IL-12 and gm-csf fusion proteins

Abstract

The present application relates to a fusion protein comprising IL-12 and GM-CSF covalently linked, which can further comprise a transmembrane domain at the C-terminus of IL-12 and GM-CSF. The present application also relates to the delivery of the fusion protein by mRNA-lipid nanoparticle system. The present application also relates to a method for expressing a membrane-bound IL-12 and GM-CSF fusion protein with a transmembrane domain in cells, comprising the following steps: using an RNA polymerase to prepare mRNA in vitro transcription with a DNA sequence encoding the fusion protein as a template; encapsulating the mRNA in a lipid nanoparticle (LNP); transfecting the mRNA-encapsulated lipid nanoparticle into cells; and translating the mRNA in the cells to express the membrane-bound IL-12 and GM-CSF fusion protein, thereby activating immune cells and regulating the tumor microenvironment.

Description

Technical Field

[0001] This invention relates to a fusion protein composed of IL-12 and GM-CSF, wherein the IL-12 and GM-CSF are covalently linked. The invention also relates to the preparation of mRNA-lipid nanoparticles (mRNA-LNPs) and their delivery into cells to obtain cells expressing a membrane-bound IL-12-GM-CSF fusion protein. This membrane-bound IL-12-GM-CSF protein can be used to induce immune responses and activate the tumor microenvironment in vivo. Background Technology

[0002] IL-12 Interleukin-12 is a heterodimeric protein reported in 1989, composed of two subunits, p40 and p35 (1). IL-12 regulates the immune system response and plays an important role in adaptive immunity (1). Phagocytes secrete this heterodimeric protein in response to pathogens (2). IL-12 binds to the IL-12 receptor (IL-12R) on the surface of T cells and natural killer (NK) cells, activating transcription activator 4 (STAT4) and stimulating the secretion of interferon-γ (IFN-γ) (2). Downstream signaling pathways of IFN-γ include activation of the T-box transcription factor TBX21 (Tbet) and induction of pro-inflammatory function of helper T cells 1 (Th1 cells), thereby linking innate immunity with adaptive immune responses (2). IL-12 is also known to suppress tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), which have immunosuppressive effects. IL-12 has demonstrated antitumor activity in multiple preclinical studies (3). Although IL-12 has antitumor effects, systemic administration is too toxic. The ideal targets for IL-12 immunotherapy are immune cells in the tumor and adjacent lymph nodes, including activated but exhausted T cells, NK cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs).

[0003] GM-CSF Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important factor regulating myeloid hematopoiesis and mobilizing hematopoietic stem cells. GM-CSF can induce hematopoietic progenitor cells in the bone marrow to differentiate into various immune cells such as granulocytes, monocytes-macrophages, T cells, and natural killer (NK) cells. This cytokine was identified as myeloid growth factor in the 1960s, purified in the 1970s, molecularly cloned in the 1980s, and entered clinical development in the 1990s (5). Sargramostim (trade name Leukine®; Partner Therapeutics, Inc., Lexington, Massachusetts, USA) is a glycosylated recombinant human granulocyte-macrophage colony-stimulating factor. With its safe and effective hematopoietic growth factor activity, it has been approved by the US FDA for the treatment of 6 diseases. It differs from natural human GM-CSF by only one amino acid, namely, arginine at position 23 is replaced by leucine (5). In addition to its myeloid hematopoietic function, GM-CSF also has an anti-apoptotic effect, which can promote the proliferation, mobilization and activation of hematopoietic stem cells, mesenchymal stromal cells, endothelial progenitor cells, pericytes, neural stem cells and oligodendrocyte progenitor cells (5). In this application, we fused GM-CSF with IL-12 membrane-binding protein through a linker peptide and co-expressed it as a single protein to induce activation of immune cells. Attached Figure Description

[0004] Figure 1 shows a schematic diagram (Figure A) of the DNA vector template (for in vitro transcription of IL-12-linker peptide-GM-CSF-transmembrane domain (TM) mRNA and its mRNA structure (Figure B). This mRNA can be used to translate target proteins expressed on the cell surface. TM: transmembrane domain; 5'-UTR: 5' untranslated region; 3'-UTR: 3' untranslated region; poly A tail is used to improve mRNA stability.

[0005] Figure 2 shows the structure of membrane-bound IL-12 linked to GM-CSF. L: linker peptide; TM: transmembrane domain; C: intracellular domain. IL-12 consists of the IL-12 p40 domain, linker peptide, and IL-12 p35 domain.

