Breast cancer cell proliferation inhibitor and breast cancer treatment agent

Abstract

To provide a breast cancer cell proliferation inhibitor and a breast cancer therapeutic agent that utilize an mRNA vaccine that encodes an analogue of the spike protein of the novel coronavirus SARS-CoV-2. The present invention includes an RNA molecule having a nucleotide sequence as set forth in SEQ ID NO: 1, the RNA molecule including a 5' cap. SEQ ID NO: 1 is the base sequence of Zapomelan, an mRNA vaccine of spike glycoprotein that binds to the ACE2 receptor present on the surface of human cells. The RNA molecule is preferably encapsulated in a lipid nanoparticle (LNP).

Description

[Technical field]

[0001] The present invention relates to an agent for inhibiting the proliferation of breast cancer cells and an agent for treating breast cancer. [Background technology]

[0002] COV1D-19 is an infectious disease caused by the novel coronavirus SARS-CoV-2 that emerged in 2019, and vaccine development was undertaken early on as a measure to prevent the spread of infection (Patent Document 1, etc.). Coronaviruses are enveloped viruses whose viral genome consists of positive-sense single-stranded RNA that codes for four structural proteins. Of these four structural proteins, the spike glycoprotein that binds to the ACE2 receptor present on the surface of human cells was the target of vaccine development.

[0003] One of the mRNA vaccines developed in this way is Zapomeran. Zapomeran is a self-replicating mRNA that encodes the Venezuelan equine encephalitis virus RNA replicase (nsP1, nsP2, nsP3, nsP4) and the full-length spike protein analogue of SARS-CoV-2 (D614G, R682G, R683S, R685S, K986P, V987P), and is a single-stranded RNA consisting of 11,861 nucleotide residues, including a 5' cap structure, a subgenomic promoter sequence, and a polyA sequence (see Figures 1 and 2 and SEQ ID NO: 1).

[0004] Meanwhile, in recent years, drug repositioning (DR) has been attracting attention as a method for developing therapeutic drugs for diseases. Drug repositioning (DR) is a development method that utilizes existing drugs, drugs and compounds under development or discontinued, and repurposes them as therapeutic drugs for diseases other than those originally intended.

[0005] Drug repositioning (DR) has the advantage of being able to move quickly to clinical trials since basic safety has already been confirmed, and it also has the advantage of reducing research and development costs since there is no need to develop new drugs from scratch.

[0006] However, there are no known cases of developing anticancer drugs by drug repositioning (DR) targeting mRNA encoding the spike protein analogue of SARS-CoV-2. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2024-15475 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a breast cancer cell proliferation inhibitor and a breast cancer therapeutic agent that utilize an mRNA vaccine that encodes a spike protein analogue of the novel coronavirus SARS-CoV-2. [Means for solving the problem]

[0009] The inventors discovered that, among mRNA vaccines encoding SARS-CoV-2 spike protein analogues, zapomeran has a significant proliferation-inhibitory effect on human breast cancer cells, thereby completing the present invention.

[0010] That is, the breast cancer cell proliferation inhibitor of the present invention comprises an RNA molecule having a nucleotide sequence set forth in SEQ ID NO: 1, and is characterized in that the RNA molecule comprises a 5' cap.

[0011] The term 5' cap refers to the structure found on the 5' end of an mRNA molecule and consists of a guanosine nucleotide linked to the mRNA via a 5'-5' triphosphate bond. The inventors have confirmed that the 5' cap with the following chemical structure reliably exerts a significant growth inhibitory effect on human breast cancer cells. [ka]

[0012] Also, the RNA molecule is preferably encapsulated in a lipid nanoparticle (LNP). The lipid nanoparticle (LNP) may be, for example, encapsulated in an LNP or associated with an LNP. The LNP may include any lipid capable of forming a particle to which one or more nucleic acid molecules are bound or in which one or more nucleic acid molecules are encapsulated. The LNP may include a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and an RNA.

[0013] The breast cancer therapeutic agent of the present invention comprises an RNA molecule having a nucleotide sequence set forth in SEQ ID NO: 1, and is characterized in that the RNA molecule comprises a 5' cap.

[0014] The inventors have confirmed that by making the 5' cap have the chemical structure shown below, a significant proliferation-inhibitory effect is reliably exerted on human breast cancer cells. [ka]

[0015] Also, the RNA molecule is preferably encapsulated in a lipid nanoparticle (LNP). The lipid nanoparticle (LNP) may be, for example, encapsulated in an LNP or associated with an LNP. The LNP may include any lipid capable of forming a particle to which one or more nucleic acid molecules are bound or in which one or more nucleic acid molecules are encapsulated. The LNP may include a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and an RNA. [Brief description of the drawings]

