Composition used for introducing messenger RNA into tumor tissue, and medicine for treating cancer

Abstract

Provided are: a composition which is used for introducing messenger RNA into a tumor tissue, the composition comprising the messenger RNA and phosphate-buffered saline containing calcium ions; and a medicine for treating cancer, the medicine being characterized by containing, as an active ingredient, messenger RNA encoding an apolipoprotein, and being also characterized in that the medicine is used in such a manner that the medicine is dissolved in phosphate-buffered saline containing calcium ions and is topically administered to a tumor tissue.

Description

Composition used for introducing messenger RNA into tumor tissue and drug for cancer treatment

[0001] The present invention relates to a composition used for introducing messenger RNA into tumor tissue and a medicament for treating cancer.

[0002] Messenger RNA (mRNA) holds great promise as a modality for expressing proteins in humans to treat pathogenic conditions. mRNA therapeutics are attractive because the mRNA sequence can be easily modified and because the mRNA is not incorporated into the genome, avoiding the risk of cancer. Furthermore, unlike other virus-based therapies, the mRNA does not elicit antibodies, allowing for repeated administration.

[0003] Clinical trials based on mRNA are currently underway in the field of cancer. These clinical trials use two types of mRNA: mRNA encoding tumor-specific mutant antigens to induce immunity against tumor-specific mutant antigens, and mRNA encoding several cytokines to activate anti-tumor immunity. These mRNAs exert their therapeutic effects by expressing the encoded proteins in several sites in the human body. Systems for tumor-specific delivery of mRNA are needed to alter the characteristics of cancer cells or directly kill them.

[0004] The development of mRNA-based therapeutics requires mRNA modifications, such as pseudouridine and 1-methylpseudouridine, to suppress immunogenicity, and a delivery system for delivering mRNA into cells in vivo. For example, lipid nanoparticles (LNPs) are used for mRNA delivery in COVID-19 vaccines. Although several LNPs have been reported to efficiently deliver mRNA to the liver and lungs (Non-Patent Documents 1 and 2), it is difficult to create LNPs with unique lipids in a conventional laboratory.

[0005] Several studies have reported that saline and sucrose can be used instead of LNPs for mRNA delivery to the heart and tumors (Non-Patent Documents 3 and 4). Plasmid transfection using calcium ions has been performed in cell culture systems using the calcium phosphate method. In this method, calcium ions combine with serum in the culture medium to form nanoparticles, which are known to be involved in the uptake of oligonucleotides. It has also been reported that calcium ions aid in the transfer of mRNA into mouse skin (Non-Patent Document 5). These studies suggest the potential of common materials as mRNA delivery systems. However, little is known about preferred delivery methods for mRNA delivery to tumors.

[0006] Kauffman KJ, Dorkin JR, Yang JH, Heartlein MW, DeRosa F, Mir FF, Fenton OS, Anderson DG. Nano Lett (2015) 15, 7300-7306.Li B, Manan RS, Liang SQ, Gordon A, Jiang A, Varley A, Gao G, Langer R, Xue W, Anderson D. Nat Biothechnol (2023) 41, 1410-1415.Sultana N, Magadum A, Hadas Y, Kondrat J, Singh N, Youssef E, Calderon D, Chepurko E, Dubois N, Hajjar RJ, Zangi L. Molecular Therapy (2017) 25, 1306-1315.Hotz C et al., Science Translational Medicine (2021) 13, eabc7804.Probst J, Weide B, Scheel B, Pichler BJ, Hoerr I, Rammensee HG, Pascolo S. Gene Ther (2007) 14, 1175-1180.

[0007] An objective of the present invention is to provide a composition used for introducing messenger RNA into tumor tissue, and a pharmaceutical and immunostimulant for cancer treatment in which the active ingredient is administered in the form of messenger RNA.

[0008] In order to solve the above-mentioned problems, the present invention encompasses the following inventions. [1] A composition used for introducing messenger RNA into tumor tissue, comprising messenger RNA and phosphate-buffered saline containing calcium ions. [2] The composition according to item [1], wherein the calcium ion concentration is 100 to 1000 μg / mL. [3] The composition according to item [1], which is for local administration to tumor tissue. [4] The composition according to any one of items [1] to [3], wherein the messenger RNA encodes an apolipoprotein or a T-cell costimulatory factor agonist. [5] The composition according to item [4], wherein the T-cell costimulatory factor agonist is OX40 ligand-fused ferritin or 4-1BB ligand-fused ferritin. [6] A pharmaceutical for cancer treatment, comprising messenger RNA encoding an apolipoprotein as an active ingredient, and being dissolved in phosphate-buffered saline containing calcium ions and used for local administration to tumor tissue. [7] The medicament for cancer treatment according to item [6], wherein the calcium ion concentration is 100 to 1000 μg / mL. [8] The medicament for cancer treatment according to item [6] or [7], wherein the medicament is used in combination with messenger RNA encoding a T cell costimulatory factor agonist. [9] The medicament for cancer treatment according to item [6] or [7], wherein the medicament is used in combination with a T cell costimulatory factor agonist antibody.

[10] The medicament for cancer treatment according to item [8], wherein the T cell costimulatory factor agonist is OX40 ligand-fused ferritin or 4-1BB ligand-fused ferritin.

[11] The medicament for cancer treatment according to item [8] or [9], wherein the medicament is further used in combination with a CD4-positive T cell depleting antibody or a CD25-positive T cell depleting antibody.

[12] An immunostimulant comprising messenger RNA encoding an apolipoprotein and messenger RNA encoding a T cell costimulatory factor agonist as active ingredients, the immunostimulant being dissolved in calcium ion-containing phosphate buffered saline and administered locally to tumor tissue.

[13] An immunostimulatory agent comprising, as active ingredients, messenger RNA encoding an apolipoprotein and a T-cell costimulatory factor agonist antibody, the agent being dissolved in calcium ion-containing phosphate-buffered saline and administered locally to tumor tissue.

[14] The immunostimulatory agent according to item

[12] , wherein the T-cell costimulatory factor agonist is OX40 ligand-fused ferritin or 4-1BB ligand-fused ferritin.

[15] The immunostimulatory agent according to item

[12] or

[13] , wherein the calcium ion concentration is 100 to 1,000 μg / mL.

[16] A method for introducing messenger RNA into tumor tissue, comprising contacting tumor cells with messenger RNA dissolved in calcium ion-containing phosphate-buffered saline.

[17] The method according to item

[16] , wherein the calcium ion concentration is 100 to 1,000 μg / mL.

[18] The method according to item

[16] , wherein the contacting is carried out by locally administering messenger RNA dissolved in phosphate buffered saline containing calcium ions to the tumor tissue.

[19] The method according to any one of items

[16] to

[18] , wherein the messenger RNA encodes an apolipoprotein or a T-cell costimulatory factor agonist.

[20] The method according to item

[19] , wherein the T-cell costimulatory factor agonist is OX40 ligand-fused ferritin or 4-1BB ligand-fused ferritin.

[0009] The present invention provides a composition for use in introducing messenger RNA into tumor tissue, as well as a cancer treatment drug and an immunostimulant in which an active ingredient is administered in the form of messenger RNA.

[0010] Figure 1 shows a schematic diagram (A) and electrophoresis results (B) of mRNA encoding luciferase 2 (luc2) prepared by in vitro transcription. Figure 2 shows the results of Example 1. (A) shows the results of measuring the region of interest (ROI) value of the luciferase signal after intratumoral administration of luc2 mRNA dissolved in each buffer. (B-E) are representative photographs showing the luciferase signal of mice administered luc2 mRNA intratumorally using ultrapure water (MilliQ) (B), phosphate-buffered saline (PBS) (C), sucrose (D), or an in vivo mRNA transfection reagent (in vivoJET) (E). Figure 3 shows the results of electrophoresis of mRNA supplemented with serum from female or male mice. Figure 4 shows the results of comparing the transfection efficiency of mRNA using an in vivo mRNA transfection reagent (in vivoJET) or phosphate-buffered saline (PBS). (A) shows the results of intratumoral administration to MC38 tumors, (B) to LL2 tumors, and (C) to 4T1 tumors. 1 shows the results of Reference Example 1. (A) is a representative photograph showing luciferase signals in mice intratumorally administered with lipid nanoparticles (LNP) and mRNA encoding luc2 used in Example 1, and (B, C) are representative photographs showing luciferase signals in the tumor, liver, draining lymph nodes, spleen, lungs, skin, and heart for CT26 tumors in Balb / c mice (B) and B16F10 tumors in C57BL / 6 mice (C).

[0034] FIG. 1 shows the results of Example 2. (A) shows the results after a single administration, and (B) shows the results after three administrations.

[0035] FIG. 1 shows the results of Example 3.(A) shows the results of measuring the ROI value of luciferase signals after intratumoral injection of 1, 5, 10, or 20 μg of mRNA and 500 μg / mL of calcium ions, magnesium ions, or zinc ions in PBS into B16F10 tumors in C57BL / 6 female mice. (B) shows the results of intratumoral injection of 20 μg or 40 μg of mRNA into B16F10 tumors in C57BL / 6 female mice. (C) shows the results of measuring luciferase signals after intratumoral injection of mRNA dissolved in PBS containing 200, 500, or 1000 μg / mL of calcium ions into B16F10 tumors in C57BL / 6 female or C57BL / 6 male mice. (A) shows the results of measuring luciferase signals after intratumoral injection of 20, 30, or 40 μg of mRNA dissolved in each solvent. (B) shows the results of examining the time-dependent change in ROI value after intratumoral injection of mRNA dissolved in PBS. (C) shows the results of examining calcium ion concentration-dependent enhancement of luciferase signals. Figure 1 shows the results of Example 4. Figure 2 shows representative photographs showing luciferase signals in the tumor, liver, draining lymph nodes, spleen, lung, skin, and heart excised from mice intratumorally injected with mRNA dissolved in calcium ion-containing PBS. Figure 3 shows the results of Example 5. (A) shows the z-average diameter (nm) measured by particle size analysis using dynamic light scattering. (B) shows the circular dichroism (CD) spectrum. (C) shows the optical absorption spectrum. Figure 4 shows the results of Example 5. (A, B) are representative transmission electron microscope (TEM) images of mRNA dissolved in PBS (A) or calcium ion-containing PBS (B), and (C) shows the results of electrophoresis of mRNA dissolved in each buffer after RNase treatment and subsequent purification. This figure shows the results of Example 6. The results show the results of three intratumoral injections of mRNA encoding OX40 ligand (OX40L), IL36γ, and IL23 dissolved in calcium ion-containing PBS into CT26 tumors in BALB / c mice.1A and 1B are schematic diagrams of the mRNA encoding apolipoprotein D (ApoD), mRNA encoding apolipoprotein L9 (ApoL9), and mRNA encoding OX40 ligand-fused ferritin (OX40L-ferritin) used in Example 6.

