Lipid-based nanoparticles comprising membrane il-35

Abstract

The invention relates to lipid-based nanoparticles comprising mRNA molecule(s) encoding membrane interleukin 35 (IL-35), and their use to treat inflammatory and / or auto-immune diseases.

Description

[0001] LIPID-BASED NANOPARTICLES COMPRISING MEMBRANE IL-35

[0002] FIELD OF THE INVENTION

[0003] The invention pertains to the field of immunotherapy. The invention relates to lipid-based nanoparticles comprising mRNA molecule(s) encoding a membrane IL-35, and its use to treat inflammatory and / or auto-immune diseases.

[0004] BACKGROUND OF THE INVENTION

[0005] Both autoimmune diseases and inflammatory conditions find their origin in an inadequate response of the immune system to either cells of the self or byproducts of the inflammation process, resulting in an excessive activation of immune cells.

[0006] The balance between inflammatory and anti-inflammatory immune responses is maintained through immunoregulatory cell populations and immunosuppressive cytokines. Interleukin-35 (IL-35), an inhibitory cytokine that belongs to the IL-12 family, is capable of potently suppressing T cell proliferation and inducing IL-35 -producing induced regulatory T cells to limit inflammatory responses. Over the past decade, a growing number of studies have indicated that IL-35 plays an important role in controlling immune-related disorders, including autoimmune diseases, infectious diseases, and cancer (Ye C, et al., J Interferon Cytokine Res. 2021 Nov;41(l l):391-406). However, half-life of IL-35 in the human body is very short, impairing the efficiency of treatment, as IL-35 is often cleared before reaching the site, cell, tissue or organ of interest.

[0007] On the other hand, vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new delivery platform. LNPs are used to deliver mRNA to cells and have led to the expression of the encoded proteins, thus providing immune-protection to the body. Expressing a protein by delivering the encoding mRNA has many benefits over methods that use proteins, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viralbased vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery.

[0008] However, even if LNPs have been successfully used by leading vaccines against COVID-19, and are considered as possible therapeutic tools to treat cancers, many questions remain regarding the risk of systemic mRNA-LNP distribution and off-target expression of immunogens that could generate systemic cytokines, activate complement, amplify the frequency or severity of adverse events (observed in recent clinical trials) and / or impair immune response generation. Pro- inflammatory concerns with lipid nanoparticles have been raised (Lee et al., Experimental & Molecular Medicine (2023) 55:2085-2096; Moghimi et al., Mol Ther. 2022 Jun 1; 30(6): 2109- 2110), strongly diminishing the attractiveness of their use in the treatment of inflammatory and auto-immune diseases.

[0009] There is therefore a need to overcome these drawbacks for the development of effective treatments for inflammatory and autoimmune diseases.

[0010] The invention seeks to meet this need.

[0011] SUMMARY OF THE INVENTION

[0012] To this end, the inventors have developed lipid-based nanoparticles which comprise mRNA molecule(s) encoding a membrane IL-35.

[0013] A strong advantage of the LNP technology is to obtain a targeting of certain specific tissues, organs, areas, populations of cells, in order to activate more selectively such mechanisms, as compared to free / naked IL-35. For instance IL-35, eventually long-acting IL-35 (Fc-IL35), administered intravenously (i.v) in the systemic circulation, will reach non-selectively undesired areas, whereas the LNP will target appropriate areas.

[0014] The targeting of LNP can be obtained through appropriate compositions of the lipids of the LNPs, e.g., hepatic targeting for inhibiting the inflammation locally in the liver. Numerous LNP compositions (in particular lipidic compositions such as included in the present application) are known to allow such liver capture and targeting of the LNP. Similarly, a targeted delivery notably to the lungs or the brain can be controlled by adjusting the LNP composition.

[0015] IL-35 delivered by LNP is likely to have different effects as regards to immune cells :

[0016] - Binding to immune effector cells, notably T Effector and / or B Effector cells, leading notably to the inhibition of T Effector cells and of their cytotoxic / pro-inflammatory effect;

[0017] - Binding to immune regulatory cells, notably T Regulatory and B Regulatory cells, leading notably (via consuming the IL-35 to the amplification of T Regulatory cells population leading (via notably secretion of IL-10 and IL-35) to immunosuppression of T cells (T Effector, T Helper, etc. . .) and / or of B Effector cells.

[0018] An even more specific targeting of the LNP can be obtained by the inclusion of targeting agents (notably antibodies) to the LNP, to target specifically and locally cells of interest: - T Regulatory cells, in order to amplify their regulatory effect ; for example by using an anti-

[0019] CTLA4 antibody,

[0020] - activated immune cells, notably activated T Effector cells, in order to inhibit their T Effector effect ; for example by using an anti-PDl antibody.

[0021] In a first aspect, the invention concerns a pharmaceutical composition comprising a lipid-based nanoparticle (LNP) comprising one or more mRNA molecule(s) encoding a membrane interleukin 35 (IL-35) molecule comprising a membrane anchoring domain and optionally a pharmaceutically acceptable carrier, for use in the treatment of an inflammatory and / or autoimmune disease.

[0022] The composition of the invention is not only capable of treating inflammation induced by an auto-immune and / or inflammatory condition, but is capable to reverse the inflammation caused by the use of a LNP, allowing to use the convenient and efficient medium that is LNPs for the localized expression of IL-35, a use that was not considered before because of the inflammatory response to LNPs.

[0023] In particular, the IL-35 molecule is a mammalian IL-35 molecule, preferably a human IL-35 molecule.

[0024] The membrane anchoring domain of IL-35 can be a transmembrane domain and / or a Glycosylphosphatidylinositol (GPI)-anchor domain.

[0025] The membrane IL-35 is typically a single-chain polypeptide comprising an EBB subunit and a p35 subunit and, optionally a linker in between.

[0026] In some embodiments, the LNP comprises a mRNA molecule encoding a membrane IL-35 molecule comprises or consists of from N-terminus to C-terminus:

[0027] - optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a membrane anchoring domain; or

[0028] - optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, a membrane anchoring domain.

[0029] In some embodiments, the LNP comprises a mRNA molecule encoding a membrane IL-35 molecule comprises or consists of from N-terminus to C-terminus:

[0030] - optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a transmembrane domain and optionally a cytosolic domain; or - optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, a transmembrane domain and optionally a cytosolic domain; or

[0031] - optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a GPI-anchor domain; or

[0032] - optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, and a GPI-anchor domain.

[0033] Particularly the membrane IL-35 comprises a peptide linker, preferably between the EBB subunit and a p35 subunit of IL-35, said peptide linker being selected from the group consisting of i) a cleavable linker, preferably a 2A self-cleaving peptide, ii) a flexible linker, preferably selected from the group consisting of Glycine- Serine linkers, Glycine-Proline linkers and Proline-Rich linkers, and iii) a rigid linker, preferably selected from the group consisting of a-Helical Linkers, coiled-coil linkers and P-Sheet linkers.

[0034] Optionally, the membrane IL-35 comprises a peptide signal, said peptide signal being p35 or IL- 2 peptide signal, preferably comprising or consisting of a sequence of SEQ ID NO: 42 or 44.

[0035] The membrane IL-35 may further comprise a hinge domain, said hinge domain being preferably selected from the group consisting of a CD8 hinge domain, a CD28 hinge domain, an IgGl hinge domain, an IgG4 hinge domain, a CH2-CH3 domain of an IgGl, a CH2-CH3 domain of an IgG4, a hinge-CH2-CH3 of an IgGl, a hinge-CH2-CH3 domain of an IgG4 and a peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:22-35, 50 and 54.

[0036] In a particular aspect, the membrane IL-35 molecule comprises a hinge domain and a transmembrane domain or a fragment thereof, and optionally a cytosolic domain or a fragment thereof. In some embodiments, the membrane IL-35 further comprises a cytosolic domain or a fragment thereof, preferably selected from the group consisting of a cytokine receptor cytosolic domain, a type I transmembrane protein cytosolic domain and a type II transmembrane protein cytosolic domain.

[0037] In some embodiments, the transmembrane domain is selected from the group consisting of a cytokine receptor transmembrane domain, a chemokine receptor transmembrane domain, a type I transmembrane protein transmembrane domain, a type II transmembrane protein transmembrane domain, preferably from the group consisting of the transmembrane domain is selected from the group consisting of a CD80 transmembrane domain, a MHC1 transmembrane domain, a FAS transmembrane domain, a TNF receptor transmembrane domain or a B cell receptor (BCR) transmembrane domain, preferably is a CD80 transmembrane domain or a BCR transmembrane domain.

[0038] Alternatively, the membrane IL-35 molecule comprises a GPLanchor domain and optionally a hinge domain.

[0039] Typically, the membrane anchoring domain is a GPLanchor domain of a protein selected from the group consisting of ACHE, ALPG, ALPI, ALPL, ALPP, ART3, ART4, BST1, BST2, CD109, CD14, CD160, CD48, CD52, CD55, CD59, CDH13, CEACAM5, CEACAM7, CNTN3, CNTN4, CNTN5, CPM, CPO, CRIPTO, DPEP1, EFNA4, EFNA5, FCGR3B, FOLR1, FOLR2, GP2, GPC1, GPIHBP1, HYAL2, IZUMO1R, LSAMP, LY6D, LY6E, LY6G6C, LY6G6D, LY6H, LY6K, LYPD1, LYPD2, LYPD3, LYPD4, LYPD5, LYPD6B, LYPD8, MDGA1, MDGA2, MELTF, MMP17, MMP25, MSLN, NEGRI, NRN1, NRN1L, NTM, NTNG1, NTNG2, OPCML, OTO A, PLAUR, PLET1, PRND, PRNP, PRSS21, PRSS41, PRSS55, PSCA, RAET1G, RAET1L, RECK, RGMB, RTN4RL1, RTN4RL2, SEMA7A, SPACA4, SPRN, TECTA, TECTB, TEX101, THY1, ULBP2, VNN1, VNN2, XPNPEP2, GPCR3, more preferably from the group consisting of CD48, CD52, CD55, CD59, PRNP, FOLR1, LSAMP, MSLN, DPEP1, THY1 and PLET1, even more preferably comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-151, preferably SEQ ID NOs: 57-145.

[0040] Preferably, the membrane IL-35 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 162-169 and 172-173, or a variant thereof having at least 85% sequence identity thereto.

[0041] In some embodiments, the mRNA molecule comprises or consists of a nucleic acid sequence as set forth in SEQ ID Nos: 46-49, 154-161 and 170-171 or a variant thereof having at least 85% sequence identity thereto.

[0042] The mRNA molecule particularly comprises a 5’UTR, a 3’UTR, 5' cap structure, a Kozak sequence, a chain terminating nucleotide, a stem loop, a poly-A sequence and / or a polyadenylation signal.

[0043] The lipid-based nanoparticle particularly comprises a cationic or ionizable lipid, a helper lipid, a sterol and a PEG-lipid.

[0044] Preferably, the lipid-based nanoparticle comprises from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG- lipid. In some embodiments, the lipid composition of the lipid-based nanoparticle comprises or consists of:

[0045] ALC-0315, DDAB, MC3-DLin-DMA or any mixture thereof, preferably from about 20 mol% to about 60 mol% of the total lipids of the lipid-based nanoparticle;

[0046] DSPC and / or DOPE, preferably from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle;

[0047] Cholesterol, preferably from about 30 mol% to about 70 mol% of the total lipids of the lipid-based nanoparticle; and

[0048] DMG-PEG, preferably from about 0.1 mol% to about 10 mol% of the total lipids of the lipid-based nanoparticle.

[0049] Particularly, the lipid composition of the lipid-based nanoparticle comprises or consists of:

[0050] ALC-0315 or a derivative thereof from about 45 mol% to about 55 mol% of the total lipids of the lipid-based nanoparticle;

[0051] DOPE from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle;

[0052] Cholesterol from about 30 mol% to about 70 mol% of the total lipids of the lipid-based nanoparticle; and

[0053] DMG-PEG from about 0.1 mol% to about 10 mol% of the total lipids of the lipid-based nanoparticle.

[0054] In some embodiments, the LNP comprises an antigen-binding domain capable of specifically binding to a target expressed on activated immune cells surface preferably on activated T cells, activated B cells, activated myeloid cells including activated macrophages, activated dendritic cells and activated neutrophils. In particular, the target is selected from the group comprising CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD154, CD183, CD3, CD4, CD8, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL / TNFSF6, PD1 / PDCD1, GITR / TNFRSF18, GPR32, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LY108 / SlamF6, OX40 / TNFRSF4, RGS1, LTBR / CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3 / TNFRSF6B, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, ICOSL, CD160, CD19, CD20, CD24, CD38 TIM-1, TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R, CLEC-1A, CDl lb, CD14, CD66b, CD163, CD206, SIGLEC 6, BCMA / TNFRSF17, CD150, CD86, OX40L, LOX1, TACETNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74 and / or CD 165.

[0055] The disease envisioned herein is preferably selected from the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoathetosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti -N-Methyl-D- Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, membranous nephropathy, allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune urticaria, Behcet's disease, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune disfunction syndrome, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barre syndrome, graft-versus-host disease (GVHD), hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (FTP), IgA neuropathy, IgM polyneuropathies, juvenile arthritis, Kawasaki's disease, lichen plantus, lichen sclerosus, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes mellitus, Multifocal motor neuropathy (MMN), pemphigoid gestationis, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynauld's phenomenon, Reiter's syndrome, sarcoidosis, solid organ transplant rejection, Takayasu arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteristis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, anti-neutrophil cytoplasmic antibody-associated vasculitides, vitiligo, asthma, autoimmune pancreatitis, IgA nephropathy and Wegner's granulomatosis; optionally selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoathetosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti-N-Methyl-D-Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, and membranous nephropathy, preferably selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, antiphospholipid syndrome, Hashimoto's thyroiditis, Graves' disease and graft rejection.

[0056] Preferably, the disease is an inflammatory and / or auto-immune disease of the liver, preferably selected from the group consisting of Autoimmune hepatitis (AIH), Primary biliary cholangitis (PBC), Primary sclerosing cholangitis (PSC) and non-alcoholic fatty liver disease.

[0057] In some embodiments, the inflammatory and / or auto-immune is a skin inflammatory and / or autoimmune disease, preferably selected from the group consisting of eczema (atopic dermatitis), contact dermatitis, discoid dermatitis, psoriasis, seborrheic dermatitis, lichen simplex chronicus, pityriasis rosea, rosacea, urticaria, erythema multiforme, erythroderma, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, dermatitis herpetiformis, epidermolysis bullosa acquisita, cutaneous lupus erythematosus, discoid lupus erythematosus, dermatomyositis, scleroderma Henoch-Schbnlein purpura, vitiligo, alopecia areata, lichen planus, lichen sclerosis, morphea, and psoriasis, preferably atopic dermatitis or psoriasis, even more preferably psoriasis.

[0058] When the inflammatory and / or auto-immune is a skin inflammatory and / or auto-immune disease, pharmaceutical composition of the invention is formulated for topical, intradermal, transdermal, epicutaneous or subcutaneous administration, preferably topical administration.

[0059] In particular embodiments, the pharmaceutical composition is a topical pharmaceutical composition; the inflammatory and / or auto-immune is psoriasis or atopic dermatitis; and the lipid composition of the lipid-based nanoparticle consists of about 48%-52% ALC-0315, 8%-12% DOPE, 38%-42% cholesterol and about 0.5% and 2% DMG-PEG of the total lipids of the lipid- based nanoparticle. In another aspect, the invention concerns a lipid-based nanoparticle comprising mRNA molecule(s) encoding a membrane interleukin 35 (IL-35) molecule such as described herein.

[0060] The invention also relates to a pharmaceutical composition comprising the lipid-based nanoparticle comprising mRNA molecule(s) encoding a membrane interleukin 35 (IL-35) molecule such as described herein, and optionally a pharmaceutically acceptable carrier.

[0061] The invention concerns finally concerns the use of the lipid-based nanoparticle or the pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment of an autoimmune disease or an inflammatory disease, preferably a skin autoimmune or inflammatory disease. The invention also relates to a method for treating an autoimmune disease or an inflammatory disease, preferably a skin autoimmune or inflammatory disease, wherein the method comprises administering a therapeutic amount of the lipid-based nanoparticle or pharmaceutical composition of the invention.

[0062] DETAILED DESCRIPTION OF THE INVENTION

[0063] Definitions

[0064] In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.

[0065] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.

[0066] As used herein, "heterodimeric cytokine" or "cytokine heterodimer" refers to a cytokine consisting of two distinct protein subunits, such as Ebi3 and p35 subunits of IL-35.

[0067] As used herein, the terms “disorder” or “disease” refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavourable environmental factors. Preferably, these terms refer to a health disorder or disease e.g., an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to immune and / or inflammatory diseases that affect animals and / or humans.

[0068] As used herein, the term "isolated" indicates that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an "isolated" antibody is one which has been identified and separated and / or recovered from a component of its natural environment. As used herein, the “sequence identity” between two sequences is described by the parameter "sequence identity", “sequence similarity” or “sequence homology”. For purposes of the present invention, the "percentage identity" between two sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. The percent identity between the two sequences is particularly a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Said alignment of sequences can be carried out by well-known methods in the art, for example, using the algorithm for global alignment of Needleman- Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool “Emboss needle” for pairwise sequence alignment of proteins providing by EMBL-EBI and available on: www.ebi.ac. uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, for example using default settings: (I) Matrix: BLOSUM62, (ii) Gap open: 10, (iii) gap extend: 0.5, (iv) output format: pair, (v) end gap penalty: false, (vi) end gap open: 10, (vii) end gap extend: 0.5.

[0069] The percent identity between two amino acid sequences or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences or nucleotide sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as the BLASTN program for nucleic acid or amino acid sequences using as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. Alternatively, sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM- HMM comparison'. Bioinformatics 21, 951-960, with the default settings.

[0070] "Eu numbering" (also known as Eu index) refers to the antibody numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda), which is based on the sequential numbering of the first human IgGl sequenced (the Eu antibody; Edelman, et al., 1969, Proc Natl Acad Sci USA 63: 78-85).

[0071] By "amino acid change" or “amino acid modification” is meant herein a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitution, insertion and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). As used herein, “amino acid position” or “amino acid position number” are used interchangeably and refer to the position of a particular amino acid in an amino acids sequence, generally specified with the one letter codes for the amino acids. The first amino acid in the amino acids sequence (i.e., starting from the N terminus) should be considered as having position 1.

