Immunostimulatory MRNA Compositions and Uses Thereof

Abstract

The present invention is located in the field of immunotherapy. More specifically, the present invention relates to a composition of one or more nucleic acid molecules encoding functional immunostimulatory proteins such as IL-21, encoding co-stimulatory molecules, optionally in combination with cytokines and / or chemokines, to enhance the host immune response against tumor cells present in the body. The present invention further relates to a nucleic acid-containing composition or pharmaceutical formulation for use in the treatment of patients suffering from tumors and / or cancer diseases.

Description

[Technical field]

[0001] The present invention is in the field of immunotherapy. More specifically, the present invention relates to compositions of one or more nucleic acid molecules encoding functional immunostimulatory proteins and co-stimulatory molecules, such as IL-21, optionally in combination with cytokines and / or chemokines, to enhance the host immune response against tumor cells present in the body.

[0002] The present invention further relates to a nucleic acid-containing composition or pharmaceutical formulation for use in treating a patient suffering from a tumor and / or cancer disease. [Background technology]

[0003] Immunotherapy, which aims to activate the immune response against tumor cells, has revolutionized cancer treatment, providing clinical benefit to previously untreatable patients. The long-term benefit of immunotherapy requires the induction, expansion, and maintenance of memory T cells that recognize cancer-associated antigens.

[0004] Although systemic delivery of checkpoint inhibitors (CPIs) - antibodies that block T cell inhibitory receptors such as CTLA-4 and PD-1 / PD-L1 - has led to durable clinical responses by reactivating / expanding preexisting T cells, many tumors face immunosuppressive strategies that render them resistant to CPIs, necessitating the development of complementary strategies that lead to enhanced T cell immunity.

[0005] The induction of optimal T cell responses requires antigen presentation to the T cell receptor (TCR) in combination with co-stimulatory ligands and additional T cell activation signals mediated by cytokines or chemokines. Summary of the Invention [Problem to be solved by the invention]

[0006] Nucleic acid-based approaches, including viral vectors, plasmid DNA and messenger mRNA, have been used to locally encode these immunostimulatory proteins in the tumor microenvironment, resulting in the induction and amplification of a systemic immune response against antigens present in the tumor bed.

[0007] These nucleic acids encoding immunostimulatory proteins can also be used in the context of vaccination to enhance the generation of T cell responses against co-delivered antigens.

[0008] Although individual immunostimulatory proteins can induce some level of antitumor efficacy, multiple immunostimulatory pathways need to be targeted simultaneously to enhance therapeutic benefit.

[0009] However, determining which factors to combine to obtain optimal efficacy remains a major challenge, and is the subject of this invention. [Means for solving the problem]

[0010] The inventors have demonstrated that (local) delivery of a composition comprising a nucleic acid encoding at least the functional immunostimulatory protein IL-21 induces a systemic immune response that results in tumor reduction or tumor growth delay.

[0011] Furthermore, the combination of said composition comprising a nucleic acid encoding a functional immunostimulatory protein IL-21 with one or more co-stimulatory molecules, cytokines, and / or chemokines further enhances the observed effects.

[0012] The present invention particularly relates to compositions comprising one or more isolated, non-viral nucleic acid molecules that encode a functional immunostimulatory protein, IL-21, and that encode a co-stimulatory molecule.

[0013] In certain embodiments, the composition may further comprise one or more nucleic acid molecules encoding a cytokine and / or a chemokine.

[0014] The present invention provides proof of the inventive concept that such compositions are capable of inducing / enhancing antitumor effects, inter alia, by reducing tumor volume after delivery, thus forming a promising new approach for antitumor immunotherapy.

[0015] Thus, the present invention provides a solution for improving the immunostimulatory properties of a composition comprising the introduction of at least one nucleic acid, in particular an mRNA or DNA molecule, encoding a protein that modifies the tumor microenvironment, characterized in that the encoded functional immunostimulatory protein is at least IL-21, optionally combined with one or more co-stimulatory molecules, cytokines and / or chemokines.

[0016] In a particular embodiment, the composition comprises a nucleic acid molecule encoding an IL-21 isoform, in particular an IL-21 isoform selected from the list comprising IL-21 isoform 1 and IL-21 isoform 2, more particularly IL-21 isoform 1.

[0017] In certain embodiments, the composition further comprises one or more of a target specific antigen, and / or a checkpoint inhibitor.

[0018] In some embodiments, the composition comprises a co-stimulatory molecule selected from the list comprising 4-1BBL, OX40L, ICOS ligand and CD40L.

[0019] In a particular embodiment, the composition comprises a cytokine selected from the list comprising IL-7, IL-12, IL-18, IFN lambda, IL-15sushi IL-23, type I IFN, type II IFN, type III IFN.

[0020] In another embodiment, the composition comprises a chemokine selected from the list comprising CCL19, CCL20, CCL21, CXCL9, CXCL10, CCL4, CCL5 and XCL1.

[0021] In certain embodiments, the composition comprises a target-specific antigen, such as a tumor-associated or tumor-specific antigen or a neoantigen.

[0022] The target-specific antigen may be derived from any one of total mRNA isolated from a target cell (a), one or more specific target mRNA molecules of a target cell (a), a protein lysate of a target cell (a), a specific protein from a target cell (a), or a synthetic target-specific peptide or protein, and synthetic mRNA or DNA encoding the target-specific antigen or a peptide derived therefrom.

[0023] In a particular embodiment, the composition of the invention comprises a checkpoint inhibitor, such as a checkpoint inhibitor polypeptide selected from the list comprising in particular an inhibitory molecule of PD-1, PD-L1, CTLA4, TIGIT, TIM3, LAG-3 and VISTA; preferably an inhibitory molecule of PD-1, PD-L1 or CTLA4.

[0024] A preferred combination of immunostimulatory factors for use in the methods of the present invention is IL-21 and 4-1BBL.

[0025] In another preferred embodiment, a combination of IL-21, 4-1BBL and IL-7 immunostimulatory molecules is used.

[0026] In yet other preferred embodiments, a combination of IL-21, 4-1BBL and IFNlamda immunostimulatory molecules; or a combination of IL-21, 4-1BBL and IL-18 is used.

[0027] In another preferred embodiment, a combination of IL-21, 4-1BBL and IL-15 is used.In another preferred embodiment, a combination of IL-21, 4-1BBL and IL-15sushi is used.

[0028] In certain embodiments, the mRNA or DNA molecule is a separate nucleic acid molecule.

[0029] In another embodiment, the mRNA or DNA molecules are part of a single nucleic acid molecule, which can simultaneously express two or more immunostimulatory proteins, for example, two or more mRNA or DNA molecules encoding immunostimulatory proteins can be linked in a single mRNA or DNA molecule by an internal ribosome entry site (IRES) or a self-cleaving 2a peptide coding sequence.

[0030] In a particular embodiment, the nucleic acid molecule in the composition is selected from the group comprising RNA, DNA, preferably RNA, more preferably mRNA.

[0031] In certain embodiments, the nucleic acid molecules of the composition comprise one or more of the following: a 5'CAP, a poly(A) tail, and / or a modified nucleoside(s), which may be N1-methylpseudouridine.

[0032] In a particular embodiment, the nucleic acid molecule of the composition is in the form of a naked nucleic acid molecule or a nucleic acid encapsulated in a nanoparticle, such as a lipid nanoparticle or a polymeric nanoparticle, in particular a lipid nanoparticle.

[0033] The present invention further provides lipid nanoparticles comprising the compositions described by the present invention.

[0034] In another embodiment, the composition or lipid nanoparticle comprises an ionizable lipid, cholesterol, a phospholipid, and a PEGylated lipid.

[0035] In a preferred embodiment, the composition or lipid nanoparticle comprises about 35 mol% to 65 mol% ionizable lipid; about 5 mol% to 25 mol% phospholipid; about 0.5 mol% to 3.0 mol% PEG lipid; balanced by the amount of sterol; in particular, 50 mol% ionizable lipid, 10 mol% phospholipid, 1.5 mol% PEG lipid, and 38.5 mol% sterol.

[0036] The present invention further provides a pharmaceutical formulation comprising the composition or lipid nanoparticles of the present invention and at least one pharma- ceutically acceptable carrier or excipient.

[0037] The pharmaceutical formulations of the present invention are particularly suitable as vaccines.

[0038] In a further aspect, the present invention provides a composition, lipid nanoparticle or pharmaceutical formulation as defined herein for use in injection; more particularly intravenous, intratumoral, intradermal, intraperitoneal, intramuscular or intranodal administration, preferably intratumoral administration.

[0039] In certain embodiments, the compositions, lipid nanoparticles or pharmaceutical formulations of the invention are provided for use in human or veterinary medicine.

