Application of RNA molecule and composition thereof in tumor immunotherapy

Abstract

The invention discloses an application of an RNA molecule and a composition thereof in preparation of a medicine for treating tumors. These compositions include mRNA molecules encoding proteins for the treatment of cancer, such as IL-12, OX40 agonists, and may be selectively used in combination with FADD, MLKL, or MLKL-4HB. The mRNAs may comprise a 5'untranslated region (UTR) and a 3 'UTR as well as a 5' cap structure that improves the molecular stability and / or therapeutic effect of the mRNAs.

Description

background

[0001] Tumor immunotherapy is a treatment method that regulates the immune system to identify and kill tumor cells, including immune checkpoint inhibition and adoptive cell therapy, etc. For example, it can enhance the effector function of tumor-specific effector T cells or weaken the inhibitory function of tumor-specific regulatory T cells.

[0002] Classic tumor immunotherapy involves the use of antibodies targeting checkpoint proteins or tumor-associated antigens, or the construction of immune cells expressing targeted receptors. However, the use of antibodies has certain limitations, and the production cost of cell therapy is extremely high.

[0003] There is an emerging technology that delivers mRNA encoding functional proteins into the body for treatment. However, mRNA molecules are often unstable and highly immunogenic, so this technology needs to be further optimized and developed. summary

[0004] The present invention proposes that certain structural elements can help improve the stability and / or therapeutic activity of mRNA molecules. For example, compared with other untranslated region (UTR) combinations, the expression level of mRNA composed of combination H (combination of 5'UTR of SEQ ID NO: 19 and 3'UTR of SEQ ID NO: 20) is higher. In addition, among different 5' cap structures, Cap1 shows the best anti-tumor effect. These structural elements can be included in mRNA molecules encoding therapeutic proteins, such as IL-12 and OX40 agonists, and can be selectively used with FADD or MLKL (or four-helix bundle structure MLKL-4HB).

[0005] The present invention provides a messenger RNA (mRNA), comprising a 5' untranslated region (UTR), a 3'UTR domain and a coding sequence, wherein the 5'UTR comprises a nucleic acid sequence of SEQ ID NO: 19, and the 3'UTR comprises a nucleic acid sequence of SEQ ID NO: 20.

[0006] In certain embodiments, the 5' end of the mRNA comprises a Cap-1 structure. In certain embodiments, the Cap-1 structure consists of Reagents were co-transcriptionally synthesized.

[0007] In certain embodiments, the mRNA is transcribed by T7 RNA polymerase. In certain embodiments, the mRNA further comprises a polyadenine (polyA) tail, preferably prepared by enzymatic synthesis or co-transcription. In certain embodiments, the polyadenine (polyA) tail comprises at least 50 adenine bases, preferably at least 80 adenine bases, and more preferably at least 100 adenine bases.

[0008] In certain embodiments, the mRNA sequence encodes IL-12 or an OX40 agonist alone, or encodes IL-12 and an OX40 agonist simultaneously. In certain embodiments, the OX40 agonist is an OX40 ligand (OX40L), a polypeptide comprising the extracellular domain of OX40L, or an agonist anti-OX40 antibody or an antigen-binding fragment thereof. In certain embodiments, the coding sequence also encodes FADD (FAS-associated death domain protein), MLKL (mixed lineage kinase domain-like pseudokinase) or a four-helix bundle structure MLKL-4HB.

[0009] In certain embodiments, the coding sequence of IL-12 is selected from a group consisting of nucleic acid sequences from SEQ ID NOs: 21 to 26. In certain embodiments, the coding sequence of OX40L is selected from a group consisting of nucleic acid sequences from SEQ ID NOs: 27 to 33, preferably selected from the group consisting of SEQ ID NOs: 27, 28, 30, 32 and 33.

[0010] The present invention provides a pharmaceutical composition, comprising mRNA and ionizable lipids, phospholipids, structural lipids, polyvinyl alcohol (PEG) lipids or a combination thereof. In certain embodiments, the ionizable lipid is SM102 or ALC0315.

[0011] The present invention provides an application in preparing a drug for treating tumors, comprising administering the mRNA or pharmaceutical composition disclosed herein to a patient.

[0012] The present invention provides an application in preparing a drug for treating tumors, comprising administering to a patient a first mRNA encoding an OX40 agonist, a second mRNA encoding IL-12, and a third mRNA encoding FADD or MLKL (or a four-helix bundle structure MLKL-4HB).

[0013] In certain embodiments, the third mRNA is on the same RNA molecule as the first mRNA or the second mRNA.In certain embodiments, the OX40 agonist is an OX40 ligand (OX40L), a polypeptide comprising the extracellular domain of OX40L, or an anti-OX40 agonistic antibody or antigen-binding fragment thereof.

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

[0015] In certain embodiments, the administration route is subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathoracic injection, intravenous injection, intraarterial injection, or a combination thereof.

[0016] In certain embodiments, the tumor is selected from the following group: 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, penile cancer, melanoma, superficial spreading melanoma, lentigo 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 disorder, brain tumor, brain cancer and head and neck cancer, preferably colon cancer, breast cancer and lung cancer.

[0017] The present invention further provides a pharmaceutical composition comprising a first mRNA encoding an OX40 agonist and / or IL-12 and a second mRNA encoding FADD or MLKL (or a four-helix bundle structure MLKL-4HB). In certain embodiments, the first mRNA encodes an OX40 agonist, and the second mRNA encodes FADD or MLKL (or MLKL-4HB), wherein the composition may optionally further comprise a third mRNA encoding IL-12. In certain embodiments, the first mRNA encodes IL-12, and the second mRNA encodes FADD or MLKL (or MLKL-4HB), wherein the composition may optionally further comprise a third mRNA encoding an OX40 agonist.

[0018] The present invention provides an mRNA, in certain embodiments, selected from a group comprising nucleic acid sequences from SEQ ID NO: 21 to 26. The present invention also provides an mRNA selected from a group comprising nucleic acid sequences from SEQ ID NO: 27 to 33, preferably selected from the group of SEQ ID NO: 27, 28, 30, 32 and 33. Graphical Description

[0019] Figure 1 Results of screening single or combined mRNA molecules for anti-tumor effects in an animal model of colon cancer are shown.

[0020] Figure 2 The therapeutic effect of OX40L in combination with IL-12 mRNA was shown to extend from the injection site to distant tumor sites.

[0021] Figure 3 Shown are the tumor weights of animals after intratumoral injection of mRNA of the test drug or drug combination.

[0022] Figure 4Shown are changes in body weight of animals after intratumoral injection of mRNA of a test drug or drug combination.

[0023] Figure 5 Shown are the changes in tumor volume on the injected side for the different treatment groups at days 8, 11, 14, and 17 after tumor implantation.

