Fusion protein comprising il-12 and GM-csf

Abstract

The present invention is directed a fusion protein comprising IL-12 and GM-CSF, which are covalently attached. The fusion protein may further comprise a transmembrane domain C-terminal to IL-12 and GM-CSF. The present invention is also directed to mRNA-LNP delivery of the fusion protein. The present invention is also directed to a method for producing membranous IL-12 and GM-CSF, and transmembrane domain in cells. The method comprises the steps of transcribing mRNA from DNA sequence encoding the fusion protein with RNA polymerase, encapsulating the mRNA in lipid nanoparticles (LNPs), transfecting the mRNA-encapsulated LNPs into cells, and translating the mRNA in the cells to produce membrane bound IL-12 fused to GM-CSF cytokine protein to stimulate immune cells and tumor microenvironment.

Description

[0001] FUSION PROTEIN COMPRISING IL-12 AND GM-CSF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a fusion protein comprising IL-12 and GM-CSF, which are covalently attached to each other. The present invention also relates to generating mRNA-LNPs (lipid nanoparticles) and delivering them into cells to generate cells expressing membrane-bound IL- 12 and GM-CSF fusion protein. This membrane bound I1-12-GM-CSF protein can be used for inducing immune response and activating tumor microenvironment in vivo.

[0004] BACKGROUND OF THE INVENTION 11-12

[0005] Interleukin- 12 is a heterodimer protein which was described in 1989 and consists of two subunits: p40 and p35 (1). L-12 regulates immune system response and is important for adaptive immunity (1). In response to pathogens, the heterodimeric protein is secreted by phagocytic cells (2). Binding of IL-12 to the IL-12 receptor (IL-12R) on T cells and natural killer (NK) cells leads to activator of transcription 4 (STAT4) and stimulates secretion of interferon gamma (IFN-y) (2). Signaling downstream of IFN-y includes activation of T-box transcription factor TBX21 (Tbet) and induces pro-inflammatory functions of T helper 1 (Tul) cells, linking innate and adaptive immune responses(2). II- 12 is also known to inhibit immunosuppressive tumor associated macrophages and MDSC (myeloid-derived suppressor cells. 11-12 showed anti-tumor activity in many pre- clinical studies (3). In spite of the anti-tumor effects of IL-12, systemic administration of IL- 12 has been too toxic. The ideal targets of IL- 12 immunotherapy are immune cells within the tumor and nearby lymph nodes, including activated but exhausted T cells, NK cells, TAMs, and MDSCs.

[0006] GM-CSF

[0007] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a well-known player of myelopoiesis and mobilization of hematopoietic stem cells (4). GM-CSF can induce the differentiation of HPCs into different immune cells, including granulocytes, monocytemacrophages, T-cells, and natural killer (NK) cells in the bone marrow (4). GM-CSF was identified in the 1960s as a myeloid growth factor, purified in the 1970s, molecularly-cloned in the 1980s, and clinically developed in the 1990s (5). Sargramostim (Leukine®; Partner Therapeutics, Inc., Lexington, MA) is a glycosylated, recombinant human granulocytemacrophage colony-stimulating factor (rhu GM-CSF), FDA-approved for six different diseases based on its safe and efficacious hematopoietic growth factor function, differing from human GM-CSF by one amino acid at position 23, where leucine is substituted for arginine(5). In addition to myelopoietic actions, GM-CSF possesses anti-apoptotic effects and is reported to induce 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 added GM-CSF to IL- 12 membrane bound protein to express as a single protein with linker in between to induce immune cells.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows the scheme of DNA vector template (panel A) used for in vitro transcription to mRNA of IL-12-linker-GM-CSF-TM (panel B). The mRNA is then used to generate translated protein that is expressed on the cell surface. TM; transmembrane domain; 5’-UTR: 5’ untranslated region; 3’-UTR: 3 ’untranslated region; poly A tail for increased stability.

[0010] FIG. 2 shows a structure of membrane-bound IL- 12 linked to GM-CSF. L: linker; TM: transmembrane domain; C: a cytoplasmic tail. IL-12 consists of IL-12 p40 domain- linker-IL12 p45 domain.

[0011] FIGs. 3A and 3B show protein expression 72 hours after delivery of IL-12-1-GM-CSF mRNA-LNP into different cell lines using mRNA-LNP. 3A. Expression of GM-CSF; 3B. Expression of IL-12. FACS was done with mouse anti-GM-CSF or anti-IL-12 antibody, and then secondary APC-conjugated anti-mouse IgG antibody was used at 1:100 dilution.

[0012] FIG. 4 shows transfection of IL-12-GM-CSF LNPs to primary T cells resulted in increased expression of IFN-gamma. T cells were used 3 days after activation with CD3 / CD28 antibodies and 2 days post- transfection with mRNA-LNP. The cells were stained for intracellular IFN-g (Thl marker). The cells were treated with Brefeldin A for 2.5 hours before FACS to accumulate cytokines within the cells. CD3 staining is shown on X-axis, IFN-gamma expression is shown on Y-axis.

[0013] FIG. 5 shows that transfection of IL-12-GM-CSF to macrophages increased survival and morphology of macrophages. Morphology of cells is shown under X400 magnification. FIG. 6 shows IL-12-GM-CSF mRNA-LNP transfection of monocytes with addition of IL-4 increases dendritic cell phenotype by expression of CD la dendritic marker and decreasing expression of monocyte marker CD 14.

[0014] FIG. 7 shows that IL-12-GM-CSF mRNA decreased OVCAR-5 xenograft tumor growth in vivo. *p<0.05 (i) IL-12-GM-CSF mRNA-LNP, (ii) IL-12-GM-CSF+EpCAM-CD3 mRNA-LNP, versus GFP mRNA as a negative control.