[0040] In a preferred embodiment, the compositions, lipid nanoparticles or pharmaceutical formulations defined herein are provided for use in combination with standard of care cancer therapies, including radiation therapy and chemotherapy.

[0041] In a more preferred embodiment, the composition, lipid nanoparticle or pharmaceutical formulation of the invention is provided for use in the prevention and / or treatment of a cell proliferative disorder.

[0042] In yet another embodiment, a composition, lipid nanoparticle or pharmaceutical formulation as defined herein is provided for use in eliciting an immune response against tumors and / or cancer in a subject. [Brief description of the drawings]

[0043] [Figure 1]FIG. 1 shows mRNA monotherapy with IL-21, IL-7, IL-15sushi or 4-1BBL in an MC38 colon (colon cancer) model. Tumor volumes (mm3) are shown for each time point at which tumors were measured after randomization. Panel A shows treatment with Fluc mRNA control (3 doses of 15 μg mRNA / dose, formulated in LNPs) on days 0, 3 and 7 after randomization, while panels B, C, D, E show treatment with the same doses and treatment regimens with mRNA encoding IL-21 iso2, IL-7, 4-1BBL and IL-15sushi, respectively. For control and IL-15sushi, complete responses ("CR") were observed in 2 of 8 mice (20%), whereas treatment with IL-21 mRNA induced complete responses in 6 of 8 mice (75%). Monotherapy treatment with IL-7 or 4-1BBL showed no effect on tumor volume. [Diagram 2] FIG. 2 shows mRNA combination therapy with IL-7 and IL-21 in an MC38 (colon cancer) model. Tumor volumes (mm3) are shown for each time point at which tumors were measured after randomization. Panel A shows treatment with NanoLuc mRNA control (10 μg mRNA / dose, formulated in LNPs, three times) on days 0, 3, and 7 after randomization, and panels B and C show treatment with NanoLuc control mRNA with mRNA encoding IL-21 or IL-7, respectively (three times at reduced doses of 5 μg for each mRNA / dose). Panel D represents combination therapy with mRNA encoding IL-7 and IL-21 (three times at doses of 5 μg for each mRNA / dose). [Diagram 3]Figure 3 shows treatment with mRNA combinations of IL-15sushi and IL-21 in an MC38 (colon cancer) model. Tumor volumes (mm3) are shown for each time point tumors were measured after randomization. Panel A shows monotherapy with mRNA encoding IL-21 (15 μg total mRNA / dose, 3 times), Panel B shows combination therapy with mRNA encoding IL-21 and IL-15sushi mRNA (3×7.5 μg each mRNA / dose), Panel C shows combination therapy with mRNA encoding IL-21, IL-7 and 4-1BBL mRNA (3×5 μg each mRNA / dose), Panel D shows combination therapy with mRNA encoding IL-21, 4-1BBL and IL-15sushi (3×5 μg each mRNA / dose). [Figure 4] FIG. 4 shows the development of a systemic memory immune response after treatment with IL-21 monotherapy or IL-based combination therapy. Tumor volumes (mm3) are shown for each time point at which tumors were measured after randomization. The presence of memory immune responses was evaluated in animals treated with IL-21 monotherapy (panels A and B) or combination therapy (panels C and D). Panel A shows tumor growth in untreated animals (n = 12) newly injected with MC238 cancer cell line. Panel B shows tumor growth in rechallenged mice (n = 6) previously treated with IL-21 monotherapy, and Panel C shows tumor growth in rechallenged mice (n = 6) previously treated with Il-21 / 4-1BBL / Il-15sushi combination therapy. Panel D shows tumor growth in rechallenged mice (n = 8) previously treated with IL-21 / 4-1BBL / IL-7 combination therapy. [Figure 5-A]Figure 5 shows the development of a systemic memory immune response after treatment with IL-21 monotherapy or IL-based combination therapy. Tumor volumes (mm3) are shown for each time point tumors were measured after randomization. Panel A shows tumor growth in TBS buffer, empty LNP and non-translated IL-21 mRNA LNP treated animals. Panel B shows tumor growth in IL-21, IL-7 or 4-1BBL mRNA LNP treated animals. Panel C shows tumor growth in animals treated with IL-21 / IL-7 mRNA LNP combination, IL-21 / 4-1BBL mRNA LNP combination or IL-7 / 4-1BBL mRNA LNP combination. Panel D shows tumor growth of IL-21 / IL-7 / 4-1BBL mRNA LNP combination. [Figure 5-B] Same as above [Figure 5-C] Same as above [Figure 5-D] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other aspect, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0045] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" means one compound or more than one compound.

[0046] As used herein, when referring to a measurable value such as a parameter, amount, duration, and the like, the term "about" or "approximately" is meant to encompass variations from the particular value of within + / - 10%, preferably within + / - 5%, more preferably within + / - 1%, and even more preferably within + / - 0.1%, as appropriate for practice in the disclosed invention.

[0047] It is to be understood that the modifier "about" or "approximately" also specifically, and preferably, discloses the value to which it refers.

[0048] In searching for new methods for inducing a systemic anti-tumor immune response, the inventors investigated whether local administration of a composition comprising an LNP-encapsulated nucleic acid encoding an immune-modulating molecule would induce an effective anti-tumor response.

[0049] The present inventors have unexpectedly discovered that a composition comprising a nucleic acid encoding a functional immunostimulatory protein, IL-21, upon direct intratumoral injection results in a potent and durable anti-tumor response.

[0050] It was further demonstrated that efficacy was further enhanced by combining IL-21 mRNA with mRNA molecules encoding 4-1BBL and IL-7.

[0051] The present invention is suitable for activating and expanding anti-tumor immunity by direct modulation of the tumor microenvironment upon intratumoral administration, but may also be suitable for enhancing T cell responses against co-delivered antigens in vaccination situations (e.g., intramuscular, intranodal or intravenous immunization).

[0052] The term "target" as used throughout this specification is not limited to the specific examples that may be described herein, but may include tumor or cancer cells that are targeted.

[0053] The term "target-specific antigen" as used throughout this specification is not limited to the specific examples that may be described herein.

[0054] It will be apparent to one of skill in the art that the present invention relates to enhancing anti-tumor responses by providing factors that modify the tumor microenvironment.

[0055] Without wishing to limit the scope of protection of the invention, some examples of possible markers are listed below.

[0056] The term "antigen presenting cells" as used throughout this specification includes all antigen presenting cells. Specific non-limiting examples are dendritic cells, dendritic cell lines, b cells, or B cell lines. Dendritic cells or B cells can be isolated or generated from the blood of a patient or a healthy subject.

[0057] The patient or subject may or may not have been the subject of a prior vaccination.

[0058] The terms "cancer" and / or "tumor" as used throughout this specification are not intended to be limited to the types of cancer or tumor exemplified. Thus, the terms encompass all proliferative disorders, such as neoplasia, dysplasia, precancerous or precancerous lesions, abnormal cell proliferation, benign tumors, malignant tumors, cancer, or metastasis, where cancer is: Leukemia, non-small cell lung cancer, small cell lung cancer, CNS cancer, melanoma, Ovarian cancer, kidney cancer, prostate cancer, breast cancer, glioma, Colon cancer, bladder cancer, sarcoma, pancreatic cancer, colorectal cancer, Head and neck cancer, liver cancer, bone cancer, bone marrow cancer, stomach cancer, The cancer is selected from the group consisting of duodenal cancer, esophageal cancer, thyroid cancer, blood cancer, and lymphoma.

[0059] The specific antigen for the cancer can be, for example, MelanA / MART1, cancer germline antigen, gplOO, tyrosinase, CEA, PSA, Her-2 / neu, survivin, telomerase.

[0060] Thus, the present invention provides compositions comprising a nucleic acid molecule encoding a functional immunostimulatory protein, IL-21, and optionally encoding one or more co-stimulatory molecules, cytokines, and / or chemokines.

[0061] In particular, the present invention provides compositions comprising one or more isolated non-viral nucleic acid molecules that encode a functional immunostimulatory protein, IL-21, and that encode a co-stimulatory molecule.

[0062] The composition may optionally further comprise one or more nucleic acid molecules encoding cytokines and / or chemokines.

[0063] In the context of the present invention, the term "immunostimulatory protein" is to be understood as any protein capable of stimulating the immune system by inducing or increasing the activation of any of its components.

[0064] As used herein, the immunostimulatory protein provides antigen specificity in an immune response, for example as a result of vaccination.

[0065] In the context of the present invention, the term "interleukin 21" is to be understood as a cytokine that has a powerful regulatory effect on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells, which are capable of destroying virally infected or cancerous cells.

