WO2019231188A1 - Pharmaceutical composition for preventing or treating cancer, containing cd300c expression inhibitor or activity inhibitor - Google Patents
Pharmaceutical composition for preventing or treating cancer, containing cd300c expression inhibitor or activity inhibitor Download PDFInfo
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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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- 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
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- 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
The present invention relates to: a pharmaceutical composition for preventing or treating cancer, containing a CD300C expression inhibitor or activity inhibitor; and the like. A CD300c expression inhibitor or activity inhibitor, according to the present invention, in a cancer environment, increases the number of tumor-infiltrating lymphocytes and cytotoxic T cells, reduces the number of myeloid-derived suppressor cells and can effectively inhibit the growth and development of cancer, and thus is expected to be effectively usable as an immunotherapeutic agent in the treatment of various cancers.
Description
본 발명은 CD300c 단백질의 발현 억제제 또는 활성 억제제를 포함하는 약학적 조성물 등에 관한 것이다. The present invention relates to pharmaceutical compositions and the like comprising inhibitors or activity inhibitors of the CD300c protein.
암은 현대인의 사망원인에서 가장 큰 비중을 차지하고 있는 질환 중 하나로서 여러가지 원인에 의하여 발생된 유전자의 돌연변이로 인하여 정상세포가 변화하여 발생된 질병으로서, 정상적인 세포의 분화, 증식, 성장 형태 등을 따르지 않는 종양 중 악성인 것을 지칭한다. 암이란, “제어되지 않은 세포성장”으로 특징지어지며, 이러한 비정상적인 세포 성장에 의해 종양(tumor)이라고 불리는 세포 덩어리가 형성되어 주위의 조직으로 침투되고, 심한 경우에는 신체의 다른 기관으로 전이되기도 한다. 암은 수술, 방사선 및 약물 요법 등으로 치료를 하더라도 많은 경우에 근본적인 치유가 되지 못하고 환자에게 고통을 주며 궁극적으로는 죽음에 이르게 하는 난치성 만성질환이다.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.
암의 약물 치료, 즉, 항암제는 일반적으로 세포 독성을 가지고 있는 화합물로서 암세포를 공격해 사멸시키는 방식으로 암을 치료하는데, 암세포뿐만 아니라 정상세포에도 손상을 주기 때문에 높은 부작용을 나타낸다. 따라서 부작용을 감소시키기 위하여 표적 항암제들이 개발되었다. 그러나 이러한 표적 항암제들의 경우에는 부작용은 낮출 수 있었으나, 높은 확률로 내성이 생긴다는 한계점을 나타내었다(한국공개특허 10-2018-0099557). 따라서, 최근에는 체내의 면역체계를 이용하여 독성 및 내성으로 인한 문제점을 감소시키는 면역 항암제에 대한 관심이 급증하고 있는 추세이다. 이러한 면역 항암제의 일례로서 암세포 표면의 PD-L1에 결합하여 T 세포의 PD-1과의 결합을 억제하여 T 세포를 활성화시키고, 암세포를 공격하도록 만드는 면역관문억제제가 개발되었다. 그러나 이러한 면역관문억제제의 경우에도 효과를 나타내는 암의 종류가 다양하지 않기 때문에, 다양한 암에서 동일하게 치료 효과를 나타내는 새로운 면역관문억제제의 개발이 필요한 실정이다.Drug treatment of cancer, that is, 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. Thus, targeted anticancer agents have been developed to reduce side effects. However, in the case of these target anticancer drugs, 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. As an example of such an immune anticancer agent, 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. However, 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.
따라서, 본 발명자들은 다양한 암에서 PD-L1과 같이 암 세포의 표면에 발현되어 T 세포의 발현을 억제하는 단백질에 대하여 예의 연구한 결과 본 발명을 완성하였다.Therefore, 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.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로, 다양한 암 세포의 표면에 존재하는 CD300c 단백질의 발현 또는 활성을 억제시킴으로써 T 세포의 활성을 증가시키고, 암 세포의 증식을 억제할 수 있다는 것을 확인하고, CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물 등을 제공하는 것을 그 목적으로 한다.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.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명은 CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.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.
본 발명의 일 구체예에 있어서, 상기 CD300c의 발현 억제제는 바람직하게는 CD300c 유전자의 mRNA에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide; ASO), 짧은 헤어핀 RNA(small hairpin RNA), 작은 간섭 RNA(small interfering RNA; siRNA), 리보자임(ribozyme) 등이나, CD300c 유전자의 발현을 감소 또는 억제하는 물질이라면 이에 제한되지 않는다. 상기 물질이 CD300c의 발현을 억제시키는 기작은 특별히 제한되지 않으며, 일례로 전사, 번역 등의 유전자 발현을 억제하는 기작으로 작용할 수 있다.In one embodiment of the invention, 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. The mechanism by which the substance inhibits the expression of CD300c is not particularly limited. For example, the substance may act as a mechanism of inhibiting gene expression such as transcription and translation.
본 발명의 다른 구체예에 있어서, 상기 CD300c의 활성 억제제는 CD300c 단백질에 상보적으로 결합하는 화합물, 펩티드, 펩티드미메틱스(peptide mimetics), 기질유사체, 압타머(aptamer), 항체 등이나, CD300c 단백질에 결합하여 CD300c의 활성을 감소 또는 억제하는 물질이라면 이에 제한되지 않는다. 상기 물질이 CD300c 단백질의 활성을 억제시키는 기작은 특별히 제한되지 않으며, 일례로 활성형을 비활성형으로 전환시키는 기작으로 작용할 수 있다. 상기 항체의 일례로는 다클론 항체일 수도 있고, 단일클론 항체일 수도 있으며, 바람직하게는 인간 단일클론 항-CD300c 항체일 수 있으며, 또는 항체 단편일 수도 있으나, CD300c에 특이적으로 결합하는 항체라면 이에 제한되지 않는다. 상기 가용성 수용체는 CD300c에 결합하는 수용체로서, 바람직하게는 서열번호 1의 아미노산 서열에 특이적으로 결합하는 서열을 포함하나, CD300c에 결합하는 수용체라면 이에 제한되지 않는다.In another embodiment of the present invention, 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. For example, 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. This is not restrictive. 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.
본 발명의 또 다른 구체예에 있어서, 상기 암은 바람직하게는 대장암, 직장암, 결장암, 갑상선암, 구강암, 인두암, 후두암, 자궁경부암, 뇌암, 폐암, 난소암, 방광암, 신장암, 간암, 췌장암, 전립선암, 피부암, 혀암, 유방암, 자궁암, 위암, 골암, 혈액암 등이나, 암 세포의 표면에 CD300c 단백질을 발현하는 암의 종류라면 이에 제한되지 않는다.In another embodiment of the present invention, 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.
본 발명의 또 다른 구체예에 있어서, 상기 약학적 조성물은 다른 기존의 항암제를 추가로 포함할 수 있으며, 상기 항암제는 바람직하게는 독소루비신, 시스플라틴, 젬시타빈, 옥살리플라틴, 5-FU, 세툭시맙, 파니투무맙, 니모투주맙, 네시투무맙, 암 항원, 항암 바이러스 등이나 현재 항암제로 사용되고 있는 물질이라면 이에 제한되지 않는다. 상기 암 항원은 암종에 특이적인 암 백신(cancer vaccine)으로서, 바람직하게는 방광암 특이적인 암 항원으로서 NY-ESO-1, 유방암 특이적인 암 항원으로서 HER2, 대장암 특이적인 암 항원으로서 CEA, 폐암 특이적인 암 항원으로서 VEGFR1, VEGFR2 등일 수 있으나, 암 백신으로 알려져 있는 암 항원의 종류라면 이에 제한되지 않는다. 항암 바이러스의 일례로 임리직, 펙사벡 등이 있으나, 알려져 있는 항암 바이러스라면 이에 제한되지 않는다. 상기 항암제를 추가로 포함하는 것은 바람직하게는 병용투여이거나, 본원 발명의 억제제에 결합된 형태일 수도 있으며, 항암제의 전달체 내에 함께 포함되어 있는 형태일 수도 있다.In another embodiment of the present invention, 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 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 As the cancer antigen, it may be VEGFR1, VEGFR2, etc., but is not limited to any kind of cancer antigen known as a cancer vaccine. Examples of 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.