[0024] Figure 6 Shown are the changes in distal tumor volumes of the different treatment groups at days 8, 11, 14, and 17 after tumor implantation.

[0025] Figure 7 Shown are the tumor weights of animals after intratumoral injection of mRNA of the test drug or drug combination.

[0026] Figure 8 Shown are changes in body weight of animals after intratumoral injection of mRNA of a test drug or drug combination.

[0027] Fig. 9 Shown are the changes in tumor volume on the injected side for the different treatment groups at days 9, 12, 15, and 18 after tumor implantation.

[0028] Fig.10 Shown are the changes in distal tumor volumes for the different treatment groups at days 9, 12, 15, and 18 after tumor implantation.

[0029] Fig.11 The antitumor effects of OX40L and IL-12 mRNA delivered with different ionizable lipids are shown.

[0030] Fig.12 Shown is the distribution of luciferase mRNA expression delivered with different ionizable lipids.

[0031] Fig.13 The antitumor effects of OX40L and IL-12 mRNA produced using different RNA polymerases and capping methods are shown.

[0032] Fig.14 The translation of EGFP mRNA transcribed by T7 or VSW-3 RNA polymerase is shown.

[0033] Fig.15 The antitumor effects of OX40L and IL-12 mRNAs with different cap structures were shown.

[0034] Fig.16 The translation of EGFP mRNA with different UTR combinations is shown.

[0035] Fig.17 The translation of codon-optimized OX40L and IL-12 mRNAs is shown.

[0036] Fig.18 The translation of EGFP mRNA with different poly(A) lengths is shown. Detailed Description Definitions and general terms

[0037] 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.

[0038] 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.

[0039] 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.

[0040] A representative nucleic acid sequence of human OX40L (splice isoform 1) is shown in NCBI reference number NM_003326, and the related protein sequence is NP_003317. Another representative nucleic acid sequence of human OX40L (splice isoform 2) is shown in NCBI reference number NM_001297562, and the related protein sequence is NP_001284491. Splice isoform 1 has a longer N-terminus than splice isoform 2, but is otherwise identical.

[0041] Interleukin 12 (IL-12) is an interleukin 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).

[0042] 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.

[0043] IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12Rβ1 and IL-12Rβ2. After binding, IL-12Rβ2 undergoes tyrosine phosphorylation, providing binding sites for kinases Tyk2 and Jak2.

[0044] The representative nucleic acid sequence of human IL-12A (splicing isoform 1) is shown in NCBI reference number NM_000882, and the related protein sequence is NP_000873. Another representative nucleic acid sequence of human IL-12A (splicing isoform 2) is shown in NCBI reference number NM_001354582, and the related protein sequence is NP_001341511. Another representative nucleic acid sequence of human IL-12A (splicing isoform 3) is shown in NCBI reference number NM_001354583, and the related protein sequence is NP_001341512. Another representative nucleic acid sequence of human IL-12A (splicing isoform 4) is shown in NCBI reference number NM_001397992, and the related protein sequence is NP_001384921.

[0045] 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.

[0046] FAS-associated death domain protein (FADD), also known as MORT1, is an adaptor protein that connects members of the tumor necrosis factor receptor superfamily, such as the Fas receptor, to form a death-inducing signaling complex (DISC) with procaspases 8 and 10, which participate in apoptosis. FADD has also been observed to play a role in other processes such as proliferation, cell cycle regulation, and development.

[0047] The representative nucleic acid sequence of human FADD is given in NCBI reference number NM_003824, and the related protein sequence is NP_003815.

[0048] Mixed lineage kinase domain-like pseudokinase (MLKL) plays a role in tumor necrosis factor (TNF)-induced necroptosis through interaction with receptor interacting protein 3 (RIP3), a key signaling molecule in the necroptosis pathway.

[0049] The representative nucleic acid sequence of human MLKL is given in NCBI reference number NM_152649, and the related protein sequence is NP_689862. Improved mRNA structure and formulation

[0050] Through testing and comparative studies, researchers have found that certain structural elements can improve the stability, translation efficiency and / or overall anti-tumor efficacy of mRNA molecules. For example, when the UTR combination H is included in the mRNA molecule and is located on both sides of the coding sequence, it shows the highest translation level (see Fig.16 ). As shown in Table 1, UTR combination H includes the 5'UTR of SEQ ID NO: 19 and the 3'UTR of SEQ ID NO: 20.

[0051] The transcription and capping methods also have an impact on the stability and potency of mRNA. Fig.15 As shown in Figure 2, the anti-tumor effect produced by the Cap-1 structure is more persistent than that of the Cap-0 structure. The AG co-transcription system is not compatible with the VSW-3 RNA polymerase using optimal IVT conditions (see Xia, H. et al., (2022), RNA Biol 19, 1130-1142.10.1080 / 15476286.2022.2139113). Therefore, T7 RNA polymerase is used in AG co-transcription system is a better choice.

[0052] Another structural element is the polyadenine tail (polyA). Fig.18 As shown, longer polyadenine tails (eg, 120A) have higher translation efficiency than shorter polyadenine tails (eg, 30A).

[0053] Various lipid nanoparticle formulations were tested for the mRNA molecules disclosed herein. Both ionizable cationic lipids tested, SM102 and ALC0315, provided high quality formulations. The mRNA delivered by SM102 showed greater exposure, while the mRNA delivered by ALC0315 showed a more efficient delivery rate.

[0054] According to embodiments of the present disclosure, the present invention provides an mRNA molecule having one or more similar structural elements. In one embodiment, the present invention provides a messenger RNA (mRNA) having a 5' untranslated region (UTR), a 3'UTR and a coding sequence. In certain embodiments, the 5'UTR includes a nucleic acid sequence of SEQ ID NO: 19. In certain embodiments, the 3'UTR includes a nucleic acid sequence of SEQ ID NO: 20. In certain embodiments, the 5'UTR is located at the 5' side of the coding sequence. In certain embodiments, the 3'UTR is located at the 3' side of the coding sequence.

[0055] According to an embodiment of the present disclosure, the present invention provides a messenger RNA (mRNA) having a 5' cap structure. In one embodiment, the 5' cap is Cap-0. In another embodiment, the 5' cap is Cap-1. In another embodiment, the 5' cap is Cap-2.

[0056] Cap-0 is an N7-methylguanosine linked to the 5' nucleotide via a 5' to 5' triphosphate, also known as m7G cap or m7Gppp-. Additional methylation at the 2'O position of the start nucleotide generates Cap-1, or m7GpppNm-, where Nm represents any nucleotide with 2'O methylation. Similarly, Cap-2 has the structure m7G-ppp-Nm-Nm, as shown in Formula I below. Formula I

[0057] In certain embodiments, the Cap-1 structure of the mRNA is The co-transcriptional capping reagents are provided by TriLink Biotechnologies (San Diego, CA). In certain embodiments, the mRNA is transcribed using T7 RNA polymerase, but is not limited thereto.