[0072] A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:

[0073] Table A - Amino Acid Residue

[0074] Table B - Alternative Conservative Amino Acid Residue Substitution Groups

[0075] Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues The term “and / or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.

[0076] The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.

[0077] The term “about” as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to + / -

[0078] 10% (e.g., + / - 0.5%, + / -1 %, + / -1.5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / -9.5%). The use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).

[0079] The term “essentially” as used herein in connection with any given biological sequence means said biological sequence varies from the reference sequence contained in the sequence listing by up to 10% of the biological sequence length. In particular, by “consists essentially of’ is intended that the biological sequence consists of that sequence, but it may also include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions, deletions or a mixture thereof, preferably 1, 2, 3, 4, or 5 substitutions, additions, deletions or a mixture thereof, with the proviso that said biological sequence varies from the reference sequence contained in the sequence listing by up to 10% of the biological sequence length.

[0080] IL-35

[0081] The LNP of the invention comprises one or more mRNA molecule(s) encoding a transmembrane interleukin 35 molecule.

[0082] Interleukin-35 (IL-35) is a member of the IL-12 family of heterodimeric cytokines and is composed of Ebi3, a b chain subunit encoded by the Epstein-Barr virus (EBV)-induced gene 3 (also known as IL27b), and the IL 12 p35 a subunit encoded by IL- 12a. IL-35 is produced by regulatory T cells and is involved in the immunosuppressive activities of Tregs.

[0083] As used herein, the terms “p35”, “IL 12- A”, “Cytotoxic lymphocyte maturation factor 35 kDa subunit (CLMF p35)”, “IL- 12 subunit p35’ and “NK cell stimulatory factor chain 1 (NKSF1)” are used interchangeably and refer to the p35 subunit of IL-35. Examples of p35 sequences are provided in Uniprot P29459 and in GenBank Accession NP_000873.2, NM_000882.3, NM_008351.3, NM_001159424.3, NM_001397992.1, NM_001354582.2, NM_001354583.2 and NM_000882.4.

[0084] Preferably, the p35 subunit is a human or mouse p35 subunit.

[0085] Preferably, the p35 subunit comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 5 or 6.

[0086] Preferably, the p35 subunit comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 7 or 8. As used herein, the terms “EBi3”, “EBI3”, “IL-27 subunit beta”, “IL-27B”, and “Epstein-Barr virus-induced gene 3 protein (EBV-induced gene 3 protein)” are used interchangeably and refer to the Ebi3 subunit of IL-35. Examples of Ebi3 sequences are provided in Uniprot Q14213, ENSMUST00000003274.8, and in GenBank Accession NP_005746.2, NM_005755.2, NM_005755.3 and NM_015766.2.

[0087] Preferably, the Ebi3 subunit is a human or mouse Ebi3 subunit.

[0088] Preferably, the Ebi3 subunit comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 1 or 2.

[0089] Preferably, the Ebi3 subunit comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 3 or 4.

[0090] In some embodiments, the IL-35 molecule described herein is derived from any organism, such as mammals, including, but are not limited to, livestock animals (e.g., cows, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., human and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters).

[0091] Preferably, the two subunits are from the same origin, preferably both of human origin.

[0092] Preferably, the IL-35 molecule, the Ebi3 subunit and / or the p35 subunit is / are not or does / do not derive from a canine IL-35.

[0093] Preferably, the IL-35 molecule is or derives from a human IL-35. Alternatively, the IL-35 molecule is or derives from a murine IL-35.

[0094] In some cases, the LNP of the invention comprise a mRNA molecule encoding a Ebi3 subunit or a variant or fragment thereof and a mRNA molecule encoding a p35 subunit or a variant or fragment thereof.

[0095] In some embodiments, the IL-35 molecule comprises a full-length Ebi3 and a full-length p35 subunit. Alternatively, the IL-35 molecule comprises a functional fragment of Ebi3 and / or p35 subunit(s) that is / are capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or some or full receptor binding activity of the full-length IL-35 molecule.

[0096] In some embodiments, the IL-35 molecule is an IL-35 variant or mutant. Such variant typically comprises mutations such as deletion(s), addition(s) and / or substitution(s) in the Ebi3 and / or p35 subunit(s), in particular in the amino acid sequence as set forth in SEQ ID NO: 1 or 2 and / or SEQ ID NO: 5 or 6, preferably SEQ ID NO : 1 and / or 5, respectively. In some aspects, the p35 subunit is a variant or mutant of SEQ ID NO 5 or 6 and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Particularly, the p35 subunit is a variant or mutant of SEQ ID NO 5 or 6 comprising 1, 2, 3, 4 or 5 mutations such as substitution(s), addition(s) and / or deletion(s), preferably substitution(s).

[0097] In some aspects, the p35 subunit is a variant or mutant of SEQ ID NO 7 or 8 and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Particularly, the p35 subunit is a variant or mutant of SEQ ID NO 7 or 8 comprising 1, 2, 3, 4 or 5 mutations such as substitution(s), addition(s) and / or deletion(s).

[0098] Alternatively or additionally, the Ebi3 subunit is a variant or mutant of SEQ ID NO 1 or 2 and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Particularly, the Ebi3 subunit is a variant or mutant of SEQ ID NO 1 or 2 comprising 1, 2, 3, 4 or 5 mutations such as substitution(s), addition(s) and / or deletion(s), preferably substitution(s).

[0099] Alternatively or additionally, the Ebi3 subunit is a variant or mutant of SEQ ID NO 3 or 4 and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Particularly, the Ebi3 subunit is a variant or mutant of SEQ ID NO 3 or 4 comprising 1, 2, 3, 4 or 5 mutations such as substitution(s), addition(s) and / or deletion(s).

[0100] In some aspects, the IL-35 molecule is an IL-35 mutant comprising one or more amino acid substitutions selected from the group consisting of K23, R74, R195, R197, C320, C497, or combinations thereof, for example such as described in WO2023250459. In some aspects, the IL-35 molecule is as described in WO2023250459, the disclosure of which being incorporated herein by reference.

[0101] Preferably, the IL-35 variant or mutant is capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or IL-35 receptor binding activity of a wild-type IL-35. In some embodiments, the IL-35 variant is a modified IL- 35, such as glycosylated IL-35.

[0102] An IL-35 variant, mutant or molecule comprising a functional fragment of Ebi3 and / or p35 subunit(s), is considered capable of producing some or full biological activity, typically when: the binding to IL-35 receptor is conserved or similar to a wild-type IL-35

[0103] - the 11-35 variant reduces the expression of one or more pro-inflammatory cytokine selected from the group comprising IL12p40, IL2 and IL6, typically by 10%, preferably 20%, more preferably 30%, more preferably 40%, more preferably 60%, more preferably 70%, even more preferably 75%, in particular in comparison to the expression of said pro-inflammatory cytokine without IL35; and / or

[0104] - the 11-35 variant allows to decrease the expression of one or more transaminase enzyme selected from the group comprising Aspartate aminotransferase (ASAT) and Alanine aminotransferase (ALAT), typically by 10%, preferably 20%, more preferably 30%, more preferably 40%, more preferably 60%, more preferably 70%, even more preferably 75%, in particular in comparison to the expression of said transaminase enzyme without IL35.

[0105] Preferably, an IL-35 variant, mutant or molecule comprising a functional fragment of Ebi3 and / or p35 subunit(s), is considered capable of producing some (e.g., at least about 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 97% or 99%) or full biological activity and / or IL-35 receptor binding activity of a wild-type IL-35 when it reduces the expression of one or more pro-inflammatory cytokine selected from the group comprising IL12p40, IL2 and IL6, and / or reduces the expression of one or more transaminase enzyme selected from the group comprising Aspartate aminotransferase (ASAT) and Alanine aminotransferase (ALAT) by 10%, preferably 20%, more preferably 30%, more preferably 40%, more preferably 60%, more preferably 70%, even more preferably 75%, in particular in comparison to a condition wherein the LNP is administered without mRNA coding for IL-35.

[0106] In some embodiments, the IL-35 molecule comprises a signal peptide at the N-terminus of the Ebi3 and / or p35 subunit(s), the signal peptide being preferably either from a different molecule or from the native IL-35 molecule.

[0107] In some embodiments, the IL-35 molecule is devoid of a signal peptide. Preferably, the Ebi3 subunit is devoid of a signal peptide. Alternatively, the p35 subunit is devoid of a signal peptide.

[0108] In some aspects, the mRNA molecule encodes a recombinant IL-35 protein.

[0109] “Recombinant” when referring to a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and / or (2) is linked to a polynucleotide other than that to which it is linked in nature and / or (3) is linked to a polynucleotide to encode for a fusion protein. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.

[0110] In some embodiments, the IL-35 molecule is a human IL-35 molecule, such as recombinant human IL-35 molecule. In some embodiments, the IL-35 molecule is a murine cytokine, such as recombinant murine IL-35 molecule. A recombinant IL-35 protein can contain all or a portion of a native IL-35 protein. For example, a recombinant IL-35 protein can contain an entire native IL- 35 human protein or an entire IL-35 native mouse protein. In another embodiment, a recombinant IL-35 protein can contain a portion of a native protein from a human and a portion of a native protein from a mouse (i.e., a “chimeric” protein).

[0111] Preferably, the recombinant IL-35 molecule is a single chain IL-35. Preferably, the membrane IL-35 is a single-chain polypeptide comprising an EBB subunit and a p35 subunit and, optionally a linker in between.

[0112] In some embodiments, the IL-35 molecule is a single chain fusion of the two subunits of IL-35, i.e., Ebi3 and p35. Preferably, the recombinant IL-35 protein is a fusion protein comprising p35 and Ebi3 subunits or any variants or fragments of said subunits. Therefore, in some cases, the LNP molecules of the invention comprise mRNA molecules encoding an IL-35 fusion protein, said fusion protein comprising p35 and Ebi3 subunits of IL-35 or any variants or fragments of said subunits.

[0113] One of ordinary skill in the art will appreciate that a recombinant IL-35 protein can contain other elements that optimize the expression and / or stability of the IL-35 protein. Particularly, the IL- 35 molecule of the invention comprises p35 and Ebi3 subunits and a linker in between. Optionally, when IL-35 is encoded by a mRNA molecule as a fusion construct, the mRNA molecule also comprises a nucleic sequence encoding a linker between the sequences encoding the two subunits of IL-35 (i.e., between the p35 and the Ebi3 subunits).

[0114] Therefore, in some instances, the mRNA molecule encodes a single-chain mammalian polypeptide that includes an EBB subunit and a p35 subunit; and a linker therebetween.

[0115] As used herein, the term "linker" refers to a sequence of at least one nucleic acid or amino acid that links the p35 and the Ebi3 subunits of IL-35. Such a linker may be useful to prevent steric hindrances.

[0116] Preferably, the linker is a flexible linker, a rigid linker or a cleavable linker. In some embodiments, the linker is selected from a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0117] In some embodiments, the IL-35 molecule comprises or consists of a p35 and a Ebi3 subunit and a flexible linker in between.

[0118] As used herein, a “flexible linker” refers to a sequence of amino acids or nucleotides that connect two functional domains or regions within a biomolecule. This linker is typically characterized by its structural flexibility, allowing the connected domains to fold and / or move independently of each other, facilitating conformational changes or interactions with other molecules. A flexible linker can enable the individual domains of fusion constructs to maintain their structural and functional integrity. The purpose of incorporating a flexible linker in a biomolecular structure is generally to provide freedom of movement and spatial separation between functional domains, such as the p35 and Eib3 subunits of IL-35.

[0119] Examples of flexible linkers are Glycine-Serine linker, Glycine-Proline linker, Proline-Rich linker, (GGGS)n linker and (GGGGS)n linker (e.g., SEQ ID NO: 22-25), wherein n indicates the number of repeats of the motif and is an integer selected from 1-10. In some embodiments, the linker is selected from the group consisting of (GGGGS)e (SEQ ID NO: 32), (GGGGS)s (SEQ ID NO: 33), (GGGGS)4(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 23), (GGGGS)2(SEQ ID NO: 24) and GGGGS (SEQ ID NO: 25) linker. Preferably, the linker is a (GGGGS)3linker.

[0120] In some embodiments, the IL-35 molecule comprises or consists of a p35 and a Ebi3 subunit and a rigid linker in between.

[0121] In comparison to a flexible linker, a “rigid linker’ refers to a sequence of amino acids or nucleotides that provides limited flexibility and maintains a more rigid structure. The purpose of a rigid linker is often to restrict the movement or maintain a specific orientation between two functional domains or regions within a biomolecule. Rigid linkers are commonly used in protein engineering to influence the relative positioning of connected domains.

[0122] Examples of rigid linkers are a-Helical Linkers such as (EAAAK)n linker (e.g., SEQ ID NO: 26), (EAAAR)n linker (e.g., SEQ ID NO: 27) and coiled-coil linker such as (AALAA)n (e.g., SEQ ID NO: 28), P-Sheet linkers such as (GAGAGA)n (e.g., SEQ ID NO: 29), KLAKLAKKLAKLAK (SEQ ID NO: 30) and AEAAAKEAAAKA (SEQ ID NO: 31), wherein n indicates the number of repeats of the motif and is an integer selected from 1-10.

[0123] Preferably, the flexible or rigid linker is a non-cleavable linker.

[0124] In some embodiments, the IL-35 molecule comprises or consists of a p35 and a Ebi3 subunit and a cleavable linker in between.

[0125] By “cleavable linker” or “self-cleavable linker” it is meant a type of linker that enables the co- translational "self-cleavage" of a polyprotein during the process of protein synthesis. Such linker is generally engineered to achieve the expression of multiple proteins from a single mRNA transcript. Examples of cleavable linkers are typically from the class of sequences called "2A peptides", such as P2A (porcine teschovirus-1 2 A), T2A (Thosea asigna virus 2A-like peptide), E2A (Equine rhinitis A virus 2A-like peptide), F2A (Foot-and-mouth disease virus 2A-like peptide), A2A (Autographa californica multicapsid nucleopolyhedrovirus 2A-like peptide) and M2A (Medicago sativa 2A-like peptide). Preferably, the linker is a P2A linker comprising or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 21.

[0126] Optionally, the linker may be short or long in term of nucleic acid or amino acid sequence. Preferably, the linker has 3-30 nucleic acids or amino acids residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleic acid residues.

[0127] In some aspects, the mRNA molecule encodes for a fusion protein comprising from N-terminus to C-terminus: a Ebi3 subunit or a variant or fragment thereof, a linker, a p35 subunit or a variant or fragment thereof. Preferably, the mRNA molecule encodes for a fusion protein comprising from N-terminus to C-terminus: a Ebi3 subunit or a variant or fragment thereof, a cleavable or flexible linker, a p35 subunit or a variant or fragment thereof. Even more preferably, the mRNA molecule encodes for a fusion protein comprising from N-terminus to C-terminus: a Ebi3 subunit or a variant or fragment thereof, a 2A or a (GGGGS)3 linker, a p35 subunit or a variant or fragment thereof.

[0128] In some aspects, when the IL-35 molecule is designed as a fusion protein, the IL-35 molecule further comprises a peptide signal, preferably in N-terminus. Preferably, the fusion protein comprises a p35 or a Ebi3 peptide signal. Alternatively, the fusion protein comprises a p35 peptide signal and a Ebi3 peptide signal.

[0129] Particularly, the mRNA encodes for a fusion protein comprising or consisting of a nucleic acid sequence as described in SEQ ID NO: 11 or 12 or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the mRNA encodes for a fusion protein comprising or consisting of a nucleic acid sequence as described in SEQ ID NO 11 or 12, preferably SEQ ID NO: 11.

[0130] Alternatively, in some aspects, the mRNA molecule encodes for a fusion protein comprising from N-terminus to the C-terminus: a p35 subunit or a variant or fragment thereof, a linker, a Ebi3 subunit or a variant or fragment thereof. Preferably, the mRNA molecule encodes for a fusion protein comprising from N-terminus to C-terminus: a p35 subunit or a variant or fragment thereof, a cleavable or flexible linker, a Ebi3 subunit or a variant or fragment thereof. Even more preferably, the mRNA molecule encodes for a fusion protein comprising from N-terminus to C- terminus: a p35 subunit or a variant or fragment thereof, a 2A or a (GGGGS)3 linker, a Ebi3 subunit or a variant or fragment thereof.

[0131] Particularly, the mRNA encodes for a fusion protein and comprises a nucleic acid sequence as described in SEQ ID NO: 9 or 10 or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the mRNA encodes for a fusion protein and comprises or consists of a nucleic acid sequence as described in SEQ ID NO 9 or 10, preferably SEQ ID NO: 9.

[0132] Alternatively, the mRNA encodes for a fusion protein and comprises a nucleic acid sequence as described in SEQ ID NO: 13 or 14 or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the mRNA encodes for a fusion protein and comprises or consists of a nucleic acid sequence as described in SEQ ID NO: 13 or 14, preferably SEQ ID NO: 13.

[0133] The IL-35 molecule of the invention is particularly designed to be inserted in a lipid bilayer or to be addressed or anchored to a cell membrane, preferably a human cell membrane. Typically, said IL-35 molecule comprises a membrane bound signal peptide and / or a membrane anchoring domain. The membrane anchoring domain refers to a mean for maintaining IL-35 molecule near the membrane. It is preferably a domain that enables a polypeptide or protein to be associated with or embed in a cellular membrane, such as the human cellular membrane, especially the cytoplasmic membrane of human cells. For example, said membrane anchoring domain is a polypeptide or protein. For instance, the membrane anchoring domain can be a transmembrane domain or a GPLanchor domain. Therefore, the IL-35 molecule of the invention is a membrane IL-35 molecule.

[0134] In some aspects, the LNP of the invention does not comprise a mRNA molecule encoding for a wild type or native IL35.

[0135] In some aspects, the LNP of the invention does not comprise a mRNA molecule encoding for a soluble IL-35, especially a single chain soluble IL-35, in particular an IL-35 devoid of a membrane anchoring domain.

[0136] As used herein, the terms “membrane IL-35” or “membranous IL-35” are used interchangeably and refer to a membrane-associated form of IL-35 subunits. Typically, membrane IL-35 benefits from an extended half-life compared to the already significant soluble IL-35 half-life. Preferably, the membrane IL-35 molecule is a single chain IL-35 molecule such as described herein, preferably a fusion protein such as described herein. In the context of the invention, the membrane IL-35 comprises any protein, peptide or domain that allows an IL-35 molecule of the invention to bound or be inserted in a lipid bilayer such as a cell membrane. Preferably, the membrane is plasma membrane.