본 발명의 또 다른 구체예에 있어서, 상기 약학적 조성물은 암 또는 암 줄기세포의 증식, 생존, 전이, 재발, 항암제 내성 등을 억제하는 것을 특징으로 하나, 본 발명의 약학적 조성물에 의하여 발생되는 효과라면 이에 제한되지 않는다.In another embodiment of the present invention, 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.
또한, 본 발명은 (a) CD300c 단백질이 발현되는 암 세포를 배양하는 단계; (b) 상기 배양된 암 세포에 후보 물질을 처리하는 단계; (c) 상기 후보 물질이 처리된 세포의 CD300c 발현량을 측정하는 단계; 및 (d) 상기 CD300c 발현량을 감소시킨 후보 물질을 선별하는 단계를 포함하는, 암의 예방 또는 치료용 물질을 선별하는 방법을 제공한다.In addition, 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.
또한, 본 발명은 (a) CD300c 단백질에 후보 물질을 처리하는 단계; 및 (b) 상기 CD300c 단백질에 결합된 후보 물질을 선별하는 단계를 포함하는, 암의 예방 또는 치료용 물질을 선별하는 방법을 제공한다.In addition, 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.
본 발명의 일 구체예에 있어서, 상기 발현량을 측정하는 단계란 mRNA 및/또는 단백질의 발현 수준을 측정하는 것으로서, mRNA의 발현 수준을 측정하는 것은 생물학적 시료에서 CD300c mRNA 존재 여부 및 발현 정도를 확인하는 것으로서, mRNA의 양을 측정함으로써 확인할 수 있다. 이를 위한 분석 방법으로는, RT-PCR, 경쟁적 RT-PCR(competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(northern blotting), DNA 마이크로어레이 칩 등이 있으나, 이들로 한정되는 것은 아니다. 또한, 단백질의 발현 수준을 측정하는 것은 생물학적 시료로부터 CD300c 단백질의 존재 여부 및 발현 수준을 확인하는 것으로서, 상기 CD300c 단백질에 대하여 특이적으로 결합하는 항체를 이용해 단백질의 양을 확인하거나, 단백질의 활성을 측정함으로써 알 수 있다. 이를 위한 분석방법으로는 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(radioimmunoassay), 방사면역확산법(radioimmunodiffusion), 오우크레로니(Ouchterlony) 면역확산법, 로케트(Rocket) 면역전기영동, 면역조직화학염색, 면역침전분석(immunoprecipitation assay), 보체고정분석(complete fixation assay), FACS, 단백질 칩(protein chip) 등이 있으나, 이들로 한정되는 것은 아니다.In one embodiment of the present invention, 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. As an analysis method for this, 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. In addition, 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. It can know by measuring. Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunity Electrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto.
본 발명의 다른 구체예에 있어서, 상기 후보 물질에 결합하는 물질을 선별하는 단계는 CD300c 단백질에 결합하는 물질을 선별하는 방법으로서, 이를 위한 분석방법으로는 웨스턴 블랏팅(western blotting), ELISA(enzyme linked immunosorbent assay), 방사선면역분석법(radioimmunoassay), 방사면역확산법(radioimmunodiffusion), 오우크레로니(Ouchterlony) 면역확산법, 로케트(Rocket) 면역전기영동, 면역조직화학염색, 면역침전분석(immunoprecipitation assay), 보체고정분석(complete fixation assay), FACS, 단백질 칩(protein chip) 등이 있으나, 이들로 한정되는 것은 아니다.In another embodiment of the present invention, 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. 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.
본 발명의 또 다른 구체예에 있어서, 상기 후보 물질은 뉴클레오티드, DNA, RNA, 아미노산, 압타머, 단백질, 화합물, 천연물, 천연추출물, 벡터 등으로 제한이 없다.In another embodiment of the present invention, the candidate material is not limited to nucleotides, DNA, RNA, amino acids, aptamers, proteins, compounds, natural products, natural extracts, vectors and the like.
또한 본 발명은 CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는 암 치료방법을 제공한다.In another aspect, 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.
또한 본 CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는 약학적 조성물의 암 예방 또는 치료 용도를 제공한다.In addition, 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.
본 발명에 따른 CD300c의 발현 억제제 또는 활성 억제제는 다양한 암의 표면에서 발현되는 CD300c에 결합하거나, CD300c의 발현을 억제함으로써 T 세포를 활성화시키는 동시에 암 세포의 증식을 억제함으로써 다양한 암의 면역 치료제로서 효과적으로 사용될 수 있다. 이러한 억제제는 암 환경 내에서 종양내침윤림프구 및 세포독성 T 림프구의 수는 증가시키고, 골수유래억제세포의 수는 감소시킬 뿐만 아니라, 암의 성장 및 발달을 효과적으로 억제할 수 있기 때문에, 본 발명의 CD300c의 발현 억제제 또는 활성 억제제는 새로운 면역 치료제로서 암의 치료에 효과적으로 사용될 수 있을 것으로 기대된다.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.
도 1은 본 발명의 일 실시예 따른 sCD300c-Fc를 SDS-PAGE로 확인한 결과를 나타낸 도면이다.1 is a view showing the result of confirming sCD300c-Fc by SDS-PAGE according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 sCD300c-Fc가 종양내침윤림프구에 미치는 영향을 확인한 결과를 나타낸 도면이다.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.
도 3은 본 발명의 일 실시예에 따른 sCD300c-Fc가 NF-κB의 신호 전달 기작에 미치는 영향을 확인한 결과를 나타낸 도면이다.3 is a view showing the results of confirming the effect of sCD300c-Fc on the signal transduction mechanism of NF-κB according to an embodiment of the present invention.
도 4는 본 발명의 일 실시예에 따른 항-CD300c 항체가 인간 T 세포에 미치는 영향을 확인한 결과를 나타낸 도면이다.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.
도 5는 본 발명의 일 실시예에 따른 항-CD300c 항체의 폐암 성장 억제 효과를 확인한 결과를 나타낸 도면이다.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.
도 6은 본 발명의 일 실시예에 따른 항-CD300c 항체의 유방암 성장 억제 효과를 확인한 결과를 나타낸 도면이다.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.
도 7은 본 발명의 일 실시예에 따른 항-CD300c 항체의 대장암 성장 억제 효과를 확인한 결과를 나타낸 도면이다.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.
도 8은 본 발명의 일 실시예에 따른 항-CD300c 항체가 in vivo에서 암 성장 억제 효과를 확인한 결과를 나타낸 도면이다.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.
도 9는 본 발명의 일 실시예에 따른 항-CD300c 항체가 in vivo에서 암 성장 억제 효과를 확인한 결과를 나타낸 도면이다.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.
도 10은 본 발명의 일 실시예에 따른 CD300c siRNA의 암 성장 억제 효과를 확인한 결과를 나타낸 도면이다.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.
도 11은 본 발명의 일 실시예에 따른 항-CD300c 항체가 항암 면역 반응에 미치는 영향을 확인한 결과를 나타낸 도면이다.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.
도 12는 CD300c의 기능 및/또는 발현을 억제함으로써 항암 효과를 나타내는 기작을 간략하게 나타낸 개략도이다.12 is a schematic diagram briefly showing the mechanism of anticancer effect by inhibiting the function and / or expression of CD300c.
