Inhibitor of breast cancer cell proliferation and breast cancer therapeutic agent

Abstract

To provide an mRNA vaccine encoding the full length of a spike protein analog of the novel coronavirus SARS-CoV-2 as an inhibitor of breast cancer cell proliferation.SOLUTION: This inhibitor of breast cancer cell proliferation comprises an RNA molecule having a specific nucleotide sequence, the RNA molecule including a 5' cap. It is preferable that the RNA molecule is encapsulated in a lipid nanoparticle (LNP).SELECTED DRAWING: Figure 3

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] COVID-19 is an infectious disease caused by the novel coronavirus SARS-CoV-2 that emerged in 2019, and vaccine development was initiated 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 encodes four structural proteins. Of these four structural proteins, the spike protein, which binds to the ACE2 receptor present on the surface of human cells, was the target of vaccine development.

[0003] The mRNA vaccines developed in this way include Tojinamelan (Figure 1) and Famtojinamelan (Figure 2). Tojinamelan is a modRNA that encodes the full-length spike protein analogue of SARS-CoV-2 (Lys 986Pro, Val 987Pro). It is a single-stranded RNA consisting of 4,284 nucleotide residues, containing a 5' cap structure and poly(A) sequence, with all uridine residues replaced with N'-methylpseudouridine residues.

[0004] In addition, famtodinamelan is an mRNA that encodes the full-length spike protein analogue (K981P, V982P) common to the variant strains (Omicron strains) of the B.1.1.529.4 and B.1.1.529.5 lineages of SARS-CoV-2. It is a single-stranded RNA consisting of 4,269 nucleotide residues, containing a 5' cap structure and a poly(A) sequence, with all uridine residues replaced by N'-methylpseudouridine residues.

[0005] 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 drugs and compounds whose development has been discontinued, and repurposes them as therapeutic drugs for diseases other than those originally intended.

[0006] Drug repositioning (DR) has the advantage of allowing for rapid clinical trials since basic safety has already been confirmed, and also reduces research and development costs because there is no need to develop new drugs from scratch.

[0007] However, there are no known cases of anticancer drugs being developed using drug repositioning (DR) targeting mRNA encoding SARS-CoV-2 spike protein analogues. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication No. 2024-15475 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a breast cancer cell proliferation inhibitor and a breast cancer therapeutic agent that utilizes an mRNA vaccine encoding a spike protein analogue of the novel coronavirus SARS-CoV-2. [Means for solving the problem]

[0010] The inventors discovered that among mRNA vaccines encoding the full-length spike protein analogues (Lys986Pro, Va1987Pro) of SARS-CoV-2, drugs containing todinameran and famtodinameran exert a significant growth-inhibitory effect on human breast cancer cells, leading to the completion of the present invention.

[0011] That is, the breast cancer cell proliferation inhibitor of the present invention comprises an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 1 are replaced with N1-methyl-pseudouridine, and an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 2 are replaced with N1-methyl-pseudouridine, and is characterized in that the RNA molecule contains a 5' cap.

[0012] The chemical structure of N1-methyl-pseudouridine is as follows: [ka]

[0013] The term 5'-cap refers to the structure found on the 5'-end of an mRNA molecule, consisting of a guanosine nucleotide linked to the mRNA via a 5'-5' triphosphate bond. The inventors have confirmed that the use of a 5'-cap with the following chemical structure reliably exerts a significant growth-inhibitory effect on human breast cancer cells. [ka]

[0014] Furthermore, the RNA molecule is preferably encapsulated in a lipid nanoparticle (LNP). Examples of the lipid nanoparticle (LNP) include a form encapsulated in an LNP or a form 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. Examples of LNP include cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA.

[0015] Preferably, the polyA sequence comprises at least 100 nucleotides.

[0016] The breast cancer therapeutic agent of the present invention comprises an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 1 are substituted with N1-methyl-pseudouridine, and an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 2 are substituted with N1-methyl-pseudouridine, wherein the RNA molecule contains a 5' cap;

[0017] The present inventors have confirmed that by making the 5' cap have the following chemical structure, a significant growth inhibitory effect is reliably exerted on human breast cancer cells. [ka]

[0018] Furthermore, the RNA molecule is preferably encapsulated in a lipid nanoparticle (LNP). Examples of the lipid nanoparticle (LNP) include a form encapsulated in an LNP or a form 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. Examples of LNP include cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA.