[0066] IL-21 has antitumor effects through sustained and increased CD8+ cell responses to achieve sustained tumor immunity.

[0067] In a particular embodiment, the composition comprises a nucleic acid molecule encoding an IL-21 isoform, in particular an IL-21 isoform selected from the list comprising IL-21 isoform 1 and IL-21 isoform 2, more particularly IL-21 isoform 1.

[0068] In the context of the present invention, the term "costimulatory molecules" is to be understood as a heterogeneous group of cell surface molecules that act to amplify or counteract the initial activation signal provided to a T cell by the T cell receptor (TCR) after its interaction with the antigen / major histocompatibility complex (MHC), thereby influencing T cell differentiation and fate.

[0069] As used herein, the co-stimulatory molecule may be one of the classes of molecules of the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily, and the T-cell Ig and mucin (TIM) domain family.

[0070] In some embodiments, the co-stimulatory molecule is selected from the list including 4-1BBL, OX40L, ICOS ligand, and CD40L.

[0071] In the context of the present invention, the term "cytokine" should be understood as a protein important in cell signaling. As used herein, cytokines are immune-regulating cell signaling proteins and include interferons, interleukins, lymphokines, and tumor necrosis factors, but do not include hormones or growth factors.

[0072] Cytokines act through cell surface receptors and are particularly important in the immune system, where they regulate the balance between humoral and cell-based immune responses and modulate the maturation, growth, and responsiveness of specific cell populations.

[0073] In particular, the cytokines defined in the present invention may in particular be selected from the list comprising IL-7, IL-12, IL-18, IFN lambda, IL-15sushi, IL-23, type I IFN, type II IFN, type III IFN. In a particular embodiment, the cytokines are selected from the list comprising IL-7, IL-12, IL-18, IFN lambda and IL-15sushi.

[0074] In a particular embodiment, the composition of the invention comprises a nucleic acid molecule encoding an IL-21 isoform and a nucleic acid molecule encoding an IL-7 isoform selected from the list comprising the cytokine IL-7 isoform, in particular IL-7 isoform 1 and IL-7 isoform 2, more in particular IL-7 isoform 1.

[0075] In another specific embodiment, the composition of the invention comprises a nucleic acid molecule encoding IL-21 isoform-1 and IL-7 isoform-1.

[0076] In yet another specific embodiment, a composition of the invention comprises a nucleic acid molecule encoding IL-21 isoform 1 and IL-15sushi.

[0077] In the context of the present invention, the term "chemokine" is to be understood as a signaling protein secreted by cells that induces the directional movement of leukocytes and other cell types, including endothelial and epithelial cells. Chemokines interact with G protein-coupled transmembrane receptors.

[0078] As used herein, said chemokines may be selected from the group comprising the CXC, CC, CX3C and C motifs. In some embodiments, the chemokine is CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, You can choose from a list that includes XCL1-V21C / V59C, XCL2 CX3CL1

[0079] It may be beneficial to deliver nucleic acids encoding additional chemokines to the tumor environment, such as using dendritic cell-attracting chemokines (e.g., CCL4, CCL5, CCL19, CCL20, CCL21, XCL1) or using T cell / NK-attracting chemokines (e.g., CCL3, CCL5, CXCL9-10_CCL19, XCL1).

[0080] In a preferred embodiment, the chemokine is selected from the list comprising CCL19, CCL20, CCL21, CXCL9, CXCL10, CCL4, CCL5 and XCL1.

[0081] In certain embodiments, the composition further comprises one or more of a target specific antigen and / or a checkpoint inhibitor.

[0082] In a particular embodiment, the composition comprises a target-specific antigen that is a tumor antigen. The target-specific antigen may be provided in the form of either total mRNA isolated from the target cell (a), one or more target-specific mRNA molecules, a protein lysate of the target cell (a), a specific protein derived from the target cell (a), or a synthetic target-specific peptide or protein, and synthetic mRNA or DNA encoding the target-specific antigen or a peptide derived therefrom.

[0083] In the context of the present invention, the term "checkpoint inhibitor" is to be understood as a type of drug or molecule that blocks immune checkpoints utilized by tumor cells to reduce immune activation and antigen recognition.

[0084] Therefore, these checkpoint inhibitors may also be powerful tools for improving cancer treatment.

[0085] In certain embodiments, the composition comprises one or more nucleic acid molecules encoding any compound that can directly or indirectly affect the regulation of said checkpoint inhibitor, for example, by reducing the expression of said checkpoint inhibitor (i.e., transcription and / or translation) or its natural ligand, or checkpoint inhibitor activity.

[0086] Such inhibitors include, but are not limited to, proteins, peptides, small molecules, antibodies, etc. that block said checkpoint inhibitor-associated signaling molecules or pathways.

[0087] In a particular embodiment, the checkpoint inhibitor is selected from the list comprising an inhibitory molecule of PD-1, PD-L1, CTLA4, TIGIT, TIM3, LAG-3 and VISTA; preferably an inhibitory molecule of PD-1, PD-L1 or CTLA4.

[0088] For example, the composition may comprise an anti-PD1 antibody against PD-1, such as nivolumab (BMS-936558 / MDX1106), pidilizumab (CT-011) or pembrolizumab (MK-3475). Alternatively, PD-L1 inhibitors, such as atezolizumab or durvalumab, may also be suitably used within the context of the present invention.

[0089] Exemplary human anti-CTLA4 antibodies are described in detail, e.g., in WO 00 / 37504, and include, but are not limited to, 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, ticilimumab, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1, as well as tremelimumab and ipilimumab.

[0090] The above-mentioned anti-checkpoint inhibitor antibodies, such as anti-PD-1 and / or anti-PD-L1 / PD-L2 antibodies, are well known in the art.

[0091] Other blocking antibodies can be readily identified and prepared by one of skill in the art.

[0092] In very specific implementations, the invention includes one or more isolated, non-viral nucleic acid molecules that encode a functional immunostimulatory protein IL-21, and optionally encode one or more of the following: - a co-stimulatory molecule selected from the list comprising 4-1BBL, OX40L, ICOS ligand, and CD40L; - a cytokine selected from the list comprising IL-7, IL-12, IL-18, IFN lambda, IL-15sushi, IL-23, type I IFN, type II IFN, type III IFN; - a chemokine selected from the list comprising CCL19, CCL20, CCL21, CXCL9, CXCL10, CCL4, CCL5 and XCL1; - inhibitory molecules of PD-1, PD-L1, CTLA4, TIGIT, TIM3, LAG-3 and / or VISTA; preferably inhibitory molecules of PD-1, PD-L1, or CTLA4.

[0093] In another very specific embodiment, the present invention provides a composition comprising one or more isolated non-viral nucleic acid molecules encoding the functional immunostimulatory protein IL-21, and optionally encoding one or more of IL-7, 4-1BBL, IFN lambda, IL-18, IL-15sushi and / or CCL5.

[0094] A preferred combination of immunostimulatory factors used in the methods of the present invention is IL-21 and 4-1BBL. In another preferred embodiment, a combination of IL-21, 4-1BBL and IL-7 immunostimulatory molecules is used. In yet another preferred embodiment, a combination of IL-21, 4-1BBL and IL-15sushi immunostimulatory molecules is used.

[0095] In certain embodiments, the composition comprises a nucleic acid molecule encoding IL-21 isoform 1 and 4-1BBL.

[0096] In another specific embodiment, the composition of the invention comprises a nucleic acid molecule encoding IL-21 isoform 2, 4-1BBL, and IL-7 isoform 1.

[0097] In yet another specific embodiment, a composition of the invention comprises a nucleic acid molecule encoding IL-21 isoform 2, 4-1BBL, and IL-15sushi.

[0098] In yet another embodiment, the composition of the invention comprises nucleic acid molecules encoding IL-21 isoform 2, 4-1BBL and IFN lambda.

[0099] In yet another embodiment, a composition of the invention comprises nucleic acid molecules encoding IL-21 isoform 2, 4-1BBL and IL-18.

[0100] The SEQ ID NOs for the human amino acid sequences of the immunostimulatory proteins defined in the present invention are listed in Table 1.

[0101] It should be understood that the amino acid sequences set forth herein are not limiting and can include sequence variations (i.e., having a percentage sequence identity to the described sequences).

[0102] Thus, in certain embodiments, the human amino acid sequences described herein are at least, and / or about, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence numbers listed in Table 1.

[0103] The present invention can also provide compositions comprising nucleic acids encoding immunostimulatory proteins, costimulatory molecules, cytokines, and / or chemokines, wherein the human amino acid sequences are replaced by the respective mouse amino acid sequences, such as those shown in Table 2, e.g., those used in the Examples section disclosed herein.

