Application of IL-12 and OX40L composition in tumor immunotherapy

Abstract

The invention discloses application of a composition in preparation of a medicine for treating tumors. The invention proves that when the mRNA molecule for coding the OX40 agonist protein and the mRNA molecule for coding the IL-12 protein are combined for use, a synergistic anti-tumor effect can be realized. This synergistic effect is further enhanced when a soluble portion of the OX40 ligand (OX40L) is used as an agonist rather than the full-length OX40L protein. In preferred cases, these mRNA molecules are synthetic molecules and are packaged in lipid nanoparticles for delivery. The mRNA molecules can be used for effectively inhibiting local and far-end tumor growth no matter through intratumor injection or other administration routes. Furthermore, when the mass ratio of IL-12 to OX40L is between 1: 1 and 1: 3, the composition shows lower toxicity along with the enhancement of the anti-tumor effect. When the GM-CSF is further added into the composition, the anti-tumor effect is further improved.

Description

background

[0001] Tumor immunity is a self-iterative cycle. Through continuous cycles, immune stimulatory molecules continue to accumulate, thereby enhancing and amplifying T cell responses. At the same time, inhibitory factors also play a role in it, suppressing the immune response through immune regulatory feedback mechanisms.

[0002] The tumor immune cycle can be divided into several key steps, starting with the release of antigens by tumor cells and ending with the killing of tumor cells. Tumor-associated antigens are presented by dendritic cells or antigen presenting cells (APCs), activating APCs and T cells. Activated T cells migrate to the tumor site, infiltrate tumor tissue, and recognize tumor cells, thereby executing a killing effect.

[0003] Each step of the tumor immune cycle involves the synergistic action of multiple molecules. Immune checkpoint proteins are a class of regulatory proteins present on the surface of immune cells that control the intensity and duration of immune responses by transmitting inhibitory or activating signals. These proteins act as "brakes" or "accelerators" in immune responses, ensuring that the immune system can effectively fight cancer while avoiding attacking normal tissues and triggering autoimmune diseases. For example, CTLA4 mainly inhibits immune responses by regulating T cell differentiation and proliferation. PD-L1 exerts an inhibitory effect by regulating activated immune responses in the tumor microenvironment.

[0004] Tumor immunotherapy (such as immune checkpoint blockade therapy and adoptive cell therapy) recognizes and attacks tumor cells by regulating the immune system, for example, by enhancing the effector function of tumor-specific effector T cells or weakening the inhibitory function of tumor-specific regulatory T cells.

[0005] Classic cancer immunotherapy uses antibodies that target checkpoint proteins or tumor-associated antigens, or modifies immune cells to express targeted receptors. However, antibodies usually have a short half-life, and adoptive cell therapy is extremely expensive to manufacture. summary

[0006] This study showed that mRNA encoding the OX40 ligand (OX40L) protein and mRNA encoding the IL-12 protein can achieve a synergistic antitumor effect when used in combination. This synergistic effect was further enhanced when the soluble portion of the OX40L protein was used as an agonist compared to the full-length OX40L protein. These mRNA molecules were synthesized and packaged in lipid nanoparticles for easy delivery. Whether injected intratumorally or by other routes, these mRNA molecules were able to effectively inhibit tumor growth in local and distant sites. In addition, when the mass ratio of IL-12 to OX40L was 1:1 to 1:3, this combination enhanced the antitumor effect while reducing toxicity. When GM-CSF was further added, the antitumor effect was further improved.

[0007] The present invention discloses an application in preparing a drug for treating tumors, comprising administering to a patient a first mRNA, the mRNA encoding an OX40 agonist, and a second mRNA, the mRNA encoding IL-12, wherein the OX40 agonist may be an OX40 ligand (OX40L), a polypeptide comprising an extracellular domain of OX40L, or an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

[0008] In some embodiments, the first mRNA and the second mRNA are contained in the same RNA molecule. In some embodiments, the polypeptide encoded by the RNA molecule can be a fusion protein comprising an OX40 agonist and IL-12, or encode different polypeptides respectively. In some embodiments, the first mRNA and the second mRNA are separate molecules.

[0009] In some embodiments, the OX40 agonist is OX40L. In some embodiments, the first The protein encoded by the mRNA comprises the amino acid sequence of SEQ ID NO: 1 or 2, or has at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the first The mRNA comprises the nucleic acid sequence of SEQ ID NO: 3 or 4, or its sequence has at least 85% sequence similarity to SEQ ID NO: 3 or 4.

[0010] In some embodiments, the extracellular domain of OX40L includes amino acid residues 52-183 of SEQ ID NO: 1, or has at least 85% sequence similarity with amino acid residues 52-183 of SEQ ID NO: 1. In some embodiments, the polypeptide further includes an oligomerization domain or a transmembrane domain. In some embodiments, the OX40 agonist is a fusion protein of the extracellular domain of OX40L and an Fc domain, wherein the extracellular domain of OX40 is a soluble protein without a transmembrane domain.

[0011] In some embodiments, the second mRNA encodes IL-12A. In some embodiments, the amino acid sequence of IL-12A is selected from the group consisting of amino acid residues 57-253 of SEQ ID NO:5, amino acid residues 57-239 of SEQ ID NO:6, amino acid residues 57-215 of SEQ ID NO:7, and Amino acid residues 23-219 of SEQ ID NO: 8, or an amino acid sequence having at least 85% sequence similarity to any of these groups. In some embodiments, the second mRNA comprises SEQ ID NO: NO:9, 10, 11 or 12, or a nucleic acid sequence having at least 85% sequence similarity to SEQ ID NO:9, 10, 11 or 12.

[0012] In some embodiments, the use further comprises administering a third mRNA to the patient, wherein the third mRNA encodes amino acid residues 23-328 of SEQ ID NO:13.

[0013] In some embodiments, the second mRNA encodes IL-12B. In some embodiments, IL-12B comprises amino acid residues 23-328 of SEQ ID NO: 13, or has at least 85% sequence similarity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the second mRNA comprises the nucleic acid sequence of SEQ ID NO: 14, or has at least 85% sequence similarity to the nucleic acid sequence of SEQ ID NO: 14.

[0014] In some embodiments, the mass ratio of the first mRNA to the second mRNA is 4: 1 to 0.5: 1. In some embodiments, the mass ratio of the first mRNA to the second mRNA is 3: 1 to 0.75: 1. In some embodiments, the mass ratio of the first mRNA to the second mRNA is 2: 1 to 0.9: 1.

[0015] In some embodiments, the use further comprises administering to the patient a fourth mRNA encoding GM-CSF (granulocyte-macrophage colony stimulating factor). In some embodiments, the use further comprises administering to the patient a fourth mRNA encoding TNFR (tumor necrosis factor receptor). In some embodiments, the use further comprises administering to the patient a fourth mRNA encoding GSDMD (GasderminD).

[0016] In some embodiments, the application does not include the administration of immune checkpoint inhibitors, interferons, other IL-12 family members, other cytokines, or nucleic acids encoding them. In some embodiments, immune checkpoint inhibitors are PD-1, PD-L1, or CTLA-4 inhibitors. In some embodiments, interferons are IFN-α, IFN-β, or IFN-γ. In some embodiments, other IL-12 family members include IL-23, IL-27, and IL-35. In some embodiments, other cytokines are IL-18.

[0017] Another embodiment also provides a use in the preparation of a drug for treating tumors, comprising administering to a patient a first agent comprising mRNA encoding IL-12 and a second agent comprising an OX40 agonist, wherein the OX40 agonist can be an anti-OX40 agonist antibody or an antigen-binding fragment thereof, an OX40 ligand (OX40L), or a polypeptide comprising an extracellular domain of OX40L. In some embodiments, the OX40 agonist is an anti-OX40 agonist antibody.

[0018] In some embodiments, each mRNA is a linear mRNA or a circular mRNA. In some embodiments, each mRNA further comprises a miRNA binding site. In some embodiments, each mRNA does not include a chemical modification that reduces immunogenicity. In some embodiments, the main chain of the mRNA does not include a chemical modification. In some embodiments, each mRNA only includes natural nucleosides.

[0019] In some embodiments, at least a portion of the uridine nucleosides in the mRNA are chemically modified. In some embodiments, the chemically modified uridine nucleosides are N1-methyl pseudouridine. In some embodiments, the first mRNA and the second mRNA are prepared together with a pharmaceutically acceptable carrier.

[0020] In some embodiments, the carrier comprises a lipid nanoparticle (LNP). In some embodiments, the LNP comprises (a) a molar ratio of 40-60% cationic lipid, a molar ratio of 8-16% phospholipid, a molar ratio of 30-45% cholesterol lipid and a molar ratio of 1-5% PEG-modified lipid; (b) a molar ratio of 45-65% cationic lipid, a molar ratio of 5-10% phospholipid, a molar ratio of 25-40% cholesterol lipid and a molar ratio of 0.5-5% PEG-modified lipid; (c) a molar ratio of 40-60% cationic lipid, a molar ratio of 8-16% phospholipid, a molar ratio of 30-45% cholesterol lipid and a molar ratio of 1-5% PEG-modified lipid. Modified lipids; (d) a molar ratio of 45-65% cationic lipids, a molar ratio of 5-10% phospholipids, a molar ratio of 25-40% cholesterol lipids and a molar ratio of 0.5-5% PEG-modified lipids; (e) a molar ratio of 40-60% cationic lipids, a molar ratio of 8-16% phospholipids, a molar ratio of 30-45% cholesterol lipids and a molar ratio of 1-5% PEG-modified lipids; or (f) a molar ratio of 45-65% cationic lipids, a molar ratio of 5-10% phospholipids, a molar ratio of 25-40% cholesterol lipids and a molar ratio of 0.5-5% PEG-modified lipids.

[0021] In some embodiments, each mRNA is packaged in a liposome. In some embodiments, the liposome comprises a cationic lipid, a non-cationic phospholipid, a cholesterol lipid, and a PEG-modified lipid.

[0022] In some embodiments, the cationic lipid is selected from the following group of compounds: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadienyl)bis(dodecan-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptatriac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)alanine (DLinDMA), 2-(2,2-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)alanine (DLinDMA), 1-(4-(6-(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl 3-(1H-imidazol-5-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecene-9,12-dien-1-yl)tetradecene-15,1 8-Dien-1-amine (HGT5000), (4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecene-9,12-dien-1-yl)tetradecano-4,15,18-trien-1-amine (HGT5001), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dimyristyloxypropionyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), dioleyloxy-N-[2-argininecarboxamido]ethyl-N,N-dimethyl-1-propylamino trifluoroacetate (DOSPA), dioctadecylamido glycine arginine ( DOGS), 1,2-dioleyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), N,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA), 1,2-dilinolenic acid-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadecene-9,1,2-diene-1-oxy)alanine (CLinDMA), 2-[5'-(cholesterol-5-ene-3[β]-oxy)-3'-oxopentyloxy]-3-dimethyl-1-1(cis,cis-9',12'-octadecene-1-yloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxyphenylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadecene-9,12-dienoate), (DLinDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)propyl)disulfide)-N,N-dimethylethylamine (HGT4003) and combinations thereof.

[0023] In some embodiments, the cholesterol lipid comprises cholesterol or PEG-modified cholesterol. In some embodiments, the cationic lipid accounts for about 30-50% of the liposome by molar ratio. In some embodiments, the ratio of cationic lipid: non-cationic phospholipid: cholesterol lipid: PEG-modified lipid is about 40:30:25:5 (molar ratio). In some embodiments, the liposome comprises a combination selected from the following combination groups: cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2K); C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K.

[0024] In some embodiments, the administration is by subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathoracic injection, intravenous injection, arterial injection, or a combination thereof.

[0025] In some embodiments, the dosing frequency is 3 times per week, 2 times per week, once per week, once every two weeks, once every three weeks, once every four weeks, once per month, or once every 3-6 months.

[0026] In some embodiments, the tumor includes the following categories: squamous cell carcinoma, lung cancer, peritoneal cancer, liver cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, soft tissue sarcoma, neuroblastoma, penile cancer, melanoma, superficial spreading melanoma, lentigo melanoma, acral melanoma, nodular melanoma, multiple myeloma, B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloid leukemia, acute myeloid leukemia, post-transplant lymphoproliferative disease, brain tumors, brain cancer and head and neck cancer, preferably colon cancer, breast cancer and lung cancer.