[0015] FIGs. 8A and 8B show the efficacy of IL-12-GM-CSF in tumor in mice at a local treated side (8A), and at a distant non-treated site (8B). PC3 tumor cells were injected subcutaneously into left and right side. mRNA-LNP were injected intratumorally only to the left tumors. Tumor size was measured at left and right tumors with calipers. *p<0.05 EpCAM-CD3+IL-12-GM-CSF vs GFP-LNP control, Student’s t-test.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is directed to a fusion protein comprising IL-12 and GM-CSF, which are covalently attached. In one embodiment, IL-12 is N-terminal to GM-CSF. In another embodiment, IL- 12 is C-terminal to GM-CSF. In a preferred embodiment, IL- 12 and GM-CSF are attached through a linker. Preferably, the linker has 5-30 or 5-20 amino acids in length.

[0018] In one embodiment, the fusion protein further comprises a transmembrane domain C- terminal to IL- 12 and GM-CSF. In another embodiment, the fusion protein also comprises a cytoplasmic tail at the C-terminal end of the fusion protein.

[0019] In general, mRNA is transient and short-lived when delivered in vivo. The present invention provides a method for producing IL-12-linker-GM-CSF protein attached to membrane by delivering lipid nanoparticle-encapsulated mRNA in cells. By encapsulating mRNA in lipid nanoparticles (LNPs), the stability of mRNA is improved.

[0020] The anti-tumor delivery of membrane-bound IL-12 and GM-CSF provides better tolerated IL-12 with higher efficacy. In this application, we designed construct using mRNA-LNP technology with membrane-bound IL-12 and GM-CSF to decrease potential toxicity compared with secreted IL-12 and GM-CSF.

[0021] In the present fusion protein, the transmembrane domain is selected from the group consisting of: a transmembrane domain of PGFRB receptor (platelet-derived growth factor receptor beta), a T cell receptor a or P chain, a CD3 zeta chain, CD28, CD3s., CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD 154, and GITR.

[0022] The present invention is also directed to lipid nanoparticles have mRNA encapsulated, wherein the mRNA is transcribed from a DNA sequence comprising: (a) a promoter coding sequence, e.g., a T7 promoter coding sequence, (b) 5'-UTR (untranslated region) coding sequence, (c) a sequence encoding the present fusion protein as described above, (d) a 3'- UTR coding sequence, and (e) a poly A tail sequence.

[0023] The present invention is further directed to a method for producing an IL-12-linker- GM-CSF protein expressed on the surface of cells. The method comprises the steps of: (i) obtaining a DNA sequence comprising: (a) a promoter coding sequence, (b) 5'-UTR coding sequence, (c) a sequence to encode the fusion protein of the present invention, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence, (ii) transcribing the DNA sequence to mRNA with RNA polymerase in vitro, (iii) encapsulating the mRNA in lipid nanoparticles (LNPs), (iv) transfecting the mRNA-encapsulated LNPs into cells, and (v) translating the mRNA in the cells to produce cytokines of IL-12 and GM-CSF which are expressed on the surface of the cells.

[0024] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino] -octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol-2000 (DMG-PEG2000). [LNP-102 (ii)]

[0025] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl|amino]-octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-MAL). [LNP-102 (i)]

[0026] In one embodiment, the lipid nanoparticles comprise 2-hexyl-decanoic acid, 1, l'-[[(4- hydroxybutyl)imino]di-6,l -hexanediyl] ester (ALC-0315), DSPC, Cholesterol, and a-[2- (ditetradecylamino)-2-oxoethyl]-co-methoxy-poly(oxy-l,2-ethanediyl) (ALC-0159). [LNP- 315]

[0027] In one embodiment, the lipid nanoparticles comprise 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino] -octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000). LNP-102 is formulated at molar ratio of SM-102, DSPC, Cholesterol, and PEG2000 (50:10:38.5: 1.5 mol%). Incorporation of mRNA into LNP nanoparticles provides protection of mRNA from degradation and increases the stability mRNA; mRNA is then released from LNPs into cells in vivo to generate protein. mRNA-lipid nanoparticle preparation is described in Schoenmaker (International J. Pharmaceutics, 601: 120856, 2021), the article is incorporated herein by reference in its entirety, regarding the LNPs.

[0028] FIG. 1 shows linearized DNA template to be used for in vitro transcription with RNA polymerase and nucleotide triphosphate to generate mRNA. The DNA template contains a promoter such as T7 promoter or SP6 promoter, then 5’UTR (untranslated region), the coding region of IL-12-linker-GM-CSF- transmembrane (TM) domain, then 3’UTR and poly A tail (for example, about 152 nucleotides) for RNA stability. The generated RNA may contain 5’ cap ([m7G(5’)ppp(5')G], capl for increased stability. The mRNA can be electroporated or transfected to cells. For in vivo use, mRNAs are preferably to be added to lipid nanoparticles, LNP-nanoparticles can be prepared according to the formula above.

[0029] In the DNA sequence, the promoter may be T7 AG promoter. Poly A tail sequence is from 20-170 nucleotides. Poly A tail sequence optionally comprises one or more linkers in between the poly A segments. If poly A tail is longer than 60 nucleotides, then it typically contains a linker which includes non-adenosine nucleotides. A linker is 5-30 or 5-25 nucleotides, e.g., 10 nucleotides or 20 nucleotides. In one example, poly A tails is 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10- nucleotide linker sequence, and another 70 adenosine residues. In another example, poly A tails is 90 nucleotides in length, consisting of a stretch of 40 adenosine residues, followed by a 30-nucleotide linker sequence, and another 30 adenosine residues. Poly A tail can be up to 150-160 nucleotides in length, interrupted with the linker sequences.

[0030] DNA expression is finely regulated at the post-transcriptional level. Untranslated regions are not translated into amino acids, however, 5’ and 3’ UTRs of mRNAs may control their regulation, degradation and localization include stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites and various cis-acting elements that are bound by RNA-binding proteins. UTRs are important in the post- transcriptional regulation of RNA expression, including modulation of the transport of mRNAs out of the nucleus and of translation efficiency, subcellular localization, and stability.

