Combinatorial immunomodulation and uses thereof

Abstract

The present disclosure relates to compositions and methods for modulating the immune system and for treating cancer and other immune disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 823,184, filed March 25, 2019, which is expressly incorporated herein by reference. Statement on Federally Sponsored Research

[0002] This invention was made with United States government support under grant R35GM119679 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present disclosure relates to compositions and methods for modulating the immune system and for treating cancer and other immune disorders. [Background technology]

[0004] Immunotherapy has become an innovative strategy for treating a wide range of diseases, including various cancers. As key immunoregulatory molecules and immune signals are identified and tailored as therapeutic agents, the clinical efficacy of such agents can be tested using well-known cancer models. Immunotherapeutic strategies include administering vaccines, activated cells, antibodies, cytokines, and chemokines.

[0005] Tumor growth and metastasis depend heavily on their ability to evade host immune surveillance and overcome host defenses. Most tumors express antigens that can be recognized to varying degrees by the host's immune system, but the immune response is often insufficient. The inability to induce potent activation of effector T cells may be due to weak immunogenicity of tumor antigens or inadequate or absent expression of costimulatory molecules by tumor cells. Most T cells require costimulatory signals during T cell receptor engagement for proliferation and IL-2 production. Without these signals, T cells may enter a functionally unresponsive state.

[0006] To date, many therapeutic agents and antibodies have been developed as immunotherapeutic agents to modulate the immune system. What is needed are new compositions and methods for stimulating the immune system to treat cancer and other immune disorders. Summary of the Invention

[0007] Disclosed herein are compositions and methods for modulating the immune system to treat cancer and other immune disorders. The inventors surprisingly discovered that when mRNA encoding a costimulatory molecule is administered together with an antibody that specifically binds to the costimulatory molecule, the combination results in improved tumor therapy and overall survival.

[0008] In some aspects, disclosed herein are compositions comprising nanoparticles comprising an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and mRNA encoding the costimulatory molecule.

[0009] In some embodiments, mRNA encoding a costimulatory molecule is encapsulated by the nanoparticle.

[0010] In some embodiments, the costimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, or LFA3. In some embodiments, the costimulatory molecule comprises OX40. In some embodiments, the costimulatory molecule comprises 4-1BB (CD137).

[0011] In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR).

[0012] In some aspects, disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0013] In some aspects, disclosed herein are methods of stimulating T cells, comprising administering to a subject an effective amount of a composition comprising an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0014] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0015] In some aspects, disclosed herein are methods of treating cancer comprising administering to a subject in need thereof an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0016] In some embodiments, the cancer comprises colorectal cancer or melanoma. In some embodiments, the compositions herein are used to treat both localized and metastatic tumors.

[0017] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0018] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an additional immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. [Brief explanation of the drawings]

[0020] [Figure 1] EG.7-OVA cells treated with phosphate-buffered saline (PBS) control or nanoparticle (NP)-OX40 mRNA. OX40-positive cells were quantified by flow cytometry cell sorting analysis. [Figure 2] Figure 2A shows the change in tumor volume in B16 melanoma-implanted mice after different treatments (Figure 2A) and the mouse survival curve (Figure 2B). NP + OX40 antibody and NP / OX40 mRNA + OX40 antibody: P = 0.0010, log-rank test. PBS vs. NP / OX40 mRNA + OX40 antibody: P = 0.0002, log-rank test. NP represents blank NP, and NP / OX40 represents NP containing OX40 mRNA. [Figure 3] This figure shows the change in tumor volume in a CT26 colon cancer mouse tumor model after different treatments. Mice were treated with PBS, nanoparticles (NPs) + anti-OX40 antibody, nanoparticles (NPs) / OX40 mRNA + anti-OX40 antibody, and nanoparticles (NPs) / OX40 mRNA + anti-OX40 antibody. Nanoparticles (NPs) / OX40 mRNA were injected 6 hours after anti-OX40 antibody (injection interval: 6 hours), or nanoparticles (NPs) / OX40 mRNA were injected simultaneously with anti-OX40 antibody (injection interval: 0 hours). NP represents blank NP, and NP / OX40 represents NP containing OX40 mRNA. [Figure 4]Figures 4A-4D illustrate the stimulation of T cell-mediated cancer immunotherapy. Figure 4A shows antibody immunotherapy enhanced via nanoparticles delivering costimulatory receptor mRNA followed by injection of an agonist antibody to the costimulatory receptor (e.g., PL1-OX40 mRNA + anti-OX40 antibody). Figure 4B shows representative synthetic routes to biomimetic compounds: phospholipid and glycolipid derivatives. i. EtN, toluene, RT. ii. EtN, DMF, RT. iii. TFA, CHCl, RT. iv. aldehyde, EtN, THF, NaBH(OAc) . Figures 4C-4D show the structures of phospholipid derivatives PL1-PL18 (Figure 4C) and glycolipid derivatives GL1-GL16 (Figure 4D). [Figure 5] Figures 5A-5G show biomimetic phospholipid- and glycolipid-derived nanoparticles for mRNA delivery. Figure 5A shows the luminescence intensity of phospholipid- and glycolipid-derived nanoparticles delivering firefly luciferase (Fluc) mRNA to E.G7 cells. Figure 5B shows cryo-electron microscopy images of PL1-OX40 nanoparticles. Scale bar = 50 nm. Figure 5C shows the delivery of GFP mRNA to E.G7 cells by PL1 nanoparticles. Figure 5D shows PL1-CD137-induced CD137 expression in E.G7 cells. Figure 5E shows PL1-OX40-induced OX40 expression in EG.7 cells. Figure 5F shows a scheme of GFP expression in B16F10 tumors after a single injection of free GFP mRNA or PL1-GFP. Figure 5G shows GFP expression in CD4+ and CD8+ T cells after a single intratumoral injection of GFP mRNA (n = 4) or PL1-GFP mRNA (n = 5) in B16F10 tumors. Data in Figures 5A-5E are from n = 3 biologically independent samples. All data are expressed as mean ± SEM. Statistical significance in Figures 5C, 5D, 5E, and 5G was analyzed by two-tailed Student's t-test. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, not significant. [Figure 6]Figures 6A-6D show regression of B16F10 and A20 tumors after treatment with PL1-CD137 mRNA plus anti-CD137 antibody. Figures 6A and 6B show B16F10 melanoma cells implanted sc into C57BL / 6 mice. Tumor volume (Figure 6A) and survival (Figure 6B) of mice (n = 10 / group) after treatment with PBS, PL1 plus anti-CD137 Ab, or PL1-CD137 plus anti-CD137 Ab. PL1-CD137 (10 μg mRNA / mouse) and anti-CD137 Ab (16 μg / mouse) were administered it six times every other day. Figures 6C and 6D show A20 lymphoma cells implanted sc into BALB / c mice. Tumor volume (Figure 6C) and survival (Figure 6D) of mice treated with PBS (n=10), PL1 + anti-CD137 Ab (n=12), or PL1-CD137 + anti-CD137 Ab (n=12). PL1-CD137 (10 μg mRNA / mouse) and anti-CD137 Ab (16 μg / mouse) were administered every other day for six it doses. Data in Figures 6A and 6C are presented as mean ± SEM. Statistical significance in a and c was analyzed by two-way analysis of variance. Statistical significance in Figures 6B and 6D was analyzed by log-rank (Mantel-Cox) test. **P<0.01; ***P<0.001; ns, not significant. [Figure 7]Figures 7A-7D show regression of B16F10 and CT26 tumors after treatment with PL1-OX40 plus anti-OX40 Ab. Figures 7A-7B show C57BL / 6 mice bearing B16F10 melanoma cells. Tumor volume (Figure 7A) and survival (Figure 7B) of mice treated with PBS, PL1 plus anti-OX40 Ab, or PL1-OX40 plus anti-OX40 Ab (n = 10 / group) were compared. PL1-OX40 (10 μg mRNA / mouse) and anti-OX40 Ab (8 μg / mouse) were administered it six times every other day. Figures 7C-7D show BABL / c mice subcutaneously implanted with CT26 colon cancer cells. Tumor volume (Figure 7C) and survival (Figure 7D) of mice treated with PBS, PL1 plus anti-OX40 Ab, or PL1-OX40 plus anti-OX40 Ab (n = 10-11 / group) were compared. PL1-OX40 (10 μg mRNA / mouse) and anti-OX40 Ab (8 μg / mouse). Six it doses were administered every other day. Data in Figures 7A and 7C are presented as mean ± SEM. Statistical significance in Figures 7A and 7C was analyzed by two-way analysis of variance. Statistical significance in Figures 7B and 7D was analyzed by log-rank (Mantel-Cox) test. **P<0.01; ***P<0.001; ****P<0.0001. [Figure 8]Figures 8A-8H show A20 tumor regression after treatment with PL1-OX40 plus anti-OX40 antibodies. Figure 8A shows a schematic diagram of the A20 mouse tumor model and treatment regimen. Figure 8B shows tumor volumes of individual mice (n = 8-10) after six it administrations of PBS, PL1-OX40 (10 μg mRNA / mouse), PL1 plus anti-OX40 Ab (8 μg / mouse), or PL1-OX40 plus anti-OX40 Ab. Figures 8C and 8D show tumor volume (Figure 8C) and overall survival (Figure 8D). Figure 8E shows rechallenge of mice with a complete response (n = 6) after treatment with PL1-OX40 plus anti-OX40 Ab. Figure 8F shows the treatment plan for assessing OX40 expression on CD8+ T cells after a single it injection of PBS (n = 5), OX40 mRNA (n = 5), or PL1-OX40 (n = 6). Figures 8G and 8H show immune cell analysis (CD8+, CD4+ T cells, macrophages, DCs) after six it injections of PBS (n = 5), PL1 + anti-OX40 Ab (n = 4), or PL1-OX40 + anti-OX40 Ab (n = 6), respectively. Data in Figures 8C, 8F, and 8H are presented as mean ± SEM. Statistical significance in Figure 8C was analyzed by two-way ANOVA. Statistical significance in Figure 8D was analyzed by the log-rank (Mantel-Cox) test. Statistical significance in Figures 8F and 8H was analyzed by a two-tailed Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. [Figure 9]Figures 9A-9J show the antitumor effects of PL1-OX40 mRNA plus anti-OX40 Ab in combination with surgery or checkpoint inhibitors. Figure 9A shows a schematic of PL1-OX40 mRNA plus anti-OX40 Ab treatment combined with surgery (tumor volume <500 mm). Figure 9B shows the tumor volume of individual mice (n = 10 / group) after six it injections of PBS, anti-OX40 (40 μg), or PL1-OX40(ψ) plus anti-OX40 Ab (n = 10). Figures 9C and 9D show the tumor volume (Figure 9C) and survival (Figure 9D) of mice. Figure 9E shows the tumor volume of mice that received PL1-OX40(ψ) plus anti-OX40 (40 μg) and then underwent surgery (n = 2) to remove residual tumors versus control (n = 5) mice after rechallenge. Figure 9F shows a schematic diagram of PL1-OX40 mRNA + anti-OX40 Ab treatment in combination with anti-PD-1 + anti-CTLA-4 Ab. Figure 9G shows tumor volumes for individual mice that received six doses of PBS (n = 10), anti-mouse PD-1 + anti-mouse CTLA-4 Ab (n = 10), or PL1-OX40(ψ) + anti-OX40 (40 μg), anti-PD-1 Ab, and anti-CTLA-4 Ab (n = 10) every other day. Anti-mouse PD-1 + anti-mouse CTLA-4 Ab was injected i.p. six times every three days. Figures 9H and 9I show tumor volumes (Figure 9H) and survival (Figure 9I) for mice. Figure 9J shows rechallenge tumor volumes for mice that achieved a complete response to treatment with PL1-OX40(ψ) + anti-OX40 (40 μg) + anti-PD-1 + anti-CTLA-4 Ab (n = 6) versus control (n = 7). Data in Figures 9C, 9E, 9H, and 9J are presented as mean ± SEM. Figures 9C and 9H were analyzed by two-way analysis of variance. Statistical significance in Figures 9D and 9I was analyzed by the log-rank (Mantel-Cox) test. ***P<0.001; ****P<0.0001; ns, not significant. [Figure 10]Figures 10A-10F show the antitumor effects in a mouse lung metastasis model. Figure 10A shows a schematic diagram of lung metastasis of B16F10 cells treated with PBS, anti-PD-1 + anti-CTLA-4 Ab, or PL1-OX40 mRNA + anti-OX40 Ab + anti-PD-1 + anti-CTLA-4 Ab. Mice received ip injections of PBS (n = 7), anti-mouse PD-1 + anti-mouse CTLA-4 Ab (n = 8), or PL1-OX40 (ψ) + anti-OX40 (100 μg) + anti-PD-1 Ab + anti-CTLA-4 Ab (n = 9) every 3 days, as indicated by the arrows. Figure 10B shows representative melanoma metastases in mouse lungs. Figure 10C shows lung weights. Figures 10D-10F show immune cell analysis of CD8+ T cells, CD4+ T cells, and Foxp3+ CD4+ (Treg) cells in the lungs of different treatments (n = 4, 5, and 5, respectively). Data in Figures 10C-10F are expressed as mean ± SEM. Statistical significance in Figures 10C-10F was analyzed by two-tailed Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. [Figure 11] Figure 1 shows the structures of biomimetic lipids and phospholipid and glycolipid derivatives. Representative examples, PL1 and GL1, consist of a biomimetic head (phosphate or sugar), an ionizable amino core, and multiple hydrophobic tails. [Figure 12] Figures 12A-12C show the properties of phospholipid- and glycolipid-derived nanoparticles. Figure 12A shows particle size (nm) and PDI. Figure 12B shows zeta potential (mV). Figure 12C shows the capture efficiency of Fluc mRNA. All data are from n=3 biologically independent samples and are expressed as mean ± SEM. [Figure 13]Figure 1 shows the endocytic pathway of PL1 nanoparticles. E.G7-OVA cells were treated with 5-(N-methyl-N-isopropyl) amiloride (EIPA), chlorpromazine hydrochloride (CPZ), or methyl-β-cyclodextrin (MβCD). After 0.5 hours, the cells were treated with PL1-Alexa-Fluor 647-labeled RNA nanoparticles. After 3 hours, the cells were analyzed by flow cytometry. All data are from n=3 biologically independent samples and are expressed as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.05. [Figure 14] Figures 14A-C show GFP expression in B16F10 tumors after a single injection of GFP mRNA or PL1-GFP mRNA. Macrophages (Figure 14A) and dendritic cells (Figure 14B) in the tumor microenvironment after a single intratumoral injection of GFP mRNA (n=4) and PL1-GFP mRNA (n=5). Data in Figure 14C are presented as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. **P<0.01, ***P<0.001. [Figure 15] Figures 15A and 15B show tumor growth curves. Figure 15A shows C57BL / 6 mice were subcutaneously implanted with B16F10 melanoma cells. Tumor volumes of individual mice treated with PBS (n=10), PL1 + anti-CD137 Ab (n=10), or PL1-CD137 + anti-CD137 Ab (n=10). PL1-CD137 (10 μg mRNA / mouse) and anti-CD137 Ab (16 μg / mouse) were injected intratumorally every other day for six doses. Figure 15B shows BALB / c mice were subcutaneously implanted with A20 lymphoma cells. Tumor volumes of individual mice treated with PBS (n = 10), PL1 + anti-CD137 Ab (n = 12), and PL1-CD137 + anti-CD137 Ab (n = 12), PL1-CD137 (10 μg mRNA / mouse), and anti-CD137 (16 μg / mouse) intratumoral injections given every other day for six doses. [Figure 16]Figures 16A and 16B show tumor growth curves. Figure 16A shows the tumor volumes of individual animals treated with PBS (n=10), PL1 + anti-OX40 Ab (n=10), or PL1-OX40 + anti-OX40 Ab (n=10). PL1-OX40 (10 μg mRNA / mouse) and anti-OX40 Ab (8 μg) were injected intratumorally every other day for six doses. Figure 16B shows the tumor volumes of individual animals treated with PBS (n=10), PL1 + anti-OX40 Ab (n=11), or PL1-OX40 + anti-OX40 Ab (n=11). PL1-OX40 (10 μg mRNA / mouse) and anti-OX40 Ab (8 μg / mouse). Intratumoral injection for six doses every other day. [Figure 17] Figures 17A-B show analyses of immune cell populations and cytokine levels. Figure 17A shows OX40 expression on the surface of CD4+ T cells, macrophages, and dendritic cells after a single intratumoral injection of PBS (n=5), OX40 mRNA (n=5), or PL1-OX40 mRNA (n=6) in the tumor microenvironment. Figure 17B shows mouse plasma cytokine levels after a single intratumoral injection of PBS (n=5), OX40 mRNA (n=4), or PL1-OX40 mRNA (n=6). All data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. *P<0.01; **P<0.001; ****P<0.0001; ns, not significant. [Figure 18] Figure 1 shows the effect of CD4+ or CD8+ T cell depletion on immunotherapy with PL1-OX40 + anti-OX40 Ab treatment. Tumor volume for IgG + PL1-OX40 + anti-OX40 (n=9), anti-mouse CD8α + PL1-OX40 + anti-OX40 (n=9), or anti-mouse CD4 + PL1-OX40 + anti-OX40 (n=9). All data are expressed as mean ± SEM. Statistical significance was analyzed by two-way ANOVA. ***P<0.001. [Figure 19]Figure 1 shows plasma cytokines after six doses of intratumoral treatment: PBS (n=5), PL1 + anti-OX40 (n=6), and PL1-OX40 + anti-OX40 (n=6). Data are presented as mean ± SEM. Statistical significance was analyzed by two-tailed Student's t-test. ns, not significant. [Figure 20] Figures 20A-20B show antitumor effects in a mouse lung metastasis model. 2 × 10 B16F10 cells were intravenously injected into C57BL / 6 mice. Mice received ip injections of PBS (n = 7), anti-mouse PD-1 + anti-mouse CTLA-4 Ab (n = 8), or PL1-OX40(ψ) + anti-OX40 (100 μg) + anti-PD-1 Ab + anti-CTLA-4 Ab (n = 9) every 3 days. Figure 20A shows the body weight of the mice. Data are expressed as mean ± SD. Figure 20B shows images of melanoma metastases in the mouse lungs 19 days after iv injection of B16F10 cells. [Figure 21] Figures 21A-21D show regression of B16F10 tumors after treatment with it injection of PL1-OX40 and ip injection of anti-OX40 antibody. Figure 21A shows a schematic diagram of the B16F10 mouse tumor model and treatment regimen. Figure 21B shows tumor volumes of individual mice (n=10 / group) after six it administrations of PBS, PL1-OX40(ψ) (10 μg mRNA / mouse), and two ip administrations of anti-OX40 Ab (150 μg / mouse). Figures 21C-21D show tumor volumes (Figure 21C) and overall survival (Figure 21D). Data in Figure 21C are presented as mean ± SEM. Statistical significance in Figure 21C was analyzed by two-way analysis of variance. Statistical significance in Figure 21D was analyzed by log-rank (Mantel-Cox) test. ***P<0.001; ****P<0.0001; ns, not significant. [Figure 22]Figure 1 shows the gating strategy for flow cytometry analysis. Cells were first gated by FSC / SSC to define single cells. Next, CD45-positive cells, CD3-positive cells, CD4 / CD8-positive cells, and OX40 / GFP-positive cells were gated. CD45-positive cells, CD11b-positive cells, CD11c / F4 / 80-positive cells, and OX40 / GFP-positive cells were also gated. DETAILED DESCRIPTION OF THE INVENTION

[0021] Disclosed herein are compositions and methods for modulating the immune system to treat cancer and other immune disorders.