[0058] In certain embodiments, the mRNA further comprises a polyadenine tail. In certain embodiments, the polyadenine tail comprises at least 50 adenine bases, or at least 60, 70, 80, 90, 100, 110 or 120 adenine bases.

[0059] In one embodiment, a pharmaceutical composition is also provided, the composition comprising the mRNA disclosed herein and an ionizable cationic lipid, phospholipid, structural lipid, polyvinyl alcohol (PEG) lipid or a combination thereof. In certain embodiments, the ionizable lipid is SM102 (9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate; CAS No.: 2089251-47-6) or ALC0315 ([(4-hydroxybutyl)imino]bis(hexene-6,1-diyl)bis(2-hexadecanoate); CAS No.: 2036272-55-4). In certain embodiments, the composition is presented in the form of lipid nanoparticles.

[0060] The term "lipid nanoparticle" refers to particles having at least one dimension in the nanometer range (e.g., 1-1000 nanometers) that contain one or more compounds of Formula I, or a pharmaceutically acceptable salt, stereoisomer, or mixture of stereoisomers thereof (e.g., SM102, ALC0315, or a pharmaceutically acceptable salt, stereoisomer, or mixture of stereoisomers thereof). Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In certain embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by water. The lipid bilayers may be functionalized and / or cross-linked to each other. The lipid bilayer may include one or more ligands, proteins, or channels.

[0061] As used herein, "phospholipid" refers to a lipid comprising a phosphate group and one or more carbon chains (such as unsaturated fatty acid chains). A phospholipid may comprise one or more multiple (eg, double or triple) bonds. Non-limiting examples of phospholipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-eicosanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-OT-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholestholesterol hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-arachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphenanthenoyl-sn-glycero-3-phosphoethanolamine (ME In some embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid is DSPC.

[0062] In certain embodiments, the composition includes a structured lipid. Non-limiting examples of structured lipids include cholesterol, coprostanol, phytosterol, ergosterol, nut sterol, sterol, cabbage sterol, tomato sterol, ursolic acid, alpha-tocopherol and combinations thereof. In one embodiment, the structured lipid is cholesterol.

[0063] In certain embodiments, the composition includes PEG lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof. Antitumor protein and protein composition

[0064] Tumor immunotherapy shows great promise by targeting multiple factors involved in the tumor-immune cycle using small molecules, antibodies, or engineered immune cells. A typical strategy is to activate stimulatory factors that promote immunity or inhibit factors that reduce immune activity and / or prevent autoimmunity. Some prominent examples include anti-CTLA4 antibodies and anti-PD-1 or anti-PD-L1 antibodies.

[0065] Delivery of cancer therapeutics via mRNA encoding them is an emerging technology that has shown some promise. However, there are still some unexpected obstacles. For example, when mRNA encoding a soluble PD1 fragment (sPD1, a known PD-1 / PD-L1 inhibitor) was delivered via intratumoral injection, tumor growth was not inhibited at all (Example 1, Figure 1 ).

[0066] 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% ( Figure 1 ). When both mRNAs were delivered simultaneously to the same animal, the inhibition rate reached about 90%. This significantly enhanced therapeutic effect clearly demonstrated a synergistic effect between these molecules.

[0067] The results showed that adding other seemingly therapeutic mRNA molecules did not further improve the efficacy. For example, although the single small molecule drug R848 (a ligand for Toll-like receptor 7 / 8) had some anti-tumor effect as an activator of innate immunity, it actually reduced the anti-tumor effect of other drugs when used in combination ( Figure 1 ).

[0068] The results of the experiment further illustrate the therapeutic effect of these combined methods. Figure 2 As shown, intratumoral injection inhibited tumor growth not only on the treated side but also on the distant untreated side.

[0069] The experimental results showed that when a soluble homologue of the OX40L protein was used in combination, the therapeutic effect was further improved (Example 3, Figure 3-6 ), especially on the side of the animal that is farther from the injected side ( Figure 3 and 6 ). The soluble homologue is a fusion protein of the extracellular fragment of the OX40L protein and the IgG Fc fragment (Fc-OX40L).

[0070] In addition, when FADD or MLKL (or MLKL-4HB) was further added to the combination, the anti-tumor effect was further improved. Moreover, the improvement effect of FADD or MLKL (or MLKL-4HB) exceeded that of GSDMD and TNFR, two immunomodulators commonly used in cancer therapy.

[0071] According to one embodiment of the present disclosure, there is provided an application in the preparation of a drug for treating tumors, which involves the use of mRNA encoding an OX40 agonist (e.g., OX40L or a protein / peptide / antibody or a combination thereof that activates 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 (e.g., IL-12 or IL-23 mRNA / protein / peptide / antibody). 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 into the body in the form of DNA and then transcribed into mRNA in the body.

[0072] In certain embodiments, OX40L is a human protein. In certain embodiments, OX40L is a complete OX40L protein, rather than a fragment or domain thereof, such as a soluble portion. In certain embodiments, OX40L is a different splice complete OX40L protein, rather than a fragment or domain thereof, such as a soluble portion.

[0073] In certain embodiments, the OX40 agonist is a polypeptide comprising at least the complete extracellular domain of OX40L, which may be fused to a transmembrane domain and optionally to an intracellular fragment of another protein.

[0074] In certain embodiments, the OX40 agonist is a polypeptide comprising an extracellular domain, which exists alone or is fused to a connecting 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.

[0075] In certain embodiments, the oligomerization domain is capable of forming homotrimers (ie, a "trimerization domain"). Trimerization domains are known in the art, such as domains in trimeric proteins that are responsible for mediating polymerization of trimeric proteins.

[0076] Exemplary trimerization domains include T4 bacteriophage fibrin trimerization motif (T4F), GCN4 trimer leucine zipper motif (GCN4), and homologous trimerization domain (TIE) derived from human type XVIII collagen. In certain embodiments, the length of the trimerization domain exceeds 100 amino acids, or does not exceed 90, 80, 70, 60 or 50 amino acids.

[0077] 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.

[0078] 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.

[0079] In certain embodiments, the OX40 agonist is an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

[0080] In certain embodiments, the OX40 agonist mRNA comprises an RNA sequence corresponding to the coding sequence of NM_003326 (SEQ ID NO: 3). In certain embodiments, the OX40L mRNA comprises The RNA sequence corresponding to the coding sequence of NM_001297562 (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).