[0031] 5’-UTR typically has 10-1000 nucleotides, or 20-500 nucleotides, or 30-200 nucleotides, or 30-100 nucleotides. For example, 5’-UTR is 50 nucleotides. 3’-UTR typically has 10-3000 nucleotides, for example, 50-500 nucleotides, or 100-300 nucleotides. Preferred 5’-UTRs and 3’-UTRs are UTRs of P-globin, or UTRs of Pfizer CO VID vaccine.

[0032] [3-Globin gene is shown in: https: / / www.ncbi.nlm.nih.gov / nucleotide / V00497. l?report=genbank&log$=nuclalign&blast rank=5&RID=TDDZlK98016

[0033] In Pfizer CO VID vaccine, the 5 '-untranslated region is derived from human alphaglobin RNA with an optimized Kozak sequence. The 3 ' untranslated region comprises two sequence elements derived from the amino-terminal enhancer of split (AES) mRNA and the mitochondrial encoded 12S ribosomal RNA to confer RNA stability and high total protein expression.

[0034] Any suitable vector, such as Vector pSP64 Poly(A) (Promega) or pGEM3Z-Vektor (Promega) can be used as a cloning vector for the DNA sequence described above.

[0035] For example, to engineer the pEM3Z- -globin UTR-UTR-poly A tail, the 3’-UTR of the P-globin molecule flanked by restriction enzyme site can be amplified from human bone marrow. For example, a single (pEM3Z-ip-globin-UTR-A

[0120] ) or 2 serial fragments (pEM3Z-2p-globin-UTR-A

[0120] ) can be inserted in front of the poly(A) tail.

[0036] In the DNA sequence, it preferably contains a transmembrane domain-coding sequence, which encodes the transmembrane domain that may be derived from a natural polypeptide, or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a transmembrane domain of a T cell receptor a or P chain, PGFRB receptor (platelet-derived growth factor receptor beta), a CD3 zeta chain, CD28, CD3e., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD 154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or a polypeptide linker, for example, a linker having a length of 2 to 10 amino acids can be arranged between the transmembrane domain and the intracellular domain.

[0037] In the DNA sequence, it may contain a cytoplasmic tail-coding sequence, which encodes a short cytoplasmic tail.

[0038] In this application, we connect IL- 12 cytokine sequence with a signaling peptide, a linker, GM-CSF, a transmembrane domain, and a small cytoplasmic tail to express IL- 12 and GM-CSF on the cell surface or tethered (membrane-bound). In one embodiment, the structure of 11-12 linked to GM-CSF with transmembrane domain and small cytoplasmic tail is shown in FIG. 2.

[0039] In one embodiment, we connect the IL- 12 with a signaling peptide, a linker, GM- CSF, transmembrane peptide from PDGFR to express 11-12 and GM-CSF linked proteins on the cell surface. This approach can be used to make other cytokines on the cell surface. The linker may be VPGVGVPGVGA or (GGGGS)n, where n= 1, 2, 3, 4, or 5; for example, n=3.

[0040] We generated mRNA of IL- 12 and GM-CSF that are membrane-bound. The advantage of IL-12 and GM-CSF to be expressed on the surface of cells is that proteins are more stable, less toxic and can activate T cells and other immune cells.

[0041] 11-12 and GM-CSF can be expressed on tumor cells to activate immune cells or tumor microenvironment.

[0042] Method for treating cancer

[0043] The present invention is directed to a method for producing a fusion protein on the surface of cells. The method comprises the steps of: obtaining a DNA sequence comprising: (a) a promoter coding sequence, (b) 5'-UTR coding sequence, (c) a sequence encoding the fusion protein of IL-12 and GM-CSF, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence; transcribing the DNA sequence to mRNA with RNA polymerase in vitro, encapsulating the mRNA in lipid nanoparticles (LNPs), transfecting the mRNA-encapsulated LNPs into cells, and translating the mRNA in the cells to produce membrane-bound fusion protein on the surface of the cells. In one embodiment, the cells are cancer cells or blood cells, and the mRNA is transfected into the cancer cells or blood cells.

[0044] The present invention is directed to a method for treating cancer, comprising the step of administering a fusion protein comprising IL- 12 and GM-CSF to a subject suffering from cancer by intratumor injection, wherein the cancer is selected from the group consisting of breast, ovarian, colon cancer, prostate cancer, sarcoma, melanoma, hepatocellular carcinoma, thyroid, multiple myeloma and other cancers.

[0045] The method comprises the steps of obtaining an mRNA sequence comprising a coding sequence to encode a fusion protein comprising IL- 12 and GM-CSF; mixing the mRNA with lipid nanoparticles to form a mRNA-lipid nanoparticle complex; and injecting the mRNA- lipid nanoparticle complex into the cancer cells. In one preferred embodiment, the fusion protein further comprises a transmembrane domain C-terminal to the two cytokines.

[0046] In the present method, IL- 12 and GM-CSF are expressed on the surface of the cancer cells and are membrane bound. By using intratumoral injection of LNP-mRNA and expressing IL- 12 and GM-CSF on the surface of the cancer cells, the toxicity of IL- 12 and GM-CSF are reduced.

[0047] In the present method, mRNA is embedded into LNPs and transfected inside tumors. The nanoparticle-based drug delivery has many advantages, such as high bioavailability, solubility, stability, passage through the blood-brain barrier, and low toxicity and minimal side effects.

[0048] In one embodiment, the mRNA or its template D A has a sequence as described in this application.

[0049] The present disclosure describes a safe local intratumoral delivery using mRNA-LNP technology to deliver IL- 12 and GM-CSF cytokine which are membrane tethered. The present disclosure combines a cell therapy approach by using cytokine RNA-LNP cells to activate T cells and monocytes, macrophages, and other immune cells surrounding tumors to kill tumors. The expressed cytokine is membrane tethered, which prevents it from circulating inside cells and causing toxicity. This approach can be used similarly to stimulate expansion of NK cells or gamma-delta T cells. The immune cells that express IL- 12 receptors will bind to IL-12-GM-CSF, and in intratumoral delivery, tumor cells will be lysed. The lysed tumor releases neoantigens (natural vaccine), which can be recognized in the presence of immunomodulators by antigen-presenting cells or dendritic cells and promote the activity of memory T and B cells.