[0019] The polyA sequence comprises at least 100 nucleotides. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a sequence listing of tojinamelan (all uridines in SEQ ID NO: 1 are replaced with N1-methylpseudouridines). [Figure 2] 1 is a sequence listing of femtodinamelan (all uridines in SEQ ID NO: 2 are replaced with N1-methylpseudouridines). [Figure 3] 1 is a graph showing the viability of BT-474 cells in the control group (saline injection group) and the test substance (iABC2010A) addition group. DETAILED DESCRIPTION OF THE INVENTION

[0021] 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. [Example]

[0022] The following test was conducted to evaluate the cell proliferation inhibitory effect of iABC2010A on the human breast cancer cell line BT-474. iABC2010A is Pfizer's COMIRNATY RTU intramuscular injection (Bivalent:Original / Omicron BA.4-5) (English name: COMIRNATY RTU intramuscular injection (Bivalent:Original / Omicron BA.4-5)), which contains todinameran and famtodinameran in an RNA mass ratio of 1:1.

[0023] (Description of materials used in the evaluation test) iABC2010A Container: 1 x 2mL vial Storage conditions: shielded from light, 2~8℃ Human breast cancer cell line BT-474 cells Storage conditions: frozen (stored in liquid nitrogen) Complete medium Complete medium was prepared using Hybri-Care Medium (ATCC) supplemented with inactivated fetal bovine serum (FBS) to a final concentration of approximately 10%, penicillin-S and peptomycin to final concentrations of approximately 100 μg / mL penicillin G, and 100 μg / mL streptomycin.

[0024] (Description of the equipment and instruments used in the evaluation test) Electronic balance for measuring reagents: ME235S (S / N. 19102898, Sartorius Co., Ltd.) Ultrapure water production system: Mi1li-Q IQ 7003 (Merck Ltd.) Water Pass: T RS (SIN.1710012, AS ONE) Safety cabinet: BCM-843SS (S / N. 254270201, Japan Air Tech) Carbon dioxide incubator: BL-42CD (S / N. CP030369, Tosk) Refrigerated centrifuge: H-60R (SIN.1.53193, Kokusan) Inverted phase contrast microscope: TE2000-S (S / N. 520361, Nikon) CMIC Pharma Science Microplate reader: SpectraMax iD3 (SIN. 373704763, Molecular Device Japan) Electronic Pipettor::Pipette Mate NEO (NICHIRYO)

[0025] (Reagents used in the evaluation test) The reagents used in the evaluation test are shown in Table 1.

[0026] [Table 1]

[0027] (Complete medium preparation method) 1) One packet (full amount) of Hybri-Care Medium was added to 950 mL of Milli-Q water (collected from an ultrapure water production system) and gently stirred to dissolve. This process also dissolved all of the Hybri-Care Medium that had adhered to the inside of the packaging bag, washing it in with the water. 2) 1.5 g of sodium bicarbonate was added to the solution prepared in 1) and stirred until dissolved, and then the solution was diluted to 1 L with Milli-Q water (collected from an ultrapure water production system). 3) The solution was sterilized by filtration using a filter with a pore size of 0.22 μm. 4) After filtration sterilization, 110 mL of inactivated fetal bovine serum (FBS) (final concentration: approximately 1 (010)) and 11 mL of penicillin-streptomycin (final concentrations: approximately 100 units / mL penicillin G, approximately 100 μg / mL streptomycin) were added to the medium, and the mixture was gently stirred to mix uniformly to prepare a complete medium. 5) Store in a refrigerator at 2-80°C when not in use.

[0028] (Thawing frozen stock 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) The thawed cells were dispersed in 9 mL of complete medium. 4) The mixture was centrifuged at approximately 125 g, 20°C, for 5 minutes, 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) The cells were cultured in a carbon dioxide incubator set at 37°C and 5% CO2. 7) The medium was completely replaced two or three times a week until subculture.