[0006] Figures 3A and 3B show the protein expression of IL-12-linker peptide-GM-CSF mRNA-LNP 72 hours after transfection into different cell lines. 3A shows GM-CSF expression; 3B shows IL-12 expression. For flow cytometry analysis, the primary antibody was either mouse anti-GM-CSF antibody or anti-IL-12 antibody, and the secondary antibody was APC-labeled anti-mouse IgG diluted 1:100.

[0007] Figure 4 shows that transfection of primary T cells with IL-12-linker peptide-GM-CSF mRNA-LNP upregulates IFN-γ expression. T cells were activated with CD3 / CD28 antibody for 3 days before transfection, and intracellular IFN-γ (a Th1 marker) was detected 2 days after transfection. Cells were treated with brevidin A for 2.5 hours before flow cytometry to retain the cytokine intracellularly. The X-axis represents CD3 staining, and the Y-axis represents IFN-γ expression.

[0008] Figure 5 shows that transfection of macrophages with IL-12-linker peptide-GM-CSF mRNA-LNP improves macrophage survival and cell morphology. The cell morphology image is magnified 400 times.

[0009] Figure 6 shows that in the presence of IL-4, after monocytes were transfected with IL-12-linker peptide-GM-CSF mRNA-LNP, the dendritic cell marker CD1a was expressed and the expression of the monocyte marker CD14 was reduced, exhibiting a dendritic cell phenotype.

[0010] Figure 7 shows that IL-12-linking peptide-GM-CSF mRNA-LNP can inhibit the growth of OVCAR-5 xenograft tumors in vivo. p<0.05: (i) IL-12-linker peptide-GM-CSF mRNA-LNP group, (ii) IL-12-linker peptide-GM-CSF+EpCAM-CD3 mRNA-LNP group, both compared with the GFP mRNA-LNP negative control group.

[0011] Figures 8A and 8B show the inhibitory effects of IL-12-linker peptide-GM-CSF mRNA-LNP on locally injected tumors (8A) and distally uninjected tumors (8B) in mice, respectively. PC3 tumor cells were subcutaneously seeded into the left and right sides of mice, with mRNA-LNP injected intratumorally only into the left tumor. The tumor volume of both sides was then measured using calipers. p<0.05, EpCAM-CD3+IL-12-GM-CSF group compared with GFP-LNP control group, Student's t test. Detailed Implementation

[0012] This invention relates to a fusion protein comprising IL-12 and GM-CSF covalently linked together. In one embodiment, IL-12 is located at the N-terminus of GM-CSF; in another embodiment, IL-12 is located at the C-terminus of GM-CSF. In a preferred embodiment, IL-12 and GM-CSF are linked by a linker peptide. The linker peptide is preferably 5–30 or 5–20 amino acids in length.

[0013] In one embodiment, the fusion protein further includes a transmembrane domain at the C-terminus of IL-12 and GM-CSF. In another embodiment, the fusion protein further includes an intracellular tail region at its C-terminus.

[0014] Typically, mRNA is transient and short-lived after delivery in vivo. This invention provides a method for expressing an IL-12-linker peptide-GM-CSF fusion protein located on the cell membrane by delivering lipid nanoparticle-encapsulated mRNA into cells. Encapsulating the mRNA in lipid nanoparticles (LNPs) improves its stability.

[0015] The antitumor effect achieved by delivering membrane-bound IL-12-linker peptide-GM-CSF can improve IL-12 tolerance and enhance efficacy. Compared with secreted IL-12 and GM-CSF, this application uses mRNA-LNP technology to design and construct a vector expressing membrane-bound IL-12-linker peptide-GM-CSF to reduce potential toxicity.

[0016] In the fusion protein of the present invention, the transmembrane domain is selected from the transmembrane domains of proteins such as platelet-derived growth factor receptor β (PDGFRβ), T cell receptor α or β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

[0017] The present invention also relates to lipid nanoparticles encapsulating mRNA, wherein the mRNA is transcribed from a DNA sequence comprising the following elements: (a) a promoter sequence, such as the T7 promoter sequence; (b) a 5'-untranslated region (UTR) sequence; (c) a sequence encoding the fusion protein of the present invention described above; (d) a 3'-UTR sequence; and (e) a poly A-tail sequence.

[0018] The present invention further relates to a method for preparing an IL-12-linker peptide-GM-CSF protein expressed on the cell surface, the method comprising the following steps: (i) obtaining a DNA sequence comprising: (a) a promoter sequence; (b) a 5'-UTR sequence; (c) a sequence encoding the fusion protein of the present invention; (d) a 3'-UTR sequence; (e) a poly A-tail sequence; (ii) transcribing the DNA sequence into mRNA in vitro using an RNA polymerase; (iii) encapsulating the mRNA in lipid nanoparticles (LNPs); (iv) transfecting the LNPs encapsulating the mRNA into target cells; and (v) translating the mRNA into cells to produce an IL-12-linker peptide-GM-CSF cytokine expressed on the cell surface.