[0050] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

[0051] EXAMPLES

[0052] Example 1. Preparation of linearized DNA template for in vitro transcription.

[0053] DNA was digested with appropriate restriction enzyme, which cuts DNA right 3’ after poly A tail, at 37°C overnight following manufacturer’s protocol. The digested DNA was treated with 50-100 pg / mL Proteinase K and 0.5% SDS for 30 minutes at 50°C. Then phenol / chloroform extraction and ethanol precipitation of DNA was performed. The DNA was used for in vitro RNA transcription reaction.

[0054] Example 2. Preparation of RNA by in vitro transcription reaction.

[0055] 2.1. The in vitro transcription reaction was done by the protocol below: The DNA template for generating RNA had T7AG promoter in front of the coding sequence of protein. The reaction was the following:

[0056] Standard RNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080) was used as described below: 1. Set up the following reaction at room temperature in the following order for RNA without pseudo-UTP:

[0057] 20 pl Final cone, or

[0058] Components reaction amount

[0059] Nuclease-free water X pl

[0060] 10X T7 CleanCap Reagent AG Reaction

[0061] 2 pl

[0062] Buffer

[0063] ATP (60 mM) 2 pl 6 mM final

[0064] UTP (50 mM) 2 pl 5 mM final

[0065] CTP (50 mM) 2 pl 5 mM final

[0066] GTP (50 mM) 2 pl 5 mM final

[0067] Cap Analog (40 mM) 2 pl 4 mM final

[0068] Template DNA X pl 1 pg

[0069] T7 RNA Polymerase Mix 2 pl

[0070] 2. Gently mix the reaction by pipetting up and down and microfuge briefly briefly. Incubate at 37°C for 2 hours.

[0071] 3. Bring the reaction volume up to 50 pl with nuclease-free water. Add 2 pl of DNase I, mix well and incubate at 37°C for 15 minutes.

[0072] 4. Proceed with mRNA purification

[0073] For reaction with pseudo-UTP we use the following protocol:

[0074] Thaw the necessary components, keep the T7 RNA Polymerase Mix on ice.

[0075] Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes. Set up the reaction at room temperature in the following order: Final

[0076] Volume concentration

[0077] Nuclease-free Water

[0078] 10X Reaction Buffer 2 0.5x

[0079] 100 mM ATP 2 5 mM final

[0080] 100 mM GTP 2 5 mM final

[0081] 100 mM methyl-Pseudo-UTP

[0082] (N-1081) 2 5 mM final

[0083] 100 mM CTP 2 5 mM final

[0084] 100 mM CleanCapAG (3’ OMe

[0085] N-7413) 1.6 4 mM final

[0086] Linear Template DNA 1 pg total

[0087] T7 RNA Polymerase Mix 4

[0088] Total 40 pL

[0089] Gently mix the reaction by pipetting up and down and microfuge briefly. Incubate at 37°C for 2 hours.

[0090] Optional: The reaction volume can be up to 50 pl with nuclease-free water. Add 2 pl of DNase I, mix well and incubate at 37°C for 15 minutes.

[0091] 2.2. Cleaning in vitro transcribed RNA

[0092] For cleaning RNA, we used NEB The Monarch RNA Cleanup Kit (T2050) according to the manufacturer’ s protocol.

[0093] 2.3. Assessing RNA yield

[0094] The concentration of RNA can be determined by diluting an aliquot of the preparation (usually a 1:50 to 1: 100 dilution) in IxTE (10 mM Tris-HCl 1 mM EDTA, pH 8) buffer, and reading the absorbance in a spectrophotometer at 260 nm. The concentration (pg / mL) of RNA is therefore calculated as follows: A260 x dilution factor x 40 pg / mL.

[0095] Example 3. Preparation of LNP

[0096] The following components were used to prepare LNP: SM-102, DSPC, Cholesterol, and PEG2000 (50:10:38.5: 1 .5 mol%) mix. Instead of SM-102 ionizing lipid, MC3 or other ionizing lipid can be used.

[0097] Nanoparticles were prepared using the below stocks dissolved in Ethanol: SM-102 (Cayman) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1- octylnonyl ester;

[0098] CAS numbr: 2089251-47-6

[0099] MW:710 (lOOmg / ml)

[0100] DSPC (Avanti)

[0101] Distearoylphosphatidylcholine

[0102] MW:790.14 (lOmg / ml)

[0103] Cholesterol (Sigma, 500mg)

[0104] MW:386.65 (25mg / ml)

[0105] DMG-PEG-2000 (Cayman) l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000

[0106] MW:2509.26, (18.82mg / ml)

[0107] Example 4. mRNA encapsulation into LNP

[0108] 4.1 PreciGenome Flex S Nanosystem

[0109] Use 40 pl of Lipid Mix of Example 3 with 160ul of mRNA in lOOmM sodium acetate (pH:4.0) (mix 32 pg of mRNA with sodium acetate solution to bring final volume to 160 pl)

[0110] Settings:

[0111] -Flow rate ratio: 4:1 (Aqueous: organic)

[0112] -Total flow rate: 3ml / min

[0113] -N / P ratio: 11.0

[0114] -Outlet PBS buffer dilution volume equal to total input volume (200 pl for the above example)

[0115] 4.2. Precigenome Flex M System:

[0116] (organic solution): 625 pl of Lipid Mix

[0117] (Aqueous Solution): 1875 pl of mRNA in lOOmM sodium acetate (pH:4.0) (mix 500 pg of mRNA with sodium acetate solution to bring final volume to 1875 pl)

[0118] Settings: -Flow rate ratio: 3:1 (Aqueous :organic) -Total flow rate: 3ml / min -N / P ratio: 11.0

[0119] Buffer exchange and filtration of RNA-LNP

[0120] After formulation is complete, collected samples are filtered and buffer exchange into PBS is performed using Amicon® Ultra-15 Centrifugal Filter Units (30kDA-100kDA). Fill the filter unit with ~14 ml PBS and directly add your sample to the filtration unit. Spin at 1000g for 20 minutes. Remove the flow-through solution, repeat it one more time. Collect the sample from the upper compartment of the filter unit. This collected sample is nanoparticles in PBS.