[0137] « Membrane IL-35 » particularly comprises the p35 and / or Ebi3 subunit(s) of IL-35 covalently linked to a membrane anchoring domain, such as a transmembrane domain or a GPI-anchor domain and optionally to a membrane-bound signal peptide. Preferably, « Membrane IL-35 » particularly comprises the p35 and / or Ebi3 subunit(s) covalently linked in C-terminal to a membrane anchoring domain, such as a transmembrane domain or a GPI-anchor domain, transmembrane domain and optionally in N-terminal to a membrane-bound signal peptide.

[0138] In some preferred aspects, the membrane IL-35 environed herein comprises or consists of a human IL-35 PGE construct, covalently linked to a membrane anchoring domain as described herein. Typically, said human IL-35 PGE comprises or consists of an amino acid sequence as described in SEQ ID NO: 13.

[0139] In some aspects, the membrane IL-35 of the invention comprises a membrane-bound signal peptide.

[0140] As used herein, the term “membrane-bound signal peptide” refers to a short amino acid sequence typically found at the N-terminus of proteins, such as IL-35 molecule, destined for membrane insertion. They are also known as signal membrane anchor sequences.

[0141] In particular, the membrane-bound signal peptide is selected from the group consisting of signal peptides of secreted protein, signal peptides of Type I transmembrane protein and signal peptides of Type II transmembrane protein. Type II membrane proteins typically have a specific class of signal sequences called type II signal anchor sequences (SASs).

[0142] Preferably, the membrane-bound signal peptide is the signal peptide of interleukin 2 (IL2) or the signal peptide of IL-35 subunit p35, more preferably the signal peptide of IL2.

[0143] In some embodiments, the IL-35 molecule comprises a membrane-bound signal peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 42 or 44, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0144] In some embodiments, the IL-35 molecule comprises a membrane-bound signal peptide comprising or consisting of an nucleic acid sequence as set forth in SEQ ID NO: 43 or 45, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0145] In some embodiments, the IL-35 molecule comprises a membrane-bound signal peptide having a sequence as set forth in SEQ ID NO: 42 or 43 or SEQ ID NO: 44 or 45, respectively. In some aspects, the membrane IL-35 of the invention comprises a transmembrane domain.

[0146] As used herein, the term “transmembrane domain” or “transmembrane moiety’, when related to IL-35, refers to a portion of a protein that allow anchoring of said protein in the membrane of a cell, especially a eucaryotic cell such as a mammal cell. It typically consists of hydrophobic amino acids that allow the protein to embed itself within the membrane. In the context of the invention, this term preferably encompasses any protein, peptide or domain that allows an IL-35 molecule to bound or inserted in a lipid bilayer such as a cell membrane.

[0147] Typically, in single chain IL-35 of the invention, the transmembrane domain is covalently linked to the C-terminus of the fusion protein, e.g., in C-terminus of the Ebi3 or p35 subunit.

[0148] The transmembrane domain may be selected from any known from the art. The transmembrane domain can be selected from the group consisting of cytokine receptor transmembrane domain, chemokine receptor transmembrane domain, type I transmembrane protein and type II transmembrane protein.

[0149] Preferentially, the transmembrane domain selected has no activating capacity. The transmembrane domain selected does not trigger a signaling pathway that activates the cell expressing the membrane IL-35. Preferentially, the transmembrane domain selected has either an inhibiting activity, a stabilizing activity or no activity.

[0150] In some aspects, the transmembrane domain is a B cell receptor (BCR) transmembrane domain, preferably comprising or consisting of SEQ ID NO: 152 or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0151] In an alternative aspects, the membrane anchoring domain is a GPI-anchor domain. Typically, in single chain IL-35 of the invention, the GPLanchored protein or domain is covalently linked to the C-terminus of the fusion protein, e.g., in C-terminus of the Ebi3 or p35 subunit.

[0152] A wide range of cell- surface proteins, including enzymes, coat proteins, surface antigens, and adhesion molecules, are attached to plasma membranes via GPI anchors (Burikofer et al. 2002 FASEB J 15:545). Glycosylphosphatidylinositol (GPI) is a post-translationally added lipid anchor; therefore, GPI anchors use a lipid structure to attach to the membrane (Englund et al., Annul Rev. Biochem. 62: 121 (1993)). GPI-anchored proteins are synthesized as precursors with a cleavable, hydrophobic signal sequence at or near the N-terminus that targets the protein to the lumen of the endoplasmic reticulum (ER), and a cleavable signal sequence (GPI anchor domain) at or near the C-terminus that directs GPI anchoring. A GPI anchor domain includes a hydrophobic region separated from the GPI-attachment site (omega-site) by a hydrophilic spacer region. GPI anchored proteins have been identified throughout a broad range of eukaryotic species ranging from humans to insects, yeasts, bacteria, and fungi (for example, see Cares et al., Science 243 : 1 196 (1989)) and have been successfully engineered onto the C-terminus of proteins of interest for their attachment to lipid bilayer such as cell membranes.

[0153] As used herein, the term “Glycosylphosphatidylinositol (GPI)-anchor domain” or “GPI anchor” refers to a polypeptide sequence that undergoes post-translational modification during the transfer from the endoplasmic reticulum to the cell membrane, said post-translational modification allowing the anchorage of a protein (e.g., IL-35) in a lipid bilayer, typically of a cell membrane, preferably a mammal cell membrane, more preferably a human cell membrane. Typically, the protein comprising a GPI anchor domain is anchored at the outer surface of the cell membrane.

[0154] In some instances, the GPI anchor domain is a synthetic GPI anchor domain. Such GPI anchor domain is a peptide sequence or domain added to recombinant proteins (such as single chain IL- 35 molecule) to direct their attachment to the outer leaflet of a cell membrane through a GPI anchor.

[0155] GPI-anchored protein typically comprises a hydrophilic oligosaccharide, a lipophilic fatty acid and a GPI anchor domain, which allow their anchorage in a cell membrane.

[0156] The linkage of a GPI anchor domain or GPI-anchored protein to a protein or polypeptide of interest (e.g., an IL-35 of the invention) allows the anchoring of said protein to a lipid bilayer, typically a cell membrane, preferably a mammal cell membrane, more preferably a human cell membrane.

[0157] The GPI anchored domain typically comprises or consists of a conserved core glycan, phosphatidylinositol, and glycan branch chains. The core structure of glycan is preferably EtNP- 6Mana2-Mana6-(EtNP)2Mana4-GlNa6-inositol-P-lipid.

[0158] Typically, in a GPI anchor domain, the amino acid to which a GPI becomes linked is referred to as the “w” residue, with amino acids N-terminal to it designated w- and those C-terminal to it designated w+. The general features of a signal anchor sequence are as follows: i) a stretch of about 10 polar amino acids (w-10 to w-1) that form a flexible linker region; ii) the w amino acid, typically G, A, S, N, D, or C; iii) the w+2 amino acid, the most restrictive position, with predominantly G, A, or S; iv) a spacer region of moderately polar amino acids (w+3 to w+9 or more); and v) a stretch of hydrophobic amino acids variable in length but capable of spanning the membrane.

[0159] Examples of suitable GPI anchor domains include, but are not limited to, those disclosed in thOrlean P, Menon AK.. J Lipid Res. 2007;48(5):993-1011. doi: 10.1194 / jlr.R700002-JLR200, Doering, T. L. et al. (1990) J. Biol. Chem. 265:61 1-614; McConville, M. J. et al. (1993) Biochem. J. 294:305-324; and PCT Publication W02003017944; all of which are hereby incorporated by reference in their entirety.

[0160] In one embodiment, the GPI anchor domain is a GPI-anchor domain of a protein selected from the group consisting of ACHE, ALPG, ALPI, ALPL, ALPP, ART3, ART4, BST1, BST2, CD109, CD14, CD160, CD48, CD52, CD55, CD59, CDH13, CEACAM5, CEACAM7, CNTN3, CNTN4, CNTN5, CPM, CPO, CRIPTO, DPEP1, EFNA4, EFNA5, FCGR3B, FOLR1, FOLR2, GP2, GPC1, GPIHBP1, HYAL2, IZUM01R, LSAMP, LY6D, LY6E, LY6G6C, LY6G6D, LY6H, LY6K, LYPD1, LYPD2, LYPD3, LYPD4, LYPD5, LYPD6B, LYPD8, MDGA1, MDGA2, MELTF, MMP17, MMP25, MSLN, NEGRI, NRN1, NRN1L, NTM, NTNG1, NTNG2, OPCML, OTOA, PLAUR, PLET1, PRND, PRNP, PRSS21, PRSS41, PRSS55, PSCA, RAET1G, RAET1L, RECK, RGMB, RTN4RL1, RTN4RL2, SEMA7A, SPACA4, SPRN, TECTA, TECTB, TEX101, THY1, ULBP2, VNN1, VNN2, XPNPEP2, GPCR3, preferably from CD48, CD52, CD55, CD59, PRNP, FOLR1, LSAMP, MSLN, DPEP1, THY1 and PLET1.

[0161] In one embodiment, the GPI anchor domain is selected from GPI anchor domains of GPI anchor proteins selected from the group consisting of ACHE, ALPG, ALPI, ALPL, ALPP, ART3, ART4, BST1, BST2, CD109, CD14, CD160, CD48, CD52, CD55, CD59, CDH13, CEACAM5, CEACAM7, CNTN3, CNTN4, CNTN5, CPM, CPO, CRIPTO, DPEP1, EFNA4, EFNA5, FCGR3B, FOLR1, FOLR2, GP2, GPC1, GPIHBP1, HYAL2, IZUMO1R, LSAMP, LY6D, LY6E, LY6G6C, LY6G6D, LY6H, LY6K, LYPD1, LYPD2, LYPD3, LYPD4, LYPD5, LYPD6B, LYPD8, MDGA1, MDGA2, MELTF, MMP17, MMP25, MSLN, NEGRI, NRN1, NRN1L, NTM, NTNG1, NTNG2, OPCML, OTOA, PLAUR, PLET1, PRND, PRNP, PRSS21, PRSS41, PRSS55, PSCA, RAET1G, RAET1L, RECK, RGMB, RTN4RL1, RTN4RL2, SEMA7A, SPACA4, SPRN, TECTA, TECTB, TEX101, THY1, ULBP2, VNN1, VNN2, XPNPEP2 or GPCR3, preferably selected from GPI anchor domain of CD48, CD52, CD55, CD59, PRNP, FOLR1, LSAMP, MSLN, DPEP1, THY1 and PLET1.

[0162] In some embodiments, the GPI anchor domain is selected from the group consisting of ACHE GPI anchor domain, ALPG GPI anchor domain, ALPI GPI anchor domain, ALPL GPI anchor domain, ALPP GPI anchor domain, ART3 GPI anchor domain, ART4 GPI anchor domain, BST1 GPI anchor domain, BST2 GPI anchor domain, CD109 GPI anchor domain, CD14 GPI anchor domain, CD 160 GPI anchor domain, CD48 GPI anchor domain, CD52 GPI anchor domain, CD55 GPI anchor domain, CD59 GPI anchor domain, CDH13 GPI anchor domain, CEACAM5 GPI anchor domain, CEACAM7 GPI anchor domain, CNTN3 GPI anchor domain, CNTN4 GPI anchor domain, CNTN5 GPI anchor domain, CPM GPI anchor domain, CPO GPI anchor domain, CRIPTO GPI anchor domain, DPEP1 GPI anchor domain, EFNA4 GPI anchor domain, EFNA5 GPI anchor domain, FCGR3B GPI anchor domain, FOLR1 GPI anchor domain, FOLR2 GPI anchor domain, GP2 GPI anchor domain, GPC1 GPI anchor domain, GPIHBP1 GPI anchor domain, HYAL2 GPI anchor domain, IZUMO1R GPI anchor domain, LSAMP GPI anchor domain, LY6D GPI anchor domain, LY6E GPI anchor domain, LY6G6C GPI anchor domain, LY6G6D GPI anchor domain, LY6H GPI anchor domain, LY6K GPI anchor domain, LYPD1 GPI anchor domain, LYPD2 GPI anchor domain, LYPD3 GPI anchor domain, LYPD4 GPI anchor domain, LYPD5 GPI anchor domain, LYPD6B GPI anchor domain, LYPD8 GPI anchor domain, MDGA1 GPI anchor domain, MDGA2 GPI anchor domain, MELTF GPI anchor domain, MMP17 GPI anchor domain, MMP25 GPI anchor domain, MSLN GPI anchor domain, NEGRI GPI anchor domain, NRN1 GPI anchor domain, NRN1L GPI anchor domain, NTM GPI anchor domain, NTNG1 GPI anchor domain, NTNG2 GPI anchor domain, OPCML GPI anchor domain, OTO A GPI anchor domain, PLAUR GPI anchor domain, PLET1 GPI anchor domain, PRND GPI anchor domain, PRNP GPI anchor domain, PRSS21 GPI anchor domain, PRSS41 GPI anchor domain, PRSS55 GPI anchor domain, PSCA GPI anchor domain, RAET1G GPI anchor domain, RAET1L GPI anchor domain, RECK GPI anchor domain, RGMB GPI anchor domain, RTN4RL1 GPI anchor domain, RTN4RL2 GPI anchor domain, SEMA7A GPI anchor domain, SPACA4 GPI anchor domain, SPRN GPI anchor domain, TECTA GPI anchor domain, TECTB GPI anchor domain, TEX101 GPI anchor domain, THY1 GPI anchor domain, ULBP2 GPI anchor domain, VNN1 GPI anchor domain, VNN2 GPI anchor domain, XPNPEP2 GPI anchor domain and GPCR3 GPI anchor domain, preferably from CD48 GPI anchor domain, CD52 GPI anchor domain, CD55 GPI anchor domain, CD59 GPI anchor domain, PRNP GPI anchor domain, FOLR1 GPI anchor domain, LSAMP GPI anchor domain, MSLN GPI anchor domain, DPEP1 GPI anchor domain, THY 1 GPI anchor domain and PLET1 GPI anchor domain.

[0163] Preferably, the GPLanchor domain is selected from the group consisting of CD48 (e.g., as set forth in SEQ ID NO: 69), CD52 (e.g., as set forth in SEQ ID NO: 70), CD55 (e.g., as set forth in SEQ ID NO: 71), CD59 (e.g., as set forth in SEQ ID NO: 145), PRNP (e.g., as set forth in SEQ ID NO: 123 or 147), FOLR1 (e.g., as set forth in SEQ ID NO: 85 or 148), LSAMP (e.g., as set forth in SEQ ID NO: 92 or 149), MSLN (e.g., as set forth in SEQ ID NO: 111 or 150), DPEP1 (e.g., as set forth in SEQ ID NO: 81 or 151), THY1 (e.g., as set forth in SEQ ID NO: 140) and PLET1 (e.g., as set forth in SEQ ID NO: 121).

[0164] In one embodiment, the GPI anchor domain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID Nos: 57-145, preferably a sequence as set forth in SEQ ID NO: 69-71, 121 and 145, even more preferably of SEQ ID NO: 145.

[0165] In some embodiments, the GPI anchor domain comprises a variant / mutant of the GPI anchor domain described herein. Said mutant typically comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 57-145, preferably any one of SEQ ID NOs: 69, 71, 121 and 145, even more preferably SEQ ID NO: 145 and is able to form a bond to GPI. In some embodiments, the GPI-anchor domain is a variant of any one of SEQ ID NOs: 57-145, comprising one, two or three amino acid modification(s) selected from the group consisting of substitution, addition or deletion. Preferably, said variant is able to form a bond to GPI.

[0166] In some embodiments, the GPI-anchor domain comprises a Glycine (G) residue followed by a protein selected from the group consisting of ACHE, ALPG, ALPI, ALPL, ALPP, ART3, ART4, BST1, BST2, CD109, CD14, CD160, CD48, CD52, CD55, CD59, CDH13, CEACAM5, CEACAM7, CNTN3, CNTN4, CNTN5, CPM, CPO, CRIPTO, DPEP1, EFNA4, EFNA5, FCGR3B, FOLR1, FOLR2, GP2, GPC1, GPIHBP1, HYAL2, IZUMO1R, LSAMP, LY6D, LY6E, LY6G6C, LY6G6D, LY6H, LY6K, LYPD1, LYPD2, LYPD3, LYPD4, LYPD5, LYPD6B, LYPD8, MDGA1, MDGA2, MELTF, MMP17, MMP25, MSLN, NEGRI, NRN1, NRN1L, NTM, NTNG1, NTNG2, OPCML, OTO A, PLAUR, PLET1, PRND, PRNP, PRSS21, PRSS41, PRSS55, PSCA, RAET1G, RAET1L, RECK, RGMB, RTN4RL1, RTN4RL2, SEMA7A, SPACA4, SPRN, TECTA, TECTB, TEX101, THY1, ULBP2, VNN1, VNN2, XPNPEP2, GPCR3, preferably from the group consisting of CD48, CD52, CD55, CD59, PRNP, FOLR1, LSAMP, MSLN, DPEP1, THY1 and PLET1.

[0167] In some embodiments the GPI anchor domain is selected from GPI anchor domains of GPI anchor proteins selected from the group consisting of CD48, CD52, CD55, CD59, PRNP, FOLR1, LSAMP, MSLN, DPEP1, THY1 and PLET1. In said embodiments, the GPI anchor domain may additionally comprise a Glycine (G) residue in N-terminus of the amino acid sequence.

[0168] In some embodiments the GPI anchor domain is selected from GPI anchor domains of GPI anchor proteins selected from the group consisting of CD55, PRNP, FOLR1, LSAMP, MSLN and DPEP1. In said embodiments, the GPI anchor domain may additionally comprise a Glycine (G) residue in N-terminus of the amino acid sequence. Typically, said GPI-anchor domain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 146- 151.

[0169] In all embodiments where a GPI anchor domain is envisioned, a Glyicine (G) can optionally be added in N-terminus of the amino acid sequence of the GPI.

[0170] In some embodiments, the GPI anchor domain comprises a variant / mutant of the GPI anchor domain described herein. Said mutant typically comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 146-151, and is able to form a bond to GPI. In some embodiments, the GPI-anchor domain is a variant of any one of SEQ ID NOs: 146-151, comprising one, two or three amino acid modification(s) selected from the group consisting of substitution, addition or deletion, preferably substitution, more preferably silent substitution. Preferably, said variant is able to form a bond to GPI.

[0171] In some embodiments, the GPI-anchor domain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-145, optionally with an additional Glycine (G) in N- terminus of the amino acid sequence.