[0022] Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the drawings and examples, but this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof and are open, non-limiting terms. While the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide more specific embodiments, and are disclosed. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] The following definitions are provided for a full understanding of terms used herein.

[0025] term As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases where the condition occurs and cases where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include cases where the formulation includes an excipient as well as cases where the formulation does not include an excipient.

[0026] The term "promoter" or "regulatory element" refers to a region or sequence determinant located upstream or downstream from the start of transcription and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin; for example, promoters derived from viruses or other organisms can be used in the compositions, systems, or methods described herein. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell-type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters.Examples of pol II promoters include, but are not limited to, the retroviral Ruth's sarcoma virus (RSV) LTR promoter (optionally containing the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally containing the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also includes enhancer elements such as WPRE, the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA, Vol. 78(3), pp. 1527-31, 1981). It will be understood by those skilled in the art that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc.

[0027] The term "recombinant" refers to a human-engineered nucleic acid (e.g., polynucleotide) or a copy or complement of a human-engineered nucleic acid (e.g., polynucleotide), or, with respect to a protein (i.e., a "recombinant protein"), to the protein (e.g., polynucleotide) encoded by the recombinant nucleic acid. In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) can include a promoter that is heterologous to the second nucleic acid (e.g., polynucleotide) as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette can include nucleic acids (e.g., polynucleotides) that are combined such that the nucleic acids (e.g., polynucleotides) are highly unlikely to be found in nature. For example, a human-engineered restriction site or a plasmid vector sequence may flank or separate a promoter from a second nucleic acid (e.g., polynucleotide). Those skilled in the art will recognize that nucleic acids (e.g., polynucleotides) can be engineered in many ways and are not limited to the above examples.

[0028] The terms "expression cassette" and "vector" refer to nucleic acid constructs that, when introduced into a host cell, result in the transcription and / or translation of RNA or polypeptide, respectively. In embodiments, an expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., a polynucleotide) can include a promoter that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In some embodiments, an expression cassette comprising a terminator (or termination sequence) operably linked to a second nucleic acid (e.g., a polynucleotide) can include a terminator that is heterologous to the second nucleic acid (e.g., a polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette comprises a promoter operably linked to a second nucleic acid (e.g., polynucleotide) and a terminator operably linked to a second nucleic acid (e.g., polynucleotide) as a result of human manipulation. In some embodiments, the expression cassette comprises an endogenous promoter. In some embodiments, the expression cassette comprises an endogenous terminator. In some embodiments, the expression cassette comprises a synthetic (or non-naturally occurring) promoter. In some embodiments, the expression cassette comprises a synthetic (or non-naturally occurring) terminator.

[0029] The term "identity" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences is determined using the BLAST or BLAST 2.0 sequence comparison algorithms using the default parameters described below, or by manual alignment and visual inspection (e.g., NCBI "Substantially identical" refers to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region. Such sequences are said to be "substantially identical." This definition refers to or can apply to the complement of a test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably, over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent amino acid sequence identity (%) is defined as the percentage of amino acids in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0030] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and subsequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence with reference sequence based on program parameters.

[0031] A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms (described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is halted when: the cumulative alignment score falls by an amount X from its maximum performance value; the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses by default a word length of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (Henikoff and Henikoff (1989), Proc Natl Acad Sci USA, 89:10915) of 50 for alignment (B), an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0032] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0033] The phrase "codon optimization," when referring to genes or coding regions of a nucleic acid molecule intended for transformation into various hosts, refers to modifying the codons in the genes or coding regions of that polynucleic acid molecule to reflect the typical codon usage of a selected organism, without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, more than one, or a significant number of codons with one or more codons that are more frequently used in the genes of the selected organism.

[0034] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous with each other and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g., enhancer and coding sequence) need not be contiguous. Linkage is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked to a coding sequence if it is capable of affecting expression of a protein from that coding sequence (e.g., regulating relative to the absence of the promoter) (i.e., when the coding sequence is under the transcriptional control of the promoter).

[0035] The term "nucleobase" refers to the portion of a nucleotide that possesses the Watson / Crick base pairing function. The most common naturally occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), possess the hydrogen-bonding function to link one nucleic acid strand to another in a sequence-specific manner.

[0036] As used throughout, "subject" (or "host") means an individual. Thus, a "subject" can include, for example, domestic animals such as cats, dogs, etc., livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animals. The subject can be a mammal, such as a primate or a human. Administration of a therapeutic agent can be at a dosage and for a duration effective to treat the subject.

[0037] As used herein, the term "about" when referring to a measurable value, such as an amount, percentage, etc., is meant to encompass a variation of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0038] A nucleic acid sequence is "heterologous" to a second nucleic acid sequence if it is derived from a foreign species, or if derived from the same species, is modified by human action from its original form. For example, a heterologous promoter (or heterologous 5' untranslated region (5'UTR)) operably linked to a coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, and, if from the same species, refers to a coding sequence that differs from a naturally occurring allelic variant (e.g., a 5'UTR or 3'UTR from a different gene is operably linked to a nucleic acid encoding a costimulatory molecule).

[0039] As used herein, the term "treating" or "treatment" of a subject includes administering a drug to a subject for the purpose of curing, healing, alleviating, relieving, altering, treating, mitigating, improving, stabilizing, or affecting a disease or disorder, or the symptoms of a disease or disorder. The terms "treating" and "treatment" can also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, and ameliorating or repairing damage.

[0040] As used herein, the term "prevention" of a disease, disorder, or undesired physiological event in a subject refers to the prevention of the disease, disorder, or undesired physiological event, or the prevention of the symptoms of the disease, disorder, or undesired physiological event.

[0041] An "effective amount" of an agent refers to an amount of the agent sufficient to provide the desired effect. The amount of an agent that is "effective" will vary from subject to subject, depending on many factors, such as the subject's age and general condition, the specific agent(s), and so forth. Therefore, it is not always possible to specify a quantified "effective amount." However, an appropriate "effective amount" for any subject can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of an agent can refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of an agent required to achieve a therapeutic effect may vary depending on factors such as the subject's age, sex, and weight. Dosage regimens can also be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0042] A "pharmaceutically acceptable" ingredient can refer to an ingredient that is not biologically or otherwise undesirable, i.e., the ingredient can be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is included. When used in reference to human administration, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing or is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0043] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations, and as further described herein.

[0044] A "therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as, for example, treating a disorder or other undesirable physiological condition, and prophylactic effects, such as, for example, preventing a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0045] As used herein, the terms "controlled release" or "controlled release drug delivery" or "sustained release" refer to the release or administration of a drug from a given dosage form in a controlled manner to achieve a desired pharmacokinetic profile in vivo. An aspect of "controlled" drug delivery is the ability to manipulate the formulation and / or dosage form to establish desired kinetics of drug release.

[0046] As used herein, the phrases "concurrent administration," "co-administration," "simultaneous administration," or "administered at the same time" mean that the compounds are administered at the same time or shortly after each other.

[0047] The term "polypeptide" refers to a compound composed of a single chain of D- or L-amino acids, or a mixture of D- and L-amino acids linked by peptide bonds.

[0048] The term "antibody" is used broadly herein and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules or fragments thereof. Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic and / or prophylactic activity is tested according to known clinical trial methods. There are five major types of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the murine equivalents. The heavy chain constant domains corresponding to the different types of immunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), respectively.

[0049] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired antagonistic activity.

[0050] The disclosed monoclonal antibodies can be produced using any procedure that produces monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, such as that described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0051] Monoclonal antibodies can also be produced by recombinant DNA methods. DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display technology, as described, for example, in U.S. Pat. No. 5,804,440 (Burton et al.) and U.S. Pat. No. 6,096,441 (Barbas et al.).

[0052] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques well known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94 / 29348 published December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments and Fc fragments, each of which has a single antigen-binding site. Pepsin treatment produces fragments that have two antigen-binding sites and are still capable of cross-linking antigen.

[0053] As used herein, the term "antibody or antigen-binding fragment thereof" or "antibody or fragment thereof" encompasses chimeric and hybrid antibodies having dual or multiple antigen or epitope specificities, as well as hybrid fragments such as F(ab')2, Fab', Fab, Fv, sFv, and scFv fragments. Thus, fragments of antibodies that retain the ability to bind to their specific antigens are provided. For example, fragments of antibodies that retain binding activity are included within the meaning of the term antibody or antigen-binding fragment thereof. Such antibodies and fragments can be produced by techniques well known in the art and screened for specificity and activity according to the methods described in the Examples and general methods for generating and screening antibodies for specificity and activity (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988)).

[0054] The meaning of "antibody or antigen-binding fragment thereof" also includes conjugates of antibody fragments and antigen-binding proteins (single-chain antibodies). The meaning of "antibody or antigen-binding fragment thereof" also includes immunoglobulin single variable domains, such as, for example, nanobodies.

[0055] Fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, so long as the activity of the antibody or fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment. These modifications can provide additional properties, such as removing / adding amino acids capable of disulfide bonding, extending its biological lifespan, or altering its secretion characteristics. In either case, the antibody or antibody fragment will retain biological activity, such as specific binding to its cognate antigen. Functional or active regions of an antibody or antibody fragment can be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and can include site-directed mutagenesis of nucleic acids encoding the antibody or antibody fragment (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0056] As used herein, the terms "antibody" or "antibodies" can also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and, therefore, can provoke an unwanted immune response when administered to humans. Therefore, the use of human or humanized antibodies in these methods helps to reduce the chance that an antibody administered to a human will provoke an unwanted immune response.

[0057] As used herein, the term "nucleic acid" means a polymer composed of nucleotides, eg, deoxyribonucleotides or ribonucleotides.

[0058] As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

[0059] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

[0060] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers.

[0061] Compositions and Methods In some aspects, disclosed herein are compositions comprising nanoparticles comprising an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and mRNA encoding the costimulatory molecule.

[0062] In some aspects, disclosed herein are compositions comprising nanoparticles comprising an antibody, or antigen-binding fragment thereof, that specifically binds to a costimulatory molecule and mRNA encoding the costimulatory molecule.

[0063] In some embodiments, mRNA encoding a costimulatory molecule is encapsulated by the nanoparticle.

[0064] In some embodiments, the nanoparticles comprise a phospholipid or a glycolipid. In some embodiments, the nanoparticles comprise a phospholipid. In some embodiments, the nanoparticles comprise a glycolipid. In some embodiments, the phospholipid is selected from the group consisting of PL1-PL18. In some embodiments, the phospholipid is PL1. In some embodiments, the glycolipid is selected from the group consisting of GL1-GL16. In some embodiments, the glycolipid is GL4.

[0065] In some embodiments, the costimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, or LFA3.

[0066] In some embodiments, the costimulatory molecule comprises OX40. In some embodiments, the costimulatory molecule comprises 4-1BB (CD137). In some embodiments, the costimulatory molecule comprises CD30. In some embodiments, the costimulatory molecule comprises CD2. In some embodiments, the costimulatory molecule comprises B7-H2. In some embodiments, the costimulatory molecule comprises B7-1. In some embodiments, the costimulatory molecule comprises B7-2. In some embodiments, the costimulatory molecule comprises CD70. In some embodiments, the costimulatory molecule comprises CD40. In some embodiments, the costimulatory molecule comprises 4-1BBL. In some embodiments, the costimulatory molecule comprises OX40L.

[0067] Examples of costimulatory molecule sequences (in the case of human sequences) include ICOS (NCBI Reference Sequence: NM_012092.3), CD28 (NCBI Reference Sequence: NM_006139.4), CD27 (NCBI Reference Sequence: NM_001242.4), HVEM (NCBI Reference Sequence: NM_003820.3), LIGHT (NCBI Reference Sequence: NM_003807.4), CD40L (NCBI Reference Sequence: NM_000074.2), 4-1BB (NCBI Reference Sequence: NM_001561.5), OX40 (NCBI Reference Sequence: NM_003331.6), and OX40 (NCBI Reference Sequence: NM_003334.6). 327.4), DR3 (NCBI reference sequence: NM_148965.1), GITR (NCBI reference sequence: NM_004195.3), CD30 (GenBank: M83554.1), SLAM (NCBI reference sequence: NM_003037.4), CD2 (NCBI reference sequence: NM_003037.4), Reference sequence: NM_001328609.1), CD226 (NCBI reference sequence: NM_006566.3), Galectin-9 (GenBank: AB040130.2), TIM1 (GenBank: U02082.1), B7-H2 (NCBI reference sequence: NM_01 5259.5), B7-1 (NCBI reference sequence: NM_005191.4), B7-2 (NCBI reference sequence: NM_175862.5), CD70 (NCBI reference sequence: NM_001252.5), CD40 (NCBI reference sequence: NM_001250.5), 4-1 BBL (NCBI reference sequence: NM_003811.4), OX40L (NCBI reference sequence: NM_003326.5), TL1A (NCBI reference sequence: NM_005118.4), GITRL (GenBank:AY358868.1), CD30L (NCBI reference sequence Sequence: NM_001244.3), SLAM (GenBank: U33017.1), CD48 (NCBI Reference Sequence: NM_001778.4), CD58 (NCBI Reference Sequence: NM_001779.3), CD155 (NCBI Reference Sequence: NM_006505.5), CD112 (NCBI Reference Sequence: NM_001042724.2), TIM3 (GenBank: AF450242.1), TIM4 (NCBI Reference Sequence: NM_138379.3), and ICAM1 (NCBI Reference Sequence: NM_000201.3).

[0068] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is BMS986178. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is GSK3174998. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is PF-04518600. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is MOXR0916. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is PF-04518600. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is MEDI6383. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is MEDI0562. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is INCAGN01949. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule is InVivoPlus anti-mouse OX40 (clone OX-86) (company: BioXcell, catalog: BP0031).

[0069] Additional antibodies or antigen-binding fragments thereof that specifically bind to costimulatory molecules include, for example, InVivoPlus anti-mouse 4-1BB in mice; (CD137) (clone LOB12.3) (company: BioXcell, catalog: BP0169), InVivoPlus anti-mouse CD40 (clone FGK4.5 / FGK45) (company: BioXcell, catalog: BP0016-2); for humans, examples include anti-human OX40, BMS986178, GSK3174998, PF-04518600, MOXR0916, PF-04518600, MEDI6383, MEDI0562, INCAGN01949; anti-human 4-1BB, utomilumab, urelumab; anti-human CD40, CP-870893, APX005M, ADC-1013, JNJ-64457107, SEA-CD40, RO7009789.

[0070] In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR).

[0071] In some embodiments, a nucleic acid (e.g., an mRNA encoding a costimulatory molecule) disclosed herein comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

[0072] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

[0073] In one embodiment, the chemically modified nucleobase is 5-formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxycytidine (5moC), 5-hydroxycytidine (5hoC), 5-hydroxymethylcytidine (5hmC), 5-formyluridine (5fU), 5-methyluridine (5-meU), 5-methoxyuridine (5moU), 5-carboxymethylesteruridine (5camU), pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1 Ψ), N 6 -Methyladenosine (me 6 A), or thienoguanosine ( th G).