[0121] Flex(M) and Flex(S) samples are prepared 2-fold diluted in PBS from their initial formulation volume.

[0122] The size of Nanoparticles was confirmed using Dynamic Light Scattering (DLS) system. The size of RNA-LNP nanoparticles was in the range of 90-105 nm for Flex S system and 75-100 nm for FlexM system.

[0123] Example 5. mRNA-LNP delivery to cells

[0124] 1-3 pg of encapsulated mRNA was added per 5xl05-IxlO6293 cells. After transfection is completed, the cells were maintained at 37°C, 5.0% CO2. After 2 days their morphology and markers were checked by microscope and FACS.

[0125] Example 6: Sequence of DNA template to generate tethered (membranous) IL-12- linker-GM-CSF m-RNA

[0126] A. Nucleic acid Sequences

[0127] The DNA sequence of template for membranous IL-12-Flag tag-linker-GM-CSF is shown in bold below:

[0128] T7 AG promoter is underlined at 5’ ; 5’UTR is shown in small font, the IL-12-Flag tag-linker-GM-CSF coding sequence is shown in bold font; 3’UTR in small font, and poly A tail is shown in italics, capital letters. The RNA is prepared by in vitro transcription using this template with 5’ capl for higher stability of RNA. The sequence of DNA template to generate IL-12 mRNA is shown below (PMC2389). T7 AG promoter-5 ’UTR- IL- 12 (P40-Flag tag-linker-P35)-linker-GM-CSF-PDGFR beta

[0129] TM- 3’UTR-polyA tail:

[0130] TAATACGACTCACTATAAGGAGAAAGCTTacatttgcttctgacacaactgtgttcactagcaacctcaaac agacaccATGTGTCATCAGCAGCTCGTTATATCATGGTTTAGTCTTGTTTTTTTG

