WO2024242545A1 - Composition de vaccin contre le cancer comprenant du lactate de calcium en tant que principe actif et son utilisation - Google Patents

Composition de vaccin contre le cancer comprenant du lactate de calcium en tant que principe actif et son utilisation Download PDF

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WO2024242545A1
WO2024242545A1 PCT/KR2024/095840 KR2024095840W WO2024242545A1 WO 2024242545 A1 WO2024242545 A1 WO 2024242545A1 KR 2024095840 W KR2024095840 W KR 2024095840W WO 2024242545 A1 WO2024242545 A1 WO 2024242545A1
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cancer
antigen
cells
meti
phosphate
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Korean (ko)
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김환묵
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Metimedi Pharmaceuticals Co Ltd
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Metimedi Pharmaceuticals Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • adjuvants such as alum
  • DAMPs DAMPs
  • adjuvants 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.
  • 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.
  • a STING activator 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.
  • 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.
  • DAMPs damage-associated molecular patterns
  • tumor necrosis factor cytokines including TRAIL, pan-caspase inhibitors including zVAD, and MLKL interfering RNA were developed, their efficacy was limited.
  • necroptosis-inducing agents that selectively act on cancer cells.
  • 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.
  • the present invention comprises: a) calcium lactate and
  • 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.
  • the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA)
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • 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
  • the phosphate is preferably, but not limited to, monosodium phosphate.
  • the composition preferably further comprises a carbonate, but is not limited thereto.
  • 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.
  • 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.
  • 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.
  • the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA)
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • 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
  • the phosphate is preferably, but not limited to, monosodium phosphate.
  • the composition preferably further comprises a carbonate, but is not limited thereto.
  • 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.
  • 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.
  • 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 provides a cancer vaccine composition
  • 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.
  • the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA)
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • 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
  • the phosphate is preferably, but not limited to, monosodium phosphate.
  • the composition preferably further comprises a carbonate, but is not limited thereto.
  • 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.
  • the composition is preferably administered intratumoral, but is not limited thereto.
  • 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.
  • 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 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.
  • 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.
  • the cancer cell line-derived antigen is a tumor specific antigen (TSA) or a tumor associated antigen (TAA)
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • 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
  • the phosphate is preferably, but not limited to, monosodium phosphate.
  • the composition preferably further comprises a carbonate, but is not limited thereto.
  • 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.
  • the composition is preferably administered intratumoral, but is not limited thereto.
  • 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.
  • 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
  • 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.
  • pharmaceutically acceptable 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the present invention may be formulated as sustained release vehicles or depot preparations.
  • 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.
  • compositions of the present invention may be administered by infusion or injection (e.g., intravenously, intramuscularly, intradermally, subcutaneously, intrathecally, intraduodenal, intraperitoneally, etc.).
  • the compositions of the present invention may be administered intranasally, vaginally, rectally, orally, or transdermally.
  • the compositions of the present invention may be administered by a "needle-less" delivery system.
  • 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.
  • the dose of the immunogen is preferably, but not necessarily, equally distributed in each separate injection.
  • 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.
  • compositions of the present invention may be delivered using delayed release systems, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a booster can be administered to support and/or maintain protective immunity.
  • the vaccine composition can be administered to the patient once, twice or more times per month.
  • the vaccine administration schedule may continue for as long as the patient requires, for example over the course of several years, to prolong the patient's life.
  • 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.
  • the vaccine composition is for treating and preventing cancer.
  • 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 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.
  • 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),
  • Fig. 5 is A graph showing the mRNA expression levels of Interferon ⁇ and ⁇ 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 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),
  • Figure 10 shows the inhibition of angiogenesis in HUVEC cells by inhibition of VEGF production.
  • 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.
  • Figure 15 shows the histological analysis of VEGF changes and CD31 in the tumor (**p ⁇ 0.005 vs Control).
  • 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).
  • 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.
  • 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
  • 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
  • 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
  • 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
  • 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.
  • DAMPs immune-induced cytotoxic peptides
  • 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.
  • HCT-116 Human colon cancer
  • AsPC-1 pancreatic cancer
  • MDA-MB-231 mouse colon cancer
  • MC-38, CT-26 mouse colon cancer