[0034] Figures 1A and 1B show the results of Example 6. (A) shows the results of three intratumoral injections of mRNA encoding Luc2, ApoD / ApoL9a / OX40L, or ApoD / ApoL9a / OX40L-ferritin in a CT26 tumor. (B) shows the results of three intratumoral injections of control mRNA, mRNA encoding ApoD / ApoL9 / OX40L, mRNA encoding ApoD / ApoL9 / OX40L-ferritin, or mRNA encoding ApoD / ApoL9, together with an OX40 antibody, in a 4T1 tumor.

[0035] Figures 1A and 1B show the results of three intratumoral injections of control mRNA, mRNA encoding ApoD / ApoL9 / OX40L, mRNA encoding ApoD / ApoL9 / OX40L-ferritin, or mRNA encoding ApoD / ApoL9, together with an OX40 antibody. 1 shows the results of FACS analysis of the number of CD4-positive T cells and CD8-positive T cells per 100,000 cells in target tumors after intratumoral administration of mRNA encoding Luc2 (A), mRNA encoding ApoD / ApoL9a / OX40L (B), or mRNA encoding ApoD / ApoL9a / OX40L-ferritin (C). Figure 1 shows the results of Example 6. Figure 1 shows the results of FACS analysis of the number of NKG2D-positive cells in target tumors after intratumoral administration of control mRNA, mRNA encoding ApoD / ApoL9a / OX40L, mRNA encoding ApoD / ApoL9a / OX40L-ferritin, or mRNA encoding ApoD / ApoL9a, together with OX40 antibody (OX40 ab). (A) shows the percentage of NKG2D-positive cells among CD4-positive T cells, and (B) shows the percentage of NKG2D-positive cells among CD8-positive T cells. Figure 1 shows the results of Example 7. (A) shows the results of evaluating OX40 stimulatory activity using HT1080 cells expressing OX40, and (B) shows the results of administering a combination of mRNA encoding OX40 ligand-fused ferritin (OX40L-Fr) and mRNA encoding ApoD and mRNA encoding Apol9a into one of the tumors in a mouse bearing two CT26 tumors.

[0023] Figure 1 shows the results of Example 8. Mice bearing two CT26 tumors were administered a combination of mRNA encoding OX40 ligand-fused ferritin (OX40L-Fr), mRNA encoding ApoD, and mRNA encoding Apol9a into one tumor, and the numbers of CD4+ T cells and CD8+ T cells per 100,000 cells in the target tumor (A) and non-target tumor (B) were analyzed by FACS. Figure 1 shows the results of Example 8. Mice bearing two CT26 tumors were administered a combination of mRNA encoding Luc2 (A), ApoD mRNA and ApoL9a mRNA with an OX40 agonist antibody (B), ApoD mRNA and ApoL9a mRNA with a 4-1BB agonist antibody (C), or ApoD mRNA and ApoL9a mRNA with an mRNA encoding BM40-4-1BB-L-ferritin (D) into one tumor, and tumor growth in the target tumor was compared.

[0024] Figure 1 shows the results of Example 8.

[0033] Figure 1 shows the results of Example 9. Mice bearing two CT26 tumors were administered intratumorally with mRNA encoding Luc2 (A), a combination of ApoD mRNA and ApoL9a mRNA with an OX40 agonist antibody (B), a combination of ApoD mRNA and ApoL9a mRNA with a 4-1BB agonist antibody (C), or a combination of ApoD mRNA and ApoL9a mRNA with an mRNA encoding BM40-4-1BB-L-ferritin (D), and tumor growth in non-target tumors was compared. Figure 2 shows the results of Example 9. Mice bearing two MC38 tumors were administered intratumorally with mRNA encoding Luc2 (A, C), or a combination of ApoD mRNA, ApoL9a mRNA, a 4-1BB agonist antibody, and a CD4+ T cell-depleting antibody (B, D), and tumor growth in target tumors (A, B) and non-target tumors (C, D) was compared. Figure 3 shows the results of Example 9.10 shows the results of Example 9. Mice bearing two MC38 tumors were administered intratumorally with Luc2-encoding mRNA (A), a combination of ApoD mRNA, ApoL9a mRNA, a 4-1BB agonist antibody, and a CD25-positive T cell-depleting antibody (B), a combination of ApoD mRNA, a 4-1BB agonist antibody (C), a combination of ApoL9a mRNA, a 4-1BB agonist antibody (D), or a combination of ApoD mRNA, ApoL9a mRNA, a 4-1BB agonist antibody, and a CD8-positive T cell-depleting antibody (E), and the tumor growth in the target tumors was compared.

[0039] Figure 1 shows the results of comparing tumor growth in non-target tumors in mice bearing two MC38 tumors after intratumoral administration of mRNA encoding Luc2 (A), a combination of ApoD mRNA, ApoL9a mRNA, a 4-1BB agonist antibody, and a CD25-positive T cell-depleting antibody (B), a combination of ApoD mRNA and a 4-1BB agonist antibody (C), a combination of ApoL9a mRNA and a 4-1BB agonist antibody (D), or a combination of ApoD mRNA, ApoL9a mRNA, a 4-1BB agonist antibody, and a CD8-positive T cell-depleting antibody (E). Figure 1 shows the results of Example 10. MC38 cells were transfected with mRNA encoding a fusion protein of 4-1BB-L and a DPS-like protein derived from Aeropyrum pernix (ap), Pyrococcus furiosus (pf), Sulfolobus solfataricus (ss), or Thermotoga maritima (tm), and the secretion of each protein into the supernatant was confirmed.

[0033] Figure 1 shows the results of Example 10. mRNA encoding ferritin fused with CD70, GITRL, or TNF, mRNA encoding 4-1BB ligand (4-1BBL), or mRNA encoding 4-1BB-L fused ferritin was introduced into MC38 cells, and the proteins secreted into the supernatant were confirmed.

[0011] [Composition Used for Introducing Messenger RNA into Tumor Tissue] The present invention provides a composition used for introducing messenger RNA (mRNA) into tumor tissue (hereinafter referred to as the "composition of the present invention"). In the composition of the present invention, the mRNA may be modified mRNA (mmRNA) in which some nucleotides have been modified. Preferably, the mRNA is modified mRNA in which uridine has been substituted with pseudouridine or 1-methylpseudouridine. It is known that the substitution of uridine with pseudouridine or 1-methylpseudouridine can have useful characteristics, including a significant reduction in the innate immune response of cells into which the mRNA is introduced, or a substantial lack of induction of such response. In the composition of the present invention, the mRNA may be commercially available mRNA or mRNA synthesized by known techniques. For example, the mRNA can be produced by amplifying the sequence of a target gene by PCR using cDNA as a template, introducing it into a desired plasmid, and purifying it by known methods after in vitro transcription.

[0012] In the compositions of the present invention, mRNA may be synthesized to contain components for regulating intracellular stability and protein production. Examples of such components include a 5'-end cap structure, an untranslated region (UTR), and a poly(A) tail. Therefore, in the compositions of the present invention, mRNA may be synthesized to incorporate a 5'-end cap structure, a 5' UTR, a 3' UTR, and / or a poly(A) sequence. In the compositions of the present invention, the 5'-end cap structure of the mRNA may be an endogenous cap, a cap analog attached during an in vitro transcription reaction, or a cap structure generated by enzymatic capping after transcription. The poly(A) sequence can be appropriately designed based on the overall length of the mRNA and the length of the final product expressed from the mRNA.

[0013] The composition of the present invention comprises mRNA and phosphate-buffered saline (PBS) containing calcium ions. The calcium ion concentration in the composition of the present invention is not particularly limited and may be 1 to 10,000 μg / mL, 50 to 5,000 μg / mL, 100 to 1,000 μg / mL, 150 to 750 μg / mL, or 200 to 500 μg / mL. It is preferably 100 to 1,000 μg / mL, more preferably 150 to 750 μg / mL, and even more preferably 200 to 500 μg / mL. The method for introducing mRNA into tumor cells in the composition of the present invention is not particularly limited. For example, the composition of the present invention can be administered locally to tumor tissue. Local administration to tumor tissue can be achieved, for example, by injecting the composition of the present invention into the tumor.

[0014] In the composition of the present invention, the amount of mRNA to be introduced into tumor tissue is appropriately determined taking into consideration the type and volume of the tumor. 3 The dose may be 150 μg or more, 300 μg or more, 450 μg or more, or 600 μg or more, or 1000 μg or less, 800 μg or less, 650 μg or less, 480 μg or less, or 320 μg or less per tumor volume of 1000 mm. 3 More preferably, the dose is 150 μg or more per tumor volume of 1000 mm 3 per unit of blood is 300 μg or more.

