WO2019231188A1 - Composition pharmaceutique permettant la prévention ou le traitement du cancer, contenant un inhibiteur de l'expression, ou un inhibiteur de l'activité, de cd300c - Google Patents

Composition pharmaceutique permettant la prévention ou le traitement du cancer, contenant un inhibiteur de l'expression, ou un inhibiteur de l'activité, de cd300c Download PDF

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WO2019231188A1
WO2019231188A1 PCT/KR2019/006307 KR2019006307W WO2019231188A1 WO 2019231188 A1 WO2019231188 A1 WO 2019231188A1 KR 2019006307 W KR2019006307 W KR 2019006307W WO 2019231188 A1 WO2019231188 A1 WO 2019231188A1
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cancer
cd300c
cells
pharmaceutical composition
expression
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PCT/KR2019/006307
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English (en)
Korean (ko)
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전재원
정준구
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주식회사 센트릭스바이오
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Priority claimed from KR1020190061067A external-priority patent/KR102320280B1/ko
Application filed by 주식회사 센트릭스바이오 filed Critical 주식회사 센트릭스바이오
Priority to EP19811930.7A priority Critical patent/EP3808375A4/fr
Priority to CN201980035806.5A priority patent/CN112384241A/zh
Priority to CA3101974A priority patent/CA3101974A1/fr
Priority to AU2019279311A priority patent/AU2019279311B2/en
Priority to US17/059,995 priority patent/US20210238596A1/en
Priority to JP2021517168A priority patent/JP7301264B2/ja
Publication of WO2019231188A1 publication Critical patent/WO2019231188A1/fr
Priority to JP2023034155A priority patent/JP2023071898A/ja

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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 pharmaceutical compositions and the like comprising inhibitors or activity inhibitors of the CD300c protein.
  • Cancer is one of the biggest causes of mortality in modern people. It is a disease caused by changes in normal cells caused by mutations in genes caused by various causes. It does not follow normal cell differentiation, proliferation, and growth patterns. Non-tumor refers to malignant. Cancer is characterized by "uncontrolled cell growth," and this abnormal cell growth forms a mass of cells called tumors that infiltrate surrounding tissues and, in severe cases, metastasize to other organs in the body. . Cancer is a refractory chronic disease that, even if treated with surgery, radiation and drug therapy, in many cases does not heal fundamentally, suffers the patient and ultimately leads to death.
  • Anticancer drugs are generally cytotoxic compounds that treat cancer by attacking and killing cancer cells, and have high side effects because they damage not only cancer cells but also normal cells.
  • targeted anticancer agents have been developed to reduce side effects.
  • the side effects could be lowered, but showed a high probability of developing resistance (Korean Patent Publication No. 10-2018-0099557). Therefore, in recent years, there is a trend of increasing interest in immune anticancer drugs that reduce problems caused by toxicity and resistance by using the body's immune system.
  • an immune gateway inhibitor has been developed that binds PD-L1 on the surface of cancer cells to inhibit T-1 binding to PD-1 to activate T cells and to attack cancer cells.
  • immune check inhibitors even in the case of such immune check inhibitors, there is no need for the development of new immune check inhibitors that have the same therapeutic effect in various cancers.
  • the present inventors have completed the present invention as a result of intensive studies on proteins that are expressed on the surface of cancer cells such as PD-L1 and inhibit T expression in various cancers.
  • the present invention has been made to solve the above-mentioned problems in the prior art, by increasing the activity of T cells by inhibiting the expression or activity of CD300c protein present on the surface of various cancer cells, it is possible to inhibit the proliferation of cancer cells After confirming that it can be, the object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the inhibitor or activity inhibitor of CD300c as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of CD300c expression or an activity inhibitor as an active ingredient.
  • the expression inhibitor of the CD300c is preferably an antisense oligonucleotide (ASO), short hairpin RNA (small hairpin RNA), small interfering RNA that complementarily binds to the mRNA of the CD300c gene (small interfering RNA; siRNA), ribozyme (ribozyme) and the like, or any substance that reduces or inhibits the expression of the CD300c gene is not limited thereto.
