothelial cells (HUVEC).

Human colon cancer cells, HCT-116, were seeded in a 60 mm cell culture dish at a cell number of 3.5 x 10 ⁵ cells/well, and cultured for 24 hours with 1% FBS to avoid interference of growth factors. 2.5 mM Meti-101 was treated to confirm the drug effect after 24 hours of culture. After 24 hours of treatment, the cell culture media under normal culture conditions and under Meti-101 treatment conditions were collected using a centrifugal filter device (Millipore) (Conditioned Media, CM). Human umbilical vein endothelial cells (HUVEC) were seeded in a 96-well culture dish, and the cell culture media (CM) was treated. The following experiment used a tube formation assay kit (Cultrex), and dose-dependent changes in cell morphology were captured through a fluorescence microscope.

As a result, it was confirmed that angiogenesis occurred well in HUVECs that were not treated with Meti-101, but angiogenesis did not occur in HUVECs that were treated with Meti-101 (Figure 10).

Production of tumor-active fibroblasts and inhibition of fibrosis

The generation of tumor-activated fibroblasts that block immune cells entering the tumor and the extracellular matrix (ECM) and molecular biological markers produced by these cells were quantified to confirm that differentiation into immune-suppressive fibroblasts was inhibited. Through experiments, it was confirmed that the entry of immune cells into the tumor was facilitated by inhibiting the production of rigid fibers on the tumor surface. To this end, the transformation into tumor-activated fibroblasts was confirmed using two types of human fibroblasts, and the protein expression levels of various factors were confirmed by immunomagnetic blotting (WB).

Human normal lung cell lines (MRC-5 and WI-38) were seeded in 60 mm cell culture dishes at a cell number of 3.5 × 105 cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C, 5% CO ₂ for cell stabilization. For pretreatment, the cells were washed with PBS and cultured for an additional 8 h in DMEM medium containing 10% FBS and 5 ng/ml TGF-β. After pretreatment, the cells were treated with 5 ng/ml TGF-β and 2.5 mM Meti-101 in DMEM medium without FBS and then cultured for 24 h under low-oxygen conditions (37°C, 5% CO ₂ , 1% O ₂ ). The cultured cells were collected with RIPA buffer and proteins were extracted. The extracted proteins were quantified through protein assay, separated by size, and then the fluorescence level of the proteins was confirmed by attaching a fibrosis-related antibody, thereby comparing the protein expression levels of the extracellular matrix and fibroblast differentiation caused by Meti-101 in cancer cells.

We confirmed that the expression of HIF-1α increases in fibroblasts under low oxygen conditions. Meti-101 decreases the expression of HIF-1α not only in cancer cells but also in fibroblasts under low oxygen conditions. When fibroblasts under low oxygen conditions were treated with TGF-β, the phosphorylation of the TGF-β receptor and the signaling protein Smad2 was confirmed through protein immunoblotting. As a result, the phosphorylation of Smad2 was decreased by METI-101, and the TGF-β receptor on the cell surface was also decreased. Similarly, it was found that low oxygen conditions and TGF-β treatment in fibroblasts increased the expression of α-SMA, Col1a1, and Fibronectin, which are markers of fibrosis. Meti-101 can inhibit tumor fibrosis by decreasing the synthesis of extracellular matrix proteins that cause fibrosis in a concentration-dependent manner (Fig. 11).

Confirmation of the effect of suppressing the production of TGF-β, an immunosuppressive factor in cancer cells

By confirming the production of TGF-β, which suppresses the activity of immune cells attacking cancer cells within the tumor and enhances the activity of immunosuppressive immune cells, we confirmed the change in the immunosuppressive tumor microenvironment by calcium lactate pentahydrate. To this end, we used human colon cancer, lung cancer, pancreatic cancer, and breast cancer cells and confirmed the expression level of TGF-β protein produced from them by immunomagnetic spectroscopy (WB).

Human colon cancer (HCT-116), pancreatic cancer (AsPC-1), non-small cell lung cancer (H1975), and breast cancer (MDA-MB-231) cell lines were seeded in 60 mm cell culture dishes at a cell number of 3.5 × 10 ⁵ cells/well, respectively. After seeding, the cells were cultured for 24 h at 37°C and 5% CO ² to stabilize them. The stabilized cells were washed with PBS and cultured in RPMI-1640 medium containing 10% FBS and 2.5 mM Meti-101 under low-oxygen conditions (37°C, 5% CO ² , 1% O ² ) for 24 h. The cultured cells were collected with RIPA buffer and proteins were extracted. The extracted proteins were quantified using a protein assay, separated by size, and then a TGF-β-specific antibody was attached to the protein to determine the level of fluorescence, thereby comparing the amount of TGF-β protein expression induced by Meti-101 in cancer cells.