[0015] In the compositions of the present invention, the mRNA may be mRNA encoding a protein from any organism. Preferably, the mRNA encodes a mammalian protein. Examples of mammals include, but are not limited to, humans, mice, rats, cows, horses, pigs, dogs, and cats. Particularly preferred is mRNA encoding a human protein. The amino acid sequence of a protein and the nucleotide sequence of the gene encoding it can be obtained from known databases such as NCBI. In the compositions of the present invention, the mRNA may be mRNA encoding a fusion protein or mRNA derived from a codon-optimized gene. In the compositions of the present invention, the mRNA encoding a fusion protein may be mRNA encoding two or more proteins linked via a sequence encoding a linker. Furthermore, in the compositions of the present invention, the mRNA may be mRNA to which a sequence encoding a signal peptide has been added or substituted. In the compositions of the present invention, the mRNA dissolved in PBS containing calcium ions may be one type of mRNA or two or more types of mRNA. In the compositions of the present invention, the mRNA may be mRNA to which a sequence encoding a tag has been added.

[0016] In the compositions of the present invention, the mRNA may be mRNA encoding an apolipoprotein or mRNA encoding a T cell costimulatory factor agonist. Examples of human apolipoproteins include, but are not limited to, APOA1BP (also known as NAXE), APOOL, APOO, APOD, APOE, APOL1, APOL2, APOL3, APOL4, APOL5, APOL6, APOC1, APOC2, and APOC4. Preferably, the mRNA encodes APOD, or a combination of an mRNA encoding APOD and an mRNA encoding an apolipoprotein other than APOD. Preferred apolipoproteins other than APOD include APOL1, APOL3, and APOL6. The amino acid sequences of apolipoproteins and the nucleotide sequences of the genes encoding them can be obtained from known databases such as NCBI. Examples include, but are not limited to, the amino acid sequences and nucleotide sequences listed in Table 1.

[0017]

[0018] Examples of T cell costimulatory factor agonists include ligands of various T cell costimulatory factors, such as OX40, 4-1BB, GITR, CD28, CD40, ICOS, HVEM, CD27, CD30, DR3, TNFR2, LTαβ, and LFA1, as well as agonist antibodies of various T cell costimulatory factors. An OX40 agonist is preferred. Examples of OX40 agonists include, but are not limited to, OX40 ligand (hereinafter referred to as OX40L) and OX40 antibodies. The amino acid sequences of the ligands of various T cell costimulatory factors and the nucleotide sequences of the genes encoding them can be obtained from known databases such as NCBI. For example, the sequence (NCBI RefSeq) of human OX40L (accession number: X79929) includes, but is not limited to, NP_001284491.1 (amino acid sequence) and NM_001297562.2 (nucleotide sequence). The sequence of the mRNA encoding the OX40 antibody is not limited, as long as it is an mRNA that encodes the OX40 antibody.

[0019] Examples of the sequence (NCBI RefSeq) of the human 4-1BB ligand (accession number: U03398.1) include, but are not limited to, NP_003802.1 (amino acid sequence) and NM_003811.4 (nucleotide sequence). The sequence of the mRNA encoding the 4-1BB antibody is not limited, as long as it encodes the 4-1BB antibody.

[0020] In the compositions of the present invention, the T cell costimulatory factor agonist may be OX40L fusion ferritin, which is an OX40 agonist; OX40L (BM40-OX40L) fusion ferritin in which the signal peptide sequence has been replaced with a BM40 sequence; or 4-1BB ligand (4-1BB-L) fusion ferritin, which is a 4-1BB agonist. In the compositions of the present invention, the ferritin fused to the ligand may be derived from any biological species. The mRNA sequences of OX40L fusion ferritin and 4-1BB-L fusion ferritin are not limited as long as they encode OX40L fusion ferritin or 4-1BB-L fusion ferritin, and examples thereof include the sequences set forth in SEQ ID NOs: 1, 3, and 5 herein.

[0021]

[0022] OX40L fusion ferritin amino acid sequence (SEQ ID NO: 2) MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSRHHHHHHGGGSGGMQLSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPLPGSGGESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS*

[0023]

[0024] BM40-OX40L fusion ferritin amino acid sequence (SEQ ID NO: 4) MRAWIFFLLCLAGRALAASRHHHHHHGGGSGGMQLSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPLPGSGGESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS*

[0025]

[0026] 4-1BB-L fusion ferritin amino acid sequence (SEQ ID NO: 6) MRAWIFFLLCLAGRALAASRHHHHHHGGGSGGRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHG AQDAYRDWELSYPNTTSFGLFLVKPDNPWEPGSGGESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNE NNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS*

[0027] In the composition of the present invention, the T cell costimulatory factor agonist may be 4-1BB-L (BM40-4-1BB-L) fusion ferritin in which the signal peptide sequence has been replaced with a BM40 sequence. The mRNA sequence of BM40-4-1BB-L fusion ferritin is not limited as long as it is an mRNA encoding BM40-4-1BB-L fusion ferritin, and examples thereof include the sequence shown in SEQ ID NO:9 herein.

[0028]

[0029] BM40-4-1BB-L fusion ferritin amino acid sequence (SEQ ID NO: 10) MRAWIFFLLCLAGRALAASRHHHHHHGGGSGGRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHG AQDAYRDWELSYPNTTSFGLFLVKPDNPWEPGSGGESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNE NNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS*

[0030] In the compositions of the present invention, the T cell costimulatory factor agonist may be fused to a DNA-binding protein (DPS: DNA-binding protein from starved cells)-like protein. In the compositions of the present invention, the DPS-like protein fused to the ligand may be derived from any biological species. Examples include the DPS-like protein (2CLB) derived from Sulfolobus solfataricus (ss) or the DPS-like protein (1VLG) derived from Thermotoga maritima (tm). In the compositions of the present invention, the T cell costimulatory factor agonist may be a 4-1BB-L fusion DPS-like protein. The mRNA of the 4-1BB-L fusion DPS-like protein is not limited as long as it encodes the 4-1BB-L fusion DPS-like protein, and examples thereof include the sequences set forth in SEQ ID NOs: 11 and 13 herein.

[0031]

[0032] 4-1BB-L fused ssDPS amino acid sequence (SEQ ID NO: 12) MRAWIFFLLCLAGRALAASRHHHHHHGGGSGGRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAY LHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWEPGGSGGPKVVGVEILEKSGLDIKKLVDKLVKATAAEFTTYYYYTILRMHLTGMEGEGLKEIAEDARLEDRL HFELMTQRIYELGGGLPRDIRQLADISACSDAYLPENWKDPKEILKVLLEAEQCAIRTWKEVCDMTYGKDPRTYDLAQRILQEEIEHEAWFLELLYGRPSGH*

[0033]

[0034] 4-1BB-L fused tmDPS amino acid sequence (SEQ ID NO: 14) MRAWIFFLLCLAGRALAASRHHHHHHGGGSGGRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRA YLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWEPGSGGMMVISEKVRKALNDQLNREIYSSYLYLSMATYFDAEGFKGFAHWMKKQAQEELTHAMKFYEYI YERGGRVELEAIEKPPSNWNGIKDAFEAALKHEEFVTQSIYNILELASEEKDHATVSFLKWFVDEQVEEEDQVREILDLLEKANGQMSVIFQLDRYLGQRE*

[0035] In the composition of the present invention, the mRNA may be mRNA encoding a TNF superfamily member, such as CD70, GITRL (Glucocorticoid-induced TNF-related ligand), or TNF. The amino acid sequence of each TNF superfamily member and the nucleotide sequence of the gene encoding it can be obtained from known databases such as NCBI. For example, sequences of human CD70 (accession number: L08096.1) (NCBI RefSeq) include NP_001243.1 (amino acid sequence) and NM_001252.5 (nucleotide sequence); sequences of human GITRL (accession number: BC069319.1) (NCBI RefSeq) include NP_005083.3 (amino acid sequence) and NM_005092.4 (nucleotide sequence); and sequences of human TNF (accession number: X01394.1) (NCBI RefSeq) include, but are not limited to, NP_000585.2 (amino acid sequence) and NM_000594.4 (nucleotide sequence).

[0036] [Method for Introducing Messenger RNA into Tumor Tissue] The present invention provides a method for introducing messenger RNA (mRNA) into tumor tissue (hereinafter referred to as the "method of the present invention"). In the method of the present invention, the mRNA can be prepared and used in the same manner as in the composition of the present invention described above.

[0037] The method of the present invention comprises a step of contacting tumor cells with mRNA dissolved in calcium ion-containing phosphate buffered saline (PBS). The concentration of calcium ions in the method of the present invention is not particularly limited, and can be carried out in the same manner as in the composition of the present invention. The method of contacting tumor cells with mRNA dissolved in calcium ion-containing PBS in the method of the present invention is not particularly limited. For example, the method can be carried out by locally administering mRNA dissolved in calcium ion-containing PBS to tumor tissue. Local administration to tumor tissue can be carried out, for example, by injecting mRNA dissolved in calcium ion-containing PBS into the tumor.

[0038] In the method of the present invention, the amount of mRNA to be introduced into tumor tissue is appropriately determined taking into consideration the type and volume of the tumor. 3 The dose may be 150 μg or more, 300 μg or more, 450 μg or more, or 600 μg or more, or 1000 μg or less, 800 μg or less, 650 μg or less, 480 μg or less, or 320 μg or less per tumor volume of 1000 mm. 3 More preferably, the dose is 150 μg or more per tumor volume of 1000 mm 3 per unit of blood is 300 μg or more.

[0039] [Drug for Cancer Treatment] The present invention provides a drug for cancer treatment containing, as an active ingredient, mRNA encoding an apolipoprotein (hereinafter referred to as the "drug of the present invention"). The apolipoprotein encoded by the active ingredient of the drug of the present invention may be an apolipoprotein from any organism, but mammalian apolipoproteins are preferred. Mammals include, but are not limited to, humans, mice, rats, cows, horses, pigs, dogs, and cats. It is particularly preferred to use mRNA encoding human apolipoproteins. Examples of human apolipoproteins include, but are not limited to, APOA1BP (also known as NAXE), APOOL, APOO, APOD, APOE, APOL1, APOL2, APOL3, APOL4, APOL5, APOL6, APOC1, APOC2, and APOC4. Preferably, the drug is a combination of mRNA encoding apolipoprotein D (APOD) or mRNA encoding APOD with mRNA encoding an apolipoprotein other than APOD. Preferred apolipoproteins other than APOD include APOL1, APOL3, and APOL6. The amino acid sequences of apolipoproteins and the nucleotide sequences of the genes encoding them can be obtained from known databases such as NCBI. Examples include, but are not limited to, the amino acid sequences and nucleotide sequences listed in Table 1.