  • ASO antisense oligonucleotide
  • small hairpin RNA small hairpin RNA
  • small interfering RNA small interfering RNA
  • siRNA small interfering RNA
  • ribozyme ribozyme
  • the CD300c activity inhibitor is a compound, peptide, peptide mimetics, substrate analogs, aptamers, antibodies, etc. that complementarily bind to the CD300c protein, CD300c
  • Any substance that binds to a protein and reduces or inhibits the activity of CD300c is not limited thereto.
  • the mechanism by which the substance inhibits the activity of the CD300c protein is not particularly limited.
  • the substance may act as a mechanism for converting the active form into an inactive form.
  • An example of the antibody may be a polyclonal antibody, a monoclonal antibody, preferably a human monoclonal anti-CD300c antibody, or an antibody fragment, but any antibody that specifically binds to CD300c.
  • the soluble receptor is a receptor that binds to CD300c, and preferably includes a sequence that specifically binds to the amino acid sequence of SEQ ID NO: 1, but is not limited thereto as long as it is a receptor that binds to CD300c.
  • the cancer is preferably colon cancer, rectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer Prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, blood cancer and the like, or any kind of cancer expressing CD300c protein on the surface of cancer cells is not limited thereto.
  • the pharmaceutical composition may further comprise another existing anticancer agent, wherein the anticancer agent is preferably doxorubicin, cisplatin, gemcitabine, oxaliplatin, 5-FU, cetuximab, Panitumumab, nimotuzumab, necitumumab, cancer antigens, anti-cancer viruses and the like or any substance that is currently used as an anticancer agent is not limited thereto.
  • the anticancer agent is preferably doxorubicin, cisplatin, gemcitabine, oxaliplatin, 5-FU, cetuximab, Panitumumab, nimotuzumab, necitumumab, cancer antigens, anti-cancer viruses and the like or any substance that is currently used as an anticancer agent is not limited thereto.
  • the cancer antigen is a cancer vaccine specific for cancer (cancer vaccine), preferably bladder cancer-specific cancer antigen NY-ESO-1, breast cancer-specific cancer antigen HER2, colorectal cancer-specific cancer antigen CEA, lung cancer specific
  • cancer antigen it may be VEGFR1, VEGFR2, etc., but is not limited to any kind of cancer antigen known as a cancer vaccine.
  • anti-cancer viruses include Imrijik, Pexabeck, and the like, but known anti-cancer viruses are not limited thereto.
  • Further comprising the anticancer agent is preferably in combination, or may be in the form of binding to the inhibitor of the present invention, may also be a form that is included together in the carrier of the anticancer agent.
  • the pharmaceutical composition is characterized by inhibiting the proliferation, survival, metastasis, recurrence, anticancer drug resistance, etc. of cancer or cancer stem cells, but is produced by the pharmaceutical composition of the present invention Effect is not limited to this.
  • the present invention comprises the steps of (a) culturing a cancer cell expressing CD300c protein; (b) treating the cultured cancer cells with a candidate substance; (c) measuring the CD300c expression level of the cells treated with the candidate substance; And (d) provides a method for selecting a substance for the prevention or treatment of cancer, comprising the step of selecting a candidate substance for reducing the expression level of CD300c.
  • the present invention comprises the steps of (a) treating the candidate substance to the CD300c protein; And (b) selecting a candidate substance bound to the CD300c protein, the method for selecting a substance for preventing or treating cancer.
  • the step of measuring the expression level is to measure the expression level of mRNA and / or protein, and measuring the expression level of mRNA confirms the presence and expression level of CD300c mRNA in the biological sample. By measuring the amount of mRNA can be confirmed.
  • RT-PCR competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting ( northern blotting), DNA microarray chips and the like, but are not limited thereto.
  • measuring the expression level of the protein confirms the presence and expression level of the CD300c protein from a biological sample, and confirms the amount of the protein using an antibody specifically binding to the CD300c protein, or the activity of the protein.
  • the step of selecting a substance that binds to the candidate substance is a method of selecting a substance that binds to the CD300c protein.
