Patent Application
20210115139
The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising a CD300E inhibitor as an active ingredient.
T cells play an important role in immunity of the human body. T cells are divided into killer T cells, helper T cells, regulatory T cells, and memory T cells. In particular, killer T cells express CD8 on the cell surface; helper T cells express CD4 on the cell surface; and regulatory T cells express CD4 and CD25 on the cell surface.
When an antigen such as a bacterium enters from the outside, helper T cells secrete substances such as cytokines to activate killer T cells and B cells. The activated killer T cells kill pathogen-infected cells, and the activated B cells secrete antibodies to inhibit activity of the antigen. Recently, attempts have been made to treat diseases such as cancer by activating such immunoregulatory abilities of T cells.
In addition, a T cell-mediated disease is recognized as a disease representing various immune-system diseases. In particular, T cells are considered as causing and perpetuating autoimmune diseases. Immune responses to self-antigens are caused by continuous or periodic activation of autoreactive T cells. In addition, the autoreactive T cells are attracting attention as a cause for characteristic tissue injury and tissue destruction which are directly or indirectly identified in autoimmune diseases.
Meanwhile, programmed cell death ligand 1 (PD-L1) is a type 1 transmembrane protein which is a ligand for programmed cell death-1 (PD-1). PD-L1 is expressed in hematopoietic cells such as T lymphocytes, B lymphocytes, dendritic cells, or macrophages. PD-1 is known as an immune checkpoint factor or immunomodulator which regulates secondary signaling activity of T cells. In addition, it has been reported that PD-1 is capable of acting to inhibit functions of T cells, such as inhibiting proliferation of T cells and decreasing expression of cytokines, by binding to PD-L1 or the like which is expressed on the surface of cells such as activated T cells or dendritic cells (Krzysztof M. Zak, et al., 2015).
Recently, attempts have been made to develop anticancer agents and immunomodulators using substances that regulate immune functions of T cells, such as PD-1 and PD-L1.
In the course of studying substances capable of inhibiting or increasing activity of immune cells, the present inventors have found that CD300E cell signaling system can regulate activity of T cells, and thus have completed the present invention.
In an embodiment, there is provided an immunopotentiator comprising, as an active ingredient, a substance that binds to CD300E protein or a substance that inhibits expression of CD300E gene.
In addition, in an embodiment, there is provided a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a substance that binds to CD300E protein or a substance that inhibits expression of CD300E gene.
In addition, in an embodiment, there is provided a method for treating cancer, comprising a step of administering the pharmaceutical composition to an individual.
The CD300E inhibitor according to the present invention can increase activity of immune cells, and thus can be used as an immunopotentiator. In addition, the CD300E inhibitor according to the present invention can potentiate immunity of an individual, and thus can be used to effectively prevent or treat cancer.
In an embodiment, there is provided an immunopotentiator comprising, as an active ingredient, a substance that binds to CD300 antigen like family member E (CD300E) protein or a substance that inhibits expression of CD300E gene.
As used herein, the term “CD300E” is the abbreviation of “CD300 antigen like family member E”, and may be a protein having the amino acid sequence of SEQ ID NO: 1. The polypeptide having the amino acid sequence of SEQ ID NO: 1 may be encoded by a polynucleotide having the base sequence of SEQ ID NO: 2.
Specifically, the sequence of 205 amino acids represented by SEQ ID NO: 1 may be comprised of an extracellular domain consisting of amino acids at positions 1 to 173 (SEQ ID NO: 3), a transmembrane domain consisting of amino acids at positions 174 to 194, and an intracellular domain consisting of amino acids at positions 195 to 205. The extracellular domain of CD300E having the amino acid sequence of SEQ ID NO: 3 may be encoded by a polynucleotide having the base sequence of SEQ ID NO: 4.
In addition, in the present disclosure, CD300E may be in a form in which a human IgG constant kappa tag is bound. The CD300E to which the human IgG constant kappa tag is bound for isolation and purification may be represented by the amino acid sequence of SEQ ID NO: 5, and the polypeptide having the amino acid sequence of SEQ ID NO: 5 may be encoded by a polynucleotide having the base sequence of SEQ ID NO: 6. Specifically, the fusion protein of SEQ ID NO: 5 may be a protein in which the extracellular domain of CD300E (at positions 1 to 173) and the human IgG constant kappa tag (at positions 179 to 285) are bound to each other.