[0027] The present invention also provides a composition for implementing the above application. In one embodiment, the composition is a pharmaceutical composition, comprising two components: the first component is an mRNA encoding an OX40 agonist, and the second component is an mRNA encoding IL-12. The OX40 agonist is an OX40 ligand (OX40L), a polypeptide comprising an OX40L extracellular domain, or an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

[0028] Another embodiment provides a pharmaceutical composition comprising two components: the first component is mRNA encoding IL-12, and the second component is an OX40 agonist, wherein the OX40 agonist comprises an anti-OX40 agonist antibody or an antigen-binding fragment thereof, an OX40 ligand (OX40L), or a polypeptide comprising an extracellular domain of OX40L. Legend Brief Description

[0029] Figure 1 The results of screening single mRNA molecules or their combinations with anti-tumor effects in an animal model of colon cancer are presented.

[0030] Figure 2 The therapeutic effect of the OX40L and IL-12 mRNA combination was demonstrated to spread from the injection site to distant tumor sites.

[0031] Figure 3 It was demonstrated that either intratumoral or distal subcutaneous injection of OX40L and IL-12 mRNA combination could inhibit tumor growth.

[0032] Figure 4 The antitumor effect of the combination of OX40L and IL-12 mRNA in a lung cancer model was demonstrated.

[0033] Figure 5 showed that in a lung cancer model, the therapeutic effect of the combination of OX40L and IL-12 mRNA spread from the injection site to distant tumor sites.

[0034] Figure 6 demonstrated the antitumor effect of the combination of OX40L and IL-12 mRNA in a triple-negative breast cancer model.

[0035] Figure 7 demonstrated that in a breast cancer model, the therapeutic effect of the OX40L and IL-12 mRNA combination spread from the injection site to distant tumor sites.

[0036] Figure 8 The therapeutic effect of human OX40L and IL-12 mRNA combination on breast cancer in tree shrews was demonstrated.

[0037] Fig. 9 Shows the tumor weight of animals after intratumoral injection of different mRNA drugs.

[0038] Fig.10 Shows the changes in animal body weight after intratumoral injection of different mRNA drugs.

[0039] Fig.11 Shown are the changes in tumor volume on the injected side of the different treatment groups at days 8, 11, 14, and 17 after tumor implantation.

[0040] Fig.12 Shown are the changes in distal tumor volumes of the different treatment groups at days 8, 11, 14, and 17 after tumor implantation.

[0041] Fig.13 Shown are the tumor weights of animals after intratumoral injection of mRNA for IL-12 and the antibody being tested.

[0042] Fig.14 Shown are changes in overall animal body weight following intratumoral injection of the indicated mRNA / antibodies.

[0043] Fig.15 Shown are the changes in tumor volume on the injected side of the different mRNA / antibody treatment groups at days 8, 11, 14, and 17 after tumor implantation.

[0044] Fig.16 Shown are the changes in distal tumor volumes at days 8, 11, 14, and 17 after tumor implantation for different mRNA / antibody treatment groups.

[0045] Fig.17 The tumor weight of animals after intratumoral injection of mRNA molecules or compositions thereof is shown.

[0046] Fig.18 Shown are the body weights of animals after intratumoral injection of mRNA molecules or compositions thereof.

[0047] Fig.19 Shown are the changes in tumor volume on the injected side of the different treatment groups at 9, 12, 15, and 18 days after tumor implantation.

[0048] Fig. 20 Shown are the changes in distal tumor volumes of the different treatment groups at days 9, 12, 15, and 18 after tumor implantation.

[0049] Fig.21 The tumor weight of animals after intratumoral injection of mRNA molecules or compositions thereof is shown.

[0050] Fig. 22 Shown are the body weights of animals after intratumoral injection of mRNA molecules or compositions thereof.

[0051] Fig.23 Shown are the changes in tumor volume on the injected side of the different treatment groups at 9, 12, 15, and 18 days after tumor implantation.

[0052] Fig.24 Shown are the changes in distal tumor volumes of the different treatment groups at days 9, 12, 15, and 18 after tumor implantation.

[0053] Fig.25 Shows the tumor weight of animals after intratumoral injection of different doses of mRNA molecules.

[0054] Fig.26 Shows the animal body weight after intratumoral injection of different doses of mRNA molecules.

[0055] Fig. 27 The changes in tumor volume on the injected side of the different dose groups at days 8, 11, 14, and 17 after tumor implantation are shown.

[0056] Fig.28 Shown are the changes in distal tumor volumes of different dose groups at days 8, 11, 14, and 17 after tumor implantation. Detailed Description Definitions and general terms

[0057] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "an mRNA" is understood to mean one or more mRNAs. Therefore, the terms "a" (or "a"), "one or more" and "at least one" can be used interchangeably herein.

[0058] OX40, also known as CD134 and tumor necrosis factor receptor superfamily member 4 (TNFRSF4), is a member of the TNFR superfamily of receptors. Unlike CD28, which is continuously expressed on naive T cells in the resting state, OX40 is a secondary immune checkpoint co-stimulatory molecule that is expressed within 24 to 72 hours after activation.

[0059] OX40L, also known as CD252, is the ligand of OX40 and is stably expressed on many antigen-presenting cells, such as DC2 (a subtype of dendritic cells), macrophages, and activated B lymphocytes. OX40L is also present on the surface of many non-immune cells, such as endothelial cells and smooth muscle cells. Surface expression of OX40L can be induced by many pro-inflammatory factors, such as TNF-α, IFN-γ, and PGE2.

[0060] The representative nucleic acid sequence of human OX40L (splicing isoform 1) is shown in NCBI reference number NM_003326, and the corresponding protein sequence is NP_003317. Another representative nucleic acid sequence of human OX40L (splicing isoform 2) is shown in NCBI reference number NM_001297562, and the corresponding protein sequence is NP_001284491. The N-terminus of splicing isoform 1 is longer than that of splicing isoform 2, but the rest of the parts are exactly the same.

[0061] Interleukin 12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 is a heterodimeric cytokine encoded by two different genes, IL12A (p35) and IL12B (p40).

[0062] IL-12 is involved in the differentiation of initial T cells into Th1 cells. It stimulates T cells and natural killer (NK) cells to produce interferon gamma (IFN-γ) and tumor necrosis factor-α (TNF-α), and weakens the inhibitory effect of IL-4 on IFN-γ. IL-12 plays an important role in the activity of natural killer cells and T lymphocytes. IL-12 can enhance the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes.

[0063] IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor composed of IL-12Rβ1 and IL-12Rβ2. Upon binding, IL-12R-β2 undergoes tyrosine phosphorylation and provides binding sites for tyrosine kinases Tyk2 and Jak2.

[0064] The representative nucleic acid sequence of human IL-12A (splicing isomer 1) is shown in NCBI reference number NM_000882, and the corresponding protein sequence is NP_000873. Another representative nucleic acid sequence of human IL-12A (splicing isomer 2) is shown in NCBI reference number NM_001354582, and the corresponding protein sequence is NP_001341511. Another representative nucleic acid sequence of human IL-12A (splicing isomer 3) is shown in NCBI reference number NM_001354583, and the corresponding protein sequence is NP_001341512. Another representative nucleic acid sequence of human IL-12A (splicing isomer 4) is shown in NCBI reference number NM_001397992, and the corresponding protein sequence is NP_001384921.

[0065] The representative nucleic acid sequence of human IL-12B is shown in NCBI reference number NM_002187, and the related protein sequence is NP_002178.

[0066] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts that functions as a cytokine. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. Monocytes leave the circulation and migrate into tissues, maturing into macrophages and dendritic cells. It is therefore part of the immune / inflammatory cascade and can rapidly increase the number of macrophages by activating a small number of them, a process that is critical for protection against infection. GM-CSF also has some effects on mature immune system cells, such as inhibiting neutrophil migration and causing changes in cell surface receptors.

[0067] A representative nucleic acid sequence of human GM-CSF is provided in NCBI reference number NM_000758, and the corresponding protein sequence is NP_000749. OX40L and IL-12 mRNA Combination

[0068] Tumor immunotherapy shows broad application prospects by targeting multiple key molecules in the tumor immune cycle using small molecules, antibodies or engineered immune cells. Its typical strategies include activating stimulatory factors that promote immunity or inhibiting factors that suppress immune activity and prevent autoimmunity. Representative therapies include anti-CTLA4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies.

[0069] The delivery of tumor therapeutic drugs through protein-encoding mRNA is an emerging technology that has shown some promise. However, there are still some unexpected obstacles. For example, soluble PD1 fragment (sPD1) is a known PD-1 / PD-L1 inhibitor. When mRNA encoding sPD1 is injected into the tumor, it does not show an inhibitory effect on tumor growth (Example 1, Figure 1 ).

[0070] In addition, intratumoral injection of mRNA encoding OX40L showed only a slight inhibitory effect on tumor growth. In contrast, intratumoral injection of mRNA encoding IL-12 significantly inhibited tumor growth by about 50% (see Figure 1 ). When both mRNAs were delivered simultaneously to the same animal, inhibition reached nearly 90%. This significantly enhanced therapeutic effect clearly demonstrated a synergistic effect between these molecules.

[0071] In addition, the addition of other mRNA molecules that appear to have therapeutic effects did not further improve the efficacy. For example, although the single small molecule drug R848 (a ligand for Toll-like receptor 7 / 8) has a certain anti-tumor effect as an innate immune activator, it actually reduces the anti-tumor effect of other drugs when used in combination with other drugs (see Figure 1 ).

[0072] The experimental data in the examples further demonstrate the therapeutic effects of these molecules when used in combination. Figure 2 As shown, intratumoral injection not only inhibited local tumor growth but also tumor growth in the distant untreated side. Figure 3 As shown, a significant therapeutic effect occurred even when subcutaneous injection was administered away from the injection site.

[0073] In addition, in colon cancer ( Figure 1-3 )、Lung cancer( Figure 4-5 ) and breast cancer ( Figure 6-7 The efficacy of this combination was tested in multiple tumor models, including . In addition, the mRNA encoding the human protein was tested in a tree shrew breast cancer model ( Figure 8 ). Therefore, the current data provides a new application for the composition in the preparation of drugs for treating various types of tumors.

[0074] In addition, when the soluble part of OX40L protein was used in combination, the efficacy of the drug in inhibiting tumors was further improved from the injected tumor to the distal tumor of the animal (Example 7, Figure 9-12 The soluble part is a protein (Fc-OX40L) in which the extracellular fragment of the OX40L protein is fused with the IgG Fc fragment.

[0075] In addition, as in Examples 8 and 9 and Figure 13-20 As shown, the agonist effect of OX40L can be replaced with an agonistic antibody with comparable results. The antibody can be delivered directly to the patient as a protein or encoded for expression after mRNA delivery into the body.

[0076] Furthermore, the toxicity of these combinations is reduced as the antitumor effect is enhanced. Fig.25 As shown in Figure 2, the combination of 0.3 μg IL-12 and 0.3 μg OX40L had a tumor suppressive effect similar to that of 2.0 μg IL-12 alone. However, treatment with 2.0 μg IL-12 alone resulted in a significant decrease in body weight ( Fig.26 Even at a dose of 1 μg, treatment with IL-12 alone completely suppressed body weight gain ( Fig.26 Through these experiments, the best mass ratio between IL-12 and OX40L was obtained in Example 11, which was approximately 1:1 to 1:3.

[0077] In addition, when GM-CSF was added to the combination, the anti-tumor effect was further improved. The magnitude of improvement achieved by GM-CSF exceeded that achieved by GSDMD and TNFR, two immunomodulators commonly used in cancer treatment.

[0078] According to one embodiment of the present disclosure, a use of the composition in the preparation of a drug for treating tumors is provided, comprising administering mRNA encoding an OX40 agonist (OX40L or a protein / peptide / antibody or a combination thereof that can activate OX40, such as an OX40L protein or an anti-OX40 agonist antibody), and mRNA encoding IL-12 or a protein / peptide / antibody or a combination thereof that can activate an IL-12 receptor similar to IL-12 (e.g., mRNA / protein / peptide / antibody of IL-12 or IL-23). ​​In certain embodiments, the mRNA molecule is directly injected into the subject. In certain embodiments, one or more or all of the mRNA molecules are delivered as DNA and subsequently transcribed into mRNA in vivo.

[0079] In certain embodiments, OX40L is a human protein. In certain embodiments, OX40L is a full-length protein of OX40L, rather than a fragment or domain thereof, such as a soluble portion. In certain embodiments, OX40L is a full-length protein of OX40L with different splice isomers, rather than a fragment or domain thereof, such as a soluble portion.

[0080] In certain embodiments, the OX40 agonist is a polypeptide comprising at least the extracellular domain of full-length OX40L, possibly fused to a transmembrane domain, and optionally to an intracellular fragment of another protein.

[0081] In certain embodiments, the OX40 agonist is a polypeptide comprising an extracellular domain, either alone or fused to a linker fragment (e.g., an oligomerization domain) that promotes the formation of homodimers, homotrimers, or homooligomers thereof. Protein domains such as the Fc fragment of an immunoglobulin are often used to promote the formation of homodimers.