  • 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.
  • 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.
  • HCT-116 Human colon cancer
  • AsPC-1 pancreatic cancer
  • H1299 non-small cell lung cancer
  • MDA-MB-231 mouse colon cancer
  • MC-38, CT-26 mouse colon cancer
  • 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 ⁇ l was taken to count the number of living cells using a hemocytometer.
  • Meti-101 treatment induced cell death and no living cells could be found.
  • 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).
  • HCT-116 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.
  • 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).
  • 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.
  • STING Stimulator of Interferon Genes
  • 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).
  • type I interferons IFN- ⁇ and IFN- ⁇ 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.
  • HCT-116 Human colon cancer
  • MDA-MB-231 breast 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- ⁇ and IFN- ⁇ 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).
  • IFNAR1 and IFNAR2 which are Type I interferon receptors produced by tumor cells.
  • WB immunomagnetic spectroscopy
  • HCT-116 Human colon cancer
  • AsPC-1 pancreatic cancer
  • MDA-MB-231 breast cancer
  • HCT-116 Human colon cancer
  • AsPC-1 pancreatic cancer
  • MDA-MB-2311 breast cancer
  • 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.
  • 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).
  • 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.
  • HCT-116 and HT29 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.
  • 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).
  • HCT-116 Human colon cancer
  • AsPC-1 pancreatic cancer
  • MDA-MB-231 breast cancer
  • HCT-116 Human colon cancer
  • AsPC-1 pancreatic cancer
  • MDA-MB-2311 breast cancer
  • 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.
  • 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.
  • 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.
  • human cancer cells treated with METI-101 induce an immune response, which can make the immune system sensitive to cancer cells (Fig. 8).
  • 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.
  • HCT-116 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.
  • 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).
  • the expression level of the angiogenic factor VEGF mRNA was measured using RT-PCR for two types of human colon cancer cells (Fig. 12).
  • 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.
  • Cultrex tube formation assay kit
  • 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- ⁇ . After pretreatment, the cells were treated with 5 ng/ml TGF- ⁇ 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.
  • HIF-1 ⁇ increases in fibroblasts under low oxygen conditions.
  • Meti-101 decreases the expression of HIF-1 ⁇ not only in cancer cells but also in fibroblasts under low oxygen conditions.
  • TGF- ⁇ the phosphorylation of the TGF- ⁇ receptor and the signaling protein Smad2 was confirmed through protein immunoblotting.
  • the phosphorylation of Smad2 was decreased by METI-101, and the TGF- ⁇ receptor on the cell surface was also decreased.
  • ⁇ -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).
  • TGF- ⁇ which suppresses the activity of immune cells attacking cancer cells within the tumor and enhances the activity of immunosuppressive immune cells
  • WB immunomagnetic spectroscopy
  • HCT-116 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- ⁇ -specific antibody was attached to the protein to determine the level of fluorescence, thereby comparing the amount of TGF- ⁇ protein expression induced by Meti-101 in cancer cells.
  • TGF- ⁇ Human cancer cell lines treated with Meti-101 showed a decrease in protein synthesis of TGF- ⁇ .
  • 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.
  • 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.
  • 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.
  • H&E hematoxylin and eosin
  • MSC mesenchymal stem cells
  • MSC mesenchymal stem cells
  • 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).
  • MSC 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).
  • 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.
  • 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).
  • mice were necropsied.
  • the spleen was removed to confirm the white pulp, an immune-related factor in the spleen.
  • 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).
  • TC-1-Luc a mouse-derived lung cancer cell line
  • control group (10 mice)
  • Meti-101 10 mice
  • peptide vaccine 10 mice
  • co-administration of peptide vaccine 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).
  • 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.
  • the control group was evenly distributed from 500 to 2000 mm 3
  • the IL-7 group was evenly distributed from 300 to 2000 mm 3
  • the distribution was from 500 to 1500 mm 3 , but no tumors larger than 1500 mm 3 were observed.
  • 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).
  • 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°C, 5% CO 2 and 1% O 2 and 37°C, 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.
  • 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).
  • 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).
  • 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°C, 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).
  • 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.
  • 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.
  • 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 IFN ⁇ , GM-CSF, IL-1 ⁇ 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.
  • 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.
  • 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.
  • GM-SCF granulocyte-macrophage colony-stimulating factor
  • 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 tumor-associated antigen protein
  • CA9 tumor marker
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • HMGB1 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.
  • 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
  • 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
  • METI-101 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.
  • PRRs pattern-recognition receptors
  • 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)
  • Example 12 Apoptosis and lymphocyte recruitment of METI-101 via intratumoral injection in mice xenografted with human colon cancer cells, HCT-116
  • 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.
  • 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
  • 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
  • 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 .
  • 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)
  • 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
  • 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.
  • 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).