[0040] In the pharmaceutical of the present invention, mRNA encoding the apolipoprotein can be synthesized by known methods. For example, the apolipoprotein sequence can be amplified by PCR using cDNA from mammalian cells, introduced into any plasmid, and purified by known methods after in vitro transcription. The pharmaceutical of the present invention may also contain a pharmaceutically acceptable carrier.

[0041] The pharmaceutical of the present invention may contain an mRNA encoding a T cell costimulatory factor agonist as an additional active ingredient. Examples of T cell costimulatory factor agonists encoded by the active ingredient of the pharmaceutical of the present invention include ligands for each T cell costimulatory factor, such as OX40, 4-1BB, GITR, CD28, CD40, ICOS, HVEM, CD27, CD30, DR3, TNFR2, LTαβ, and LFA1, as well as agonist antibodies for each T cell costimulatory factor. An OX40 agonist is preferred. Examples of OX40 agonists include, but are not limited to, OX40 ligand (hereinafter referred to as OX40L) and OX40 antibodies. The amino acid sequences of the ligands for each T cell costimulatory factor and the nucleotide sequences of the genes encoding them can be obtained from known databases such as NCBI. For example, the sequence of human OX40L (accession number: X79929) (NCBI RefSeq) includes, but is not limited to, NP_001284491.1 (amino acid sequence) and NM_001297562.2 (nucleotide sequence). The sequence of the mRNA encoding the OX40 antibody is not limited as long as it is an mRNA encoding the OX40 antibody.

[0042] A 4-1BB agonist can also be suitably used as the T cell costimulatory factor agonist encoded by the active ingredient of the pharmaceutical of the present invention. Examples of 4-1BB agonists include, but are not limited to, 4-1BB ligand (hereinafter referred to as 4-1BB-L) and 4-1BB antibodies. Examples of sequences (NCBI RefSeq) for human 4-1BB-L (accession number: U03398.1) include, but are not limited to, NP_003802.1 (amino acid sequence) and NM_003811.4 (nucleotide sequence). The sequence of the mRNA encoding the 4-1BB antibody is not limited, as long as it encodes the 4-1BB antibody.

[0043] The pharmaceutical of the present invention may contain a T cell costimulatory factor agonist antibody as an additional active ingredient. Examples of the T cell costimulatory factor agonist antibody that is the active ingredient of the pharmaceutical of the present invention include agonist antibodies of various T cell costimulatory factors, such as OX40, 4-1BB, GITR, CD28, CD40, ICOS, HVEM, CD27, CD30, DR3, TNFR2, LTαβ, and LFA1. An OX40 agonist antibody or a 4-1BB agonist antibody is preferred.

[0044] The pharmaceutical of the present invention may contain, as an additional active ingredient, mRNA encoding OX40L fusion ferritin, an OX40 agonist. The mRNA sequence of OX40L fusion ferritin is not limited as long as it is mRNA encoding OX40L fusion ferritin, and examples thereof include the sequence shown in SEQ ID NO: 1 herein. The pharmaceutical of the present invention may contain, as an additional active ingredient, mRNA encoding 4-1BB-L fusion ferritin. The mRNA sequence of 4-1BB-L fusion ferritin is not limited as long as it is mRNA encoding 4-1BB-L fusion ferritin, and examples thereof include the sequence shown in SEQ ID NO: 5 herein. In the pharmaceutical of the present invention, the ferritin fused to the ligand may be derived from any biological species.

[0045] The pharmaceutical of the present invention may contain, as an additional active ingredient, mRNA encoding a protein in which a T cell costimulatory factor agonist is fused with a DPS-like protein. Examples include 4-1BB-L fusion DPS-like proteins. The mRNA for the 4-1BB-L fusion DPS-like protein is not limited as long as it encodes a 4-1BB-L fusion DPS-like protein, and examples thereof include the sequences set forth in SEQ ID NOs: 11 and 13 herein. In the pharmaceutical of the present invention, the DPS-like protein fused to the ligand may be derived from any biological species.

[0046] When used in combination with a T-cell costimulatory factor agonist, the medicament of the present invention can exert an anticancer effect even on cancer tissues located at sites distant from the target cancer tissue. Specifically, for example, when the medicament of the present invention and a T-cell costimulatory factor agonist are locally administered into a tumor, the medicament not only inhibits the growth of the tumor to which the drug is administered (target tumor), but also exerts a growth-inhibitory effect on tumors located at sites distant from the target tumor that have not been administered anything (non-target tumor) (see Examples).

[0047] The mRNA serving as the active ingredient of the pharmaceutical of the present invention may be synthesized to contain components for regulating intracellular stability and protein production. Examples of such components include a 5'-end cap structure, an untranslated region (UTR), and a poly(A) tail. Therefore, the pharmaceutical of the present invention may contain as an active ingredient an mRNA synthesized to incorporate a 5'-end cap structure, a 5' UTR, a 3' UTR, and / or a poly(A) sequence. The 5'-end cap structure of the mRNA may be an endogenous cap, a cap analog added during an in vitro transcription reaction, or a cap structure generated by enzymatic capping after transcription. The poly(A) sequence can be appropriately designed based on the overall length of the mRNA and the length of the final product expressed from the mRNA. The mRNA serving as the active ingredient of the pharmaceutical of the present invention may be an mRNA encoding a fusion protein, an mRNA derived from a codon-optimized gene, or an mRNA to which a sequence encoding a signal peptide has been added or substituted. In the pharmaceutical of the present invention, the mRNA encoding a fusion protein may be an mRNA encoding two or more proteins linked via a sequence encoding a linker. In the pharmaceutical of the present invention, the mRNA may be an mRNA to which a sequence encoding a tag has been added.

[0048] The pharmaceutical composition of the present invention may be used in combination with a T cell costimulatory factor agonist and a CD4+ T cell depleting antibody or a CD25+ T cell depleting antibody. Examples of CD4+ T cell depleting antibodies include the humanized anti-CD4 antibody "IT1208" (J Immunother Cancer. 2019 Jul 24;7(1):195. doi: 10.1186 / s40425-019-0677-y), "MAX.16H5" (Front Immunol. 2019 May 24;10:1035. doi: 10.3389 / fimmu.2019.01035), and "cM-T412" (J Clin Invest. 1997 May 1;99(9):2225-31. doi: 10.1172 / JCI119396). Examples of CD25-positive T cell depleting antibodies include humanized anti-CD25 antibodies Daclizumab, RG6292, CHT-25, Inolimomab, and H3F1-4V2. Combining CD4-positive T cell depleting antibodies or CD25-positive T cell depleting antibodies can enhance the anti-cancer effect on target and non-target cancer tissues.

[0049] Cancers to be treated by the medicament of the present invention are not particularly limited, and examples include melanoma (malignant melanoma), Merkel cell carcinoma, lung cancer, mesothelioma, head and neck cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, pancreatic cancer, colon cancer, prostate cancer, kidney cancer, bladder cancer, urothelial cancer, breast cancer, uterine cancer, ovarian cancer, brain tumor, thyroid cancer, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, synovial sarcoma, liposarcoma, neuroendocrine tumor, lymphoma, leukemia, myeloma, etc. Preferably, the cancer is one that forms a solid tumor.

[0050] The pharmaceutical compositions of the present invention can be formulated according to conventional methods. For example, injections are used as formulations for local administration. Injections include intravenous, subcutaneous, intradermal, intramuscular, infusion, and intra-articular injections. Such injections are prepared according to known methods, for example, by dissolving, suspending, or emulsifying the active ingredient in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50). Examples of oily solutions include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The preparations thus obtained are safe and have low toxicity, and can be administered topically to, for example, humans and mammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

[0051] The medicament of the present invention is preferably dissolved in phosphate-buffered saline (PBS) containing calcium ions and administered locally to tumor tissue. The concentration of calcium ions in the medicament of the present invention is not particularly limited, and may be 1 to 10,000 μg / mL, 50 to 5,000 μg / mL, 100 to 1,000 μg / mL, 150 to 750 μg / mL, or 200 to 500 μg / mL. It is preferably 100 to 1,000 μg / mL, more preferably 150 to 750 μg / mL, and even more preferably 200 to 500 μg / mL.

[0052] The medicament of the present invention may contain 0.001 to 50% by mass, preferably 0.01 to 10% by mass, and more preferably 0.1 to 1% by mass of the active ingredient. The dosage of the medicament of the present invention is appropriately determined by a doctor or medical professional, taking into consideration the type of cancer, severity of the disease, age, weight, sex, medical history, etc. of the patient. The total daily dosage may be a single dose or divided doses. For example, for a tumor with a volume of 1000 mm, 3 The amount may be 150 μg or more, 300 μg or more, 450 μg or more, or 600 μg or more, or 1000 μg or less, 800 μg or less, 650 μg or less, 480 μg or less, or 320 μg or less per unit area. Preferably, it is 150 μg or more. More preferably, it is 300 μg or more.

[0053] [Immunostimulating agent] The present invention provides an immunostimulating agent containing, as active ingredients, mRNA encoding an apolipoprotein and mRNA encoding a T cell costimulatory factor agonist (hereinafter referred to as "the immunostimulating agent of the present invention").