  • a method of selecting a substance that binds to the CD300c protein As an analysis method for this, Western blotting, ELISA (enzyme) linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement Complete fixation assay, FACS, protein chip, etc., but are not limited to these.
  • the candidate material is not limited to nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, natural extracts, vectors and the like.
  • the present invention provides a method for treating cancer, comprising administering to a subject a pharmaceutical composition comprising an expression inhibitor or an activity inhibitor of CD300c as an active ingredient.
  • the present invention provides a prophylactic or therapeutic use of a pharmaceutical composition comprising an expression inhibitor or an activity inhibitor of the CD300c as an active ingredient.
  • CD300c expression inhibitors or activity inhibitors according to the present invention effectively binds to CD300c expressed on the surface of various cancers, or by inhibiting the expression of CD300c by activating T cells at the same time effectively inhibits the proliferation of cancer cells effectively as an immunotherapy agent of various cancers Can be used. Since such inhibitors can increase the number of intratumorally infiltrating lymphocytes and cytotoxic T lymphocytes in the cancer environment, reduce the number of myeloid derived inhibitory cells, as well as effectively inhibit cancer growth and development, Expression inhibitors or activity inhibitors of CD300c are expected to be effectively used in the treatment of cancer as new immunotherapeutic agents.
  • 1 is a view showing the result of confirming sCD300c-Fc by SDS-PAGE according to an embodiment of the present invention.
  • FIG. 2 is a view showing the results of confirming the effect of sCD300c-Fc on the tumor infiltrating lymphocytes according to an embodiment of the present invention.
  • FIG. 3 is a view showing the results of confirming the effect of sCD300c-Fc on the signal transduction mechanism of NF- ⁇ B according to an embodiment of the present invention.
  • Figure 4 shows the results confirming the effect of the anti-CD300c antibody on human T cells according to an embodiment of the present invention.
  • FIG. 5 is a view showing the results confirming the lung cancer growth inhibitory effect of the anti-CD300c antibody according to an embodiment of the present invention.
  • FIG. 6 is a view showing the results confirming the breast cancer growth inhibitory effect of the anti-CD300c antibody according to an embodiment of the present invention.
  • FIG. 7 is a view showing the results of confirming the effect of inhibiting the growth of colorectal cancer of the anti-CD300c antibody according to an embodiment of the present invention.
  • FIG. 8 is a view showing the results of confirming the cancer growth inhibitory effect of the anti-CD300c antibody according to an embodiment of the present invention in vivo.
  • FIG. 9 is a view showing the results of confirming the cancer growth inhibitory effect of the anti-CD300c antibody according to an embodiment of the present invention in vivo.
  • FIG. 10 is a view showing the results of confirming the cancer growth inhibitory effect of CD300c siRNA according to an embodiment of the present invention.
  • 11 is a view showing the results confirming the effect of the anti-CD300c antibody on the anti-cancer immune response according to an embodiment of the present invention.
  • FIG. 12 is a schematic diagram briefly showing the mechanism of anticancer effect by inhibiting the function and / or expression of CD300c.
  • CD300c expression inhibitor or activity inhibitor of the present invention not only increases the number of intratumoral infiltrating lymphocytes and cytotoxic T lymphocytes in the cancer environment, but also reduces the number of myeloid derived inhibitory cells, and effectively inhibits the growth and development of various cancers. Since it can suppress, it can be effectively used for the treatment of various cancers which express CD300c on the surface.
  • antibody includes immunoglobulin molecules that are immunologically reactive with specific antigens, and include polyclonal antibodies, monoclonal antibodies, and functional fragments thereof.
  • the term may also include forms produced by genetic engineering, such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).
  • Dual monoclonal antibodies are highly specific antibodies directed against a single antigenic site (epitope), unlike polyclonal antibodies that include different antibodies directed against different epitopes, monoclonal antibodies are antigens. Since only a single epitope of the top is indicated, quality control as a therapeutic is easy.