[0131] GCGAGTCCGCTCGTAGCAATTTGGGAACTCAAGAAGGACGTATATGTAGTG

[0132] GAGCTTGACTGGTATCCAGACGCCCCTGGTGAAATGGTGGTCTTGACGTGT

[0133] GACACGCCCGAAGAGGATGGCATCACATGGACGCTCGACCAAAGCTCAGAA

[0134] GTATTGGGGAGCGGCAAGACGCTCACTATTCAAGTAAAAGAGTTTGGAGAT

[0135] GCAGGGCAATACACGTGCCACAAAGGAGGAGAGGTTCTTAGCCATAGTCTG

[0136] CTCCTGCTGCACAAGAAGGAGGACGGGATCTGGTCCACTGATATCCTGAAA

[0137] GACCAGAAAGAACCCAAGAACAAAACTTTTCTTCGATGTGAAGCGAAAAATT

[0138] ATTCAGGCCGGTTCACATGCTGGTGGCTTACTACAATATCTACTGACCTGAC

[0139] GTTCAGCGTCAAGTCTAGTCGAGGGTCTTCTGATCCGCAAGGTGTCACGTG

[0140] CGGGGCCGCCACTCTGTCTGCAGAACGGGTTAGGGGGGACAACAAGGAATA

[0141] TGAATATTCCGTGGAGTGCCAAGAAGATAGCGCTTGCCCTGCGGCGGAAGA

[0142] GAGCCTCCCCATCGAGGTTATGGTtGACGCCGTTCATAAACTCAAGTACGAA

[0143] AACTATACTTCCTCATTTTTTATAAGGGACATCATAAAGCCGGACCCACCTA

[0144] AGAATTTGCAATTGAAGCCTTTGAAGAATAGTgGACAAGTTGAGGTGTCCTG

[0145] GGAGTACCCGGACACTTGGAGCACTCCCCATTCATATTTCTCCCTGACTTTC

[0146] TGCGTTCAGGTTCAGGGCAAATCTAAACGAGAGAAAAAAGACCGCGTATTC

[0147] ACGGACAAGACTTCTGCCACAGTAATCTGTCGAAAGAATGCTTCAATCTCCG

[0148] TGAGGGCCCAAGACAGATACTATTCATCTAGCTGGTCCGAATGGGCCAGTG

[0149] TTCCGTGCTCAgccgcagactacaaagacgatgacgacaagGTTCCTGGAGTAGGGGTACCT

[0150] GGGGTGGGCgccAGAAACTTGCCAGTTGCGACGCCTGATCCCGGCATGTTTC

[0151] CTTGCTTGCACCATTCACAGAATCTGCTTCGAGCTGTCTCCAATATGCTGCA

[0152] AAAAGCCAGACAAACCCTGGAGTTCTACCCATGTACTAGCGAGGAGATAGA

[0153] TCACGAGGATATCACGAAGGATAAAACTTCAACAGTCGAAGCTTGTCTTCCC

[0154] CTGGAGTTGACTAAGAATGAGTCCTGTCTCAACTCTCGCGAAACATCTTTCA

[0155] TCACAAATGGTAGCTGTCTCGCGTCTCGGAAAACGTCTTTCATGATGGCGCT

[0156] GTGCCTGTCCTCCATATACGAAGACTTGAAAATGTATCAGGTGGAATTCAAG

[0157] ACCATGAACGCCAAGCTCTTGATGGACCCAAAGAGACAGATCTTTCTTGACC

[0158] AGAACATGCTTGCAGTGATAGATGAGTTGATGCAAGCTTTGAACTTCAACTC

[0159] AGAAACAGTGCCGCAAAAATCATCACTTGAGGAGCCTGATTTTTACAAAACG

[0160] AAAATCAAGTTGTGCATTCTGTTGCATGCTTTCCGCATCAGGGCAGTAACTA TCGATAGGGTGATGTCCTACCTTAACGCTAGTGTTCCTGGAGTAGGGGTACC TGGGGTGGGCgccatgTGGCTTCAGTCCCTGTTGCTCCTCGGAACGGTCGCAT GTTCAATAAGCGCACCTGCGAGAAGTCCGAGTCCTAGCACGCAACCCTGGG AACACGTCAACGCTATACAGGAGGCGCGACGGCTGCTCAATCTGAGCAGGG ACACGGCGGCTGAAATGAACGAGACAGTGGAAGTTATCAGCGAAATGTTTG ATTTGCAGGAGCCTACGTGCCTTCAAACAAGGCTTGAACTGTATAAGCAAGG GCTTAGGGGTAGTCTTACGAAGCTTAAAGGACCATTGACGATGATGGCTAG TCACTATAAGCAACACTGTCCACCGACGCCCGAGACGAGTTGCGCTACTCA GATAATCACGTTTGAAAGCTTTAAAGAGAATCTCAAGGATTTTCTGCTGGTC ATTCCTTTCGATTGTTGGGAGCCCGTGCAAGAGgctgtgggccaggacacgcaggaggtcat cgtggtgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcat cctcatcatgctttggcagaagaagccacgtctcgagggcagcggaTAAGctcgctttcttgctgtccaattctataaaggttc ctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattg cagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagc atctggattctgcctaataaaaaacatttattttcattgcaGTCGACTCTAGAA4AA4AAAAAA4AAA4A4AA4A AAAAAAAAAAAAAAAAAAGGATCCCCGGGCGAGCTCCCAAAAAAAAAAAAAAAAAAAAA AAAAAAAAACCGAATTCCTGCAGCTCGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ ID NO: 1)

[0161] T7AG promoter:

[0162] TAATACGACTCACTATAAG_(SEQ ID NO: 2)

[0163] 5’-UTR:

[0164] GAGAAAGCTTacatttgcttctgacacaactgtgttcactagcaacctcaaacagacacc (SEQ ID NO: 3)

[0165] IL- 12 (P40-Flag tag-linker-P35)-linker-GM-CSF-PDGFR beta TM:

[0166] ATGTGTCATCAGCAGCTCGTTATATCATGGTTTAGTCTTGTTTTTTTGGCGA

[0167] GTCCGCTCGTAGCAATTTGGGAACTCAAGAAGGACGTATATGTAGTGGAGC

[0168] TTGACTGGTATCCAGACGCCCCTGGTGAAATGGTGGTCTTGACGTGTGACA

[0169] CGCCCGAAGAGGATGGCATCACATGGACGCTCGACCAAAGCTCAGAAGTAT

[0170] TGGGGAGCGGCAAGACGCTCACTATTCAAGTAAAAGAGTTTGGAGATGCAG

[0171] GGCAATACACGTGCCACAAAGGAGGAGAGGTTCTTAGCCATAGTCTGCTCC

[0172] TGCTGCACAAGAAGGAGGACGGGATCTGGTCCACTGATATCCTGAAAGACC AGAAAGAACCCAAGAACAAAACTTTTCTTCGATGTGAAGCGAAAAATTATTC AGGCCGGTTCACATGCTGGTGGCTTACTACAATATCTACTGACCTGACGTTC AGCGTCAAGTCTAGTCGAGGGTCTTCTGATCCGCAAGGTGTCACGTGCGGG GCCGCCACTCTGTCTGCAGAACGGGTTAGGGGGGACAACAAGGAATATGAA TATTCCGTGGAGTGCCAAGAAGATAGCGCTTGCCCTGCGGCGGAAGAGAGC CTCCCCATCGAGGTTATGGTtGACGCCGTTCATAAACTCAAGTACGAAAACT ATACTTCCTCATTTTTTATAAGGGACATCATAAAGCCGGACCCACCTAAGAA TTTGCAATTGAAGCCTTTGAAGAATAGTgGACAAGTTGAGGTGTCCTGGGAG TACCCGGACACTTGGAGCACTCCCCATTCATATTTCTCCCTGACTTTCTGCG TTCAGGTTCAGGGCAAATCTAAACGAGAGAAAAAAGACCGCGTATTCACGG ACAAGACTTCTGCCACAGTAATCTGTCGAAAGAATGCTTCAATCTCCGTGAG GGCCCAAGACAGATACTATTCATCTAGCTGGTCCGAATGGGCCAGTGTTCC GTGCTCAgccgcagactacaaagacgatgacgacaagGTTCCTGGAGTAGGGGTACCTGGG GTGGGCgccAGAAACTTGCCAGTTGCGACGCCTGATCCCGGCATGTTTCCTTG CTTGCACCATTCACAGAATCTGCTTCGAGCTGTCTCCAATATGCTGCAAAAA GCCAGACAAACCCTGGAGTTCTACCCATGTACTAGCGAGGAGATAGATCAC GAGGATATCACGAAGGATAAAACTTCAACAGTCGAAGCTTGTCTTCCCCTGG AGTTGACTAAGAATGAGTCCTGTCTCAACTCTCGCGAAACATCTTTCATCAC AAATGGTAGCTGTCTCGCGTCTCGGAAAACGTCTTTCATGATGGCGCTGTGC CTGTCCTCCATATACGAAGACTTGAAAATGTATCAGGTGGAATTCAAGACCA TGAACGCCAAGCTCTTGATGGACCCAAAGAGACAGATCTTTCTTGACCAGAA CATGCTTGCAGTGATAGATGAGTTGATGCAAGCTTTGAACTTCAACTCAGAA ACAGTGCCGCAAAAATCATCACTTGAGGAGCCTGATTTTTACAAAACGAAAA TCAAGTTGTGCATTCTGTTGCATGCTTTCCGCATCAGGGCAGTAACTATCGA