Human cancer cell lines treated with Meti-101 showed a decrease in protein synthesis of TGF-β. The decrease in TGF-β changes the microenvironment inside the tumor, preventing immune cell dysfunction and allowing them to attack cancer cells normally (Figure 12).

Confirmation of the effect of suppressing the production of immunosuppressive chemicals in cancer cells

In order to confirm the production of lactate, proton, and other chemicals that suppress the activity of immune cells that attack cancer cells inside the tumor, the concentration of intracellular lactate and the change in intracellular pH were measured. For this purpose, human colon cancer HCT-116 cell line was used, and fluorescent cell analysis, enzymatic lactate quantification, and pH measurement by dye were performed. Through this, the inhibition effect of the production of immunosuppressive chemicals by calcium lactate pentahydrate was confirmed.

To measure the concentration of intracellular lactate, HCT-116 cells were seeded in 60 mm cell culture dishes at a density of 5 x 10 ⁵ cells/well and cultured at 37°C for 24 hours. After culture, the cells were treated with Meti-101 in RPMI1640 medium for 24 hours. The cells were then washed and collected using DPBS, and the cells and culture medium were homogenized to extract proteins. The protein concentration of the extracted proteins was measured using the BCA assay. The lactate concentration was measured by spectrophotometry and normalized to total protein, and the sample solution was incubated with the reagent for 30 minutes at room temperature. The absorbance of the sample was then measured at 450 nm using a microplate spectrophotometer. The above experiment was analyzed and performed using the Lactate Assay Kit (Abcam). Samples for measuring intracellular pH concentration were collected using the same method as the above experiment. The collected samples were cultured using pH-sensitive pHrodo TM Red AM (Molecular Probes; Thermo Fisher Scientific). After incubation, fluorescence was measured and analyzed at a wavelength of 555–580 nm, and converted to intracellular pH concentration using a nigericin calibration curve.

As a result, it was found that the concentration of intracellular lactate significantly increased when treated with Meti-101, and it was confirmed that the intracellular pH concentration significantly decreased under conditions treated with Meti-101 (Fig. 13).

Confirmation of the effect of suppressing the expression of immunosuppressive immune checkpoint proteins in cancer cells

We measured the expression level of immunosuppressive immune checkpoint proteins expressed on the surface of cancer cells, which block the tumor attack of immune cells and induce the death and fatigue of immune cells. The amount of PD-L1, a representative immune checkpoint protein, was confirmed in human lung cancer cells expressing the normal and mutant forms of EGFR by immunofluorescence, and through this, the inhibitory effect of calcium lactate pentahydrate on the production of immunosuppressive immune checkpoint proteins was confirmed.

After ^3x103 of human lung cancer cells A549 (EGFR wild type) and H1975 (EGFR mutant type) were inoculated onto a bio-coated cover slip, 2.5 mM Meti-101 was treated when the cancer cells were stably established and cultured for 24 hours under normoxic and hypoxic conditions (37℃, 5% _CO2 , 1% _O2 ). After culture, each cell was fixed using a formalin solution and then bound with PD-L1 antibody and fluorescence was expressed using a secondary antibody. After this process, the cells were placed on a slide and observed using a confocal microscope at a wavelength of 570-620 nm.

In A549 and H1975 lung cancer cell lines, cells treated with Meti-101 showed a significant decrease in PD-L1 compared to untreated cells under both normoxic and hypoxic conditions (Fig. 14).

Example 4 .In Vivo Immunopharmacological effects

Inhibition of intratumoral immunosuppressive VEGF expression and angiogenesis in a colon cancer transplantation model

In a xenograft model using human-derived cells, HCT-116, the tumor was extracted at the time of autopsy after the experiment was completed. The extracted tumor was cut in half, and one half was fixed in formalin, and the other half was homogenized in RIPA buffer, and proteins were extracted and quantified using the BCA method. The extracted proteins were subjected to immunomagnetic banding (WB) experiments using an electroporation device, and the bands were quantified and data was converted into representative images using the ImmageJ program. The fixed tumor was cryosectioned after making a paraffin block, and slides were made. The manufactured slides were stained with hematoxylin and eosin (H&E) using a CD31 antibody and immunofluorescence was performed.

As a result, it was found that tumor cells were significantly reduced in mice treated with Meti-101 (H&E), and the immunofluorescence results confirmed that CD31 was significantly reduced (Fig. 15).

Anticancer immunity and immune memory effects by combination with mesenchymal stem cells (MSC)

1) Confirmation of antitumor activity through combined use with mesenchymal stem cells (MSC)

To explore the pharmacological effects of Meti-101, a xenograft model was prepared using MC-38, a mouse-derived colon cancer cell line, divided into three groups: control group (10 mice), mesenchymal stem cell group (10 mice), and co-administration of mesenchymal stem cells and Meti-101 (10 mice). ^1x105 of the MC-38 cell line was subcutaneously administered into the flank of the mice, and from the 7th day after tumor cell administration until the end of the experiment, tumor occurrence was confirmed (palpated) twice a week and the long and short axis sizes of the tumors were measured using an electric caliper.