본 발명의 CD300c의 발현 억제제 또는 활성 억제제는 암 환경 내에서 종양내침윤림프구 및 세포독성 T 림프구의 수는 증가시키고, 골수유래억제세포의 수는 감소시킬 뿐만 아니라, 다양한 암의 성장 및 발달을 효과적으로 억제할 수 있기 때문에, CD300c를 표면에 발현하고 있는 다양한 암의 치료에 효과적으로 사용될 수 있다.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.
본 명세서에 있어서, "항체(antibody)"란 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하며, 다클론 항체(polyclonal antibody), 단일클론 항체(monoclonal antibody)및 이의 기능적인 단편(fragment)를 모두 포함한다. 또한, 상기 용어는 키메라성 항체(예를 들면, 인간화 뮤린 항체) 및 이종결합항체(예를 들면, 양특이성 항체)와 같은 유전공학에 의해 생산된 형태를 포함할 수 있다. 이중 단일클론항체는 단일 항원성 부위(에피톱, epitope)에 대해서 지시되는 고도의 특이적인 항체로서, 상이한 에피톱들에 대해 지시되는 상이한 항체들을 포함하는 다클론 항체와 다르게, 단일클론 항체는 항원 상의 단일 에피톱에 대해서만 지시되기 때문에, 치료제로서의 품질 관리가 용이하다. 상기 항체는 면역 글로불린 분자를 구성 중 중쇄(heavy chain) 및/또는 경쇄(light chain)의 가변 영역(variable region)을 포함하며, 상기 가변 영역은 그 1차 구조로서 항체 분자의 항원 결합 부위를 형성하는 부분을 포함하며, 본 발명의 항체는 상기 가변 영역을 포함하는 일부 단편으로 구성될 수 있으며, 바람직하게는 상기 가변 영역은 CD300c에 대한 가용성 수용체로 대체될 수 있으나, 본 발명의 항-CD300c 항체와 동일한 효과를 나타낸다면 이에 제한되지 않는다.As used herein, "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.
본 명세서에 있어서,“예방(prevention)”이란 본 발명에 따른 약학적 조성물의 투여에 의해 암 등의 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.In the present specification, "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.
본 명세서에 있어서, "치료(treatment)"란 본 발명에 따른 약학적 조성물의 투여에 의해 암 등의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. In the present specification, "treatment" means any action that improves or advantageously changes the symptoms of cancer by administration of the pharmaceutical composition according to the present invention.
본 명세서에 있어서, “개체(individual)”란 본 발명의 약학적 조성물이 투여될 수 있는 대상을 말하며, 그 대상에는 제한이 없다.In the present specification, "individual" refers to a subject to which the pharmaceutical composition of the present invention can be administered, and the subject is not limited.
본 명세서에 있어서, “약학적 조성물(pharmaceutical composition)”이란 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학적 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. 상기 약학적 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학적 조성물은 약제적으로 허용가능한 담체를 포함할 수 있다. 약제학적으로 허용되는 담체는 경구투여시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 약학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형할 수 있다.In the present specification, 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. Can be. 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. In the case of injections, 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. For example, 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. have. And others, solutions, suspensions, tablets, capsules, sustained release preparations and the like.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Examples of 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. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
본 발명에 따른 약학적 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. 본원에 사용된 용어 "비경구"는 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학적 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.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. As used herein, the term “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.
본 발명의 약학적 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여시간, 투여경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학적 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 500 mg/kg 또는 0.001 내지 500 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 약학적 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.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.
이하, 본 발명의 이해를 돕기 위하여 하기 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, the following examples are presented to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예Example
실시예 1: sCD300c-Fc제작Example 1 Preparation of sCD300c-Fc
암 세포 및 면역계에서 CD300c의 기능을 분석하기 위하여, soluble CD300c에 항체의 중쇄 영역 중 Fc 부분이 결합된 sCD300c-Fc를 제작하였다. sCD300c-Fc 제작을 위하여, 서열번호 3의 아미노산 서열을 코딩하는 유전자(서열번호 4)를 pcDNA3.1에 삽입하고, 이를 HEK293T 세포주에 형질전환시켰다. 그리고 sCD300c-Fc 생산을 위하여, 형질전환된 세포, 및 세포 내 유전자 전달 효율을 증가시키는 폴리머를 IgG 함량이 매우 낮은 우태아혈청(fetal bovine serum with ultra-low IgG)이 첨가된 RPMI 배지에 첨가하여 4 일간 세포 배양기에서 배양하였다. 배양이 완료된 후에는 원심분리기를 이용하여 sCD300c-Fc가 포함된 상층액을 분리하고, 0.22 μm 필터를 이용하여 1 회 여과시켰다. 그리고 재조합 단백질-A 세파로즈 컬럼(GE healthcare)을 이용하여 sCD300c-Fc를 분리 및 정제하였고, 정제된 sCD300c-Fc는 흡광도를 측정하여 농도를 결정하였다. 그리고 정제된 sCD300c-Fc 2 μg을 각각 Reducing sample buffer와 Non-reducing sample buffer에 첨가한 후에, pre-made SDS-PAGE gel(Invitrogen)을 이용하여 전기영동하였다. 이후 쿠마지 블루를 이용하여 단백질을 염색하였다. 그 결과는 도 1에 나타내었다.To analyze the function of CD300c in cancer cells and immune system, sCD300c-Fc was prepared in which the Fc portion of the heavy chain region of the antibody was bound to soluble CD300c. For sCD300c-Fc production, 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. For sCD300c-Fc production, 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. After the incubation was completed, the supernatant containing sCD300c-Fc was separated using a centrifuge, and filtered once using a 0.22 μm filter. And 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. And 2 μg 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.
도 1에 나타난 바와 같이, 순도 90 % 이상의 sCD300c-Fc가 정제되었음을 확인하였다.As shown in FIG. 1, it was confirmed that sCD300c-Fc having a purity of 90% or more was purified.
실시예 2: sCD300c-Fc가 종양내침윤림프구에 미치는 영향 확인Example 2: Confirmation of the effect of sCD300c-Fc on intratumoral infiltrating lymphocytes
sCD300c-Fc가 종양내침윤림프구(tumor infiltrating lymphocytes; TIL)에 미치는 영향을 확인하기 위하여, 실시예 1과 동일한 방법으로 제작된 sCD300c-Fc를 사용하여 실험을 진행하였다. 보다 자세하게는, 6웰-플레이트에 1 %의 우태아혈청(fetal bovine serum; FBS)이 첨가된 EBM 배지(endothelial basal medium, Lonza)를 분주하고 1X10
6 cells/mL의 인간혈관내피세포(Human Umbilical Vein Endothelial Cells; HUVEC, PromoCell)와 1X10
5 cells/mL의 말초혈액단핵구(peripheral blood mononuclear cell; PBMC, CTL)를 접종하고 sCD300c-Fc를 10, 1, 0.1, 0.01, 및 0.001 nM의 농도로 처리하였다. 그리고 37 ℃, 5 % CO
2 조건에서 16 시간 동안 배양한 후에 OD 450nm에서 흡광도를 측정하여 종양내침윤림프구를 측정하였다. 그 결과는 도 2에 나타내었다.In order to confirm the effect of sCD300c-Fc on tumor infiltrating lymphocytes (TIL), experiments were performed using sCD300c-Fc prepared in the same manner as in Example 1. In more detail, EBM medium (Lonza) with 1% fetal bovine serum (FBS) added to 6-well plate was dispensed and human vascular endothelial cells (Human Umbilical) at 1 × 10 6 cells / mL. Inoculate Vein Endothelial Cells (HUVEC, PromoCell) with 1 × 10 5 cells / mL of peripheral blood mononuclear cells (PBMC, CTL) and treat sCD300c-Fc at concentrations of 10, 1, 0.1, 0.01, and 0.001 nM It was. And after incubating for 16 hours at 37 ℃, 5% CO 2 conditions, the absorbance at OD 450nm was measured to measure the tumor infiltrating lymphocytes. The results are shown in FIG.