[0074] In some embodiments, the chemically modified nucleobase is 5-methoxyuridine (5moU). In some embodiments, the chemically modified nucleobase is pseudouridine (Ψ). In some embodiments, the chemically modified nucleobase is N 1 -Methylpseudouridine (m 1 Ψ).

[0075] The structures of these modified nucleobases are shown below: [ka]

[0076] In one embodiment, the at least one chemically modified nucleotide is a chemically modified ribose.

[0077] In one embodiment, the chemically modified ribose is selected from 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro-beta-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'-SFANA, 2'-azido, UNA, 2'-O-methoxy-ethyl (2'-O-ME), 2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, locked nucleic acid (LAN), methylene-cLAN, N-MeO-amino BNA, or N-MeO-aminooxy BNA. In one embodiment, the chemically modified ribose is 2'-O-methyl (2'-O-Me). In one embodiment, the chemically modified ribose is 2'-fluoro (2'-F).

[0078] The structures of these modified riboses are shown below: [ka]

[0079] In one embodiment, at least one chemically modified nucleotide is a chemically modified phosphodiester linkage.

[0080] In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boranophosphate, phosphodithioate (PS2), 3',5'-amide, N3'-phosphoramidate (NP), phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester linkage is phosphorothioate.

[0081] The structures of these modified phosphodiester linkages are shown below: [ka]

[0082] In some embodiments, the composition further comprises an immunotherapeutic agent, hi some embodiments, the immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0083] In some aspects, disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0084] In some aspects, disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to a nanoparticle comprising a costimulatory molecule and mRNA encoding the costimulatory molecule.

[0085] In some aspects, disclosed herein are methods of treating cancer comprising administering to a subject in need thereof an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0086] In some aspects, disclosed herein are methods of treating cancer, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof that specifically binds a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0087] In some embodiments, mRNA encoding a costimulatory molecule is encapsulated by the nanoparticle.

[0088] The nanoparticles used can be any nanoparticles useful for nucleic acid delivery. In some embodiments, the nanoparticles comprise lipid-like nanoparticles. See, for example, WO / 2016 / 187531A1, WO / 2017 / 176974, WO / 2019 / 027999, or Li, B et al. An Orthogonal Array Optimization of Lipid-Like Nanoparticles for mRNA Delivery in Vivo. Nano Lett. 2015, 15, 8099-8107; these are incorporated herein by reference. In some embodiments, the nanoparticles (or delivery agents) can comprise lipid bilayers or liposomes. In some embodiments, the nanoparticles can comprise polymers, such as biodegradable polymers. The polymers can include both biostable and biodegradable polymers such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyalkylene oxides such as polyethylene oxide (PEG), polyanhydrides, poly(ester anhydrides), polyhydroxy acids such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0089] In some embodiments, the costimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, or LFA3. In some embodiments, the costimulatory molecule comprises OX40. In some embodiments, the costimulatory molecule comprises 4-1BB (CD137).

[0090] In some embodiments, the mRNA encoding the costimulatory molecule is isolated. In some embodiments, the mRNA encoding the costimulatory molecule is recombinant. In some embodiments, the antibody or antigen-binding fragment thereof is isolated. In some embodiments, the antibody or antigen-binding fragment thereof is recombinant. In some embodiments, the antibody is a monoclonal antibody.

[0091] In some embodiments, the cancer comprises melanoma, colorectal cancer, lung cancer, colon cancer, or lymphoma. In some embodiments, the cancer comprises colorectal cancer or melanoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is melanoma. In some embodiments, the compositions herein are used to treat both localized and metastatic tumors.

[0092] In some embodiments, the compositions and methods described herein are useful for treating or preventing metastasis or recurrence of cancer. In some embodiments, the compositions and methods described herein are useful for preventing recurrence of resected solid tumors. In some embodiments, the compositions and methods described herein are useful for preventing metastasis of resected solid tumors.

[0093] In one aspect, the methods described herein are directed to the treatment of cancer, such as, among others, melanoma, lung cancer (such as lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma, etc.); breast cancer (such as ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serous cavity breast carcinoma, etc.); colorectal cancer (such as colon carcinoma, rectal carcinoma, colorectal adenocarcinoma); anal cancer; pancreatic cancer (such as pancreatic adenocarcinoma, pancreatic islet cell carcinoma, neuroendocrine tumors, etc.); prostate cancer; prostate adenocarcinoma; ovarian cancer (such as ovarian epithelial or surface epithelial-stromal tumors, including serous tumors, endometrioid tumors and mucinous cystadenocarcinomas, sex cord-stromal tumors, Tumors); Liver and bile duct cancer (hepatocellular carcinoma, cholangiocarcinoma, hemangioma, etc.); Esophageal cancer (esophageal adenocarcinoma and squamous cell carcinoma, etc.); Oral cavity and oropharyngeal squamous cell carcinoma; Salivary gland adenoid cystic carcinoma; Bladder cancer; Bladder carcinoma; Uterine cancer (including endometrial adenocarcinoma, eye, uterine serous carcinoma, uterine clear cell carcinoma, uterine sarcoma, leiomyosarcoma, mixed Mullerian tumor); Glioma, glioblastoma, medulloblastoma, and other tumors of the brain; Kidney cancer (renal cell carcinoma, clear cell carcinoma, Wilms' tumor, etc.); Head and neck cancer (squamous cell carcinoma, etc.); Gastric cancer (gastric carcinoma, gastric adenocarcinoma, gastrointestinal stromal tumor); Testicular tumor; Germ cell tumor; Neuroendocrine tumor Tumors; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; mesenchymal tumors such as sarcomas, fibrosarcoma, hemangiomas, hemangiomatosis, hemangiopericytomas, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin, melanoma, neck, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, testicular, kidney, bladder, soft tissue, adrenal, urethral, ​​penile cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma Used to treat malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumor, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, adrenal nephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, anaplastic glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant neurilemmoma, neurofibrosarcoma, parathyroid carcinoma, medullary thyroid carcinoma, bronchial carcinoid, pheochromocytoma, pancreatic islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phyllodes, salivary carcinoma, thymic carcinoma, and vaginal carcinoma.

[0094] In some embodiments, the compositions and methods described herein are useful for treating or preventing cancer. In some cases, the cancer is a circulating cancer cell (circulating tumor cell). In some cases, the cancer is a metastatic cancer cell.

[0095] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof and the nanoparticles are administered by intramuscular injection or are administered systemically.

[0097] In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises an additional immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

[0098] In one embodiment, the immunotherapeutic agent is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is selected from atezolizumab, durvalumab, or avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab (MPDL3280A) (Roche). In one embodiment, the anti-PD-L1 antibody is durvalumab (MEDI4736). In one embodiment, the anti-PD-L1 antibody is avelumab (MS0010718C).

[0099] In one embodiment, the immunotherapeutic agent is a programmed death protein 1 (PD-1) inhibitor or an inhibitor of programmed death protein ligand 1 or 2. PD-1 inhibitors are known in the art and include, for example, nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS), and MEDI4736 (Roche / Genentech).

[0100] In one embodiment, the immunotherapeutic agent is an anti-PD1 antibody. In one embodiment, the anti-PD1 antibody is nivolumab. In one embodiment, the anti-PD1 antibody is pembrolizumab.

[0101] In one embodiment, the immunotherapeutic agent is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is ipilimumab.

[0102] In some embodiments, the additional therapeutic agent is an anti-neoplastic agent, such as avilason acetate, avitrexate (metrecelate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), Abraxane, ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, trastuzumab emtansine (Ado-Trastuzumab Emtansine), Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib maleate, Afinitor (everolimus), Aquinzeo (netupitant / palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), ambochlorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitan , Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), asparaginase erwinia chrysanthemum, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, Besenam (carmustine), Beleodac (belinostat), belinstat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Bilincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib s-malate, CAF, Camptosar (alemtuzumab), Camptosar Taxol (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, Carmbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CeeNU (lomustine), ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab,Chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), chloral (clofarabine), CMF, Cometriq (cabozantinib s-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), Crizolib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine, liposomal, Cytosar-U (cytarabine) , Cicloxane (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (doxorubicin hydrochloride liposomal), DTIC-Dome (dacarbazine), Efudex (fluorouracil), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase), Erwinia chrysanthesin Chimy), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), exemestane, Fairston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab,FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivert vaccine), Gardasil 9 (recombinant HPV non-valent vaccine), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gliotrif (afatinib dimaleate), Glivec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV non- valent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hyper CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, IFEX (ifosfamide), ifosfamide, ifosfamid, imatinib mesylate, Imbruvica (ibrutinib), imiquimod, interferon alfa-2b, recombinant, Intron A (recombinant interferon alfa-2b), iodine I 131 Thiositomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, ISTODAX (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakavi (ruxolitinib), Jevtana (cabazitaxel), Kadcyla (trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kyprolis (carfilzomib), lanreotide acetate, lapatinib tosilate hydrate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Revlan (aminolevulinic acid), Linfolizin (chlorambucil),Lipodox (doxorubicin hydrochloride liposomal), liposomal cytarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot-3 Month (leuprolide acetate), Lupron Depot-4 Month (leuprolide acetate) Month (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposomal), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megase (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), metazolastone (temozolomide), methotrexate Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozilex (Mitomycin C), MOPP, Mosvir (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine) , Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), nelarabine, Neosar (cyclophosphamide), netupitant and palonosetron hydrochloride, Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, nivolumab, Nolvadex (tamoxifen citrate), N-plate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuxinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Ontak (denileukin diftitox), OPPA oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin,Palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaspargase, PEG-interferon alfa-2b, PEG-Intron (PEG-interferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine) , Provenge (sipuleucel-T), Purintor (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonvalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, R-EPOCH, Revlimid (lenamidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intrapleural aerosol Intrapleural Aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alfa-2b), Sylvant (siltuximab), Synovir (thalidomide), Synribo (omacetaxine mepesuxinate), TAC, Tafinlar (dabrafenib), talc,Tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thiotepa, Toposa, (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I 131, tositumomab, Totect (dexrazoxane hydrochloride), TPF, tramelenib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tycarb (lapatinib ditosilate), Unituxin (dinutuximab), vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Versal (vin Vinblastine sulfate), vemurafenib, Bepcid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP vismodegib, Voraxaze (glucarpidase), vorinostat, Votrie (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xeliri, Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumumab) The agent may be selected from the group consisting of Mabtiuxetan, Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0103] In some embodiments, the costimulatory molecule is ICOS. In some embodiments, the costimulatory molecule is CD28. In some embodiments, the costimulatory molecule is CD27. In some embodiments, the costimulatory molecule is HVEM. In some embodiments, the costimulatory molecule is LIGHT. In some embodiments, the costimulatory molecule is CD40L. In some embodiments, the costimulatory molecule is 4-1BB. In some embodiments, the costimulatory molecule is DR3. In some embodiments, the costimulatory molecule is GITR. In some embodiments, the costimulatory molecule is CD30. In some embodiments, the costimulatory molecule is SLAM. In some embodiments, the costimulatory molecule is CD2. In some embodiments, the costimulatory molecule is CD226. In some embodiments, the costimulatory molecule is galectin 9. In some embodiments, the costimulatory molecule is TIM1. In some embodiments, the costimulatory molecule is LFA1. In some embodiments, the costimulatory molecule is B7-H2. In some embodiments, the costimulatory molecule is B7-1. In some embodiments, the costimulatory molecule is B7-2. In some embodiments, the costimulatory molecule is CD70. In some embodiments, the costimulatory molecule is LIGHT. In some embodiments, the costimulatory molecule is HVEM. In some embodiments, the costimulatory molecule is CD40. In some embodiments, the costimulatory molecule is 4-1BBL. In some embodiments, the costimulatory molecule is OX40L. In some embodiments, the costimulatory molecule is TL1A. In some embodiments, the costimulatory molecule is GITRL. In some embodiments, the costimulatory molecule is CD30L. In some embodiments, the costimulatory molecule is SLAM. In some embodiments, the costimulatory molecule is CD48. In some embodiments, the costimulatory molecule is CD58. In some embodiments, the costimulatory molecule is CD155. In some embodiments, the costimulatory molecule is CD112. In some embodiments, the costimulatory molecule is CD80. In some embodiments, the costimulatory molecule is CD86. In some embodiments, the costimulatory molecule is ICOSL.In some embodiments, the costimulatory molecule is TIM3. In some embodiments, the costimulatory molecule is TIM4. In some embodiments, the costimulatory molecule is ICAM1. In some embodiments, the costimulatory molecule is LFA3.

[0104] In some embodiments, the costimulatory molecule is OX40. In some embodiments, the OX40 costimulatory molecule comprises the mRNA sequence of SEQ ID NO: 1. In some embodiments, the OX40 costimulatory molecule comprises the mRNA sequence of SEQ ID NO: 2. In some embodiments, the OX40 costimulatory molecule comprises the mRNA sequence of SEQ ID NO: 5. In some embodiments, the costimulatory molecule is OX40. In some embodiments, the OX40 costimulatory molecule comprises the mRNA sequence of SEQ ID NO: 6.

[0105] In some embodiments, the OX40 costimulatory molecule comprises a nucleic acid sequence or a variant or fragment thereof that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 1. In some embodiments, the OX40 costimulatory molecule comprises a nucleic acid sequence or a variant or fragment thereof that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 2. In some embodiments, the OX40 costimulatory molecule comprises a nucleic acid sequence or a variant or fragment thereof that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 5. In some embodiments, the OX40 costimulatory molecule comprises a nucleic acid sequence or a variant or fragment thereof that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 6.

[0106] In some embodiments, the costimulatory molecule is selected from the group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80 , CD86, ICOSL, TIM3, TIM4, ICAM1, LFA3, or a variant or fragment thereof.

[0107] In some embodiments, the mRNA encoding the costimulatory molecule comprises a modified 5' untranslated region (5'UTR). In some embodiments, the mRNA encoding the costimulatory molecule comprises a modified 3' untranslated region (3'UTR). For example, the modified sequence can include an insertion, deletion, or nucleotide substitution.

[0108] In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR) comprising the mRNA sequence of SEQ ID NO: 3. In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR) comprising mRNA SEQ ID NO: 4. In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR) comprising a nucleic acid sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 3, or a variant or fragment thereof. In some embodiments, the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR) comprising a nucleic acid sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO:4, or a variant or fragment thereof.

[0109] In some aspects, disclosed herein are methods of stimulating T cells, comprising administering to a subject an effective amount of a composition comprising an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0110] In some aspects, disclosed herein are methods of stimulating T cells, comprising administering to a subject an effective amount of a composition comprising an antibody, or antigen-binding fragment thereof, that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0111] In some embodiments, the antigen-binding fragment that specifically binds to a costimulatory molecule comprises an OX40 ligand or a functional fragment thereof that binds to OX40. In some embodiments, the OX40 ligand is encoded by a nucleic acid sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 13 or 14.

[0112] In some embodiments, the antigen-binding fragment that specifically binds to a costimulatory molecule comprises an ICOS ligand or a functional fragment thereof that binds to ICOS. In some embodiments, the ICOS ligand is encoded by a nucleic acid sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 15 or 16.

[0113] In some embodiments, the antigen-binding fragment that specifically binds to a costimulatory molecule comprises a CD137 ligand or a functional fragment thereof that binds to CD137. In some embodiments, the CD137 ligand is encoded by a nucleic acid sequence that is at least 60% (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 19 or 20. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the T cells comprise CD4+ T cells, CD8+ T cells, or a combination thereof. In some embodiments, the T cells comprise CD8+ T cells. CD8+ T cells, also known as cytotoxic T cells, can function to kill specifically recognized cells (such as tumor cells).

[0114] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and the nanoparticles comprising mRNA encoding the costimulatory molecule are administered simultaneously (simultaneously or shortly thereafter). In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and the nanoparticles comprising mRNA encoding the costimulatory molecule are administered sequentially.

[0115] Disclosed herein are methods for treating a disease or condition, such as an inflammatory disease (such as an autoimmune disease) or a lymphoproliferative disease, the method comprising administering to a subject in need thereof an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0116] Also disclosed herein are methods for treating a disease or condition, such as an inflammatory disease (such as an autoimmune disease) or a lymphoproliferative disease, the method comprising administering to a subject in need thereof an effective amount of an antibody, or antigen-binding fragment thereof, that specifically binds to a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule.

[0117] In one embodiment, provided herein is a method of treating an inflammatory disorder, such as an autoimmune disease, in a subject. The method comprises administering to the subject a therapeutically effective amount of a compound, a combination of compounds, or a composition provided herein, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition provided herein. Examples of autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune skin diseases, celiac disease, Crohn's disease, diabetes mellitus (type 1), Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, lupus erythematosus, multiple sclerosis, myasthenia gravis, myoclonus syndrome (OMS), optic neuritis, and orthothyroiditis. These disorders include adenitis, emphysema, polyarthritis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), warm autoimmune hemolytic anemia, Wegener's granulomatosis, alopecia areata (e.g., inflammatory alopecia), Chagas' disease, chronic fatigue syndrome, autonomic neuropathy, endometriosis, hidradenitis suppurativa, interstitial cystitis, neuromyotonia, sarcoidosis, scleroderma, ulcerative colitis, vitiligo, and vulvodynia. Other disorders include bone resorption disorders and thrombosis.