In the present disclosure, the substance that binds to CD300E protein may be a compound, an aptamer, a peptide, or an antibody or a fragment thereof which specifically binds to the CD300E protein. The antibody or a fragment thereof may be any one selected from the group consisting of a monoclonal antibody, scFv, Fab, Fab′, and F(ab)′.
In the present disclosure, the substance that inhibits expression of CD300E gene may be an antisense nucleic acid, siRNA, shRNA, miRNA, or ribozyme which complementarily binds to DNA or mRNA of the CD300E gene. The CD300E siRNA may be any one of the base sequences of SEQ ID NOS: 7 to 18.
In one embodiment, it was intended to identify whether an anti-CD300E antibody or CD300E siRNA, which targets CD300E and inhibits activity thereof, can increase cytotoxicity of peripheral blood mononuclear cells (PBMCs) against cancer cells. As a result, in a mixture of PBMCs and a cancer cell line which had been treated with a CD300E inhibitor, cytotoxicity of the PBMCs against the cancer cell line was exhibited at a higher level than a control group. In another embodiment of the present disclosure, it was intended to identify whether CD300E siRNA that inhibits activity of CD300E inhibits growth of tumors in mice. As a result, in mice in which CD300E had been knocked down by treatment with CD300E siRNA, the tumor growth rate was remarkably inhibited as compared with a control group. From the above results, it is found that the anti-CD300E antibody or CD300E siRNA, which targets CD300E and inhibits activity thereof, can delay or stop the progress of cancer by blocking or knocking down CD300E to inhibit activity or expression thereof.
In addition, in an embodiment, there is provided a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a substance that binds to CD300E protein or a substance that inhibits expression of CD300E gene.
The substance that binds to CD300E protein or the substance that inhibits expression of CD300E gene is as described above. The pharmaceutical composition may further comprise a pharmaceutically acceptable additive.
In the present disclosure, the cancer may be, but is not limited to, any one selected from the group consisting of bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, central nervous system tumor and liver cancer.
A preferred daily dose of the pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a substance that binds to CD300E protein or a substance that inhibits expression of CD300E gene according to the present disclosure may be in a range of 0.01 ug/kg to 10 g/kg and preferably 0.01 mg/kg to 1 g/kg, depending on the patient's condition, body weight, sex, age, severity of disease, and route of administration. Administration may be carried out once or several times a day.
In addition, in an embodiment, there is provided a method for treating cancer, comprising a step of administering, to an individual, a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, a substance that binds to CD300E protein or a substance that inhibits expression of CD300E gene.
The route of administration may be, but is not limited to, any one selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraarteriole, intraventricular, intralesional, intrathecal, topical, and combinations thereof.
Hereinafter, the present invention will be described in more detail by way of the following examples. However, the following examples are intended to only illustrate the present invention, and the scope of the present invention is not limited thereto.
Buffers used in the examples were prepared as follows:
1×PBS (Thermo Fisher gibco #10010) was prepared by mixing 155 mM sodium chloride, 2.96 mM sodium phosphate solution, and 1.05 mM potassium phosphate solution, pH 7.4.
FACS buffer was prepared by mixing 1×PBS (Thermo Fisher gibco #10010), 10 ml of 2% FBS (Thermo Fisher gibco #16000-044), and 1 ml of 1 mM EDTA (Fisher 15575020).
1×RBC lysis buffer was prepared by diluting 10×RBC solution (Biolegend #420301) in triple-distilled water at 1:10.
10% FBS RPMI1640 was prepared by mixing RPMI medium (Cellgrow #10-040-CVR), 50 ml of 10% FBS (Thermo Fisher gibco #16000-044), 5 ml of 1% antibiotics (Thermo Fisher gibco #15140-122), and 0.5 ml of 2-mercaptoethanol (Thermo Fisher gibco #21985-023).
MACS buffer was prepared by mixing 1×PBS (Thermo Fisher gibco #10010), 0.5% bovine serum albumin (BSA) (Millipore #82-100-6), and 2 ml of 2 mM EDTA (Fisher 15575020).
In the present example, it was intended to identify whether CD300E protein inhibits proliferation and activity of T cells, and thus causes cancer cells to avoid the T cell-mediated immune system.