Tumor size (mm ³ ) = (long axis) x (short axis ² ) / 2

Mesenchymal stem cells were administered intratumorally, 1 x 10 ⁶ cells were inoculated five times twice a day for a total of three days, and Meti-101 was administered subcutaneously at 20 mg/kg twice a day. After 10 days of administration, the tumor size was significantly reduced in the mesenchymal stem cell and combination administration groups, and after 30 days, no tumors were observed in 9 mice in the mesenchymal stem cell group, and all 10 mice in the combination administration group showed tumor disappearance (Fig. 16).

2) Confirmation of anti-tumor immune memory effect through combined use with mesenchymal stem cells (MSC)

Based on the experiment in Fig. 16, a re-challenge experiment was conducted to confirm the anti-tumor immune memory effect through combination with mesenchymal stem cells.

The control group was euthanized in accordance with the Animal Ethics Act, and a re-experiment was conducted with a total of 29 animals: 9 animals from the mesenchymal stem cell group, 10 animals from the combined administration group, and 10 new control groups. After a 15-day rest period, the mesenchymal stem cell group and the combined administration group were re-inoculated with 1 x 10 ⁵ of the mouse colon cancer cell line MC-38 to the control group (10 animals), mesenchymal stem cell group (9 animals), and combined administration group (10 animals).

After administering tumor cells, the tumor size was measured, and the tumor volume of the control group grew the largest, while the mesenchymal stem cell group showed a tendency to slightly decrease. In the combined administration group, the tumors grew in 4 mice, but the size was not large, and in 6 mice, no tumor growth was observed from the first tumor measurement date to the end date. Significance was confirmed in the mesenchymal stem cell group and the combined administration group (Fig. 17).

After tumor measurement, the mice were necropsied. The spleen was removed to confirm the white pulp, an immune-related factor in the spleen. After the spleen was removed, it was fixed in 10% formalin and paraffin blocks were made. Slides were made using a microtome, and the made slides were stained with H&E. When the spleen of each group was compared, it was found that the white pulp was significantly increased in the combination treatment group. White pulp was written in white letters as WP (Fig. 18).

In order to confirm the correlation between tumor volume and white pulp through a re-challenge experiment, an immune distribution map was created (Fig. 19). The immunized individuals indicated by the red circles are individuals in which no tumors were observed and the distribution showed a further increase in white pulp. In addition, it was confirmed that the white pulp became smaller as the tumor size increased.

Confirmation of antitumor activity by combined administration with peptide vaccine

To explore the pharmacological effects of Meti-101, a xenograft model was prepared using TC-1-Luc, a mouse-derived lung cancer cell line, divided into four groups: control group (10 mice), Meti-101 (10 mice), peptide vaccine (10 mice), and co-administration of peptide vaccine and Meti-101 (10 mice). TC-1-Luc cell line ^1x105 was subcutaneously administered into the flank of the mice, and from the time the tumor size reached ^150mm3 after tumor cell administration until the end of the experiment, tumor occurrence was confirmed (palpated) twice a week and the long and short axis sizes of the tumors were measured using an electric caliper.

Tumor size (mm ³ ) = (long axis) x (short axis ² ) / 2

Meti-101 was administered subcutaneously at 20 mg/kg twice daily for 2 weeks, and the peptide vaccine was administered intramuscularly at 6-day intervals. From 7 days after administration, the tumor size difference between the peptide vaccine and co-administration group increased compared to the control group, and from 12 days, a difference in the tumor size between the control group and Meti-101 was confirmed (56%). From 15 days, a significant decrease in the tumor size was confirmed in the peptide vaccine and co-administration group (61%) (Fig. 20).

Confirmation of antitumor activity by combined administration of IL-7 and Meti-101

To explore the pharmacological effects of Meti-101, a xenograft model was prepared using MC-38, a mouse-derived colon cancer cell line, divided into four groups: control (10 mice), Meti-101 (10 mice), IL-7 (10 mice), and co-administration of IL-7 and Meti-101 (10 mice). ^1x105 of the MC-38 cell line was subcutaneously administered into the flank of the mice, and from 7 days after tumor cell administration until the end of the experiment, tumor occurrence was confirmed (palpated) twice a week and the long and short axis sizes of the tumors were measured using an electric caliper.