[0054] The immunostimulant of the present invention is preferably dissolved in PBS containing calcium ions and administered locally to tumor tissue. The concentration of calcium ions in the immunostimulant of the present invention is not particularly limited, and may be 1 to 10,000 μg / mL, 50 to 5,000 μg / mL, 100 to 1,000 μg / mL, 150 to 750 μg / mL, or 200 to 500 μg / mL. It is preferably 100 to 1,000 μg / mL, more preferably 150 to 750 μg / mL, and even more preferably 200 to 500 μg / mL.

[0055] The immunostimulant of the present invention can be used to treat cancer. For example, the immunostimulant of the present invention can be suitably used to treat melanoma (malignant melanoma), Merkel cell carcinoma, lung cancer, mesothelioma, head and neck cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, pancreatic cancer, colon cancer, prostate cancer, kidney cancer, bladder cancer, urothelial cancer, breast cancer, uterine cancer, ovarian cancer, brain tumor, thyroid cancer, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, synovial sarcoma, liposarcoma, neuroendocrine tumor, lymphoma, leukemia, myeloma, and the like. Cancers that form solid tumors are preferred. The immunostimulant of the present invention can also be suitably used to treat infectious diseases. For example, it can be suitably used for the treatment of contagious impetigo, necrotizing fasciitis, cellulitis, osteomyelitis, meningitis, intractable skin ulcers (such as diabetic ulcers), tuberculosis, tinea, sexually transmitted diseases, herpes (herpes labialis, herpes genitalis), shingles, etc.

[0056] The immunostimulant of the present invention can be produced and used in the same manner as the above-mentioned medicament of the present invention.

[0057] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0058] 1. Materials and Methods 1-1 Preparation of Plasmid The pmRVac3-Luc2 plasmid was synthesized by VectorBuilder. The pmRVac3-Luc2 plasmid contained a T7 promoter with an AGG sequence (TAATACGACTCACTATAAGGAGA; SEQ ID NO: 7), Cap-1 (Clean Cap Reagent AG-(N-7113), TriLink BioTechnologies), a β-globulin leader sequence as the 5'UTR, codon-optimized luciferase 2 (Luc2), the 3'UTR of the amino terminal enhancer of split (AES) and mtRNR1 (Orlandini von Niessen AG et al., Molecular Therapy 2019, 27, 824-836), and a poly(A) sequence (AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA; SEQ ID NO: 8). Furthermore, a SapI recognition sequence was incorporated at the end of the poly(A) sequence to allow linearization without additional nucleotides. Luc2, OX40 ligand (OX40L), IL36γ, IL23, apolipoprotein D (ApoD), ApoL9a, OX40L-fused ferritin (OX40L-ferritin), BM40-OX40L-fused ferritin (BM40-OX40L-Fr), BM40-4-1BB ligand-fused ferritin (BM40-4-1BB-L-Fr), 4-1BB ligand-fused DPS-like protein (4-1BB-L-DPS), CD70-fused ferritin, GITRL-fused ferritin, and TNF-fused ferritin were synthesized by standard methods and introduced into the pmRVac3 plasmid.

[0059] 1-2 In vitro transcription: The pmRVac3 plasmid was linearized with SapI. The linearized plasmid was purified using the Monarch PCR & DNA cleanup kit (T1030L, New England BioLabs). mRNA was generated using the CUGA 7 in vitro Transcription Kit (307-13531, Nippon Gene Co., Ltd.) and N1-Methylpseudouridine-5'-Triphosphate (N-1081, TriLink BioTechnologies). After in vitro transcription, the template DNA was digested with DNase solution, and mRNA was purified using the PureLink RNA Mini Kit (12183018A, Thermo Fisher Scientific). The quality of the synthesized mRNA was analyzed using an Agilent 4150 TapeStation system (G2992AA, Agilent Technologies).

[0060] 1-3 Preparation of mRNA for Intratumoral Injection. After washing the mRNA twice with 70% ethanol, 2 μg of mRNA was mixed with 2 μl of in vivo-jetRNA (Polyplus, 101000013) in 50 μl of mRNA buffer. The mRNA was used in combination with ion-supplemented PBS, sucrose-citrate, calcium phosphate, cationic gelatin, or serum. Ion-supplemented PBS was prepared by mixing 10x PBS with 5 mg / mL CaCl2, MgCl2, or Zn(CH3COO)2. Sucrose-citrate was prepared to a final concentration of 0.1 g / mL sucrose and 33.3 mM citric acid (pH 7.0). Calcium phosphate was prepared to contain 124 mM CaCl2, 25 mM HEPES (pH 7.0), 140 mM NaCl, and 0.75 mM Na2HPO4. Ten μg of cationic gelatin was mixed with 5 μg or 10 μg of mRNA in 50 μl of PBS. Serum concentrations were measured using a BioDrop (BioDrop). 200 μg of serum was mixed with mRNA and 2 U / μl of RNase inhibitor (F83923-1, BIOSEARCH TECHNOLOGIES).

[0061] 1-4 Cell Culture B16F10 (CRL-6475), LL / 2 (CRL-1642), 4T1 (CRL-2539), and CT26 (CRL-2638) cells were purchased from the American Type Culture Collection. MC38 (ENH204) cells were purchased from Kerafast. B16F10, LL / 2, and MC38 cells were cultured in DMEM medium (08458-45, Nacalai Tesque) containing 10% FBS (172012, Sigma), 100 U / mL penicillin, and 100 μg / mL streptomycin (penicillin-streptomycin mixed solution, 26253-84, Nacalai Tesque). 4T1 and CT26 cells were cultured in RPMI 1640 medium (30264-56, Nacalai Tesque, Inc.) containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. HT1080 cells (ECACC, catalog number: 85111505) were cultured in MEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cell culture was performed at 37°C, 95% humidity, and 5% CO2.

[0062] 1-5 In vivo studies All animal experiments were approved by the Animal Experiment Committee of Osaka University and the Animal Experiment Committee of Gunma University and were conducted in accordance with their guidelines. 6Cancer cells were injected intradermally into C57BL / 6N or BALB / cA mice. Five days after cancer cell inoculation, mRNA was injected intratumorally. CT26, MC38, or 4T1 cells were inoculated into 6-week-old BALB / c female mice. Five days later, 20 μg of the mRNA mixture was injected intratumorally into the CT26, MC38, or 4T1 tumors in combination with PBS and 200 μg / mL calcium ion. The following mRNA mixtures were used for intratumoral injection: mRNA encoding luciferase 2 (Luc2); a mixture of mRNAs encoding OX40 ligand (OX40L), IL36γ, and IL23; a mixture of mRNAs encoding apolipoprotein D (ApoD) and ApoL9a with mRNA encoding OX40L; mRNAs encoding ApoD and ApoL9a with OX40 antibody; and a mixture of mRNAs encoding ApoD and ApoL9a with mRNA encoding OX40L-ferritin. For repeated administration, the same mRNA was injected into the tumor again 2 and 6 days after the initial mRNA administration, or 2 and 4 days after the initial mRNA administration. Luciferase signals were measured using an IVIS Lumina II imaging system (PerkinElmer). D-luciferin (XLF-1, SPI Summit Pharmaceuticals International) was dissolved in PBS at a concentration of 30 mg / mL and 200 μL was administered intraperitoneally to mice to achieve a concentration of 150 μg of D-luciferin per gram of body weight. Luciferase signals were measured after incubating the mice at 37°C for 3 minutes under anesthesia and analyzed using Living Image 4.2 (PerkinElmer). Tumor size was measured every other day for 14 days using a digital caliper. Tumor volume was calculated using the following formula: tumor volume (mm 3) = major diameter × minor diameter × minor diameter × 1 / 2. The following antibodies were used: APC / Cyanine7-labeled anti-mouse CD45 antibody (product number 103116, BioLegend), Brilliant Violet 421-labeled anti-mouse CD45 antibody (product number 103134, BioLegend), FITC-labeled anti-mouse CD3 antibody (product number 100204, BioLegend), PerCP / Cyanine5.5-labeled anti-mouse CD3 antibody (product number 100218, BioLegend), Brilliant Violet 421-conjugated anti-mouse CD3 antibody (Cat. No. 100228, BioLegend), APC / Cyanine7-conjugated anti-mouse CD4 antibody (Cat. No. 100414, BioLegend), PE / Cyanine7-conjugated anti-mouse CD8a antibody (Cat. No. 100722, BioLegend), FITC-conjugated anti-mouse CD8a antibody (Cat. No. 100706, BioLegend), Brilliant Violet 510-conjugated anti-mouse CD8a antibody (Cat. No. 100752, BioLegend), PE-conjugated anti-mouse CD8a antibody (Cat. No. 100708, BioLegend), APC-conjugated anti-mouse CD314 (NKG2D) antibody (Cat. No. 130212, BioLegend).

[0063] The following mRNA mixtures were also used for intratumoral injection: a mixture of mRNA encoding ApoD and ApoL9a, respectively, and mRNA encoding BM40-OX40L fusion ferritin (BM40-OX40L-ferritin); mRNA encoding ApoD and ApoL9a, respectively, and 4-1BB antibody; mRNA encoding ApoD and 4-1BB antibody; mRNA encoding ApoL9a and 4-1BB antibody; a mixture of mRNA encoding ApoD and ApoL9a, respectively, and mRNA encoding 4-1BB-L fusion DPS; mRNA encoding ApoD and ApoL9a, respectively, and mRNA encoding CD70 fusion ferritin; mRNA encoding ApoD and ApoL9a, respectively, and mRNA encoding GITRL fusion ferritin; and mRNA encoding ApoD and ApoL9a, respectively, and mRNA encoding TNF fusion ferritin.