[0081] 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 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 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.

[0082] In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_003317 (i.e., 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 residues 52-183 of SEQ ID NO: 1). In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_001284491 (i.e., 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 residues 2-133 of SEQ ID NO: 2).

[0083] 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 on the plasma membrane. The transmembrane domain and the intracellular fragment can be derived from any protein, such as a human protein, especially those cell membrane proteins in tissues where OX40L is desired to be expressed.

[0084] The transmembrane domain can be 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, activated NK cell receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27 6(B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96(Tactile), CEACAM1, CRTAM, 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, ligands 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), lymphocyte activation signaling molecules (SLAM family proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll-like receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations or combinations of these proteins.

[0085] 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.

[0086] 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 mature protein (residues 57-253 of SEQ ID NO: 5).

[0087] 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 mature protein (residues 57-239 of SEQ ID NO: 6).

[0088] 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 mature protein (residues 57-215 of SEQ ID NO: 7).

[0089] 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 mature protein (residues 23-219 of SEQ ID NO: 8).

[0090] 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 mature protein (residues 23-328 of SEQ ID NO: 13). Table A. Sequence

[0091] In certain embodiments, OX40L mRNA (or mRNA encoding a protein / polypeptide / antibody or a combination thereof that activates OX40) and IL-12 mRNA are independent 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, and the mRNA can be translated into a fusion protein / polypeptide or an independent protein / polypeptide.

[0092] 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 encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similarity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, a fragment of the encoded protein has at least 90%, 95%, 98% or 99% similarity to 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 encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similarity to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, a fragment of the encoded protein has at least 90%, 95%, 98% or 99% similarity to the amino acid sequence of a fragment in SEQ ID NO: 2. (Perhaps someone will construct a fusion protein containing the extracellular domain of OX40L and the intracellular domain and transmembrane domain of other proteins).

[0093] 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.

[0094] 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% similarity to the amino acid sequence of SEQ ID NO:5. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO:6. In certain embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similarity to the amino acid sequence of SEQ ID NO:6. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO:7. In certain embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similarity to the amino acid sequence of SEQ ID NO:7. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO:8. In certain embodiments, the encoded protein has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% similarity to the amino acid sequence of SEQ ID NO:8.

[0095] 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 having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:12.

[0096] 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% similarity to the amino acid sequence of SEQ ID NO: 13.

[0097] 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.

[0098] 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.

[0099] When the mRNA molecules encoding IL-12 and OX40L (or their soluble counterparts) are present as separate molecules, their ratios can be adjusted as needed. In a certain embodiment, the mass ratio between IL-12 mRNA and OX40L mRNA ranges from 1:0.5 to 1:6, without limitation. In a certain embodiment, the mass ratio is from 1:0.5 to 1:5. In a certain embodiment, the mass ratio is from 1:0.5 to 1:4. In a certain embodiment, the mass ratio is from 1:0.75 to 1:6. In a certain embodiment, the mass ratio is from 1:0.75 to 1:5. In a certain embodiment, the mass ratio is from 1:0.75 to 1:4. In a certain embodiment, the mass ratio is from 1:0.8 to 1:5. In a certain embodiment, the mass ratio is from 1:0.8 to 1:4. In a certain embodiment, the mass ratio is from 1:0.8 to 1:3.

[0100] 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 is 1:0.9 to 1:4. In one embodiment, the mass ratio is 1:0.9 to 1:3.5. In one embodiment, the mass ratio is 1:0.9 to 1:3. In one embodiment, the mass ratio is 1:0.9 to 1:2.5.

[0101] 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 is 1:1 to 1:4. In one embodiment, the mass ratio is 1:1 to 1:3. In one embodiment, the mass ratio is 1:1 to 1:2.5. In one embodiment, the mass ratio is 1:1 to 1:2.

[0102] In some embodiments, OX40L is a full-length OX40L protein. OX40L is Fc-OX40L (soluble fragment).

[0103] The present invention also provides and tests the codon-optimized coding sequences of IL-12 and OX40L. In some embodiments, the mRNA sequence of IL-12 is selected from the group consisting of SEQ ID NOs: 21-26. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 27-33. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NOs: 27, 28, 30, 32, and 33.

[0104] As shown in the examples, adding other potential therapeutic agents may not increase the therapeutic effect of the mRNA of the present combination. Therefore, in a certain embodiment, the method of treatment, use or composition does not include other types of mRNA.

[0105] 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 members of the IL-12 family, 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.

[0106] In some cases, one or more of these molecules may still be included. In some embodiments, one or more of these molecules, or one or more of other molecules, are included. For example, in a certain embodiment, the method, use or composition further comprises a partial or full-length mRNA encoding an immunomodulatory factor, such as FADD, MLKL, 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, IFN-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.

[0107] In certain embodiments, the agent used is FADD or mRNA encoding FADD. In certain embodiments, the agent used is MLKL (or MLKL-4HB) or mRNA encoding MLKL (or MLKL-4HB). For example, in certain embodiments, the method, use or composition further comprises a small molecule agent, a recombinant protein, an antibody.

[0108] In some embodiments, FADD comprises the amino acid sequence of SEQ ID NO: 15, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 15. In some embodiments, the mRNA encoding FADD comprises SEQ ID NO: 16, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence similarity to SEQ ID NO: 16.

[0109] In some embodiments, MLKL comprises the amino acid sequence of SEQ ID NO: 17, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to SEQ ID NO: 17 (or residues 1-125 of SEQ ID NO: 17). In some embodiments, the mRNA encoding MLKL comprises SEQ ID NO: 18, or has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence similarity to SEQ ID NO: 18.

[0110] In some embodiments, the agent used includes both FADD and MLKL, which can be encoded in the same mRNA or in different mRNA molecules.

[0111] 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.

[0112] 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.

[0113] 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. 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.

[0114] 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.

[0115] 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.

[0116] 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-(2-thiouracil), 5-(6-(2-thiouracil), 5-(7-(2-thiouracil), 5-(7-(2-thiouracil), 5-(8-(4-thiouracil), 5-(9-(2-thiouracil), 5-(9-(2-thiouracil), 5-(7-(2-thiouracil), 5-(8-(4-thiouracil), 5-(9-(2-thiouracil), 5-(7-thiouracil), 5-(8-(2-thiouracil), 5-(9-(2-thiouracil), 5-(7-thiouracil), 5-(8-(2-thiouracil), 5-(9-(2-thiouracil), 5-(8-(4-thiouracil), 5-(9-(2-thiouracil), 5-(8-(2-thiouracil), 5-(9-(2-thiouracil), 5-(8-(4-thiouracil), 5-(8-(2-thiouracil), 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.