Tumor size (mm ³ ) = (long axis) x (short axis ² ) / 2

Meti-101 was administered subcutaneously at 20 mg/kg twice daily for two weeks, and IL-7 was administered once via intramuscular injection 7 days after tumor cell administration. The difference in tumor size between the IL-7 and Meti-101 groups increased from 7 days after administration, and from 10 days, the tumor size increased in the control, IL-7, and Meti-101 groups, but most tumors in the control group grew to 500 to 1000 mm ³ , while in the IL-7 group, some tumors grew to 500 to 1000 mm ³ but decreased significantly. Meti-101 also increased like the above two groups. It was confirmed that the tumor size in the combination administration group decreased significantly compared to the three groups. At the end of the experiment, the control group was evenly distributed from 500 to 2000 mm ³ , and the IL-7 group was evenly distributed from 300 to 2000 mm ³ . In the Meti-101 group, the distribution was from 500 to 1500 mm ³ , but no tumors larger than 1500 mm ³ were observed. In the combination treatment group, the tumor size was evenly distributed from 300 to 1000 mm ³ , and it was found that the tumor size was significantly reduced (Fig. 21).

Example 5: Apoptosis induced by calcium lactate

HCT116 colon cancer cells were cultured in RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin, serum-free RPMI1640 medium without FBS, and serum-free RPMI1640 medium treated with 2.5 mM calcium lactate at 37°C, 5% CO ₂ and 1% O ₂ for 2 hours. After completion of the culture, cell images were obtained using an optical microscope.

Figures 22 to 24 are photographs of colon cancer cells cultured in a medium containing 10% FBS (Figure 22), colon cancer cells cultured in a serum-free medium (Figure 23), and colon cancer cells cultured in a serum-free medium treated with 2.5 mM calcium lactate (Figure 24). It was confirmed that the presence or absence of serum made no difference in the 2-hour culture under low-oxygen conditions. It was found that colon cancer cells cultured in a serum-free medium containing 2.5 mM calcium lactate underwent apoptosis necrosis.

Example 6: Apoptosis and necrosis caused by calcium lactate according to composition

HCT-116 colon cancer cell lines were cultured in serum-free RPMI1640 medium and serum-free RPMI1640 medium treated with 2.5 mM calcium lactate at 37℃, 5% CO ₂ and 1% O ₂ and 37℃, 5% CO ₂ for 2 hours. After culture, colon cancer cells were photographed with an optical microscope to identify them. To confirm that apoptosis had occurred in the serum-free RPMI1640 medium treated with 2.5 mM calcium lactate, the medium was removed, washed once with saline, treated with 1 ml of trypsin-EDTA, and cultured for 1 minute. After that, colon cancer cells were detached. The colon cancer cells were collected with 1 ml of saline and transferred to an Enpendorf tube. The clumped colon cancer cells were released, and 10 ul of the cell suspension was mixed with 10 ml of trypan blue for staining. 10 ml of the dyed colon cancer cell suspension was placed on a hemocytometer plate and the number of viable cells was measured.

When the experiment was conducted under the above conditions, colon cancer cells in the serum-free medium containing 2.5 mM calcium lactate underwent apoptosis regardless of the general culture conditions (Fig. 25, upper right) and hypoxic conditions (Fig. 25, lower right). Colon cancer cells cultured in the serum-free medium without 2.5 mM calcium lactate survived under both the general culture conditions (Fig. 25, upper left) and hypoxic conditions (Fig. 25, lower left). When the survival of colon cancer cells treated with 2.5 mM calcium lactate was measured to some extent compared to the control group using trypan blue staining, it was found that more than 99% of the colon cancer cells were dead (Fig. 26).

The above results confirmed that treating 2.5 mM calcium lactate in serum-free medium could induce apoptosis in almost all colon cancer cells regardless of atmospheric conditions.

Example 7: Apoptosis and necrosis phenomenon according to calcium lactate and representative cancer cell culture media

Colon cancer cells, HCT-116, were cultured in three types of media (DMEM, IMEM, and RPMI1640) mainly used for culturing cancer cells, treated with 2.5 mM calcium lactate under serum-free conditions. The cells were cultured for 2 hours under the conditions of 37℃, 5% _CO2 , and 1% _O2, and photographs were taken with an optical microscope to observe apoptosis. To quantify the death of colon cancer cells, the medium was removed, washed once with saline, treated with 1 ml of trypsin-EDTA, and cultured for 1 minute, and colon cancer cells were detached. The colon cancer cells were collected with 1 ml of saline and transferred to an EP tube. The clumped colon cancer cells were dissolved, and 10 ul of the cell suspension and 10 ml of trypan blue were mixed for staining. 10 ml of the stained colon cancer cell suspension was placed on a hemocytometer, and the number of living cells was measured.

Figure 27 shows that when HCT-116 colon cancer cell lines were cultured for 2 hours in serum-free DMEM, IMEM, and RPMI1640 media containing 2.5 mM calcium lactate, apoptosis was confirmed in RPMI1640 medium (Figure 27 right). When 2.5 mM calcium lactate is treated in DMEM and IMEM, apoptosis of colon cancer cells does not occur (Figure 27 left, Figure 27 middle).