[0082] In certain embodiments, the oligomerization domain is capable of forming homotrimers (hence the term "trimerization domain"). Trimerization domains are known in the art, for example, domains in trimeric proteins that are responsible for mediating the association of proteins into trimers.

[0083] Common trimerization domains include T4 bacteriophage fibrin trimerization motif (T4F), GCN4 trimerization leucine zipper motif (GCN4), and homologous trimerization domain derived from human type XVIII collagen (TIE). In certain embodiments, the trimerization domain length does not exceed 100 amino acids, or does not exceed 90, 80, 70, 60 or 50 amino acids.

[0084] In certain embodiments, the fusion protein further comprises a peptide linker between the OX40L extracellular domain and the trimerization domain. In certain embodiments, the peptide linker is flexible.

[0085] In certain embodiments, the distance between the OX40L extracellular domain and the trimerization domain is no more than 100 amino acids, or no more than 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or 10 amino acids. In certain embodiments, the length of the peptide linker is 5 to 50 amino acid residues, preferably 5 to 20 amino acid residues.

[0086] In certain embodiments, the OX40 agonist is an anti-OX40 agonistic antibody or antigen-binding fragment thereof.

[0087] In certain embodiments, the OX40 agonist mRNA comprises an RNA sequence corresponding to the NM_003326 coding sequence (SEQ ID NO:3). In certain embodiments, the OX40L mRNA comprises an RNA sequence corresponding to the NM_001297562 coding sequence (SEQ ID NO:4). In certain embodiments, the OX40 agonist mRNA encodes the protein sequence of NP_003317 (SEQ ID NO:1). In certain embodiments, the OX40L mRNA encodes the protein sequence of NP_001284491 (SEQ ID NO:2).

[0088] In certain embodiments, the protein sequence encoded by the OX40 agonist mRNA has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 1 or amino acid residues 52-183 of SEQ ID NO: 1. In certain embodiments, the protein sequence encoded by the OX40 agonist mRNA has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to amino acid residues 2-133 of SEQ ID NO: 2 or SEQ ID NO: 2. In certain embodiments, the protein sequence retains the activity of human OX40L or the ability to activate OX40.

[0089] In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_003317 (i.e., amino acid residues 52-183 of SEQ ID NO: 1, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to amino acid residues 52-183 of SEQ ID NO: 1). In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_001284491 (i.e., amino acid residues 2-133 of SEQ ID NO: 2, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to amino acid residues 2-133 of SEQ ID NO: 2).

[0090] In certain embodiments, the extracellular domain of OX40L can be fused with the transmembrane domain and optional intracellular fragment of another protein, so that the fusion protein can be anchored on the plasma membrane. The transmembrane domain and intracellular fragment can be from any protein, such as human proteins, especially those cell membrane proteins in tissues where OX40L is desired to be expressed.

[0091] The transmembrane domain can be derived from any membrane-bound or transmembrane protein, such as the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD45, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, C D49D, CD49f, CD69, CD84, CD96 (Tactile), CD5, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulatory molecule (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC Class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll-like receptor ligand, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncated forms or combinations thereof.

[0092] In certain embodiments, IL-12 is human IL-12. In certain embodiments, IL-12 comprises IL-12A (p35). In certain embodiments, IL-12 comprises IL-12B (p40). In certain embodiments, IL-12 mRNA comprises mRNA encoding IL-12A and mRNA encoding IL-12B. In certain embodiments, IL-12 mRNA comprises mRNA encoding IL-12A and IL-12B at the same time.

[0093] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_000882 (SEQ ID NO: 9). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_000873 (SEQ ID NO: 5), or the protein sequence of its mature form (amino acid residues 57-253 of SEQ ID NO: 5).

[0094] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_001354582 (SEQ ID NO: 10). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_001341511 (SEQ ID NO: 6), or the protein sequence of its mature form (amino acid residues 57-239 of SEQ ID NO: 6).

[0095] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_001354583 (SEQ ID NO: 11). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_001341512 (SEQ ID NO: 7), or the protein sequence of its mature form (amino acid residues 57-215 of SEQ ID NO: 7).

[0096] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_001397992 (SEQ ID NO: 12). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_001384921 (SEQ ID NO: 8), or the protein sequence of its mature form (amino acid residues 23-219 of SEQ ID NO: 8).

[0097] In certain embodiments, the IL-12B mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_002187 (SEQ ID NO: 14). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_002178 (SEQ ID NO: 13), or the protein sequence of its mature form (amino acid residues 23-328 of SEQ ID NO: 13). Table 1. Sequences

[0098] In certain embodiments, OX40L mRNA (or mRNA encoding a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12 mRNA are separate mRNA molecules. In certain embodiments, the OX40L (or mRNA encoding a protein / polypeptide / antibody or a combination thereof that activates OX40) coding sequence and the IL-12 coding sequence are contained in the same mRNA molecule, which can be translated into a fusion protein / polypeptide or a separate protein / polypeptide.

[0099] In certain embodiments, one of the mRNAs encodes OX40L, such as human OX40L. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 1. In certain embodiments, a fragment of the encoded protein has at least 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of a fragment of SEQ ID NO: 1. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the amino acid sequence of the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 2. In certain embodiments, a fragment of the encoded protein has at least 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of a fragment of SEQ ID NO: 2. (One could potentially construct a fusion protein containing the extracellular domain of OX40L and the intracellular and transmembrane domains from other proteins).

[0100] In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 3. In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 4.

[0101] In certain embodiments, the mRNA encodes IL-12A, such as human IL-12A. In certain embodiments, the encoded protein has the amino acid sequence of SEQ ID NO:5. In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:5. In certain embodiments, the encoded protein comprises In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 6. In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 7. In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 7. In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 8. In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 8.

[0102] In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO:9. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:9. In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO:10. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:10. In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO:11. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:11. In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO:12. In certain embodiments, the mRNA comprises a nucleic acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:12.

[0103] In certain embodiments, one of the mRNAs encodes IL-12B, such as human IL-12B. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the encoded protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 13.

[0104] In certain embodiments, the mRNA comprises the nucleic acid sequence of SEQ ID NO: 14. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:14.

[0105] In certain embodiments, one or more mRNAs may collectively encode OX40L (or a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12A. In certain embodiments, one or more mRNAs may collectively encode OX40L (or a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12B. In certain embodiments, one or more mRNAs may collectively encode OX40L (or a protein / polypeptide / antibody or a combination thereof that activates OX40), IL-12A, and IL-12B.

[0106] When different mRNAs encode IL-12 and OX40L (or their soluble counterparts), respectively, their ratios can be adjusted as needed. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.5 to 1:6, and is not limited thereto. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.5 to 1:5. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.5 to 1:4. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.75 to 1:6. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.75 to 1:5. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.75 to 1:4. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.8 to 1:5. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.8 to 1:4. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.8 to 1:3.

[0107] In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.9 to 1:5. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.9 to 1:4. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.9 to 1:3.5. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.9 to 1:3. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.9 to 1:2.5.

[0108] In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:0.9 to 1:2. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:1 to 1:4. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:1 to 1:3. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:1 to 1:2.5. In one embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA is 1:1 to 1:2.

[0109] In certain embodiments, OX40L is a full-length protein of OX40L. In certain embodiments, OX40L is Fc-OX40L (soluble fragment of OX40L).

[0110] As shown in the examples, the addition of other potential therapeutic agents may not increase the therapeutic effect of this mRNA combination. Therefore, in one embodiment, the method of treatment or use or composition does not include other types of mRNA.

[0111] In one embodiment, mRNA encoding immune checkpoint inhibitors, such as PD-L1, PD-1, and CTLA-4, are not included. In one embodiment, mRNA encoding interferons, such as IFN-α, IFN-β, or IFN-γ, are not included. In one embodiment, mRNA encoding other IL-12 family members, such as IL-23, IL-27, and IL-35, are not included. In one embodiment, mRNA encoding other cytokines, such as IL-18, are not included.

[0112] Still can comprise one or more of these factors in some cases.In some embodiments, can comprise one or more of these factors, or one or more other factors. For example, in one embodiment, the method, use or composition further comprises mRNA encoding a partial or full-length immunomodulatory factor, such as CD27, CD28, CD40, CD122, CD137, GITR, GSDMD, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, SIGLEC9, IL-2, IL-15, IL-6, IL-18, IL-23, IFN-α, TNF-β, IFN-γ, GM-CSF, M-CSF, RIG-I, MDA5, cGAS, Toll-like receptor, MAVS / VISA, STING / MITA, TRIF, TBK1, IRF3, IRF7, IRF1, JAK1, JAK2, Tyk2, STAT1, STAT2, STAT3, TNFR and any combination thereof.

[0113] In a specific embodiment, the added agent is GM-CSF or mRNA encoding GM-CSF. In a specific embodiment, the added agent is TNFR or mRNA encoding TNFR. In a specific embodiment, the added agent is GSDMD (Gasdermin D) or mRNA encoding GSDMD. For example, in one embodiment, the method, use or composition further comprises a small molecule agent, a recombinant protein, an antibody. In some embodiments, the use further comprises administering to the patient a fourth mRNA encoding TNFR (tumor necrosis factor receptor). In some embodiments, the use further comprises administering to the patient a fourth mRNA encoding GSDMD.

[0114] In some embodiments, GM-CSF comprises amino acid residues 18-144 of SEQ ID NO: 15, or comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence similarity to amino acid residues 18-144 of SEQ ID NO: 15. In some embodiments, GM-CSF comprises SEQ ID NO: 15, or comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence similarity to amino acid residues 18-144 of SEQ ID NO: 15. In certain embodiments, the mRNA encoding GM-CSF comprises SEQ ID NO: 16, or comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence similarity to amino acid residues 18-144 of SEQ ID NO: 15. SEQ ID NO: 16 has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity.

[0115] mRNA can be synthesized using a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a set of ribonucleoside triphosphates, a buffer system containing DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, SP6 or other RNA polymerases), DNase I, pyrophosphatase, and / or RNase inhibitors. The specific conditions will vary depending on the specific application.

[0116] In some embodiments, to prepare mRNA, a DNA template is transcribed in vitro. The DNA template typically has a promoter, such as T3, T7, SP6 or other RNA polymerase promoters for in vitro transcription, followed by the desired nucleotide sequence and termination signal.

[0117] The desired mRNA sequence can be determined and integrated into the DNA template by conventional methods. For example, starting from the desired amino acid sequence, a virtual reverse translation is performed based on the degenerate genetic code. The appropriate codons are selected by an optimization algorithm. Generally, the G / C content can be optimized, on the one hand, the G / C content is increased as much as possible, and on the other hand, the frequency of tRNA is considered as much as possible in combination with the codon usage. For example, the optimized RNA sequence can be established and displayed with the help of a suitable display device and compared with the original (wild type) sequence. At the same time, the secondary structure can be analyzed to determine the properties of the stability and instability of the RNA or its corresponding region.

[0118] mRNA includes linear RNA, circular RNA and any other form of RNA. mRNA can be synthesized in unmodified or modified form. In some embodiments, mRNA is modified to enhance stability. In some embodiments, mRNA is modified to reduce immunogenicity. In some embodiments, mRNA is modified to improve translation efficiency.

[0119] In certain embodiments, in view of the special application of the prior art, the mRNA used is not modified for reducing immunogenicity, which is beneficial to the therapeutic effect. In certain embodiments, each mRNA does not include chemical modifications that reduce immunogenicity. In certain embodiments, each mRNA does not include chemical modifications to the backbone. In certain embodiments, each mRNA only includes natural nucleosides.

[0120] The modification of mRNA includes modification of RNA nucleotides. Therefore, the modified mRNA may include, for example, backbone modification, glycosylation modification or base modification. In some embodiments, mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentenyladenine, N6-methyladenine, N6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, hypoxanthine, 1-methylhypoxanthine, pseudouracil (5-uracil), and the like. uracil, 5-(4-(2-thiouracil), 5-(4-(2-thiouracil), 5-(5-(2-thiouracil), 5-(6-(4-thiouracil), 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methylpseudouracil, quinoline, 13-D-mannosylquinoline, wybutoxosine, phosphoamide, phosphorothioate, peptide nucleotides, methylphosphonic acid, 7-deazaguanosine, 5-methylcytosine and hypoxanthine. In some embodiments, at least one uridine nucleoside in the mRNA is chemically modified. In some embodiments, the chemically modified uridine nucleoside is N1-methyl pseudouridine.