[0117] 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, methylphosphamides, phosphoramides, 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.

[0118] 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).

[0119] 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.

[0120] According to different embodiments, the expression time of the mRNA delivered can be adjusted according to specific medical needs. In certain embodiments, after 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and / or 72 hours of a single administration of the liposome or composition provided, the expression of the OX40L protein encoded by the mRNA delivered can be detected in serum or target tissue. In certain embodiments, after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and / or 7 days of a single administration of the liposome or composition provided, the expression of the OX40L protein encoded by the mRNA delivered can be detected in serum or target tissue. In certain embodiments, after 1 week, 2 weeks, 3 weeks and / or 4 weeks of a single administration of the liposome or composition provided, the expression of the protein encoded by the mRNA delivered can be detected in serum or target tissue. In certain embodiments, after a single administration of one month or more of the liposome or composition provided, the expression of the protein encoded by the mRNA delivered can be detected in serum or target tissue.

[0121] In some embodiments, the expression of the IL-12A protein encoded by the delivered mRNA can be detected 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 administration of the provided liposomes or compositions. In some embodiments, the expression of the IL-12A protein encoded by the delivered mRNA can be detected in serum or target tissues 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and / or 7 days after a single administration of the provided liposomes or compositions. In some embodiments, the expression of the IL-12A protein encoded by the delivered mRNA can be detected in serum or target tissues 1 week, 2 weeks, 3 weeks and / or 4 weeks after a single administration of the provided liposomes or compositions. In some embodiments, the expression of the protein encoded by the delivered mRNA can be detected after a single administration of the provided liposomes or compositions for one month or longer. In some embodiments, the expression of the IL-12B protein encoded by the delivered mRNA can be detected in serum or target tissues after 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and / or 72 hours of a single administration of the liposomes or compositions provided. In some embodiments, the expression of the IL-12B protein encoded by the delivered mRNA can be detected in serum or target tissues after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and / or 7 days of a single administration of the liposomes or compositions provided. In some embodiments, the expression of the IL-12B protein encoded by the delivered mRNA can be detected in serum or target tissues after 1 week, 2 weeks, 3 weeks and / or 4 weeks of a single administration of the liposomes or compositions provided. In some embodiments, the expression of the protein encoded by the delivered mRNA can be detected after one month or more after a single administration of the liposomes or compositions provided.

[0122] 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, 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 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 can be determined according to known pharmacological and pharmacokinetic principles.

[0123] The method of administration of mRNA includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and epidural administration. The term "parenteral" as used herein refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, 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 injection. In certain embodiments, the administration method is subcutaneous injection, intramuscular injection, intraperitoneal injection, chest injection, intravenous injection, arterial injection or a combination thereof.

[0124] In some embodiments, the drug is injected into the tumor tissue. In some embodiments, the drug is injected into a 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.

[0125] In certain embodiments, the frequency of administration is 3 times per week, 2 times per week, once per week, once every two weeks, once every 3 weeks, once every 4 weeks, once per month, or once every 3-6 months.

[0126] 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 cancer 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, 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, post-transplant lymphoproliferative disease, brain tumor, brain cancer and head and neck cancer.

[0127] In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a metastatic cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is breast cancer, including triple negative breast cancer. In certain embodiments, the cancer is lung cancer. Composition and composition

[0128] Also provided herein are combinations, packages, kits, and compositions useful for practicing the methods of the invention.

[0129] One embodiment provides a combination, package, kit or composition 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 mRNA encoding IL-12 or a protein / peptide / antibody similar to IL-12 that can be used to stimulate an IL-12 receptor (e.g., mRNA / protein / peptide / antibody of IL-12 or IL-23) or a combination thereof. In certain embodiments, the mRNA molecules are injected directly into a subject. In certain embodiments, one or more or all of the mRNA molecules are delivered as DNA and subsequently transcribed into mRNA in vivo.

[0130] Another embodiment provides a combination, package, kit or composition 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 an anti-OX40 agonist antibody)), and IL-12 or a protein / peptide / antibody similar to IL-12 that can be used to stimulate an IL-12 receptor (e.g., mRNA / protein / peptide / antibody of IL-12 or IL-23) or a combination thereof.

[0131] Another embodiment provides a combination, package, kit or composition comprising an OX40 agonist (e.g., OX40L or a protein / peptide / antibody that activates OX40 or a combination thereof (e.g., OX40L protein or an anti-OX40 agonist antibody)), and mRNA encoding IL-12 or a protein / peptide / antibody similar to IL-12 that can be used to stimulate an IL-12 receptor (e.g., mRNA / protein / peptide / antibody of IL-12 or IL-23) or a combination thereof.

[0132] Another embodiment provides a combination, package, kit or composition comprising 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 IL-12 or a protein / peptide / antibody similar to IL-12 that can be used to stimulate the IL-12 receptor (e.g., a protein / peptide / antibody of IL-12 or IL-23) or a combination thereof.

[0133] In any of the above embodiments, the combination, package, kit or composition further comprises FADD protein, mRNA encoding FADD or a DNA vector encoding FADD. In any of the above embodiments, the combination, package, kit or composition further comprises MLKL protein, mRNA encoding MLKL or a DNA construct encoding MLKL.

[0134] In any of the above embodiments, the combination, package, kit or composition further comprises TNFR protein, mRNA encoding TNFR or a DNA vector encoding TNFR. In any of the above embodiments, the combination, package, kit or composition further comprises GSDMD protein, mRNA encoding GSDMD or a DNA vector encoding GSDMD.

[0135] In certain embodiments, OX40L is a human protein. In certain embodiments, OX40L is a full-length OX40L protein, rather than a fragment or domain thereof, such as a soluble portion. In certain embodiments, OX40L is a full-length OX40L protein with different isoforms, rather than a fragment or domain thereof, such as a soluble portion.

[0136] In certain embodiments, the OX40 agonist is a polypeptide comprising at least the extracellular domain of OX40L, which may be fused to the transmembrane domain and intracellular fragment of another protein.

[0137] In certain embodiments, the OX40 agonist is a polypeptide comprising an extracellular domain, which may exist alone or be fused to a connecting fragment (e.g., a polymerization domain) that promotes the formation of homodimers, homotrimers, or homooligomers. Protein domains such as the Fc fragment of an immunoglobulin are often used to promote the formation of homodimers.

[0138] Exemplary trimerization domains include the T4 bacteriophage fibrin trimerization motif (T4F), the GCN4 trimerization leucine zipper motif (GCN4), and the homologous trimerization domain (TIE) derived from human collagen XVIII. In some embodiments, the length of the trimerization domain does not exceed 100 amino acids, or does not exceed 90, 80, 70, 60, or 50 amino acids.