[0172] In some embodiments, the GPI-anchor domain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 146-151.

[0173] In some embodiments, the GPI-anchor domain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-151.

[0174] Advantageously, the membrane IL-35 molecule of the invention comprises a GPI anchor domain selected among the GPI anchor domain that do not trigger a signal in the cell harboring the membrane cytokine.

[0175] Preferably, the membrane IL-35 molecule of the invention comprises a GPI anchor domain of a protein such as disclosed in Table D.

[0176] Table D. List of GPI anchor domain

[0177]

[0178]

[0179]

[0180]

[0181]

[0182] The applicant has further investigated the use of GPI anchor domains with other membrane cytokines, including anti-inflammatory cytokines such as IL4, IL 10, IL 13, IL37 or IL38. In some aspects, the membrane IL-35 comprises or consist of, from N-terminus to C-terminus: optionally a signal peptide, an EBB subunit; a linker, preferably a flexible linker, more preferably (G4S)3; a p35 subunit; and a GPI anchor domain, preferably selected from the group consisting of CD48 GPI anchor domain, CD52 GPI anchor domain, CD55 GPI anchor domain, CD59 GPI anchor domain, PRNP GPI anchor domain, FOLR1 GPI anchor domain, LSAMP GPI anchor domain, MSLN GPI anchor domain, DPEP1 GPI anchor domain, THY1 GPI anchor domain and PLET1 GPI anchor domain, more preferably CD59 GPI anchor domain; or optionally a signal peptide, a p35 subunit, a linker, preferably a flexible linker, more preferably (G4S)3; a p35 subunit and a GPI anchor domain, preferably selected from the group consisting of CD48 GPI anchor domain, CD52 GPI anchor domain, CD55 GPI anchor domain, CD59 GPI anchor domain, PRNP GPI anchor domain, FOLR1 GPI anchor domain, LSAMP GPI anchor domain, MSLN GPI anchor domain, DPEP1 GPI anchor domain, THY1 GPI anchor domain and PLET1 GPI anchor domain, more preferably CD59 GPI anchor domain.

[0183] The membrane IL-35 may alternatively comprise transmembrane domain of known transmembrane protein. Preferably the transmembrane domain is selected from the group consisting of cytokine receptor transmembrane domain, chemokine receptor transmembrane domain, type I transmembrane protein and type II transmembrane protein.

[0184] In some embodiments, the transmembrane domain is of a type II transmembrane protein, preferably selected from the group consisting of FasL, TRAIL, TNF, APRIL, CD30, BTL-II, TGFRII, DC-SIGN and MINCLE, preferably a member of the TNF family.

[0185] In some embodiments, the transmembrane domain is of a type I transmembrane protein, preferably selected from the group consisting of B7H4, B7H3, PDL2, PDL1, CTLA-4, Lag-3, TIGIT, BTLA, CD101, nectin-2, nectin-3, TIM3, TIM4, LAIR1, IL10R, IL6RA, IL6RB, TGFBRII, IL22R, CD 112, CD 113, VISTA, SIRPalpha, CD80, Siglec-10, Fas, LTBR and IL21R.

[0186] In some embodiments, the transmembrane domain is of a protein selected from the group consisting of CD4, CD8, CD28, ICOS, CD3, CD3zeta, CD3epsilon, CD3gamma, CD3delta, TCRalpha, TCRbeta, TCRgamma, TCRdelta, CD9, CD16, CD22, CD37, CD64, CD123, CD134, CD99, CD137 and CD154. Preferably, the transmembrane domain is selected from the group consisting of CD80 transmembrane domain, MHC1 transmembrane domain, FAS transmembrane domain and TNF receptor transmembrane domain, preferably the transmembrane domain of CD80.

[0187] In some embodiments, the membrane IL-35 comprises a transmembrane domain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 38, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0188] In some embodiments, the membrane IL-35 comprises a transmembrane domain comprising or consisting of an nucleic acid sequence as set forth in SEQ ID NO: 39, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0189] In some aspects, the membrane IL-35 further comprises a hinge domain.

[0190] As used herein, the term “hinge domain” when related to IL-35 molecules, refers to a flexible region within a protein, such as IL-35, that connects two or more structural domains and allows for relative movement between them. Typically, the hinge domain as envisioned herein covalently links an IL-35 subunit (i.e., p 35 or EBi3) to a transmembrane domain.

[0191] The hinge domain may be selected from any known from the art. In particular, the hinge domain is selected from the group consisting of a CD8 hinge domain, a CD28 hinge domain, a hinge domain of an immunoglobulin (IgG), for example an IgGl or IgG4, a CH2-CH3 domain derived from an IgG, for example an IgGl or IgG4, a hinge-CH2-CH3 domain derived from an IgG, for example an IgGl or IgG4.

[0192] In some embodiments, the hinge domain comprises or consist of a sequence as set forth in SEQ ID 34, 35, 50, 54 and a linker as described here above.

[0193] In some embodiments, the hinge domain comprises or consist of an amino acid sequence 5’ - GGI -3’ or a nucleic acid sequence of 5’ - GGCGGCATC -3’.

[0194] In some embodiments, the hinge domain comprises or consist of an amino acid sequence 5’- GGG -3’ or a nucleic acid sequence of 5’- GGCGGCGGC -3’.

[0195] In some embodiments, the membrane IL-35 comprises a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 23, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0196] In some aspects, the membrane IL-35 comprises a hinge domain and a transmembrane domain.

[0197] Preferably, the membrane IL-35 comprises a hinge domain and a transmembrane domain having a sequence as set forth in SEQ ID NO: 23 and 38, respectively. In some embodiments, the membrane IL-35 comprises a peptide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 36, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto. Preferably, the N-terminal of this peptide is covalently linked to the C-terminal of the p35 or Ebi3 subunit.

[0198] In some embodiments, the membrane IL-35 comprises a peptide encoded by an nucleic acid sequence as set forth in SEQ ID NO: 37, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0199] In some aspect, the membrane IL-35 molecule further comprises a cytosolic domain or a fragment thereof. Preferably, the cytosolic domain is linked to the C-terminus of the transmembrane domain.

[0200] As used herein, the term “cytosolic domain” and “intracellular domain” are used interchangeably refers to a portion of a protein that is located in the cytosol of a cell, typically a eucaryotic cell such as a mammalian cell. Cytosolic domain are typically found on the intracellular side of membrane proteins. Typically, the N-terminal end of the cytosolic domain as envisioned herein is covalently linked to the transmembrane domain disclosed herein.

[0201] The cytosolic domain may be selected from any know from the art. In particular, the cytosolic domain is selected from the group consisting of cytokine receptor cytosolic domain, type I transmembrane protein cytosolic domain and type II transmembrane protein cytosolic domain.

[0202] Preferably, the cytosolic domain is of the same protein type as the transmembrane domain. For example, when the transmembrane domain is from a type I transmembrane protein, the cytosolic domain is a type I transmembrane protein cytosolic domain. More preferably, the cytosolic domain is of the same protein as the transmembrane domain. For example, when the transmembrane domain is the transmembrane domain of CD80, the cytosolic domain is the cytosolic domain of CD80.

[0203] Preferentially, the cytosolic domain selected has no activating capacity. The cytosolic domain selected does not trigger a signaling pathway that activates the cell expressing the membrane IL- 35. Preferentially, the cytosolic domain selected has either an inhibiting activity, a stabilizing activity or no activity.

[0204] In some embodiments, the cytosolic domain is a type II transmembrane protein cytosolic domain preferably selected from the group consisting of FasL, TRAIL, TNF, APRIL, CD30, BTL-II, TGFRII, DC-SIGN and MINCLE, preferably a member of the TNF family. In some embodiments, the cytosolic domain is a type I transmembrane protein cytosolic domain preferably selected from the group consisting of, B7H4, B7H3, PDL2, PDL1, CTLA-4, Lag-3, TIGIT, BTLA, CD101, nectin-2, nectin-3, TIM3, TIM4, LAIR1, IL10R, IL6RA, IL6RB, TGFBRII, IL22R, CD112, CD113, VISTA, SIRPalpha, CD80, Siglec-10, Fas, LTBR and IL21R.

[0205] Preferably, the cytosolic domain is selected from the group consisting of CD80 cytosolic domain, CD59 cytosolic domain, MHC1 cytosolic domain, FAS transmembrane domain and TNF receptor cytosolic domain, preferably selected from the group consisting of MHC1 cytosolic domain, FAS transmembrane domain and TNF receptor cytosolic domain.

[0206] In some preferred embodiments, the cytosolic domain is CD80 cytosolic domain.

[0207] In some embodiments, the membrane IL-35 comprises a cytosolic domain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 40, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0208] In some embodiments, the membrane IL-35 comprises a cytosolic domain comprising or consisting of an nucleic acid sequence as set forth in SEQ ID NO: 41, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0209] In some embodiments, the membrane IL-35 comprises a transmembrane domain and a cytosolic domain having a sequence as set forth in SEQ ID NO: 38 and 40, respectively.

[0210] In some embodiments, the membrane IL-35 comprises a hinge domain, a transmembrane domain and a cytosolic domain having a sequence as set forth in SEQ ID NO: 23, 38 and 40, respectively.

[0211] In some aspects, the membrane IL-35 molecule comprises or consists of, from N-terminal to C- terminal: optionally a membrane-bound signal peptide, optionally a hinge domain, a transmembrane domain and optionally a cytosolic domain. Preferably, the membrane IL-35 molecule comprises or consists of, from N-terminal to C-terminal: a membrane-bound signal peptide, IL-35 subunits and a transmembrane domain. Alternatively, the membrane IL-35 molecule comprises or consists of, from N-terminal to C-terminal: a membrane-bound signal peptide, a hinge domain, a transmembrane domain and optionally a cytosolic domain.

[0212] In some embodiments, the mRNA molecule of the invention encodes for a membrane IL-35, comprising or consisting of from N-terminal to C-terminal: a) optionally a signal peptide, preferably an IL2 signal peptide, more preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 42, b) a p35 subunit, preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 5, c) a peptide linker, preferably a (GGGGS)3 linker (SEQ ID NO: 23), d) a Ebi3 subunit, preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 1, e) optionally a peptide linker or hinge domain, preferably selected from the group consisting of E15 (SEQ ID NO: 153); GGI ; hinge FcGILALAPG (SEQ ID NO 35); hinge FcGl (SEQ ID NO 34); (EAAAK)3 (SEQ ID NO: 54) and (GGGGS)6 (SEQ ID NO: 32); f) a transmembrane domain or GPI anchor domain, preferably selected from the group consisting of a CD80 transmembrane domain, a BCR transmembrane domain and a CD59 anchor domain; more preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 38, 145 or 152 and g) optionally a cytosolic domain, preferably a CD80 cytosolic domain, more preferably comprising or consisting of SEQ ID NO: 40.

[0213] In some embodiments, the mRNA molecule of the invention encodes for a membrane IL-35, comprising or consisting of from N-terminal to C-terminal: a) optionally a signal peptide, preferably an IL2 signal peptide, more preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 42, b) a Ebi3 subunit, preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 1, c) a peptide linker, preferably a (GGGGS)3 linker (SEQ ID NO: 23), d) a p35 subunit, preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 5, e) optionally a peptide linker or hinge domain, preferably selected from the group consisting of E15 (SEQ ID NO: 153); GGI ; hinge FcGILALAPG (SEQ ID NO 35); hinge FcGl (SEQ ID NO 34); (EAAAK)3 (SEQ ID NO: 54) and (GGGGS)6 (SEQ ID NO: 32); f) a transmembrane domain or GPI anchor domain, preferably selected from the group consisting of a CD80 transmembrane domain, a BCR transmembrane domain and a CD59 anchor domain; more preferably encoding an amino acid sequence comprising or consisting of SEQ ID NO: 38, 145 or 152 and g) optionally a cytosolic domain, preferably a CD80 cytosolic domain, more preferably comprising or consisting of SEQ ID NO: 40.

[0214] In some embodiments, the mRNA molecule of the invention encodes for a membrane IL-35, and comprises or consists of from N-terminal to C-terminal: a) A membrane bound peptide signal, preferably comprising or consisting of SEQ ID NO: 42 or 44; b) An IL-35 molecule, preferably an IL-35 fusion protein such as disclosed herein; c) A hinge domain, preferably comprising or consisting of SEQ ID NO: 23 or 50; d) A transmembrane domain, preferably a CD80 transmembrane domain, even more preferably comprising or consisting of SEQ ID NO: 38; and e) Optionally, a cytosolic domain, preferably a CD80 cytosolic domain, even more preferably comprising or consisting of SEQ ID NO: 40.

[0215] In some embodiments, the mRNA molecule of the invention encodes for a membrane IL-35, and comprises or consists of from N-terminal to C-terminal: a) A peptide signal, preferably comprising or consisting of SEQ ID NO: 43 or 45; b) An IL-35 molecule comprising ebi3 and p35, preferably comprising or consisting of SEQ ID NO: 9-14, preferably SEQ ID NO: 9, 11 or 13; c) A hinge domain, preferably comprising or consisting of SEQ ID NO: 37; d) A transmembrane domain, preferably comprising or consisting of SEQ ID NO: 39; and e) Optionally, a cytosolic domain, preferably comprising or consisting of SEQ ID NO: 41.

[0216] In some embodiments, the mRNA molecule of the invention encodes for a IL-35 fusion protein further comprising a hinge domain and a membrane-anchoring domain. Preferably, the mRNA molecule encodes for a fusion protein comprising or consisting of, from N-terminus to C- terminus: i) a p35 subunit or a variant or fragment thereof, a linker, a Ebi3 subunit or a variant or fragment thereof, a hinge domain and a membrane-anchoring domain, or ii) a Ebi3 subunit or a variant or fragment thereof, a linker, a p35 subunit or a variant or fragment thereof, a hinge domain and a membrane-anchoring domain. In some embodiments, the mRNA molecule of the invention encodes for a IL-35 fusion protein further comprising a hinge domain and a membrane-anchoring domain. Preferably, the mRNA molecule encodes for a fusion protein comprising or consisting of, from N-terminus to C- terminus: i) a p35 subunit or a variant or fragment thereof, a linker, a Ebi3 subunit or a variant or fragment thereof, a hinge domain and a GPI-anchor domain, or ii) a Ebi3 subunit or a variant or fragment thereof, a linker, a p35 subunit or a variant or fragment thereof, a hinge domain and a GPI-anchor domain.

[0217] Preferably, the GPI anchor domain is a GPI-anchor domain of a protein selected from the group consisting of CD48 (e.g., as set forth in SEQ ID NO: 69), CD52 (e.g., as set forth in SEQ ID NO: 70), CD55 (e.g., as set forth in SEQ ID NO: 71), CD59 (e.g., as set forth in SEQ ID NO: 145), PRNP (e.g., as set forth in SEQ ID NO: 123 or 147), FOLR1 (e.g., as set forth in SEQ ID NO: 85 or 148), LSAMP (e.g., as set forth in SEQ ID NO: 92 or 149), MSLN (e.g., as set forth in SEQ ID NO: 111 or 150), DPEP1 (e.g., as set forth in SEQ ID NO: 81 or 151), THY1 (e.g., as set forth in SEQ ID NO: 140) and PLET1 (e.g., as set forth in SEQ ID NO: 121).

[0218] In some embodiments, the mRNA molecule encodes for a fusion protein comprising or consisting of, from N-terminus to C-terminus: a) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a hinge domain and a CD59 GPI-anchor domain; b) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a El 5 linker (i.e., 15 consecutive E residues, SEQ ID NO: 153) and a CD59 GPI-anchor domain; c) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a GGI hinge domain and a CD 59 GPI-anchor domain; d) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a hinge FcGILALAPG and a CD59 GPI-anchor domain; e) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a (GGGGS)4 linker (SEQ ID NO: 22) and a CD59 GPI-anchor domain; f) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a (GGGGS)6 linker (SEQ ID NO: 32) and a CD59 GPI-anchor domain, or g) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit and a CD59 GPI-anchor domain.

[0219] In some embodiments, the mRNA molecule of the invention encodes for a membrane IL-35, comprising or consisting of from N-terminal to C-terminal: a) A membrane bound peptide signal, preferably comprising or consisting of SEQ ID NO: 42; b) A p35 subunit, preferably comprising or consisting of SEQ ID NO: 5, a linker, preferably comprising or consisting of SEQ ID NO: 23, and an Ebi3 subunit, preferably comprising or consisting of SEQ ID NO: 1; c) A hinge domain, preferably consisting of El 5 (i.e., 15 consecutive E residues, SEQ ID NO: 153); GGI ; hinge FcGILALAPG (SEQ ID NO 35); (EAAAK)3 (SEQ ID NO: 54) or (GGGGS)6 (SEQ ID NO: 32); d) A GPI anchor domain of a protein selected from the group consisting of CD48 (e.g., as set forth in SEQ ID NO: 69), CD52 (e.g., as set forth in SEQ ID NO: 70), CD55 (e.g., as set forth in SEQ ID NO: 71), CD59 (e.g., as set forth in SEQ ID NO: 145), PRNP (e.g., as set forth in SEQ ID NO: 123 or 147), FOLR1 (e.g., as set forth in SEQ ID NO: 85 or 148), LSAMP (e.g., as set forth in SEQ ID NO: 92 or 149), MSLN (e.g., as set forth in SEQ ID NO: 111 or 150), DPEP1 (e.g., as set forth in SEQ ID NO: 81 or 151), THY1 (e.g., as set forth in SEQ ID NO: 140) and PLET1 (e.g., as set forth in SEQ ID NO: 121), preferably CD59 (e.g., as set forth in SEQ ID NO: 145).

[0220] In some aspects, the mRNA molecules encodes for a membrane IL-35 comprising a CD59 GPI anchor domain, wherein the amino acid sequence of said membrane IL-35 comprises or consists of SEQ ID NO: 168, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0221] In some embodiments, the mRNA molecule of the invention encodes for a IL-35 fusion protein further comprising a hinge domain and a transmembrane domain or a fragment thereof. Preferably, the mRNA molecule encodes for a fusion protein comprising or consisting of, from N-terminus to C-terminus: i) a p35 subunit or a variant or fragment thereof, a linker, a Ebi3 subunit or a variant or fragment thereof, a hinge domain and a transmembrane domain or a fragment thereof, or ii) a Ebi3 subunit or a variant or fragment thereof, a linker, a p35 subunit or a variant or fragment thereof, a hinge domain and a transmembrane domain or a fragment thereof. In some aspect, the mRNA encodes for a fusion protein further comprising a cytosolic domain or a fragment thereof, preferably in C-terminus of the transmembrane domain.