[0121] In some embodiments, mRNA may include RNA backbone modifications. Generally, backbone modifications refer to situations where the backbone phosphates of nucleotides in RNA are chemically modified. Typical backbone modifications include, but are not limited to, modifications in the following groups, such as methylphosphonates, methylphosphoamidites, phosphoamidites, phosphosulfates (e.g., cytidine 5'-O-(1-sulfate)), borate phosphates, positively charged guanidine groups, etc., which means that the phosphodiester linkage is replaced by other anions, cations, or neutral groups.

[0122] In some embodiments, the mRNA may contain glycosylation modifications. Typical glycosylation modifications are chemical modifications of the nucleotide sugars it contains, including but not limited to glycosylation modifications selected from the following groups, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-amino-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2' -O-alkyl-oligoribonucleotides, 2'-deoxy-2'-C-alkyl-oligoribonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl-oligoribonucleotides and isomers thereof (2'-ala-cytidine 5'-triphosphate, 2'-ala-uridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0123] In some embodiments, the mRNA may contain modifications of nucleotide bases (base modifications). Modified nucleotides containing base modifications are also referred to as base modified nucleotides. Examples of such base modified nucleotides include, but are not limited to, 2-amino-6-chloropurine nucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate. , 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole nucleoside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthine 5'-triphosphate.

[0124] In some embodiments, mRNA synthesis includes adding a "cap structure" at the N-terminus (5' end) and a "tail structure" at the C-terminus (3' end). The presence of the cap structure is very important for resisting nucleases present in most eukaryotic cells, while the tail structure is used to protect mRNA from degradation by exonucleases.

[0125] Thus, in some embodiments, the mRNA comprises a 5' cap structure. The 5' cap structure is typically added by first removing one terminal phosphate group of the 5' nucleotide by an RNA terminal phosphatase, leaving two terminal phosphates; then adding guanosine triphosphate (GTP) to the terminal phosphate by a guanylate transferase, resulting in a 5'5'5 triphosphate linkage; and finally, methylating the 7-nitrogen of guanosine by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5)A and G(5)ppp(5')G).

[0126] In some embodiments, the mRNA comprises a 3'poly (A) tail structure. The poly (A) at the 3' end of the mRNA generally comprises about 10 to 300 adenine nucleotides (e.g., about 10 to 200 adenine nucleotides, about 10 to 175 adenine nucleotides, about 10 to 150 adenine nucleotides, about 10 to 125 adenine nucleotides, 10 to 100 adenine nucleotides, about 10 to 75 adenine nucleotides, about 20 to 70 adenine nucleotides, or about 20 to 60 adenine nucleotides). In some embodiments, the mRNA comprises a 3'poly (C) tail structure. Suitable poly (C) tails generally include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides) at the 3' end of the mRNA. The poly (C) tail can be added to the poly (A) tail, or can replace the poly (A) tail.

[0127] In some embodiments, mRNA comprises 5' and / or 3' untranslated regions. In some embodiments, 5' untranslated regions include one or more elements that affect mRNA stability or translation, for example, iron response elements. In some embodiments, the length of 5' untranslated regions is between about 50 and 500 nucleotides (e.g., about 50 and 400 nucleotides in length, about 50 and 300 nucleotides in length, about 50 and 200 nucleotides in length, or about 50 and 100 nucleotides in length).

[0128] In some embodiments, the 5' region of the mRNA includes a sequence encoding a signal peptide, such as those described herein. Typically, the sequence encoding the signal peptide is linked directly or indirectly to the N-terminus of the coding sequence.

[0129] In some embodiments, the 3' untranslated region includes one or more polyadenylation signals, protein binding sites that affect the stability of the mRNA in the cell or the localization in the cell, or one or more miRNA binding sites. In some embodiments, the length of the 3' untranslated region may be between 50 and 500 nucleotides or longer.

[0130] In some embodiments, the mRNA is packaged with a delivery vehicle. In some embodiments, the delivery vehicle comprises a liposome, a lipoplex, a lipid nanoparticle (LNP), a polymer compound, a peptide, a protein, a cell, a nanoparticle imitation, a nanotube, a conjugate, or any other delivery material. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNPs). In some embodiments, the lipid nanoparticle comprises a lipid selected from the following group: DLin-DMA, DLin-K-DMA, 98N12-5, C12- 200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, polyvinyl alcohol (PEG), PEG-DMG, PEG-modified lipids, amino alcohol lipids, KL22, and combinations thereof.

[0131] In one embodiment, the LNP comprises an ionizable cationic lipid in a molar ratio of 40-60%, a phospholipid in a molar ratio of 8-16%, a cholesterol lipid in a molar ratio of 30-45%, and a PEG-modified lipid in a molar ratio of 1-5%. In one embodiment, the LNP comprises an ionizable cationic lipid in a molar ratio of 45-65%, a phospholipid in a molar ratio of 5-10%, a cholesterol lipid in a molar ratio of 25-40%, and a PEG-modified lipid in a molar ratio of 0.5-5%. In one embodiment, the LNP comprises an ionizable cationic lipid in a molar ratio of 40-60%, a phospholipid in a molar ratio of 8-16%, a cholesterol lipid in a molar ratio of 30-45%, and a PEG-modified lipid in a molar ratio of 1-5%. In one embodiment, the LNP comprises an ionizable cationic lipid in a molar ratio of 45-65%, a phospholipid in a molar ratio of 5-10%, a cholesterol lipid in a molar ratio of 25-40%, and a PEG-modified lipid in a molar ratio of 0.5-5%. In one embodiment, LNP comprises ionizable cationic lipids in a molar ratio of 40-60%, phospholipids in a molar ratio of 8-16%, cholesterol lipids in a molar ratio of 30-45%, and PEG-modified lipids in a molar ratio of 1-5%. In one embodiment, LNP comprises ionizable cationic lipids in a molar ratio of 45-65%, phospholipids in a molar ratio of 5-10%, cholesterol lipids in a molar ratio of 25-40%, and PEG-modified lipids in a molar ratio of 0.5-5%.

[0132] In certain embodiments, mRNA is packaged in liposomes. Liposomes can be prepared by known various techniques. For example, multilamellar vesicles (MLV) can be prepared according to conventional techniques, such as by dissolving the selected lipid in an appropriate solvent, then depositing it on the inner wall of a suitable container or container, and evaporating the solvent to leave a film, or by spray drying. Subsequently, an aqueous phase can be added to the container, accompanied by vortex motion, to form MLV. Unilamellar vesicles (ULV) can be formed by homogenizing, ultrasonicating or extruding multilamellar vesicles. In addition, unilamellar vesicles can also be formed by removing the technology of surfactant.

[0133] In certain embodiments, the mRNA is bound to the surface of the liposome and is encapsulated in the same liposome. For example, during the preparation of the composition of the present invention, the cationic liposome can bind to the mRNA through electrostatic interaction.

[0134] In some embodiments, mRNA is encapsulated in liposomes. In some embodiments, one or more mRNAs can be encapsulated in the same liposome. In some embodiments, one or more mRNAs can be encapsulated in different liposomes. In some embodiments, mRNA is encapsulated in one or more liposomes, and these liposomes are different in their lipid composition, the molar ratio of lipid components, size, charge (Zeta potential), targeting ligands and / or its combination. In some embodiments, one or more liposomes may be composed of different cationic lipids, neutral lipids, PEG-modified lipids and / or its combination. In some embodiments, one or more liposomes may include different molar ratios of cationic lipids, neutral lipids, cholesterol and PEG-modified lipids for building liposomes.

[0135] In some embodiments, the liposomes include cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids. In some embodiments, the cationic lipid is selected from the following group: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadienyl)bis(dodecan-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptatriac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)alanine (DLinDMA), 2-(2,2-bis((9Z,12Z)- Octadecene-9,12-dien-1-yl)-1,3-dioxol-4-yl)-N,N-dimethylethylamine (DLinKC2DMA, [XTC2]), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl 3-(1H-imidazol-5-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)- Octadecene-9,12-dien-1-yl)tetracon-15,18-dien-1-amine (HGT5000), (4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecene-9,12-dien-1-yl)tetracon-4,15,18-trien-1-amine (HGT5001), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dimyristyloxypropionyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), dioleyloxy-N-[2-argininecarboxamido]ethyl-N ,N-dimethyl-1-propylamino trifluoroacetate (DOSPA), dioctadecylamido glycine arginine (DOGS), 1,2-dioleyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), N,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA), 1,2-dilinolenic acid-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadecene-9,12-diene-1-oxy)alanine (CLinDMA), 2-[5'-(cholesterol-5-ene-3[β]-oxy)-3'-oxopentyloxy]-3-dimethyl-1-1(cis,cis-9',12'-octadecene-dienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxyphenylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadecene-9,12-dienoate), (DLinDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)propyl)disulfide)-N,N-dimethylethylamine (HGT4003) and combinations thereof.

[0136] In some embodiments, the cholesterol-based lipid is cholesterol or PEG-modified cholesterol. In some embodiments, the molar ratio of cationic lipid to liposome is about 30-50%. In some embodiments, the ratio of cationic lipid: non-cationic lipid: cholesterol lipid: PEG-modified lipid is about 50:10:35:5.

[0137] In some embodiments, the liposomes comprise a combination selected from the following groups: cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dilauroyl-sn-glycerol, methoxypolyvinyl alcohol (DMG-PEG2K); C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K.

[0138] Suitable liposomes can be made into various sizes. In certain embodiments, the liposomes provided can be smaller than previously known mRNA encapsulation liposomes. In certain embodiments, the reduced size of the liposomes is associated with more effective delivery of the mRNA. When selecting a suitable liposome size, the location of the target cell or tissue needs to be considered, and the application purpose of the liposomes is considered to a certain extent.

[0139] In some embodiments, suitable liposomes are no larger than about 250 nanometers in size (e.g., no larger than about 225 nanometers, 200 nanometers, 175 nanometers, 150 nanometers, 125 nanometers, 100 nanometers, 75 nanometers, or 50 nanometers). In some embodiments, suitable liposomes are in a size range of about 10-250 nanometers (e.g., in a range of about 10-225 nanometers, 10-200 nanometers, 10-175 nanometers, 10-150 nanometers, 10-125 nanometers, 10-100 nanometers, 10-75 nanometers, or 10-50 nanometers). In some embodiments, suitable liposomes are in a size range of about 100-250 nanometers (e.g., in a range of about 100-225 nanometers, 100-200 nanometers, 100-175 nanometers, 100-150 nanometers). In some embodiments, suitable liposome sizes range from about 10-100 nm (eg, in the range of about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10-5 nm).

[0140] According to various embodiments, the expression time of the delivered mRNA can be adjusted to adapt to specific medical needs. In some embodiments, the expression of the OX40L protein encoded by the delivered mRNA is detectable in serum or target tissues 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and / or 72 hours after a single dose. In some embodiments, the expression of the OX40L protein encoded by the delivered mRNA is detectable in serum or target tissues 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and / or 7 days after a single dose. In some embodiments, the expression of the protein encoded by the delivered mRNA is detectable in serum or target tissues 1 week, 2 weeks, 3 weeks and / or 4 weeks after a single dose. In some embodiments, the expression of the protein encoded by the delivered mRNA is detectable in serum or target tissues one month or longer after a single dose.

[0141] In some embodiments, the expression of the IL-12A protein encoded by the delivered mRNA is detectable in serum or target tissues 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and / or 72 hours after a single dose. In some embodiments, the expression of the IL-12A protein encoded by the delivered mRNA is detectable in serum or target tissues 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days after a single dose. In some embodiments, the expression of the IL-12A protein encoded by the delivered mRNA is detectable in serum or target tissues 1 week, 2 weeks, 3 weeks, and / or 4 weeks after a single dose. In some embodiments, the expression of the protein encoded by the delivered mRNA is detectable one month or more after a single dose. In some embodiments, the expression of the IL-12B protein encoded by the delivered mRNA is detectable in serum or target tissues 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and / or 72 hours after a single dose. In some embodiments, the expression of the IL-12B protein encoded by the delivered mRNA is detectable in serum or target tissues 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and / or 7 days after a single dose. In some embodiments, the expression of the IL-12B protein encoded by the delivered mRNA is detectable in serum or target tissues 1 week, 2 weeks, 3 weeks and / or 4 weeks after a single dose. In some embodiments, the expression of the protein encoded by the delivered mRNA is detectable in serum or target tissues one month or longer after a single dose.

[0142] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific mRNA used and its variants or derivatives thereof, the patient's age, weight, overall health, sex and diet, administration time, excretion rate, drug combination and the severity of the disease being treated. The judgment of these factors by medical care personnel belongs to the routine skills in this area. The dosage used will also depend on the individual patient to be treated, the route of administration, the type of preparation, the characteristics of the mRNA used, the severity of the disease and the desired effect. The dosage used can be determined by the pharmacology and pharmacokinetic principles recognized in the field.