[0139] 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 a flexible structure.

[0140] 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 chain is 5 to 50 amino acid residues, preferably 5 to 20 amino acid residues.

[0141] In certain embodiments, the OX40 agonist is an anti-OX40 agonist antibody or an antigen-binding fragment thereof.

[0142] In certain embodiments, the OX40 agonist mRNA comprises an RNA sequence corresponding to the coding sequence of NM_003326 (SEQ ID NO: 3). In certain embodiments, the OX40L mRNA comprises The RNA sequence corresponding to the coding sequence of NM_001297562 is (SEQ ID NO:4). In certain embodiments, the protein sequence encoded by the OX40 agonist mRNA is NP_003317 (SEQ ID NO:1). In certain embodiments, the protein sequence encoded by the OX40L mRNA is NP_001284491 (SEQ ID NO:2).

[0143] 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 residues 52-183 of SEQ ID NO: 1 or 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 residues 2-133 of SEQ ID NO: 2 or SEQ ID NO: 2. In certain embodiments, the protein sequence has the activity of human OX40L or the ability to activate OX40.

[0144] In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_003317 (i.e., 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 residues 52-183 of SEQ ID NO: 1). In certain embodiments, the OX40 agonist mRNA encodes the extracellular domain of NP_001284491 (i.e., residues 2-133 of SEQ ID NO: 2, or a peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to residues 2-133 of SEQ ID NO: 2).

[0145] In certain embodiments, the extracellular domain of OX40L can be fused to a transmembrane domain and optionally to 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, especially a cell membrane protein on a tissue where OX40L is expected to be expressed.

[0146] 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 both IL-12A and IL-12B.

[0147] 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 protein sequence encoded by the IL-12A mRNA is NP_000873 (SEQ ID NO: 5), or encodes a mature protein (residues 57-253 of SEQ ID NO: 5).

[0148] 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 protein sequence encoded by the IL-12A mRNA is NP_001341511 (SEQ ID NO: 6), or encodes a mature protein (residues 57-239 of SEQ ID NO: 6).

[0149] 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 protein sequence encoded by the IL-12A mRNA is NP_001341512 (SEQ ID NO: 7), or encodes a mature protein (residues 57-215 of SEQ ID NO: 7).

[0150] 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 protein sequence encoded by the IL-12A mRNA is NP_001384921 (SEQ ID NO: 8), or encodes a mature protein (residues 23-219 of SEQ ID NO: 8).

[0151] 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 a protein sequence of NP_002178 (SEQ ID NO: 13), or encodes a mature protein (residues 23-328 of SEQ ID NO: 13).

[0152] 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 an independent protein / polypeptide.

[0153] In some embodiments, one of the mRNAs encodes OX40L, such as human OX40L. In some embodiments, the encoded protein contains the amino acid sequence of SEQ ID NO: 1. 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: 1. In some 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 in SEQ ID NO: 1. In some embodiments, the encoded protein contains the amino acid sequence of SEQ ID NO: 2. 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: 2. In some 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 in SEQ ID NO: 2. (It is possible that one could create fusion proteins containing the extracellular domain of OX40L and the intracellular and transmembrane domains from other proteins).

[0154] 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.

[0155] 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 the amino acid sequence of SEQ ID NO:6. 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:6. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO:7. In certain embodiments, the encoded protein has an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:7. In certain embodiments, the encoded protein comprises the amino acid sequence of SEQ ID NO:8. In certain embodiments, the encoded protein has an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:8.

[0156] 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 having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to SEQ ID NO:12.

[0157] 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.

[0158] 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.

[0159] In certain embodiments, one or more mRNAs collectively encode OX40L (or a protein / peptide / antibody or a combination thereof that activates OX40) and IL-12A. In certain embodiments, one or more mRNAs collectively encode OX40L (or a protein / peptide / antibody or a combination thereof that activates OX40) and IL-12B. In certain embodiments, one or more mRNAs collectively encode OX40L (or a protein / peptide / antibody or a combination thereof that activates OX40), IL-12A, and IL-12B.

[0160] When the mRNA molecules encoding IL-12 and OX40L (or their soluble counterparts) are provided separately, their ratios can be adjusted as needed. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA ranges from 1:0.5 to 1:6, and is not limited. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.5 and 1:5. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.5 and 1:4. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.75 and 1:6. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.75 and 1:5. In a certain embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.75 and 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.8 and 1:5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.8 and 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.8 and 1:3.

[0161] In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.9 and 1:5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.9 and 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.9 and 1:3.5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.9 and 1:3. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.9 and 1:2.5.

[0162] In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:0.9 and 1:2. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:1 and 1:4. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:1 and 1:3. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:1 and 1:2.5. In one embodiment, the mass ratio of IL-12 mRNA to OX40L mRNA is between 1:1 and 1:2.

[0163] In some embodiments, OX40L is a full-length OX40L protein. OX40L is Fc-OX40L (soluble fragment).

[0164] As shown in the examples, adding other potential therapeutic agents may not increase the therapeutic effect of the current mRNA combination. Therefore, in one embodiment, the method of treatment, use or combination does not include other types of mRNA.

[0165] 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 members of the IL-12 family, 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.

[0166] In some embodiments, each mRNA is a linear mRNA or a circular mRNA. In some embodiments, each mRNA also includes 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 is not chemically modified. In some embodiments, each mRNA only includes natural nucleotides.

[0167] 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. In some embodiments, the first mRNA and the second mRNA are formulated with a pharmaceutically acceptable carrier. Example Example 1: Combination of IL-12 and OX40L mRNA synergistically inhibits tumors

[0168] This example evaluates the effects of various factors delivered by mRNA in inhibiting tumor growth.

[0169] These factors included soluble PD1 (sPD1, as a PD-1 / PD-L1 inhibitor), OX40 ligand (OX40L, splice isoform 1), IL-12 (IL12B-IL12A fusion protein), interferon β (IFN-β), resiquimod (R-848, a ligand for Toll-like receptor 7 / 8), and GFP as a control.

[0170] 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, a single factor, a single factor mRNA, and a mixture of multiple factor mRNAs (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 Table 1. Figure 1 shown. Table 1. mRNA samples

[0171] Interestingly, sPD1 alone did not show antitumor effects. Although OX40L showed some antitumor effects, R848 and IFN-β had stronger antitumor effects, while IL-12 showed the strongest antitumor effect as a single dose.

[0172] Among all the combination treatments, the combination of OX40L and IL-12 (treatment number 10) was the most effective, reducing tumor size by about 10-fold. This combination achieved a synergistic effect compared to the respective single agents (treatment numbers 4 and 5).