[0029] (Subculture of BT-474 cells) The following describes the method used to subculture the cells onto one 100 mm x 20 mm cell culture dish. 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 D-PBS(-). 4) The entire volume of D-PBS(-) was removed, and 2 mL of TrypLE Express Enzyme was added and allowed to permeate the entire cell culture, 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 125 g, 20°C, and 5 minutes, and the entire supernatant was removed. 8) The cells were suspended in 10 mL of complete medium and the number of cells was counted. 9) 1 / 2 to 1 / 3 of the cell suspension from 8) was seeded onto one 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. 11) If there was a long time until the next passage, the medium was completely replaced two or three times a week.

[0030] (Cell cryopreservation) Frozen stocks were prepared from the cells remaining after passage and stored in liquid nitrogen until the end of the study. 1) After passage (step 9) of passage of BT-474 cells), the remaining cells were centrifuged at 125 g, 20° C., for 5 minutes, and the entire supernatant was removed. 2) 2 mL of Bambanker was added to each cell pellet and suspended thoroughly. 3) Each cell suspension was dispensed into two cryotubes at 1 mL / tube and frozen and stored at -80°C. 4) After the test was completed, the materials were disposed of as infectious waste.

[0031] <Evaluation> (Drug sensitivity test of BT-474 cells) The test substance was evaluated for its growth inhibitory effect on BT474 cells. 1) 1.67 x 10 in complete medium 4 cells / mL, 5.56 × 10 4 cells / mL, 1.67 × 10 5 BT-474 cells were suspended at 180 μL / well in a 96-well plate in the arrangement shown in Table 2.

[0032] [Table 2]

[0033] Three 96-well plates were prepared in the same way to correspond to the number of culture days (3, 5, and 7 days) between the addition of the test substance and the measurement. The number of cells per well after seeding the cell suspension was 3 × 10 3 , 1×10 4 Or 3 x 10 3 The cells / well were used as blank wells. 200 μL of complete medium was dispensed into wells in rows A and H, columns 1, 11, and 12, where no cell suspension was seeded, and these wells were not used for analysis. 180 μL of complete medium was dispensed into wells in columns 2 to 10 of row G, and these wells were used as blank wells during analysis.

[0034] 2) The cells were cultured in a carbon dioxide incubator set at 37°C and 5% CO2 until the next day. 3) Using saline as the dilution medium, repeated 2-fold dilutions were made to prepare 1x (undiluted), 2x, 4x, and 8x diluted solutions of the test substance, and these were added at 20 μL / well to the 96-well plate after the incubation in 2) according to Table 3.

[0035] [Table 3]

[0036] 4) Three 96-well plates were cultured in a carbon dioxide incubator set at 37°C and 5% CO2 for 3 days, 5 days, and 7 days. 5) After the culture was completed, Cell Counting Kit-8 was added at 20 μL / well to the 96-well plate from 4), and the plate was incubated in a carbon dioxide incubator set at 37°C and 5% CO2. 6) Approximately 1 hour, 2 hours, and 4 hours after adding Cell Counting Kit-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 (1). The cell viability was plotted against various drug concentrations.

[0037]

number

[0038] (Result) The viability of BT-474 cells in the control group (saline-added group) and the test substance (iABC2010A)-added group is shown in Figure 3. As can be seen from this figure, the minimum viability of BT-474 cells after iABC2010A addition was 18.7%, and the maximum was 71.4%, demonstrating that iABC2010A clearly has a cell proliferation inhibitory effect compared to the control group (cell viability 100%. iABC2010A is an intramuscular injection drug containing tosinameran and famtosinameran in an RNA mass ratio of 1:1, and it was found that the mixture of tosinameran and famtosinameran clearly exhibited a cell proliferation inhibitory effect on breast cancer cells. Although the details of this mechanism of action are unknown, since this test only used breast cancer cells and there were no T cells or B cells involved in the immune system, it was clear that the cell proliferation inhibitory effect was at least exhibited by a mechanism other than immune function. [Industrial Applicability]

[0039] 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. The present invention includes an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 1 are replaced with N1-methyl-pseudouridine, and an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 2 are replaced with N1-methyl-pseudouridine, 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. The present invention includes an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 1 are replaced with N1-methyl-pseudouridine, and an RNA molecule in which all uridines in the nucleotide sequence set forth in SEQ ID NO: 2 are replaced with N1-methyl-pseudouridine, 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).

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