[0064] The antibodies administered intratumorally were Ultra-LEAF Purified anti-mouse CD134 (OX-40) clone: ​​OX-86 (Biolegend 119431), an agonist antibody against the T cell costimulator OX40, and InVivoMAb anti-mouse 4-1BB (CD137) clone LOB12.3 (BioXCell BE0169). The anti-CD4 antibody used to deplete CD4+ T cells was InVivoMAb anti-mouse CD4 clone GK1.5 (BioXCell BE0003-1), the anti-CD25 antibody used to deplete CD25+ T cells was anti-mouse CD25 (IL-2Rα)-InVivo (Selleck A2107), and the anti-CD8 antibody used to deplete CD8+ T cells was InVivoMAb anti-mouse CD8α (2.43) clone LOB12.3 (BioXCell BE0061). The dose of each antibody was 10 μg.

[0065] 1-6 Measurement of z-average, circular dichroism (CD), and optical absorption spectra of nanoparticles. Five μL of a mixture of 0.4 μg mRNA (200 μg / mL) and 1×PBS was diluted 100-fold with 200 μg / mL or 500 μg / mL CaCl2 and 1×PBS. After incubation at room temperature for 15 minutes, measurements were performed using a Zetasizer nano ZS (Malvern Panalytical).

[0066] 1-7 Analysis of nanoparticles using transmission electron microscopy. This was performed at the Hanaichi Institute of Electron Microscopy. 5 μL of mRNA suspended in PBS or mRNA suspended in a mixture of PBS and 200 μg / mL CaCl2 was added to a 400-mesh copper grid coated with a carbon support film and incubated at room temperature for 30 seconds. Excess liquid was removed with filter paper, and the grid was washed twice for 10 seconds on a droplet of distilled water. Negative staining of the grid was performed on a droplet of 2% uranyl acetate solution for 10 seconds. The grid was then air-dried after removing excess liquid. Sample analysis was performed using a transmission electron microscope (H-7600, Hitachi High-Tech Corporation) at an accelerating voltage of 100 kV.

[0067] 1-8 RNase protection assay: 200 ng of mRNA was incubated with PBS, PBS and 200 μg / mL CaCl2, or saline for 10 minutes. The mixture was then incubated with 0.25 ng / μl RNase A at room temperature for 10 minutes. The mRNA was purified with TRIzol (Thermo Fisher Scientific) and analyzed using an Agilent 4150 TapeStation system (G2992AA, Agilent Technologies).

[0068] 1-9 Statistical Analysis Normality of data was assessed using the Shapiro-Wilk test. Equality of variance between two samples was confirmed using a two-tailed F test. Nonparametric data were analyzed using the Wilcoxon rank sum test to compare two samples. Multigroup comparisons were performed using one-way analysis of variance and Tukey's HSD test. Error bars indicate standard deviations, and calculations were performed in triplicate using data analysis software (JMP Pro 13, JMP).

[0069] 2. Results [Example 1: Evaluation of Materials for Introducing mRNA into Tumor Cells] Figure 1A shows a schematic diagram of mRNA encoding the luciferase 2 (luc2) gene. mRNA encoding the Luc2 gene was produced by in vitro transcription with Cap-1 and 1-methylpseudouridine (Figure 1A). A poly(A) sequence was encoded in the template plasmid. Figure 1B shows the results of electrophoresis of the mRNA produced by in vitro transcription to confirm its integrity. Figure 2A shows the results of measuring the region of interest (ROI) values ​​using an in vivo imaging system. The numbers in parentheses indicate the number of tumors. P values ​​were calculated using the Wilcoxon test. Several studies have reported that saline and sucrose promote mRNA transfection into cells in vivo. Therefore, we investigated the effects of ultrapure water (MILLI-Q®, Merck), glycerol, cationized gelatin, saline, PBS, sucrose, serum, and the in vivo mRNA transfection reagent in vivo-jetRNA (Polyplus) (Figure 2A–E). The luciferase signal using MILLI-Q® was less than 10,000 ROIs. While 15% glycerol allowed mRNA delivery into tumors, 30% glycerol prevented it. Cationized gelatin did not promote mRNA transfection. Saline, PBS, and sucrose promoted mRNA transfection, reaching approximately 100,000 ROIs. PBS, in particular, provided more stable mRNA delivery than saline or sucrose. Serum degraded a portion of the mRNA (Fig. 3), preventing its delivery into tumors. Although in vivo-jetRNA demonstrated high efficiency in mRNA transfection, luciferase expression was not observed in some tumors.

[0070] Figure 4 shows the results of comparing the mRNA transfection efficiency of in vivo-jetRNA and PBS in MC38 tumors (C57BL / 6 mice), LL2 tumors (C57BL / 6 mice), and 4T1 tumors (BALB / c mice). In MC38 tumors (Figure 4A), LL2 tumors (Figure 4B), and 4T1 tumors (Figure 4C), administration of PBS and mRNA resulted in stronger luciferase signals than administration of in vivo-jetRNA and mRNA. However, the transfection efficiency of in vivo-jetRNA was suggested to be tumor type-dependent. Furthermore, administration of 2 μg or more of mRNA using in vivo-jetRNA induced an inflammatory response. These results demonstrate that PBS is suitable for mRNA transfection in a variety of tumors.

[0071] Reference Example 1: Evaluation of Lipid Nanoparticles Constituting COMIRNATY (Registered Trademark) 1. Materials The lipid nanoparticles constituting COMIRNATY (Registered Trademark, BioNTech SE), a vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are disclosed in JP 2023-526178 and other publications. ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldocanoate) (ALC-0315, SINOPEG), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC, Nippon Fine Chemicals), cholesterol (Nippon Fine Chemicals), and MPEG2000-DMG (SINOPEG) were dissolved in ethanol at a molar ratio of 50:10:39.5:1.5. mRNA was prepared in 25 mM acetate buffer (pH 4.0). LNP lipid solution (20 mg / mL), DOTAP solution (10 mg / mL), and mRNA solution (0.26 mg / mL) were mixed in a volume ratio of 1:0.14:3 using a microfluidic mixer. The final flow rate was 14.18 mL / min (3.6 mL / min LNP lipid solution, 0.5 mL / min DOTAP solution, and 10.08 mL / min mRNA solution). The LNP mixture was concentrated to approximately 0.2 mg / mL by dialysis using Amicon Ultra (UFC910024, 100 kD MWCO, Merck) and filtered through a 0.22 μm PES filter (16532--K, Sartorius) for intratumoral administration.

[0072] 2. Results Figure 5 shows the results of measuring luciferase signals after intratumoral injection of lipid nanoparticles (LNPs) comprising COMIRNATY® and mRNA encoding luciferase 2, as used in Example 1, into BALB / c mice bearing CT26 tumors and C57BL / 6 mice bearing B16F10 tumors. The white outline indicates the location of the tumor. While a significant amount of luciferase signal was observed at the tumor site, signals were also detected in the draining lymph nodes (BALB / c mice, C57BL / 6N mice), liver (BALB / c mice, C57BL / 6N mice), and spleen (BALB / c mice) (Figures 5A, 5B, and 5C). These results suggest that target-cell-specific delivery of mRNA using LNPs comprising COMIRNATY® is difficult.

[0073] Example 2: Evaluation of repeated intratumoral injections. The effect of repeated intratumoral injections of mRNA on intratumoral luciferase signal enhancement was examined. Figure 6 shows the results of measuring ROI values ​​in B16F10 tumors in C57BL / 6N mice using an in vivo imaging system. The gray line indicates the signal in each tumor, and the black line indicates the average. Following a single administration of mRNA and PBS, the luciferase signal level was unstable (Figure 6A). Following three administrations of mRNA and PBS, the luciferase signal level stabilized and was detected within the same intensity range (Figure 6B). These results suggest that repeated administration into the tumor stabilizes expression levels more than a single administration.

[0074] Example 3: Evaluation of luciferase expression efficiency in B16F10 tumors. Calcium ions have been reported to be involved in mRNA transfection in vivo (Probst J et al., Gene Ther 2007, 14, 1175-1180). The effect of divalent ions on mRNA transfection efficiency in B16F10 tumors was examined. The results of measuring ROI values ​​in B16F10 tumors in C57BL / 6 female mice are shown in Figure 7A. The numbers in parentheses indicate the number of tumors. P values ​​were calculated using the Wilcoxon test. PBS containing 1, 5, 10, or 20 μg of mRNA and any divalent ion (500 μg / mL calcium, magnesium, or zinc ions) was injected intratumorally. The strongest signal was measured when 20 μg of mRNA was administered (Figure 7A). Regarding the combination of any divalent ion, the strongest signal was measured when the mRNA was administered in combination with PBS containing calcium ions (Figure 7A). Figure 7B shows the results of intratumoral injection of 20 μg or 40 μg of mRNA into B16F10 tumors in C57BL / 6 female mice. The numbers in parentheses indicate the number of tumors. P values ​​were calculated by T-test. Equivalent luciferase signals were measured after administration of 20 μg and 40 μg of mRNA (Figure 7B). This result suggests that mRNA expression in B16F10 tumors was nearly saturated after administration of 20 μg of mRNA. These results suggest that the combination of calcium ions and PBS is preferable for mRNA delivery into tumor cells. The tumor volume was 62.5 mm 3 Therefore, the maximum amount of mRNA for intratumoral injection was 1000 mm2. 3 It was suggested that the mean value was 320 μg per 1000 mg / kg.

[0075] We investigated the optimal calcium ion concentration for mRNA transfection into B16F10 tumors. Figure 7C shows the ROI values ​​measured in B16F10 tumors from C57BL / 6 female or male mice. The numbers in parentheses indicate the number of tumors. P values ​​were calculated using the Wilcoxon test. Intratumoral injection of 20 μg of mRNA in PBS containing calcium ions resulted in a stronger luciferase signal than that observed in PBS alone (Figure 7C). When compared with PBS containing 200, 500, or 1000 μg / mL of calcium ions, the combination with PBS containing 500 μg / mL consistently produced a stronger signal. The calcium phosphate method is a well-known in vitro DNA transfection technique. However, intratumoral injection of mRNA using the calcium phosphate method in B16F10 tumors did not result in efficient induction of luciferase activity. On the other hand, when combined with PBS containing 500 μg / mL of calcium ions, a stronger signal was measured compared to the control (PBS only) (FIG. 7C).