[0118] Inflammation takes many forms, including but not limited to acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotizing, obstructive, parenchymal, plastic, productive, proliferative, pseudomembranous, suppurative, sclerosing, seroplastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and / or ulcerative inflammation.

[0119] Exemplary inflammatory conditions include, but are not limited to, acne, anemia (e.g., aplastic anemia, hemolytic autoimmune anemia), asthma, arteritis (e.g., polyarteritis, temporal arteritis, periarteritis nodosa, Takayasu's arteritis), arthritis (e.g., crystalline arthritis, osteoarthritis, psoriatic arthritis, gout flare, gouty arthritis, reactive arthritis, rheumatoid arthritis, and Reiter's arthritis), ankylosing spondylitis, amylosis, amyotrophic lateral sclerosis, autoimmune diseases, allergies or allergic reactions, atherosclerosis nodosa, tracheal ulcers, and ulcers. bronchiolitis, bursitis, chronic prostatitis, conjunctivitis, Chagas' disease, chronic obstructive pulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., type 1 diabetes, type 2 diabetes), skin conditions (e.g., psoriasis, eczema, burns, dermatitis, pruritus (itching)), endometriosis, Guillain-Barre syndrome, infections, ischemic heart disease, Kawasaki disease, glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g., migraine, tension headache), intestinal obstruction (e.g., idiopathic thrombocytopenic purpura, interstitial cystitis (painful bladder syndrome), gastrointestinal disorders (e.g., peptic ulcer, regional enteritis, diverticulitis) , gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., selected from eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, and eosinophilic colitis), gastritis, diarrhea, gastroesophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, fecal diversion colitis, Behcet's syndrome, and atypical colitis) and inflammatory bowel syndrome (IBS)), lupus, multiple sclerosis, morphea, myasthenia gravis, myocardial ischemia, nephropathic syndrome, pemphigus vulgaris, pernicious anemia, peptic ulcer, multiple sclerosis, urinary tract infection ... These include myositis, primary biliary cirrhosis, encephalopathy-associated neuroinflammation (e.g., Parkinson's disease, Huntington's disease, and Alzheimer's disease), prostatitis, chronic inflammation associated with cranial radiation injury, pelvic inflammatory disease, polymyalgia rheumatica, reperfusion injury, regional enterocolitis, rheumatic fever, systemic lupus erythematosus, scleroderma, scleroderma (scierodoma), sarcoidosis, spondyloarthritis Sjogren's syndrome, thyroiditis, transplant rejection, tendonitis, trauma or injury (e.g., frostbite, chemical irritants, toxins, scars, burns, physical injuries), vasculitis, vitiligo, and Wegener's granulomatosis.In certain embodiments, the inflammatory disorder is selected from arthritis (e.g., rheumatoid arthritis), inflammatory bowel disease, inflammatory bowel syndrome, asthma, psoriasis, endometriosis, interstitial cystitis, and prostatitis. In certain embodiments, the inflammatory condition is an acute inflammatory condition (e.g., inflammation resulting from infection). In certain embodiments, the inflammatory condition is a chronic inflammatory condition (e.g., conditions resulting from asthma, arthritis, and inflammatory bowel disease). These compounds may also be useful in treating inflammation associated with trauma and non-inflammatory muscle pain.

[0120] Immune disorders, such as autoimmune disorders, include, but are not limited to, arthritis (degenerative joint diseases such as rheumatoid arthritis, spondyloarthropathy, gouty arthritis, osteoarthritis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, undifferentiated spondyloarthritis), Behcet's disease, hemolytic autoimmune anemia, multiple sclerosis, amyotrophic lateral sclerosis, amylosis, acute painful shoulder, psoriasis, and juvenile degenerative diseases. age-related arthritis), asthma, atherosclerosis, osteoporosis, bronchitis, tendonitis, bursitis, skin conditions (e.g., psoriasis, eczema, burns, dermatitis pruritus (itching)), bedwetting, eosinophilic diseases, gastrointestinal disorders (e.g., peptic ulcer, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic gastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic gastroenteropathy), gastritis, diarrhea, stomach These include esophageal reflux disease (GORD, or its synonym GERD), inflammatory bowel disease (IBD) (e.g., Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, fecal diversion colitis, Behcet's syndrome, inflammatory bowel disease) and irritable bowel syndrome (IBS)), relapsing polychondritis (e.g., atrophic polychondritis and systemic polychondritis), and disorders that are improved by gastrokinetic agents (e.g., ileus, postoperative ileus, and ileus during sepsis; gastroesophageal reflux disease (GORD, or its synonym GERD); eosinophilic esophagitis, gastroparesis such as diabetic gastroparesis; food intolerances and food allergies, and other functional bowel disorders such as non-ulcer dyspepsia (NUD) and non-cardiac chest pain (NCCP, costochondritis, etc.)). [Example]

[0121] The following examples are set forth below to illustrate compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention that would be apparent to one skilled in the art. The present disclosure relates, for example, to the following: [Section 1] an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule; and nanoparticles comprising mRNA encoding the costimulatory molecule. [Section 2] Item 1. The composition according to Item 1, wherein the mRNA encoding the costimulatory molecule is encapsulated by the nanoparticles. [Section 3] Item 3. The composition according to item 1 or 2, wherein the nanoparticles comprise a phospholipid or a glycolipid. [Section 4] Item 4. The composition according to Item 3, wherein the phospholipid is selected from the group consisting of PL1 to PL18. [Section 5] Item 5. The composition according to Item 4, wherein the phospholipid is PL1. [Section 6] Item 4. The composition according to Item 3, wherein the glycolipid is selected from the group consisting of GL1 to GL16. [Section 7] Item 7. The composition according to Item 6, wherein the glycolipid is GL4. [Section 8] The composition of any one of Items 1 to 7, wherein the costimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, and LFA3. [Section 9] The composition of paragraph 8, wherein the costimulatory molecule comprises OX40 or 4-1BB. [Section 10] Item 10. The composition of any one of Items 1 to 9, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). [Section 11] Item 10. The composition of any one of Items 1 to 9, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR). [Section 12] Item 12. The composition according to any one of Items 1 to 11, wherein the mRNA comprises a chemically modified nucleic acid base. [Section 13] Item 13. The composition of claim 12, wherein the chemically modified nucleobase is pseudouridine. [Section 14] Item 14. The composition according to any one of Items 1 to 13, further comprising an immunotherapeutic agent. [Section 15] The composition of paragraph 14, wherein the immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof. [Section 16] 16. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the composition according to any one of items 1 to 15. [Section 17] A method for stimulating T cells, comprising administering an effective amount of the composition according to any one of items 1 to 15 or the pharmaceutical composition according to item 16 to a subject. [Section 18] Item 18. The method of Item 17, wherein the subject is a mammal. [Section 19] Item 19. The method of item 18, wherein the mammal is a human. [Section 20] 1. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds a costimulatory molecule and nanoparticles comprising mRNA encoding the costimulatory molecule. [Section 21] 21. The method of claim 20, wherein the mRNA encoding the costimulatory molecule is encapsulated by the nanoparticle. [Section 22] Item 22. The method according to item 20 or 21, wherein the nanoparticles comprise phospholipids or glycolipids. [Section 23] Item 23. The method according to Item 22, wherein the phospholipid is selected from the group consisting of PL1 to PL18. [Section 24] Item 24. The method of Item 23, wherein the phospholipid is PL1. [Section 25] Item 23. The composition according to Item 22, wherein the glycolipid is selected from the group consisting of GL1 to GL16. [Section 26] Item 26. The composition according to Item 25, wherein the glycolipid is GL4. [Section 27] 27. The method of any one of items 20 to 26, wherein the costimulatory molecule is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, CD226, galectin 9, TIM1, LFA1, B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, SLAM, CD48, CD58, CD155, CD112, CD80, CD86, ICOSL, TIM3, TIM4, ICAM1, and LFA3. [Section 28] 28. The method of paragraph 27, wherein the costimulatory molecule comprises OX40 or 4-1BB. [Section 29] Item 29. The method of any one of Items 20 to 28, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR). [Section 30] 30. The method of any one of Items 20 to 29, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR). [Section 31] Item 31. The method according to any one of Items 20 to 30, wherein the chemically modified nucleic acid base is pseudouridine. [Section 32] Item 32. The method of any one of items 20 to 31, wherein the cancer comprises melanoma, colorectal cancer, lung cancer, colon cancer, or lymphoma. [Section 33] Item 33. The method according to any one of Items 20 to 32, wherein the subject is a mammal. [Section 34] 34. The method of claim 33, wherein the mammal is a human. [Section 35] Item 35. The method according to any one of Items 20 to 34, wherein the antibody or antigen-binding fragment thereof and the nanoparticles are administered by intramuscular injection or systemic administration. [Section 36] Item 36. The method according to any one of items 20 to 35, further comprising administering an additional therapeutic agent. [Section 37] 37. The method of paragraph 36, wherein the additional therapeutic agent comprises an additional immunotherapeutic agent. [Section 38] 38. The method of clause 37, wherein the additional immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof. [Section 39] Item 39. The method according to any one of Items 20 to 38, wherein the antibody or antigen-binding fragment thereof that specifically binds to the costimulatory molecule and the nanoparticle containing mRNA encoding the costimulatory molecule are simultaneously administered.

[0122] Example 1. Nanoparticle (NP)-OX40 mRNA induced an increase in the expression level of OX40 OX40 expression was characterized in EG.7-OVA cells. Nanoparticle (NP)-OX40 mRNA induced significantly higher OX40 expression than the control group (Figure 1). 5'UTR and 3'UTR modifications are widely applicable to mRNAs encoding cytokines, immune checkpoint regulators such as ICOS, 4-1BB, GITR, and CD40. The nanoparticles were formulated with lipids, DOPE, cholesterol, DMG-PEG, and mRNA (see Li, B et al. An Orthogonal Array Optimization of Lipid-Like Nanoparticles for mRNA Delivery in Vivo. Nano Lett. 2015, 15, 8099-8107).

[0123] Example 2. NP / OX40 mRNA + anti-OX40 antibody combination therapy improved tumor therapy in a B16 melanoma tumor model. We established a B16 melanoma mouse tumor model (Triplett, TA, et al. Reversal of IDO-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme, Nat Biotechnol. 2018 Sep;36(8):758-764). Mice were treated with PBS, NP plus anti-OX40 antibody (InVivoPlus anti-mouse OX40 (clone OX-86) (Company: BioXcell, Catalog: BP0031)), or NP / OX40 mRNA plus anti-OX40 antibody. The combination of these mRNAs and their associated antibodies significantly improved tumor therapy (Figure 2A) and prolonged overall survival (Figure 2B) in this mouse tumor model.

[0124] Example 3. Combination therapy of NP / OX40 mRNA plus anti-OX40 antibody improved tumor therapy in the CT26 tumor model. A CT26 mouse tumor model was established (Malvicini, M, et al., "Tumor Microenvironment Remodeling by 4-Methylumbelliferone Boosts the Antitumor Effect of Combined Immunotherapy in Murine Colorectal Carcinoma," Molecular Therapy. Vol. 23 No. 9, pp. 1444-1455, September 2015). Mice were treated with PBS, nanoparticles (NPs) plus anti-OX40 antibody, nanoparticles (NPs) / OX40 mRNA plus anti-OX40 antibody, and nanoparticles (NPs) / OX40 mRNA plus anti-OX40 antibody (injection interval: 6 h); and nanoparticles (NPs) / OX40 mRNA plus anti-OX40 antibody (injection interval: 0 h). The OX40 antibody used was InVivoPlus anti-mouse OX40 (clone OX-86) (company: BioXcell, catalog: BP0031). The combination of these mRNAs and their associated antibodies significantly extended overall survival in mouse tumor models and improved tumor therapy.

[0125] Example 4. Nanoparticles containing mRNA encoding costimulatory molecules. Cancer immunotherapy employs various approaches to stimulate antitumor immune responses, including cancer vaccines, cell-based therapies, immune checkpoint blockers, monoclonal antibodies, mRNA-based immunotherapies, and other nanoparticle-mediated immunotherapies. In particular, the use of immune checkpoint inhibitors has improved overall survival in cancer patients by targeting T cell co-inhibitory pathways such as PD-1 and CTLA-4. Although these antibodies are routinely used in the clinic, only approximately 25% of patients experience meaningful tumor responses. Therefore, there is an urgent need to develop new immunotherapeutic strategies for cancer treatment.

[0126] In recent years, researchers have discovered a series of costimulatory molecules on T cells for cancer immunotherapy. Interaction of costimulatory molecule ligands with costimulatory receptors on the T cell surface activates the expansion and differentiation of clonal T cells, resulting in increased antitumor efficacy in several human cancers. CD137 (also known as 4-1BB) and OX40 (also known as CD134) are T cell costimulatory receptors that provide activation signals to CD8 and CD4 T cells. CD137 plays an important role in T cell proliferation and cytokine secretion. Recently, two anti-CD137 antibodies (urelumab and utomilumab) have been investigated in clinical trials. OX40 is involved in stimulating CD8+ T cells to generate antitumor immune responses. Anti-OX40 antibodies enhance T cell differentiation, cytolytic function, and antitumor immunity in various cancer types. Several agonistic anti-OX40 antibodies are currently in clinical trials. Costimulatory signals are important for T cell stimulation, but are poorly expressed in the tumor microenvironment, hindering the efficacy of immunotherapy. Therefore, delivery of costimulatory receptor mRNA to tumor-infiltrating T cells, combined with the use of agonistic antibodies against the receptor, can directly activate T cells and improve cancer immunotherapy (Figure 4A).

[0127] To deliver costimulatory receptor mRNA to T cells, we used phospholipids and glycolipids, which are natural components of the cell membrane. Based on the chemical structures of phospholipids and glycolipids, we designed and synthesized a library of phospholipid- and glycolipid-mimetic materials (Figures 4B-4D). These compounds were formulated into phospholipid- and glycolipid-derived nanoparticles for mRNA delivery. One phospholipid-derived nanoparticle, PL1, efficiently delivered mRNA to T cells both in vitro and in vivo. Next, we used PL1 nanoparticles to deliver costimulatory receptor CD137 or OX40 mRNA to tumor-infiltrating T cells in combination with anti-CD137 or anti-OX40 antibodies in multiple tumor models. Furthermore, this therapeutic approach significantly improved the immunotherapeutic efficacy of anti-PD-1 plus anti-CTLA-4 antibodies. This example provides a new and urgently needed biomaterial for delivering costimulatory receptor mRNA to activate T cells and enhance antitumor immunity.

[0128] Example 5. Design and synthesis of phospholipid and glycolipid derivatives (PL and GL) for mRNA delivery. The biomimetic compounds, phospholipids and glycolipids, consist of a biomimetic head (phosphate or sugar head), an ionizable amino core, and multiple hydrophobic tails (Figure 11). These phospholipid and glycolipid derivatives (PL and GL) were synthesized according to previously reported procedures. See, for example, WO / 2019 / 027999. Figure 4B shows a representative synthetic route to PL1 and GL1. Following this synthetic route, PL1-18 and GL1-16 materials were synthesized (Figure 4C), which are 1The nanoparticles were characterized by H nuclear magnetic resonance (NMR) and mass spectrometry (MS) (Figure 4C). Next, PL and GL nanoparticles were formulated with firefly luciferase mRNA (FLuc mRNA) and characterized according to size, surface charge, and mRNA encapsulation efficiency (Figures 12A-12C). Next, the mRNA delivery efficiency of PL1-18 and GL1-16 nanoparticles was investigated in E.G7 cells (a T-lymphocyte cell line), and PL1 nanoparticles were found to exhibit the highest delivery efficiency of FLuc mRNA (Figure 5A). Furthermore, PL1 delivered GFP mRNA to approximately 94% of E.G7 cells, demonstrating its function as a T-cell delivery vehicle (Figure 5C). The endocytic pathway of PL1 nanoparticles was further investigated using endocytosis inhibitors, such as 5-(N-methyl-N-isopropyl)amiloride (EIPA) for macropinocytosis, chlorpromazine hydrochloride (CPZ) for clathrin-mediated endocytosis, and methyl-beta-cis-rhodextrin (MβCD) for caveolae-mediated endocytosis. Treatment with EIPA, CPZ, and MβCD significantly inhibited cellular uptake of PL1 nanoparticles by 50%, 56%, and 39%, respectively (Figure 13), indicating that PL1 nanoparticles were internalized via multiple endocytic pathways. T cell costimulatory receptor CD137 mRNA and OX40 mRNA were also delivered into E.G7 cells. Figure 5B shows cryo-electron microscopy images of PL1-OX40 nanoparticles. Flow cytometry results showed that both PL1-CD137 (27.8%) and PL1-OX40 (47.4%) significantly increased the cell surface expression of CD137 and OX40, respectively (Figures 5D and 5E). Next, we investigated intratumoral (IT) delivery of PL1-GFP in tumor-infiltrating lymphocytes in a mouse melanoma model (B16F10 melanoma cells grown sc in C57BL / 6 mice) (Figure 5F). Following PL1-GFP treatment, increased GFP expression was observed in tumor-infiltrating CD4+ and CD8+ T cells (Figure 5G), as well as macrophages and dendritic cells (DCs) (Figures 14A–14C). Based on these results, PL1 nanoparticles were selected for in vivo delivery of CD137 and OX40 mRNA.