도 2에 나타난 바와 같이, sCD300c-Fc의 농도가 증가할수록 종양내침윤림프구의 수가 증가되는 것을 확인하였고, 이를 통하여 sCD300c-Fc의 처리를 통하여 말초혈액단핵구가 분화되어 인간혈관내피세포에 침윤되는 림프구의 수가 증가되었으며, 림프구 수는 처리된 sCD300c-Fc의 농도에 의존적으로 증가되는 것을 확인하였다. 상기 결과를 통하여, sCD300c-Fc의 처리를 통하여 면역세포를 활성화시킬 수 있다는 것을 확인할 수 있었다.As shown in Figure 2, as the concentration of sCD300c-Fc increased the number of intratumorally infiltrating lymphocytes, it was confirmed that through the treatment of sCD300c-Fc peripheral blood monocytes are differentiated and lymphocytes infiltrating human vascular endothelial cells The number of cells was increased, and lymphocyte count was confirmed to increase depending on the concentration of treated sCD300c-Fc. Through the above results, it was confirmed that the immune cells can be activated through the treatment of sCD300c-Fc.
실시예 3: sCD300c-Fc가 NF-κB 신호 전달 기작에 미치는 영향 확인Example 3 Identification of sCD300c-Fc on NF-κB Signaling Mechanisms
sCD300c-Fc가 NF-κB의 신호(signaling)에 미치는 영향을 확인하기 위하여, 실시예 1과 동일한 방법으로 제작된 sCD300c-Fc를 사용하여 실험을 진행하였다. 보다 자세하게는, THP-1 blue 세포(monocyte cells, Invivogen)를 96 웰 플레이트에 웰당 5,000 cells가 되도록 접종한 후에 12 시간 동안 배양하여 세포를 안정화시켰다. 그리고 sCD300c-Fc, LPS(lipopolysaccharide) 및/또는 IgG를 각각의 웰에 처리한 후에 37 ℃, 5 % CO
2 조건에서 48 시간 동안 배양하였다. 그리고 배양 상등액을 분리하여 SEAP 발색 시약(Invivogen)을 처리하고 1 시간 동안 반응시킨 후에, 650 nm에서 흡광도를 측정하여 NF-κB의 신호 정도를 측정하였다. 그 결과는 도 3에 나타내었다. In order to confirm the effect of sCD300c-Fc on the signaling of NF-κB, experiments were conducted using 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 ℃, 5% CO 2 conditions. In addition, the culture supernatant was separated, treated with SEAP coloring reagent (Invivogen) and reacted for 1 hour, and then the absorbance was measured at 650 nm to measure the signal level of NF-κB. The results are shown in FIG.
도 3 에 나타난 바와 같이, LPS 만을 처리한 대조군에서는 NF-κB의 신호가 0.4 정도를 나타내며, sCD300c-Fc를 처리한 대조군에서는 0.8 정도의 기본값을 나타내는 것을 확인하였다. 반면 LPS 및 sCD300c-Fc를 함께 처리한 실험군에서는 NF-κB의 신호가 현저히 증가되는 것을 확인하였다. 또한, 가열하여 비활성화시킨 sCD300c-Fc(h.i. sCD300c-Fc; heat inactivated sCD300c-Fc)의 경우에는 NF-κB의 신호에 영향을 미치지 않는 것을 확인하였다. 상기 결과들을 통하여, sCD300c-Fc가 NF-κB의 신호 전달 기작을 활성화시켜, 면역 세포인 THP-1 monocyte가 활성화되고, macrophage로 분화를 촉진시켜 선천성 면역계를 효율적으로 활성화시킬 수 있다는 것을 확인할 수 있었다.As shown in FIG. 3, the control group treated with LPS alone showed a signal of NF-κB of about 0.4, and the control group treated with sCD300c-Fc showed a default value of 0.8. On the other hand, the experimental group treated with LPS and sCD300c-Fc confirmed that the signal of NF-κB is significantly increased. In addition, it was confirmed that sCD300c-Fc (h.i.sCD300c-Fc; heat inactivated sCD300c-Fc) that was inactivated by heating did not affect the signal of NF-κB. Through these results, it was confirmed that sCD300c-Fc activates the signal transduction mechanism of NF-κB, activates THP-1 monocyte, which is an immune cell, and promotes differentiation into macrophage to efficiently activate the innate immune system. .
실시예 4: 항-CD300c 항체 제작Example 4: Anti-CD300c Antibody Construction
4.1. 항-CD300c 폴리클로날 항체 제작4.1. Anti-CD300c Polyclonal Antibody Construction
CD300c에 대한 폴리클로날 항체를 제작하기 위하여, 항원으로 사용할 CD300c의 Extracellular domain의 유전자(서열번호 2)를 6x 히스티딘(Histidine)을 발현하도록 pET28a(Novagen) 발현 벡터에 삽입하고 대장균에 형질전환시켰다. 그리고 형질전환된 대장균을 100 mg/mL의 암피실린이 첨가된 100 mL의 LB 배지에 접종한 후에, 흡광도가 OD
600nm에서 0.8 - 1.0이 되도록 배양하고 IPTG를 첨가하였다. 그리고 25 ℃에서 16 시간 동안 배양하여 His-tag을 가지는 CD300c의 발현을 유도한 후에, 배양액을 원심분리하여 상층액을 제거하고 세포를 획득하였다. 획득된 세포는 50 mM의 NaH
2PO
4 및 500 mM NaCl(pH 8.0)이 혼합되어 있는 용액을 이용하여 재현탁시키고, 초음파를 이용하여 세포를 파쇄하였다. 그리고 원심분리 및 여과 과정을 거쳐서 단백질의 정제를 위해 사용할 세포 용해물을 확보하였다. 세포 용해물 내에 포함되어 있는 His-tag을 가지는 CD300c(서열번호 5; CD300c-His)는 Ni-NTA Sepharose column(GE healthcare)을 이용한 affinity chromatography 방법을 이용하여 정제하였으며, step gradient elution을 통하여 His-tag을 가지는 CD300c를 용출시켰다. 정제된 His-tag을 가지는 CD300c는 실시예 1과 동일한 방법으로 SDS-PAGE를 실시하였으며, 순도 90 % 이상의 His-tag을 가지는 CD300c가 정제되었다는 것을 확인하였다.To prepare a polyclonal antibody against CD300c, 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.
그리고 정제된 His-tag을 가지는 CD300c를 이용하여 뉴질랜드 백토끼에 항원 면역을 실시하였다. 보다 자세하게는, 정제된 His-tag을 가지는 CD300c 항원을 0.5 mg/400 μL의 농도로 인산염완충용액(phosphate buffered saline; PBS)을 이용하여 희석하고 동량의 complete Freund's adjuvant(Sigma)와 혼합하여 피하주사하는 방법으로 1차 면역을 수행하였다. 그리고 2 주 후에 0.5 mg/400 μL의 항원과 동량의 incomplete Freund's adjuvant(Sigma)를 혼합하여 동일한 방법으로 2차 면역을 실시한 후에, 2 주 간격으로 동일한 방법으로 최종 4차 면역까지 실시하였다. 3 차 면역 후에는 꼬리 정맥에서 채혈하여 혈액을 획득하고, 획득된 혈액을 1/1,000으로 희석하여 ELISA를 실시하여 항체의 활성도를 평가하였으며, 최종 4차 면역 후에는 마취 후 토끼의 전혈을 채혈하여 혈장을 획득하였다.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 μL 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 μL 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. After the 3rd immunization, blood was collected from the tail vein, and the obtained blood was diluted to 1 / 1,000, and ELISA was performed to evaluate the activity of the antibody. After the final 4th immunization, the whole blood of the rabbit was collected after anesthesia. Plasma was obtained.