Human blood was collected and placed in a 10-mL tube coated with EDTA (or heparin). The human blood was mixed with PBS at a ratio of 1:1. Then, Ficoll-Paque PLUS was placed in a 50-mL tube, and the above blood sample was added thereto. After centrifugation, human PBMCs were collected. The collected product was centrifuged to remove the supernatant. Subsequently, RBC lysis (1×) buffer was added thereto, pipetting was performed, and then the resultant was kept on ice for 3 minutes. Subsequently, 50 ml of 10% FBS RPMI1640 was added thereto and the mixture was centrifuged to remove supernatant. Then, FACS buffer was added thereto and centrifugation was performed to remove the supernatant. Thereafter, 50 ml of MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) was added thereto, the number of cells was counted, and centrifugation was performed to remove the supernatant.
CD4+ T cells and CD8+ T cells were resuspended using 40 μl of MACS buffer per 1×107 cells. 10 μl of each of anti-CD4 and anti-CD8 biotin antibodies was placed in the tube, and then the tube was kept in a refrigerator for 5 minutes. Thereafter, 30 μl of MACS buffer per 1×107 cells was added to the resulting product. 20 μl of anti-biotin microbeads was added thereto and mixing was performed. Subsequently, CD4+ T cells and CD8+ T cells were isolated using LS column and the number of the respective cells was counted.
Each of the prepared CD4+ T cells and CD8+ T cells was mixed with 1 μl of carboxyfluorescein succinimidyl ester (CF SE) per 2×106 cells and each mixture was kept at 37° C. for 3 minutes. Then, FBS was added to each tube containing each of CD4+ T cells and CD8+ T cells, and each tube was kept on ice for 10 minutes. Thereafter, centrifugation was performed to remove the supernatant. 30 ml of FACS buffer was added to the resulting product, and then pipetting was performed. Centrifugation was performed to remove the supernatant. Then, 10% FBS RPMI1640 was added thereto, and then pipetting was performed. Centrifugation was performed to remove the supernatant. Thereafter, the resulting product was mixed with 10 ml of 10% FBS RPMI1640, and then the number of cells was counted.
Recombinant human IgG1 protein (Cat. No. 110-HG) and recombinant human PD-L1/B7-H1 protein (Cat. No. 156-B7) were purchased from R&D Systems. Recombinant human CD300E protein (human CD300E with human IgG constant kappa tag) was produced by the following method. Specifically, using pCEP4 (Invitrogen) plasmid DNA as a vector, a polynucleotide represented by SEQ ID NO: 6 was cloned into the multiple cloning site (MCS) region thereof. The resulting DNA construct was transfected into human embryonic kidney (HEK) 293F cells, and the recombinant human CD300E protein (human CD300E with human IgG constant kappa tag) contained in the culture medium was purified and obtained using kappa select resin.
7.5 μg/ml or 10 μg/ml of each of the proteins was mixed with 2.5 μg/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325). Each of the mixtures was used to coat a 96-well plate at 4° C., and washing was performed three times with PBS. The CD4+ T cells prepared in Example 1.1 were added at 200 μl in an amount of 2×106 cells per each well of the 96-well plate and incubation was performed. Activation of the CD4+ T cells using the anti-CD3 antibody was allowed to occur for 72 hours.
In addition, 5 μg/ml or 7.5 μg/ml of each of the proteins was mixed with 2.5 μg/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325). Each of the mixtures was used to coat a 96-well plate at 4° C., and washing was performed three times with PBS. The CD8+ T cells prepared in Example 1.1 were added at 200 μl in an amount of 2×106 cells per each well of the 96-well plate and incubation was performed. Activation of the CD8+ T cells using the anti-CD3 antibody was allowed to occur for 72 hours.
Here, proliferation of the CD4+ T cells and the CD8+ T cells was measured by a level of CFSE staining, and was analyzed by flow cytometry using FACSDiVa software (BD Biosciences). The results are illustrated in
In addition, in the group treated with CD300E, proliferation of the CD4+ T cells and the CD8+ T cells was remarkably inhibited as compared with the control group treated with IgG1, and proliferation of the CD4+ T cells and the CD8+ T cells was inhibited even as compared with the control group treated with PD-L1. From these results, it can be seen that neutralization of CD300E due to its blocking or knockdown decreases an inhibitory ability of CD300E on proliferation of T cells, and thus enables effective treatment of cancer.