[0019] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester (SM-102), distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl racemic glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000). [LNP-102 (ii)] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester (SM-102), distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000-MAL). [LNP-102 (i)] In one embodiment, the lipid nanoparticles comprise 2-hexyldecanoate-1,1'-[[(4-hydroxybutyl)imino]di-6,1-hexadiyl] ester (ALC-0315), distearate phosphatidylcholine (DSPC), cholesterol, and -[2-(di-tetradecylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethylidene) (ALC-0159). [LNP-315] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester (SM-102), distearylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl racemic glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000). The molar ratio of LNP-102 is SM-102:distearylphosphatidylcholine:cholesterol:polyethylene glycol 2000 = 50:10:38.5:1.5 (molar %).

[0020] Encapsulating mRNA in lipid nanoparticles protects it from degradation and enhances its stability; the mRNA is then released from the lipid nanoparticles into cells in vivo to achieve protein expression. The preparation method of mRNA-LNP in this invention is based on the research of Schoenmaker et al. (International Journal of Pharmaceutics, 601:120856, 2021), and all content regarding lipid nanoparticles in that literature is incorporated herein by reference.

[0021] Figure 1 shows a linearized DNA template for in vitro transcription, which generates mRNA in the presence of RNA polymerase and nucleoside triphosphate. The DNA template contains a promoter (e.g., the T7 or SP6 promoter), followed by a 5' untranslated region (5'UTR), a coding region for the IL-12-linker peptide-GM-CSF-transmembrane (TM) domain, and a 3' untranslated region (3'UTR) and a polyadenylated tail (e.g., approximately 152 polyadenylates) to enhance RNA stability. The generated RNA may have a 5' cap structure (e.g., [m7G(5')ppp(5')G], i.e., a cap 1 structure, which further enhances RNA stability). The mRNA can be introduced into cells via electroporation or transfection. For in vivo applications, the mRNA is preferably encapsulated in lipid nanoparticles prepared according to the above formulation.

[0022] In the DNA sequence, the promoter may be a T7 AG promoter. The polyadenylated tail sequence is 20-170 nucleotides in length, and may optionally include one or more linker sequences between the polyadenylated segments. If the polyadenylated tail is longer than 60 nucleotides, it typically contains a linker sequence comprising non-adenosine nucleotides; the linker sequence is 5-30 or 5-25 nucleotides in length, for example, 10 or 20 nucleotides. In one example, the polyadenylated tail is 110 nucleotides long, consisting of a 30-adenosine residue segment, a linker sequence containing 10 nucleotides, and another 70-adenosine residue segment in sequence. In another example, the polyadenylated tail is 90 nucleotides long, consisting of a 40-adenosine residue segment, a linker sequence containing 30 nucleotides, and another 30-adenosine residue segment in sequence. The polyadenylated tail can be up to 150-160 nucleotides long, with the aforementioned linker sequences inserted as spacers.

[0023] DNA expression is finely regulated at the posttranscriptional level. While untranslated regions (UTRs) are not translated into amino acids, the 5' and 3' UTRs of mRNA regulate mRNA expression, degradation, and subcellular localization through stem-loop structures, upstream start codons and open reading frames, internal ribosome entry sites, and various cis-regulatory elements that bind to RNA-binding proteins. UTRs play a crucial role in the posttranscriptional regulation of RNA expression, including regulating mRNA nuclear export, translation efficiency, subcellular localization, and stability.

[0024] The length of the 5' untranslated region is typically 10-1000 nucleotides, or 20-500 nucleotides, or 30-200 nucleotides, or 30-100 nucleotides, for example, 50 nucleotides; the length of the 3' untranslated region is typically 10-3000 nucleotides, for example, 50-500 nucleotides or 100-300 nucleotides. Preferably, the 5' and 3' untranslated regions of this invention are the untranslated regions of β-globin, or the untranslated regions used in Pfizer's COVID-19 vaccine.

[0025] For sequence information of the β-globin gene, please refer to: https: / / www.ncbi.nlm.nih.gov / nucleotide / V00497.1?report=genbank&log$= nuclalign&blast_rank=5&RID=TDDZ1K98016 .

[0026] The 5' untranslated region of Pfizer's COVID-19 vaccine is derived from human α-globin RNA and contains an optimized Kozak sequence; its 3' untranslated region contains two sequence elements, derived from the mRNA of the amino-terminal enhancer (AES) of the split protein and the mitochondrial-encoded 12S ribosomal RNA, which can confer RNA stability and achieve efficient total protein expression.