4.2. 항-CD300c 항체의 항체 특이성 확인4.2. Antibody Specificity Identification of Anti-CD300c Antibodies
실시예 4.1과 동일한 방법으로 제작된 항-CD300c 항체의 특이성을 확인하기 위하여, 1 ng, 10 ng, 100 ng, 500 ng, 및 1 μg의 농도로 희석된 CD300c 항원에 대한 특이성을 1,000배로 희석된 혈장을 이용하여 ELISA 및 웨스턴 블롯팅을 실시하였다. 그 결과, 항-CD300c 항체가 CD300c 항원에 대하여 특이적으로 반응하는 것을 확인하였다. In order to confirm the specificity of the anti-CD300c antibody prepared in the same manner as in Example 4.1, the specificity for the CD300c antigen diluted at concentrations of 1 ng, 10 ng, 100 ng, 500 ng, and 1 μg 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.
4.3. 항-CD300c 항체의 정제4.3. Purification of Anti-CD300c Antibodies
실시예 4.1과 동일한 방법으로 획득된 혈장으로부터 항-CD300c 항체를 정제하기 위하여, affinity purification 방법을 이용하였다. 보다 자세하게는, CD300c 항원과 NHS activated sepharose Fast Flow resin(GE Healthcare)을 coupling buffer(0.2 M NaHCO
3, 0.5 M NaCl, pH 8.3)를 이용하여 함께 혼합하여 affinity gel을 제작하고, 이를 폴리프로필렌 컬럼에 packing하였다. 그리고 컬럼에 획득된 혈장을 첨가하여 항원에 특이적으로 결합하는 항체 만을 컬럼에 남긴 후에, 0.1 M Glycine(pH 2.5) 및 0.1 M Citric acid(pH 3.0)가 혼합된 용출완충용액(elution buffer)을 사용하여 항체를 정제하였다. 그리고 정제된 항체는 인산염완충용액을 이용하여 투석시킨 후에 농축하여, 1.0 mg/mL의 농도로 나누어 분주하였고, -80 ℃에서 사용 전까지 보관하였다.In order to purify the anti-CD300c antibody from the plasma obtained in the same manner as in Example 4.1, 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.
실시예 5: 항-CD300c 항체가 T 세포에 미치는 영향 확인Example 5: Identification of Anti-CD300c Antibody on T Cells
항-CD300c 항체가 인간 T 세포에 미치는 영향을 확인하기 위하여 실험을 진행하였다. 보다 자세하게는, 일차적으로 인간의 말초 혈액 단핵세포(peripheral blood mononuclear cell, PBMC)로부터 T 세포 분리 키트(130-096-534, Milltenyi)를 이용하여 전체 T 세포(pan T cells)를 분리하였다. 그리고 분리된 T 세포를 96 웰 플레이트에 웰 당 5,000 cells가 되도록 접종한 후에 6 시간 동안 배양하여 세포를 안정화시키고, 항-CD3 항체(Biolegend) 및 항-CD28 항체(Biolegend)를 처리하였다. 그리고 실시예 4.3과 동일한 방법으로 정제된 항-CD300c 폴리클로날 항체를 농도별로 처리하였다. 그리고 37 ℃에서 48 시간 동안 배양한 후에, 배양 상등액을 분리하여 IL-2의 양을 측정하였다. IL-2의 양은 IL-2 Quantikine 키트(R&D systems)를 이용하여 확인하였다. 그 결과는 도 4에 나타내었다.Experiments were conducted to determine the effect of anti-CD300c antibody on human T cells. More specifically, 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 ℃, 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.
도 4에 나타난 바와 같이, 항-CD300c 항체를 처리한 실험군의 경우에는 처리 농도에 따라 IL-2의 양이 증가되는 것을 확인하였다. 이를 통하여, 항-CD300c 항체의 경우에는 IL-2의 분비량을 증가시켜 적응성 면역계인 T 세포를 활성화시키는 것을 확인할 수 있었다.As shown in FIG. 4, in the experimental group treated with the anti-CD300c antibody, the amount of IL-2 was increased according to the treatment concentration. Through this, in the case of 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.
실시예 6: 항-CD300c 항체의 in vitro에서의 항암 효과 확인Example 6: Confirmation of anti-cancer effect of anti-CD300c antibody in vitro
6.1. 항-CD300c 항체의 폐암 성장 억제 효과 확인6.1. Anti-CD300c Antibody Confirms Lung Cancer Growth Inhibition
항-CD300c 항체가 폐암의 성장을 억제하는 효과가 있는지 확인하기 위하여, 일차적으로 A549 세포 표면에 CD300c 항원을 가지고 있는지 확인하였다. 보다 자세하게는, 인간 폐암 세포주인 A549 세포를 4 % 포름알데히드로 고정시킨 후에, 5 % Normal goat serum을 이용하여 블록킹을 하였다. 그리고 1 μg의 항-CD300c 항체를 처리하고 반응시킨 후에, FITC-labeled 항-rabbit IgG 항체를 이용하여 염색하였다. 그리고 형광표지된 세포를 유세포분석기(FACS)를 이용하여 확인하였다. 그 결과는 도 5A에 나타내었다.In order to determine whether 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 μg 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.
도 5A에 나타난 바와 같이, 인간 폐암 세포주에는 CD300c 항원을 가지고 있다는 것을 확인하였다.As shown in Figure 5A, it was confirmed that the human lung cancer cell line has a CD300c antigen.
또한, 폐암의 성장을 억제하는지 확인하기 위하여, 암세포의 증식 억제 효과를 확인하였다. 보다 자세하게는, 96 웰 플레이트에 A549 세포를 웰 당 10,000 cells가 되도록 접종한 후에 18 시간 동안 배양하여 안정화시켰다. 그리고 0.1, 1, 및 10 μg/mL의 항-CD300c 항체를 처리하고, 72 시간 동안 배양하였다. 그리고 현미경을 이용하여 사진을 촬영하였다. 그 결과는 도 5B에 나타내었다. 또한, 각각의 웰에 CCK-8(Dijindo)을 10 μL씩 첨가하고 4 시간 동안 반응시킨 후에, 450 nm에서 흡광도를 측정하였다. 그 결과는 도 5C에 나타내었다.In addition, in order to confirm whether the growth of lung cancer is inhibited, the effect of inhibiting the proliferation of cancer cells was confirmed. In more detail, A549 cells were inoculated in a 96 well plate to 10,000 cells per well, and then stabilized by incubating for 18 hours. And 0.1, 1, and 10 μg / mL of anti-CD300c antibody were treated and incubated for 72 hours. And a picture was taken using a microscope. The results are shown in Figure 5B. In addition, after adding 10 μL of CCK-8 (Dijindo) to each well and reacting for 4 hours, absorbance was measured at 450 nm. The results are shown in Figure 5C.
도 5B 및 5C에 나타난 바와 같이, 항-CD300c 항체의 처리 농도가 증가될수록 폐암 세포의 증식이 억제되는 것을 확인하였으며, 이를 통하여, 항-CD300c 항체가 폐암의 성장을 억제한다는 것을 확인할 수 있었다.As shown in Figures 5B and 5C, it was confirmed that as the treatment concentration of the anti-CD300c antibody was increased, the proliferation of lung cancer cells was inhibited, through which the anti-CD300c antibody inhibited the growth of lung cancer.