[0016] [Figure 1] 1 is a sequence listing of zapomelan (SEQ ID NO:1). [Diagram 2] 1 is a chemical structure of the 5' cap portion of zapomelan. [Diagram 3] 1 is a graph showing the viability of BT-474 cells in the test substance (iABC2024K)-added group and the saline-added group in plate 1. [Figure 4] 13 is a graph showing the viability of BT-474 cells in the test substance (iABC2024K)-added group and the saline-added group in plate 3. [Diagram 5] 13 is a graph showing the viability of BT-474 cells in the test substance (iABC2024K)-added group and the saline-added group in plate 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The breast cancer cell proliferation inhibitor and breast cancer therapeutic agent of the present invention will be described in detail below with reference to examples. EXAMPLES

[0018] We conducted an evaluation test on the cell proliferation inhibitory effect of iABC2024K on human breast cancer cell line BT-474 cells. iABC2024K is an intramuscular injection of Costibe manufactured by Meiji Seika Pharma Co., Ltd., and contains the active ingredient zapomelan, which was developed as an mRNA vaccine.

[0019] <Materials and methods used in the evaluation test> (Test substance) ·i ABC2024K Container: Vial Properties: White dry preparation Storage conditions: -20℃, tolerance range ±5.0℃ (-25.0~-15.0℃), store in dark Test substance stock solution For iABC2024K, the vial was returned to room temperature, and a total of 9 mL of saline was added in three separate portions to dissolve the dry preparation in the vial, to prepare the test substance stock solution. - Preparation of dilution series of test substance For each test substance stock solution, two-fold dilution was repeated using saline as a medium to prepare 2-fold (relative concentration: 0.5), 4-fold (relative concentration: 0.25), 8-fold (relative concentration: 0.125), and 16-fold (relative concentration: 0.0625) diluted solutions of the test substance stock solution (relative concentration: 1). These were prepared just before use.

[0020] <Reagents and materials> The reagents and materials used are shown in Table 1.

[0021] [Table 1]

[0022] <Equipment / Appliances> The equipment and instruments used are shown in Table 2.

[0023] [Table 2]

[0024] <Cell culture> Test culture Name: Human breast cancer cell line BT-474 cells (hereafter referred to as BT-474) Complete medium Hybri-Care Medium was prepared by adding inactivated Fetal Bovine Serum (FBS) to a final concentration of approximately 10%, Penicillin-Streptomycin to a final concentration of approximately 50 units / mL, Penicillin G, and 50 μg / mL Streptomycin to prepare a complete medium. It was stored in a refrigerator at 2.0 to 8.0°C. Thawing frozen stocks of BT-474 cells 1) The complete medium was warmed in a thermostatic water bath set at 37°C. 2) Frozen cell stocks were thawed in a thermostatic water bath set at 37°C. 3) After thawing, the cells were dispersed in 9 mL of complete medium. 4) The mixture was centrifuged at 200 g for 5 minutes at 20° C., and the entire supernatant was removed. 5) The cells from one cryotube were dispersed in 10 mL of complete medium and seeded onto a 100 mm x 20 mm cell culture dish. 6) After step 5), the cells were immediately observed under a microscope. Since the cell density was appropriate, no operations such as thinning out the cells were performed. 7) The cells were cultured in a carbon dioxide incubator set at 37°C and 5% CO2. 8) Until subculture, the medium was completely replaced two or three times a week.

[0025] Passaging of BT-474 cells The following describes the method for subculturing cells on one 100 mm x 20 mm cell culture dish. When changing the size or number of culture dishes, the amount of medium and reagents used was increased or decreased proportionally. 1) The complete medium was warmed in a thermostatic water bath set at 37°C. 2) The cell density and state of the cells before passage were observed under a microscope. 3) The old medium was completely removed from the culture vessel, and the cells were washed with 10 mL of DPBS, no calcium, no magnesium (hereinafter referred to as D-PBS(-)). 4) The entire amount of D-PBS(-) was removed, and 2 mL of TrypLE Select Enzyme was added and allowed to permeate throughout the cells, followed by incubation at 37°C for approximately 5 minutes until the cells detached. 5) The culture vessel was gently tapped to detach the cells from the bottom of the vessel. 6) 5 mL of complete medium was added, and the cells were suspended by pipetting and then collected in a 15 mL tube. 7) The mixture was centrifuged at 200 g for 5 minutes at 20° C., and the entire supernatant was removed. 8) The cells were suspended in 5 mL of complete medium and the cell number was counted. 9) 1 / 2 to 1 / 3 of the cell suspension from 8) was seeded onto a 100 mm x 20 mm cell culture dish, and complete medium was added to bring the culture volume to 10 mL. 10) The cells were cultured in a carbon dioxide incubator set at 37°C and 5% CO2. If there was a long time until the next passage, the medium was replaced with a full volume exchange two or three times a week.

[0026] ·Cell cryopreservation Frozen stocks were prepared from the cells remaining after passage and stored in liquid nitrogen until the end of the study. 1) 200 g of the cells remaining after the subculture were centrifuged at 20° C. for 5 minutes, and the entire supernatant was removed. 2) Added 2 mL of STEM-CELLBANKER GMP grade to the cell pellet and suspended well. 3) The cell suspension was dispensed into 2 cryotubes at 1 mL / tube and stored frozen at -80°C. After freezing at -80°C for more than 1 day, the cells were transferred to a liquid nitrogen storage container. The remainder of the cell frozen stock was discarded as infectious waste after the test.