[0173] TAGGGTGATGTCCTACCTTAACGCTAGTGTTCCTGGAGTAGGGGTACCTGG GGTGGGCgccatgTGGCTTCAGTCCCTGTTGCTCCTCGGAACGGTCGCATGTTC AATAAGCGCACCTGCGAGAAGTCCGAGTCCTAGCACGCAACCCTGGGAACA CGTCAACGCTATACAGGAGGCGCGACGGCTGCTCAATCTGAGCAGGGACAC GGCGGCTGAAATGAACGAGACAGTGGAAGTTATCAGCGAAATGTTTGATTT GCAGGAGCCTACGTGCCTTCAAACAAGGCTTGAACTGTATAAGCAAGGGCT TAGGGGTAGTCTTACGAAGCTTAAAGGACCATTGACGATGATGGCTAGTCA CTATAAGCAACACTGTCCACCGACGCCCGAGACGAGTTGCGCTACTCAGAT AATCACGTTTGAAAGCTTTAAAGAGAATCTCAAGGATTTTCTGCTGGTCATT CCTTTCGATTGTTGGGAGCCCGTGCAAGAGgctgtgggccaggacacgcaggaggtcatcgtgg tgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctcccttatcatcctca tcatgctttggcagaagaagccacgtctcgagggcagcggaTAA

[0174] (SEQ ID NO: 4)

[0175] 3’UTR:

[0176] Gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcat ctggattctgcctaataaaaaacatttattttcattgcagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaact actaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaGTCGACTCTAG (SEQ ID NO: 5)

[0177] Poly A tail:

[0178] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGATCCCCGGGCGAGCTC CCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCGAATTCCTGCAGCTCGAGAAAAAAAA

[0179] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6)

[0180] B. Amino acid Sequences

[0181] Amino Acid Sequence of Signal Peptide-IL-12 (P40-Flag tag-linker-P35)-Linker-GMCSF-

[0182] PDGFR beta transmembrane domain-cytoplasmic tail:

[0183] MCHOQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTP EEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVLSHSLLLLHK KEDGIWSTDILKDOKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPOGVTCGAATLSAERVRGDNKEYEYSVECOEDSACPAAEESLPIEVMVDA VHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSGOVEVSWEYPDTWSTPHSYF SLTFCVOVOGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWA

[0184] SVPCSAADYKDDDDKVPGVGVPGVGARNLPVATPDPGMFPCLHHSQNLLRAVSNM LQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCL ASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELM OALNFNSETVPOKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASVPGVGV PGVGAMWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAE MNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTP

[0185] ETSCATQIITFESFKENLKDFLLVIPFDCWEPVQEAVGQDTQEVIVVPHSLPFKVVVI SAILALVVLTIISLIILIMLWQKKPRLEGSG (SEQ ID NO: 7)

[0186] <Signaling peptide>

[0187] MCHQQLVISWFSLVFLASPLVA (SEQ ID NO: 8)

[0188] <IL-12 P40 amino-acid>

[0189] IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTL TIQVKEFGDAGOYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTF LRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRG DNKEYEYSVECOEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPP KNLQLKPLKNSGOVEVSWEYPDTWSTPHSYFSLTFCVOVOGKSKREKKDRVFT DKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 9) <AA>

[0190] <Flag tag>

[0191] DYKDDDDK (SEQ ID NO: 10)

[0192] Linker

[0193] VPGVGVPGVGA (SEQ ID NO: 11)

[0194] <IL-12 p35 amino-acid sequence>

[0195] LCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 12)

[0196] <Linker>

[0197] VPGVGVPGVGA (SEQ ID NO: 11)

[0198] <GM-CSF sequence>

[0199] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETV

[0200] EVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCA

[0201] TQIITFESFKENLKDFLLVIPFDCWEPVQE (SEQ ID NO: 13)

[0202] <PDGFR beta transmembrane domain>

[0203] AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0204] (SEQ ID NO: 14)

[0205] <CytopIasmic tail>

[0206] LEGSG (SEQ ID NO: 15)

[0207] Flag tag was used after IL-12 p40 for detection of IL-12. Other tags such as transferrin tag can be added after IL- 12 protein sequence and before transmembrane domain to test expression of this protein.

[0208] Example 7. Expression of membranous IL-12 -linker-GM-CSF after transfecting IL- 12- linker-GM-CSF mRNA-LNP to 293 cells We used IL-12-linker-GM-CSF mRNA generated from DNA template PMC2389 (see Example 6) with CleanCap Reagent AG (NEB #E2080), and pseudo-UTP, incorporated into LNP, and delivered to 293, Daudi, Jurkat, Nalm-6 and Raji cell lines by transfection.

[0209] Transfected cells were shown GM-CSF positive and IL- 12-positive when tested with anti-GM-CSF antibody (FIG. 3A) and with anti-IL-12 antibody (FIG. 3B) at 72 posttransfections by FACS, which confirms expression of cytokines on the cell surface. There was variation in expression between cell lines from 15 to 95.7% of expression of GM-CSF (FIG. 3A) and from 22% to 96.8% of IL- 12 expression (FIG. 3B).

[0210] To test the functional effect on immune cells, the effects of IL-12-GM-CSF mRNA- LNP on T cells, monocytes, macrophages, and dendritic cells were tested after transfection to these cells.

[0211] Transfection of IL-12-GM-CSF to T cells activated with CD3-CD28 beads increased Thl (effector) phenotype of T cells, which was confirmed by increased expression of IFN- gamma (FIG. 4). IFN-gamma expression in transfected cells increased from 26% to 53% (Y- axis) (FIG. 4). This confirms the functional effect of IL- 12, which is known to stimulate Thl T cell subtype activation with increased IFN-gamma secretion.

[0212] Transfection of monocytes with IL-12-GM-CSF mRNA-LNP caused expression of IL-12 in 50% of LNP-treated cells, which was shown by staining with IL-12 Antibody, and expression of GM-CSF in 12% of cells, which was shown by staining with GM-CSF antibody.

[0213] Treatment of monocyte-derived macrophages with 1L-12-GM-CSF mRNA-LNP increased survival of macrophages with distinct macrophage morphology (FIG. 5). It is known that GM-CSF increases survival of macrophages. The results show that the functional effect of IL-12-GM-CSF mRNA-LNP is the same as GM-CSF added to the medium (FIG. 5).