[0027] Any suitable vector can be used as a cloning vector for the above DNA sequence, such as the pSP64 Poly (A) vector (Promega) or the pGEM3Z vector (Promega).

[0028] For example, when constructing the pEM3Z-β-globin-untranslated-untranslated-polyadenylate tail vector, a β-globin 3' untranslated region fragment with restriction enzyme sites on both sides can be amplified from human bone marrow; this fragment can be inserted upstream of the polyadenylate tail in a single copy form (pEM3Z-1β-globin-untranslated-A

[120] ) or in two tandem copies form (pEM3Z-2β-globin-untranslated-A

[120] ).

[0029] The DNA sequence preferably contains a transmembrane domain coding sequence, which can be derived from natural polypeptides or artificially designed. Transmembrane domains derived from natural polypeptides can be derived from any membrane-binding protein or transmembrane protein, such as the transmembrane domains of T cell receptor α or β chains, platelet-derived growth factor receptor β (PDGFRβ), CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR. Artificially designed transmembrane domains are mainly composed of hydrophobic amino acid residues such as leucine and valine, and preferably contain a phenylalanine-tryptophan-valine triplet at each end of the artificially synthesized transmembrane domain. Short oligopeptide linkers or polypeptide linkers, such as linkers with a length of 2 to 10 amino acids, can be optionally placed between the transmembrane domain and the intracellular domain.

[0030] The DNA sequence may also contain an intracellular tail coding sequence for encoding a short intracellular tail region.

[0031] In this application, we sequentially link the IL-12 cytokine coding sequence with a signal peptide, a linker peptide, GM-CSF, a transmembrane domain, and a short intracellular tail region, so that IL-12 and GM-CSF are expressed on the cell surface in a membrane-bound form. In one embodiment, the structure of IL-12 and GM-CSF linked by the linker peptide and having a transmembrane domain and a short intracellular tail region is shown in Figure 2.

[0032] In one embodiment, IL-12 is linked to a signal peptide, a linker peptide, GM-CSF, and a transmembrane peptide derived from PDGFR, thereby expressing the IL-12-linker peptide-GM-CSF fusion protein on the cell surface. This method can also be used to express other cytokines on the cell surface. The linker peptide can be VPGVGVPGVGA or (GGGGS)n, where n = 1, 2, 3, 4, or 5, for example, n = 3.

[0033] We prepared mRNA expressing the membrane-bound IL-12-linker peptide-GM-CSF. The advantages of expressing IL-12 and GM-CSF on the cell surface are: higher protein stability, lower toxicity, and effective activation of T cells and other immune cells.

[0034] IL-12 and GM-CSF can be expressed on the surface of tumor cells and are used to activate immune cells or regulate the tumor microenvironment.

[0035] Cancer treatment methods This invention relates to a method for expressing a fusion protein on the cell surface, the method comprising the following steps: obtaining a DNA sequence comprising: (a) a promoter sequence; (b) a 5' untranslated region sequence; (c) a sequence encoding an IL-12 / GM-CSF fusion protein; (d) a 3' untranslated region sequence; and (e) a polyadenylate tail sequence; transing the DNA sequence into mRNA in vitro using an RNA polymerase; encapsulating the mRNA in lipid nanoparticles (LNPs); transfecting cells with the encapsulated mRNA lipid nanoparticles; and translating the mRNA intracellularly to express a membrane-bound fusion protein on the cell surface. In one embodiment, the cells are cancer cells or blood cells, and the mRNA is transfected into the cancer cells or blood cells.

[0036] This invention relates to a method for treating cancer, comprising administering an mRNA-LNP encoding a fusion protein comprising IL-12 and GM-CSF to a subject suffering from cancer via intratumoral injection, wherein the cancer is selected from breast cancer, ovarian cancer, colon cancer, prostate cancer, sarcoma, melanoma, hepatocellular carcinoma, thyroid cancer, multiple myeloma, and other cancers.

[0037] The method includes the following steps: obtaining an mRNA sequence containing a coding sequence for an IL-12 and GM-CSF fusion protein; mixing the mRNA with lipid nanoparticles to form an mRNA-lipid nanoparticle complex; and injecting the mRNA-lipid nanoparticle complex into tumor tissue. In a preferred embodiment, the fusion protein further includes a transmembrane domain present at the C-terminus of both cytokines.

[0038] In the method of this invention, the IL-12 and GM-CSF fusion protein is expressed on the surface of cancer cells in a membrane-bound form. By injecting mRNA-LNP intratumorally and inducing the expression of the IL-12 and GM-CSF fusion protein on the surface of cancer cells, the toxicity of IL-12 and GM-CSF can be reduced.