  • the antibody comprises a variable region of the heavy chain and / or light chain of the immunoglobulin molecule, which variable region forms the antigen binding site of the antibody molecule as its primary structure.
  • the antibody of the present invention may be composed of some fragments including the variable region, and preferably, the variable region may be replaced with a soluble receptor for CD300c, but the anti-CD300c antibody of the present invention If it shows the same effect as is not limited thereto.
  • prevention means any action that inhibits or delays the onset of a disease such as cancer by administration of the pharmaceutical composition according to the present invention.
  • treatment means any action that improves or advantageously changes the symptoms of cancer by administration of the pharmaceutical composition according to the present invention.
  • “individual” refers to a subject to which the pharmaceutical composition of the present invention can be administered, and the subject is not limited.
  • the "pharmaceutical composition” may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, wherein the pharmaceutical composition may be characterized as targeting humans.
  • the pharmaceutical composition is not limited to these, but can be used in the form of oral dosage forms, such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.
  • the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as oral administration binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc.
  • buffers, preservatives, analgesic Topical agents, solubilizers, isotonic agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used.
  • the formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carrier as described above.
  • oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in unit dosage ampoules or multiple dosage forms.
  • solutions, suspensions, tablets, capsules, sustained release preparations and the like may be used.
  • suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
  • Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , Sublingual or rectal. Oral or parenteral release is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
  • the pharmaceutical composition of the present invention is dependent on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed.
  • the dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, extent of disease, drug form, route of administration, and duration, and may be appropriately selected by those skilled in the art and may be 0.0001 to 500 mg per day. / kg or 0.001-500 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical compositions according to the invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
  • sCD300c-Fc was prepared in which the Fc portion of the heavy chain region of the antibody was bound to soluble CD300c.
  • a gene encoding the amino acid sequence of SEQ ID NO: 3 (SEQ ID NO: 4) was inserted into pcDNA3.1 and transformed into a HEK293T cell line.
  • transformed cells and polymers that increase the efficiency of intracellular gene transfer were added to RPMI medium supplemented with fetal bovine serum with ultra-low IgG. The cells were cultured in the cell incubator for 4 days.
  • sCD300c-Fc was separated using a centrifuge, and filtered once using a 0.22 ⁇ m filter.
  • sCD300c-Fc was isolated and purified using a recombinant protein-A Sepharose column (GE healthcare), the purified sCD300c-Fc was determined by measuring the absorbance to determine the concentration.
  • 2 ⁇ g of purified sCD300c-Fc was added to Reducing sample buffer and Non-reducing sample buffer, respectively, and then electrophoresed using pre-made SDS-PAGE gel (Invitrogen). The protein was then stained using Coomassie Blue. The results are shown in FIG.
  • sCD300c-Fc tumor infiltrating lymphocytes
  • experiments were performed using sCD300c-Fc prepared in the same manner as in Example 1.
  • EBM medium Lionza
  • FBS fetal bovine serum
  • human vascular endothelial cells Human Umbilical
  • VEC Vein Endothelial Cells
  • PBMC peripheral blood mononuclear cells
  • sCD300c-Fc concentrations of 10, 1, 0.1, 0.01, and 0.001 nM It was.
  • the absorbance at OD 450nm was measured to measure the tumor infiltrating lymphocytes. The results are shown in FIG.
  • sCD300c-Fc prepared in the same manner as in Example 1. More specifically, THP-1 blue cells (monocyte cells, Invivogen) was inoculated to 5,000 cells per well in a 96 well plate and then cultured for 12 hours to stabilize the cells. And sCD300c-Fc, LPS (lipopolysaccharide) and / or IgG was treated in each well and incubated for 48 hours at 37 °C, 5% CO 2 conditions.
  • the control group treated with LPS alone showed a signal of NF- ⁇ B of about 0.4
  • the control group treated with sCD300c-Fc showed a default value of 0.8
  • the experimental group treated with LPS and sCD300c-Fc confirmed that the signal of NF- ⁇ B is significantly increased.