In order to compare the degree of apoptosis of colon cancer cells in three types of media in Fig. 28, the number of live colon cancer cells was measured using a hemocytometer and presented in a graph. The number of live colon cancer cells cultured in serum-free DMEM and IMEM media treated with 2.5 mM calcium lactate was measured to be around 1 million per ml, but the amount of live cells cultured in RPMI1640 medium was significantly reduced to 4,000 per ml. Through the above experiment, it can be confirmed that calcium lactate specifically causes apoptosis of cancer cells in RPMI1640 medium.

Example 8: Immune factor activation of human colon cancer cells by METI-101 treatment in serum-free RPMI1640 medium

Human colon cancer cells, HCT-116, were cultured in serum-free RPMI1640 medium sold by Welgene and treated with METI-101. In experiments according to changes in time, the concentration of METI-101 was treated at 2.5 mM, and in experiments according to changes in concentration, the incubation time was set to 1 hour. Proteins secreted outside the cell (IFNα, GM-CSF, IL-1β) were analyzed by enzyme-linked immunosorbent assay using cell culture fluid. Intracellular signaling proteins and membrane proteins were analyzed by collecting total cell proteins and performing immunoblotting. Finally, the interferon family, which plays a key role in the immune response, was identified at the molecular biological level using real-time polymerase chain reaction.

Interferon alpha secreted by cells was secreted about twice as much as cells without METI-101 when exposed to 2.5 mM METI-101 for 1 hour in serum-free medium. (Fig. 29, first left) IFNAR1, a transmembrane receptor that recognizes interferon, also significantly increased in mRNA expression level at 1 hour. (Fig. 31, first left) Although the mRNA expression level of IFNAR2, which forms a heterodimer with IFNAR1, did not increase, the intracellular protein significantly increased at 1 hour. (Fig. 31, second left, Fig. 30, first left) In addition, it was confirmed that phosphorylation of STING protein, which promotes interferon and pro-inflammatory cytokines, occurred and changed into an active form, and the protein level of major histocompatibility complex 1 (MHC class 1), an antigen-presenting protein, increased. (Fig. 30, first left, Fig. 30, right)

Treatment with 2.5 mM METI-101 in serum-free medium induced phosphorylation of STING, which in turn increased the synthesis of interferon, as evidenced by the mRNA synthesis levels of IFNα and IFNβ. When cultured with METI-101 in serum-free medium for 2 hours, interferon mRNA synthesis increased two-fold (Fig. 31, third from the left, fourth from the left, and fifth from the left).

When METI-101 was treated in a concentration-dependent manner on human cancer cells, the protein secretion amounts of granulocyte-macrophage colony-stimulating factor (GM-SCF), which functions as a growth factor for leukocytes, and IL-1β, which promotes immunity, began to increase significantly from 2.5 mM of METI-101. (Figure 29, second from the left, third from the left)

In cancer cells treated with 2.5 mM or more of METI-101 in serum-free medium, interferon and pro-inflammatory cytokines are produced and secreted through intracellular STING signaling, and antigen markers and interferon receptors are increased to activate the immune response.

Example 9: Reduction of biomarkers by tumor-associated antigen presentation and cancer cell death due to treatment with METI-101 at different concentrations in serum-free RPMI1640 medium

Human colon cancer cells, HCT-116, human pancreatic cancer cells, AsPC-1, and human non-small cell lung cancer cells were cultured in serum-free RPMI1640 medium sold by Welgene and treated with METI-101 at various concentrations. The culture time was fixed to 1 hour, and enzyme-linked immunosorbent assay was performed on the cell culture for tumor-associated antigen protein (TSPAN8) and tumor marker (CA9) specifically produced by cancer cells for 1 hour.

TSPAN8, a tumor-associated antigen presentation protein, increased to a very high level under the condition of treating HCT-116 cells with 2.5 mM METI-101, and when treated with 5.0 mM METI-101, it decreased by about half, but still maintained a high level compared to the control group. (Fig. 32, first from the left) Unlike HCT-116, AsPC-1 cells and H1299 cells maintained the expression of TSPAN8 at a very high level starting from a low concentration of 1.5 mM METI-101. Similar to HCT-116, the secretion of tumor-associated antigen presentation protein decreased as the concentration increased. (Fig. 32, second from the left, third from the left)

Cancer cells maintain high or low levels of specific proteins or enzymes depending on their type and characteristics. CA9 enzyme is used as one of the representative tumor markers. A high level of CA9 was expressed in human colon cancer cells, HCT-116, and it was confirmed that apoptosis of cancer cells occurred when METI-101 was treated, and CA9 decreased in the cell culture medium. (First from the left in Figure 33) H1299 cells showed the same pattern as HCT-116, and when METI-101 was treated, apoptosis caused a rapid decrease in CA9 secretion from METI-101 1.5 mM, and AsPC-1 cells showed a concentration-dependent decrease in CA9. (Second and third from the left in Figure 33)

The results above show that when METI-101 is treated in serum-free medium, tumor cells are killed as shown by CA9 results and present the tumor-associated antigen TSPAN8.