[0039] In the method of this invention, mRNA is encapsulated in LNPs and transfected into the tumor. Nanoparticle-based drug delivery has many advantages, such as high bioavailability, high solubility, high stability, ability to cross the blood-brain barrier, low toxicity, and minimized side effects.

[0040] In one embodiment, the mRNA or its template DNA has the sequence described in this application.

[0041] This invention discloses a method for the safe and local intratumoral delivery of a membrane-bound IL-12 fusion cytokine with GM-CSF using mRNA-LNP technology. This invention incorporates cell therapy principles, activating T cells, monocytes, macrophages, and other immune cells surrounding the tumor through cytokine mRNA-LNP to kill the tumor. The expressed cytokine is membrane-bound, preventing it from circulating in vivo and contacting cells, thus avoiding toxicity. This method can also be used to stimulate the proliferation of NK cells or γδ T cells. After intratumoral delivery, immune cells expressing the IL-12 receptor can bind to the IL-12-linker peptide-GM-CSF and become activated, thereby causing tumor cell lysis. The lysed tumor releases neoantigens (natural vaccines), which can be recognized by antigen-presenting cells or dendritic cells in the presence of immunomodulators, promoting the activity of memory T cells and B cells.

[0042] The following embodiments further illustrate the present invention. These embodiments are for illustrative purposes only and should not be construed as limiting the present invention to these embodiments.

[0043] Example Example 1: Preparation of linearized DNA template for in vitro transcription Following the manufacturer's instructions, the DNA was digested overnight at 37°C with a suitable restriction endonuclease that cleaves the DNA immediately downstream of the 3' end of the polyadenylated (poly A) tail. The digested DNA was then treated with 50–100 μg / mL proteinase K and 0.5% sodium dodecyl sulfate (SDS) at 50°C for 30 minutes. Subsequently, the DNA was extracted with phenol / chloroform and precipitated with ethanol. The resulting DNA was used for in vitro RNA transcription.

[0044] Example 2: Preparation of RNA by in vitro transcription reaction 2.1 The in vitro transcription reaction was performed according to the following protocol: The DNA template used for RNA transcription contains the T7AG promoter upstream of the protein-coding sequence. The reaction system is as follows: The standard RNA synthesis protocol of the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080) was used, as follows: 1. Prepare the reaction solution (RNA without pseudouridine triphosphate) in the following order at room temperature:

[0045] If pseudouridine triphosphate (pseudo-UTP) is used for the reaction, the following scheme shall be adopted: Remove the required components and thaw them. The T7 RNA polymerase mixture should be placed on ice.

[0046] After mixing, centrifuge briefly using a microcentrifuge to concentrate the solution to the bottom of the tube.

[0047] Prepare the reaction system in the following order at room temperature:

[0048] 2.2 Purification of in vitro transcribed RNA RNA purification was performed using the NEB Monarch RNA Purification Kit (T2050), following the manufacturer's instructions.

[0049] 2.3 RNA yield determination Take an appropriate amount of RNA sample (usually diluted 1:50 to 1:100), dilute it with 1×TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and measure the absorbance at 260 nm using a spectrophotometer.

[0050] The formula for calculating RNA concentration (μg / mL) is as follows: Concentration = A260 × Dilution factor × 40 μg / mL Example 3: Preparation of LNP The components used to prepare the lipid nanoparticles are as follows: a mixture of SM-102, DSPC, cholesterol, and PEG2000 in a molar ratio of 50:10:38.5:1.5. SM-102 can be replaced by MC3 or other ionizable lipids.

[0051] All lipids are soluble in ethanol, and the stock solutions are as follows: SM-102 (Cayman): 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoic acid-1-octylnonyl ester; CAS No.: 2089251-47-6; Molecular weight: 710 (concentration 100 mg / mL) DSPC (Avanti): Distearylphosphatidylcholine; Molecular weight: 790.14 (concentration 10 mg / mL) Cholesterol (Sigma, 500mg) Molecular weight: 386.65 (concentration 25 mg / mL) DMG-PEG-2000 (Cayman): 1,2-Dimyristicoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000; Molecular weight: 2509.26 (concentration 18.82 mg / mL) Example 4: mRNA encapsulated in LNP 4.1 Precigenome Flex S Nanosystem Take 40 μL of the lipid mixture from Example 3 and mix it with 160 μL of mRNA solution (dissolved in 100 mM sodium acetate, pH 4.0, containing 32 μg mRNA).