[0129] Example 6. Regression of tumor growth by treatment with PL1-CD137 mRNA plus anti-CD137 antibody. PL1-CD137 was combined with anti-CD137 antibody and injected intratumorally six times every other day in a B16F10 melanoma mouse model. Administration of PL1-CD137 plus anti-CD137 dramatically reduced tumor growth rate (5-fold lower than control, 18 days after inoculation) (Figures 6A and 15A). Treatment also significantly increased overall survival compared with PBS and PL1 (empty nanoparticles) plus anti-CD137 Ab (Figure 6B). Similar experiments were performed in an A20 lymphoma tumor model. Treatment with PL1-CD137 plus anti-CD137 Ab resulted in a 2-fold reduction in tumor growth rate (18 days after inoculation) compared with PBS and PL-1 plus anti-CD137 Ab (Figures 6C and 15B). However, no significant increase in overall survival was observed with PL1-CD137 plus anti-CD137 Ab compared with PL-1 plus anti-CD137 Ab treatment (Figure 6D). Thus, PL1 nanoparticle delivery of costimulatory receptor CD137 mRNA improved the outcome of immunotherapy with anti-CD137 Ab to some extent in both tumor models, with better results in the B16F10 melanoma model compared with the A20 lymphoma model.

[0130] Example 7. Regression of tumor growth by treatment with PL1-OX40 mRNA plus anti-OX40 antibody. We also investigated the therapeutic effect of costimulatory receptor OX40 delivery in a B16F10 melanoma tumor model. PL1-OX40 + anti-OX40 Ab treatment (it) significantly reduced tumor growth and prolonged survival compared with PBS and PL1 + anti-OX40 Ab treatment (Figures 7A, 7B, and 16A). Next, we established a CT26 mouse tumor model in BABL / c mice. A significant therapeutic effect was observed after treatment with PL1-OX40 + anti-OX40 Ab (Figures 7C, 7D, and 16B).

[0131] Next, we evaluated the therapeutic efficacy of PL1-OX40 plus anti-OX40 Ab treatment in the A20 B cell lymphoma model. Mice received injections of PBS, PL1-OX40, PL1 plus anti-OX40 Ab, or PL1-OX40 plus anti-OX40 Ab. Tumor growth was monitored for 60 days (Figures 8A and 8B). Treatment with PL1-OX40 plus anti-OX40 Ab significantly reduced tumor growth (Figure 8C) and prolonged survival (Figure 8D) compared with controls. Importantly, 6 of 10 mice (60%) treated with PL1-OX40 plus anti-OX40 Ab exhibited a complete response (Figure 8D) and were resistant to rechallenge with A20 tumor cells (Figure 8E). These results indicated that PL1 nanoparticles delivering costimulatory OX40 mRNA could enhance the immunotherapeutic efficacy of anti-OX40 Ab therapy in three different mouse models.

[0132] Tumor-infiltrating lymphocytes (TILs) play an important role in antitumor immunity. mRNA delivery to intratumoral T cells was investigated in an A20 B-cell lymphoma model. OX40 expression on tumor-infiltrating CD8+ T cells significantly increased after PL1-OX40 treatment (Figure 8F), whereas changes in OX40 expression on infiltrating CD4+ T cells or macrophages were minimal (Figure 17A). OX40 expression on infiltrating dendritic cells (DCs) also significantly increased (Figure 17A). Cytokine and chemokine levels were also investigated. Plasma levels of IFN-γ significantly increased after PL1-OX40 treatment compared with the control group, and levels of chemokine ligand 12 (CCL12), neutrophil chemoattractant (CXCL1), and macrophage colony-stimulating factor (M-CSF) also increased 2- to 10-fold with PL1-OX40 treatment (Figure 17B).

[0133] We also investigated the infiltrating T cell population in A20 B cell lymphoma tumors. Using the same dosing strategy as described in Figure 6A, immune cell populations were analyzed 24 h after the last treatment (Figure 8G). A significant increase in CD8+ T cells was observed, but there was no change in the levels of CD4+ T cells, macrophages, or dendritic cells with PL1-OX40 + anti-OX40 Ab treatment compared with PL1 + anti-OX40 Ab (Figure 8H). Interestingly, when T cells were depleted with either anti-CD8 or anti-CD4 Ab, the efficacy of the combination treatment was significantly impaired compared with administration of control Ab (Figure 18). Cytokine levels of IFN-γ, CCL12, M-CSF, and CXCL1 in mouse plasma were similar in the different groups after six treatment doses (Figure 19).

[0134] Example 8. Enhancement of antitumor effect of PL1-OX40 mRNA + anti-OX40 antibody. Although PL1-OX40 plus anti-OX40 Ab treatment significantly reduced B16F10 tumor growth and prolonged survival (Figures 7A and 7B), complete eradication of the tumor burden remains an important goal of immune-based therapy. To improve the antitumor efficacy of PL1-OX40 + anti-OX40 Ab therapy, OX40 mRNA was modified from its wild type (OX40(WT)) to a pseudouridine (ψ)-modified (OX40(ψ)) and the dose of anti-OX40 Ab was increased from 8 μg to 40 μg. Treatment of B16F10 tumor-bearing mice with PL1-OX40(ψ) + anti-OX40 Ab (40 μg) significantly reduced tumor growth and prolonged survival compared with PBS and PL1 + anti-OX40 treatment (Figures 9A–9D). On day 35, five mice developed tumors less than 500 mm3 in size, and surgery was performed to remove tumors from these mice. Two mice remained tumor-free for more than 50 days, indicating delayed tumor growth compared with control mice rechallenged with B16F10 tumor cells (Figure 9E).

[0135] In another treatment regimen, treatment with anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitor Abs was added to treatment with PL1-OX40(ψ) anti-OX40 Ab (40 μg) (Figure 9F). The combination of PL1-OX40(ψ) and anti-PD-1 and anti-CTLA-4 Abs dramatically inhibited tumor growth and prolonged survival compared with PBS or anti-PD-1 and anti-CTLA-4 Ab treatment (Figures 9G-I). At day 45, six mice were tumor-free, and one mouse had a small tumor (approximately 50 mm3). Surviving mice tolerated rechallenge with B16F10 tumor cells (Figure 9J). Among them, one mouse's primary tumor regrew and met the early tumor clearance criteria by day 58. The remaining five mice remained bilaterally tumor-free. These results indicate that the PL1-OX40 + anti-OX40 Ab treatment regimen improved the response to anti-PD-1 + anti-CTLA-4 Ab therapy.

[0136] Next, we evaluated the therapeutic efficacy of this treatment regimen using a B16F10 lung metastasis mouse model by systemic administration of anti-PD-1 + anti-CTLA-4 antibody and PL1-OX40(ψ) + anti-OX40 antibody (100 μg) (Figure 10A). The results showed that this treatment regimen dramatically reduced tumor metastasis in mouse lungs compared with anti-PD-1 + anti-CTLA-4 antibody and PBS treatment (Figures 10B-C, 20A-B). Compared with anti-PD-1 + anti-CTLA-4 Ab treatment, PL1-OX40(ψ) + anti-OX40 Ab and anti-PD-1 + anti-CTLA-4 Ab groups showed a significant increase in CD8+ and CD4+ T cells in mouse lungs (Figures 10D-E). Furthermore, the number of Foxp3+CD4+ cells (Treg cells) was reduced in the lungs of the PL1-OX40(ψ) + anti-OX40 Ab and anti-PD-1 + anti-CTLA-4 Ab groups (Figure S10F). These results demonstrate that this systemic treatment regimen exhibits potent antitumor activity in a mouse model of lung metastasis.

[0137] Agonist antibodies can be replaced with moieties having similar functions, such as endogenous ligands. For example, the OX40 costimulatory receptor can interact with the OX40 ligand. The coding sequence of the OX40 ligand is shown in SEQ ID NO: 13.

[0138] Example 9. Discussion T cell-based immunotherapy for cancer is a rapidly developing field. In recent years, nanotechnology has been developed to improve T cell therapy, including ex vivo engineering of T cells and in vivo modulation of T cells. Despite these important advances, a significant challenge remains: stimulating antitumor immunity of primary T cells in vivo.

[0139] In this study, we designed and synthesized a library of phospholipid and glycolipid derivatives (PL and GL) to explore nanoparticles for delivering mRNA to T cells. These materials were used to formulate biomimetic nanoparticles for mRNA delivery. PL1 nanoparticles were not only able to deliver costimulatory receptor mRNA to T cell lines in vitro, but also to T cells within tumors, providing a useful delivery tool for modulating T cell function.

[0140] In recent years, agnostic antibodies (mAbs) specific for costimulatory receptors (CORs) have been developed for cancer therapy, with the ability to enhance antitumor T cell immunity. For example, anti-OX40 antibodies can activate T cells, enabling them to eliminate tumor cells. However, low OX40 expression has hindered the efficacy of anti-OX40 antibody immunotherapy in many tumor models (e.g., B16F10). In this study, PL1 nanoparticles were used to deliver OX40 mRNA to tumor-infiltrating T cells, which increased OX40 expression and consequently improved the antitumor efficacy of anti-OX40 antibodies. Combination treatment of PL1-OX40 and anti-OX40 antibodies demonstrated significant antitumor activity compared with antibodies alone in multiple tumor models. To further enhance the antitumor activity of PL1-OX40 and anti-OX40 antibodies, anti-PD-1 and anti-CTLA-4 antibodies were added to the treatment regimen. This therapeutic approach resulted in approximately 50% complete responses in the B16F10 tumor model. Notably, these mice were resistant to rechallenge with B16F10 tumor cells, demonstrating that this treatment regimen effectively induces antitumor immunity in vivo.

[0141] Furthermore, this therapeutic strategy is compatible with multiple administration routes. For example, combination treatment of PL1-OX40 and anti-OX40 antibodies exhibited significant antitumor activity not only through local administration but also through systemic administration of anti-OX40 antibodies (Figures 21A-21D). More importantly, systemic administration of anti-PD-1 + anti-CTLA-4 Abs and PL1-OX40(ψ) + anti-OX40 Abs dramatically reduced tumor metastasis in a lung metastasis model. These results demonstrate the broad applicability of this therapeutic regimen in diverse therapeutic settings.