6.2. 항-CD300c 항체의 유방암 성장 억제 효과 확인6.2. Anti-CD300c Antibody Confirms Breast Cancer Growth Inhibition
항-CD300c 항체가 유방암의 성장을 억제하는 효과가 있는지 확인하기 위하여, 실시예 6.1과 동일한 방법으로 유방암 세포주인 MDA-MB-231 세포를 사용하여 세포 증식 억제 효과를 확인하였다. 그 결과는 도 6에 나타내었다.In order to determine whether the anti-CD300c antibody has an effect of inhibiting the growth of breast cancer, the effect of inhibiting cell proliferation was confirmed using MDA-MB-231 cells, which are breast cancer cell lines, in the same manner as in Example 6.1. The results are shown in FIG.
도 6에 나타난 바와 같이, 항-CD300c 항체의 처리 농도가 증가될수록 유방암 세포의 증식이 억제되는 것을 확인하였으며, 이를 통하여, 항-CD300c 항체가 유방암의 성장을 억제한다는 것을 확인할 수 있었다.As shown in FIG. 6, it was confirmed that as the treatment concentration of the anti-CD300c antibody was increased, the proliferation of breast cancer cells was inhibited. Through this, it was confirmed that the anti-CD300c antibody inhibited the growth of breast cancer.
6.3. 항-CD300c 항체의 대장암 성장 억제 효과 확인6.3. Anti-CD300c Antibody Confirmation of Colorectal Cancer Growth Inhibition
항-CD300c 항체가 대장암의 성장을 억제하는 효과가 있는지 확인하기 위하여, 실시예 6.1과 동일한 방법으로 전이성 대장암 세포주인 CT-26 세포를 사용하여 세포 증식 억제 효과를 확인하였다. 그 결과는 도 7에 나타내었다.In order to determine whether the anti-CD300c antibody has an effect of inhibiting the growth of colorectal cancer, the effect of inhibiting cell proliferation was confirmed using CT-26 cells, which are metastatic colorectal cancer cell lines, in the same manner as in Example 6.1. The results are shown in FIG.
도 7에 나타난 바와 같이, 항-CD300c 항체의 처리 농도가 증가될수록 대장암 세포의 증식이 억제되는 것을 확인하였으며, 이를 통하여, 항-CD300c 항체가 대장암의 성장을 억제한다는 것을 확인할 수 있었다.As shown in FIG. 7, it was confirmed that as the treatment concentration of the anti-CD300c antibody was increased, the proliferation of colorectal cancer cells was suppressed. Through this, it was confirmed that the anti-CD300c antibody inhibited the growth of colorectal cancer.
실시예 7: 항-CD300c 항체의 Example 7: Anti-CD300c Antibodies
in vivoin vivo
에서의 항암 효과 확인Anticancer effects
7.1. 항-CD300c 항체의 대장암 성장 억제 효과 확인7.1. Anti-CD300c Antibody Confirmation of Colorectal Cancer Growth Inhibition
항-CD300c 항체가
in vivo에서도 암 세포의 성장을 억제하는 효과가 있는지 확인하기 위하여, 8 주령의 BALB/c 마우스에 전이성 대장암 세포주인 CT-26 세포주를 5X10
5 cells가 되도록 마우스의 오른쪽 측면에 피하 주사하고 먹이와 물을 주며 사육하였다. 동물 사육 및 모든 실험 절차는 동물 실험에 대한 법칙 및 규제에 의거하여 진행하였다. 그리고 종양의 크기가 약 70 mm
3에 도달하였을 때 0.1, 1, 및 10 μg 농도의 항-CD300c 항체를 0, 1, 및 5 일차에 복강주사로 투여한 후에 종양의 크기를 확인하였다. 대조군으로는 인산염완충용액을 주사하였다. 그 결과는 도 8에 나타내었다.To determine whether anti-CD300c antibody is effective in inhibiting cancer cell growth in vivo , the CT-26 cell line, which is a metastatic colorectal cancer cell line, is 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. When the tumor size reached about 70 mm 3 , anti-CD300c antibodies at concentrations of 0.1, 1, and 10 μg 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.
도 8에 나타난 바와 같이, 항-CD300c 항체의 농도가 증가될수록 전이성 대장암의 크기의 증가가 감소될 뿐만 아니라, 10 μg을 처리한 실험군에서는 15 일까지 종양의 성장이 완전히 억제되어 더 이상 종양이 성장되지 않는 것을 확인하였다.As shown in FIG. 8, as the concentration of the anti-CD300c antibody was increased, not only the increase in the size of metastatic colorectal cancer decreased, but in the experimental group treated with 10 μg, the growth of tumor was completely inhibited until 15 days so that the tumor was no longer present. It was confirmed that it did not grow.
7.2. 항-CD300c 항체의 폐암 성장 억제 효과 확인7.2. Anti-CD300c Antibody Confirms Lung Cancer Growth Inhibition
폐암 동물 모델을 이용하여 실시예 7.1과 동일한 실험을 진행하였다. 보다 자세하게는, 4 내지 6 주령의 BALB/c 마우스에 인간 폐암세포주인 A549 세포주를 5X10
6 cells가 되도록 마우스의 오른쪽 겨드랑이에 피하 주사하고 먹이와 물을 주며 사육하였다. 그리고 종양의 직경이 약 3 내지 5 mm에 도달하였을 때 1, 10, 및 100 mg/kg 농도의 항-CD300c 항체를 0, 1, 및 5 일차에 복강주사로 투여한 후에 종양의 크기를 확인하였다. 대조군으로는 인산염완충용액을 주사하였다. 그 결과는 도 9에 나타내었다. 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.
도 9에 나타난 바와 같이, 항-CD300c 항체의 농도가 증가될수록 폐암의 증식이 억제되어 종양이 성장되지 않는 것을 확인하였다. 상기 결과를 통하여, 본 발명의 항-CD300c 항체가
in vivo에서도 암의 성장을 효과적으로 억제한다는 것을 확인할 수 있었다.As shown in Figure 9, as the concentration of the anti-CD300c antibody increased it was confirmed that the proliferation of lung cancer is suppressed tumor growth. Through the above results, wherein -CD300c antibodies of the invention have confirmed that inhibit the growth of cancer in in vivo efficiently.
상기 결과들을 통하여, 항-CD300c 항체는 다양한 암의 증식을 효과적으로 억제할 수 있으며, 항-CD300c 항체를 항암제로 사용가능하다는 것을 확인할 수 있었다.Through the above results, it was confirmed that 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.
실시예 8: CD300c siRNA의 항암 효과 확인Example 8: Confirmation of anticancer effect of CD300c siRNA
CD300c가 암 세포 증식에 미치는 영향을 추가적으로 확인하기 위하여, CD300c의 발현 억제가 항암 효과를 나타내는지 확인하였다. 보다 자세하게는, CD300c에 대한 siRNA(sc-93646, SantaCruz)를 이용하여 제공되는 매뉴얼에 따라 폐암 세포주인 A549 세포에서 CD300c의 발현을 억제하였다. 대조군으로는 Scrambled RNA를 사용하였다. siRNA를 Lipofectamine RNAiMax(Life Technologies)를 이용하여 세포에 형질감염시킨 후에 30 시간을 배양하였다. 그리고 배양된 세포에 세포 용해 용액(20 mM의 Tris-HCl(pH 7.5), 150 mM의 NaCl, 1mM의 Na
2EDTA, 1 mM의 EGTA, 1 %의 Triton, 2.5 mM의 sodium pyrophosphate, 1 mM의 glycerophosphate, 1 mM의 Na
3VO
4, 1 μg/mL의 leupeptin, 및 1 mM의 PMSF)을 처리하여 세포 용해물을 회수하였다. 그리고 용해물 내에 포함되어 있는 단백질의 양을 BCA 방법을 이용하여 정량하였다. 그리고 동량의 CD300c에 특이적으로 결합하는 항체(PA5-87097, Thermofisher)를 이용하여 웨스턴 블롯팅을 실시하였다. 그 결과는 도 10A에 나타내었다. 그리고 암 세포 증식 억제 효과를 확인하기 위하여, 96 웰 플레이트에 A549 세포를 웰 당 10,000 cells가 되도록 접종한 후에 18 시간 동안 배양하여 안정화시켰다. 그리고 siRNA를 처리한 후에 5 일 동안 배양하며 CCK-8을 이용하여 흡광도를 측정하였다. 그 결과는 도 10B에 나타내었다.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. And cultured cells with cell lysis solution (20 mM Tris-HCl, pH 7.5), 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM Cell lysates were recovered by treatment with glycerophosphate, 1 mM Na 3 VO 4 , 1 μg / mL leupeptin, and 1 mM PMSF). And the amount of protein contained in the lysate was quantified using the BCA method. And Western blotting was performed using an antibody (PA5-87097, Thermofisher) that specifically binds the same amount of CD300c. The results are shown in Figure 10A. In order to confirm the cancer cell proliferation inhibitory effect, 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.