[0143] The administration method of mRNA includes, but is not limited to, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, nasal administration and epidural injection. The term "parenteral administration" as used herein refers to administration methods including intravenous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, subcutaneous injection and intraarticular injection and infusion. In some embodiments, the administration method is intratumoral injection. In some embodiments, the administration method is subcutaneous injection. In some embodiments, the administration method is intramuscular injection or intravenous injection. In some embodiments, the administration method can be subcutaneous injection, intramuscular injection, intraperitoneal injection, chest injection, intravenous injection, arterial injection or a combination thereof.

[0144] In some embodiments, the drug is injected into the tumor tissue. In some embodiments, the drug is injected into one side of the tumor tissue, such as an end or a portion of the tumor tissue. In some embodiments, the drug is not injected into the tumor tissue.

[0145] In some embodiments, the frequency of dosing is 3 times per week, 2 times per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once per month, or once every 3-6 months.

[0146] The present invention provides an application in the preparation of a drug for treating a tumor, wherein the tumor to be treated includes solid tumors, leukemia and lymphoma. In certain embodiments, the tumor is squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, soft tissue sarcoma, neuroblastoma, penis cancer, melanoma, superficial spreading melanoma, freckle melanoma, acral melanoma, nodular melanoma, multiple myeloid neoplasms, B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloid leukemia, acute myeloid leukemia, post-transplant lymphoproliferative disease, brain tumor, brain cancer and head and neck cancer.

[0147] In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor is a metastatic tumor. In one embodiment, the tumor is colon cancer. In one embodiment, the tumor is breast cancer, including triple negative breast cancer. In one embodiment, the tumor is lung cancer. Direct delivery of protein or a mixture with mRNA

[0148] In some of the above embodiments, multiple (or more) drugs (e.g., IL-12 and OX40L) are delivered as encoded mRNA molecules. In some embodiments, one or more drugs can be delivered directly in the form of protein. For example, in Example 9, whether the anti-OX40 agonist antibody is delivered in the form of protein or mRNA, similar therapeutic effects are obtained. Therefore, IL-12 protein can also be delivered in the form of protein.

[0149] Therefore, according to one embodiment of the present disclosure, an application in the preparation of a drug for treating tumors is provided, which includes delivering mRNA encoding an OX40 agonist (e.g., OX40L or a protein / peptide / antibody or a combination thereof that activates OX40 (e.g., OX40L protein or anti-OX40 agonistic antibody)), and IL-12 or an IL-12-like protein / peptide / antibody or a combination thereof that can activate an IL-12 receptor (e.g., IL-12 or IL-23 mRNA / protein / peptide / antibody).

[0150] In another embodiment, an application in the preparation of a drug for treating tumors is provided, which includes delivering an OX40 agonist (e.g., OX40L or a protein / peptide / antibody or a combination thereof that activates OX40 (e.g., OX40L protein or anti-OX40 agonistic antibody)), and mRNA encoding IL-12 or a protein / peptide / antibody or a combination thereof that is similar to IL-12 and can activate an IL-12 receptor (e.g., IL-12 or IL-23 mRNA / protein / peptide / antibody).

[0151] In another embodiment, an application in the preparation of a drug for treating tumors is provided, which includes delivering an OX40 agonist (e.g., OX40L or a protein / peptide / antibody or a combination thereof that activates OX40 (e.g., OX40L protein or anti-OX40 agonist antibody)), and IL-12 or an IL-12-like protein / peptide / antibody or a combination thereof that can activate an IL-12 receptor (e.g., IL-12 or IL-23 protein / peptide / antibody).

[0152] In certain embodiments, OX40L is a human protein. In certain embodiments, OX40L is a full-length protein of OX40L, rather than a fragment or domain thereof, such as a soluble portion. In certain embodiments, OX40L is a full-length protein of a different splice isoform of OX40L, rather than a fragment or domain thereof, such as a soluble portion.

[0153] In certain embodiments, the OX40 agonist is a polypeptide comprising at least the extracellular domain of full-length OX40L, which may be fused to a transmembrane domain, and optionally to an intracellular fragment of another protein.

[0154] In certain embodiments, the OX40 agonist is a polypeptide comprising an extracellular domain, which can exist alone or be fused with a connecting fragment (e.g., an oligomerization domain) that can promote the formation of homodimers, homotrimers, or homooligomers thereof. Protein domains such as the Fc fragment of an immunoglobulin are generally used to promote the formation of homodimers.

[0155] Embodiment trimerization domain comprises T4 phage fibrin trimerization motif (T4F), GCN4 trimer leucine zipper motif (GCN4) and homologous trimerization domain (TIE) derived from human collagen XVIII.In certain embodiments, trimerization domain is no more than 100 amino acids, or no more than 90, 80, 70, 60 or 50 amino acids.

[0156] In certain embodiments, the fusion protein further comprises a peptide linker located between the OX40L extracellular domain and the trimerization domain. In certain embodiments, the peptide linker is flexible.

[0157] In certain embodiments, the distance between the OX40L extracellular domain and the trimerization domain is no more than 100 amino acids, or no more than 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or 10 amino acids. In certain embodiments, the length of the peptide linker is 5 to 50 amino acid residues, preferably 5 to 20 amino acid residues.

[0158] In certain embodiments, the OX40 agonist is an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

[0159] In certain embodiments, the extracellular domain of OX40L can be fused to a transmembrane domain and optionally an intracellular fragment of another protein, so that the fusion protein can be anchored to the plasma membrane. The transmembrane domain and the intracellular fragment can be from any protein, such as a human protein, particularly a membrane protein on tissues where OX40L expression is desired.

[0160] The transmembrane domain can be derived from any membrane-bound or transmembrane protein, such as the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD45, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD13 7. Activate NK cell receptors, immunoglobulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD5, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC Class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncated forms or combinations thereof.

[0161] In certain embodiments, IL-12 is human IL-12. In certain embodiments, IL-12 includes IL-12A (p35). In certain embodiments, IL-12 includes IL-12B (p40).

[0162] In certain embodiments, the OX40 agonist is OX40L, such as human OX40L. In certain embodiments, the OX40L protein comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the OX40L protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the sequence of SEQ ID NO: 1. In certain embodiments, a fragment of the OX40L protein has at least 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of a fragment in SEQ ID NO: 1. In certain embodiments, the OX40L protein comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the amino acid sequence of the OX40L protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the sequence of SEQ ID NO: 2. In certain embodiments, a fragment of the OX40L protein has at least 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of a fragment of SEQ ID NO: 2. (One may construct a fusion protein comprising the extracellular domain of OX40L and the intracellular and transmembrane domains from other proteins.)

[0163] In certain embodiments, IL-12 includes IL-12A, such as human IL-12A. In certain embodiments, the IL-12A protein comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the amino acid sequence of the IL-12A protein is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similar to the sequence of SEQ ID NO: 5. In certain embodiments, the IL-12A protein comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the amino acid sequence of the IL-12A protein is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similar to the sequence of SEQ ID NO: 6. In certain embodiments, the IL-12A protein comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the amino acid sequence of the IL-12A protein is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similar to the sequence of SEQ ID NO: 7. In certain embodiments, the IL-12A protein comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the IL-12A protein comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similar to SEQ ID NO: 8.

[0164] In certain embodiments, the IL-12 protein includes IL-12B, such as human IL-12B. In certain embodiments, the IL-12B protein comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the amino acid sequence comprised by the IL-12B protein has a sequence similarity of at least 70%, 80%, 85%, 90%, 95%, 98% or 99% to SEQ ID NO: 13.

[0165] In certain embodiments, OX40L is a full-length OX40L protein. OX40L is Fc-OX40L (soluble fragment of OX40L).

[0166] As shown in the experimental examples, the addition of other potential therapeutic agents may not improve the therapeutic effect of the current combination. Therefore, in one embodiment, the method of treatment or use or combination does not include certain other types of drugs.

[0167] In one embodiment, immune checkpoint inhibitors, such as PD-L1, PD-1, and CTLA-4, are not included. In one embodiment, interferons, such as IFN-α, IFN-β, or IFN-γ, are not included. In one embodiment, proteins of the IL-12 family, such as IL-23, IL-27, and IL-35, are not included. In one embodiment, other cytokines, such as IL-18, are not included.

[0168] In some cases, one or more of these molecules may still be included. In some embodiments, one or more of these molecules may be included, or one or more other molecules may be included. For example, in one embodiment, the method, use or combination further includes an immunomodulatory factor, such as CD27, CD28, CD40, CD122, CD137, GITR, GSDMD, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, SIGLEC9, IL-2, IL-15, IL-6, IL-18, IL-23, IFN-α, INF-β, IFN-γ, GM-CSF, M-CSF, RIG-I, MDA5, cGAS, Toll-like receptors, MAVS / VISA, STING / MITA, TRIF, TBK1, IRF3, IRF7, IRF1, JAK1, JAK2, Tyk2, STAT1, STAT2, STAT3, TNFR and any combination thereof. In certain embodiments, the added drug is GM-CSF or mRNA encoding GM-CSF. In certain embodiments, the added drug is TNFR or mRNA encoding TNFR. In certain embodiments, the added drug is GSDMD or mRNA encoding GSDMD. For example, in one embodiment, the method, use or combination further comprises a small molecule agent, a recombinant protein, an antibody.

[0169] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific protein, mRNA, variant or derivative used, the patient's age, weight, overall health, sex and diet, as well as the administration time, excretion rate, drug combination and the severity of the disease being treated. The judgment of these factors by medical care personnel belongs to the routine skills in this area. The dosage used will also depend on the individual patient to be treated, the route of administration, the type of preparation, the characteristics of the protein / mRNA used, the severity of the disease and the desired effect. The dosage used can be determined by pharmacology and pharmacokinetic principles well-known in the art.

[0170] Methods of administration of proteins / mRNA include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and epidural injections. The term "parenteral administration" as used herein refers to administration methods including intravenous, intramuscular, intraperitoneal, intrathoracic, subcutaneous, and intraarticular injections and infusions. In certain embodiments, the administration method is intratumoral injection. In certain embodiments, the administration method is subcutaneous injection. In certain embodiments, the administration method is intramuscular or intravenous. In certain embodiments, the administration method is subcutaneous, intramuscular, intraperitoneal, intrathoracic, intravenous, intraarterial, or a combination thereof.

[0171] In certain embodiments, the drug is injected into the tumor tissue. In certain embodiments, the drug is injected into one side of the tumor tissue, such as an end or a portion of the tumor tissue. In certain embodiments, the drug is not injected into the tumor tissue.

[0172] In certain embodiments, the dosing frequency is 3 times per week, 2 times per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once per month, or once every 3-6 months.

[0173] Tumors suitable for treatment with the present technology include solid tumors, leukemias, and lymphomas. In certain embodiments, the tumor is squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, soft tissue sarcoma, neuroblastoma, penis cancer, melanoma, superficial spreading melanoma, senile melanoma, acral melanoma, nodular melanoma, multiple myeloid neoplasms, B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute lymphocytic leukemia, hairy cell leukemia, chronic myeloid leukemia, acute myeloid leukemia, post-transplant lymphoproliferative disease, brain tumors, brain cancer, and head and neck cancer.

[0174] In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor is a metastatic tumor. In one embodiment, the tumor is colon cancer. In one embodiment, the tumor is breast cancer, including triple negative breast cancer. In one embodiment, the tumor is lung cancer. Composition and formulation

[0175] The present invention provides compositions, packages, kits and formulations for practicing the disclosed applications.

[0176] One embodiment provides a composition, package, kit or formulation comprising mRNA encoding an OX40 agonist (e.g., OX40L or a protein / peptide / antibody or a combination thereof that activates OX40 (e.g., OX40L protein or anti-OX40 agonist antibody)), and mRNA encoding IL-12 or a protein / peptide / antibody or a combination thereof that is similar to IL-12 and can activate an IL-12 receptor (e.g., IL-12 or IL-23 mRNA / protein / peptide / antibody). In certain embodiments, the mRNA molecules are injected directly into the subject. In certain embodiments, one or more or all of the mRNA molecules are delivered as DNA and subsequently transcribed into mRNA in vivo.

[0177] Another embodiment provides a composition, package, kit or formulation comprising mRNA encoding an OX40 agonist (e.g., OX40L or a protein / peptide / antibody that activates OX40 or a combination thereof (e.g., OX40L protein or anti-OX40 agonist antibody)), and an IL-12 or an IL-12-like protein / peptide / antibody that can activate an IL-12 receptor or a combination thereof (e.g., IL-12 or IL-23 mRNA / protein / peptide / antibody).