  • sCD300c-Fc h.i.sCD300c-Fc; heat inactivated sCD300c-Fc
  • sCD300c-Fc activates the signal transduction mechanism of NF- ⁇ B, activates THP-1 monocyte, which is an immune cell, and promotes differentiation into macrophage to efficiently activate the innate immune system.
  • a gene of the extracellular domain of CD300c to be used as an antigen (SEQ ID NO: 2) was inserted into a pET28a (Novagen) expression vector to express 6x Histidine and transformed into E. coli.
  • the transformed Escherichia coli was inoculated into 100 mL of LB medium to which 100 mg / mL of ampicillin was added, followed by incubation of absorbance at 0.8-1.0 at OD 600 nm and addition of IPTG. After incubating at 25 ° C. for 16 hours to induce the expression of CD300c having His-tag, the culture was centrifuged to remove supernatant and cells were obtained.
  • the obtained cells were resuspended using a solution containing 50 mM NaH 2 PO 4 and 500 mM NaCl (pH 8.0), and the cells were disrupted by using ultrasonic waves.
  • the cell lysate used for purification of the protein was obtained by centrifugation and filtration.
  • CD300c having His-tag contained in the cell lysate (SEQ ID NO: 5; CD300c-His) was purified by affinity chromatography using a Ni-NTA Sepharose column (GE healthcare), and his-tag through step gradient elution. CD300c with a tag was eluted.
  • CD300c having a purified His-tag was subjected to SDS-PAGE in the same manner as in Example 1, and it was confirmed that CD300c having a His-tag having a purity of 90% or more was purified.
  • Antigen immunization was performed on New Zealand white rabbits using CD300c having a purified His-tag. More specifically, the CD300c antigen with purified His-tag was diluted with phosphate buffered saline (PBS) at a concentration of 0.5 mg / 400 ⁇ L and mixed with the same amount of complete Freund's adjuvant (Sigma) for subcutaneous injection. Primary immunization was carried out by the method. After 2 weeks, 0.5 mg / 400 ⁇ L of antigen and the same amount of incomplete Freund's adjuvant (Sigma) were mixed and subjected to the second immunization in the same manner, followed by the same method every two weeks until the final fourth immunization.
  • PBS phosphate buffered saline
  • the specificity for the CD300c antigen diluted at concentrations of 1 ng, 10 ng, 100 ng, 500 ng, and 1 ⁇ g was diluted 1,000-fold.
  • ELISA and Western blotting were performed using plasma. As a result, it was confirmed that the anti-CD300c antibody specifically reacts with the CD300c antigen.
  • the affinity purification method was used. More specifically, the CD300c antigen and NHS activated sepharose Fast Flow resin (GE Healthcare) were mixed together using a coupling buffer (0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3) to prepare an affinity gel, which was then added to a polypropylene column. packing. After adding the obtained plasma to the column, only the antibody that specifically binds to the antigen was left on the column, and then the elution buffer containing 0.1 M Glycine (pH 2.5) and 0.1 M Citric acid (pH 3.0) was mixed. The antibody was purified using. The purified antibody was dialyzed with phosphate buffer solution, concentrated, divided into 1.0 mg / mL concentrations, and stored at -80 ° C until use.
  • a coupling buffer 0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3
  • T cells total T cells (pan T cells) were first isolated from human peripheral blood mononuclear cells (PBMC) using T cell separation kit (130-096-534, Milltenyi). The isolated T cells were inoculated to 5,000 cells per well in 96 well plates, and then cultured for 6 hours to stabilize the cells, and treated with anti-CD3 antibody (Biolegend) and anti-CD28 antibody (Biolegend). And the anti-CD300c polyclonal antibody purified in the same manner as in Example 4.3 was treated by concentration. After incubation for 48 hours at 37 °C, the culture supernatant was separated to measure the amount of IL-2. The amount of IL-2 was confirmed using the IL-2 Quantikine kit (R & D systems). The results are shown in FIG.
  • the amount of IL-2 was increased according to the treatment concentration.
  • the anti-CD300c antibody was confirmed to activate the T cells, which is an adaptive immune system by increasing the secretion amount of IL-2.