도 10A에 나타난 바와 같이, CD300c siRNA를 이용하여 세포에서 CD300c의 발현을 억제할 수 있다는 것을 확인하였다. 또한, 도 10B에 나타난 바와 같이, 암 세포주에 siRNA를 처리하면 세포 증식이 억제되는 것을 확인하였다.As shown in FIG. 10A, it was confirmed that CD300c siRNA can be used to inhibit the expression of CD300c in cells. In addition, as shown in Figure 10B, the treatment of siRNA to the cancer cell line was confirmed that cell proliferation is inhibited.
상기 결과들을 통하여, CD300c의 발현을 억제시킴으로써 항암 효과를 나타낼 수 있다는 것을 확인할 수 있었으며, siRNA(small interfering RNA), 안티센스 올리고뉴클레오티드(ASO, antisenseoligonucleotide), miRNA(micro RNA) 등을 이용하여 CD300c의 발현을 억제하거나, CD300c에 특이적으로 결합하는 항체, 압타머(aptamer) 등을 이용하여 CD300c의 활성을 억제함으로써 다양한 암에서 항암 치료 효과를 나타낼 수 있다는 것을 확인할 수 있었다.Through the above results, it was confirmed that 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.
실시예 9: CD300c 기능 및/또는 발현 억제를 통한 항암 면역 효과 확인Example 9: Confirmation of anti-cancer immune effect through inhibition of CD300c function and / or expression
CD300c의 기능 및/또는 발현을 억제함으로써 항암 면역 효과를 나타내는지 확인하기 위하여, 실시예 7.1과 동일한 방법으로 대장암 세포주가 이식된 BALB/c 마우스에 10 μg 농도의 항-CD300c 항체를 0, 1, 및 5 일차에 복강주사로 투여하고, 7 일 차에 안락사시킨 후에 마우스의 골수를 분리하였다. 그리고 분리된 골수로부터 골수유래억제세포(Myeloid- derived suppressor cells; MDSC), 림프구(lymphocyte), 및 세포독성T림프구(cytotoxic T lymphocyte)를 유세포분석기(FACS)를 이용하여 확인하였다. 그 결과는 도 11에 나타내었다.In order to confirm the anti-cancer immune effect by inhibiting the function and / or expression of CD300c, 10 μg of anti-CD300c antibody was injected into BALB / c mice transplanted with colon cancer cell lines in the same manner as in Example 7.1. , And 5th day by intraperitoneal injection and 7th day after euthanasia, the bone marrow of the mouse was isolated. And bone marrow-derived suppressor cells (MDSC), lymphocytes (lymphocytes), and cytotoxic T lymphocytes (cytotoxic T lymphocytes) were identified from the isolated bone marrow using a flow cytometer (FACS). The results are shown in FIG.
도 11에 나타난 바와 같이, 항-CD300c 항체를 처리한 마우스의 경우 림프구 및 세포독성 T 림프구(CD8+)의 경우에는 수가 증가되고, 림프구는 분화 유도(CD4+)가 촉진된 것을 확인하였다. 반면, 골수유래억제세포의 수는 감소된 것을 확인하였다. 상기 결과들을 통하여, CD300c의 기능 및/또는 발현을 억제함으로써 암 동물 모델의 혈액 내에서 면역계의 활성화가 유도되며, T 면역세포의 활성 또는 증식을 억제시키는 골수유래억제세포의 수는 감소시킴으로써, 항암 면역 효과를 현저히 증가시켜, 암 치료 효과를 나타낸다는 것을 확인할 수 있었다.As shown in FIG. 11, the 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 +). On the other hand, the number of myeloid-derived suppressor cells was confirmed to be reduced. These results suggest that by inhibiting the function and / or expression of CD300c, the activation of the immune system in the blood of a cancer animal model is induced, and by reducing the number of myeloid derived inhibitory cells that inhibit the activity or proliferation of T immune cells, It was confirmed that the immune effect was remarkably increased to show a cancer treatment effect.
도 12는 CD300c의 활성 및/또는 발현을 억제함으로써 항암 효과를 나타내는 기작에 대하여 간략하게 나타낸 도면으로서, 기존에 알려져 있는 면역관문(immune checkpoint)을 형성하고 있는 B7 패밀리(예를 들어, PD-1/PD-L1 상호작용 등)와 같이 종양의 표면에 발현되는 CD300c는 T 세포 표면의 상호 결합 분자(Binding Partner)와 결합하여 T 세포의 활성화를 억제하는 작용을 하나, CD300c에 대하여 특이적으로 결합하는 항-CD300c 항체를 처리함으로써 T 세포의 활성 및 증식을 자극하여 항암 효과를 나타냄과 동시에 종양 세포 표면의 CD300c에 결합하여 직접적으로 종양의 증식을 억제하는 것으로 확인된다. 이는 CD300c의 발현을 억제하는 올리고뉴클레오티드를 이용하여, 종양 표면의 CD300c가 발현되지 못하도록 하여도 동일한 효과를 나타내는 것으로 확인된다. 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. By treating 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.
따라서 상기 결과들을 통하여, CD300c의 발현 및/또는 기능을 억제함으로써, 암 환경 내에서 종양내침윤림프구 및 세포독성 T 림프구의 수는 증가시키고, 골수유래억제세포의 수는 감소시켜 체내의 항암 면역 반응은 효과적으로 활성화시킬 뿐만 아니라, 세포의 증식을 억제하여 암의 성장 및 발달을 효과적으로 억제할 수 있다는 것을 확인하였기 때문에, CD300c의 발현 및/또는 기능을 억제하는 물질을 다양한 암의 증식, 재발 전이 등을 억제하는데 효과적으로 사용가능하다는 것을 확인할 수 있었다.Thus, through the above results, by inhibiting the expression and / or function 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. Not only effectively activates the cells, but also inhibits the growth and development of cancer by inhibiting the proliferation of cells. Thus, 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.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The description of the present invention set forth above is for illustrative purposes, and it will be understood by those skilled in the art that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명은 다양한 암 세포의 표면에 존재하는 CD300c 단백질의 신규한 용도에 관한 것으로, CD300c 단백질의 발현 또는 활성을 억제시킴으로써 T 세포의 활성을 증가시키고, 암 세포의 증식을 억제할 수 있다는 것을 확인하였기 때문에, 본 발명의 CD300c의 발현 억제제 또는 활성 억제제는 다양한 암에 적용 가능할 뿐만 아니라, 암의 예방 및/또는 치료 효과를 현저히 증가시킬 수 있으므로, 다양한 암 치료제에 적용되어 폭넓게 사용가능할 것으로 기대된다. 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.
Claims (10)
- CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는, 암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, comprising CD300c expression inhibitor or activity inhibitor as an active ingredient.