[0104] Thus, in certain embodiments, the mouse amino acid sequences described herein are at least, and / or about, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence numbers listed in Table 2.

[0105] For purposes of comparing two or more nucleotide or amino acid (AA) sequences, the percentage of "sequence identity" between a first nucleotide / AA sequence and a second nucleotide / AA sequence is defined as: [the number of nucleotides / AA in a first nucleotide / AA sequence that are identical to a nucleotide / AA at the corresponding position in a second nucleotide / AA sequence] of [total number of nucleotides / AA in first nucleotide / AA sequence] Divide by [100%], Here, each deletion, insertion, substitution or addition of a nucleotide / AA in a second nucleotide / AA sequence compared to a first nucleotide / AA sequence is considered as a difference at a single nucleotide / AA (position). Alternatively, the degree of sequence identity between two or more nucleotide / AA sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. A particular method utilizes the BLAST module of WU-BLAST-2 set to default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0106] In determining the degree of sequence identity between two amino acid sequences, one skilled in the art can also take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and has little or no effect on the function, activity or other biological properties of the polypeptide. Such conservative substitutions are preferably those in which one amino acid within the following groups (a) to (e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) Aromatic residues: Phe, T. Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asp; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; From Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or From Phe to Val, Ile, or Leu. Thus, in one embodiment, a sequence having a given percentage sequence identity as given herein above is a sequence that has one, two, three or more conservative amino acid substitutions compared to the reference sequence.

[0107] On the other hand, variant antigens / polypeptides encoded by the nucleic acids of the present disclosure may contain amino acid changes that confer any of a number of desirable properties, e.g., enhancing their immunogenicity in a subject, enhancing their expression, and / or improving their stability or PK / PD properties. Mutant antigens / polypeptides can be made using routine mutagenesis techniques and appropriately assayed to determine whether they have the desired properties.

[0108] As will be appreciated by those of skill in the art, protein fragments (nucleic acid encoding), functional protein domains, and homologous proteins are also considered to be within the scope of tumor antigens of interest. For example, any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference antigen sequence but is otherwise identical) of a reference protein is provided herein, provided that the fragment is immunogenic and confers a protective immune response against the tumor-associated antigen. In addition to variants that are identical to the reference protein but are truncated, in some embodiments, the antigen may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. Antigens / antigenic polypeptides can range in length from about 4, 6, or 8 amino acids up to the full-length protein.

[0109] The present invention also provides mouse-specific nucleic acid molecules encoding the immunostimulatory proteins, costimulatory molecules, cytokines, and / or chemokines listed in Table 3.

[0110] Thus, in certain embodiments, the mouse nucleic acid sequences described herein are at least, and / or about, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence numbers listed in Table 3.

[0111] In the context of the present invention, it is important to understand that the composition comprises at least one nucleic acid molecule encoding the immunostimulatory protein IL-21. Additionally, the composition may include at least one or more nucleic acid sequences encoding additional immunostimulatory proteins, such as co-stimulatory molecules, cytokines, and / or chemokines. Thus, the composition may contain one or more nucleic acid sequences encoding only IL-21, or may contain two or more nucleic acid molecules where additional immunostimulatory proteins are encoded. Alternatively, IL-21 and at least one other immunostimulatory protein may be encoded by one single nucleic acid molecule.

[0112] When a composition comprises nucleic acid encoding more than one protein as defined herein, these may be encoded from a single nucleic acid molecule or from more than one nucleic acid molecule.

[0113] In a particular embodiment, the two or more nucleic acid molecules, in particular the mRNA or DNA molecules, encoding an immunostimulatory protein are therefore separate nucleic acid molecules. In another embodiment, said mRNA or DNA molecules are part of one single nucleic acid molecule, which single nucleic acid molecule is capable of expressing two or more immunostimulatory proteins simultaneously. This single mRNA or DNA molecule is preferably capable of independently expressing two or more proteins. In a preferred embodiment, two or more mRNA or DNA molecules encoding immunostimulatory proteins are linked in a single mRNA or DNA molecule by an internal ribosome entry site (IRES) that allows separate translation of each of the two or more mRNA sequences into their respective amino acid sequences. Alternatively, the autocleaving 2a peptide coding sequence is incorporated between the coding sequences of the different immunostimulatory factors. In this way, two or more factors can be encoded by one single mRNA or DNA molecule.

[0114] Thus, the present invention further provides an mRNA molecule encoding two or more immunostimulatory factors, which are translated separately from a single mRNA molecule by the use of an IRES between the two or more coding sequences. Alternatively, the invention provides an mRNA molecule encoding two or more immunostimulatory factors separated by a self-cleaving 2a peptide coding sequence, allowing cleavage of the two protein sequences after translation.

[0115] In a particular embodiment, the nucleic acid molecule in the composition is selected from the group comprising RNA, DNA, preferably RNA, more preferably mRNA.

[0116] "Nucleic acid" in the context of the present invention is deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA, but may also include cDNA, recombinantly produced molecules and chemically synthesized molecules. According to the present invention, nucleic acids may be single-stranded or double-stranded and may be in the form of linear or closed covalently circular molecules. The nucleic acid can be used for introduction into a cell, ie, transfection of a cell, for example in the form of RNA which can be prepared by in vitro transcription from a DNA template. Furthermore, the RNA can be modified by stabilizing sequences, capping, and / or polyadenylation prior to application.

[0117] In the context of the present invention, the term "RNA" relates to molecules comprising, and preferably consisting entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2'-position of a βD-ribofuranosyl group. The term includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, for example to the end(s) of the RNA, or internally, for example at one or more nucleotides of the RNA. Nucleotides in an RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs. The nucleic acid can be contained in a vector. The term "vector" as used herein includes any vector known to those skilled in the art, including plasmid vector, cosmid vector, phage vector such as lambda phage, viral vector such as adenovirus or baculovirus vector, or artificial chromosome vector such as bacterial artificial chromosome (BAC), yeast artificial or naturally occurring RNA analogue.

[0118] According to the present application, "RNA" includes "mRNA" which means "messenger RNA" and preferably relates to "transcripts" which can be produced using DNA as a template and which code for a peptide or protein. mRNA typically includes a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-time in cells and in vitro. Preferably, the mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the present invention, the RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, there are various in vitro transcription kits available commercially.

[0119] In certain embodiments, the nucleic acid molecules of the composition comprise one or more of the following: a 5' CAP, a poly(A) tail and / or a modified nucleoside(s); wherein said modified nucleoside is N1-methylpseudouridine.

[0120] In the context of the present invention, the term "5 prime cap (5CAP)" should be understood as a specifically modified nucleotide on the 5' end of some primary transcripts, such as precursor messenger RNAs, that are synthesized during the mRNA capping process. In certain embodiments, the in vitro transcribed mRNA molecule may have a 5'CAP-1, 5'CAP-2, 5'm6Am structure, or derivatives thereof. The eukaryotic 5' cap contains a 7-methylguanosine (m7G) attached to the first base by a triphosphate bridge, forming a structure known as the ARCA cap analog (5' CAP-0 analog). In the context of the present invention, the term "5' CAP-1" (CleanCap) means a CAP-0 structure having an additional methyl group (2' monomethylation) on the second carbon of the ribose sugar of the first cap-proximal nucleotide, as represented herein below: [ka]

[0121] In the context of the present invention, the term "5' CAP-2" refers to a CAP-1 structure with an additional methyl group (2' dimethylation) on the second carbon of the ribose sugar of the second cap-proximal nucleotide. In the context of the present invention, the term "5'm6Am" refers to a CAP-1 structure in which the first nucleotide is an adenosine with a methyl group on the sixth nitrogen, forming N6-methyladenosine (m6Am). As used herein, capping can include capping strategies during mRNA production. In certain embodiments, capping can refer to "co-transcriptional capping" in which a cap analog is incorporated during transcription. For example, CleanCap® technology is a proprietary, co-transcriptional 5' capping solution that generates a native Cap 1 structure. In another embodiment, capping can refer to "post-transcriptional capping" in which a cap analog is incorporated after mRNA synthesis using an enzyme-based method.

[0122] The term "nucleoside" refers to a nucleotide that does not have a phosphate group. In a further embodiment, one or more of the mRNA molecules of the present invention may further comprise at least one modified nucleoside. In another specific embodiment, two, three, four, ... or all of the mRNA molecules used in the present invention have at least one modified nucleoside.

[0123] In another particular embodiment of the invention, said mRNA molecule further comprises at least one modified nucleoside, such as selected from the list comprising pseudouridine, 5-methoxyuridine, 5-methylcytidine, 2-thiouridine, and N6-methyladenosine.