[0052] Parameter settings: Flow rate ratio: 4:1 (aqueous phase: organic phase) Total flow rate: 3 mL / min N / P ratio: 11.0 The volume of the PBS diluent at the sample outlet should be equal to the total volume of lipids and mRNA loaded (200 μL in this example). 4.2 Precigenome Flex M System Organic phase: 625 μL lipid mixture Aqueous phase: mRNA solution dissolved in 100 mM sodium acetate (pH 4.0), total volume 1875 μl (Preparation method: take 500 μg mRNA, add sodium acetate solution, and adjust the volume to 1875 μl). Parameter settings: Flow rate ratio (aqueous phase: organic phase): 3:1 Total flow rate: 3 mL / min N / P ratio: 11.0 RNA-LNP medium change and filtration After preparation, the sample was replaced with PBS buffer using an Amicon® Ultra-15 centrifugal ultrafiltration tube (30 kDa–100 kDa). Approximately 14 mL of PBS was added to the ultrafiltration tube, followed by the sample. The tube was centrifuged at 1000×g for 20 minutes, and the eluent was discarded. This process was repeated once. The sample was collected from the top layer of the ultrafiltration tube and filtered through a membrane to obtain the LNP preparation dissolved in PBS.

[0053] Flex (M) and Flex (S) samples were diluted twice with PBS at their initial volume.

[0054] Nanoparticle size was determined using dynamic light scattering (DLS). RNA-LNP particle size prepared by the Flex (S) system: 90–105 nm RNA-LNP particle size prepared by the Flex (M) system: 75–100 nm Example 5: mRNA-LNP cell transfection Every 5×10 5 ~1×10 6 1–3 μg of encapsulated mRNA-LNP was added to each 293 cells. After transfection, the cells were cultured at 37°C and 5.0% CO2. After 2 days of culture, cell morphology was observed under a microscope and marker expression was detected by flow cytometry.

[0055] Example 6: DNA template sequence for preparing membrane-bound IL-12-linker peptide-GM-CSF mRNA A. Nucleic acid sequence The DNA template sequence used to prepare the membrane-bound IL-12-Flag tag-linker peptide-GM-CSF is as follows (the bolded portion is the coding region): the underlined 5' end indicates the T7 AG promoter; the 5' untranslated region is represented by lowercase letters; the IL-12-Flag tag-linker peptide-GM-CSF coding sequence is shown in bold; the 3' untranslated region is represented by lowercase letters; the poly A tail is represented by italicized uppercase letters. RNA was prepared by in vitro transcription using this template, and a type 1 cap structure (cap1) was added to the 5' end to improve RNA stability. The DNA template sequence (PMC2389) used to prepare IL-12 mRNA is as follows: T7 AG promoter - 5'UTR - IL-12 (p40-Flag tag - linker peptide - p35) - linker peptide - GM-CSF-PDGFRβ transmembrane region - 3'UTR - poly A tail:

[0056]

[0057] T7AG promoter: TAATACGACTCACTATAAG (SEQ ID NO: 2) 5'-UTR: GAGAAAGCTTacatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc (SEQ IDNO: 3) IL-12 (P40-Flag-linking peptide-P35)-linking peptide-GM-CSF-PDGFRβ transmembrane: (SEQ ID NO: 4) 3'UTR: GctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaGTCGACTCTAG (SEQ ID NO: 5) Poly A tail: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGATCCCCGGGCGAGCTCCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCGAATTCCTGCAGCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6) B. amino acid sequence Amino acid sequence of signal peptide-IL-12 (p40-Flag tag-linker peptide-p35)-linker peptide-GM-CSF-PDGFRβ transmembrane domain-intracellular segment:

[0058] <signal peptide> MCHQQLVISWFSLVFLASPLVA(SEQ ID NO: 8) <IL-12 P40 amino acid>

[0059] <aa> <Flag tag> DYKDDDDK (SEQ ID NO: 10) Linker peptide VPGVGVPGVGA (SEQ ID NO: 11) <IL-12 p35 amino acid sequence>

[0060] <linker peptide> VPGVGVPGVGA (SEQ ID NO: 11) <GM-CSF sequence> MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO: 13) <PDGFRβ transmembrane domain> AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 14) <intracellular segment> LEGSG (SEQ ID NO: 15) A Flag tag is used downstream of IL-12 p40 for the detection of IL-12. Other tags such as a transferrin tag can also be added after the IL-12 protein sequence and before the transmembrane domain for detecting the expression of the protein.

[0061] Example 7: Expression of membrane-bound IL-12-linker peptide-GM-CSF fusion protein after transfection of 293 cells with IL-12-linker peptide-GM-CSF mRNA-LNP IL-12-linker peptide-GM-CSF mRNA was prepared by in vitro transcription using DNA template PMC2389 (see Example 6) in the presence of CleanCap Reagent AG (NEB #E2080) and pseudouridine UTP, then encapsulated into LNP and delivered via transfection into 293, Daudi, Jurkat, Nalm-6 and Raji cell lines.