[0142] Example 10. Chemical synthesis of phospholipid and glycolipid derivatives (PL and GL) Phospholipid and glycolipid compounds and their analogs were synthesized according to previously reported methods. See, for example, WO / 2019 / 027999. In the general method for PL1–PL18 and GL1–GL16, an excess of trifluoroacetic acid (1 mL) was added to a solution of compound i or an analog (0.5 mmol) in CHCl (2 mL). The mixture was stirred at room temperature for 2 h and monitored by thin-layer chromatography. Upon completion of the reaction, the solvent was evaporated to give an oily intermediate. The intermediate was dissolved in 10 mL of anhydrous tetrahydrofuran, followed by the addition of triethylamine (0.2 mL). The mixture was stirred at room temperature for 30 min. After the addition of aldehyde (3 mmol) and NaBH(OAc) (3 mmol), the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was purified by column chromatography using a CombiFlash RF system equipped with a RediSep Gold Resolution silica column (Teledyne Isco) with a gradient elution (CHCl and HCl) from 100% to 70% CHCl (CHCl / MeOH / NHOH = 75 / 22 / 3 (volume)) to give the corresponding product. PL1, 34% yield. 1 H NMR (400 MHz, CDCl3) δ = 4.86-4.80 (3H, m), 4.16-4.08 (6H, m), 2.53-2.50 (2H, t, J = 8), 2.42-2.37 (8H, m), 2.31-2.28 (6H, t, J = 8), 1.83-1.80 (2H, m), 1.63-1.51 (21H, m), 1.37-1.28 (54H, m), 0.90-0.87 (18H, t, J = 8). MS (m / z): [M+H] + C 61 H 122 N2O 10 Calculated value for P: 1073.88, Measured value: 1073.88 PL2, 64% yield. 1H NMR (400 MHz, CDCl3) δ = 4.13-4.08 (2H, m), 3.79 (3H, s), 3.76 (3H, s), 2.53-2.50 (2H, t, J = 8), 2.42-2.37 (9H, m), 1.85-1.78 (2H, m), 1.62-1.57 (2H, m), 1.45-1.43 (6H, m), 1.27 (54H, s), 0.91-0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 44 H 94 Calculated value of N2O4P, 745.70, measured value, 745.69 PL3, yield 50%. 1 H NMR (400 MHz, CDCl3) δ = 4.86-4.80 (3H, m), 4.08-4.03 (2H, m), 2.54-2.51 (2H, t, J = 8), 2.46-2.38 (9H, m), 1.83-1.80 (2H, m), 1.62-1.60 (2H, m), 1.45 (8H, m), 1.35-1.34 (12H, m), 1.28 (49H, s), 0.91-0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 48 H 102 Calculated value of N2O4P, 801.76, measured value, 801.76 PL4, yield 48%. 1 H NMR (400 MHz, CDCl3) δ = 4.16-4.07 (6H, m), 2.54-2.50 (2H, t, J = 8), 2.43-2.37 (9H, m), 1.84-1.80 (2H, m), 1.59-1.56 (2H, m), 1.45 (6H, m), 1.38-1.28 (49H, m), 0.91-0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 40 H 86 Calculated value of N2O4P, 689.63, measured value, 689.63 PL5, yield 40%.1 1H NMR (400 MHz, CDCl3) δ = 4.14 - 4.07 (6H, m), 2.54 - 2.50 (2H, t, J = 8), 2.43 - 2.37 (8H, m), 1.84 - 1.80 (2H, m), 1.59 - 1.56 (2H, m), 1.45 (6H, m), 1.37 - 1.28 (55H, m), 0.91 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 43 H 92 Calculated value for C 1 H 1 N2O4P, 731.68, Measured value, 731.68 PL6, yield 48%. 1 1H NMR (400 MHz, CDCl3) δ = 4.16 - 4.07 (6H, m), 3.72 - 3.69 (2H, m), 2.54 - 2.50 (2H, t, J = 8), 2.44 - 2.38 (8H, m), 1.86 - 1.79 (2H, m), 1.72 - 1.69 (2H, m), 1.44 (6H, m), 1.37 - 1.34 (6H, m), 1.27 (54H, s), 0.91 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + C<0OO0032>H 98 [[ID=I7]]Calculated value for C 1 H 1 N2O4P, 773.73, Measured value, 773.73 PL7, yield 41%. 1 1H NMR (400 MHz, CDCl3) δ = 4.16 - 4.07 (6H, m), 3.72 - 3.69 (2H, m), 2.54 - 2.50 (2H, t, J = 8), 2.44 - 2.38 (8H, m), 1.86 - 1.79 (2H, m), 1.72 - 1.69 (2H, m), 1.44 (6H, m), 1.37 - 1.34 (6H, m), 1.27 (54H, s), 0.91 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 49 H 104 Calculated value for C 1 H 1 N2O4P, 815.77, Measured value, 815.77 PL8, yield 26%. 1 H NMR (400 MHz, CDCl3) δ = 4.14-4.08 (6H, m), 3.24-3.22 (2H, m), 2.80-2.77 (1H, t, J = 8), 2.54-2.50 (2H, t, J = 8), 2.46-2.32 (14H, m), 2.22 (3H, s), 1.83-1.80 (2H, m), 1.65-1.60 (6H, m), 1.44 (5H, m), 1.37-1.28 (50H, m), 0.91-0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 44 H 95 Calculated value of N3O4P, 760.71, measured value, 760.71 PL9, yield 24%. 1 H NMR (400 MHz, CDCl3) δ = 4.16-4.07 (6H, m), 2.80-2.76 (2H, t, J = 8), 2.74-2.70 (4H, m), 2.61-2.58 (2H, m), 2.53-2.44 (8H, m), 2.31 (3H, s), 1.87-1.81 (4H, m), 1.69-1.66 (2H, m), 1.56 (4H, m), 1.44 (2H, m), 1.37-1.27 (54H, m), 0.91-0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 47 H 101 Calculated value of N3O4P, 802.75, Calculated value, 802.75 PL10, yield 41%. 1 H NMR (400 MHz, CDCl3) δ = 4.12-4.06 (6H, m), 2.51-2.50 (2H, t, J = 4), 2.43-2.32 (14H, m), 2.22 (3H, s), 1.83-1.79 (2H, m), 1.62-1.60 (2H, m), 1.43 (6H, m), 1.37-1.27 (62H, m), 0.91-0.87 (9H, t, J = 8). MS (m / z): [M+H]+ C 50 H 107 Calculated value for C3H11N3O4P, 844.80, measured value, 844.80 PL11, yield 33%. 1 1H NMR (400 MHz, CDCl3) δ = 4.15 - 4.06 (6H, m), 2.53 - 2.50 (2H, t, J = 4), 2.44 - 2.40 (9H, m), 2.37 - 2.32 (5H, m), 2.22 (3H, s), 1.83 - 1.79 (2H, m), 1.71 - 1.68 (lH, m), 1.64 - 1.60 (4H, m), 1.43 (6H, m), 1.37 - 1.27 (66H, m), 0.91 - 0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 53 H 113 Calculated value for C3H11N3O4P, 886.85, measured value, 886.85 PL12, yield 32%. 1 1H NMR (400 MHz, CDCl3) δ = 4.15 - 4.06 (6H, m), 2.52 - 2.31 (22H, m), 1.84 - 1.77 (2H, m), 1.65 - 1.60 (4H, m), 1.42 - 1.41 (6H, m), 1.37 - 1.27 (49H, m), 0.91 - 0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 47 H 100 Calculated value for C3H11N4O4P, 815.75, measured value, 815.75 PL13, yield 30%. 1 1H NMR (400 MHz, CDCl3) δ = 4.16 - 4.06 (6H, m), 2.52 - 2.32 (22H, m), I.82 - 1.79 (2H, m), 1.66 - 1.60 (4H, m), 1.42 - 1.41 (6H, m), 1.37 - 1.27 (55H, m), 0.91 - 0.87 (9H, t, J = 8).MS (m / z): [M+H] + C 50 H 106Calculated value of N4O4P, 857.80, measured value, 857.79. PL14, yield 36%. 1 H NMR (400 MHz, CDCl3) δ = 4.16-4.07 (6H, m), 2.53-2.32 (22H, m), 1.83-1.80 (2H, m), 1.66-1.61 (4H, m), 1.42 (6H, m), 1.37-1.28 (61H, m), 0.91-0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 53 H 112 Calculated value of N4O4P is 899.84, measured value is 899.84. PL15, yield 21%. 1 H NMR (400 MHz, CDCl3) δ = 4.13-4.08 (6H, m), 2.53-2.33 (24H, m), 1.85-1.80 (4H, m), 1.66-1.63 (5H, m), 1.42 (9H, m), 1.38-1.28 (72H, m), 0.91-0.88 (9H, t, J = 8).MS (m / z): [M+H] + C 56 H 118 Calculated value of N4O4P is 941.89, measured value is 941.89. PL16, yield 23%. 1 H NMR (400 MHz, CDCl3) δ = 5.69-5.63 (3H, m), 5.57-5.51 (3H, m), 4.65-4.63 (6H, d, J = 8), 4.16-4.08 (6H, m), 2.55-2.40 (10H, m), 2.34-2.30 (6H, m), 2.14-2.09 (6H, m), 1.85-1.80 (2H, m), 1.65-1.62 (9H, m), 1.42 (9H, m), 1.38-1.31 (62H, m), 0.92-0.89 (9H, t, J = 8).MS (m / z): [M+H] + C 64 H 122 N2O 10Calculated value for P, 1109.87, measured value, 1109.89. PL17, yield 23%. 1 H NMR (400 MHz, CDCl3) δ = 5.41 - 5.28 (12H, m), 4.15 - 4.06 (6H, d, J = 8), 3.15 - 3.03 (2H, m), 2.97 - 2.89 (7H, m), 2.78 - 2.75 (7H, m), 2.07 - 2.00 (22H, m), 1.62 - 1.55 (5H, m), 1.35 - 1.29 (51H, m), 0.90 - 0.86 (9H, t, J = 8). MS (m / z): [M+H] + C 64 H 122 Calculated value for N2O4P, 1013.91, measured value, 1013.91. PL18, yield 24%. 1 H NMR (400 MHz, CDCl3) δ = 4 .22 - 4.10 (7H, m), 2.43 - 2.39 (11H, m), 2.34 - 2.30 (2H, t, J = 8), 2.04 - 2.01 (2H, t, J = 8), 1.67 - 1.63 (4H, m), 1.38 - 1.28 (71H, m), 0.91 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 52 H 107 Calculated value for N2O6P, 887.79, measured value, 887.79. GL1, yield 26%. 1 H NMR (400 MHz, CDCl3) δ = 4.17 - 4.11 (2H, m), 2.70 - 2.57 (11H, m), 2.33 - 2.29 (2H, t, J = 8), 1.78 (2H, s), 1.69 - 1.62 (3H, m), 1.54 - 1.48 (8H, m), 1.28 (59H, s), 0.92 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 50 H 95 N2O 10Calculated value for, 883.70; Measured value, 883.70. GL2, Yield 35%. 1 H NMR (400 MHz, CDCl3) δ = 5.40 (1H, m), 5.24 - 5.19 (1H, m), 5.04 - 5.00 (1H, m), 4.48 - 4.46 (1H, d, J = 8), 4.17 (2H, m), 3.91 (2H, m), 3.54 - 3.51 (1H, m), 2.40 - 2.38 (12H, m), 2.16 (3H, s), 2.06 (6H, s), 1.99 (4H, s), 1.74 (2H, m), 1.73 - 1.70 (2H, m), 1.57 (6H, m), 1.27 (52H, s), 0.89 (9H, t, J = 8). MS (m / z): [M+H] + C 53 H 101 N2O 10 Calculated value for, 925.75, Measured value, 925.74. GL3, Yield 64%. 1 H NMR (400 MHz, CDCl3) δ = 5.41 - 5.40 (1H, m), 5.32 - 5.20 (1H, m), 5.04 - 5.01 (1H, m), 4.48 - 4.46 (1H, d, J = 8), 4.22 - 4.13 (2H, m), 3.94 - 3.90 (2H, m), 3.54 - 3.52 (1H, m), 2.46 - 2.37 (12H, m), 2.16 (3H, s), 2.06 (6H, s), 2.00 (4H, s), 1.73 - 1.72 (2H, m), 1.58 - 1.56 (2H, m), 1.42 (6H, m), 1.28 (55 H, s), 0.89 (9H, t, J = 8). MS (m / z): [M+H] + C 56 H 107 N2O 10 Calculated value for, 967.79, Measured value, 967.79. GL4, Yield 35%. 11H NMR (400 MHz, CDCl3) δ = 5.39 (1H, m), 5.19 - 5.15 (1H, m), 5.03 - 5.01 (1H, m), 4.47 - 4.45 (1H, m), 4.15 - 4.14 (2H, m), 3.93 - 3.92 (2H, m), 3.53 - 3.51 (1H, m), 2.84 - 2.74 (6H, m), 2.64 - 2.59 (4H, m), 2.55 - 2.51 (2H, m), 2.10 (3H, s), 2.05 (6H, s), 1.98 (6H, s), 1.83 - 1.78 (4H, m), 1.58 (4H, m), 1.46 (2H, m), 1.26 (63H, s), 0.89 - 0.88 (9H, t, J = 4). MS (m / z): [M+H] + Calculated for C59H113N2O10, 1009.84; found, 1009.84. GL5, yield 35%. 1 1H NMR (400 MHz, CDCl3) δ = 5.41 - 5.40 (1H, m), 5.22 - �.19 (1H, m), 5.04 (1H, m), 4.48 - 4.46 (1H, d, J = 8), 4.17 (2H, m), 3.91 (2H, m), 3.54 - 3.52 (1H, m), 2.84 - 2.51 (15H, m), 2.10 (3H, s), 2.16 (3H, s), 2.07 (5H, s), 2.00 (4H, s), 1.73 - 1.72 (2H, m), 1.62 - 1.60 (4H, s), 1.43 - 1.42 (6H, m), 1.26 (53H, s), 0.89 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + Calculated for C60H116N3O10, 1038.87; found, 1038.86. GL6, yield 35%. 11H NMR (400 MHz, CDCl3) δ = 5.41 - 5.40 (1H, m), 5.24 - 5.21 (1H, m), 5.04 = 5.01 (1H, m), 4.48 - 4.46 (1H, d, J = 8), 4.22 - 4.12 (2H, m), 3.95 - 3.89 (2H, m), 3.56 - 3.50 (1H, m), 2.84 - 2.33 (22H, m), 2.16 (3H, s), 2.07 - 2.06 (1H, s), 2.00 (3H, s), 1.73 - 1.63 (6H, m), 1.42 (6H, m), 1.27 (53H, s), 0.91 - 0.88 (9H, t, J = 8). MS (m / z): [M+H] + C 63 H 121 N4O 10 Calculated value for, 1093.91, measured value, 1093.91. GL7, yield 30%. 1 1H NMR (400 MHz, CDCl3) δ = 5.24 - 5.19 (1H, m), 5.13 - 5.08 (1H, m), 5.02 - 4.98 (1H, m), 4.52 - 4.50 (1H, d, J = 8), 4.32 - 4.27 (1H, m), 4.16 - 4.13 (1H, m), 3.94 - 3.89 (1H, m), 3.72 - 3.69 (1H, m), 3.56 - 3.50 (1H, m), 3.37 - 3.34 (1H, m), 2.46 - 2.35 (15H, m), 2.23 (3H, s), 2.10 - 2.02 (11H, m), 1.72 - 1.60 (8H, m), 1.45 - 1.28 (64H, s), 0.91 - 0.88 (12H, t, J = 8). MS (m / z): [M+H] + C 60 H 116 N3O 10 Calculated value for, 1038.87, measured value, 1038.87. GL8, yield 65%. 1H NMR (400 MHz, CDCl3) δ = 5.24-5.19 (1H, m), 5.12-5.08 (1H, m), 5.01-4.97 (1H, m), 4.51-4.49 (1H, d, J = 8), 4.31-4.27 (1H, m), 4.16-4.13 (1H, m), 3.92-3.88 (1H, m), 3.70-3.69 (3H, m), 3.55-3.50 (1H, m), 2.47-2.34 (25H, m), 2.10 (3H, s), 2.05-2.02 (9H, m), 1.70 (10H, m), 1.50 (10H, m), 1.27 (55H, s), 0.91-0.88 (9H, t, J = 8).MS (m / z): [M+H] + C 63 H 121 N4O 10 Calculated value: 1093.91, measured value: 1093.91. GL9, yield 49%. 1 H NMR (400 MHz, CDCl3) δ = 5.69-5.63 (1H, m), 5.57-5.51 (3H, m), 5.24-5.19 (1H, m), 5.12-5.08 (1H, m), 5.02-4.97 (1H, m), 4.64-4.63 (6H, d, J = 4), 4.52-4.50 (1H, d, J = 8), 4.31-4.27 (1H, m), 4.17-4.11 (1H, m), 3.94-3.88 (1H, m), 3.73-3.69 (1H, m), 3.54-3.51 (1H, m), 2.47-2.30 (18H, m), 2.14-2.02 (17H, m), 1.65-1.62 (11H, m), 1.40-1.31 (55H, m), 0.92-0.88 (9H, t, J = 8).MS (m / z): [M+H] + C 74 H 131 N2O 16 Calculated value: 1303.95, measured value: 1303.94. GL10, yield 33%. 11H NMR (400 MHz, CDCl3) δ = 5.64 - 5.63 (2H, m), 5.57 - 5.51 (2H, m), 5.24 - 5.19 (1H, m), 5.13 - 5.08 (1H, m), 5.02 - 4.97 (1H, m), 4.64 - 4.63 (4H, d, J = 4), 4.52 - 4.50 (1H, d, J = 8), 4.31 - 4.27 (1H, m), 4.17 - 4.13 (1H, m), 3.95 - 3.89 (1H, m), 3.72 - 3.70 (2H, m), 3.58 - 3.52 (2H, m), 2.55 - 2.38 (9H, m), 2.34 - 2.30 (5H, m), 2.22 (2H, m), 2.16 - 2.02 (16H, m), 1.79 - 1.60 (9H, m), 1.46 - 1.27 (32H, m), 0.92 - 0.88 (6H, t, J = 8). MS (m / z): [M+H] + C 57 H 101 N2O 14 Calculated value for, 1037.73, measured value, 1037.73. GL11, yield 20%. 1 1H NMR (400 MHz, CDCl3) δ = 5.25 - 5.20 (1H, m), 5.14 - 5.08 (1H, m), 5.02 - 4.97 (1H, m), 4.55 - 4.53 (1H, d, J = 8), 4.​​​​​N2O 16 Calculated value: 1183.86, measured value: 1183.85. GL12, yield 63%. 1 H NMR (400 MHz, CDCl3) δ = 5.24-5.19 (1H, m), 5.12-5.08 (1H, m), 5.02-4.97 (1H, m), 4.52-4.50 (1H, d, J = 8), 4.31-4.26 (1H, m), 4.16-4.12 (1H, m), 3.94-3.89 (1H, m), 3.71-3.68 (2H, t, J = 8), 3.58-3.52 (1H, m), 2.44-2.33 (12H, t, J = 8), 2.21 (3H, s), 2.10 (3H, s), 2.06 (3H, s), 2.04 (3H, s), 2.02 (3H, s), 1.75-1.63 (6H, m), 1.44 (4H, m), 1.27 (37H, m), 0.91-0.87 (6H, t, J = 8).MS (m / z): [M+H] + C 45 H 85 N2O 10 Calculated value: 813.62, Measured value: 813.62. GL13, yield 44%. 1 H NMR (400 MHz, CDCl3) δ = 5.42 (1H, m), 5.24-5.19 (1H, m), 5.13-5.09 (1H, m), 4.52-4.50 (1H, m), 4.21-4.17 (1H, m), 4.07-3.87 (3H, m), 3.53-3.47 (1H, m), 2.52-2.39 (10H, m), 1.77-1.66 (4H, m), 1.50-1.42 (5H, m), 1.27-1.13 (89H, m), 0.91-0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 68 H 131 N2O 10 Calculated value: 1135.98, Measured value: 1135.98. GL14, yield 37%.1 1H NMR (400 MHz, CDCl3) δ = 5.43 - 5.24 (4H, m), 5.09 - 5.04 (1H, t, J = 8), 4.89 - 4.82 (2H, m), 4.53 - 4.47 (2H, m), 4.29 - 4.22 (3H, m), 4.07 - 3.96 (4H, m), 3.90 - 3.87 (1H, m), 3.72 - 3.67 (4H, m), 3.53 - 3.50 (1H, m), 2.48 - 2.37 (10H, m), 2.16 - 2.01 (27H, m), 1.73 - 1.69 (3H, m), 1.45 - 1.41 (6H, m), 1.27 (45H, s), 0.91 - 0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 68 H 123 N2O 18 Calculated value for, 1255.88, measured value, 1255.88. GL15, yield 48%. 1 1H NMR (400 MHz, CDCl3) δ = 5.36 - 5.35 (1H, d, J = 4), 5.22 - 5.18 (1H, t, J = 8), 5.14 - 5.10 (1H, m), 4.98 - 4.95 (1H, m), 4.91 - 4.87 (1H, m), 4.50 - 4.45 (3H, m), 4.15 - 4.07 (3H, m), 3.88 - 3.78 (4H, m), 3.62 - 3.58 (1H, m), 3.52 - 3.47 (1H, m), 2.39 - 2.37 (1H, m), 2.16 (3H, s), 2.13 (3H, s), 2.07 - 2.04 (12H, m), 1.97 (3H, s), 1.71 - 1.68 (3H, m), 1.55 - 1.53 (2H, m), 1.41 (6H, m), 1.27 (51H, m), 0.91 - 0.87 (9H, t, J = 8). MS (m / z): [M+H] + C 68 H 123 N2O 18 Calculated value for, 1255.88, measured value, 1255.88. GL16, yield 55%. 