[0173] Although R848 had a modest antitumor effect as a single agent, it reduced the antitumor effect of other drugs when used in combination. For example, treatment number 18 (R848+OX40L+IL-12) was less effective than treatment number 10 (OX40L+IL-12), and treatment number 20 (R848+sPD1+OX40L+IL-12) was less effective than treatment number 16 (sPD1+OX40L+IL-12). Example 2: Anti-tumor effect at the distal side of the tumor

[0174] Based on the results of Example 1, this example further explored the anti-tumor efficacy of combined mRNA therapy in distant tumor areas.

[0175] The animal model used in this example is the same as that in Example 1. On the 10th day (18th day) after injection of LNP-packaged OX40L and IL-12 mRNA, the tumor volume on the injection side and the distal side was measured. Figure 2 As shown, even if the mRNA was injected into only one side of the tumor mass, the anti-tumor effect was still similar on both sides.

[0176] Therefore, this example shows that the injected mRNA and / or its expressed protein products can diffuse in tumor tissue, thereby effectively inhibiting the entire tumor. Example 3: Comparison of full-length OX40L and soluble OX40L fragments

[0177] This example compares the therapeutic effects of full-length OX40L and its soluble fraction in tumor animal models.

[0178] mRNA preparations were injected intratumorally. These preparations included mRNA preparations of GFP (control), IL-12, full-length OX40L, and a soluble OX40L fragment fused to an IgG Fc fragment (Fc-OX40L).

[0179] CT26 tumor cells (1×10^6) were implanted into mice, and the animals were injected with the test drug (a total of 1.5μg mRNA per animal) into the tumor on days 8 and 11. At the time of the first dose, the CT26 tumor mass was approximately 4 mm in diameter. The animals were examined on days 8, 11, 14, and 17 after implantation. Figure 3As shown, overall, the combination of IL-12 and OX40L (whether full-length or soluble) was superior to either agent alone in terms of tumor shrinkage, with better results both in the local tumor ("injected side") and in distant areas ("distal side"). Interestingly, the combination containing soluble OX40L (Fc-OX40L) was superior to the combination with full-length OX40L.

[0180] Detailed measurements of the safety and antitumor efficacy of these test agents are available in Figure 4-6 , covering days 8, 11, 14 and 17 after implantation ( Figure 5 For the injection side; Figure 6 All test agents showed good safety in animals and were comparable to the control ( Figure 4 ).

[0181] These results confirm that the combination of IL-12 and soluble Fc-OX40L is the most effective combination. Example 4: IL-12, OX40L and MLKL or FADD

[0182] This example tests whether the addition of additional drugs can further enhance the anti-tumor effect of the IL-12 / OX40L combination.

[0183] Four additional drugs were tested, including FADD (FAS-associated death domain protein), MLKL (mixed lineage kinase-like pseudokinase), GSDMD (Gasdermin D), and TNFR (tumor necrosis factor receptor). FADD is an adaptor protein that links members of the tumor necrosis factor receptor superfamily (such as the Fas receptor) to pro-caspases 8 and 10 to form the death-inducing signaling complex (DISC) during apoptosis. MLKL plays a role in tumor necrosis factor (TNF)-induced necroptosis through its interaction with receptor-interacting protein 3 (RIP3). GSDMD is a specific substrate for inflammatory caspases (caspase-1, -4, -5, and -11) and acts as an effector molecule of programmed cell death, participating in both lytic and highly inflammatory pyroptosis.

[0184] All of these drugs were injected into the same animal model in different combinations via mRNA (1.5 μg in total) on days 9 and 12 after tumor cell implantation. Although IL-12 and OX40L were expressed via different vectors, the added drug (e.g., FADD) was fused to OX40L in the same mRNA via an IRES element.

[0185] like Figure 7As shown in Figure 2, the addition of any one of MLKL, FADD, GSDMD, and TNFR further enhanced the efficacy of the IL-12 / OX40L combination, even though no anti-tumor effect was observed when they were used alone. Among them, the triple combination containing the MLKL or FADD four-helix bundle structure showed the most significant efficacy ( Figure 7 , 9 ). In particular, the IL-12 / OX40L / FADD combination showed the most significant therapeutic effect on both the injected and distal sides of the animals ( Figure 7 , 10 All tested drugs showed good safety in animals ( Figure 8 ). Example 5: Lipid Nanoparticle Formulation

[0186] This example tests the anti-tumor effect of delivering IL-12 and OX40L mRNA using lipid nanoparticles with different formulations.

[0187] The formulations included SM102-1 and SM102-2 (50% SM102, 38.5% cholesterol, 10% phospholipids and 1.5% lipid-PEG), as well as the ALC0315 formulation. Their antitumor effects were evaluated using the above-mentioned methods. Fig.11 As shown, these three preparations have comparable antitumor efficacy.

[0188] The tissue distribution of the products expressed by different formulations was detected by delivering luciferase mRNA. Equal amounts of luciferase mRNA were delivered intratumorally by SM102-1, SM102-2, and ALC0315. The activity of luciferase in different tissues was analyzed at 6 hours, 24 hours, and 48 hours after injection.

[0189] ALC0315 showed low luciferase activity 48 hours after injection, but high activity 6 hours after injection. This suggests that ALC0315-mediated mRNA delivery is fast but short-lived, which explains Fig.11 For all preparations, most of the mRNA was detected in the liver, while a large portion remained in the tumor tissue ( Fig.12 ). Example 6: RNA polymerase, cap structure and polyadenylation method

[0190] This example tests the tumor therapeutic efficiency of mRNA prepared by different RNA polymerases, different cap structures, different polyadenine tails, and different preparation methods.

[0191] In the first experiment, OX40L and IL-12 mRNAs were transcribed by VSW-3 RNA polymerase, capped with VCE (vaccinia virus capping enzyme), and poly(A)-enzymatically capped and tailed with yeast polyadenylate polymerase. In the second experiment, mRNAs were transcribed by T7 RNA polymerase and capped with AG capping and the addition of a polyadenine tail by the transcription template (co-transcriptional capping and tailing). The mRNA synthesized by the two methods was injected intratumorally into the tumor on one side of the test animal. Fig.13 As shown, OX40L and IL-12 mRNA produced using different RNA polymerases, capping methods, and polyadenylation methods all showed significant anti-tumor effects.

[0192] It has been reported that the amount of double-stranded RNA byproduct (dsRNA) contained in the RNA product synthesized by VSW-3 RNA polymerase is much lower than that in the T7 RNAP IVT product. AG compatibility. EGFP was used as a marker protein to indicate the expression of mRNA transcribed by different RNA polymerases. Equal amounts of EGFP mRNA produced by AG transcription were transfected into HEK293T cells. The fluorescence signal in HEK293T cells transfected with EGFP mRNA transcribed by VSW-3 was much lower than that of EGFP mRNA transcribed by T7 ( Fig.14 ), which indicates that VSW-3 is AG has poor compatibility.