[0222] In this embodiment, the mRNA molecule encodes for a fusion protein comprising or consisting of, from N-terminus to C-terminus: i) a p35 subunit or a variant or fragment thereof, a linker, a Ebi3 subunit or a variant or fragment thereof, a hinge domain, a transmembrane domain or a fragment thereof and a cytosolic domain or a fragment thereof, or ii) a Ebi3 subunit or a variant or fragment thereof, a linker, a p35 subunit or a variant or fragment thereof, a hinge domain, a transmembrane domain or a fragment thereof and a cytosolic domain or a fragment thereof.

[0223] Each of the components of such fusion protein are particularly as disclosed herein.

[0224] In some embodiments, the mRNA molecule of the invention encodes for a IL-35 fusion protein further comprising a hinge domain and a transmembrane domain or a fragment thereof.

[0225] In some embodiments, the IL-35 molecule of the invention comprises or consists of, from N- terminus to C-terminus: a) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a hinge domain and a CD80 transmembrane domain and optionally a cytosolic domain; b) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a El 5 linker (i.e., 15 consecutive E residues) and a CD80 transmembrane domain and optionally a cytosolic domain; c) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a GGI hinge domain and a CD80 transmembrane domain and optionally a cytosolic domain; d) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a hinge FcGILALAPG and a CD80 transmembrane domain and optionally a cytosolic domain; e) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a (GGGGS)4 linker (SEQ ID NO: 22) and a CD80 transmembrane domain and optionally a cytosolic domain; or f) optionally a signal peptide, a p35 subunit, a (GGGGS)3 linker, a Ebi3 subunit, a (GGGGS)6 linker (SEQ ID NO: 32) and a CD80 transmembrane domain and optionally a cytosolic domain. In some embodiments, the mRNA molecule of the invention encodes for a membrane IL-35, comprising or consisting of from N-terminal to C-terminal: a) A membrane bound peptide signal, preferably comprising or consisting of SEQ ID NO: 42; b) A p35 subunit, preferably comprising or consisting of SEQ ID NO: 5, a linker, preferably comprising or consisting of SEQ ID NO: 23, and an Ebi3 subunit, preferably comprising or consisting of SEQ ID NO: 1; c) A hinge domain, preferably consisting of El 5 (i.e., 15 consecutive E residues, SEQ ID NO: 153); GGI ; hinge FcGILALAPG (SEQ ID NO 35); hinge FcGl (SEQ ID NO 34); (EAAAK)3 (SEQ ID NO: 54) or (GGGGS)6 (SEQ ID NO: 32); d) A transmembrane domain, preferably a CD80 transmembrane domain, even more preferably comprising or consisting of SEQ ID NO: 38; and e) Optionally, a cytosolic domain, preferably a CD80 cytosolic domain, even more preferably comprising or consisting of SEQ ID NO: 40.

[0226] In some embodiments, the membrane IL-35 molecule of the invention comprises or consists of, from N-terminus to C-terminus: i) a p35 subunit or a variant or fragment thereof, a (GGGGS)3 linker, a Ebi3 subunit or a variant or fragment thereof, a hinge domain and a CD80 transmembrane domain or a fragment thereof, optionally a cytosolic domain or ii) a Ebi3 subunit or a variant or fragment thereof, a (GGGGS)3 linker, a p35 subunit or a variant or fragment thereof, a hinge domain and a CD80 transmembrane domain or a fragment thereof, optionally a cytosolic domain.

[0227] In some embodiments, the membrane IL-35 molecule of the invention comprises or consists of, from N-terminus to C-terminus: a membrane bound peptide signal, a p35 subunit or a variant or fragment thereof, a (GGGGS)3 linker, a Ebi3 subunit or a variant or fragment thereof, a hinge domain and a BCR transmembrane domain or a fragment thereof, optionally a cytosolic domain.

[0228] In some aspects, the mRNA molecules encodes for a membrane IL-35 comprising a CD80 transmembrane domain and optionally a cytosolic domain, wherein the amino acid sequence of said membrane IL-35 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 162-167 and 172-173, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto. In some aspects, the mRNA molecule of the invention encodes for a membrane IL-35, comprising or consisting of from N-terminal to C-terminal: a) A membrane bound peptide signal, preferably comprising or consisting of SEQ ID NO: 42; b) A p35 subunit, preferably comprising or consisting of SEQ ID NO: 5, a linker, preferably comprising or consisting of SEQ ID NO: 23, and an Ebi3 subunit, preferably comprising or consisting of SEQ ID NO: 1; c) A hinge domain, preferably consisting of hinge FcGILALAPG (SEQ ID NO 35); d) A transmembrane domain, preferably a BCR transmembrane domain, even more preferably comprising or consisting of SEQ ID NO: 152; and e) Optionally, a cytosolic domain.

[0229] In some aspects, the mRNA molecules encodes for a membrane IL-35 comprising a BCR domain, preferably comprising or consisting of SEQ ID NO: 169, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0230] In some aspects, the mRNA molecules encodes for a membrane IL-35 having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169, 172 and 173, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0231] In some aspects, the mRNA molecules encodes for a membrane IL-35 having an amino acid sequence comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 162, 163, 164, 165, 166, 167, 168, 169 and 172, or a variant thereof having at least 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0232] In some particular aspects, the LNP of the invention comprises one or more mRNA molecule(s) encoding a fusion protein comprising a membrane IL-35 molecule as described herein fused to a Fc fragment.

[0233] In some particular aspects, the LNP of the invention comprises one or more mRNA molecule(s) encoding a fusion protein comprising a membrane IL-35 molecule as described herein fused to a human serum albumin (HS A) or a HSA fragment.

[0234] In some instance, the nucleic acid sequences of p35 and Ebi3 are optimized in order to reduce uridine occurrence and generate a linear mRNA structure. mRNA In a particular aspect, the lipid-based nanoparticle comprises a messenger ribonucleic acid (mRNA) polynucleotide or a set of mRNA polynucleotides. The technology of mRNA polynucleotide is now well-known by the person skilled in the art, as illustrated in WO21159130, the disclosure thereof being incorporated herein by reference.

[0235] Preferably, the lipid-based nanoparticle of the invention comprises one or more isolated mRNA molecule(s).

[0236] The lipid-based nanoparticle of the invention particularly includes an mRNA encoding a polypeptide of interest capable of being translated by the immune cell to produce the polypeptide of interest.

[0237] The mRNA molecule of the invention particularly comprises structural elements that allows its encapsulation into the lipid-based nanoparticle and / or its expression into the targeted immune cell. The mRNA molecule is typically encapsulated within or associated with the LNPs.

[0238] Preferably, the mRNA molecule contains stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5'-end (5' UTR) and / or at their 3'-end (3' UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. The mRNA molecule may comprise RNA Secondary Structures such as hairpin loops or stem-loop structures. mRNA envisioned herein typically include a region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5'-terminus of the region of linked nucleosides (e.g., a 5’-UTR), a second flanking region located at the 3'-terminus of the region of linked nucleosides (e.g., a 3’-UTR), at least one 5'-cap region, and a 3 '-stabilizing region. In some aspects, the mRNA of the invention includes a poly-A region or a Kozak sequence (e.g., in the 5’-UTR). In some cases, the mRNA of the invention comprises one or more intronic nucleotide sequences capable of being excised from the polynucleotide.

[0239] In some aspects, the mRNA of the invention includes a flanking region, a 5'-cap structure, a chain terminating nucleotide, a stem loop, a poly-A sequence and / or a polyadenylation signal.

[0240] In some aspects, the mRNA of the invention comprises a flanking region. A 5'-UTR or a 3 ’-UTR may be provided as a flanking region to the mRNA of the invention. A 5 ’-UTR may be homologous or heterologous to the coding region of the mRNA. Multiple 5'- UTRs or 3’-UTRs may be included in the flanking region and may be of the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and / or after codon optimization. Preferably, the mRNA comprises a human haemoglobin subunit beta (hHbB) 5’UTR and / or a hHbB 3’UTR, or any UTR derived from SaRS-Cov2, for example as disclosed in Leppek et al 2022 Nature Communications volume 13, Article number: 1536 ), preferably the variant named “10032218_ElNando888_Nonconformist” as described in supplemental data 1 having a nucleotide sequence as set forth in SEQ ID NO: 56.

[0241] To alter one or more properties of an mRNA, 5'-UTRs or 3’-UTRs which are heterologous to the coding region of an mRNA may be engineered. The mRNA may then be administered to cells, tissue or organisms and outcomes such as protein level, localization, and / or half- life may be measured to evaluate the beneficial effects the heterologous 5'-UTR and / or 3 ’-UTR may have on the mRNA. Variants of the 5'-UTRs and / or 3’-UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G. 5'-UTRs and / or 3 ’-UTR may also be codon-optimized, or altered in any manner described herein.

[0242] In some aspects, the mRNA comprises an Internal ribosome entry site (IRES) or a Kozak sequence in the 5 ’-UTR region. The Kozak consensus sequence (Kozak consensus or Kozak sequence) is a nucleic acid motif that functions as the protein translation initiation site. An internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in a cap-independent manner.

[0243] In some aspects, the mRNA of the invention comprises a 5 ’-capping region or structure. The 5'- cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5'-proximal introns during mRNA splicing. Endogenous polynucleotide molecules may be 5'-end capped generating a 5'-ppp-5'-triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the polynucleotide. This 5'- guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or ante-terminal transcribed nucleotides of the 5'-end of the polynucleotide may optionally also be 2'-O-methylated. 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. Preferably, mRNA polynucleotides comprise at least one base modification in the 5'-terminal cap, such as a modified guanine. Alterations to polynucleotides may generate a non-hydrolysable cap structure preventing decapping and thus increasing polynucleotide half-life. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.

[0244] 5'-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme or a Faustovirus Capping Enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Vims Capping Enzyme or a Faustovirus Capping Enzyme and a 2'-O- methyl -transferase to generate: m7G(5')ppp(5')G-2'-0-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-Omethyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-O- methyl-transferase. Enzymes may be derived from a recombinant source. Exemplary cap structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')NlmpNp (Cap 1), 7mG(5')- ppp(5')NlmpN2mp (Cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (Cap 4). Alternatively, other caps may be used, such as CleanCap structures (Trilink). Cleancap is a trinucleotide with a 5'-m7G joined by a 5'-5' triphosphate linkage to an AG sequence. The mRNA of the invention may particularly comprise a 5 ’-cap analogue. Cap analogues, which herein are also referred to as synthetic cap analogues, chemical caps, chemical cap analogues, or structural or functional cap analogues, differ from natural (i.e., endogenous, wild-type, or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogues may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / linked to a polynucleotide. Examples of cap analogues are Anti-Reverse Cap Analog (ARCA) cap and mCAP. Various cap analogues and the methods of synthesizing cap analogues are described in Kore et al. Bioorganic & Medicinal Chemistry 20132: 4570-4574; the cap structures of which are herein incorporated by reference.

[0245] In some aspects, the mRNA of the invention includes a stem loop such as, but not limited to, a histone stem loop. Preferably, the stem loop is a nucleotide sequence of about 25 or about 26 nucleotides in length. The histone stem loop may be located 3'-relative to the coding region (e.g., at the 3'-terminus of the coding region). The histone stem loop may be before and / or after the poly-A region. The mRNA including the histone stem loop and a poly-A region sequence may include a chain terminating nucleoside described herein. In other instances, the mRNA includes a histone stem loop and a 5'-cap structure such as described herein and / or known in the art. In some cases, the conserved stem loop region may include a miR sequence. As a non-limiting example, the stem loop region may include the seed sequence of a miR sequence. For example, the stem loop region may include a miR- 122 seed sequence.

[0246] As a non-limiting example, the stem loop may be located at the 3'-end of a mRNA described herein. In some cases, an mRNA includes more than one stem loop (e.g., two stem loops). A stem loop may be located in a second terminal region of a polynucleotide. As a non-limiting example, the stem loop may be located within an untranslated region (e.g., 3'-UTR) in a second terminal region. In some cases, a mRNA which includes the histone stem loop may be stabilized by the addition of a 3 ’-stabilizing region (e.g., a 3 ’-stabilizing region including at least one chain terminating nucleoside). Not wishing to be bound by theory, the addition of at least one chain terminating nucleoside may slow the degradation of a polynucleotide and thus can increase the half-life of the polynucleotide.

[0247] In other cases, the mRNA, which includes the histone stem loop is stabilized by an alteration to the 3'-region of the polynucleotide that can prevent and / or inhibit the addition of oligo(U).

[0248] In yet other cases, the mRNA, which includes the histone stem loop is stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2', 3 '-dideoxynucleoside 3'-O- m ethylnucleosides, 3’-O- ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0249] The mRNA may particularly include at least one histone stem-loop and a poly-A region or polyadenylation signal.

[0250] In some aspects, the mRNA comprised in the lipid-based nanoparticle of the invention includes a poly-A sequence and / or polyadenylation signal. A poly-A sequence may be comprised entirely or mostly of adenine nucleotides or analogues or derivatives thereof. A poly-A sequence may be a tail located adjacent to a 3 '-untranslated region of a nucleic acid. In some instances, the poly-A region may be between 80 and 160 nucleotides, between 90 and 150 nucleotides, between 100 and 140 nucleotides or between 110 and 130 nucleotides in length on an mRNA molecule described herein. In other instances, the poly-A region is of 20, 40, 80, 100, 120, 140 or 160 nucleotides in length on an mRNA molecule described herein. In some cases, the poly-A region is designed relative to the length of the overall mRNA. This design may be based on the length of the coding region, the length of a particular feature or region of the mRNA or based on the length of the ultimate product expressed from the mRNA. When relative to any feature of the mRNA (e.g., other than the mRNA portion which includes the poly-A region) the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in length than the additional feature. The poly-A region may also be designed as a fraction of the mRNA to which it belongs. In this context, the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A region.

[0251] In some instances, the mRNA includes a poly-A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A region.

[0252] In some aspects, the mRNA molecule of the invention may include one or more naturally occurring components, including any of the canonical nucleotides: A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published patent application Nos. WO2013052523; WO2014093924; W02015051173; W02015051169; W02015089511; W02015196130; WO2015196118; WO2015196128; or WO2017153936 all of which are incorporated by reference herein.

[0253] In some instances, the mRNA molecule comprises from N-terminus to C-terminus: a hHbB 5’UTR, a sequence encoding a first IL-35 subunit, a sequence encoding a linker, a sequence encoding a second IL-35 subunit, a hHbB 3’UTR and a poly-A tail.

[0254] In some instances, the mRNA molecule comprises from N-terminus to C-terminus: a hHbB 5’UTR, a sequence encoding Ebi3, a sequence encoding a linker, a sequence encoding p35, a hHbB 3’UTR and a poly-A tail.

[0255] Preferably, the mRNA molecule comprises a nucleic acid sequence of SEQ ID NO: 19 or 20, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the mRNA molecule comprises a nucleic acid sequence of SEQ ID NO: 19 or 20, even more preferably of SEQ ID NO: 19.

[0256] In some instances, the mRNA molecule comprises from N-terminus to C-terminus: a hHbB 5’UTR, a sequence encoding p35, a sequence encoding a linker (e.g., (GGGGS)s or P2A), a sequence encoding Ebi3, a hHbB 3’UTR and a poly-A tail.

[0257] Preferably, the mRNA molecule comprises a nucleic acid sequence of SEQ ID NO: 15 or 16 or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the mRNA molecule comprises a nucleic acid sequence of SEQ ID NO: 15 or 16, even more preferably of SEQ ID NO: 15. Preferably, the mRNA molecule comprises a nucleic acid sequence of SEQ ID NO: 16 or 17, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the mRNA molecule comprises a nucleic acid sequence of SEQ ID NO: 16 or 17, even more preferably of SEQ ID NO: 17.

[0258] Preferably, the mRNA molecule of the invention comprises or consists of a nucleic acid sequence as described in SEQ ID NO 46 or 47, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0259] Alternatively, the mRNA molecule of the invention comprises or consists of a nucleic acid sequence as described in SEQ ID NO: 48 or 49 or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0260] In some embodiments, the mRNA comprises or consists of a nucleic acid sequence as described in any one of SEQ ID NO 46-49, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0261] In some embodiments, the mRNA comprises or consists of a nucleic acid sequence as described in any one of SEQ ID NO 154-161, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0262] In some embodiments, the mRNA comprises or consists of a nucleic acid sequence as described in SEQ ID NO: 170 or 171, preferably SEQ ID NO: 171 or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0263] In some embodiments, the mRNA comprises or consists of a nucleic acid sequence as described in any one of SEQ ID NO: 154-161 or 170-171, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0264] In some embodiments, wherein membrane IL-35 comprises a CD80 transmembrane domain, the mRNA comprises or consists of a nucleic acid sequence as described in any one of SEQ ID NO: 154-159 or 170-171, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0265] In some embodiments, wherein membrane IL-35 comprises a CD59 GPI anchor domain, the mRNA comprises or consists of a nucleic acid sequence as described in SEQ ID NO 160, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0266] In some embodiments, wherein membrane IL-35 comprises a BCR transmembrane domain, the mRNA comprises or consists of a nucleic acid sequence as described in SEQ ID NO 161, or a variant thereof having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some aspects, the LNP of the invention comprises one or more mRNA molecule(s) encoding construct as described in the “Examples” section below.

[0267] Optionally, the mRNA can be a circular mRNA, especially as described in WO2014 / 186334 and WO2022 / 261490.

[0268] In some aspects, the lipid-based nanoparticles of the invention comprises a mRNA molecule encoding a membrane IL-35 as described herein and one or more mRNA molecules encoding an additional immunotherapeutic agent.

[0269] In particular, the lipid-based nanoparticle may comprise 1, 2, 3, 4 or 5 mRNA molecule(s) encoding 1, 2, 3, 4 or 5 different immunotherapeutic agents.

[0270] In some aspects, such immunotherapeutic agent is not a cytokine or an interleukin. Alternatively, the immunotherapeutic agent is a cytokine or an interleukin.

[0271] Additionally, the immunotherapeutic agent encoded by a mRNA molecule included in the lipid- based nanoparticle of the invention may particularly be selected based on the desired effect. For example, the immune cell inhibiting protein may be selected for a particular indication, condition, disease, or disorder.