[0178] Another embodiment provides a composition, package, kit or formulation comprising an OX40 agonist (e.g., OX40L or a protein / peptide / antibody that can activate OX40 or a combination thereof (e.g., OX40L protein or anti-OX40 agonist antibody)), and mRNA encoding IL-12 or a protein / peptide / antibody that is similar to IL-12 and can activate IL-12 receptor or a combination thereof (e.g., IL-12 or IL-23 mRNA / protein / peptide / antibody).

[0179] Another embodiment provides a composition, package, kit or formulation comprising an OX40 agonist (e.g., OX40L or a protein / peptide / antibody that can activate OX40 or a combination thereof (e.g., OX40L protein or anti-OX40 agonist antibody)), and an IL-12 or an IL-12-like protein / peptide / antibody that can activate an IL-12 receptor or a combination thereof (e.g., IL-12 or IL-23 protein / peptide / antibody).

[0180] In any of the above embodiments, the composition, package, kit or formulation further comprises GM-CSF protein, mRNA encoding GM-CSF, or a DNA vector expressing GM-CSF. In any of the above embodiments, the composition, package, kit or formulation further comprises TNFR protein, mRNA encoding TNFR, or a DNA vector expressing TNFR. In any of the above embodiments, the composition, package, kit or formulation further comprises GSDMD protein, mRNA encoding GSDMD, or a DNA vector expressing GSDMD.

[0181] In certain embodiments, OX40L is a human protein. In certain embodiments, OX40L is a full-length protein of OX40L, rather than a fragment or domain thereof, such as a soluble portion. In certain embodiments, OX40L comprises a full-length protein of OX40L of different splice isomers, rather than a fragment or domain thereof, such as a soluble portion.

[0182] In certain embodiments, the OX40 agonist is a polypeptide comprising at least the extracellular domain of full-length OX40L, which may be fused to a transmembrane domain and optionally to an intracellular fragment of another protein.

[0183] In certain embodiments, the OX40 agonist is a polypeptide comprising an extracellular domain, which can exist alone or be fused to a connecting fragment (e.g., a polymerization domain) that promotes the formation of homodimers, homotrimers, or homooligomers thereof. Protein domains such as the Fc fragment of an immunoglobulin are often used to promote the formation of homodimers.

[0184] Common trimerization domains include T4 phage fibrin trimerization motif (T4F), GCN4 trimer leucine zipper motif (GCN4), and homotrimerization domain derived from human collagen XVIII (TIE). In certain embodiments, the trimerization domain is no more than 100 amino acids, or no more than 90, 80, 70, 60 or 50 amino acids.

[0185] In certain embodiments, the fusion protein further comprises a peptide linker between the OX40L extracellular domain and the trimerization domain. In certain embodiments, the peptide linker is flexible.

[0186] In certain embodiments, the distance between the OX40L extracellular domain and the trimerization domain is no more than 100 amino acids, or no more than 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or 10 amino acids. In certain embodiments, the length of the peptide linker is 5 to 50 amino acid residues, preferably 5 to 20 amino acid residues.

[0187] In certain embodiments, the OX40 agonist is an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

[0188] In certain embodiments, the OX40 agonist mRNA comprises an RNA sequence corresponding to the NM_003326 coding sequence (SEQ ID NO: 3). In certain embodiments, the OX40L mRNA comprises an RNA sequence corresponding to the NM_001297562 coding sequence (SEQ ID NO: 4). In certain embodiments, the OX40 agonist mRNA encodes the protein sequence of NP_003317 (SEQ ID NO: 1). In certain embodiments, the OX40L mRNA encodes the protein sequence of NP_001284491 (SEQ ID NO: 2).

[0189] In certain embodiments, the protein sequence encoded by the OX40 agonist mRNA has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO: 1 or amino acid residues 52-183 of SEQ ID NO: 1. In certain embodiments, the protein sequence encoded by the OX40 agonist mRNA has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to amino acid residues 2-133 of SEQ ID NO: 2 or SEQ ID NO: 2. In certain embodiments, the protein sequence retains the activity of human OX40L or the ability to activate OX40.

[0190] In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_003317 (i.e., amino acid residues 52-183 of SEQ ID NO: 1, or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to amino acid residues 52-183 of SEQ ID NO: 1). In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_001284491 (i.e., amino acid residues 2-133 of SEQ ID NO: 2, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to amino acid residues 2-133 of SEQ ID NO: 2).

[0191] In certain embodiments, the extracellular domain of OX40L can be fused with a transmembrane domain and an intracellular fragment of another protein, so that the fusion protein can be anchored on the cell membrane. The transmembrane domain and the intracellular fragment can be from any protein, such as a human protein, especially a cell membrane protein in a tissue where OX40L is desired to be expressed.

[0192] In certain embodiments, IL-12 is human IL-12. In certain embodiments, IL-12 includes IL-12A (p35). In certain embodiments, IL-12 includes IL-12B (p40). In certain embodiments, IL-12 mRNA includes mRNA encoding IL-12A and mRNA encoding IL-12B. In certain embodiments, IL-12 mRNA includes mRNA encoding IL-12A and IL-12B at the same time.

[0193] In certain embodiments, IL-12A mRNA includes an mRNA sequence corresponding to the NM_000882 coding sequence (SEQ ID NO: 9). In certain embodiments, IL-12A mRNA encodes the protein sequence of NP_000873 (SEQ ID NO: 5), or the sequence of its mature form protein (amino acid residues 57-253 of SEQ ID NO: 5).

[0194] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_001354582 (SEQ ID NO: 10). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_001341511 (SEQ ID NO: 6), or the sequence of its mature form protein (amino acid residues 57-239 of SEQ ID NO: 6).

[0195] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_001354583 (SEQ ID NO: 11). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_001341512 (SEQ ID NO: 7), or the sequence of its mature form protein (amino acid residues 57-215 of SEQ ID NO: 7).

[0196] In certain embodiments, the IL-12A mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_001397992 (SEQ ID NO: 12). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_001384921 (SEQ ID NO: 8), or the sequence of the mature form of the protein thereof (amino acid residues 23-219 of SEQ ID NO: 8).

[0197] In certain embodiments, the IL-12B mRNA comprises an mRNA sequence corresponding to the coding sequence of NM_002187 (SEQ ID NO: 14). In certain embodiments, the IL-12A mRNA encodes the protein sequence of NP_002178 (SEQ ID NO: 13), or the sequence of the mature form of the protein thereof (residues 23-328 of SEQ ID NO: 13).

[0198] In certain embodiments, OX40L mRNA (or mRNA encoding a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12 mRNA are separate mRNA molecules. In certain embodiments, the sequence encoding OX40L (or mRNA encoding a protein / polypeptide / antibody or a combination thereof that activates OX40) and the sequence encoding IL-12 are contained in the same mRNA molecule, which can be translated into a fusion protein / polypeptide or a separate protein / polypeptide.

[0199] In certain embodiments, one of the mRNAs encodes OX40L, such as human OX40L. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the amino acid sequence comprised by the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with SEQ ID NO: 1. In certain embodiments, a fragment of the encoded protein has at least 90%, 95%, 98% or 99% sequence similarity with the amino acid sequence of a fragment in SEQ ID NO: 1. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the amino acid sequence comprised by the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with SEQ ID NO: 2. In certain embodiments, a fragment of the encoded protein has at least 90%, 95%, 98% or 99% sequence similarity with the amino acid sequence of a fragment in SEQ ID NO: 2. (One could construct a fusion protein containing the extracellular domain of OX40L and the intracellular and transmembrane domains of other proteins).

[0200] In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 3. In certain embodiments, the mRNA comprises a nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to SEQ ID NO: 4.

[0201] In certain embodiments, one of the mRNAs encodes IL-12A, such as human IL-12A. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO:5. In certain embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of SEQ ID NO:5. In certain embodiments, the encoded protein comprises In some embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 8.

[0202] In certain embodiments, the mRNA comprises the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the mRNA comprises In some embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 13. The nucleic acid sequence of SEQ ID NO: 12 has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity.

[0203] In certain embodiments, one of the mRNAs encodes IL-12B, such as human IL-12B. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the amino acid sequence of SEQ ID NO: 13.

[0204] In certain embodiments, the mRNA comprises the nucleic acid sequence of SEQ ID NO: 14. In certain embodiments, the mRNA comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to the nucleic acid sequence of SEQ ID NO: 14.

[0205] In certain embodiments, one or more mRNAs collectively encode OX40L (or a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12A. In certain embodiments, one or more mRNAs collectively encode OX40L (or a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12B. In certain embodiments, one or more mRNAs collectively encode OX40L (or a protein / polypeptide / antibody or a combination thereof that activates OX40), IL-12A, and IL-12B.

[0206] When mRNA encoding IL-12 and OX40L (or its soluble counterpart) are provided respectively, their ratios can be adjusted as needed. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.5 to 1:6, but is not limited to this range. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.5 to 1:5. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.5 to 1:4. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.75 to 1:6. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.75 to 1:5. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.75 to 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.8 to 1:5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.8 to 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.8 to 1:3.

[0207] In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.9 to 1:5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.9 to 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.9 to 1:3.5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.9 to 1:3. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.9 to 1:2.5.

[0208] In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:0.9 to 1:2. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:1 to 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:1 to 1:3. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:1 to 1:2.5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is 1:1 to 1:2.

[0209] In certain embodiments, OX40L is a full-length protein of OX40L. In certain embodiments, OX40L is Fc-OX40L (soluble fragment of OX40L).

[0210] As shown in the examples, adding other potential therapeutic agents may not improve the therapeutic effect of the mRNA combination. Therefore, in a certain embodiment, the method of treatment or application or composition does not include other types of mRNA.

[0211] In one embodiment, mRNA encoding immune checkpoint inhibitors, such as PD-L1, PD-1, and CTLA-4, are not included. In one embodiment, mRNA encoding interferons, such as IFN-α, IFN-β, or IFN-γ, are not included. In one embodiment, mRNA encoding other IL-12 family members, such as IL-23, IL-27, and IL-35, are not included. In one embodiment, mRNA encoding other cytokines, such as IL-18, are not included.

[0212] In certain embodiments, each mRNA is a linear mRNA or a circular mRNA. In certain embodiments, each mRNA also comprises a miRNA binding site. In certain embodiments, each mRNA does not comprise a chemical modification that reduces immunogenicity. In certain embodiments, mRNA does not comprise a chemical modification to the backbone. In certain embodiments, each mRNA only comprises natural nucleosides.

[0213] In certain embodiments, at least one uridine nucleoside in the mRNA is chemically modified. In certain embodiments, the chemically modified uridine nucleoside is N1-methyl pseudouridine. In certain embodiments, the first mRNA and the second mRNA are formulated in a pharmaceutically acceptable carrier.

[0214] In certain embodiments, the carrier comprises a lipid nanoparticle (LNP). In certain embodiments, the LNP comprises (a) a molar ratio of 40-60% ionizable amino lipids, 8-16% phospholipids, 30-45% sterols and 1-5% PEG-modified lipids, (b) a molar ratio of 45-65% ionizable amino lipids, 5-10% phospholipids, 25-40% sterols and 0.5-5% PEG-modified lipids, (c) a molar ratio of 40-60% ionizable amino lipids, 8-16% phospholipids, 30-45% sterols and 1-5% PEG modified lipids, (d) a molar ratio of 45-65% ionizable amino lipids, 5-10% phospholipids, 25-40% sterols and 0.5-5% PEG-modified lipids, (e) a molar ratio of 40-60% ionizable amino lipids, 8-16% phospholipids, 30-45% sterols and 1-5% PEG-modified lipids, or (f) a molar ratio of 45-65% ionizable amino lipids, 5-10% phospholipids, 25-40% sterols and 0.5-5% PEG-modified lipids.

[0215] In certain embodiments, each mRNA is packaged in a liposome. In certain embodiments, the liposome comprises a cationic lipid, a non-cationic lipid, a cholesterol-based lipid, and a PEG-modified lipid.