[0124] In a particular embodiment of the invention, the at least one modified nucleoside is 4-thiopseudouridine, 2-thiopseudouridine, 1-carboxymethyl-pseudouridine, 1-Ethyl-pseudouridine; 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, N1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, Dihydropseudouridine, 2-thiodihydropseudouridine, 4-Methoxy-pseudouridine, and The pseudouridine is selected from the list including 4-methoxy-2-thio-pseudouridine. In a very particular implementation, the at least one modified nucleoside is N1-methyl-pseudouridine.

[0125] Further nucleoside modifications suitable for use within the present invention include: pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethylpseudouridine, 5-Taurinomethyl-2-thio-uridine, 1-Taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, Dihydrouridine, Dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-Methoxyuridine, 2-Methoxy-4-thio-uridine, 4-Methoxy-pseudouridine and 4-Methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of: 5-aza-cytidine, Pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylthytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, Pyrrolo-cytidine, Pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 2-Methoxy-cytidine, 2-Methoxy-5-methyl-cytidine, 4-Methoxy pseudoisocytidine, and 4-Methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the following group: 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyladenosine, 7-methylthioadenine, 2-methylthio-adenine, and 2-Methoxyadenine. In some embodiments, the mRNA comprises at least one nucleoside selected from the following group: Inosine, 1-methylinosine, Wyosin, Wibtsin, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-Methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guaiguanosine, and N2,N2-Dimethyl-6-thio-guanosine.

[0126] The mRNA molecule used in the present invention may contain one or more modified nucleotides, and in particular embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleotides of a particular type may be replaced by modified nucleotides.It is not excluded that different nucleotide modifications are contained in the same mRNA molecule.In a very particular implementation of the present invention, about 100% of the uridines in the mRNA molecule are replaced by N1-methylpseudouridine.

[0127] As used herein, the term "poly(A) tail" should be understood as a moiety that contains multiple adenosine monophosphates and is well known in the art. Poly(A) tails are generally produced during a process called polyadenylation, a post-translational modification that commonly occurs during the production of mature messenger RNA; such poly(A) tails contribute to the stability and half-life of the mRNA and can be of variable length. In particular, the poly(A) tail can be 10 or more adenosine nucleotides, including 100 or more adenosine nucleotides, including 20 or more adenosine nucleotides, for example, about 90 to 120 adenosine nucleotides, such as about 90 or about 120 adenosine nucleotides.

[0128] The nucleic acid molecules used or referred to herein can be either naked mRNA or DNA, or protected mRNA or DNA. Protection of DNA or mRNA increases its stability but preserves the ability to use mRNA or DNA for vaccination purposes. Non-limiting examples of protection of both mRNA and DNA can be liposomal encapsulation, protamine protection, (cationic) lipid lipoplexing, lipidic, cationic or polycationic compositions, mannosylated lipoplexing, bubble liposomal, polyethyleneimine (PEI) protection, liposome-filled microbubble protection, lipid nanoparticles, etc.

[0129] The present invention also provides a composition as defined herein, wherein one or more of said mRNA molecules are comprised in a nanoparticle.

[0130] As used herein, the term "nanoparticle" refers to any particle having a diameter making the particle suitable for systemic, particularly intratumoral, intramuscular or intravenous administration, particularly nucleic acids, typically having a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, for example between 50 and 200 nm, preferably between 80 and 160 nm.

[0131] In a particular embodiment of the invention, the nanoparticles are selected from the list comprising lipid nanoparticles and polymeric nanoparticles.

[0132] The present invention further provides lipid nanoparticles comprising the compositions described by the present invention.

[0133] In the present context, the term "lipid nanoparticle" or LNP refers to a nano-sized particle composed of one or more lipids, such as a combination of various lipids. The lipids that may be used in LNPs may be, for example, but are not limited to, at least one phospholipid, at least one polymer-modified lipid, such as PEG lipid or polysarcosine lipid, at least one cationic or ionizable lipid, at least one sterol.

[0134] In the context of the present invention, the term "ionizable" (or alternatively cationic) in the context of a compound or lipid refers to any uncharged group in said compound or lipid that can dissociate by generating an ion (usually an H+ ion) and is therefore itself positively charged. Alternatively, any uncharged group in said compound or lipid can give up an electron and therefore become negatively charged. The pH sensitivity of ionizable lipids is beneficial for in vivo mRNA delivery, as neutral lipids have less interaction with the anionic membrane of blood cells, thus improving the biocompatibility of lipid nanoparticles. As used herein, any type of ionizable lipid may be used appropriately. For example, a suitable ionizable lipid is an ionizable amino lipid that includes two identical or different tails linked via S-S bonds.

[0135] In the context of the present invention, the term "PEG lipid" or alternatively "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. In the context of the present invention, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are most often linked together by a glycerol molecule, and therefore, in the phospholipids of the present invention, they are preferably glycerol phospholipids.

[0136] In the context of the present invention, the term "sterols", also known as steroid alcohols, is a subgroup of steroids that occur naturally in plants, animals and fungi, or may be produced by some bacteria. In the context of the present invention, any suitable sterol may be used, such as, for example, cholesterol.

[0137] In certain embodiments of the invention, one or more of the following apply: - the LNP comprises about and between 10 mol% and 70 mol% of the ionizable lipid; - the LNPs comprise between about 1 mol% and 40 mol% of the phospholipid; - the LNPs comprise between about 0.5 mol% and 10 mol% of the PEG-lipid; The amount of sterol is balanced.

[0138] In preferred embodiments of the invention, one or more of the following apply: - the LNPs comprise between about 35 mol% and 65 mol% of the ionic lipid; - the LNPs comprise between about 5 mol% and 25 mol% of the phospholipid; - the LNPs comprise between about 0.5 mol% and 3.0 mol% of the PEG-lipid; The amount of sterol is balanced.

[0139] In another preferred embodiment, the LNPs of the invention comprise 50 mol % ionizable lipid, 10 mol % phospholipid, 1.5 mol % PEG lipid, and 38.5 mol % sterol.

[0140] In some embodiments, the mixture of lipids forms lipid nanoparticles. In some embodiments, the compositions of the present invention are formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed as empty lipid nanoparticles and combined with the composition immediately prior to (e.g., within minutes to an hour) administration, particularly vaccine administration.

[0141] For the avoidance of doubt, the LNPs of the present invention may comprise a composition or may comprise multiple compositions, such as a combination of one or more compositions comprising nucleic acids encoding immunomodulatory proteins; and / or one or more compositions comprising nucleic acids encoding co-stimulatory molecules, cytokines, and / or chemokines.

[0142] In very specific embodiments, the LNPs of the invention may comprise a composition comprising a nucleic acid encoding an immune modulatory molecule and one or more nucleic acid molecules encoding a peptide derived from a tumor or cancer cell. For example, an LNP of the invention may comprise a composition comprising a nucleic acid encoding a tumor-derived peptide in combination with one or more nucleic acids encoding the immunostimulatory protein IL-21, optionally in combination with one or more other nucleic acid molecules encoding co-stimulatory molecules, cytokines, and / or chemokines. It is further understood that the LNP of the present invention may comprise said composition, said nucleic acid molecule or said pharmaceutical formulation according to the present invention.

[0143] In some embodiments, two or more different nucleic acid molecules (eg, mRNA) encoding immunostimulatory proteins and / or tumor antigens can be formulated in the same lipid nanoparticle. In other embodiments, two or more different nucleic acid molecules encoding immunostimulatory proteins or tumor antigens can be formulated in separate lipid nanoparticles (each nucleic acid is formulated in a single lipid nanoparticle). The lipid nanoparticles may then be administered in combination as a single composition (e.g., containing multiple immunostimulatory proteins or multiple nucleic acids encoding tumor antigens) or may be administered separately.

[0144] The compositions, nucleic acid molecules, or pharmaceutical preparations defined herein can be formulated in lipid nanoparticles (LNPs), which encapsulate the constructs, protecting them from degradation and facilitating cellular uptake.

[0145] The present invention further provides a pharmaceutical formulation comprising the composition or lipid nanoparticles and at least one pharma- ceutically acceptable carrier or excipient.

[0146] In particular, as defined herein, the term "pharmaceutical formulation" is used in the context of said LNPs, particularly comprising the composition of the invention in combination with at least one pharma- ceutically acceptable carrier or excipient. However, the term "pharmaceutical formulation" may also refer to a combination of a composition of the present invention with at least one pharma- ceutically acceptable carrier or excipient (ie, not encapsulated within an LNP). This combination can be formulated, for example, in an LNP, particularly for the purpose of extending the stability and / or improving delivery of the nucleic acid.