1 H NMR (400 MHz, CDCl3) δ = 5.34-5.30 (1H, m), 5.24-5.23 (1H, m), 4.99 (1H, m), 4.35-4.28 (2H, m), 4.14-4.10 (1H, m), 3.75-3.71 (1H, t, J = 8), 3.45-3.41 (1H, t, J = 8), 2.41(12H, m), 2.12-2.05 (9H, m), 1.70(3H, m), 1.58 (3H, m), 1.42 (6H, m), 1.27 (52H, s), 0.91-0.87 (9H, t, J = 8).MS (m / z): [M+H] + C 53 H 103 Calculated value for N2O8: 895.77, Measured value: 895.77. array RNA sequencing Human OX40 mRNA (including 5'UTR and 3'UTR) (SEQ ID NO: 1) GGGAAAAGUAGAAAGAAAGAAAGAAGAGAAAAUAAAGACAAAGAGCCACCAUGUGCGUGGGAGCACGGAGACUGGGAAGGGGACCUUGCGCCGCCCUGCUGCUGCUGGGCCUGGGCCUGUCCACCGUGACAGGCCUGCACUGCGUGGGCGACACCUACCCUUCUAACGAUAGGUGCUGUCACGAGUGUCGCCCAGGCAAUGGCAUGGUGUCCAGGUGCUCCCGCUCUCAGAACACCGUGUGCCGGCCUUGUGGCCCAGGCUUCUAUAAUGACGUGGUGAGCUCCAAGCCCUGCAAGCCUUGUACAUGGUGCAACCUGCGGAGCGGCUCCGAGAGAAAGCAGCUGUGCACCGCCACACAGGAUACCGUGUGCCGGUGUAGAGCCGGCACACAGCCACUGGACUCUUACAAGCCAGGAGUGGAUUGUGCACCUUGCCCACCUGGCCACUUUAGCCCAGGCGACAACCAGGCCUGUAAGCCCUGGACCAAUUGCACACUGGCAGGCAAGCACACCCUGCAGCCAGCAUCUAAUUCUAGCGAUGCCAUCUGCGAGGACAGAGAUCCACCAGCAACCCAGCCUCAGGAGACACAGGGACCUCCAGCCAGGCCAAUCACCGUGCAGCCAACAGAGGCAUGGCCUCGGACCUCUCAGGGACCAAGCACAAGACCCGUGGAGGUGCCUGGAGGAAGGGCAGUGGCAGCUAUCUUGGGGCUCGGGUUGGUACUGGGACUGCUUGGCCCACUUGCUAUCUUGCUGGCUCUGUAUCUGCUGAGGCGCGACCAGCGCCUGCCCCCUGAUGCACACAAGCCACCAGGAGGAGGAAGCUUCCGGACCCCAAUCCAGGAGGAGCAGGCAGACGCACACUCCACACUGGCCAAGAUCUGAUUGUGUAUGCGUUAAUAAAAAGAAGGAACUCGUA Mouse OX40 mRNA (including 5’UTR and 3’UTR) (SEQ ID NO: 2) GGGAAAAGUAGAAAGAAAGAAAGAAGAGAAAAUAAAGACAAAGAGCCACCAUGUAUGUGUGGGUUCAGCAGCCCACAGCCCUUCUGCUGCUGGGACUCACACUUGGAGUUACAGCAAGGCGGCUCAACUGUGUUAAACAUACCUACCCCAGUGGUCACAAGUGCUGUCGUGAGUGCCAGCCAGGCCAUGGUAUGGUGAGCCGCUGUGAUCAUACCAGGGAUACUCUAUGUCAUCCGUGUGAGACUGGCUUCUACAAUGAAGCUGUCAAUUAUGAUACCUGCAAGCAGUGUACACAGUGCAACCAUCGAAGUGGAAGUGAACUCAAGCAGAAUUGCACACCUACUCAGGAUACUGUCUGCAGAUGUAGACCAGGCACCCAACCUCGGCAGGACAGCGGCUACAAGCUUGGAGUUGACUGUGUUCCCUGCCCUCCUGGCCACUUUUCUCCAGGCAACAACCAGGCCUGCAAGCCCUGGACCAAUUGUACCUUAUCUGGAAAGCAGACCCGCCACCCAGCCAGUGACAGCUUGGACGCAGUCUGUGAGGACAGAAGCCUCCUGGCCACACUGCUCUGGGAGACCCAGCGCCCUACAUUCAGGCCAACCACUGUCCAAUCCACCACAGUCUGGCCCAGGACUUCUGAGUUGCCCUCUCCACCCACCUUGGUGACUCCUGAGGGCCCUGCAUUUGCUGUUCUCCUAGGCCUGGGCCUGGGCCUGCUGGCUCCCUUGACUGUCCUGCUGGCCUUGUACCUGCUCCGGAAGGCUUGGAGAUUGCCUAACACUCCCAAACCUUGUUGGGGAAACAGCUUCAGGACCCCGAUCCAGGAGGAACACACAGACGCACACUUUACUCUGGCCAAGAUCUGAUUGUGUAUGCGUUAAUAAAAAGAAGGAACUCGUA Heterologous 5’UTR / 3’UTR 5’UTR: GGGAAAAGUAGAAAGAAAGAAAGAAGAGAAAAUAAAGACAAAGAGCCACC (SEQ ID NO: 3) 3’UTR: UUGUGUAUGCGUUAAUAAAAAGAAGGAACUCGUA (SEQ ID NO: 4) Human OX40 coding sequence (SEQ ID NO: 5) AUGUGCGUGGGAGCACGGAGACUGGGAAGGGGACCUUGCGCCGCCCUGCUGCUGCUGGGCCUGGGCCUGUCCACCGUGACAGGCCUGCACUGCGUGGGCGACACCUACCCUUCUAACGAUAGGUGCUGUCACGAGUGUCGCCCAGGCAAUGGCAUGGUGUCCAGGUGCUCCCGCUCUCAGAACACCGUGUGCCGGCCUUGUGGCCCAGGCUUCUAUAAUGACGUGGUGAGCUCCAAGCCCUGCAAGCCUUGUACAUGGUGCAACCUGCGGAGCGGCUCCGAGAGAAAGCAGCUGUGCACCGCCACACAGGAUACCGUGUGCCGGUGUAGAGCCGGCACACAGCCACUGGACUCUUACAAGCCAGGAGUGGAUUGUGCACCUUGCCCACCUGGCCACUUUAGCCCAGGCGACAACCAGGCCUGUAAGCCCUGGACCAAUUGCACACUGGCAGGCAAGCACACCCUGCAGCCAGCAUCUAAUUCUAGCGAUGCCAUCUGCGAGGACAGAGAUCCACCAGCAACCCAGCCUCAGGAGACACAGGGACCUCCAGCCAGGCCAAUCACCGUGCAGCCAACAGAGGCAUGGCCUCGGACCUCUCAGGGACCAAGCACAAGACCCGUGGAGGUGCCUGGAGGAAGGGCAGUGGCAGCUAUCUUGGGGCUCGGGUUGGUACUGGGACUGCUUGGCCCACUUGCUAUCUUGCUGGCUCUGUAUCUGCUGAGGCGCGACCAGCGCCUGCCCCCUGAUGCACACAAGCCACCAGGAGGAGGAAGCUUCCGGACCCCAAUCCAGGAGGAGCAGGCAGACGCACACUCCACACUGGCCAAGAUCUGA Mouse OX40 coding sequence (SEQ ID NO: 6) AUGUAUGUGUGGGUUCAGCAGCCCACAGCCCUUCUGCUGCUGGGACUCACACUUGGAGUUACAGCAAGGCGGCUCAACUGUGUUAAACAUACCUACCCCAGUGGUCACAAGUGCUGUCGUGAGUGCCAGCCAGGCCAUGGUAUGGUGAGCCGCUGUGAUCAUACCAGGGAUACUCUAUGUCAUCCGUGUGAGACUGGCUUCUACAAUGAAGCUGUCAAUUAUGAUACCUGCAAGCAGUGUACACAGUGCAACCAUCGAAGUGGAAGUGAACUCAAGCAGAAUUGCACACCUACUCAGGAUACUGUCUGCAGAUGUAGACCAGGCACCCAACCUCGGCAGGACAGCGGCUACAAGCUUGGAGUUGACUGUGUUCCCUGCCCUCCUGGCCACUUUUCUCCAGGCAACAACCAGGCCUGCAAGCCCUGGACCAAUUGUACCUUAUCUGGAAAGCAGACCCGCCACCCAGCCAGUGACAGCUUGGACGCAGUCUGUGAGGACAGAAGCCUCCUGGCCACACUGCUCUGGGAGACCCAGCGCCCUACAUUCAGGCCAACCACUGUCCAAUCCACCACAGUCUGGCCCAGGACUUCUGAGUUGCCCUCUCCACCCACCUUGGUGACUCCUGAGGGCCCUGCAUUUGCUGUUCUCCUAGGCCUGGGCCUGGGCCUGCUGGCUCCCUUGACUGUCCUGCUGGCCUUGUACCUGCUCCGGAAGGCUUGGAGAUUGCCUAACACUCCCAAACCUUGUUGGGGAAACAGCUUCAGGACCCCGAUCCAGGAGGAACACACAGACGCACACUUUACUCUGGCCAAGAUCUGA DNA sequence Human OX40 mRNA (including 5’UTR and 3’UTR) (SEQ ID NO: 7) GGGAAAAGTAGAAAGAAAGAAAGAAGAGAAAATAAAGACAAAGAGCCACCATGTGCGTGGGAGCACGGAGACTGGGAAGGGGACCTTGCGCCGCCCTGCTGCTGCTGGGCCTGGGCCTGTCCACCGTGACAGGCCTGCACTGCGTGGGCGACACCTACCCTTCTAACGATAGGTGCTGTCACGAGTGTCGCCCAGGCAATGGCATGGTGTCCAGGTGCTCCCGCTCTCAGAACACCGTGTGCCGGCCTTGTGGCCCAGGCTTCTATAATGACGTGGTGAGCTCCAAGCCCTGCAAGCCTTGTACATGGTGCAACCTGCGGAGCGGCTCCGAGAGAAAGCAGCTGTGCACCGCCACACAGGATACCGTGTGCCGGTGTAGAGCCGGCACACAGCCACTGGACTCTTACAAGCCAGGAGTGGATTGTGCACCTTGCCCACCTGGCCACTTTAGCCCAGGCGACAACCAGGCCTGTAAGCCCTGGACCAATTGCACACTGGCAGGCAAGCACACCCTGCAGCCAGCATCTAATTCTAGCGATGCCATCTGCGAGGACAGAGATCCACCAGCAACCCAGCCTCAGGAGACACAGGGACCTCCAGCCAGGCCAATCACCGTGCAGCCAACAGAGGCATGGCCTCGGACCTCTCAGGGACCAAGCACAAGACCCGTGGAGGTGCCTGGAGGAAGGGCAGTGGCAGCTATCTTGGGGCTCGGGTTGGTACTGGGACTGCTTGGCCCACTTGCTATCTTGCTGGCTCTGTATCTGCTGAGGCGCGACCAGCGCCTGCCCCCTGATGCACACAAGCCACCAGGAGGAGGAAGCTTCCGGACCCCAATCCAGGAGGAGCAGGCAGACGCACACTCCACACTGGCCAAGATCTGATTGTGTATGCGTTAATAAAAAGAAGGAACTCGTA Mouse OX40 mRNA (including 5’UTR and 3’UTR) (SEQ ID NO: 8) GGGAAAAGTAGAAAGAAAGAAAGAAGAGAAAATAAAGACAAAGAGCCACCATGTATGTGTGGGTTCAGCAGCCCACAGCCCTTCTGCTGCTGGGACTCACACTTGGAGTTACAGCAAGGCGGCTCAACTGTGTTAAACATACCTACCCCAGTGGTCACAAGTGCTGTCGTGAGTGCCAGCCAGGCCATGGTATGGTGAGCCGCTGTGATCATACCAGGGATACTCTATGTCATCCGTGTGAGACTGGCTTCTACAATGAAGCTGTCAATTATGATACCTGCAAGCAGTGTACACAGTGCAACCATCGAAGTGGAAGTGAACTCAAGCAGAATTGCACACCTACTCAGGATACTGTCTGCAGATGTAGACCAGGCACCCAACCTCGGCAGGACAGCGGCTACAAGCTTGGAGTTGACTGTGTTCCCTGCCCTCCTGGCCACTTTTCTCCAGGCAACAACCAGGCCTGCAAGCCCTGGACCAATTGTACCTTATCTGGAAAGCAGACCCGCCACCCAGCCAGTGACAGCTTGGACGCAGTCTGTGAGGACAGAAGCCTCCTGGCCACACTGCTCTGGGAGACCCAGCGCCCTACATTCAGGCCAACCACTGTCCAATCCACCACAGTCTGGCCCAGGACTTCTGAGTTGCCCTCTCCACCCACCTTGGTGACTCCTGAGGGCCCTGCATTTGCTGTTCTCCTAGGCCTGGGCCTGGGCCTGCTGGCTCCCTTGACTGTCCTGCTGGCCTTGTACCTGCTCCGGAAGGCTTGGAGATTGCCTAACACTCCCAAACCTTGTTGGGGAAACAGCTTCAGGACCCCGATCCAGGAGGAACACACAGACGCACACTTTACTCTGGCCAAGATCTGATTGTGTATGCGTTAATAAAAAGAAGGAACTCGTA Heterologous 5’UTR / 3’UTR 5'UTR: GGGAAAAGTAGAAAGAAAGAAAGAAGAGAAAATAAAGACAAAGAGCCACC (SEQ ID NO: 9) 3'UTR: TTGTGTATGCGTTAATAAAAAGAAGGAACTCGTA (SEQ ID NO: 10) Human OX40 coding sequence (SEQ ID NO: 11) ATGTGCGTGGGAGCACGGAGACTGGGAAGGGGACCTTGCGCCGCCCTGCTGCTGGGCCTGGGCCTGTCCACCGTAGACAGGCCTGCACTGCGTGGGCGACACCTACCCTTCTAACGATAGGTGCTGTCGAGTGTCGCCCAGGCAATGCATGGTGTCCAGGTGCTCCCGCTCTCAGAACACCGTGTGCCGGCCTTGTGGCCCAG GCTTCTATAATGACGTGGTGAGCTCCAAGCCCTGCAAGCCTTGTACATGGTGCAACCTGCGGAGCGGCTCCGAGAGAAAGCAGCTTGCACCGCCACAGGATACCGTGTGCCGGTGTAGAGCCGGCACACAGCCACTGGACTCTTACAAGCCAGGAGTGGATTGTGCACCTTGCCCACCTGGCCACTTTAGCCCAGGCGACAACCAG GCCTGTAAGCCCTGGACCAATTGCACACTGGCAGGCAAGCACACCCTGCAGCCAGCATCTAATTCTAGCGATGCCATCTGCGAGACAGAGATCCACCAGCAACCCAGCCTCAGGAGACACAGGGACCTCCAGCCAGGCCAATCACCGTGCAGCCAACAGAGGCATGGCCTCGGACCTTCCAGGGACCAAGCACAAGACCCGTGGAGG TGCCTGGAGGAAGGGCAGTGGCAGCTATCTTGGGGCTCGGGTTGGTACTGGGACTGCTTGGCCACTTGCTATCTTGCTGGCTCTGTATCTGCTGAGGCGCGACCAGCGCCTGCCCCCTGCACACAAGCCACCAGGAGGAGGAAGCTTCCGGACCCAATCCAGGAGGAGCAGCAGCACACTCCACACTGGCCAAGATCTGA MouseOX40 code sequence(sequence number12) ATGTATGTGTGGGTTCAGCAGCCCACAGCCCTTCTGCTGGCTGGGACTCACACTTGGAGTTACAGCAAGGCGGCTCAACTGTGTTAAACATACCTACCCCAGTGGTCACAAGTGCTGTCGTGAGTGCCAGCCAGGCCATGGTATGGTGAGCCGCTGTGATCATACCAGGGATACTCTATGTCATCCGTGTGAGACTGGCTTCTAC AATGAAGCTGTCAATTATGATACCTGCAAGCAGTGTACACAGTGCAACCATCGAAGTGGAAGTGAACTCAAGCAGAATTGCACACCTACTCAGGATACTGTCTGCAGATGTAGACCAGGCACCCAACCTCGGCAGAGCGGCTACAAGCTTGGAGTTGACTGTGTTCCCTGCCCTCCTGGCCACTTTTCTCCAGGCAACAACC AGGCCTGCAAGCCCTGGACCAATTGTACCTTATCTGGAAAGCAGACCCGCCACCCAGCCAGTCAGCTTGGACGCAGTCTGTGAGACAGAAGCCTCCTGGCCACACTGCTCTGGGAGACCCAGCGCCCTACATTCAGGCCAACCACTGTCCAATCCACCACAGTCTGGCGCAGGACTTCTGAGTTGCCCTCTCCACCCACCTT GGTGACTCCTGAGGGCCCTGCATTTGCTGTTCTCTAGGCCTGGGCCTGGGCCTGCTGCTCCCTTGACTGTCCTGCTGGCCTTGTACCTGCTCCGGAAGGCTTGGAGATTGCCTAACACTCCCAAACCTTGTTGGGGAAACAGCTTCAGGACCCCGATCCAGGAGGAACACACAGAGCACACTTTACTCTGGCCCAAGATCTGA アッスOX40リガンドのコードアショ(アショック 13)。 ATGGAAGGGGAAGGGGTTCAACCCCTGGATGAGAATCTGGAAACGGATCAAGGCCAAGATTCAAGTGGAAGAAGACGCTAAGGCTGGTGGTCTCTGGGATCAAGGGAGCAGGGATGCTTCTGTGCTTCATCTATGTCTGCCTGCAACTCTCTTCCTCTCCGGCAAAGGACCCTCAATCCAAAGACTCAGAGGAGCAGTTACCAGATGTGAGGATGGGCAACTATTCATCAGCTCATACAAGAATGAGTATCAAACTATGGAGGTGCAGAACAAATTCGGTTGCATCAAGTGCGATG GGCTTTATATCATCTACCTGAAGGGCTCCTTTTTCCAGGAGGTCAAGATTGACCTTCATTTCCGGGAGGATCATAATCCCATCTCTATTCCAATGCTGAACGATGGTCGAAGGATTGTCTTCACTGTGGTGGCCTCTTTGGCTTTCAAAGATAAAGTTTACCTGACTGTAAATGCTCTGATACTCTCTGCGAACACCTCCAGATAAATGATGGGGAGCTGATTGTTGTCCAGCTAACGCCTGGATACTGTGCTCCTGAAGGATCTACCACAGCACTGTGAACCAAGTACCACTGTGA ヒトOX40リガンドのコメーショード(ファーションフェック14) ATGGAAAGGGTCCAACCCCTGGAAGAGAATGTGGGAAATGCAGCCAGGCCAAGATTCGAGAGGAACAAGCTATTGCTGGTGGCCTCTGTAATTCAGGGACTGGGGCTGCTCCTGTGCTTCACCTACATCTGCCTGCACTTCTCTGCTCTTCAGGTATCACATCGGTATCCTCGAATTCAAAGTATCAAAGTACAATTTACCGAATATAAGAAGGAGAAAGGTTTCATCCTCACTTCCCAAAAGGAGGATGAAATCATGAAGGTGCAGAACAACTCAGTCATCATCAACTGTGATGGGTTTTATCTCATCTCCCTGAAGGGCTACTTCTCCCAGGAAGTCAACATTAGCCTTCATTACCAGAAGGATGAGGAGCCCCTCTTCCAACTGAAGAAGGTCAGGTCTGTCAACTCCTTGATGGTGGCCTCTCTGACTTACAAAGACAAAGTCTACTTGAATGTGACCACTGACAATACCTCCCTGGATGACTTCCATGTGAATGGCGGAGAACTGATTCTTATCCATCAAAATCCTGGTGAATTCTGTGTCCTTTGA Coding sequence of mouse ICOS (SEQ ID NO: 15) ATGAA GCCGTACTTCTGCCGTGTCT TTGTCTTCTG CTTCCTAATC AGACTTTTAA CAGGAGAAAT CAATGGCTCGGCCGATCATA GGATGTTTTC ATTTCACAAT GGAGGTGTAC AGATTTCTTG TAAATACCCTGAGACTGTCC AGCAGTTAAA AATGCGATTG TTCAGAGAGA GAGAAGTCCT CTGCGAACTCACCAAGACCA AGGGAAGCGG AAATGCGGTG TCCATCAAGA ATCCAATGCT CTGTCTATATCATCTGTCAA ACAACAGCGT CTCTTTTTTC CTAAACAACC CAGACAGCTC CCAGGGAAGCTATTACTTCT GCAGCCTGTC CATTTTTGAC CCACCTCCTT TTCAAGAAAG GAACCTTAGTGGAGGATATT TGCATATTTA TGAATCCCAGCTCTGCTGCCAGCTGAAGCTCTGGCTACCCGTAGGGTGTGCAGCTTTCGT TGTGGTACTC CTTTTTGGAT GCATACTTAT CATCTGGTTTTCAAAAAAGA AATACGGATCCAGTGTGCATGACCCTAATAGTGAATACATGTTCATGGCGGCAGTCAACA CAAACAAAAA GTCTAGACTT GCAGGTGTGA CCTCATAA Coding sequence of human ICOS (SEQ ID NO: 16) ATG AAGTCAGGCC TCTGGTATTT CTTTCTCTTC TGCTTGCGCA TTAAAGTTTTAACAGGAGAA ATCAATGGTT CTGCCAATTA TGAGATGTTT ATATTTCACA ACGGAGGTGT ACAAATTTTA TGCAAATATC CTGACATTGT CCAGCAATTT AAAATGCAGT TGCTGAAAGGGGGGCAAATA CTCTGCGATC TCACTAAGAC AAAAGGAAGT GGAAACACAG TGTCCATTAA GAGTCTGAAA TTCTGCCATT CTCAGTTATC CAACAACAGT GTCTCTTTTT TTCTATACAA CTTGGACCAT TCTCATGCCA ACTATTACTT CTGCAACCTA TCAATTTTTG ATCCTCCTCCTTTTAAAGTA ACTCTTACAG GAGGATATTT GCATATTTAT GAATCACAAC TTTGTTGCCAGCTGAAGTTC TGGTTACCCA TAGGATGTGC AGCCTTTGTT GTAGTCTGCA TTTTGGGATGCATACTTATT TGTTGGCTTA CAAAAAAGAA GTATTCATCC AGTGTGCACG ACCCTAACGGTGAATACATG TTCATGAGAG CAGTGAACAC AGCCAAAAAA TCTAGACTCA CAGATGTGAC CCTATAA The coding sequence of mouse CD137 (4-1BB) (SEQ ID NO: 17) ATGGGAAAC AACTGTTACA ACGTGGTGGT CATTGTGCTG CTGCTAGTGGGCTGTGAGAA GGTGGGAGCC GTGCAGAACT CCTGTGATAA CTGTCAGCCT GGTACTTTCTGCAGAAAATA CAATCCAGTC TGCAAGAGCT GCCCTCCAAG TACCTTCTCC AGCATAGGTGGACAGCCGAA CTGTAACATC TGCAGAGTGT GTGCAGGCTA TTTCAGGTTC AAGAAGTTTTGCTCCTCTAC CCACAACGCG GAGTGTGAGT GCATTGAAGG ATTCCATTGC TTGGGGCCACAGTGCACCAG ATGTGAAAAG GACTGCAGGC CTGGCCAGGA GCTAACGAAG CAGGGTTGCAAAACCTGTAG CTTGGGAACA TTTAATGACC AGAACGGTAC TGGCGTCTGT CGACCCTGGACGAACTGCTC TCTAGACGGA AGGTCTGTGC TTAAGACCGG GACCACGGAG AAGGACGTGGTGTGTGGACC CCCTGTGGTG AGCTTCTCTC CCAGTACCAC CATTTCTGTG ACTCCAGAGGGAGGACCAGG AGGGCACTCC TTGCAGGTCC TTACCTTGTT CCTGGCGCTG ACATCGGCTTTGCTGCTGGC CCTGATCTTC ATTACTCTCC TGTTCTCTGT GCTCAAATGG ATCAGGAAAA AATTCCCCCA CATATTCAAG CAACCATTTA AGAAGACCAC TGGAGCAGCT CAAGAGGAAGATGCTTGTAG CTGCCGATGT CCACAGGAAG AAGAAGGAGG AGGAGGAGGC TATGAGCTGTGA The coding sequence of human CD137 (4-1BB) (SEQ ID NO: 18) ATGGGAAAC AGCTGTTACA ACATAGTAGC CACTCTGTTGCTGGTCCTCA ACTTTGAGAGGACAAGATCATTGCAGGATCCTTGTAGTAACTGCCCAGCTGGTACATTCTGTGATAATAACAGGAATCAGATTTGCAGTCCCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGTGACATATGCAGGCAGTGTAAAGGTGTTTTCAGGACCAGGAAGGAGTGTTCCTCCACCAGCAATGCAGAGTGTGACTGCACTCCAGGGTTTCACTGCCTGGGGGCAGGATGCAGCATGTGTGAACAGGATTGTAAACAAGGTCAAGAACTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAACGATCAGAAACGTGGCATCTGTCGACCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGTGGACCATCTCCAGCCGACCTCTCTCCGGGAGCATCCTCTGTGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAAC CATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGC CGATTTCCAG AAGAAGAAGA AGGAGGATGTGAACTGTGA The coding sequence of mouse CD137 ligand (4-1BBL) (SEQ ID NO: 19) ATGGACCAGCACACACTTGATGTGGAGGATACCGCGGATGCCAGACATCCAGCAGGTACTTCGTGCCCCCTCGGATGCGGCGCTCCTCAGAGATACCGGGCTCTCGCGGACGCTGCGCTCCTCTCAGATACTGTGCGCCCCAAAATGCCGCGCTCCCCACGGATGCTGCCTACCCTGCGGTTAATGTTCGGGATCGCGAGGCCGCGTGGCCGCCTGCACTGAACTTCTGTT CCCGCCACCCAAAGCTCTATGGCCTAGTCGCTTTGGTTTTGCTGCTTCTGATCGCCGCCTGTGTTCCTATCTTCACCCGCACCGAGCCTGGCCAGCGCTCACAATCACCACTCGCCCAACCTGGGTACCCGAGAGAATAATGCAGACCAGGTCACCCCTGTTTCCACATTGGCTGCCCCAACACTACACAACAGGCTCTCCTGTGTTCGCCAAGCTACTGGCTAAAAAC CAAGCATCGTTGTGCAATACAACTCTGAACTGGCACAGCCAAGATGGAGCTGGGAGCTCATACCTATCTCAAGGTCTGAGGTACGAAGAAGACAAAAGGAGTTGGTGGTAGACAGTCCCGGGCTCTACTACGTATTTTTGGAACTGAAGCTCAGTCCAACATTCACAAACACAGGCCACAAGGTGCAGGGCTGGGTCTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATG ACTTTGACAACTTGGCCCTGACAGTGGAACTGTTCCCTTGCTCCATGGAGAACAAGTTAGTGGACCGTTCCTGGAGTCCAACTGTTGCTCCTGAAGGCTGGCCACCGCCTCAGTGTGGGTCTGAGGGCTTATCTGCATGGAGCCCAGGATGCATACAGAGACTGGGAGCTGTCTTATCCCAACACCACCAGCTTTGGACTCTTTCTTGTAGAAACCCGACAACCCATGGGAATGA CD137リガンド(4-1BBL) ATGGAATACGCCTCTGACGCTTCACTGGACCCCGAAGCCCCGTGGCCTCCCGCGCCCCGCGCTCGCGCCTGCCGCGTACTGCCTTGGGCCCTGGTCGCGGGGCTGCTGCTGCTGCTGCTGCTCGCTGCCGCCTGCGCCGTCTTCCTCGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAATAA Mouse GITR coding sequence (SEQ ID NO: 21) ATGGGGGCATGGGCCATGCTGTATGGAGTCTCGATGCTCTGTGTGCTGGACCTAGGTCAGCCGAGTGTAGTTGAGGAGCCTGGCTGTGGCCCTGGCAAGGTTCAGAACGGAAGTGGCAACAACACTCGCTGCTGCAGCCTGTATGCTCCAGGCAAGGAGGACTGTCCAAAAGAAAGGTGCATATGTGTCACACCTGAGTACCACTGTGGAGACCCTCAGTGCAAGATCTGCAAGCACTACCCCTGCCAACCAGGCCAGAGGGTGGAGTCTCAAGGGGATATTGTGTTTGGCTTCCGGTGTGTTGCCTGTGCCATGGGCACCTTCTCCGCAGGTCGTGACGGTCACTGCAGACTTTGGACCAACTGTTCTCAGTTTGGATTTCTCACCATGTTCCCTGGGAACAAGACCCACAATGCTGTGTGCATCCCGGAGCCACTGCCCACTGAGCAATACGGCCATTTGACTGTCATCTTCCTGGTCATGGCTGCATGCATTTTCTTCCTAACCACAGTCCAGCTCGGCCTGCACATATGGCAGCTGAGGAGGCAACACATGTGTCCTCGAGAGACCCAGCCATTCGCGGAGGTGCAGTTGTCAGCTGAGGATGCTTGCAGCTTCCAGTTCCCTGAGGAGGAACGCGGGGAGCAGACAGAAGAAAAGTGTCATCTGGGGGGTCGGTGGCCAT GA Coding sequence of human GITR (SEQ ID NO: 22) ATGGCACAG CACGGGGCGA TGGGCGCGTT TCGGGCCCTG TGCGGCCTGG CGCTGCTGTG CGCGCTCAGC CTGGGTCAGC GCCCCACCGG GGGTCCCGGG TGCGGCCCTG GGCGCCTCCT GCTTGGGACG GGAACGGACG CGCGCTGCTG CCGGGTTCAC ACGACGCGCT GCTGCCGCGA TTACCCGGGC GAGGAGTGCT GTTCCGAGTG GGACTGCATGTTGTCCAGC CTGAATTCCA CTGCGGAGAC CCTTGCTGCA CGACCTGCCG GCACCACCCTTGTCCCCCAG GCCAGGGGGT ACAGTCCCAG GGGAAATTCA GTTTTGGCTT CCAGTGTATCGACTGTGCCT CGGGGACCTT CTCCGGGGGC CACGAAGGCC ACTGCAAACC TTGGACAGAC TGCACCCAGT TCGGGTTTCT CACTGTGTTC CCTGGGAACA AGACCCACAA CGCTGTGTGCGTCCCAGGGT CCCCGCCGGC AGAGCCGCTT GGGTGGCTGA CCGTCGTCCT CCTGGCCGTGGCCGCCTGCG TCCTCCTCCT GACCTCGGCC CAGCTTGGAC TGCACATCTG GCAGCTGAGG AGTCAGTGCA TGTGGCCCCG AGAGACCCAG CTGCTGCTGG AGGTGCCGCC GTCGACCGAA GACGCCAGAA GCTGCCAGTT CCCCGAGGAA GAGCGGGGCG AGCGATCGGC AGAGGAGAAGGGGCGGCTGG GAGACCTGTG GGTGTGA