[0193] This example then further tested the anti-tumor effects of OX40L and IL-12 mRNAs with different cap structures. OX40L and IL-12 mRNAs with cap-0 or cap-1 were injected on days 8 and 11 after tumor implantation. On day 14, both cap0 and cap1 mRNAs showed comparable anti-tumor effects, but on day 17, the anti-tumor effect of cap1 mRNA was more stable than that of cap0 mRNA ( Fig.15 ). Example 7: mRNAs with different UTR sequences and codon optimization

[0194] This example evaluates the effects of different UTR combinations on mRNA translation.

[0195] Use EGFP mRNA as a research tool. Fig.16As shown, UTR combination H or combination R was tested by detecting the translation of EGFP mRNA with different UTRs. The sequence of combination R was adopted from PCT application number WO2023142283A1, and the 5'UTR and 3'UTR sequences of combination H are shown in SEQ ID NO: 19 and 20 (Table A). UTR combination H showed a slightly higher mean fluorescence intensity (MFI) than combination R.

[0196] In addition, the sequences of OX40L and IL-12 mRNA were codon optimized. Fig.17 As shown, except for #8 (SEQ ID NO: 29) and #10 mRNA (SEQ ID NO: 31) of OX40L which showed relatively low translation levels, most of the codon-optimized mRNAs showed excellent translation levels. Example 8: mRNAs with different poly(A) lengths

[0197] This example examines the effect of different lengths of poly(A) on the translation of EGFP mRNA. EGFP mRNA with 30 adenines or 120 adenines poly(A) tails was transfected into HEK293T cells in equal amounts, and fluorescence was measured 18 hours after transfection. Compared with 30A, 120A EGFP mRNA had a higher translation level ( Fig.18 ).

[0198] The scope of the present disclosure should not be limited to the specific embodiments described, which are only single examples of various aspects of the present disclosure, and any functionally equivalent compositions or methods are within the scope of the present disclosure. It is easy for those skilled in the art to make various modifications and changes to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover these modifications and changes as long as they are within the scope of the attached claims and their equivalents.

[0199] All publications and patent applications mentioned in this specification are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was individually and specifically identified as such.

Claims

1. A messenger RNA (mRNA), comprising a 5' untranslated region (UTR), a 3'UTR and a coding sequence, wherein the 5'UTR comprises the nucleic acid sequence of SEQ ID NO: 19, and the 3'UTR comprises the nucleic acid sequence of SEQ ID NO:

20.

2. The mRNA according to claim 1, wherein the 5' end of the mRNA comprises a Cap-1 structure.

3. according to the mRNA of claim 2, wherein the Cap-1 structure consists of AG co-transcriptional capping reagent or enzymatic cap formation.

4. mRNA according to claim 3, wherein the mRNA is transcribed by T7 RNA polymerase.

5. The mRNA according to any preceding claim, wherein the mRNA further comprises a poly(A) tail, preferably prepared enzymatically or co-transcriptionally.

6. The mRNA according to claim 5, wherein poly(A) comprises at least 50 adenine bases, preferably at least 80 adenine bases, more preferably at least 100 adenine bases.

7. The mRNA according to any preceding claim, wherein the coding sequence encodes IL-12 or an OX40 agonist, or both IL-12 and an OX40 agonist.

8. The mRNA according to claim 7, 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.

9. The mRNA according to claim 7 or 8, wherein the coding sequence further encodes FADD (FAS-associated death domain protein), MLKL (mixed lineage kinase domain-like pseudokinase) or the four-helix bundle domain of MLKL (MLKL-4HB).

10. The mRNA according to claim 7 or 8, wherein the coding sequence of IL-12 is selected from the group consisting of nucleic acid sequences from SEQ ID NO: 21-26.

11. The mRNA according to claim 7 or 8, wherein the coding sequence of OX40L is selected from a group comprising nucleic acid sequences from SEQ ID NO: 27-33, preferably from the group of SEQ ID NO: 27, 28, 30, 32 and 33.

12. A pharmaceutical composition comprising the mRNA of any one of claims 1-11, and an ionizable lipid, a phospholipid, a structural lipid, a polyvinyl alcohol (PEG) lipid, or a combination thereof.

13. The pharmaceutical composition according to claim 12, wherein the ionizable lipid is SM102 or ALC0315.

14. Use in the preparation of a drug for treating tumors, comprising administering the mRNA of any one of claims 1 to 11 or the pharmaceutical composition of claim 12 or 13 to a patient.

15. A use in preparing a drug for treating tumors, comprising administering a first mRNA (encoding an OX40 agonist), a second mRNA (encoding IL-12), and a third mRNA (encoding FADD, MLKL, or MLKL-4HB) to a patient.

16. The use according to claim 15, wherein the third mRNA is on the same RNA molecule as the first mRNA or the second mRNA.

17. The use according to claim 15 or 16, wherein the OX40 agonist is an OX40 ligand (OX40L), a polypeptide comprising the extracellular domain of OX40L, or an anti-OX40 agonistic antibody or an antigen-binding fragment thereof.

18. The use according to any one of claims 15 to 17, wherein the first mRNA and the second mRNA are administered in a mass ratio of 4:1 to 0.5:1, preferably 3:1 to 0.75:1, more preferably 2:1 to 0.9:

1.

19. The use according to any one of claims 14 to 18, wherein the administration is by subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathoracic injection, intravenous injection, arterial injection or a combination thereof.

20. The use according to any one of claims 14 to 20, wherein the tumor 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 tumors, 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, brain cancer and head and neck cancer, preferably colon cancer, breast cancer and lung cancer.

21. A pharmaceutical composition comprising a first mRNA (encoding an OX40 agonist and / or IL-12) and a second mRNA (encoding FADD, MLKL or MLKL-4HB).

22. The pharmaceutical composition according to claim 21, wherein the first mRNA encodes an OX40 agonist, the second mRNA encodes FADD, MLKL or MLKL-4HB, and wherein the composition further optionally comprises a third mRNA (encoding IL-12).

23. The pharmaceutical composition according to claim 21, wherein the first mRNA encodes IL-12, the second mRNA encodes FADD, MLKL or MLKL-4HB, and wherein the composition further optionally comprises a third mRNA (encoding an OX40 agonist).

24. An mRNA selected from the group consisting of nucleic acid sequences from SEQ ID NO: 21-26.

25. An mRNA selected from the group consisting of nucleic acid sequences from SEQ ID NO: 27-33, preferably selected from the group consisting of SEQ ID NO: 27, 28, 30, 32 and 33.

Images