[0216] In certain embodiments, the cationic lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadienyl)bis(dodecan-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptatriac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)alanine (DLinDMA), 2-(2,2-bis((9Z,12Z)-octadecene-9,12-diene- 1-yl)-1,3-dioxol-4-yl)-N,N-dimethylethylamine (DLinKC2DMA, [XTC2]), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl 3-(1H-imidazol-5-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecene-9,12-dien-1-yl)tetradecene-15,18 -diene-1-amine (HGT5000), (4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecene-9,12-dien-1-yl)tetradecanoic acid-4,15,18-trien-1-amine (HGT5001), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dimyristyloxypropionyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), dioleyloxy-N-[2-argininecarboxamido]ethyl-N,N-dimethyl-1-propylamino trifluoroacetate (DOSPA), dioctadecylamido glycine arginine ( DOGS), 1,2-dioleyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), N,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA), 1,2-dilinolenic acid-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadecene-9,1,2-dioleyl-1-oxy)alanine (CLinDMA), 2-[5'-(cholesterol-5-ene-3[β]-oxy)-3'-oxypentyloxy]-3-dimethyl-1-1(cis,cis-9',12'-octadecene-1-yloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxyphenylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), (9Z,9'Z,12Z,12'Z)- 3-(Dimethylamino)propane-1,2-diylbis(octadecene-9,12-dienoate) (DLinDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadecene-9,12-dienoate-1-oxy)propyl)disulfide)-N,N-dimethylethylamine (HGT4003) and combinations thereof.

[0217] In certain embodiments, the cholesterol-based lipid is cholesterol or PEGylated cholesterol. In certain embodiments, the molar ratio of cationic lipid in the liposome is about 30-50%. In certain embodiments, the ratio of cationic lipid: non-cationic lipid: cholesterol lipid: PEGylated lipid is about 40:30:25:5. In certain embodiments, the liposome comprises a combination selected from the following groups: cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dilauroyl-sn-glycerol, methoxypolyvinyl alcohol (DMG-PEG2K); C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K. Example Example 1: IL-12 and OX40L mRNA synergistically inhibit tumor growth

[0218] This example evaluates the effectiveness of various molecules delivered by mRNA in inhibiting tumor growth.

[0219] These molecules included soluble PD1 (sPD1, as a PD-1 / PD-L1 inhibitor), OX40 ligand (OX40L, splice isoform 1), IL-12 (IL12B-IL12A fusion protein), IFN-β, resiquimod (R-848, a ligand for Toll-like receptor 7 / 8), and GFP as a control.

[0220] This example uses a mouse colon cancer model. CT-26 is a mouse colon cancer cell line. Eight days after CT-26 cells were implanted in mice, single molecules, single molecule mRNA, and multiple molecule mRNA mixtures (10 μg mRNA per mouse) (Table 1) were packaged in lipid nanoparticles (LNPs) and these mRNA samples were injected into the tumor. On day 18, the animals were euthanized, the tumors were isolated, and the tumor volume was measured. The results are shown in Figure 1. Figure 1 shown. Table 1. mRNA samples serial number mRNA#1(or R848) mRNA#2 mRNA#3 mRNA#4 1 GFP – – – 2 GFP GFP – – 3 sPD1 – – – 4 OX40L – – – 5 IL-12 – – – 6 IFN-β – – – 7 R848 – – – 8 sPD1 OX40L – – 9 sPD1 IL-12 – – 10 OX40L IL-12 – – 11 IFN-β OX40L – – 12 IFN-β IL-12 – – 13 IFN-β OX40L IL-12 – 14 IFN-β OX40L sPD1 – 15 IFN-β sPD1 IL-12 OX40L 16 sPD1 OX40L IL-12 – 17 R848 sPD1 OX40L – 18 R848 OX40L IL-12 – 19 R848 sPD1 IL-12 – 20 R848 sPD1 OX40L IL-12 21 IFN-β IL-12 sPD1 – 22 GFP GFP GFP – 23 – – – –

[0221] sPD1 alone failed to show significant antitumor effects. Although OX40L had some antitumor activity, R848 and IFN-β showed stronger antitumor effects, and IL-12 showed the strongest antitumor effect when used as a single therapeutic dose.

[0222] Of all the combination treatments, the combination of OX40L and IL-12 (treatment number 10) was the most effective, with a 10-fold reduction in tumor volume. This combination demonstrated a clear synergistic effect compared to either drug alone (treatment numbers 4 and 5).

[0223] Although R848 has a certain anti-tumor effect as a single drug, it reduces the anti-tumor effect of other drugs when used in combination. For example, treatment number 18 (R848+OX40L+IL-12) is less effective than treatment number 10 (OX40L+IL-12), and treatment number 20 (R848+sPD1+OX40L+IL-12) is less effective than treatment number 16 (sPD1+OX40L+IL-12). Example 2. Antitumor effect on the distal tumor side

[0224] Based on the results of Example 1, this example further explores the anti-tumor efficacy of mRNA composition therapy in distal tumor areas.

[0225] The animal model used in this example is the same as that in Example 1, but the OX40L and IL-12 mRNA packaged in LNPs were injected into the tumor on only one side of the animal. Ten days after injection (18 days after tumor implantation), the volume of the tumor on both sides was measured. Figure 2 As shown, although the mRNA was only injected into the tumor on one side, the drug had almost the same anti-tumor effect on both sides.

[0226] Therefore, this example demonstrates that the injected mRNA and / or its expressed protein products can diffuse in tumor tissue, thereby effectively inhibiting the overall tumor growth. Example 3. Antitumor effects of multiple routes of administration

[0227] This example further explored whether other administration routes besides intratumoral injection can also produce effective therapeutic effects.

[0228] The animal model used was the same as in Example 1-2. In addition to intratumoral injection, this example also included subcutaneous injection (LNP-packaged OX40L and IL-12 mRNA) away from the tumor. Both routes were injected twice on days 8 and 11 after tumor implantation. Tumor volume was measured on days 3 or 6 after injection, and the results are shown in Table 1. Figure 3 shown.

[0229] Although intratumoral injection produced the most significant antitumor effect, distal subcutaneous injection also showed comparable antitumor effects. Example 4. Antitumor effect in lung cancer model

[0230] This example tests the anti-tumor effect of the combination of OX40L and IL-12 mRNA in an animal lung cancer model.

[0231] The lung cancer model used here is the TC-1 model. On days 6 and 9 after tumor implantation, LNP-packaged OX40L and IL-12 mRNA were injected into the tumor on one side of the animal. Tumor volume was measured on days 3 and 6 after treatment. Figure 4 shown.

[0232] The results showed that the mRNA combination therapy could inhibit lung cancer growth.

[0233] This example also evaluates the effect of the drug on such distal tumors after intratumoral injection, and the experimental process is as described in Example 2. The mRNA was injected into the lung tumor on one side, and the therapeutic effect of the drug on the tumors on both sides was tested. Figure 5 As shown, the antitumor effect was widespread throughout the tumor. Example 5. Anti-tumor effect in triple-negative breast cancer model

[0234] This example further evaluated the anti-tumor effect of the combination of OX40L and IL-12 mRNA in the animal 4T1 model that simulates triple-negative breast cancer.

[0235] A similar experimental procedure as in the previous embodiment was used. Figure 6 As shown, the combination of OX40L and IL-12 mRNA almost completely inhibited tumor growth (left panel) without adversely affecting the overall health status / body weight of the animals (right panel).

[0236] This example also evaluates the effect of the drug on such distal tumors after intratumoral injection, and the experimental process is as described in Example 2 and Example 4. The mRNA was injected into the breast tumor on one side, and the therapeutic effect of the drug on the tumors on both sides was tested. Figure 7 As shown, the antitumor effect was widespread throughout the tumor. Example 6. Anti-tumor effects of human OX40L and IL-12 mRNA in tree shrew breast cancer model

[0237] This example uses human OX40L and IL-12 mRNA to treat breast cancer in tree shrews (a type of primate).

[0238] Tumors were induced on both sides of the mammary glands of tree shrews, and then human OX40L and IL-12 or GFP control mRNA / LNP nanoparticles (50 micrograms of mRNA per tree shrew) were injected into the mammary tumor on one side. The same mRNA / LNP nanoparticles were injected again on the same tumor side on days 3 and 6 after injection, respectively. The tumor volume of the injected and uninjected sides was measured every three days until day 21.

[0239] like Figure 8 As shown, human OX40L and IL-12 mRNA inhibited breast tumor growth and even eliminated breast tumors. Even when mRNA was injected into only one side of the breast tumor, tumors on both sides were inhibited or eliminated. Example 7. Comparison of antitumor effects of full-length OX40L and soluble OX40L fragments

[0240] This example compares the effects of full-length OX40L and its soluble fragments in animal tumor models.

[0241] The target proteins were produced in animals by intratumoral injection of mRNA, including a control group (GFP), IL-12, full-length OX40L protein, and a soluble OX40L fragment fused to an IgG Fc fragment (Fc-OX40L).

[0242] CT26 tumor cells (1x10^6) were implanted into mice, and animals were injected intratumorally on days 8 and 11 (a total of 1.5 μg mRNA was injected per animal). At the first administration, the diameter of the CT26 tumor mass was about 4 mm. The experimental animals were observed on days 8, 11, 14, and 17 after tumor implantation. Fig. 9As shown, the combination of IL-12 and OX40L (full-length or soluble) had the most significant inhibitory effect on both local tumors ("injected side") and distant tumors ("distal side") compared to either drug alone. Notably, the combination containing soluble OX40L (Fc-OX40L) was more effective than the combination containing full-length OX40L.

[0243] Figure 10-12 Detailed data on the safety and anti-tumor effects of these drugs on days 8, 11, 14, and 17 after tumor implantation are presented ( Fig.11 , injection side; Fig.12 , distal side). All the test drugs showed good safety in animals, comparable to the control group ( Fig.10 ).

[0244] These results demonstrate that the combination of IL-12 and soluble Fc-OX40L is the most effective. Example 8. Anti-tumor effect of IL-12 mRNA combined with anti-OX40 antibody

[0245] This example tests whether anti-OX40 antibodies can synergize with IL-12 to inhibit tumor growth.

[0246] The animal model used was similar to that in Example 7. IL-12 mRNA (0.3 μg) and the antibody to be tested (20 μg) were injected into the animals on days 8 and 11 after CT26 tumor cells were implanted into the animals. On day 8, the diameter of the CT26 tumor mass was approximately 5 mm.

[0247] The combination of IL-12 mRNA and anti-OX40 antibodies (one commercial antibody and one proprietary antibody "HX") was administered on the injection side ( Fig.13 , Fig.15 ) had an anti-tumor effect comparable to that of the combination of IL-12 mRNA and anti-PD1 / PD-L1 antibodies. On the distal tumor side, the combination of anti-OX40 antibodies was far superior to the combination of anti-PD1 / PD-L1 antibodies ( Fig.13 , Fig.16 ).

[0248] In summary, the combination of IL-12 mRNA and anti-OX40 antibody can safely ( Fig.14 ) and collaboratively ( Fig.13 ) inhibited tumor growth. Example 9. Anti-tumor effect of anti-OX40 antibody mRNA combined with IL-12 mRNA

[0249] This example tests whether anti-OX40 antibodies (delivered as mRNA) can synergize with IL-12 (delivered as mRNA) to inhibit tumor growth.

[0250] The animal model used was similar to that of Example 7. On days 9 and 12 after CT26 tumor cell implantation, the tested mRNA molecules (10.5 μg in total) were injected intratumorally into the animals. On day 9, the CT26 tumor mass was approximately 62 mm in diameter. mRNA combinations are shown in Table 1. Fig.17 , the combination includes IL-12 and full-length OX40L, OX40 antibody, PD antibody or PD-L1 antibody.

[0251] As shown in Example 8, the combination of IL-12 and OX40L / OX40 antibody showed the best anti-tumor efficacy ( Fig.17 , Fig.19 ), especially in the distal region ( Fig.17 , Fig. 20 All tested agents were safe in animals ( Fig.18 ). Example 10. Anti-tumor effect of combined use of IL-12, OX40L and GM-CSF

[0252] This example tests whether the addition of other drugs can further enhance the anti-tumor effect of the IL-12 / OX40L combination.

[0253] Three additional drugs were tested, including GM-CSF (granulocyte-macrophage colony-stimulating factor), GSDMD (Gasdermin D), and TNFR (tumor necrosis factor receptor). GM-CSF stimulates monocytes and macrophages to produce proinflammatory cytokines. GSDMD is a specific substrate for inflammatory caspases (caspase-1, -4, -5, and -11) and is an effector molecule involved in the lytic and highly inflammatory type of programmed cell death, known as pyroptosis.

[0254] All of these drugs were delivered in different combinations in the same animal model as above in the form of mRNA (1.5 micrograms in total), and the drugs were injected on days 9 and 12 after the animals were implanted. Different mRNAs encode IL-12 and OX40L, respectively, and additional drugs (such as GM-CSF) are fused to OX40L on the same mRNA through an IRES linker.

[0255] like Fig.21 As shown, the addition of each of GM-CSF, GSDMD, or TNFR further enhanced the efficacy of the IL-12 / OX40L combination. However, the triple combination containing GM-CSF showed the highest efficacy ( Fig.21 , Fig.23 In particular, the IL-12 / OX40L / GM-CSF combination showed significantly higher efficacy in the distant tumor areas of the animals ( Fig.21 , Fig.24 All tested drugs were shown to be safe in animals ( Fig. 22 ). Example 11. IL-12 and OX40L ratio

[0256] This example tests whether different ratios of IL-12 mRNA to OX40L mRNA will lead to different results.