Example 10: Secretion of Damage-associated molecular patterns (DAMPs) and immunogenic cell death (ICD) due to treatment with METI-101 at different concentrations in serum-free RPMI1640 medium

Human colon cancer cells, HCT-116, were cultured in serum-free RPMI1640 medium sold by Welgene and treated with METI-101 at various concentrations. The culture time was fixed to 1 hour, and apoptosis classified as immunogenic cell death indicates damage-associated molecule type. In this experiment, the damage-associated molecule type was confirmed in the cell culture solution. DNA, a nucleic acid type damage-associated molecule type, was isolated using a kit using the silica adsorption method, and RNA and dsRNA were isolated using TRI solution. DNA and RNA were quantified using a microspectrophotometer (Nanodrop), and dsRNA was quantified using an enzyme-linked immunosorbent assay.

Other types of damage-associated molecules were assayed using enzyme-linked immunosorbent assay. When the nucleic acids were separated from the cell culture medium, the DNA and RNA in the culture medium increased when METI-101 2.5 mM was treated for 1 hour in the serum-free medium. (Fig. 34, first and second from the left) When dsRNA was quantified by treating the serum-free medium with METI-101 2.5 mM for 1 hour, it was confirmed that a high level of dsRNA was present in the medium compared to the control group. (Fig. 34, third from the left) As for the damage-related molecule types other than nucleic acids, the proteins HMGB1, Calreticulin, and HSP90 were identified by enzyme-linked immunosorbent assay, and Calreticulin and HSP90 in the cell culture medium significantly increased from 1.5 mM. (Fig. 35, second and third from the left) HMGB1 showed a tendency to increase in METI-101 1.5 mM, but no significant difference was observed, and it increased to a high level compared to the control group in METI-101 2.5 mM. (Fig. 35, left) First) Finally, the organic molecule ATP was quantified using colorimetry. A significant difference was observed from the control group at a low concentration of 1.5 mM METI-101, and the amount of ATP in the cell culture increased in a concentration-dependent manner. (Figure 35, fourth from the left)

Through quantitative analysis of damage-associated molecular types in cell culture media, METI-101 was confirmed to induce apoptosis of cancer cells. In addition, apoptosis causes intracellular substances to be released outside the cell, thereby inducing an immune response by providing specific biomolecules, which are included in immunogenic cell death. These damage-associated molecular types transmit information to pattern-recognition receptors (PRRs) of immune cells, thereby enhancing overall immunity against cancer cells.

Example 11: Induction of apoptosis by METI-101 via intratumoral injection in mice xenografted with human colon cancer cells, HCT-116

Tumors were formed by subcutaneously injecting human colon cancer cell lines, HCT-116 cells, into nude mice. Tumors were prepared by mixing serum-free RPMI1640 medium and METI-101 to 54 mM and immediately injected into the tumor using an insulin syringe. After the animal experiment, tumor tissues were stained with phosphorylated MLKL, a marker of apoptosis and necrosis, using immunohistochemistry. (Fig. 36, top)

Tissues that were not injected with METI-101 do not show phosphorylated MLKL and cancer cells are normally located. (Figure 36, bottom, first from the left) Around the tissues directly injected with METI-101, apoptotic cells are located and phosphorylated MLKL is strongly stained. This is the area where the tissue collapses and the tumor size decreases directly and rapidly. (Figure 36, bottom, second from the left) It can also be confirmed that the area surrounding the directly injected area is phosphorylated with MLKL and stained. The surrounding area slowly dies as apoptotic cells and plays a role in further enhancing the immune response. (Figure 36, bottom, third from the left)

This animal experiment demonstrates that direct intratumoral injection of METI-101 induces rapid cell death at the injection site and surrounding areas, resulting in a decrease in overall tumor size and the effect of immunogenic cell death.

Example 12: Apoptosis and lymphocyte recruitment of METI-101 via intratumoral injection in mice xenografted with human colon cancer cells, HCT-116

As a result of Example 11, the tissue was stained using hematoxylin-eosin staining, one of the tissue staining methods. The photographed tissue is the part where METI-101 was directly injected, where apoptotic cells are gathered. As shown in Fig. 29, it can be seen that lymphocytes are gathered around apoptotic cells in the tumor, along with the result of GM-CSF increasing when METI-101 is treated. (Second from the left in Fig. 29, Fig. 37)

Example 13: Induction of apoptosis and necrosis by METI-101 via intratumoral injection in mice allogeneically transplanted with colon cancer cell line CT26 and combined effect with standard radiotherapy