- 제 1 항에 있어서,The method of claim 1,상기 CD300c의 발현 억제제는 CD300c 유전자의 mRNA에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide; ASO), 짧은 헤어핀 RNA(small hairpin RNA) 작은 간섭 RNA(small interfering RNA; siRNA), 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 약학적 조성물.The expression inhibitors of CD300c include antisense oligonucleotides (ASOs), small hairpin RNAs, small interfering RNAs (siRNAs), and ribozymes that complementarily bind to mRNA of the CD300c gene. A pharmaceutical composition, characterized in that at least one selected from the group consisting of.
- 제 1 항에 있어서,The method of claim 1,상기 CD300c의 활성 억제제는 CD300c 단백질에 상보적으로 결합하는 화합물, 펩티드, 펩티드미메틱스(peptide mimetics), 기질유사체, 압타머(aptamer), 및 항체로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 약학적 조성물.The activity inhibitor of the CD300c is any one or more selected from the group consisting of compounds, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that complementarily bind to the CD300c protein , Pharmaceutical compositions.
- 제 1 항에 있어서,The method of claim 1,상기 암은 대장암, 직장암, 결장암, 갑상선암, 구강암, 인두암, 후두암, 자궁경부암, 뇌암, 폐암, 난소암, 방광암, 신장암, 간암, 췌장암, 전립선암, 피부암, 혀암, 유방암, 자궁암, 위암, 골암 및 혈액암으로 이루어진 군으로부터 선택된 어느 하나 이상인 것을 특징으로 하는, 약학적 조성물.The cancer is 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 and hematological cancer, characterized in that any one or more selected from the group consisting of, pharmaceutical composition.
- 제 1 항에 있어서,The method of claim 1,상기 약학적 조성물은 다른 항암제를 추가로 포함하는 것을 특징으로 하는, 약학적 조성물.The pharmaceutical composition is characterized in that it further comprises another anticancer agent, pharmaceutical composition.
- 제 1 항에 있어서,The method of claim 1,상기 약학적 조성물은 암의 증식, 생존, 전이, 재발 또는 항암제 내성을 억제하는 것을 특징으로 하는, 약학적 조성물.The pharmaceutical composition is characterized by inhibiting the proliferation, survival, metastasis, recurrence or anticancer drug resistance of cancer.
- (a) CD300c 단백질이 발현되는 암 세포를 배양하는 단계;(a) culturing cancer cells in which the CD300c protein is expressed;(b) 상기 배양된 암 세포에 후보 물질을 처리하는 단계;(b) treating the cultured cancer cells with a candidate substance;(c) 상기 후보 물질이 처리된 세포의 CD300c 발현량을 측정하는 단계; 및(c) measuring the CD300c expression level of the cells treated with the candidate substance; And(d) 상기 CD300c 발현량을 감소시킨 후보 물질을 선별하는 단계를 포함하는, 암의 예방 또는 치료용 물질을 선별하는 방법.(d) selecting a candidate substance for which the CD300c expression is reduced, the method for selecting a substance for preventing or treating cancer.
- (a) CD300c 단백질에 후보 물질을 처리하는 단계; 및(a) treating the candidate material with the CD300c protein; And(b) 상기 CD300c 단백질에 결합된 후보 물질을 선별하는 단계를 포함하는, 암의 예방 또는 치료용 물질을 선별하는 방법.(b) selecting a candidate substance bound to the CD300c protein, the method for selecting a substance for preventing or treating cancer.
- CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는 조성물을 개체에 투여하는 단계를 포함하는, 암의 치료방법.A method of treating cancer, comprising administering to a subject a composition comprising an expression inhibitor or an activity inhibitor of CD300c as an active ingredient.
- CD300c의 발현 억제제 또는 활성 억제제를 유효성분으로 포함하는 조성물의 암 예방 또는 치료 용도.Cancer prevention or treatment of the composition comprising a CD300c expression inhibitor or an activity inhibitor as an active ingredient.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2019279311A AU2019279311B2 (en) | 2018-05-31 | 2019-05-27 | Pharmaceutical composition for preventing or treating cancer, containing CD300C expression inhibitor or activity inhibitor |
| EP19811930.7A EP3808375A4 (en) | 2018-05-31 | 2019-05-27 | Pharmaceutical composition for preventing or treating cancer, containing cd300c expression inhibitor or activity inhibitor |
| JP2021517168A JP7301264B2 (en) | 2018-05-31 | 2019-05-27 | Pharmaceutical composition for prevention or treatment of cancer containing a CD300c expression inhibitor or activity inhibitor |
| CN201980035806.5A CN112384241A (en) | 2018-05-31 | 2019-05-27 | Pharmaceutical composition for preventing or treating cancer comprising inhibitor of expression or activity of CD300C |
| 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 |
| CA3101974A CA3101974A1 (en) | 2018-05-31 | 2019-05-27 | Pharmaceutical composition for preventing or treating cancer, containing cd300c expression inhibitor or activity inhibitor |
| JP2023034155A JP2023071898A (en) | 2018-05-31 | 2023-03-07 | PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF CANCER CONTAINING CD300c EXPRESSION INHIBITOR OR ACTIVITY INHIBITOR |
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| KR10-2018-0062620 | 2018-05-31 | ||
| KR20180062620 | 2018-05-31 | ||
| KR10-2019-0061067 | 2019-05-24 | ||
| KR1020190061067A KR102320280B1 (en) | 2018-05-31 | 2019-05-24 | Pharmaceutical composition comprising inhibitors of CD300c's expression or activation for preventing or treating of cancer |
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| WO2019231188A1 true WO2019231188A1 (en) | 2019-12-05 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160194631A1 (en) * | 2009-09-30 | 2016-07-07 | Harvard Medical School | Methods for Modulation of Autophagy Through the Modulation of Autophagy-Inhibiting Gene Products |
| WO2017069958A2 (en) * | 2015-10-09 | 2017-04-27 | The Brigham And Women's Hospital, Inc. | Modulation of novel immune checkpoint targets |
| KR20180099557A (en) | 2017-02-28 | 2018-09-05 | 한양대학교 산학협력단 | Anti-tumor composition comprising oncologic adenovirus and immune checkpoint inhibitor |
-
2019
- 2019-05-27 WO PCT/KR2019/006307 patent/WO2019231188A1/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160194631A1 (en) * | 2009-09-30 | 2016-07-07 | Harvard Medical School | Methods for Modulation of Autophagy Through the Modulation of Autophagy-Inhibiting Gene Products |
| WO2017069958A2 (en) * | 2015-10-09 | 2017-04-27 | The Brigham And Women's Hospital, Inc. | Modulation of novel immune checkpoint targets |
| KR20180099557A (en) | 2017-02-28 | 2018-09-05 | 한양대학교 산학협력단 | Anti-tumor composition comprising oncologic adenovirus and immune checkpoint inhibitor |
Non-Patent Citations (4)
| Title |
|---|
| BORREGO, F.: "The CD300 molecules: an emerging family of regulators of the immune system", BLOOD, 14 March 2013 (2013-03-14), pages 1951 - 1960, XP002794483 * |
| CUI, C.: "A CD300c-Fc fusion protein inhibits T cell immunity", FRONTIERS IN IMMUNOLOGY, 15 November 2018 (2018-11-15), XP055660033 * |
| DIMITROVA, M.: "CD300c is uniquely expressed on CD56 bright Natural Killer Cells and differs from CD300a upon ligand recognition", SCIENTIFIC REPORTS, 4 April 2016 (2016-04-04), pages 1 - 12, XP055660025 * |
| SIMHADRI, V. R.: "CD300c is an activating receptor expressed on human monocytes", JOURNAL OF INNATE IMMUNITY, 2013, XP055660026 * |
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