[0214] IL-12-GM-CSF mRNA-LNP treated macrophages were bigger, with many vacuoles, more adherent, with higher MHCII expression, and lower CD 14 expression. Treatment of monocytes with IL-12-GM-CSF mRNA-LNP and IL-4 promoted dendritic cell differentiation that was detected by FACS with CD la antibodies. IL-12-GM-CSF mRNA-LNP (PMC2389) transfected monocytes lost monocyte CD 14 marker 6 days after transfection and increased dendritic cell marker CDla from 0.66% to 23% in monocytes (FIG. 6). Monocyte CD14+ marker dropped from 67% to 1.4% (circle) in IL-12-GM-CSF mRNA-LNP+Il-4-treated cell during 6 days (FIG. 6).

[0215] The results show that IL-12-GM-CSF changed function of T cells, monocytes, macrophages and dendritic cells, which is important for anti-tumor therapy where functional Thl effector cells, monocytes and dendritic cells from tumor microenvironment play functional role.

[0216] Example 8. In vivo efficacy of IL-12-GM-CSF in mice locally

[0217] 4xl06OVCAR-5 gastrointestinal (GI) cancer cells were injected subcutaneously into left and right flanks of NSG mice. Then IL-12-GM-CSF mRNA-LNPs were injected intratumorally (20 mcl, 1 mcg) on days 7, 14, and 21. Then T cells were injected intravenously on days 9 and 16. The results show that 1L-12-GM-CSF significantly inhibited tumor growth with T cells alone and in combination with EpCAM-CD3 mRNA-LNP. (FIG. 7).

[0218] EpCAM-CD3 mRNA-LNPs were prepared with the sequences and method described in WO2024 / 148107.

[0219] Example 9. In vivo efficacy of IL-12-GM-CSF in mice at local treated and untreated distant tumors

[0220] In order demonstrate efficacy of IL-12-GM-CSF at a local treated side and at a distant untreated tumor side, PC3 prostate cancer cells were injected at the left side (6xl06cells) and right side (2x l06cells) subcutaneously. Then EpCAM-CD3 and GM-CSF mRNA or EpCAM-CD3+IL-12-GM-CSF mRNA-LNPs were injected intratumorally at days 14, 21 and 28 at the left side only. T cells were injected intravenously on days 15, 22 and 29. GFP mRNA-LNP was used as negative control.

[0221] The results (FIGs. 8A and 8B) show that IL-12-GM-CSF mRNA in combination with EPCAM-CD3 mRNA eradicated tumors not only at a local side, but also at a distant untreated side. This is important for treating metastatic tumors.

[0222] REFERENCES

[0223] 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

[0224] 2. K. A. Ullrich, L. L. Schulze, E. M. Paap, T. M. Muller, M. F. Neurath 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. M. P. Colombo and G. Trinchieri: Interleukin- 12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev , 13(2), 155-68 (2002) doi: 10.1016 / sl359- 6101(01)00032-6

[0225] 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 solid tumors. FEBS Open Bio, 12(7), 1268-1285 (2022) doi: 10. 1002 / 2211-5463.13445

[0226] 5. H. M. Lazarus, K. Pitts, T. Wang, E. Lee, E. Buchbinder, M. Dougan, D. G.

[0227] Armstrong, R. Paine, 3rd, C. E. Ragsdale, T. Boyd, E. P. Rock and 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

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising IL- 12 and GM-CSF, which are covalently attached.

2. The fusion protein of claim 1 , which further comprises a transmembrane domain C- terminal to IL- 12 and GM-CSF.

3. The fusion protein of claim 1, wherein the transmembrane domain is selected from the group consisting of: a transmembrane domain of PGFRB receptor (platelet-derived growth factor receptor beta), a T cell receptor a or chain, a CD3 zeta chain, CD28, CD3s., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR.

4. The fusion protein of claim 3, wherein the transmembrane domain is a transmembrane domain of PGFRB receptor.

5. The fusion protein of any one of claims 1-4, wherein the IL-12 comprises the amino acid sequence of SEQ ID NOs: 9 and 12, and GM-CSF comprises the amino acid sequence number of SEQ ID NO: 13.

6. The fusion protein of claim 5, having the amino acid sequence of SEQ ID NO: 7.

7. Lipid nanoparticles (LNPs) have mRNA encapsulated, wherein the mRNA is transcribed from a DNA sequence comprising: (a) a promoter coding sequence, (b) 5’-UTR (untranslated region) coding sequence, (c) a sequence encoding the fusion protein of any one of claims 1-5, (d) a 3’-UTR coding sequence, and (e) a poly A tail sequence.

8. The LNPs of claim 7, wherein the LNPs comprises (a) 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl] amino] -octanoic acid, 1-octylnonyl ester (SM-102), distearoylphosphatidylcholine (DSPC), Cholesterol, and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), or (b) SM-102, DSPC, Cholesterol, and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(poly ethylene glycol)-2000] (DSPE-PEG2000-MAL), or (c) 2-hexyl-decanoic acid, l,l'-[[(4-hydroxybutyl)imino]di-6,l- hexanediyl] ester (ALC-0315), DSPC, Cholesterol, and a-[2-(ditetradecylamino)-2- oxoethyl]-(o-methoxy-poly(oxy-l,2-ethanediyl) (ALC-0159).

9. A method for producing a fusion protein on the surface on cells, comprising the steps of: obtaining a DNA sequence comprising: (a) a promoter coding sequence, (b) 5'-UTR coding sequence, (c) a sequence encoding the fusion protein of any one of claims 1-6, (d) a 3'-UTR coding sequence, and (e) a poly A tail sequence; transcribing the DNA sequence to mRNA with RNA polymerase in vitro, encapsulating the mRNA in lipid nanoparticles (LNPs), transfecting the mRNA-encapsulated LNPs into cells, and translating the mRNA in the cells to produce membrane-bound fusion protein on the surface of the cells.

10. The method of claim 9, wherein the cells are cancer cells or blood cells and the mRNA is transfected into the cancer cells or blood cells.

11. A method for treating cancer, comprising the steps of: injecting the mRNA encapsulated -LNPs of claim 7 to a tumor lesion of a subject having cancer.

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

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

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