When METI-101 was directly injected to induce apoptosis, an experiment was conducted to determine whether systemic immunity against cancer cells was successfully completed based on the previous example. CT26 cells, a mouse-derived colon cancer, were injected subcutaneously under the skin of mice to form tumors. The formed tumors were treated with standard radiation therapy and METI-101 direct administration, either individually or simultaneously. Radiation therapy was performed once at a high dose, and METI-101 was directly injected into the tumor four times over two weeks. Once

The injection was administered at a concentration of 54 mM METI-101 in serum-free medium. (Fig. 38, top) After the above experimental process, all mice underwent surgery to remove tumors. After a short recovery period after the surgery, all mice were injected subcutaneously with CT26 cells at the same number of cells as before. The tumor formation was monitored for 2 weeks to confirm whether or not tumors were formed. (Fig. 38, bottom)

Example 14: Induction of apoptosis and necrosis by METI-101 via intratumoral injection in mice allogeneically transplanted with colon cancer cell line CT26 and combined effect with standard radiotherapy

As a result of conducting an animal experiment according to the experimental method of Example 13, the control group and the group that received direct intratumoral injection of RPMI1640 showed normal growth of cancer cells and formed tumors with an average size of 2 to 2.5 mm ³ . On the other hand, the group that received standard radiotherapy showed a significant decrease in the size of cancer cells to an average size of 0.56 mm 3 . The group that received direct intratumoral injection of METI-101 54 mM in serum-free medium showed an average tumor size of 0.22 mm ³ , which was smaller than the standard radiotherapy treatment. It can be confirmed that the group that received both treatments together showed a decrease in tumor size to an average of 0.02 mm ³ (left photo and graph of the left figure in Figure 39)

To confirm apoptosis, an immunogenic cell death, tumor tissues were stained for phosphorylated MLKL by immunohistochemistry. Phosphorylation of MLKL did not occur in tumors treated with standard radiotherapy. Phosphorylation of MLKL was clearly confirmed in tumors injected with METI-101. When the two treatments were applied together, cancer cells died and holes were formed, and phosphorylation of MLKL occurred in the remaining cells. (Right photo of the left picture of Fig. 39)

When CT26 cells were injected again, all mice in the control group and the group injected with RPMI1640 medium developed tumors. None of the mice in the group treated with standard radiotherapy or direct intratumoral injection of METI-101 developed tumors (Fig. 39, right graph).

Both standard radiation and direct intratumoral injection of METI-101 resulted in dramatic reductions in tumor size, and when administered simultaneously, tumors were reduced to the point of disappearance. As in the previous examples, METI-101 activates immunity through apoptosis, reduces tumor size, and prevents recurrence through immune memory.

Example 15: Apoptosis and necrosis induced by METI-101 in NaH ₂ PO ₄ + NaHCO ₃ solution and RPMI1640

Human colon cancer cells, HCT-116, were treated with METI-101 2.5 mM in a solution of NaH ₂ PO ₄ + NaHCO ₃ in distilled water to confirm apoptosis. PBS and PBS with METI-101 2.5 mM were treated as negative controls, and RPMI1640 from Welgene with METI-101 2.5 mM was treated as a positive control. The experimental groups were selected from a solution of NaH ₂ PO ₄ + NaHCO ₃ in distilled water at the same molar concentration as the composition of RPMI1640 and a solution of NaH ₂ PO ₄ + NaHCO ₃ in distilled water adjusted to pH 7.4, and treated with METI-101 2.5 mM. The drug-treated cells were cultured under hypoxic conditions for 2 hours. Turbidity was also measured at a wavelength of 660 nm after treating with METI-101 2.5 mM.

Compared to before treatment, cancer cells treated with PBS alone were all floating on the bottom, while cancer cells treated with METI-101 2.5 mM in PBS were stuck to the bottom due to the right osmotic pressure. (Figure 40, top) Cancer cells treated with METI-101 2.5 mM in RPMI1640 from Welgene underwent apoptosis necrosis in the same manner as in the previous example. (Figure 40, bottom left, first) When METI-101 2.5 mM was treated in a solution of NaH ₂ PO 4 + NaHCO ₃ dissolved in distilled water and a solution of NaH ₂ PO ₄ + NaHCO ₃ dissolved in distilled water with a pH adjusted to 7.4, the same phenomenon as Welgene's RPMI1640 was observed. (Figure 40, bottom left, second and third)

To confirm whether apoptosis was induced and cell death was confirmed, trypan blue staining was performed to measure the number of living cells. The control group treated with PBS and 2.5 mM METI-101 in PBS showed no significant difference in values, and there was a difference between the positive control group and the experimental group. It can be confirmed that apoptosis was induced slightly more when METI-101 2.5 mM was treated in a solution containing NaH ₂ PO ₄ + NaHCO ₃ dissolved in distilled water than in RPMI1640, and it can be seen that most cells were killed when the pH was adjusted (Fig. 41, top). Turbidity, which is one of the characteristics that appears when METI-101 is treated in RPMI1640, also showed the same level of turbidity in a solution containing NaH ₂ PO ₄ + NaHCO ₃ dissolved in distilled water (Fig. 41, bottom).