[0027] <Drug Sensitivity Test of BT-474 Cells> The growth inhibitory effect of the test substance on BT-474 cells was evaluated. 1) BT-474 cells suspended in complete medium to a concentration of 5.56 × 10 4 cells / mL were seeded into 3 96-well plates (designated as plate.1, plate.2, and plate.3) at 180 μL / well (1.00 × 10 4 cells / well) according to the layout in Table 3. For all 96-well plates, only the wells in row A and row H where no cell suspension was seeded, as well as the wells in column 1 and column 12, were dispensed with 200 μL / well of complete medium for preventing drying and were not used for analysis. On the other hand, the wells in columns 2 to 11 of row G were dispensed with 180 μL / well of complete medium and used as blank wells during analysis.

[0028]

Table 3

[0029] 2) The 6 96-well plates seeded with cells were cultured in a carbon dioxide incubator set at 37°C and 5% CO2 until the next day. 3) The dilution series of each test substance and the vehicle prepared at the time of use were added to the 96-well plates after the culture in step 2 at 20 μL / well according to Table 5. 4) Plate.1 was cultured for 3 days, plate.2 for 5 days, and plate.3 for 7 days in a carbon dioxide incubator set at 37°C and 5% CO2. 5) After incubation, Cell Counting Kit-8 was added to the 96-well plate from 4) at 20 μL / well, and the plate was incubated in a carbon dioxide incubator set at 37°C and 5% CO2. 6) Approximately 1 hour, approximately 2 hours, and approximately 4 hours after the addition of Cel1 Counting Kit.it-8 in 5), the absorbance at 450 nm (reference wavelength: 600 nm) was measured using a microplate reader. 7) The cell viability was calculated using the following formula: The cell viability was graphed against various drug concentrations.

[0030]

number

[0031] <Results and Discussion> In the drug sensitivity test, BT-474 cells were cultured at 1.00 × 10 4 A dilution series of iABC2024K was added as a test substance to 96-well plates (plate.1, plate.2, and plate.3) seeded with 100 cells / well, and the plates were cultured for 3, 5, or 7 days. Cell Counting Kit-8 was then added, and cell viability was measured after 1, 2, and 4 hours of color development. 1.00×10 4 Cells were seeded at 100 cells / well, and the cell viability was measured 3 days after addition of the test substance. The results for plate 1 are shown in FIG. 1.00 x 10 4 Cells were seeded at cell 1s / well and cell viability was measured 5 days after addition of the test substance. The results of plate 2 are shown in Figure 4. 1.00×10 4 Cells were seeded at cell 1s / well and cell viability was measured 7 days after addition of the test substance (plate 3). The results are shown in FIG.

[0032] As a result, iABC2024K showed a concentration-dependent cell proliferation inhibitory effect over the entire concentration range evaluated (relative concentration 0.0625-0.5). Since iABC2024K is an intramuscular injection drug containing zapomerane as an active ingredient, which was developed as an mRNA vaccine, it became clear that zapomerane exhibited a particularly remarkable cell proliferation inhibitory effect on breast cancer cells.

[0033] Although the details of the mechanism of action of the cell proliferation inhibitory effect have not been made clear, since T cells and B cells involved in immunity were not present in the evaluation tests of the cell proliferation inhibitory effect mentioned above, it is clear that the mechanism of action is at least based on something other than immune function.

[0034] In addition to the mRNA components, iABC2024K contains various additives. However, these additives are, for example, lipid nanoparticles for encapsulating the mRNA, or simply emulsion stabilizers, surfactants, sweeteners, pH buffers, etc., and it is highly unlikely that they will have any effect on the protein synthesis required for the proliferation of breast cancer cells. [Industrial Applicability]

[0035] The breast cancer cell proliferation inhibitor of the present invention can be suitably used for research on breast cancer cells and for treating breast cancer patients.

Claims

1. comprising an RNA molecule having the nucleotide sequence set forth in SEQ ID NO:1, A breast cancer cell proliferation inhibitor, wherein the RNA molecule comprises a 5' cap.

2. The breast cancer cell proliferation inhibitor of claim 1, wherein the 5' cap has the following chemical structure: 【Chemistry 1】

3. The breast cancer cell proliferation inhibitor according to claim 1 or 2, wherein the RNA molecule is encapsulated in a lipid nanoparticle (LNP).

4. comprising an RNA molecule having the nucleotide sequence set forth in SEQ ID NO:1, A breast cancer therapeutic agent, wherein the RNA molecule comprises a 5' cap.

5. The breast cancer therapeutic agent of claim 4, wherein the 5' cap has the following chemical structure: 【Chemistry 1】

6. The breast cancer therapeutic agent according to claim 4 or 5, wherein the RNA molecule is encapsulated in a lipid nanoparticle (LNP).

Images