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications and the materials for which they are cited herein are specifically incorporated by reference.

[0144] Those skilled in the art will understand that many changes and modifications may be made to the preferred embodiments of the invention and that such changes and modifications may be made without departing from the spirit of the invention. It is, therefore, intended by the appended claims to cover all such equivalent variations which fall within the true spirit and scope of the invention.

Claims

1. an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule; nanoparticles comprising mRNA encoding the costimulatory molecule; A composition comprising: the antibody, ligand, or antigen-binding fragment thereof comprises BMS986178, GSK3174998, PF-04518600, MOXR0916, MEDI6383, MEDI0562, INCAGN01949, InVivoPlus anti-mouse OX40 (clone OX-86) (company: BioXcell, catalog: BP0031), InVivoPlus anti-mouse 4-1BB (CD137) (clone LOB12.3) (company: BioXcell, catalog: BP0169), utomilumab, urelumab, or a ligand encoded by SEQ ID NO: 13, 14, 19, or 20; The composition, wherein the costimulatory molecule comprises OX40 or 4-1BB.

2. The composition of claim 1 , wherein the mRNA encoding the costimulatory molecule is encapsulated by the nanoparticle.

3. The composition of claim 1 or 2, wherein the nanoparticles comprise a phospholipid or a glycolipid.

4. The phospholipid is 【Chemical 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 The composition of claim 3 selected from the group consisting of:

5. The phospholipid 【Chemistry 5】 The composition of claim 4, wherein

6. The glycolipid 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 The composition of claim 3 selected from the group consisting of:

7. The glycolipid 【Chemistry 10】 The composition of claim 6, wherein

8. The composition of any one of claims 1 to 7, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR).

9. The composition of any one of claims 1 to 7, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR).

10. The composition of any one of claims 1 to 9, wherein the mRNA comprises a chemically modified nucleic acid base.

11. The composition of claim 10, wherein the chemically modified nucleobase is pseudouridine.

12. The composition of any one of claims 1 to 11, further comprising an immunotherapeutic agent.

13. The composition of claim 12, wherein the immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

14. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the composition of any one of claims 1 to 13.

15. 15. The composition of any one of claims 1 to 14 for use in a method for stimulating T cells, comprising administering to a subject an effective amount of the composition of any one of claims 1 to 13 or the pharmaceutical composition of claim 14.

16. The composition of claim 15 , wherein the subject is a mammal.

17. 17. The composition of claim 16, wherein the mammal is a human.

18. A composition comprising an effective amount of an antibody, ligand, or antigen-binding fragment thereof that specifically binds to a costimulatory molecule, and nanoparticles comprising mRNA encoding the costimulatory molecule, the composition being for use in a method of treating cancer, the method comprising administering the composition to a subject in need thereof, wherein the costimulatory molecule comprises OX40 or 4-1BB, and the antibody, ligand, or antigen-binding fragment thereof is selected from the group consisting of BMS986178, GSK3174998, and PF-045186. 00, MOXR0916, MEDI6383, MEDI0562, INCAGN01949, InVivoPlus anti-mouse OX40 (clone OX-86) (company: BioXcell, catalog: BP0031), InVivoPlus anti-mouse 4-1BB (CD137) (clone LOB12.3) (company: BioXcell, catalog: BP0169), utomilumab, urelumab, or a ligand encoded by SEQ ID NO: 13, 14, 19, or 20.

19. 20. The composition of claim 18, wherein the mRNA encoding the costimulatory molecule is encapsulated by the nanoparticle.

20. 20. The composition of claim 18 or 19, wherein the nanoparticles comprise a phospholipid or a glycolipid.

21. The phospholipid is 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 21. The composition of claim 20, selected from the group consisting of:

22. The phospholipid 【Chemistry 15】 22. The composition of claim 21, wherein:

23. The glycolipid 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 21. The composition of claim 20, selected from the group consisting of:

24. The glycolipid 【Chemistry 20】 24. The composition of claim 23, wherein:

25. 25. The composition of any one of claims 18 to 24, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 5' untranslated region (5'UTR).

26. 26. The composition of any one of claims 18 to 25, wherein the mRNA encoding the costimulatory molecule comprises a heterologous 3' untranslated region (3'UTR).

27. ​​A composition described in any one of claims 18 to 26, wherein the mRNA comprises a chemically modified nucleic acid base, and the chemically modified nucleic acid base is pseudouridine.

28. The composition of any one of claims 18 to 27, wherein the cancer comprises melanoma, colorectal cancer, lung cancer, colon cancer, or lymphoma.

29. The composition of any one of claims 18 to 28, wherein the subject is a mammal.

30. 30. The composition of claim 29, wherein the mammal is a human.

31. The composition of any one of claims 18 to 30, wherein the antibody or antigen-binding fragment thereof and the nanoparticles are administered by intramuscular injection or systemically.

32. The composition of any one of claims 18 to 31, wherein the method further comprises administering an additional therapeutic agent.

33. 33. The composition of claim 32, wherein the additional therapeutic agent comprises an additional immunotherapeutic agent.

34. 34. The composition of claim 33, wherein the additional immunotherapeutic agent is selected from an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination thereof.

35. 35. The composition of any one of claims 18 to 34, wherein the antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule and the nanoparticles comprising mRNA encoding the costimulatory molecule are administered simultaneously.

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