[0062] Seventy-two hours after transfection, flow cytometry was used to detect the expression of anti-GM-CSF antibody (Figure 3A) and anti-IL-12 antibody (Figure 3B). The results showed that the transfected cells were positive for both GM-CSF and IL-12, confirming the expression of these cytokines on the cell surface. The expression levels varied among different cell lines, with GM-CSF expression rates ranging from 15% to 95.7%. Figure 3A The expression rate of IL-12 was 22%–96.8% (Figure 3B).

[0063] To verify its effect on immune cell function, the effects of IL-12-linker peptide-GM-CSF mRNA-LNP transfection on T cells, monocytes, macrophages and dendritic cells were examined.

[0064] Transfecting IL-12-linker peptide-GM-CSF into CD3 / CD28 magnetic bead-activated T cells enhanced the Th1 effector phenotype of T cells, manifested as upregulated IFN-γ expression (Figure 4), with IFN-γ expression in transfected cells increasing from 26% to 53% (Y-axis). This result confirms the function of IL-12, namely, its known activation of Th1 subset T cells and promotion of IFN-γ secretion.

[0065] After transfection of monocytes with IL-12-linker peptide-GM-CSF mRNA-LNP, staining with IL-12 antibody showed that approximately 50% of the cells expressed IL-12, and staining with GM-CSF antibody showed that approximately 12% of the cells expressed GM-CSF.

[0066] Transfection of monocyte-derived macrophages with IL-12-linker peptide-GM-CSF mRNA-LNP improved macrophage survival and resulted in characteristic macrophage morphology (Figure 5). GM-CSF is known to promote macrophage survival. These results demonstrate that treatment with IL-12-linker peptide-GM-CSF mRNA-LNP has the same stimulatory effect as direct addition of GM-CSF to the culture medium.

[0067] Macrophages treated with IL-12-linker peptide-GM-CSF mRNA-LNP were larger, had more vacuoles, and were more adherent, with increased MHCII expression and decreased CD14 expression.

[0068] Treatment of monocytes with IL-12-linker peptide-GM-CSF mRNA-LNP in combination with IL-4 promoted dendritic cell differentiation, as confirmed by flow cytometry detection of CD1a antibody. Six days after transfection, monocytes lost the monocyte marker CD14, but the dendritic cell marker CD1a increased from 0.66% to 23% (Figure 6); the proportion of the monocyte marker CD14 decreased from 67% to 1.4% (circled area).

[0069] The above results indicate that IL-12-linker peptide-GM-CSF can alter the function of T cells, monocytes, macrophages, and dendritic cells, which is of great significance for anti-tumor therapy, as functional Th1 effector cells, monocytes, and dendritic cells play a key role in the tumor microenvironment.

[0070] Example 8: Local tumor-suppressing effect of IL-12-linking peptide-GM-CSF in mice 4×10⁴ cells were subcutaneously injected into the left and right flanks of NSG mice. 6 OVCAR-5 advanced ovarian cancer cells were treated. IL-12-linker peptide-GM-CSF mRNA-LNP (20 μL, 1 μg) was injected intratumorally into the tumor on days 7, 14, and 21, followed by intravenous injection of T cells on days 9 and 16. Results showed that IL-12-linker peptide-GM-CSF mRNA-LNP combined with T cells, and IL-12 mRNA-LNP-GM-CSF combined with EpCAM-CD3 mRNA-LNP, significantly inhibited tumor growth. Figure 7 ).

[0071] EpCAM-CD3 mRNA-LNP was prepared according to the sequence and method described in WO2024 / 148107.

[0072] Example 9: Antitumor effect of IL-12-linking peptide-GM-CSF on locally treated tumors and distal untreated tumors in mice. To verify the efficacy of IL-12-linker peptide-GM-CSF on locally treated sites and distal untreated tumors, 6 × 10⁶ cells were subcutaneously injected into the left side of mice. 6 Two PC3 prostate cancer cells were subcutaneously injected on the right side. 6 PC3 cells were used. EpCAM-CD3 mRNA-LNP, IL-12-linker peptide-GM-CSF mRNA-LNP, or EpCAM-CD3 mRNA-LNP + IL-12-linker peptide-GM-CSF mRNA-LNP were injected intratumorally into the left tumor on days 14, 21, and 28, respectively; T cells were injected intravenously on days 15, 22, and 29. GFP mRNA-LNP was used as a negative control.

[0073] The results (Figures 8A and 8B) show that the combination of IL-12-linker peptide-GM-CSF mRNA-LNP and EpCAM-CD3 mRNA-LNP can not only clear local tumors but also inhibit distant untreated tumors, which is of great significance for the treatment of metastatic tumors.