[0257] On days 8 and 11 after implantation, each animal received a total of 4.3 μg of mRNA, as Fig.25 As shown (where necessary, GFP-expressing mRNA was added to ensure a total of 4.3 μg of mRNA was delivered). At a ratio of approximately 1:1 to 1:3, the combination of IL-12 mRNA and OX40L mRNA showed the highest tumor growth inhibition effect both at the injection site and at distal sites ( Fig.25 , 27 , 28).

[0258] Although higher doses of IL-12 alone (e.g., 1 μg and 2 μg vs. 0.3 μg in the combination) showed a stronger tumor suppressive effect, it had the greatest negative impact on the animals' weight gain ( Fig.26 ), suggesting that these higher doses may be associated with toxicity. Therefore, the combination of IL-12 and OX40L not only enhances the tumor inhibitory effect but also improves the safety profile. ***

[0259] The scope of the present application is not limited to the specific embodiments described, and the contents described are only embodiments adopted for the convenience of understanding the present application, and are not intended to limit the present application. Any functionally equivalent composition or application belongs to the scope of the present application. It is obvious that any technician in the field to which the present application belongs, without departing from the spirit and scope disclosed in the present application, makes various modifications and changes to the application and composition of the present application. Therefore, the present application is intended to cover these modifications and changes, as long as they are within the scope of the claims and their equivalents.

[0260] All publications and patent applications mentioned in this specification are incorporated in their entirety by reference, and the effectiveness of this specification is equivalent to listing and explicitly citing each publication or patent application separately.

Claims

1. Use of a pharmaceutical composition in preparing a drug for treating tumors, characterized in that: The use comprises administering to the patient a first mRNA encoding an OX40 agonist and a second mRNA encoding IL-12, wherein the OX40 agonist is an OX40 ligand (OX40L), a polypeptide comprising an extracellular domain of OX40L, or an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

2. The use according to claim 1, wherein the first mRNA and the second mRNA in the composition are contained in the same RNA molecule.

3. The composition according to claim 2, wherein the polypeptides encoded by the RNA molecules include OX40 agonist and IL-12, and the polypeptides can be in the form of fusion proteins or separately expressed polypeptides.

4. The use according to claim 1, wherein the first mRNA and the second mRNA in the composition are contained in different RNA molecules respectively.

5. Use according to any preceding claim, wherein the OX40 agonist is OX40L.

6. The use according to claim 5, wherein OX40L comprises SEQ ID NO: 1 or SEQ ID The amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 85% sequence similarity to SEQ ID NO:1 or SEQ ID NO:

2.

7. The use according to claim 5 or 6, wherein the first mRNA comprises the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a nucleic acid sequence having at least 85% sequence similarity to SEQ ID NO: 3 or SEQ ID NO:

4.

8. The use according to any one of claims 1 to 4, wherein the extracellular domain of OX40L comprises amino acid residues 52 to 183 of SEQ ID NO: 1, or comprises a sequence having at least 85% sequence similarity to amino acid residues 52 to 183 of SEQ ID NO:

1.

9. The use according to any one of claims 1-4 or 8, wherein the polypeptide may comprise an oligomerization domain or a transmembrane domain.

10. The use according to claim 9, wherein the OX40 agonist comprises an Fc domain fused to the extracellular domain of OX40L, and the OX40 agonist is a soluble protein lacking the transmembrane domain of the OX40L protein.

11. Use according to any preceding claim, wherein the second mRNA encodes IL-12A.

12. The use according to claim 11, wherein the amino acid sequence of IL-12A is selected from the following group: Amino acid residues 57-253 of SEQ ID NO:5, amino acid residues 57-239 of SEQ ID NO:6, amino acid residues 57-215 of SEQ ID NO:7 and amino acid residues 23-219 of SEQ ID NO:8, or an amino acid sequence having at least 85% sequence similarity to any amino acid sequence in these groups.

13. The use according to claim 11 or 12, wherein the second mRNA comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, or a nucleic acid sequence having at least 85% sequence similarity to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

12.

14. The use according to claim 12 or 13, further comprising administering to the patient a gene encoding SEQ ID Three mRNAs for amino acid residues 23-328 of NO:

13.

15. The use according to any one of claims 1 to 10, wherein the second mRNA encodes IL-12B.

16. The use according to claim 15, wherein IL-12B comprises amino acid residues 23 to 328 of SEQ ID NO: 13, or an amino acid sequence having at least 85% sequence similarity to amino acid residues 23 to 328 of SEQ ID NO:

13.

17. The use according to claim 15 or 16, wherein the second mRNA comprises the nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence having at least 85% sequence similarity to SEQ ID NO:

14.

18. The use according to any one of claims 1 and 4-17, wherein the mass ratio of the first mRNA to the second mRNA is 4:1 to 0.5:

1.

19. The use according to claim 18, wherein the mass ratio of the first mRNA to the second mRNA is 3:1 to 0.75:

1.

20. The use according to claim 18, wherein the mass ratio of the first mRNA to the second mRNA is 2:1 to 0.9:

1.

21. Use according to any preceding claim, further comprising administering to the patient a fourth mRNA encoding GM-CSF (granulocyte-macrophage colony stimulating factor).

22. The use according to any preceding claim, excluding the administration of immune checkpoint inhibitors, interferons, other IL-12 family members, other cytokines or nucleic acids encoding the same.

23. The use according to claim 22, wherein the immune checkpoint inhibitor is a PD-1, PD-L1 or CTLA-4 inhibitor, the interferon is IFN-α, IFN-β or IFN-γ, other IL-12 family members include IL-23, IL-27 and IL-35, or the other cytokine is IL-18.

24. Use of a pharmaceutical composition in the preparation of a drug for treating tumors, comprising administering to a patient a first preparation comprising mRNA encoding IL-12 and a second preparation comprising an OX40 agonist, wherein the OX40 agonist is an anti-OX40 agonist antibody or an antigen-binding fragment thereof, an OX40 ligand (OX40L), or a polypeptide comprising the extracellular domain of OX40L.

25. The use according to claim 24, wherein the OX40 agonist is an anti-OX40 agonist antibody.

26. Use according to any preceding claim, wherein each mRNA is a linear mRNA or a circular mRNA.

27. Use according to any preceding claim, wherein each mRNA further comprises a miRNA binding site.

28. Use according to any preceding claim, wherein each mRNA does not contain a chemical modification that reduces immunogenicity.

29. Use according to any preceding claim, wherein the mRNA does not comprise chemical modifications to the backbone.

30. Use according to any preceding claim, wherein each mRNA comprises only natural nucleosides.

31. The use according to any one of claims 1 to 30, wherein at least one uridine nucleoside in the mRNA is chemically modified.

32. The use according to claim 31, wherein the chemically modified uridine nucleoside is N1-methyl pseudouridine.

33. Use according to any preceding claim, wherein the first mRNA and the second mRNA are formulated together with a pharmaceutically acceptable carrier.

34. The use according to claim 33, wherein the carrier comprises lipid nanoparticles (LNP).

35. The use according to claim 34, wherein the lipid nanoparticles (LNP) comprise: (a) an ionizable cationic lipid with a molar ratio of 40-60%, a phospholipid with a molar ratio of 8-16%, a cholesterol lipid with a molar ratio of 30-45%, and a PEG-modified lipid with a molar ratio of 1-5%; (b) an ionizable cationic lipid with a molar ratio of 45-65%, a phospholipid with a molar ratio of 5-10%, a cholesterol lipid with a molar ratio of 25-40%, and a PEG-modified lipid with a molar ratio of 0.5-5%; (c) an ionizable cationic lipid with a molar ratio of 40-60%, a phospholipid with a molar ratio of 8-16%, a cholesterol lipid with a molar ratio of 30-45%, and a PEG-modified lipid with a molar ratio of (d) a molar ratio of 45-65% ionizable cationic lipid, a molar ratio of 5-10% phospholipid, a molar ratio of 25-40% cholesterol lipid, and a molar ratio of 0.5-5% PEG-modified lipid; (e) a molar ratio of 40-60% ionizable cationic lipid, a molar ratio of 8-16% phospholipid, a molar ratio of 30-45% cholesterol lipid, and a molar ratio of 1-5% PEG-modified lipid; or (f) a molar ratio of 45-65% ionizable cationic lipid, a molar ratio of 5-10% phospholipid, a molar ratio of 25-40% cholesterol lipid, and a molar ratio of 0.5-5% PEG-modified lipid.

36. The use according to any one of claims 1 to 33, wherein each mRNA is packaged in a liposome.

37. The use according to claim 36, wherein the liposome comprises a cationic lipid, a non-cationic phospholipid, a cholesterol-based lipid and a PEG-modified lipid.

38. The use according to claim 37, wherein the cationic lipid is selected from the following group: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadienyl)bis(dodecan-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptatriac-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadecene-9,12-dien-1-yl)alanine (DLinDMA), 2-(2,2-bis((9Z,12Z)-octadecene-9,12-dien-1-yl)-1,3-dioxolane-4-ol -yl)-N,N-dimethylethylamine (DLinKC2DMA, [XTC2]), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16 ,17-tetrahydro-1H-cyclopenta[a]fluoren-3-yl 3-(1H-imidazol-5-yl) propionate (ICE),(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecene-9,12-dien-1-yl)tetradecano-15,18-dien-1-amine (HGT5000),(4Z,15Z,18Z)-N,N-dimethyl-6- ((9Z,12Z)-octadecene-9,12-dien-1-yl)tetradecanoyl-4,15,18-trien-1-amine (HGT5001), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dimyristyloxypropionyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), dioleyloxy-N-[2-argininecarboxamido]ethyl-N,N-dimethyl-1-propylamino trifluoroacetate (DOSPA), dioctadecylamidoglycine arginine (DOGS), 1,2-dioleyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), N,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA), 1,2-dilinolenic acid-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]fluorene -3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadecene-9,12-dien-1-oxy)alanine (CLinDMA), 2-[5'-(cholesterol-5-ene-3[β]-oxy)-3'-oxopentyloxy]-3-dimethyl-1-1(cis,cis-9',12'-octadecene-dienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxyphenylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadecene-9,12-dienoate) (DLinDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadecene-9,12-diene-1-oxy)propyl)disulfide)-N,N-dimethylethylamine (HGT4003), and combinations thereof.

39. The use according to claim 37 or 38, wherein the cholesterol lipid is cholesterol or PEGylated cholesterol (PEG-modified cholesterol).

40. The use according to any one of claims 37 to 39, wherein the cationic lipid accounts for about 30-50% (calculated on a molar basis) of the liposome.

41. The use according to any one of claims 37 to 40, wherein the ratio of cationic lipid: non-cationic phospholipid: cholesterol lipid: PEG-modified lipid is about 40:30:25:5 (calculated as molar ratio).

42. The use according to any one of claims 36 to 41, wherein the liposomes comprise a combination selected from the following groups: cKK-E12, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dilauroyl-sn-glycerol, methoxypolyvinyl alcohol (DMG-PEG2K); C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K.

43. The use according to any of the preceding claims, wherein the administration is by subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathoracic injection, intravenous injection, arterial injection or a combination thereof.

44. The use according to any preceding claim, wherein the administration frequency is 3 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, or once every 3-6 months.

45. The method according to any of the preceding claims, wherein the tumor type is selected from the group consisting of squamous cell carcinoma, lung cancer, peritoneal cancer, liver cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, soft tissue sarcoma, neuroblastoma, penile cancer, melanoma, superficial spreading melanoma, lentigo melanoma, acral melanoma, nodular melanoma, multiple myeloma, B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, hairy cell leukemia, chronic myeloid leukemia, acute myeloid leukemia, post-transplant lymphoproliferative disorder, brain tumors and brain cancers, head and neck cancer, preferably colon cancer, breast cancer and lung cancer.

46. ​​A pharmaceutical composition comprising a first mRNA encoding an OX40 agonist and a second mRNA encoding IL-12, wherein the OX40 agonist is OX40 ligand (OX40L), a polypeptide comprising the extracellular domain of OX40L, or an anti-OX40 agonistic antibody or an antigen-binding fragment thereof.

47. A pharmaceutical composition comprising a first preparation of mRNA encoding IL-12 and a second preparation comprising an OX40 agonist, wherein the OX40 agonist is an anti-OX40 agonist antibody or antigen-binding fragment thereof, an OX40 ligand (OX40L), or a polypeptide comprising the extracellular domain of OX40L.

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