[0076] Example 4: Evaluation of luciferase expression efficiency in CT26 tumors. Luciferase signals after intratumoral injection of mRNA were compared in CT26 tumors in BALB / c mice. The results of measuring ROI values ​​are shown in Figure 8A. The numbers in parentheses indicate the number of tumors. P values ​​were calculated using the Wilcoxon test. Compared with the combination of saline or in vivo-jetRNA, the combination of PBS and mRNA produced an enhanced luciferase signal (Figure 8A). Intratumoral injection of a combination of PBS and 20 μg mRNA produced a more stable signal than the combination of 30 or 40 μg mRNA (Figure 8A). Furthermore, the tumor volume was 62.5 mm 3 These results suggest that intratumoral injection of a combination of PBS and mRNA is effective for tumors with a tumor volume of 1000 mm. 3The results show that 320 μg of mRNA per tumor is preferable. Figure 8B shows the time-dependent changes in ROI values ​​after intratumoral injection of PBS and mRNA in CT26 tumors in female BALB / c mice. Luciferase signals were still detectable 4 days after injection, but a time-dependent decline was observed (Figure 8B).

[0077] Figure 8C shows the results of the enhancement of luciferase signal by the combination of PBS and calcium ions in CT26 tumors in BALB / c mice. The numbers in parentheses indicate the number of tumors. P values ​​were calculated using the Wilcoxon test. In female BALB / c mice, no enhancement of luciferase signal was detected by the combination of PBS and calcium ions (Figure 8C). A weaker signal was detected in CT26 tumors in male mice than in CT26 tumors in female mice. In male mice, the combination of PBS and 200 μg / mL calcium ions enhanced luciferase signal compared with PBS alone, but the combination of PBS and 500 or 1000 μg / mL calcium ions did not enhance the signal (Figure 8C). In mice receiving intratumoral injections of mRNA with the combination of PBS and calcium ions, significant luciferase signal was detected in the tumor site, but not in other tissues (Figure 9).

[0078] Example 5: Evaluation of the effect of the combination of PBS and calcium ions on mRNA structure. Hori et al. reported that calcium ions and serum promote nanoparticle formation and increase the cellular uptake of oligonucleotides, but that such nanoparticle formation was not observed when plasmid DNA was used (Hori S et al., Nucleic Acids Res 2015, 43, e128). In the present invention, we investigated whether the combination of mRNA with PBS and calcium ions promotes nanoparticle formation. The z-average diameter (nm) measured by particle size analysis using dynamic light scattering is shown in Figure 10A. ND indicates not detected. Nanoparticles were detected when mRNA was combined with PBS and calcium ions, and larger nanoparticles were observed with the addition of 500 μg / mL calcium ions than with the addition of 200 μg / mL calcium ions (Figure 10A). Nanoparticles were not detected with PBS alone, the combination of PBS and calcium ions, or the combination of PBS and mRNA. No nanoparticles were detected when PBS containing 200 or 500 μg / mL zinc ions or magnesium ions was combined with mRNA (Figure 10A). These results indicate that the combination of mRNA with PBS and calcium ions promotes the formation of nanoparticles, suggesting that such structures may be useful for mRNA uptake into cancer cells.

[0079] The structure of mRNA in buffer solution was examined using circular dichroism (CD) spectroscopy, as shown in Figures 10B and 10C. Folded RNA exhibits a high CD spectrum around 260–280 nm, while unfolded RNA exhibits a low optical absorption spectrum around 250–270 nm. It is also known that right-handed A-form RNA and left-handed Z-form RNA exhibit different CD spectra. Analysis of the mRNA CD spectrum indicated that the mRNA was unfolded in water (Figure 10B). When PBS alone, calcium ions alone, and a combination of PBS and calcium ions were used as buffers, the mRNA was folded (Figure 10B). The optical absorption spectrum of mRNA was highest in water (Figure 10C). This result is consistent with the CD spectral pattern. These results suggest that mRNA is unfolded in water and folded in other buffers.

[0080] Figure 11 shows the results of analyzing the structure of mRNA in buffer using a transmission electron microscope (TEM). Nanoparticle structures were observed when mRNA was combined with PBS and 200 μg / mL calcium ions (Figure 11B), but not when mRNA was combined with PBS alone (Figure 11A). Figure 11C shows the electrophoresis results of purified mRNA after RNase treatment. Compared to when physiological saline or PBS alone was used, when PBS and calcium ions were used, the combination was confirmed to be resistant to RNase A (Figure 11C). These results indicate that using a combination of PBS and calcium ions as a buffer for mRNA promotes the formation of nanoparticle structures.

[0081] Example 6: Analysis of Antitumor Effect. It has been reported that the administration of a combination of mRNA encoding OX40 ligand (OX40L), IL36γ, and IL23 in lipid nanoparticles (LNPs) exhibits antitumor effects (Hewitt SL et al., Science Translational Medicine, 2019, 11, eaat9143). Figure 12 shows the results of an analysis of the antitumor effect of using a combination of PBS and calcium ions as an mRNA buffer in the present invention. The number in parentheses indicates the number of samples. P values ​​were calculated using the Wilcoxon test. Error bars indicate standard deviation. Three intratumoral injections of mRNA encoding OX40L, IL36γ, and IL23 (total 20 μg) in combination with PBS and calcium ions in BALB / c mouse CT26 tumors significantly suppressed tumor growth compared to the administration of control mRNA (Figure 12, p=0.0281). These results demonstrate that intratumoral administration of mRNA in combination with PBS and calcium ions exhibits significant antitumor effects.

[0082] The present inventors have confirmed that intratumoral injection of apolipoprotein D (ApoD) and / or ApoL9 in combination with an OX40 agonist antibody results in tumor volume reduction and activation of intratumoral T cells (International Publication No. WO 2022 / 059703A). The antitumor effects of intratumoral injection of a combination of mRNA encoding ApoD and ApoL9, respectively, with mRNA encoding OX40L or OX40-ferritin, dissolved in calcium ion-containing PBS, or mRNA encoding ApoD and ApoL9, respectively, with an OX40 antibody, dissolved in calcium ion-containing PBS, were analyzed. Schematic diagrams of the mRNA encoding ApoD, mRNA encoding ApoL9, and mRNA encoding OX40L-fused ferritin (OX40L-ferritin) used in this example are shown in Figure 13. The results of tumor volume measurements are shown in Figure 14. The numbers in parentheses indicate the number of samples. P values ​​were calculated using the Wilcoxon test. Error bars indicate standard deviation. In BALB / c mouse CT26 tumors, three intratumoral injections of a mixture of mRNA encoding ApoD and ApoL9a and mRNA encoding OX40L-ferritin (total 20 μg) significantly suppressed tumor growth compared with control mRNA (Fig. 14A, p=0.02). In BALB / c mouse 4T1 tumors, three intratumoral injections of a mixture of mRNA encoding ApoD and ApoL9 and mRNA encoding OX40L-ferritin (total 20 μg) significantly suppressed tumor growth compared with control mRNA (Fig. 14B, p<0.0001).

[0083] Figure 15 shows the results of analyzing intratumoral T cell proliferation after intratumoral administration of mRNA. Luciferase 2 (Luc2)-encoding mRNA, a mixture of OX40L-encoding mRNA and OX40L-encoding mRNA, or a mixture of OX40L-ferritin-encoding mRNA and ApoD-encoding mRNA and ApoL9a-encoding mRNA were injected intratumorally in combination with PBS and calcium ions. Tumors were excised on day 14, and the numbers of CD4+ and CD8+ T cells per 100,000 cells in the target tumor were analyzed by FACS. Intratumoral administration of a mixture of OX40L-ferritin-encoding mRNA and ApoD-encoding mRNA and ApoL9a-encoding mRNA significantly increased the numbers of CD4+ and CD8+ T cells in the tumor (Figure 15).

[0084] Figure 16 shows the results of analyzing T cell activation after intratumoral administration of mRNA. Control mRNA, mRNA encoding ApoD / ApoL9a / OX40L, mRNA encoding ApoD / ApoL9a / OX40-ferritin, or mRNA encoding ApoD / ApoL9a, combined with OX40 antibody (OX40 ab), PBS, and calcium ions were injected intratumorally. Tumors were excised on day 14, and the numbers of NKG2D (natural killer group 2 member D)-positive cells in CD4+ T cells and CD8+ T cells were measured by FACS. The present inventors have previously demonstrated that the interaction between NKG2D on T cells and the NKG2 ligand on cancer cells is essential for the systemic antitumor effect of the combined use of Sendai virus envelope (hemagglutinating virus of Japan envelope; HVJ-E) and OX40 antibody, and that the systemic antitumor effect of HVJ-E and OX40 antibody was attenuated by the use of NKG2D-blocking antibodies. When the percentages of NKG2D-positive cells relative to total CD4+ or CD8+ T cells were compared, the percentage of NKG2D-positive T cells was significantly increased, particularly when a mixture of mRNA encoding ApoD and ApoL9a and mRNA encoding OX40L-ferritin was administered intratumorally (Fig. 16). These results demonstrate that intratumor administration of a mixture of mRNA encoding ApoD and ApoL9a and mRNA encoding OX40L-ferritin in combination with PBS and calcium ions exerted significant antitumor and immunostimulatory effects.

[0085] Example 7: Systemic Antitumor Effect of Apolipoprotein mRNA and T Cell Costimulatory Factor-Fused Ferritin mRNA. Ferritin, which self-assembles to form a 24-mer globular protein, was combined with the OX40 / 4-1BB ligand to design a protein carrying eight trimers of OX40 ligands (OX40L-Fr) and a protein carrying eight trimers of 4-1BB ligands (4-1BB-L-Fr). These mRNAs were then administered intratumorally. The results of evaluating the OX40-stimulating activity of OX40L-Fr mRNA are shown in Figure 17A. Control mRNA, OX40L mRNA, and OX40L-Fr mRNA were transfected into B16F10 cells, and the culture supernatants were administered in equal amounts to OX40-expressing cells (HT1080 cells expressing OX40). An OX40 antibody (1 μg / mL) was administered as a positive control. OX40-expressing cells secrete IL8 upon OX40 stimulation. When OX40-stimulating activity was evaluated using the amount of IL8 secreted as an indicator, OX40L-Fr exhibited high activating activity despite secreting less IL8 than OX40L in the B16F10 cell culture supernatant.