[0147] As used herein, a "formulation" refers to any mixture of two or more products or compounds (e.g., drugs, modulators, regulators, etc.). It can be a solution, suspension, liquid, or aqueous formulation, or any combination thereof.

[0148] In the context of this specification, by means of the term "pharmaceutical formulation" is meant a formulation having medicinal properties. In other words, reference is made to a formulation that provides a pharmacological and / or physiological effect. A pharmaceutical formulation may include one or more pharma- ceutically acceptable agents, such as excipients, carriers, diluents, etc.

[0149] In some embodiments, pharma- ceutically acceptable agents include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form.

[0150] As used herein, and unless otherwise specified, the term "excipient" should be understood as any substance formulated with an active compound that is included for the purpose of long-term stabilization, such as preventing denaturation or aggregation over the expected shelf life, bulking up liquid or solid formulations containing small amounts of potent active compounds (and thus often referred to as "bulking agents," "fillers," or "diluents"), or imparting enhancements to the active compound in the final dosage form, such as facilitating absorption, reducing viscosity, or increasing solubility.

[0151] Particular formulations of the invention include, for example: Buffer solution: phosphate buffer (potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate), Other additives: Potassium chloride, Sodium chloride, Sucrose, Water for injection, Buffer: Tris (tromethamine) Other excipients: Sodium acetate, sucrose, water for injection

[0152] The pharmaceutical preparation is particularly suitable as a vaccine.

[0153] In the context of the present invention, the term "vaccine" as used herein means any preparation intended to provide adaptive immunity (antibody and / or T cell response) against a disease. To that end, the term "vaccine" as used herein includes at least one composition or at least one lipid nanoparticle, or at least one pharmaceutical preparation, on which an adaptive immune response is mounted. In the context of the present invention, the term vaccine can be used interchangeably with the term pharmaceutical preparation.

[0154] In some embodiments, the vaccine may comprise naked nucleic acid suspended in an appropriate injection buffer, such as Ringer's lactate buffer.

[0155] The vaccines of the invention can be used prophylactically (e.g., prior to the manifestation of symptoms) or therapeutically (e.g., to actively treat or alleviate ongoing disease symptoms such as tumor growth). Administration of a vaccine is referred to as vaccination.

[0156] In a further aspect, the present invention provides compositions, lipid nanoparticles or pharmaceutical formulations for use in parenteral administration, more particularly for use in vaccine administration routes known in the art such as intravenous, intratumoral, intradermal, intraperitoneal, intramuscular or intranodal administration, preferably intratumoral administration.

[0157] The vaccine of the present invention is particularly intended for intratumoral administration, i.e., injection of liquid material directly into a tumor, where the liquid is rapidly, i.e., systematically, absorbed into the circulation.

[0158] The present invention also provides for vaccines to be administered intravenously, i.e., injection of liquid substances into a vein. The intravenous route is the fastest way to deliver fluids and drugs throughout the body, but results in widespread systemic exposure.

[0159] In the context of the present invention, the vaccine may be administered prophylactically (=preventively) or therapeutically as part of an active vaccination scheme to an individual early in the onset of the disease, after the onset of symptoms, or after the expression or detection of one or more tumor antigens.

[0160] In some embodiments, the vaccine can be administered as a monotherapy or as a combination therapy.As described in the Examples section, the term "monotherapy" should be understood as a vaccine administration that includes a nucleic acid that codes for a single immunostimulatory protein, such as IL-21.In contrast, the term "combination therapy" refers to a vaccine administration that includes a nucleic acid that codes for two or more immunostimulatory proteins, such as IL-21 combined with IL-7.

[0161] Alternatively, combination therapy may also be used in the context of the compositions of the invention encoding IL-21, in combination with or without other immunostimulatory molecules or other standard of care tumor treatments such as chemotherapy or radiation therapy.

[0162] In another embodiment, the therapeutic agents of the invention comprising nucleic acids encoding one or more immunomodulatory proteins may also be administered in combination with an antigen-specific tumor vaccine resulting in a "combination therapeutic" effect.

[0163] In some embodiments, the vaccine (as a monotherapy or combination therapy) may be administered as a single dose, two doses, three doses, four doses, preferably three doses, or repeatedly for as long as the subject requires it.

[0164] In some embodiments, the duration of administration in monotherapy or combination therapy can be, but is not limited to, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year, or may be repeated yearly for as long as the subject requires.

[0165] In another embodiment, the administration time between injections in combination therapy (i.e., mono / combination therapy with another anti-tumor vaccine) can be, but is not limited to, 1 minute to 30 minutes, 30 minutes to 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year.

[0166] For each of the above administrations, dosage may also vary, such as a higher dosage at the beginning of treatment and a lower dosage towards the end of treatment.

[0167] In certain embodiments, the present invention also provides compositions, lipid nanoparticles, pharmaceutical formulations or vaccines for use in human or veterinary medicine.

[0168] The use of the compositions, lipid nanoparticles, pharmaceutical formulations or vaccines defined herein in human or veterinary medicine is also contemplated.Finally, the present invention provides methods for the prevention and / or treatment of human and veterinary disorders by administering to a subject in need thereof a composition, lipid nanoparticle, pharmaceutical formulation or vaccine defined herein.

[0169] In a more preferred embodiment, the composition, lipid nanoparticle or pharmaceutical formulation as defined herein is provided for use in the prevention and / or treatment of a cell proliferative disorder.

[0170] In the context of this application, the terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely stabilises or cures the disease and / or the adverse effects caused by the disease. "Treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject who may be predisposed to the disease or condition, but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., halting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or condition.

[0171] In yet another embodiment, a composition, lipid nanoparticle or pharmaceutical formulation as defined herein is provided for use in eliciting an immune response against tumors and / or cancer in a subject.

[0172] The term "immune response" as used throughout this specification is not intended to be limited to the types of immune responses exemplified herein, and thus includes all tumor or cancer antigens against which vaccination would be beneficial to a subject.

[0173] The vaccine of the present invention can be used to induce an immune response, in particular an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen, such as an immune response against a tumor-associated antigen. Preferably, said immune response is a T cell response. In one embodiment, the disease-associated antigen is a tumor antigen. The antigen encoded by the nucleic acid of the present invention described herein is preferably a disease-associated antigen or induces an immune response against a disease-associated antigen or a cell expressing a disease-associated antigen.

[0174] In a preferred embodiment, the present invention provides compositions that are used for direct in vivo application.

[0175] Direct in vivo delivery provides strong activation and stimulation for DCs, resulting in the induction of a T cell attractant and stimulatory environment. Furthermore, when antigen mRNA is co-delivered with the composition of the present invention, this results in the recruitment of antigen-specific CD4+ and CD8+ T cells and CTLs against various tumor antigens. In particular, in vivo delivery of the composition causes maturation of DCs after uptake and translation of the mRNA itself, thus allowing for strong antigen expression by these DCs. Co-delivery of the composition and antigen mRNA significantly enhances the induction of antigen-specific T cells compared to direct in vivo delivery of antigen mRNA alone. These observations point to the value of co-delivering mRNA compositions for the induction of strong antigen-specific immune responses against cancer.

[0176] In a preferred embodiment, the compositions, lipid nanoparticles or pharmaceutical formulations defined herein are provided for use in combination with standard cancer treatments, including radiation therapy and chemotherapy.

[0177] The present invention further provides a method of treating a patient in need thereof with the composition, lipid nanoparticle or pharmaceutical formulation of the present invention or the vaccine of the present invention.

[0178] The present invention further provides a composition, lipid nanoparticle or pharmaceutical formulation as defined herein for treating cancer. In the case of active immunotherapy against cancer, the treatment with the composition, lipid nanoparticle or pharmaceutical formulation of the invention can be preceded, combined or followed by any non-specific treatment of immune modulation in order to improve the activity of the invention or to take advantage of synergies between different treatment modalities, for example by improving the immune response to the invention by non-specifically stimulating the patient's immune system with cytokines (e.g. interleukin-2 or interferon alpha-2b) or TLR-ligands, or for example by combining the invention with co-stimulating signal-modulating drugs such as ipilimumab or tremelimumab. The present invention also provides for combination treatment regimens, where the present invention itself and a defined number of other immunomodulatory treatments are used to provide a more aggressive treatment plan, for example sequential use of the present invention with modality 1 (e.g., cytokines), followed by use of the present invention for in vivo or ex vivo expansion of vaccine immune cells, followed by adoptive cell transfer of these cells, followed by combination treatment of the present invention with an additional modality (e.g., co-stimulatory receptor signal modulators), or any possible combination of combination and / or sequential use of the present invention and additional immunomodulatory treatment.