[0272] In some aspects, the immunotherapeutic agent is an immune checkpoint inhibitor. Particularly, the checkpoint inhibitor is an antibody or antigen-binding fragment thereof, such as a F(ab')2, a Fab, a Fab’, a F(ab)2, a crossMAb, a single-chain variable fragment (scFV) or a VHH.

[0273] In some aspects, the checkpoint inhibitor inhibits a checkpoint selected from the group consisting of PD-1, PD-L1, CTLA4, LAG-3, BTLA, TIM3 and TIGIT, or a combination thereof.

[0274] In some aspects, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof that specifically binds to a checkpoint selected from the group consisting of PD-1, PD-L1, CTLA4, LAG-3, BTLA, TIM3 and TIGIT, or a combination thereof. Numerous antibodies directed against PD-1, PD-L1, TIM-3, CTLA-4, LAG-3, BTLA and TIGIT have already been described in the art.

[0275] Lipid-based nanoparticles

[0276] The present disclosure relates to a lipid-based nanoparticle comprising one or several mRNA molecule(s) encoding a membrane IL-35 as described herein.

[0277] “Lipid-based nanoparticles” or “lipid nanoparticles” are a type of delivery system used in the field of drug delivery, particularly in the context of nucleic acid therapeutics such as RNA. LNPs are designed to encapsulate and deliver therapeutic molecules. In the context of RNA-based therapeutics, such as mRNA vaccines or RNA interference (RNAi) therapies, LNPs serve as vehicles to transport RNA molecules to the target cells, tissues or organ of interest.

[0278] Lipid-based nanoparticles of the invention typically comprise helper lipid, sterol and / or PEG lipid components along with the mRNA molecule encoding a membrane IL-35 as described herein.

[0279] The lipid-based nanoparticle according to the invention is particularly formulated either as a liposome or a lipid nanoparticle (LNP), especially a lipid nanoparticle comprising a mixture of lipids. The lipid-based nanoparticle envisioned herein also encompasses similar nanoparticles such as but not limited to micelles and nano-emulsions. Lipid based nanoparticles also include Hybrid nanoparticles comprising polymers-lipids hybrid compounds, such as polamines- polaxamers.

[0280] The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a lipid-based nanoparticle may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements. The lipid-based nanoparticles of the disclosure can particularly be generated using components, compositions, and methods as generally known in the art, for example such as disclosed in W02020160397, W02019046809, WO2017049245; WO2017112865; WO2017218704; WO2015164674; WO2017031232; WO2017099823; WO2016118724; WO2016118724; WO2017223135; WO2014152211; WO2015038892; W02017049074; W02013090648; W02017180917; WO2017075531 and WO2017117528 all of which are incorporated herein by reference herein in their entirety.

[0281] The manufacture of LNPs is well described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al.,

[0282] 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al.,

[0283] 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated herein by reference in their entirety. In some aspects, the method for obtaining the lipid-based nanoparticles of the invention is as described under the “Examples” section below. Alternatively, the method for obtaining the lipid- based nanoparticles of the invention is as described in PCT / EP2024 / 058775.

[0284] In some aspects, the lipid-based nanoparticle according to the invention comprises one or more ionizable or cationic lipid(s). As used herein, the term “ionizable or cationic lipid” refers to a lipid molecule positively charged in an acidic environment.

[0285] Examples of ionizable or cationic lipids are described in WO 2016 / 021683, WO 2015 / 011633, WO 2011 / 153493, WO 2013 / 126803, WO 2010 / 054401, WO 2010 / 042877, WO 2016 / 104580, WO 2015 / 005253, WO 2014 / 007398, WO 2017 / 117528, WO 2017 / 075531, WO 2017 / 00414, WO 2015 / 199952, US 2015 / 0239834, WO2019 / 131839 all of which are incorporated by reference herein in their entirety. In a preferred aspect, the lipid-based nanoparticle according to the invention comprises an ionizable or cationic lipid such as described in WO 2016 / 021683 or WO 2019 / 131839 which are incorporated herein by reference in their entirety.

[0286] Ionizable or cationic lipids can be selected from the group consisting of l,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dimethyl-2, 3 -di oleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N- (N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammonium propanes; l,2-dialkyloxy-3 -dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3 -di oleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9, 12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5- en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl- 3 -dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine

[0287] (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin- MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l- propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)- N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminiiim (DOBAQ), 2-({8-[(3P)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]propan-l-amine (Octyl -CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3 - aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3- bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-l-amonium bromide (DLRIE), N-(2- aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-

[0288] (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2- dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98Niz-5), 1- [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin- l-yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200), [(4-hydroxybutyl)azanediyl]di(hexane-6,l- diyl) bis(2-hexyldecanoate) (ALC-0315), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate (SM-102) and any mixtures thereof.

[0289] Additional examples of ionizable or cationic lipids are described in WO 2016 / 021683, WO 2015 / 011633, WO 2011 / 153493, WO 2013 / 126803, WO 2010 / 054401, WO 2010 / 042877, WO 2016 / 104580, WO 2015 / 005253, WO 2014 / 007398, WO 2017 / 117528, WO 2017 / 075531, WO 2017 / 00414, WO 2015 / 199952, US 2015 / 0239834, WO2019 / 131839 all of which are incorporated by reference herein in their entirety. In a preferred aspect, the lipid-based nanoparticle according to the invention comprises an ionizable or cationic lipid such as described in WO 2016 / 021683 or WO 2019 / 131839 which are incorporated herein by reference in their entirety.

[0290] In addition, synthetic ionizable or cationic lipids (e.g., K-E12, H-A12, Y-E12, G-O12, K-A12, R-A12, CKK-E12, cPK-E12, PK1K-E12, PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K- E12, CWK-E12, PK500-012, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-012, CIK-E12, cKK-ElO, cKK-E14, and cKK-E16, preferably, cKK-E12, cKK-E14) described in Dong et al. (Proc Natl Acad Sci U S A. 2014 Apr 15; 111(15):5753, the disclosure thereof being incorporated herein by reference), and the synthetic ionizable or cationic lipid (e.g., C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96 and C12-200, preferably C14- 110, C16-96 and C12-200) described in Love KT et al. (Proc Natl Acad Sci U S A. 2010 May 25; 107(21):9915, the disclosure thereof being incorporated herein by reference) can be also envisioned.

[0291] Particularly, the lipid composition of the lipide-based nanoparticle according to the invention comprises an ionizable or cationic lipid selected from the group consisting of [(4- hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ALC-0315), heptatri aconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 9-Heptadecanyl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) and any mixtures thereof.

[0292] In some aspects, the ionizable or cationic lipid represents from about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the lipid- based nanoparticle according to the invention.

[0293] In some aspects, the ionizable or cationic lipid, preferably ALC-0315, represents from about 45 mol % to about 55 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle according to the invention. Preferably, the ionizable or cationic lipid, preferably ALC-0315, represents from about 48 mol % to about 52 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle according to the invention.

[0294] In some aspects, the lipid-based nanoparticle according to the invention comprises a helper lipid. As used herein, the term “helper lipid” refers to a class of lipid molecules that increases particle stability, fluidity tolerability and / or biodistribution of lipid-based nanoparticles.

[0295] Helper Lipids are well known in the art, for example as described in Cheng X, Lee RJ. Adv Drug Deliv Rev. 2016 Apr l;99(Pt A): 129-137, PMID: 26900977. For instance, the helper lipid can be selected from the group consisting of 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0- octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn-glycero- 3 -phosphocholine (Cl 6 Lyso PC), 1, 2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), ethyl phosphatidylcholine (EPC), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphocholine (18 : 1 Lyso PC),

[0296] 1.2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16:0 PE), 1 -hexadecyl -2-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (C16— 18: 1), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME), 1- stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), l,2-dielaidoylsn-glycero-3- phosphoethanol amine (DEPE), N -(7 -nitrob enz-2-oxa- 1 , 3 -di azol -4-yl)- phosphatidylethanolamine (NBD-PE), N-(lisamineRhodamine B sulfonyl)- phosphatidylethanolamine (Rh-PE), 1 -oleoyl-2-hydroxy-sn-glycero-3 -phosphoethanolamine (18 : 1 Lyso PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (18 :1 Monomethyl PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl (18 : 1 Dimethyl PE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(hexanoylamine) (18 : 1 Caproylamine PE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (18 : 1 Biotinyl PE), sn-(3-oleoyl-2- hydroxy)-glycerol-l-phospho-sn-l '-(3 '-oleoyl -2'-hydroxy)-glycerol (BMP-S,S), sn-(3-(9Z- octadecenoyl)-2-hydroxy)-glycerol-l-phospho-sn-3'-(l'-(9Z-octadecenoyl)-2'-hydroxy)- glycerol (BMP-S,R), l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG),

[0297] 1.2-Diacyl-sn-glycero-3-phospho-L-serine (DSPS), L-a-phosphatidylserine (PS), 1,2-dioleoyl- sn-glycero-3 -phosphate (PA), l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (PG), 1,2- dioleoyl-sn-glycero-3-phosphomethanol (18 : 1 Phosphatidymethanol), 1,2-dioleoyl-sn-glycero- 3 -phosphoethanol (18 : 1 Phosphatidyethanol), l,2-dioleoyl-sn-glycero-3 -phosphopropanol (18 : 1 Phosphatidypropanol), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (18: 1 PS, DOPS), 1.2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), N-oleoyl-D-erythro-sphingosine (Ceramide), Sphingomyelin (SM), Phosphatidylinositol (PI), 9A1P9, l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), Dimethyldioctadecylammonium (18:0 DDAB), and any mixtures thereof.

[0298] Particularly, the helper lipid can be selected from the group consisting of 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0- octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn-glycero- 3 -phosphocholine (Cl 6 Lyso PC), 1, 2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), ethyl phosphatidylcholine (EPC), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphocholine (18 : 1 Lyso PC),

[0299] 1.2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16:0 PE), 1 -hexadecyl -2-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (C16— 18: 1), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME), 1- stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), l,2-dielaidoylsn-glycero-3- phosphoethanol amine (DEPE), N -(7 -nitrob enz-2-oxa- 1 , 3 -di azol -4-yl)- phosphatidylethanolamine (NBD-PE), N-(lisamineRhodamine B sulfonyl)- phosphatidylethanolamine (Rh-PE), 1 -oleoyl-2-hydroxy-sn-glycero-3 -phosphoethanolamine (18 : 1 Lyso PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (18 : 1 Monomethyl PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl (18 : 1 Dimethyl PE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(hexanoylamine) (18 : 1 Caproylamine PE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (18 : 1 Biotinyl PE), sn-(3-oleoyl-2- hydroxy)-glycerol-l-phospho-sn-l '-(3 '-oleoyl -2'-hydroxy)-glycerol (BMP-S,S), sn-(3-(9Z- octadecenoyl)-2-hydroxy)-glycerol-l-phospho-sn-3'-(l'-(9Z-octadecenoyl)-2'-hydroxy)- glycerol (BMP-S,R), l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG),

[0300] 1.2-Diacyl-sn-glycero-3-phospho-L-serine (DSPS), L-a-phosphatidylserine (PS), 1,2-dioleoyl- sn-glycero-3 -phosphate (PA), l,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (PG), 1,2- dioleoyl-sn-glycero-3-phosphomethanol (18 : 1 Phosphatidymethanol), 1,2-dioleoyl-sn-glycero- 3 -phosphoethanol (18 : 1 Phosphatidyethanol), l,2-dioleoyl-sn-glycero-3 -phosphopropanol (18 : 1 Phosphatidypropanol), l,2-dioleoyl-sn-glycero-3-phospho-L-serine (18: 1 PS, DOPS), l,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), N-oleoyl-D-erythro-sphingosine (Ceramide), Sphingomyelin (SM), Phosphatidylinositol (PI), 9A1P9, l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), Dimethyldioctadecylammonium (18:0 DDAB), l,2-dioleyloxy-3- dimethylaminopropane (DODMA), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2- dierucoyl-sn-glycero-3-phosphocholine (DEPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE) and any mixtures or combinations thereof.

[0301] Preferably, the helper lipid is selected from the group consisting of DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DOPC, DEPC and DSPE, and any combinations thereof.

[0302] Preferably, the helper lipid is selected from the group consisting of from the group consisting of DOPE, DOPC, DDAB, POPE and DSPC, and any combinations thereof.

[0303] In some embodiments, the helper lipid is DOPE. Alternatively, the helper lipid is DSPC.

[0304] In some aspects, the helper lipid represents from about 5 mol% to about 100 mol%, from about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0305] In some aspects, the helper lipid, preferably DOPE or DSPC, represents from about 5 mol% to about 15 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0306] In some aspects, the helper lipid, preferably DOPE or DSPC, represents from about 8 mol% to about 12 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0307] In some aspects, the helper lipid, preferably DOPE or DSPC, represents about 10 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0308] In some aspects, the lipid of the lipid-based nanoparticle disclosed herein comprises one or more molecules comprising polyethylene glycol (PEG). Accordingly, the lipid-based nanoparticle may comprise PEG or PEG-modified lipids.

[0309] As used herein, the term “PEG lipid” may refer to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCI4 or PEG-CerC20), PEG- modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some aspects, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some aspects, the PEG- modified lipids are a modified form of PEG-DMG. A PEG lipid may particularly be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG- modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some aspects, a PEG lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, DMG-PEG- 2000, PEG-DLPE, PEG- DMPE, PEG-DPPC, and PEG-DSPE lipid.

[0310] In some aspects, the PEG-lipid is selected from the group consisting of 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG- 1,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA), PEG-c-DOMG, PEG-DMG, DMG-PEG-2000, PEG-DLPE, PEG- DMPE, PEG-DPPC, and PEG-DSPE.In one aspect, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0311] In some aspects, the PEG-lipid includes, but is not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0312] In some aspects, the PEG-lipid includes, but is not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA) and ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG), and any mixture thereof. Preferably, the PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, PEG- c- DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG and PEG-c-DMA, ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG), and any mixture thereof; particularly from the group consisting of PEG-DMG, PEG-DSPE, ALC-0159 and any mixture thereof.

[0313] In a very particular aspect, the PEG-lipid is PEG 2000-DMG. In some aspects, the PEG-lipid is PEG 2000-DSG. In another very particular aspect, the PEG-lipid is ALC-0159.

[0314] In some aspects, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. Alternatively, in a more specific aspect, the lipid, in particular the lipid moiety of the PEG-lipids includes those having a length of from about C16 to about C22 (C16, Cl 7, Cl 8, Cl 9, C20, C21 or C22), preferably C16 to C20 (C16, C17, C18, C19 or C20), especially Cl 8.

[0315] Optionally, the PEG may present a molecular weight within the range from 0.5 to 50 kDa, more preferably from 1 to 20 kDa. In some aspects, a PEG moiety, for example a mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 Daltons. In a particular aspect, the PEG has a size of about 2000.

[0316] In a very particular aspect, the PEG-lipid is PEG 2000-DMG or PEG 2000-DSG.

[0317] Preferably, the PEG is comprised between 2000 Daltons and 5000 Daltons (i.e., PEG-2000 to PEG-5000), preferably is DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG- 5000 or any mixture thereof.

[0318] In a particular aspect, the LNP comprises a PEG that is not functionalized, which is a PEG that does not comprises any reactive species at its end, said reactive species being usable to conjugate a target moiety such as an antibody or a fragment thereof to the PEG.

[0319] In some aspects, the PEG lipid represents from about 1 mol% to about 100 mol%, from about 2 mol% to about 100 mol%, about 3 mol% to about 100 mol%, about 4 mol% to about 100 mol%, about 5 mol% to about 100 mol%, about 10 mol% to about 100 mol%, or about 15 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0320] In some aspects, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 0.5 mol% to about 5 mol% or from about 0.5 mol% to about 2.5 mol% of the total lipids present in the lipid-based nanoparticle of the invention. Optionally, the PEG lipid in the lipid-based nanoparticle is within the range from about 0.5 mol% to about 2 mol% of the total lipids present in the nanoparticle, for instance about 0.5, 0.6, 0.7,

[0321] 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mol%, especially 1.5 mol%.

[0322] In some aspects, the lipid-based nanoparticle of the invention comprises one or more sterol. The sterol can particularly be selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixtures thereof. Preferably, the sterol is cholesterol.

[0323] In some aspects, the sterol represents from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, about 50 mol% to about 100 mol%, about 60 mol% to about 100 mol%, or about 70 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0324] Particularly, the sterol, preferably cholesterol, represents from about 35 mol% to about 40 mol% of the total lipids present in the lipid-based nanoparticle of the invention. Such sterol being preferably cholesterol.

[0325] In a preferred aspect, the lipid-based nanoparticle of the invention comprises an ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid.

[0326] In a preferred aspect, the lipid composition of the lipid-based nanoparticle of the invention consists of an ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid, these lipids being preferably as described above.

[0327] Preferably, in said lipid-based nanoparticle, the ionizable or cationic lipid is from about 10 mol % to about 70 mol % of the total lipids present in the nanoparticle, the helper lipid is from about 5 mol% to about 70 mol % of the total lipids present in the nanoparticle, the sterol is from about 10 mol% to about 70 mol% of the total lipids present in the nanoparticle, and the PEG lipid is from about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticle.

[0328] Particularly, the lipid-based nanoparticle comprises or consists of from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid.

[0329] Preferably, the lipid-based nanoparticle comprises or consists of from about 45 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 15 mol % of a helper lipid from about 35 mol% to about 45 mol% of a sterol, and from about 0.5 mol% to about 2.5 mol% of a PEG-lipid. In some aspects, a polymer may be included in and / or used to encapsulate or partially encapsulate the lipid-based nanoparticle according to the invention. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. In some aspects, the lipid based particle comprises a poloxamine and / or a poloxamer. In some aspects, the lipid based particle comprises a plyethyleneimine, rotamine and / or polyaspartamide.

[0330] In some aspects, a surface altering agent may be disposed within a lipid-based nanoparticle of the invention and / or on the surface of the lipid-based nanoparticle (e.g., by coating, adsorption, covalent linkage, or other process). Surface altering agents include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin b4, domase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase).

[0331] In a very specific aspect, the lipid-based nanoparticle comprises a lipid mixture.

[0332] Preferably, the lipid mixture comprises or consists of an ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid, these lipids being preferably as described here above.

[0333] In a specific aspect, the lipid-based nanoparticle comprises [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315) as ionizable or cationic lipid, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and one or more polyethylene glycol (PEG)-modified lipid(s).