[0074] References 1. Z. Zhu, Q. Peng, X. Duan and J. Li: Interleukin-12: Structure, Function, and Its Impact in Colorectal Cancer.J Interferon Cytokine Res, 44(4), 158-169 (2024) doi:10.1089 / jir.2023.0190 2. KA Ullrich, LL Schulze, EM Paap, TM Muller, MFNeurath and S. Zundler: Immunology of IL-12: An update on functional activities and implications for disease. EXCLI J, 19, 1563-1589 (2020) doi:10.17179 / excli2020-3104 3. MP Colombo and G. Trinchieri: Interleukin-12 in anti-tumorimmunity and immunotherapy. Cytokine Growth Factor Rev, 13(2), 155-68 (2002)doi:10.1016 / s1359-6101(01)00032-6 4. K. He, X. Liu, R. D. Hoffman, R. Z. Shi, G. Y. Lv and J. L. Gao:G-CSF / GM-CSF-induced hematopoietic dysregulation in the progression of solidtumors.FEBS Open Bio, 12(7), 1268-1285 (2022) doi:10.1002 / 2211-5463.13445 5. H. M. Lazarus, K. Pitts, T. Wang, E. Lee, E. Buchbinder, M.Dougan, D. G. Armstrong, R. Paine, 3rd, C. E. Ragsdale, T. Boyd, E. P. Rockand R. P. Gale: Recombinant GM-CSF for diseases of GM-CSF insufficiency:Correcting dysfunctional mononuclear phagocyte disorders.Front Immunol, 13,1069444 (2022) doi:10.3389 / fimmu.2022.1069444< / aa>

Claims

1. A fusion protein comprising IL-12 and GM-CSF, wherein the IL-12 and GM-CSF are covalently linked.

2. The fusion protein of claim 1 further comprises a transmembrane domain located at the C-terminus of IL-12 and GM-CSF.

3. The fusion protein of claim 1, wherein the transmembrane domain is selected from: platelet-derived growth factor receptor β (PDGFRβ), T cell receptor α or β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and transmembrane domains of GITR, etc.

4. The fusion protein of claim 3, wherein the transmembrane domain is the transmembrane domain of platelet-derived growth factor receptor β.

5. The fusion protein according to any one of claims 1 to 4, wherein the IL-12 comprises the amino acid sequence shown in SEQ ID NOs:9 and 12, and the GM-CSF comprises the amino acid sequence shown in SEQ ID NO:

13.

6. The fusion protein of claim 5, having the amino acid sequence shown in SEQ ID NO:

7.

7. A lipid nanoparticle (LNP) encapsulating mRNA, wherein the mRNA is transcribed from a DNA sequence comprising the following elements: (a) Promoter sequence; (b) 5'-Untranslated Region (UTR) sequence; (c) Encoding the sequence of the fusion protein according to any one of claims 1 to 5; (d) 3'-UTR sequence; and (e) Polyadenylate (poly A) tail sequence.

8. The lipid nanoparticles of claim 7, wherein the lipid nanoparticles comprise: (a) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate 1-octylnonyl ester (SM-102), distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), or; (b) SM-102, DSPC, cholesterol, and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000-MAL), or; (c) 2-Hexyldecanoic acid 1,1'-[[(4-hydroxybutyl)imino]bis(6,1-hexadiyl)] ester (ALC-0315), DSPC, cholesterol, and α-[2-(tetracosylamino)-2-oxoethyl]-ω-methoxy-poly(oxy-1,2-ethylidene) (ALC-0159).

9. A method for expressing a fusion protein on the cell surface, comprising the following steps: Obtain a DNA sequence containing the following elements: (a) promoter sequence; (b) 5'-UTR sequence; (c) sequence encoding the fusion protein according to any one of claims 1 to 6; (d) 3'-UTR sequence; (e) poly A tail sequence; The DNA sequence was transcribed into mRNA in vitro using RNA polymerase; The mRNA was encapsulated in lipid nanoparticles; Transfect cells with lipid nanoparticles encapsulating mRNA; as well as The mRNA is translated within the cell, thereby expressing a membrane-bound fusion protein on the cell surface.

10. The method of claim 9, wherein the cell is a cancer cell or a blood cell, and the mRNA is transfected into the cancer cell or blood cell via LNP.

11. A method for treating cancer, comprising the following steps: The lipid nanoparticles encapsulating mRNA as described in claim 7 are injected into the tumor lesions of a subject with cancer.

12. The method of claim 11, which is capable of activating T cells, NK cells, monocytes, macrophages and dendritic cells in the tumor microenvironment.

13. The method of claim 11 or 12, wherein the cancer is colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, melanoma, sarcoma, or lung cancer.

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