[0179] In a preferred embodiment, the invention resides in the co-expression of IL-21 with a target-specific antigen, thereby resulting in an increase in the immunostimulatory effect of DCs in the tumor environment. In a further preferred embodiment, a specific combination of IL-21, 4-1BBL and IL-7 is used to improve the immunostimulatory effect of DCs. In another preferred embodiment, a specific combination of IL-21, 4-1BBL and IL-15sushi is used to improve the immunostimulatory effect of DCs. In all of these embodiments, any of the following markers can be introduced simultaneously: cytokines and / or chemokines.

[0180] Table 1: Human amino acid sequences of the encoded immunostimulatory proteins. SEQ ID Peptide or fragment NCBI number Amino acid sequence [Table 1]

[0181] Table 2: Mouse amino acid sequences of the encoded immunostimulatory proteins. SEQ ID Peptide or fragment NCBI number Amino acid sequence [Table 2-1] [Table 2-2]

[0182] Table 3: Mouse nucleic acid sequences of encoded immunostimulatory proteins. SEQ ID Peptide or Fragment NCBI Number Nucleotide Sequence (DNA) [Table 3-1] [Table 3-2] [Table 3-3] EXAMPLES

[0183] The invention will now be illustrated by way of the following synthetic and biological examples which in no way limit the scope of the invention.

[0184] Materials and Methods: In vitro transcription of mRNA mRNAs encoding various cytokines (muIL-21, muIL-7, and muIL-15sushi), co-stimulatory molecules (mu4-1BBL), or FireFly or Nano luciferase were prepared in vitro by T7-mediated transcription from linearized DNA templates (peTheRNAvs3 vectors) incorporating 5' and 3' UTRs and polyA tails. The final mRNAs utilize Cap1 and replace 100% of uridines with N1-methyl pseudouridine.

[0185] Synthesis and purification of mRNA-lipid-based nanoparticles Lipid-based nanoparticles were fabricated by microfluidic mixing of mRNA solution in sodium acetate buffer (100 mM, pH 4) with lipid solution at a volume ratio of 2:1 at a rate of 9 mL / min using a NanoAssemblr Benchtop (Precision Nanosystems). The lipid solution contained a mixture of suitable ionizable lipids, DSPC (Avanti), cholesterol (Sigma) and DMG-PEG2000 (Sunbright GM-020, NOF). The four lipids were mixed in a standard ratio of 50 / 10 / 38.5 / 1.5 (ionizable lipid, helper lipid, cholesterol, PEG lipid). LNPs were dialyzed against TBS (10000 times more TBS volume than LNP volume) using a slide alyzer dialysis cassette (20K MWCO, 3 mL, ThermoFisher). Size, polydispersity and zeta potential were measured using a Zetasizer Nano (Malvern) and mRNA encapsulation was measured by a standard RiboGreen RNA assay (Invitrogen).

[0186] Cell line: The most optimal culture conditions, including growth medium, subculture ratios, and medium renewal recommendations, are summarized below. To harvest adherent cells, the spent growth medium is discarded and the cells are rinsed twice with phosphate-buffered saline (PBS) (Sigma), followed by the addition of trypsin-EDTA (0.05%) (Gibco, Thermo Fisher Scientific) to loosen the cells. Medium renewal should be performed every 2–3 days, whenever the cells reach approximately 70% confluence. Cell viability was determined using a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter).

[0187] Table 4.Cell type specific culture conditions [Table 4] Abbreviations: DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum

[0188] Tumor inoculation and intratumoral injection MC38 tumor cells were inoculated into C57 / BL6 mice. Female C57BL / 6J mice were purchased from Charles River Laboratories (France) and housed in individually ventilated cages containing standard bedding material. Animals were maintained and treated in accordance with the Facility for Animal Experiments (UGhent) and European Union guidelines. Mice had free access to food and water. Experiments began when mice were approximately 7-9 weeks old. In preparation for subcutaneous tumor inoculation, mice were anesthetized with 2.5% isoflurane and the injection site was shaved. The injection site is typically on the posterior / lateral aspect of the left lower flank. For the purposes of inoculation, cells need to be approximately 1 week in culture and between passages 3-5 after thawing. Cold tumor cell solution was injected subcutaneously into the left flank at a dose of 0.5×10e6 cells / 50μl PBS. Tumor growth was measured every 2-3 days using a Caliper device. Tumor size was calculated using the following formula: (tumor width*tumor width*tumor length) / 2. Tumors with an average volume of 50-100 mm were 3When t10 was reached, mice were randomized into vehicle and mRNA treatment groups (8-10 mice per group) and treatment was initiated. Tumors were injected as monotherapy (or combination therapy) with LNPs containing mRNA (5-15 μg mRNA / dose in 20 μl TBS buffer) or control buffer (TBS) using U-100 insulin needles (BD Biosciences, San Diego, CA, USA). After injection, mice were constantly monitored for 5-10 min until fully awake without showing any signs of pain distress or complications. Mice were monitored every other day, tumor volumes were measured with calipers, and body weights were followed 2-3 times a week after treatment.

[0189] For combination therapy, mRNAs encoding different cytokines were also formulated into s-Ac7-Dog LNPs. 3 When tumor volume reached 100 mg / kg / day, animals were treated with three intratumoral doses of mRNA (10 μg or 15 μg total mRNA / dose; as indicated). Treatment was by injection on days of randomization (DR), DR+3, and DR+7. Control animals were treated with an equal amount of irrelevant mRNA (NanoLuc). Tumor volumes were measured at the indicated time points using calipers and recorded in cubic millimeters. The effect of combination therapy was tested with IL-21 in combination with other immune-modulating molecules such as Il-7, IL-15sushi, and 4-1BBL.

[0190] 4. Data Analysis All raw data were analyzed using Graph Pad Prism version 7 software.

[0191] result Example 1 Efficacy of mRNA Monotherapy in the MC38 (Colon Cancer) Model The efficacy of monotherapy with IL-21 mRNA monotherapy was evaluated in the MC38 colon adenocarcinoma model. 5MC-38 colon tumor cells were established subcutaneously in C57BL / 6 mice, and mRNA encoding IL-21 and an unrelated molecule, Fluc, were prepared and formulated in LNPs as described in M&M. 3 When tumor volume reached 100 mg / kg / day, animals were treated with three intratumoral doses of mRNA (15 μg total mRNA / dose). Treatments were administered by injection on days randomized (DR), DR+3, and DR+7. Control animals were treated with an equivalent amount of negative control mRNA. Tumor volumes were measured at the indicated time points using calipers and recorded in cubic millimeters.

[0192] Complete responses ("CR") were observed in 2 of 8 (20%) mice treated with Fluc mRNA control (3 x 15 μg total mRNA / dose) (Figure 1A). Treatment with IL-21 mRNA (3 x 15 μg total mRNA / dose) resulted in 6 / 8 (75%) complete responses (Figure 1B). Figures 1C, 1D, and 1E show the lack of efficacy for IL-7, 4-1BBL, or IL-15sushi mRNA monotherapy treatment (3 x 15 μg total mRNA / dose), respectively. IL-15sushi treated mice showed complete responses ("CR") in 2 of 8 (20%) mice, similar to the control group (Figure 1E).

[0193] Example 2 Synergistic effect of mRNA combination therapy in MC38 (colon cancer) model IL-7 and IL-21 The addition of IL-7 to the mRNA encoding IL-21 increases the efficacy of the treatment in the MC38 colon cancer model. Treatment with NanoLuc mRNA control (3 x 10 μg mRNA) showed a lack of efficacy (Figure 2A). Treatment with IL-21 monotherapy (3 x 5 μg total mRNA / dose) and mRNA encoding the filler RNA NanoLuc (3 x 5 μg total mRNA / dose) showed a complete response ("CR") in 2 of 8 subjects (20%) and a partial response in 2 of 8 animals (20%) (Figure 2B). Treatment with mRNA encoding IL-7 monotherapy (3 × 5 μg total mRNA / dose) and the filler RNA NanoLuc (3 × 5 μg total mRNA / dose) showed a complete response ("CR") in 1 of 8 subjects (20%) and a partial response in 2 of 8 animals (20%) (Figure 2C). Combination therapy with mRNA encoding IL-7 and IL-21 (3x 5 μg each, 10 μg total mRNA / dose) resulted in a complete response ("CR") in 3 of 8 animals (37.5%) and a partial response in 2 of 8 animals (20%) (Figure 2D). The data showed that the IL-21 / IL-7 combination provided improved efficacy when compared to monotherapy treatment.