[0334] In a very specific aspect, the lipid composition of the LNP according to the invention comprises:

[0335] ALC-0315 from about 35 mol % to about 55 mol % of the total lipids present in the LNP,

[0336] - DOPE from about 5 mol% to about 20 mol % of the total lipids present in the LNP, Cholesterol from about 30 mol% to about 60 mol% of the total lipids present in the LNP,

[0337] - and PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG, from about 0.5 mol% to about 4 mol% of the total lipids present in the LNP, preferably from about 0.5 mol% to about 1.5 or 2 mol% of the total lipids present in the LNP, optionally from about 0.5 mol% to about 1.0 mol% of the total lipids present in the LNP. In a very specific aspect, the lipid composition of the LNP according to the invention comprises: 50% of ALC-0315, 10% ofDOPE, 38.5% of Cholesterol, 1.5 %, of DMG-PEG-2000, preferably at a total lipid concentration of 9.1 mM.

[0338] In a very specific aspect, the lipid composition of the LNP according to the invention comprises:

[0339] ALC-0315 from about 35 mol % to about 55 mol % of the total lipids present in the LNP,

[0340] - DOPE from about 5 mol% to about 20 mol % of the total lipids present in the LNP, Cholesterol from about 30 mol% to about 60 mol% of the total lipids present in the LNP,

[0341] - and PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG, from about 0.5 mol% to about 4 mol% of the total lipids present in the LNP, preferably from about 0.5 mol% to about 1.5 or 2 mol% of the total lipids present in the LNP, optionally from about 0.5 mol% to about 1.0 mol% of the total lipids present in the LNP.

[0342] In some aspects, the lipid-based composition of the lipid-based nanoparticle comprises or consists of a lipid mixture selected from the group consisting of: a) ALC-0315, DOPE, cholesterol and DMG-PEG; b) ALC-0315, DDAB, cholesterol and DMG-PEG; c) ALC-0315, POPE, cholesterol and DMG-PEG; d) ALC-0315, DOPE, cholesterol and DSPE-PEG; e) ALC-0315, DSPC, cholesterol and DMG-PEG; f) ALC-0315, DSPC, cholesterol and ALC-0159; g) SM-102, DSPC, cholesterol and DMG-PEG; h) Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG; and i) ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG;

[0343] Preferably, in such specific aspects, the PEG has a size of 2000 or 5000 Daltons (i.e., PEG-2000 or PEG-5000).

[0344] In a very specific aspect, the lipid composition of the LNP according to the invention comprises:

[0345] ALC-00315, SM-102 or Dlin-MC3-DMA or any mixture thereof from about 45 mol % to about 55 mol %, preferably from about 48 mol % to about 52 mol %, more preferably of about 50 mol% of the total lipids present in the LNP,

[0346] DOPE, DDAB, DOPC, POPE or DSPC or any mixture thereof from about 5 mol% to about 15 mol %, preferably from about 8 mol% to about 12 mol %, more preferably of about 10 mol% of the total lipids present in the LNP, - Cholesterol from about 35 mol% to about 45 mol%, preferably from about 37 mol% to about 40 mol %, more preferably of about 38.5 mol% of the total lipids present in the LNP, and

[0347] - PEG 2000-DSG, PEG 2000-DMG, PEG 5000-DSG, PEG 5000-DMG or any mixture thereof, from about 0.5 mol% to about 2.5 mol%, preferably from about 1 mol% to about 2 mol%, more preferably of about 1.5 mol% of the total lipids present in the LNP.

[0348] In yet another aspect, the lipid composition of the LNP according to the invention comprises or consists of:

[0349] - ALC-0315, or SS-OP or any mixture thereof from about 45 mol % to about 55 mol %, preferably from about 48 mol % to about 52 mol %, more preferably of about 50 mol% of the total lipids present in the LNP,

[0350] - DOPE, or DSPC or any mixture thereof from about 5 mol% to about 15 mol %, preferably from about 8 mol% to about 12 mol %, more preferably of about 10 mol% of the total lipids present in the LNP,

[0351] - Cholesterol from about 35 mol% to about 45 mol%, preferably from about 37 mol% to about 40 mol %, more preferably of about 38.5 mol% of the total lipids present in the LNP,

[0352] - PEG 2000-DSG from about 0.5 mol% to about 2.5 mol%, preferably from about 1 mol% to about 2 mol%, more preferably of about 1.5 mol% of the total lipids present in the LNP, and

[0353] - PEG 2000-DSPE-Mal eimide from about 0.01 mol% to about 2 mol%, preferably from about 0.05 mol% to about 1 mol%, more preferably of about 0.1 mol% of the total lipids present in the LNP.

[0354] The lipid-based nanoparticle according to the invention may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction.

[0355] Lipid-based nanoparticles or a composition comprising LNPs may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a LNP or of a composition comprising LNPs. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a LNP or of a composition comprising LNPs, such as particle size, poly dispersity index, and zeta potential. Physiochemical properties of lipid-based nanoparticles may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs.

[0356] In one aspect, the mean size of the lipid-based nanoparticle of the invention may be between 10 of nm and 200 of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 200 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm or 200nm.

[0357] The lipid-based nanoparticle according to the invention is preferably a nt-LNP. A “nt-LNP” refers to a lipid nanoparticle devoid of antigen binding domain or antigen binding moiety.

[0358] Alternatively, the lipid-based nanoparticle according to the invention is a t-LNP. As used herein, the term “t-LNP” refers to a targeted lipid nanoparticle, i.e., a lipid nanoparticle comprising an antigen binding domain or a targeting moiety comprising an antigen binding domain. Preferably, the antigen binding domain is an antigen binding domain of an antibody.

[0359] Accordingly, the lipid-based nanoparticle according to the invention comprises a targeting moiety or an antigen binding domain capable of specific binding to an antigen, target, cell, tissue or organ of interest. Preferably, the antigen binding domain is an antigen binding domain of an antibody.

[0360] In some aspects, the targeting moiety is an antibody or antigen binding domain thereof.

[0361] The terms "specific binding", "specifically binds to," "specific for" or "selectively binds" a particular target or an epitope on a particular antigen mean that the antigen binding domain recognizes and binds a specific antigen or epitope, but does not substantially recognize or bind other molecules in a sample. For example, an antigen binding domain that specifically (or preferentially) binds to an antigen, is an antigen binding domain that binds said antigen for example with greater affinity, avidity, more readily, and / or with greater duration than it binds to other / different antigens. Preferably, the term "specifically binds to" or "binds specifically" refers to the ability of an antigen receptor to bind to an antigen with an affinity of at least about 1 x 1 O’6M, 1 x 10’7M,1 x 10’8M, 1 x 10’9M, 1 x 10’10M, 1 x 10’11M, 1 x 10’12M, or more, and / or bind to a target with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. The affinity can be determined by various methods well known from one skilled in the art. These methods include, but are not limited to, Biacore Analysis, Blitz analysis and Scatchard plot. In some aspects, the LNP comprises an antigen biding domain that specifically targets immune cells, in particular an antigen biding domain that specifically binds an extracellular marker expressed on immune cells. “Immune cell” as used herein includes neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells). It preferably refers to T cells, more specifically CD4+ T cells, CD8+ T cells, effector T cells and / or exhausted T cells.

[0362] Thus, in some embodiments, the LNP comprises a targeting moiety or an antigen binding domain that specifically binds to CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD154, CD183, CD3, CD4, CD8, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL / TNFSF6, PD1 / PDCD1, GITR / TNFRSF18, GPR32, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LY108 / SlamF6, OX40 / TNFRSF4, RGS1, LTBR / CD70, TNFSF14, CD112R, CD28H, CD 164, TRAF2, CDCR3 / TNFRSF6B, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, ICOSL, CD160, CD19, CD20, CD24, CD38 TIM-1, TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R, CLEC-1A, CDl lb, CD14, CD66b, CD163, CD206, SIGLEC 6, BCMA / TNFRSF17, CD150, CD86, OX40L, L0X1, TACETNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74 and / or CD 165.

[0363] Thus, in some embodiments, the LNP comprises a targeting moiety or an antigen binding domain that specifically binds to PD1 or CTLA-4.

[0364] As used herein, the terms "Programmed Death 1", "Programmed Cell Death 1", “PD-1”, "PDCD1", “PD-1 antigen”, “human PD-1”, "hPD-1" and "hPD-1" are used interchangeably and refer to the Programmed Death- 1 receptor, also known as CD279, and include variants and isoforms of human PD-1, and analogs having at least one common epitope with PD-1. PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. As an example, the amino acid sequence of a human PD-1 is disclosed under GenBank accession number NP 005009. PD-1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC). Accordingly, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD- 1, such as PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4. Unless specified otherwise, the terms include any variant and, isoform of human PD-1 that are naturally expressed by PBMC, or that are expressed by cells transfected with a PD-1 gene.

[0365] Several anti -PD-1 are already clinically approved, and others are still in clinical developments. For instance, the anti -PD-1 antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS- 936558, ONO-4538), OSE-279 (such as disclosed in WO2020 / 127366), Pidilizumab (CT-011), Cemiplimab (Libtayo), Camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (Tisleizumab), PDR001 (spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ- 63723283, Genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), JSOO1 (see Si-Yang Liu et al., J. Hematol. Oncol.10: 136 (2017)), BL754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR- 042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI- 1110 (see WO 2014 / 194302), AGEN2034 (see WO 2017 / 040790), MGA012 (see WO 2017 / 19846), or IBI308 (see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, the disclosure thereof being incorporated herein by reference), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006 / 121168, the disclosure thereof being incorporated herein by reference.

[0366] In some particular aspect, the anti -PD-1 antibody OSE-279 such as described in WO2020 / 127366, which is incorporated herein by reference.

[0367] In some embodiments, the antigen binding domain aims at Treg cells. “Treg” or “regulatory T cells”, are a subset of T lymphocytes that play a crucial role in regulating the immune system and maintaining immune tolerance. Regulatory T cells (Treg cells) are characterized by the expression of specific cell surface markers and regulatory molecules. These markers help identify and distinguish Treg cells from other T cell subsets. The most widely used markers for identifying Treg cells include CD4 (Cluster of Differentiation 4) and CD25 (Interleukin-2 Receptor Alpha Chain).

[0368] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to CD3, CD4, CD25 or CTLA4.

[0369] In some embodiments, the antigen binding domain aims at activated T cells. Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD154, CD183, CD3, CD4, CD8, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL / TNFSF6, PD1 / PDCD1, GITR / TNFRSF18, GPR32, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LY108 / SlamF6, OX40 / TNFRSF4, RGS1, LTBR / CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3 / TNFRSF6B, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TR0Y, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, ICOSL and / or CD160.

[0370] In some embodiments, the antigen binding domain aims at activated B cells. “Activated B cells” or “Activated B lymphocytes” are B cells activated when the B cell binds to an antigen via its B cell Receptor. Markers expressed by activated B cells include but are not limited to BCMA / TNFRSF17, CD 150 and CD86.

[0371] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to BCMA / TNFRSF17, CD 150, CD86, OX40L, LOX1, TACI / TNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD 180, CD59, CD54, CD71, CD35, CD218a, CD74, CD 19, CD20 and / or CD 165.

[0372] In some embodiments, the antigen binding domain aims at Breg cells. “Breg cells”, or “regulatory B cells”, are a subset of B lymphocytes that play a role in immune regulation and maintaining tolerance. Similar to Treg, Breg cells are involved in suppressing immune responses and preventing excessive inflammation. Some of the markers associated with Breg cells include: CD38 (Cluster of Differentiation 38), (Cluster of Differentiation 4), and TIM-1 (T cell immunoglobulin and mucin domain-containing protein 1).

[0373] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to CD24, CD38 and / or TIM-1.

[0374] In some embodiments, the antigen binding domain aims at myeloid cells. Myeloid cells are differentiated descendants from common progenitors derived from hematopoietic stem cells in the bone marrow. Upon pathogen invasion, myeloid cells are rapidly recruited into local tissues via various chemokine receptors, where they are activated for phagocytosis as well as secretion of inflammatory cytokines, thereby playing major roles in innate immunity.

[0375] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R and / or CLEC-1A.

[0376] In some embodiments, the antigen binding domain aims at neutrophils. “Neutrophils” are a type of white blood cells called granulocytes and are produced from stem cells in the bone marrow. They play an important role in innate immunity. “Activated Neutrophils” are neutrophils activated upon their entry into inflammatory or infected tissue site, in response to pro- inflammatory stimuli in the tissue. Neutrophils activation is characterized by the release of granule proteins, acquisition of phagocytic capabilities, and production of NETs, all of which are designed to enhance the cells’ pathogen-destruction capacity. Markers of activated neutrophils include but are not limited to CD1 lb, CD 18, CD66b, CD 177 and PRTN3.

[0377] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to CD1 lb, CD14 and CD66b.

[0378] In some embodiments, the antigen binding domain aims at macrophages. “Macrophages” are a type of white blood cells that help eliminate foreign substances by engulfing foreign materials and initiating an immune response. As used herein, this term includes for example Adipose tissue macrophages, Monocytes, Kupffer cells, Sinus histiocytes, Alveolar macrophages (dust cells), Tissue macrophages (histiocytes) leading to giant cells, Microglia, Hofbauer cells, Intraglomerular mesangial cells, Osteoclasts, Langerhans cells, Epithelioid cells, Red pulp macrophages (sinusoidal lining cells), Peritoneal macrophages, LysoMac. “Activated Macrophages” are macrophages activated either by a priming signal through IFNg followed by encountering an appropriate stimulus, such as bacterial LPS, or by direct stimulation by IL4 and / or IL13. Activated macrophages are able to kill non-self cells through phagocytosis. Markers of activated macrophages include but are not limited to TNFRSF12A, FN14 and / TWEAKR.

[0379] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to CD163, CD206 and / or SIGLEC 6.

[0380] In some embodiments, the antigen binding domain aims at dendritic cells. “Dendritic cells” or “DCs” are antigen-presenting cells of the immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. As used herein, this term includes for example plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC). “Activated Dendritic cells” are dendritic cells directly activated by conserved pathogen molecules and indirectly by inflammatory mediators produced by other cell types that recognize such molecules. Markers of activated dendritic cells include but are not limited to TRAF4.

[0381] Thus, in some embodiments, the LNP comprises an antigen binding domain that specifically binds to TRAF4.

[0382] In some embodiments, the LNP comprises an antigen binding domain that specifically binds to a target selected from the group consisting of CDlOl, CD103, CD119, CD137, CD154, CD183, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL, P-Dl, GITR, GPR32, ICOS, IL18R1, ITGAE, SlamF6, 0X40, RGS1, LTBR, TNFSF14, CD112R, CD28H, CD164, TRAF2, TNFRSF6B, CD357, TNFRSF19L, TNFRSF19, TNFRSF21, TNFRSF25, ICOSL, CD 160, CD38, TRAF1, TRAF4, TRAF7, TRAP 100, TNFRSF12A, CD301, IL4R, CLEC-1A, CD 163, CD206, SIGLEC 6, BCMA, CD 150, CD86, OX40L, LOX1, TNFRSF13B, FCRL4, CD40BP, TRAP1, BAFFR, CD21, CLEC-9A, CD 180, CD59, CD54, CD71, CD35, CD218a, CD74, CD25, CTLA4, LGR6,

[0383] CD138, CD3, CD4, CD8, CD18, CD19, CD20, CD24, CD66b, CD78, CD165, CD177, PRTN3, CD1 lb, CD14 and TIM-1, and any combination thereof.

[0384] In some embodiments, the LNP comprises an antigen binding domain that specifically binds to a target selected in Table E. Table E: Examples of targets of interest.

[0385]

[0386] In some aspects, the LNP comprises a targeting moiety, preferably comprising or consisting of an antigen biding domain of an antibody.

[0387] Preferably, the antigen biding domain specifically targets Organ-Specific Antigens. Organ- specific antigens are proteins or molecules that are expressed predominantly or uniquely in specific organs or tissues of the body.

[0388] In some aspects, the targeting moiety or antigen binding domain is an antibody, a fragment thereof or a derivative thereof. Preferably, the antigen binding domain is preferably derived from an immunoglobulin format selected from the group consisting of IgA, IgM, IgE, IgD and IgG, or a vari ant thereof.

[0389] The terms “derive from” and “derived from” as used herein refers to a compound having a structure derived from the structure of a parent compound or protein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar properties, activities and utilities as the claimed compounds.

[0390] The targeting moiety or antigen binding domain is preferably a monoclonal antibody, preferably a human, humanized, chimeric or recombinant antibody or antigen binding fragment thereof. As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The term "antibody" particularly refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding domain that specifically binds an antigen.

[0391] In a preferred aspect, the targeting moiety or antigen binding domain is an antibody, a fragment or a derivative thereof such as a Fab, a F(ab)2, a Fab’, a F(ab')2, a Fd, a Fv, a crossMAb or a single-chain variable fragment (scFV) a VHH or a single-chain Fab fragment.

[0392] The terms "antigen-binding fragment", “antibody fragment” or “antigen-binding domain” of an antibody, as used herein, refers to one or more fragments or derivatives of an antibody that retain the ability to specifically bind to an antigen. Typically, the antigen binding domain comprises the paratope of an antibody, typically at least its complementary determining regions (CDRs), preferably its heavy chain and light chain variable domains (i.e., VH and VL).

[0393] In some aspects, the targeting moiety or antigen binding domain comprises or is modified to comprise a group such as a thiol group, capable of reacting with a group carried by a lipid of the nanoparticle, such as a PEG-maleimide, so as to conjugate the antigen binding domain to a lipid of the lipid-based nanoparticle. The suitable reactive groups and the conjugation chemistry is well-known be the person skilled in the art. For instance, the conjugation can be carried out by click chemistry. Alternatively, the conjugation can be carried out by an enzyme.

[0394] In an aspect, the targeting moiety or antigen binding domain is conjugated to a lipid of the lipid- based nanoparticle, for instance through a maleimide moiety which is conjugated, attached or linked to a PEG-derivative. Preferably, the targeting moiety or antigen binding domain may comprise a thiol group capable of reacting with the PEG-maleimide to conjugate the targeting moiety or antigen binding domain to the lipid-based nanoparticle. Other methods known to the person skilled in the art may be used to conjugate the targeting moiety or antigen binding domain to a lipid of the lipid-based nanoparticle, optionally through a linker, such as disclosed in Kedmi, Ranit et al. “A modular platform for targeted RNAi therapeutics.” Nature nanotechnology vol. 13,3 (2018): 214-219. doi: 10.1038 / s41565-017-0043-5, the disclosure thereof being incorporated herein by reference. In a particular aspect, the targeting moiety or antigen binding domain is not covalently bound to any of the lipids of the LNP or does not comprise any modification for coupling or grafting the targeting moiety or antigen binding domain to a lipid. In particular, the targeting moiety or antigen binding domain does not comprise a lipophilic moiety or a grafting moiety such as free cysteine(s) or a thiol group.