Claims (21)

a) Calcium lactate and b) A pharmaceutical composition comprising, as an active ingredient, at least one substance selected from the group consisting of a phosphate, a stem cell, a cytokine, a cancer cell line, an antigen derived from the cancer cell line, a polynucleotide encoding the antigen, an antigen gene construct in which the polynucleotide is operably linked to a promoter, and an expression vector comprising the gene construct. In the first paragraph, the cancer cell line-derived antigen is a tumor specific antigen. antigen (TSA) or tumor associated antigen (TAA), The above cytokines are one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21 and GM-CSF, A pharmaceutical composition, characterized in that the above phosphate is monosodium phosphate. A pharmaceutical composition according to claim 1 or 2, characterized in that the composition further comprises a carbonate. A pharmaceutical composition according to claim 1 or 2, characterized in that in the composition, calcium lactate is 2.5 mM to 54 mM and the phosphate is 5 mM or more. a) Calcium lactate and b) A pharmaceutical composition for treating cancer, comprising as an active ingredient at least one substance selected from the group consisting of a phosphate, a stem cell, a cytokine, a cancer cell line, an antigen derived from the cancer cell line, a polynucleotide encoding the antigen, an antigen gene construct in which the polynucleotide is operably linked to a promoter, and an expression vector comprising the gene construct. In the fifth paragraph, the cancer cell line-derived antigen is a tumor specific antigen. antigen (TSA) or tumor associated antigen (TAA), The above cytokines are one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21 and GM-CSF, A pharmaceutical composition for treating cancer, characterized in that the above phosphate is monosodium phosphate. A pharmaceutical composition for treating cancer according to claim 5 or 6, characterized in that the composition further comprises a carbonate. A pharmaceutical composition for treating cancer, characterized in that in claim 5 or 6, the calcium lactate in the composition is 2.5 mM to 54 mM and the phosphate is 5 mM or more. A pharmaceutical composition for treating cancer, characterized in that in claim 5, the cancer is selected from the group consisting of colon cancer, lung cancer, stomach cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel cell carcinoma, prostate cancer, blood cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof. a) Calcium lactate and b) A cancer vaccine composition comprising, as an active ingredient, at least one substance selected from the group consisting of a phosphate, a stem cell, a cytokine, a cancer cell line, an antigen derived from the cancer cell line, a polynucleotide encoding the antigen, an antigen gene construct in which the polynucleotide is operably linked to a promoter, and an expression vector comprising the gene construct. In the 10th paragraph, the cancer cell line-derived antigen is a tumor specific antigen. antigen (TSA) or tumor associated antigen (TAA), The above cytokines are one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21 and GM-CSF, A cancer vaccine composition, characterized in that the above phosphate is monosodium phosphate. A cancer vaccine composition according to claim 10 or 11, characterized in that the composition further comprises a carbonate. A cancer vaccine composition according to claim 10 or 11, characterized in that in the composition, calcium lactate is 2.5 mM to 54 mM and the phosphate is 5 mM or more. A cancer vaccine composition according to claim 10 or 11, characterized in that the composition is administered intratumoral. A cancer vaccine composition in claim 10, characterized in that the cancer is selected from the group consisting of colon cancer, lung cancer, stomach cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel cell carcinoma, prostate cancer, blood cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof. a) Calcium lactate and b) A method for treating or preventing cancer, comprising administering to a subject having a cancer a composition comprising, as an active ingredient, at least one substance selected from the group consisting of a phosphate, a stem cell, a cytokine, a cancer cell line, an antigen derived from the cancer cell line, a polynucleotide encoding the antigen, an antigen gene construct in which the polynucleotide is operably linked to a promoter, and an expression vector comprising the gene construct. In claim 16, the cancer cell line-derived antigen is a tumor specific antigen. antigen (TSA) or tumor associated antigen (TAA), The above cytokines are one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21 and GM-CSF, A method for treating or preventing cancer, characterized in that the above phosphate is monosodium phosphate. A method for treating or preventing cancer, characterized in that the composition further comprises a carbonate in claim 16 or 17. A method for treating or preventing cancer, characterized in that in claim 16 or 17, the composition comprises calcium lactate at 2.5 mM to 54 mM and the phosphate at 5 mM or more. A method for treating or preventing cancer, characterized in that the composition according to claim 16 or 17 is administered intratumoral. A method for treating or preventing cancer, characterized in that in claim 16, the cancer is selected from the group consisting of colon cancer, lung cancer, stomach cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel cell carcinoma, prostate cancer, blood cancer, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof.

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