[0194] IL-15sushi and IL-21 Treatment with mRNA encoding IL-21 and IL-15sushi mRNA (3 × 7.5 μg each, 15 μg total mRNA / dose) did not appear to increase efficacy of treatment in the MC38 colon cancer model over that observed with IL-21 alone (see Figures 3B and 3A). Complete responses were observed in 6 of 8 mice (75%) with either treatment.

[0195] IL-7, 4-1BBL and IL-21 Combining mRNAs encoding IL-21, IL-7 and 4-1BBL (3×5 μg each mRNA / dose) resulted in a 100% response in all animals (FIG. 3C).

[0196] IL-15sushi, 4-1BBL, IL-21 Combining mRNA encoding IL-21, IL-15sushi, and 4-1BBL (3 × 5 μg each mRNA / dose) resulted in complete responses in 6 of 8 animals (75%) (Figure 3D).

[0197] Example 3 Generation of systemic memory immune responses following treatment with IL-21 monotherapy or IL-21-based combination therapy Memory immune responses were evaluated in animals treated with IL-21 monotherapy or combination API therapy. 5 Mice inoculated with MC38 tumor cells were treated with IL-21 monotherapy. 15 micrograms of total mRNA was injected intratumorally (three times) as described in the previous example. Complete responders from the treatment group were injected 80 days after tumor inoculation with 5×10 5 MC-38 colon tumors were re-injected into the contralateral flank. As a control, 12 untreated animals were also injected with MC38 carcinoma.

[0198] After IL-21 monotherapy Untreated animals (n = 12) were unable to control tumor growth (Figure 4A). In contrast, complete responders (n = 6) that received intratumoral treatment were rechallenged with the same cancer cells, but no tumor growth was observed in any of the rechallenged mice until at least day 22 post-inoculation (6 out of 6 mice) (Figure 4B). The generation of systemic anti-cancer memory post local therapy was observed for mice treated with monotherapy IL-21.

[0199] After combination API treatment Complete responder animals that received intratumoral treatment were rechallenged with the same cancer cells, and no tumor growth was observed in any of the rechallenged mice until at least day 26 post-inoculation: 6 of 6 in the IL-21 / 4-1BBL / IL-15sushi-treated group (Fig. 4C) and 8 of 8 in the IL-21 / 4-1BBL / IL-7-treated group (Fig. 4D). These results indicate that intratumoral injection of mRNA encoding IL-21 / 4-1BBL / IL-15sushi or IL-21 / 4-1BBL / IL-7 induces a memory immune response with antitumor effects.

[0200] Example 4 Synergistic effect of IL-21 with IL-7 and 4-1BBL mRNA therapy at 10 μg dose in MC38 (colon cancer) model Monotherapy versus combination therapy of 2 or 3 mRNA was evaluated in the MC38 colon adenocarcinoma model. 5 MC-38 colon tumor cells were established subcutaneously in C57BL / 6 mice. mRNAs encoding ntrIL-21 (non-translated IL-21), IL-21, IL-7 and 4-1BBL were prepared and formulated in LNP as described in M&M. Animals were cultured at 10-20°C for 12-24 h. 3 When tumor volume reached 100 μg, animals were treated with three intratumoral doses of mRNAs (1 API per dose: 3.33 μg mRNA, 2 API per dose: 6.66 μg mRNA, 3 API per dose: 10 μg total mRNA). Doses were administered by injection on days randomized (DR), DR+3, and DR+7. Control animals were treated with an equivalent amount of untranslated IL-21 mRNA. Tumor volumes were measured at the indicated time points using calipers and recorded in cubic millimeters.

[0201] Treatment with IL-21 mRNA (3 x 3.3 μg total mRNA / dose) induced a complete response ("CR") in 3 of 7 (42%) and a partial response in 1 of 7 (14%) (Figure 5B). IL-7 or IL-1BBL mRNA monotherapy treatment (3 x 3.33 μg total mRNA / dose) showed a lack of efficacy for TBS, empty LNP and non-translated IL-21 mRNA LNP treated animals (Figure 5A).

[0202] Combination therapy with mRNAs encoding IL-21 and IL-7 (3x 3.33 μg each, 6.66 μg total mRNA / dose) resulted in complete responses in 4 of 7 patients (57%) and partial responses in 2 of 7 patients (28%) (Figure 5C). The data showed that the IL-21 / IL-7 combination resulted in improved efficacy when compared to monotherapy treatment. Combinations of mRNAs encoding IL-21 and 4-1BBL, and IL-7 and 4-1BBL, did not appear to significantly increase efficacy of treatment in the MC38 nodal cancer model, with only 1 CR out of 7 patients (14%) (Figure 5C).

[0203] Combining mRNA encoding all three molecules, IL-21, IL-7, and 4-1BBL (3x 3.33 μg each mRNA / dose, 10 μg total mRNA / dose), yielded a response rate of 85% (Figure 5D).

[0204] Kariko, K., Muramatsu, H., Ludwig, J. & Weissm et al. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 39, e142 (2011). This study demonstrates the importance of purification of IVT mRNA in achieving potent protein translation and in suppressing inflammatory responses.

Claims

1. A composition comprising one or more isolated non-viral nucleic acid molecules encoding a functional immunostimulatory protein, IL-21, and encoding a co-stimulatory molecule.

2. The composition of claim 1, wherein the co-stimulatory molecule is selected from the list including 4-1BBL, OX40L, ICOS ligand, and CD40L, or is 4-1BBL.

3. The composition of claim 1 or 2, further comprising one or more nucleic acid molecules encoding cytokines and / or chemokines.

4. - said cytokines are selected from the list comprising IL-7, IL-12, IL-18, IFNlambda (also called IFN lambda), IL-15sushi, IL-23, type I IFN, type II IFN, type III IFN, and / or - the chemokine is selected from the list comprising: CCL19, CCL21, CXCL9, CXCL10, CCL5 and XCL1, 4. The composition of claim 3.

5. 10. The composition of claim 1 or 4, further comprising a target specific antigen and / or a checkpoint inhibitor. (Note: Multi-Multi)

6. The composition of claim 5, wherein the target-specific antigen is a tumor-associated antigen or a neo-antigen.

7. The checkpoint inhibitor is selected from the list comprising inhibitors of PD-1, PD-L1, CTLA4, TIGIT, TIM3, LAG6 and VISTA, or An inhibitor of PD-1, PD-L1, or CTLA4. The composition according to claim 5

8. The composition of claim 1, comprising one or more isolated, non-viral nucleic acid molecules encoding any of the following combinations: - IL-21, 4-1BBL and IL-7; - IL-21, 4-1BBL and IL-15sushi; - IL-21, 4-1BBL and IL-18; or - IL-21, 4-1BBL and IFN-lambda.

9. The composition according to any one of claims 1 to 8, wherein the one or more nucleic acid molecules are linear or circular nucleic acids.

10. The composition according to any one of claims 1 to 9, wherein the one or more nucleic acid molecules are selected from the group comprising RNA and DNA, RNA, and mRNA.

11. the one or more nucleic acid molecules comprise one or more of a 5' CAP, a poly(A) tail, and / or a modified nucleoside; or the one or more nucleic acid molecules comprise one or more of a 5'CAP, a poly(A) tail, and / or an N1-methylpseudouridine; The composition according to any one of claims 1 to 10. (Note: Multi-Multi)

12. The composition according to any one of claims 1 to 11, wherein the one or more nucleic acid molecules are in the form of nanoparticles, lipid nanoparticles or polymer nanoparticles, or naked nucleic acid molecules or nucleic acids encapsulated in lipid nanoparticles.

13. A lipid nanoparticle comprising the composition according to any one of claims 1 to 12.

14. A pharmaceutical formulation comprising the composition according to any one of claims 1 to 12 or the lipid nanoparticle according to claim 13, and at least one pharma- ceutically acceptable carrier or excipient.

15. The composition according to any one of claims 1 to 12, the lipid nanoparticle according to claim 13, or the pharmaceutical preparation according to claim 14, which is for use in injection, or for intravenous, intratumoral, intradermal, intraperitoneal, intramuscular or intranodal administration, or for use in intratumoral administration. (Note: Multi-Multi)

16. A composition according to any one of claims 1 to 12, or a lipid nanoparticle according to claim 13, or a pharmaceutical formulation according to claim 14, for use in human or veterinary medicine.

17. The composition according to any one of claims 1 to 12, the lipid nanoparticle according to claim 13, or the pharmaceutical formulation according to claim 14, for use in combination with standard of care cancer therapy, including radiation therapy, targeted therapy, and chemotherapy.

18. A composition according to any one of claims 1 to 12, a lipid nanoparticle according to claim 13, or a pharmaceutical preparation according to claim 14, for use in the prevention and / or treatment of a cell proliferation disorder.

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