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Abstract

La présente invention concerne une composition pharmaceutique comprenant, en tant que principes actifs : a) du lactate de calcium ; et b) au moins une substance choisie dans le groupe constitué par un phosphate, une cellule souche, une cytokine, une lignée cellulaire cancéreuse, un antigène dérivé de la lignée cellulaire cancéreuse, un polynucléotide codant pour l'antigène, une construction génique d'antigène dans laquelle le polynucléotide est lié fonctionnellement à un promoteur, et un vecteur d'expression comprenant la construction génique. La composition de la présente invention a un effet de mémoire immunologique antitumoral, induit l'apoptose des cellules cancéreuses, et peut donc être utilisée en tant que candidat d'immunothérapie efficace contre des cancers solides, candidat de vaccin pour le traitement in situ de cancers solides, et agent de traitement du cancer administré dans des tumeurs.
PCT/KR2024/095840 2023-05-23 2024-05-22 Composition de vaccin contre le cancer comprenant du lactate de calcium en tant que principe actif et son utilisation Ceased WO2024242545A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
KR10-2023-0066352 2023-05-23
KR20230066352 2023-05-23
US202363516952P 2023-08-01 2023-08-01
US63/516,952 2023-08-01
KR10-2023-0154511 2023-11-09
KR20230154511 2023-11-09

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WO2024242545A1 true WO2024242545A1 (fr) 2024-11-28

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011069587A1 (fr) * 2009-12-09 2011-06-16 Curevac Gmbh Lyophilisation d'acides nucléiques dans des solutions contenant du lactate
KR20160128284A (ko) * 2014-12-29 2016-11-07 가천대학교 산학협력단 락테이트 금속염을 포함하는 암 치료용 약학 조성물
KR20180113976A (ko) * 2015-11-20 2018-10-17 센화 바이오사이언시즈 인코포레이티드 암을 치료하기 위한 테트라사이클릭 퀴놀론 유사체의 병행 요법
WO2021181230A1 (fr) * 2020-03-07 2021-09-16 Arga' Medtech Sa Procédé de réalisation d'une ablation non thermique d'un tissu cible
KR20230004968A (ko) * 2016-11-30 2023-01-06 (주) 메티메디제약 칼슘 락테이트 조성물 및 사용 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011069587A1 (fr) * 2009-12-09 2011-06-16 Curevac Gmbh Lyophilisation d'acides nucléiques dans des solutions contenant du lactate
KR20160128284A (ko) * 2014-12-29 2016-11-07 가천대학교 산학협력단 락테이트 금속염을 포함하는 암 치료용 약학 조성물
KR20180113976A (ko) * 2015-11-20 2018-10-17 센화 바이오사이언시즈 인코포레이티드 암을 치료하기 위한 테트라사이클릭 퀴놀론 유사체의 병행 요법
KR20230004968A (ko) * 2016-11-30 2023-01-06 (주) 메티메디제약 칼슘 락테이트 조성물 및 사용 방법
WO2021181230A1 (fr) * 2020-03-07 2021-09-16 Arga' Medtech Sa Procédé de réalisation d'une ablation non thermique d'un tissu cible

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