  • the anti-CD300c antibody has an effect of inhibiting the growth of lung cancer, it was primarily confirmed that it has a CD300c antigen on the surface of A549 cells. More specifically, the human lung cancer cell line A549 cells were fixed with 4% formaldehyde and then blocked with 5% Normal goat serum. After 1 ⁇ g of anti-CD300c antibody was treated and reacted, staining was performed using a FITC-labeled anti-rabbit IgG antibody. Fluorescently labeled cells were identified using a flow cytometer (FACS). The results are shown in Figure 5A.
  • the human lung cancer cell line has a CD300c antigen.
  • the CT-26 cell line which is a metastatic colorectal cancer cell line
  • 5X10 5 cells on the right side of the mouse in 8-week-old BALB / c mice.
  • Subcutaneous injections were fed with food and water.
  • Animal breeding and all experimental procedures were conducted in accordance with the rules and regulations for animal testing.
  • anti-CD300c antibodies at concentrations of 0.1, 1, and 10 ⁇ g were administered by intraperitoneal injection on day 0, 1, and 5, and then the tumor size was confirmed.
  • Phosphate buffer solution was injected as a control. The results are shown in FIG.
  • Example 7.1 The same experiment as in Example 7.1 was conducted using a lung cancer animal model. More specifically, 4 to 6 weeks old BALB / c mice were injected subcutaneously with A549 cell line, a human lung cancer cell line, into the right armpit of the mouse to be 5 ⁇ 10 6 cells, and fed with water and food. When the diameter of the tumor reached about 3 to 5 mm, anti-CD300c antibodies at concentrations of 1, 10, and 100 mg / kg were administered by intraperitoneal injection on day 0, 1, and 5, and the tumor size was confirmed. . Phosphate buffer solution was injected as a control. The results are shown in FIG.
  • the anti-CD300c antibody can effectively inhibit the proliferation of various cancers, and that the anti-CD300c antibody can be used as an anticancer agent.
  • CD300c In order to further confirm the effect of CD300c on cancer cell proliferation, it was confirmed whether the inhibition of CD300c expression had anti-cancer effects. More specifically, the expression of CD300c was suppressed in lung cancer cell line A549 cells according to the manual provided using siRNA for CD300c (sc-93646, SantaCruz). Scrambled RNA was used as a control. The siRNAs were transfected into cells using Lipofectamine RNAiMax (Life Technologies) and incubated for 30 hours.
  • A549 cells were inoculated in a 96 well plate to 10,000 cells per well, and then cultured for 18 hours to stabilize. And after treatment with siRNA and incubated for 5 days, the absorbance was measured using CCK-8. The results are shown in Figure 10B.
  • CD300c siRNA can be used to inhibit the expression of CD300c in cells.
  • the treatment of siRNA to the cancer cell line was confirmed that cell proliferation is inhibited.
  • the anti-cancer effect can be exhibited by suppressing the expression of CD300c, the expression of CD300c using siRNA (small interfering RNA), antisense oligonucleotide (ASO, antisenseoligonucleotide), miRNA (micro RNA), etc. It was confirmed that by inhibiting the activity of the CD300c by inhibiting or using the antibody, aptamer (aptamer) that specifically binds to CD300c, it can be confirmed that it can exhibit an anticancer therapeutic effect in various cancers.
  • siRNA small interfering RNA
  • ASO antisense oligonucleotide
  • miRNA micro RNA
  • Example 9 Confirmation of anti-cancer immune effect through inhibition of CD300c function and / or expression
  • mice treated with the anti-CD300c antibody showed increased numbers in the case of lymphocytes and cytotoxic T lymphocytes (CD8 +), and lymphocytes were promoted to induce differentiation (CD4 +).
  • the number of myeloid-derived suppressor cells was confirmed to be reduced.
  • FIG. 12 is a simplified diagram of a mechanism showing anticancer effects by inhibiting the activity and / or expression of CD300c.
  • FIG. 12 is a B7 family (eg, PD-1) that forms a known immune checkpoint.