In the present example, it was intended to identify whether PBMCs are capable of exhibiting increased cytotoxicity (killing ability) against cancer cells when CD300E is neutralized using a CD300E inhibitor.
Human blood was collected and placed in a 10-mL tube coated with EDTA (or heparin). The human blood was mixed with PBS at a ratio of 1:1. Then, Ficoll-Paque PLUS was placed in a 50-mL tube, and the above blood sample was added thereto. After centrifugation, human PBMCs were collected.
1.0 μg/ml of anti-CD3 antibody (BioLegend, Cat. No. 317325) was used to coat a 96-well plate at 4° C. Each well of the 96-well plate was washed 3 times with PBS before addition of the PBMCs. The previously obtained PBMCs were mixed with 10% FBS RPMI1640 and added at 100 μl in an amount of 6×105 cells per each well of the 96-well plate. Activation of the PBMCs by the anti-CD3 antibody was allowed to occur for 72 hours.
The lung cancer cell line A549 (ATCC®CCL-185), the colon cancer cell line HCT-116 (ATCC®CCL-247), the breast cancer cell line MDA-MB-231 (ATCC®HTB-26), the gastric cancer cell line MKN-74 (KCLB No. 80104), and the leukemia cell line U937 (ATCC®CRL-1593.2) were each mixed with 5 μM of CFSE and kept at 37° C. for 5 minutes. Thereafter, FBS was added to each tube containing each cell line, and each tube was kept on ice for 10 minutes. Subsequently, centrifugation was performed to remove the supernatant. To the product thus obtained was added 30 ml of FACS buffer. Then, pipetting was performed and centrifugation was performed to remove the supernatant. Then, 10% FBS RPMI1640 was added thereto. Then, pipetting was performed and centrifugation was performed to remove the supernatant. The product thus obtained was mixed with 10 ml of 10% FBS RPMI1640, and then the number of cells was counted.
Each type of the cancer cells was added at 3×104 cells/100 μl per each PBMC-containing well of the 96-well plate prepared in Example 2.1, and incubation was performed.
10 μg/mL of anti-human CD300E antibody or 50 nM CD300E siRNA was added to each well of the 96-well plate, and incubation was performed for 24 hours. Table 1 below shows experimental groups, in which four types of neutralizing antibodies are used to block CD300E, and an untreated control group; and Table 2 below shows experimental groups, in which three types of siRNAs are used to knock down CD300E, and an untreated control group.
Each mixture of PBMCs and each cancer cell line was incubated with the antibody or siRNA. After 24 hours, in order to identify lysed cells, the cells were stained with 7-aminoactinomycin D (7-AAD; BD Pharmingen, San Diego, Calif., USA). Staining for CFSE and 7-AAD was measured using the FACSDiVa software (BD Biosciences), to identify the PBMC's cytolytic ability against each cancer cell line. The results are illustrated in
As illustrated in
Similar to the above results that PBMCs exhibit an increased killing ability against the lung cancer cell line, it was found that PBMCs also exhibit an increased killing ability against the colon cancer cell line, the breast cancer cell line, the gastric cancer cell line, and the leukemia cell line in the case where CD300E is neutralized using the CD300E neutralizing antibody or siRNA (
In the present example, it was intended to identify in vivo whether growth of tumors in mice is inhibited when CD300E is neutralized using a CD300E inhibitor.
The MC38 cell line derived from C57BL/6 colon adenocarcinoma cells was resuspended at a concentration of 2.0×105 cells in 50 μl of PBS and subcutaneously injected into the flank of 6-week-old female C57BL/6 mice. Table 3 below shows experimental groups, in which siRNA is used to knock down CD300E, and an untreated control group.
For all experimental groups, starting from the 11th day after injection of the MC38 cell line, each siRNA targeting mouse CD300E was injected into the mouse tumor three times in total at intervals of 5 days. Specifically, 10 μs of siRNA was mixed with 7.5 μl of Oligofectamine (Invitrogen) in PBS according to the manufacturer's instructions, and then the mixture was injected, at a dose of 0.5 mg/kg, directly into the tumor tissue induced in mice. The results obtained by measuring tumor size in mice in the untreated control group and the experimental groups, in which CD300E is knocked down, are illustrated in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0062068 | May 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/006479 | 5/30/2019 | WO | 00 |