[0086] Figure 17B shows the results of intratumoral administration of OX40L-Fr mRNA in combination with ApoD mRNA and ApoL9a mRNA. CT26 cells were implanted into both sides of mice to form two CT26 tumors. mRNA encoding ApoD, ApoL9a, and OX40L-Fr, respectively, was administered in combination with PBS and calcium ions into one tumor. Tumor growth was significantly suppressed in both the target and non-target tumors. Furthermore, the results of flow cytometry analysis of tumor-infiltrating T cells are shown in Figure 18. Intratumoral administration of OX40L-Fr mRNA, ApoD mRNA, and ApoL9a mRNA in combination with PBS and calcium ions demonstrated an increase in CD4+ and CD8+ T cells in both the target and non-target tumors.

[0087] Example 8: Examination of systemic antitumor effects of apolipoprotein mRNA and T cell costimulatory factor agonist antibody In mice bearing two CT26 tumors, ApoD mRNA and ApoL9a mRNA (10 μg each) and an OX40 agonist antibody, ApoD mRNA and ApoL9a mRNA (10 μg each) and a 4-1BB agonist antibody (10 μg), or ApoD mRNA and ApoL9a mRNA and self-assembling 4-1BB-L ferritin mRNA (BM40-4-1BB-L-Fr mRNA (total 20 μg)) were administered in combination with PBS and calcium ions into one tumor, and tumor growth was measured in the target tumor ( FIG. 19 ) and non-target tumor ( FIG. 20 ). In all combinations, both the target tumor and the non-target tumor showed no significant changes compared with the control (Luc2 These results demonstrate that intratumoral administration of a mixture of apolipoprotein-encoding mRNA and a T cell costimulatory factor agonist in combination with PBS and calcium ions exerts significant antitumor and immunostimulatory effects in both targeted and non-targeted tumors.

[0088] Example 9: Investigation of the involvement of CD4-positive T cells, CD25-positive T cells, and CD8-positive T cells in the systemic antitumor effect An experiment was conducted to clarify the roles played by CD4-positive T cells, CD25-positive T cells, and CD8-positive T cells in the antitumor effect against non-target tumors when a combination of mRNA encoding an apolipoprotein and a T-cell costimulatory factor agonist antibody was administered intratumorally in combination with PBS and calcium ions.

[0089] MC38 cells were implanted into both sides of mice to form two MC38 tumors. Luc2-encoding mRNA, or ApoD mRNA (10 μg) and ApoL9a mRNA (10 μg), 4-1BB agonist antibody (10 μg), and CD4+ T cell-depleting antibody (10 μg) were administered into one of the tumors in combination with PBS and calcium ions. The results are shown in Figure 21. Administration of the antibody in combination with the CD4+ T cell-depleting antibody significantly suppressed tumor growth in both target and non-target tumors.

[0090] In mice bearing two MC38 tumors, we administered Luc2 mRNA, ApoD mRNA, ApoL9a mRNA, a 4-1BB agonist antibody, and a CD25+ T cell-depleting antibody in combination with PBS and calcium ions into one tumor. Tumor growth was compared in the target tumor (Figure 22) and non-target tumors (Figure 23). For each administration, the total amount of mRNA was 20 μg, and the amount of antibody was 10 μg. Local depletion of CD25+ T cells was shown to enhance the anti-tumor effect in both the target tumor and non-target tumor, similar to that observed with CD4+ T cell-depleting antibodies. Anti-tumor effects were also induced when ApoD mRNA or ApoL9a mRNA and a 4-1BB antibody were administered in combination with PBS and calcium ions. On the other hand, local depletion of CD8-positive T cells with a CD8-depleting antibody abolished the antitumor effect. These results indicate that intratumoral administration of apolipoprotein-encoding mRNA and a T cell costimulatory factor agonist in combination with PBS and calcium ions induces tumor immunity in target tumors, and further transfers tumor immunity to non-target tumors, resulting in a systemic antitumor effect.

[0091] Example 10: Evaluation of expression of T cell costimulatory factor-fused DPS and TNF superfamily-fused ferritin. MC38 cells were transfected with mRNA encoding a fusion protein of 4-1BB-L with a DPS-like protein derived from Aeropyrum pernix (ap; A0A401H889), Pyrococcus furiosus (pf; 7STW), Sulfolobus solfataricus (ss; 2CLB), or Thermotoga maritima (tm; 1VLG). Western blotting of the protein secreted into the supernatant was performed using a His tag. The results are shown in Figure 24. Secretion of the 4-1BB ligand fused with a DPS-like protein derived from Sulfolobus solfataricus (denoted as "ss" in Figure 24) or Thermotoga maritima (denoted as "tm" in Figure 24) into the supernatant was confirmed.

[0092] MC38 cells were transfected with mRNA encoding ferritin fused with CD70, GITRL, or TNF, a TNF superfamily gene, and the proteins secreted into the supernatant were analyzed by Western blotting using His tags. The results are shown in Figure 25. Secretion of all proteins into the supernatant was confirmed.

[0093] Example 11: Systemic Antitumor Effect of Combinations of Apolipoprotein mRNA with T Cell Costimulator-Fused DPS mRNA or TNF Superfamily-Fused Ferritin mRNA. Mice bearing two MC38 tumors were intratumorally administered a combination of mRNA encoding ApoD and ApoL9, respectively, and mRNA encoding 4-1BB-L-DPS, or a combination of mRNA encoding ApoD and ApoL9, respectively, and mRNA encoding ferritin fused with CD70, GITRL, or TNF, in combination with PBS and calcium ions. Tumor growth in the target tumor and non-target tumor was compared. Six mice were administered to each control group and each treatment group. The total amount of mRNA in each treatment was 20 μg. As a result of three intratumoral injections, 12 days after administration, the tumor diameters of the target and non-target tumors were reduced and tumor growth was significantly suppressed in the combination of mRNA encoding ApoD and ApoL9, respectively, and mRNA encoding 4-1BB-L-DPS, compared with the control mRNA (target tumor: 371 mm in the treatment group). 3 : Control group 494mm 3 , non-target tumor; treatment group 431.7mm 3 : Control group 609mm 3 Administration of a combination of mRNA encoding ApoD and ApoL9, and mRNA encoding ferritin fused with CD70, GITRL, or TNF, resulted in a smaller tumor diameter and significantly suppressed tumor growth compared to administration of control mRNA (target tumor; ApoD / ApoL9 / CD70-Fr group: 390 mm 3 , ApoD / ApoL9 / GITRL-Fr administration group 310mm 3 , ApoD / ApoL9 / TNF-Fr administration group 313mm 3 : Control group 494mm 3 , non-target tumor; ApoD / ApoL9 / CD70-Fr group: 456mm 3 , ApoD / ApoL9 / GITRL-Fr administration group 344mm 3 , ApoD / ApoL9 / TNF-Fr administration group 339mm 3 : Control group 609mm3 While no mice in the control group showed tumor disappearance, one mouse in the ApoD / ApoL9 / GITRL-Fr group and three mice in the ApoD / ApoL9 / TNF-Fr group showed tumor disappearance.

[0094] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A composition used for introducing messenger RNA into tumor tissue, comprising messenger RNA and phosphate-buffered saline containing calcium ions.

2. The composition according to claim 1, wherein the concentration of calcium ions is 100 to 1000 μg / mL.

3. The composition according to claim 1, which is for local administration to tumor tissue.

4. The composition of any one of claims 1 to 3, wherein the messenger RNA encodes an apolipoprotein or a T-cell costimulator agonist.

5. The composition of claim 4, wherein the T cell costimulatory factor agonist is OX40 ligand fusion ferritin or 4-1BB ligand fusion ferritin.

6. A pharmaceutical for treating cancer, which contains messenger RNA encoding an apolipoprotein as an active ingredient, and is dissolved in phosphate-buffered saline containing calcium ions and administered locally to tumor tissue.

7. The pharmaceutical for cancer treatment according to claim 6, wherein the calcium ion concentration is 100 to 1000 μg / mL.

8. A pharmaceutical for cancer treatment according to claim 6 or 7, used in combination with messenger RNA encoding a T-cell costimulatory factor agonist.

9. A pharmaceutical for cancer treatment according to claim 6 or 7, used in combination with a T-cell costimulatory factor agonist antibody.

10. The pharmaceutical for cancer treatment according to claim 8, wherein the T cell costimulatory factor agonist is OX40 ligand-fused ferritin or 4-1BB ligand-fused ferritin.

11. A pharmaceutical for cancer treatment according to claim 8 or 9, further comprising a CD4-positive T cell depleting antibody or a CD25-positive T cell depleting antibody in combination.

12. An immunostimulant characterized by containing as active ingredients messenger RNA encoding an apolipoprotein and messenger RNA encoding a T-cell costimulatory factor agonist, and being dissolved in phosphate-buffered saline containing calcium ions and administered locally to tumor tissue.

13. An immunostimulant characterized by containing messenger RNA encoding an apolipoprotein and a T-cell costimulatory factor agonist antibody as active ingredients, and being dissolved in phosphate-buffered saline containing calcium ions and administered locally to tumor tissue.

14. The immunostimulatory agent according to claim 12, wherein the T cell costimulatory factor agonist is OX40 ligand-fused ferritin or 4-1BB ligand-fused ferritin.

15. The immunostimulant according to claim 12 or 13, wherein the calcium ion concentration is 100 to 1000 μg / mL.

Images