  • CD300c which is expressed on the surface of the tumor, such as / PD-L1 interaction), binds to the binding cell of the T cell surface to inhibit T cell activation, but specifically binds to CD300c.
  • the anti-CD300c antibody to stimulate the activity and proliferation of T cells to exhibit an anti-cancer effect, while binding to CD300c on the surface of tumor cells, it is confirmed that directly inhibit the growth of the tumor. It is confirmed that the same effect can be obtained by preventing the expression of CD300c on the tumor surface by using an oligonucleotide that inhibits the expression of CD300c.
  • the number of intratumoral infiltrating lymphocytes and cytotoxic T lymphocytes in the cancer environment is increased, and the number of myeloid-derived suppressor cells is decreased, thereby reducing the anticancer immune response in the body.
  • a substance that inhibits the expression and / or function of CD300c may be used to inhibit the proliferation and recurrence of various cancers. It can be confirmed that it can be effectively used to suppress.
  • the present invention relates to a novel use of the CD300c protein present on the surface of various cancer cells, it was confirmed that by inhibiting the expression or activity of the CD300c protein can increase the activity of T cells and inhibit the proliferation of cancer cells. Therefore, the expression inhibitor or activity inhibitor of the CD300c of the present invention is not only applicable to various cancers, but also can significantly increase the prophylactic and / or therapeutic effect of cancer, and therefore, it is expected to be widely applied to various cancer therapeutic agents.

Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement du cancer, contenant un inhibiteur de l'expression, ou un inhibiteur de l'activité, de CD300C ; et similaire. Dans un environnement cancéreux, un inhibiteur de l'expression, ou un inhibiteur de l'activité, de CD300c selon la présente invention augmente le nombre de lymphocytes infiltrant la tumeur et de cellules T cytotoxiques, diminue le nombre de cellules myéloïdes suppressives et peut efficacement inhiber la croissance et le développement du cancer, et il est ainsi attendu qu'il soit utilisable de manière efficace en tant qu'agent immunothérapeutique dans le traitement de divers cancers.
PCT/KR2019/006307 2018-05-31 2019-05-27 Composition pharmaceutique permettant la prévention ou le traitement du cancer, contenant un inhibiteur de l'expression, ou un inhibiteur de l'activité, de cd300c WO2019231188A1 (fr)

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EP19811930.7A EP3808375A4 (fr) 2018-05-31 2019-05-27 Composition pharmaceutique permettant la prévention ou le traitement du cancer, contenant un inhibiteur de l'expression, ou un inhibiteur de l'activité, de cd300c
CN201980035806.5A CN112384241A (zh) 2018-05-31 2019-05-27 包含cd300c表达抑制剂或活性抑制剂的预防或治疗癌症的药物组合物
CA3101974A CA3101974A1 (fr) 2018-05-31 2019-05-27 Composition pharmaceutique permettant la prevention ou le traitement du cancer, contenant un inhibiteur de l'expression, ou un inhibiteur de l'activite, de cd300c
AU2019279311A AU2019279311B2 (en) 2018-05-31 2019-05-27 Pharmaceutical composition for preventing or treating cancer, containing CD300C expression inhibitor or activity inhibitor
US17/059,995 US20210238596A1 (en) 2018-05-31 2019-05-27 Pharmaceutical composition for preventing or treating cancer, containing cd300c expression inhibitor or activity inhibitor
JP2021517168A JP7301264B2 (ja) 2018-05-31 2019-05-27 ＣＤ３００ｃの発現抑制剤または活性抑制剤を含む癌の予防または治療用薬学的組成物
JP2023034155A JP2023071898A (ja) 2018-05-31 2023-03-07 ＣＤ３００ｃの発現抑制剤または活性抑制剤を含む癌の予防または治療用薬学的組成物

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KR20180062620 2018-05-31
KR10-2018-0062620 2018-05-31
KR1020190061067A KR102320280B1 (ko) 2018-05-31 2019-05-24 CD300c의 발현 억제제 또는 활성 억제제를 포함하는 암 예방 또는 치료용 약학적 조성물
KR10-2019-0061067 2019-05-24

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