[0395] In a particular aspect, the LNP does not include any targeting moiety or antigen binding domain specific for an antigen present on the antigen binding domain capable of specifically binding to a target expressed on activated immune cells surface, in particular an antigen binding domain directed against the Fc domain of an antibody.

[0396] In some aspects, the lipid-based nanoparticle does not comprise a moiety comprising a lipidation peptide or motif. As used herein, the terms “lipidation peptide or motif’ refers to a specific sequence pattern in proteins or proteic entity (such as antibodies or fragment thereof) that is associated with the attachment or anchoring of lipid moi eties.

[0397] In a particular aspect, the LNP does not comprise a targeting moiety, an antibody or an antigen binding domain thereof.

[0398] In a specific aspect, the lipid-based nanoparticle comprises a cationic or ionizable lipid, a helper lipid, a structural lipid or sterol, a PEG-lipid that do not comprise a reactive group for conjugation to a targeting moiety and a PEG lipid comprising a reactive group for conjugation to a targeting moiety.

[0399] In a specific aspect, the lipid-based nanoparticle comprises a cationic or ionizable lipid, a helper lipid, a structural lipid or sterol, a non-conjugated PEG-lipid and a PEG lipid conjugated to a targeting moiety.

[0400] In order to have a biodistribution which favour the escape of capture organs in particular the liver, the non-conjugated PEG-lipid is preferably a C16-C22 PEG lipid, preferably a Cl 8 PEG (such as DSPE PEG, DSG PEG). In some embodiments, the PEG-lipid it is not a C14 PEG (such as DMG PEG). Said PEG lipid typically has stealth property allowing to escape said organs and this allow the LNP to reach blood circulation and targeted areas of interest, in particular inflamed tissues. The LNP of the invention is capable of very specifically targeting immune cells of interest locally in the target tissue, in particular thanks to the contribution of the PEG lipid conjugated to a targeting entity, in particular activated immune cells that are locally present in the tissue, such as PD1 positive immune cells (T cells) in an inflamed tissue when using an anti- PD1 antibody conjugated to the conjugated PEG lipid of the LNP. This will allow to target even more specifically and efficiently notably auto-reactive (activated with excessive pro- inflammatory activity) T or B cells, such as auto-reactive T cells, and this will lead to a targeted decrease of their inflammatory activity, consequently to the direct or indirect local overproduction of IL-35 (IL-35 encoded by IL-35 RNA of LNP transduced in targeted cells).

[0401] In this aspect, the lipid-based nanoparticle comprises from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, from about 0.5 mol% to about 4 mol% of a PEG-lipid that do not comprise a reactive group for conjugation to a targeting moiety and from about 0.01 mol% to about 4 mol% of a PEG lipid conjugated to a targeting moiety.

[0402] In the LNP of the invention comprising a mRNA encoding a membrane IL-35 molecule as described herein, the targeting moiety is typically coupled to the DSPE-PEG-Maleimide moiety.

[0403] Accordingly, in some aspects, the lipid composition of the LNP according to the invention comprises or consists of:

[0404] SS-OP: about 50% ; DSPC: about 10% ; Cholesterol: about 38.5 % ; DSG-PEG: about 1.4 % and DSPE-PEG-Maleimide : about 0.1 % of the total lipids present in the lipid- based nanoparticle according to the invention.; or

[0405] - ALC-0315: about 50% ; DOPE or DSPC: about 10% ; Cholesterol: about 38.5 % / DSG- PEG: about 1.5 % and DSPE-PEG-Maleimide : about 0.1 % of the total lipids present in the lipid-based nanoparticle according to the invention.

[0406] In some aspects, the lipid composition of the LNP according to the invention comprises or consists of:

[0407] ALC-0315, DDAB, MC3-DLin-DMA or any mixture thereof, preferably from about 20 mol% to about 60 mol% of the total lipids of the lipid-based nanoparticle;

[0408] - DSPC and / or DOPE, preferably from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle;

[0409] Cholesterol, preferably from about 30 mol% to about 70 mol% of the total lipids of the lipid-based nanoparticle; and

[0410] - DMG-PEG, preferably from about 0.1 mol% to about 10 mol% of the total lipids of the lipid-based nanoparticle.

[0411] In some aspects, the lipid composition of the LNP according to the invention comprises or consists of:

[0412] ALC-0315, DDAB or MC3-DLin-DMA, preferably from about 25 mol% to about 55 mol%, more preferably from about 29mol% to about 50 mol%, most preferably about 50%; DSPC or DOPE, preferably from about 5 mol% to about 15 mol%, more preferably from about 9mol% to about 10 mol%, most preferably about 38,5%;

[0413] - Cholesterol, preferably from about 30 mol% to about 70 mol%, more preferably from about 38,5mol% to about 63 mol%, most preferably about 38,5%; and

[0414] - DMG-PEG, preferably from about 1 mol% to about 10 mol%, more preferably from about 1,5 mol% to about 5 mol%, most preferably about 1,5%

[0415] In some aspects, the lipid composition of the LNP according to the invention comprises or consists of:

[0416] - ALC-0315 about 50%, DOPE about 10%, Cholesterol about 38.5%, DMG-PEG about 1.5%,

[0417] - DDAB about 29%, DSPC about 9%, Cholesterol about 63%, DMG-PEG about 5%,

[0418] - MC3-DLin-DMA about 50%, DSPC about 10%, Cholesterol about 38.5%, DMG-PEG about

[0419] 1.5%, or

[0420] - MC3-DLin-DMA about 50%, DOPE about 10%, Cholesterol about 38.5%, DMG-PEG-2000 about 1.5%.

[0421] Preferably, the DMG-PEG is DMG-PEG-2000.

[0422] In some particular aspects, the mixture of lipids in the LNP is adapted for skin delivery, in particular by a topic administration.

[0423] The lipid-based nanoparticle particularly comprises or consists of: [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315) as ionizable or cationic lipid or a derivative thereof, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and DMG-PEG (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol).

[0424] Preferably, in this lipid mixture, ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)] or a derivative thereof represents from about 45 mol % to about 55 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle according to the invention. Preferably, ALC-0315 or a derivative thereof represents from about 48 mol % to about 52 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle according to the invention. More preferably, ALC-0315 or a derivative thereof represents from about 49 mol % to about 50 mol % of the total lipids present in the lipid composition of the lipid- based nanoparticle according to the invention.

[0425] By the term “derivative of ALC-0315”, the applicant refers to lipid related to ALC-0315. Said derivatives typically comprises chemical modification. These derivatives are well-known known by the one killed in the art of LNP technology, and includes those described in W02018 / 081480, the disclosure thereof being incorporated herein by reference. Said derivatives particularly include without being limited thereto :

[0426] Said derivatives of ALC-0315 may have in particular the following formula : wherein:

[0427] -Z is ORl or NRlR2

[0428] -R1 and R2 are each independently H or an optionally substituted Ci-Cio alkyl or C2-C10 alkenyl; -R3, R4, R5, R6 are each independently H or a, optionally substituted Ce-Cis alkyl or Ce-Cis alkenyl;

[0429] -XI and X2 are each independently H or an optionally substituted C1-C10 alkyl or C2-C10 alkenyl; -Y is independently -O(C=O), -(C=O)O, -C(=O), NR1C(=O), -C(=O)NR1, R1 being as defined above.

[0430] In the lipid mixture for skin delivery, DOPE (1 -oleoyl -2-hydroxy-sn-glycero-3-phosphocholine (18 : 1 Lyso PC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine) typically represents from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle according to the invention. Preferably, DOPE represents from about 8 mol% to about 12 mol% of the total lipids of the lipid-based nanoparticle according to the invention. More preferably, DOPE represents from about 10 mol% to about 11 mol% of the total lipids of the lipid-based nanoparticle according to the invention.

[0431] In the lipid mixture for skin delivery, DMG-PEG (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol) typically represents from about 0.1 mol% to about 10 mol% of the total lipids of the lipid-based nanoparticle according to the invention. Preferably, DMG-PEG represents from about 0.5 mol% to about 2 mol% of the total lipids of the lipid-based nanoparticle according to the invention. More preferably, DMG-PEG represents from about 0.5 mol% to about 1.5 mol% of the total lipids of the lipid-based nanoparticle according to the invention. Optionally, the PEG is comprised between 2000 Daltons and 5000 Daltons (i.e., PEG-2000 to PEG-5000). Preferably, DMG-PEG is DMG-PEG-2000, DMG-PEG-5000 or any mixture thereof. In a very particular aspect, the DMG-PEG is PEG 2000-DMG.

[0432] In the lipid mixture for skin delivery, cholesterol typically represents from about 30 mol% to about 70 mol% of the total lipids present in the lipid-based nanoparticle of the invention. Preferably, cholesterol represents from about 38 mol% to about 42 mol% of the total lipids present in the lipid-based nanoparticle of the invention. More preferably, cholesterol represents from about 38 mol% to about 40 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0433] In a specific aspect, the lipid composition of the LNP, especially for skin delivery, comprises or consists of:

[0434] ALC-0315 or a derivative thereof from about 45 mol % to about 55 mol % of the total lipids present in the LNP,

[0435] - DOPE from about 1 mol% to about 20 mol % of the total lipids present in the LNP, Cholesterol from about 30 mol% to about 70 mol% of the total lipids present in the LNP, and

[0436] DMG-PEG, preferably DMG-PEG 2000, from about 0.1 mol% to about 10 mol% of the total lipids present in the LNP.

[0437] In another aspect, the lipid composition of the LNP, especially for skin delivery, comprises or consists of:

[0438] ALC-0315 or a derivative thereof from about 48 mol % to about 52 mol % of the total lipids present in the LNP,

[0439] - DOPE from about 8 mol% to about 12 mol % of the total lipids present in the LNP, Cholesterol from about 38 mol% to about 42 mol% of the total lipids present in the LNP, and DMG-PEG, preferably DMG-PEG 2000, from about 0.5 mol% to about 5 mol% of the total lipids present in the LNP.

[0440] In an additional specific aspect, the lipid composition of the LNP, especially for skin delivery, comprises or consists of:

[0441] ALC-0315 or a derivative thereof from about 49 mol % to about 50 mol % of the total lipids present in the LNP,

[0442] - DOPE from about 10 mol% to about 11 mol % of the total lipids present in the LNP, Cholesterol from about 38 mol% to about 40 mol% of the total lipids present in the LNP, and

[0443] DMG-PEG, preferably DMG-PEG 2000, from about 0.5 mol% to about 1.5 mol% of the total lipids present in the LNP.

[0444] In some aspects, the invention concerns a lipid-based nanoparticle comprising mRNA molecule(s) encoding a membrane interleukin 35 (IL-35) molecule as described herein, wherein the IL-35 is in the form of a fusion protein as described herein.

[0445] The invention particularly concerns a lipid-based nanoparticle comprising mRNA molecule(s) encoding a fusion construct comprising or consisting of a sequence as set forth in SEQ ID NO 9- 20 or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively.

[0446] The invention particularly concerns a lipid-based nanoparticle comprising mRNA molecule(s) encoding a fusion construct comprising a sequence as set forth in SEQ ID NO 9-14, preferably in SEQ ID NO: 9, 11 or 13, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively.

[0447] The invention particularly concerns a lipid-based nanoparticle comprising mRNA molecule(s) encoding a fusion construct comprising a sequence as set forth in SEQ ID NO 15-20, preferably in SEQ ID NO: 15, 17 or 19, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively.

[0448] The invention particularly concerns a lipid-based nanoparticle comprising mRNA molecule(s) encoding a fusion construct comprising or consisting of a sequence as set forth in SEQ ID NO: 13, 14, 17 or 18, preferably in SEQ ID NO: 13 or 17, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively.

[0449] The invention particularly concerns a lipid-based nanoparticle comprising mRNA molecule(s) comprising or consisting of a sequence as set forth in SEQ ID NO: 46-49, 154-161 and 170-171, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, respectively.

[0450] The amount of a mRNA molecule in a lipid-based nanoparticle may depend on the size, composition, desired target and / or application, or other properties of the lipid-based nanoparticle. For example, the amount of mRNA useful in the lipid-based nanoparticle may also depend on the size, sequence, and other characteristics of the mRNA. The relative amounts of a mRNA molecule and other elements (e.g., lipids) in a lipid-based nanoparticle may also vary. In some aspects, the wt / wt ratio of the lipid component to a mRNA molecule in a lipid-based nanoparticle may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1,30:1,35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of the lipid component to a mRNA molecule may be from about 10:1 to about 40: 1. In certain aspects, the wt / wt ratio is about 20:1. The amount of a mRNA molecule in a lipid- based nanoparticle may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0451] Alternatively, the amount of lipid and mRNA may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an mRNA. In general, a lower N:P ratio is preferred. The one or more mRNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain aspects, theN:P ratio may be from about 2:1 to about 8:1. In other aspects, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio may be between 5.0:1 and 7.0:1, preferably between 5.5:1 and 6.5:1. For example, the N:P ratio may be about 6: 1. Preferably, the N:P ratio is about 6: 1.

[0452] Pharmaceutical compositions

[0453] The present invention also relates to pharmaceutical compositions comprising any of the lipid- based nanoparticles as described hereabove, preferably as the active ingredient or compound and optionally a pharmaceutically acceptable carrier or excipient.

[0454] As used herein, a “pharmaceutical composition” refers to a preparation of one or more of the active agents, such as comprising a LNP according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism. Compositions of the present invention can be in a form suitable for any conventional route of administration or use. In one aspect, a “composition” typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally- occurring carrier, inert or active, such as an adjuvant, diluent, binder, stabilizer, buffers, preservative or the like and include pharmaceutically acceptable carriers. An "acceptable vehicle" or “acceptable carrier” as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0455] The pharmaceutical compositions can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers, excipients, salts, antioxidant and / or stabilizers which do not deleteriously interact with the lipid-based nanoparticle of the invention and does not impart any undesired toxicological effects.

[0456] Particularly, the pharmaceutical composition according to the invention can be formulated for any conventional route of administration including a topical, enteral, oral, parenteral, pulmonary, intranasal, intravenous, intramuscular, intradermal, transdermal, epicutaneous, subcutaneous or intraocular administration and the like. Preferably, the pharmaceutical composition according to the invention is formulated for intravenous, intramuscular or subcutaneous administration. In some cases, LNPs may be administered directly to specific tissues or organs. For example, LNPs designed for targeted delivery to the lungs may be administered via inhalation or intratracheal injection for respiratory diseases. For example, LNPs designed for targeted delivery to the skin, preferably comprising the lipid mixture for skin delivery as described herein, can be formulated for topical, intradermal, transdermal, epicutaneous or subcutaneous route of administration.

[0457] To facilitate administration, the lipid-based nanoparticle as described herein can particularly be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)). In yet another embodiment, the pharmaceutical composition is administered intranodally or intratum orally.

[0458] The pharmaceutical composition may be prepared by mixing a lipid-based nanoparticle having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, antioxidant, and / or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, antioxidants, and / or stabilizers are well known in the art and have been for example described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Preferably, the pharmaceutical composition comprising the lipid-based nanoparticle is relatively homogenous. A poly dispersity index may be used to indicate the homogeneity of the composition, e.g., the particle size distribution of the lipid-based nanoparticles comprised in the composition. A small (e.g., less than 0.3) poly dispersity index generally indicates a narrow particle size distribution. Preferably, the pharmaceutical composition has a poly dispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the poly dispersity index of the pharmaceutical composition is from about 0.10 to about 0.25.

[0459] Pharmaceutical compositions according to the invention may be formulated to release the active ingredients (e.g. the lipid-based nanoparticle of the invention) substantially immediately upon administration or at any predetermined time or time period after administration. The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Means known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[0460] It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer.

[0461] Pharmaceutical composition typically must be sterile and stable under the conditions of manufacture and storage. Prevention of presence of microorganisms may be ensured both by sterilization procedures (for example by microfiltration), and / or by the inclusion of various antibacterial and antifungal agents.

[0462] In some embodiments, the pharmaceutical composition includes one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surface active agents, buffering agents and / or preservatives. Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and / or combinations thereof.

[0463] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol preservatives and / or acidic preservatives.

[0464] Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite and / or sodium sulphite.

[0465] Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, eidetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and / or trisodium edetate.

[0466] Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerine, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and / or sorbic acid.

[0467] Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulphite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™ and / or EUXYL®. An exemplary free radical scavenger includes butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine.

[0468] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminium hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution and / or ethyl alcohol.

[0469] In some embodiments, the pharmaceutical composition including a lipid-based nanoparticle according to the invention further includes a salt, such as a chloride salt. In some embodiments, the pharmaceutical composition including a lipid-based nanoparticle further includes a sugar such as a disaccharide. In some embodiments, the pharmaceutical composition further includes a sugar but not a salt, such as a chloride salt. In some embodiments, a pharmaceutical composition further includes one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogues thereof).

[0470] The LNP of the invention can be formulated with amphiphilic polymers in a pharmaceutical composition. Common amphiphilic polymers used in the formulation of LNPs include polyethylene glycol (PEG) and polyvinyl alcohol (PVA). Relative amounts of the lipid-based nanoparticles, pharmaceutically acceptable excipients and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. In some embodiments, the pharmaceutical composition comprises between 0.1% and 100% (wt / wt) of one or more lipid-based nanoparticles such as disclosed herein. The amount or number of lipid- based nanoparticles which can be combined with a carrier material to produce a single dosage form will generally be that amount of the lipid-based nanoparticles which produces a therapeutic effect.

[0471] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of the pharmaceutical composition including a lipid-based nanoparticle such as disclosed herein. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and / or for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0472] Therapeutic uses

[0473] The lipid-based nanoparticle and the pharmaceutical composition comprising such as defined above have numerous in vitro and in vivo utilities and applications. Particularly, the lipid-based nanoparticles and the pharmaceutical compositions provided herein may be used in therapeutic methods and / or for therapeutic purposes.

[0474] As used herein, the term "treatment", "treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain aspects, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease, such as according to the present disclosure, the disruption or the delay in the resolution of the inflammation leading to inflammation associated disease. In other aspects, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease. The present invention relates to a lipid-based nanoparticle or a pharmaceutical composition comprising the LNP of the invention for use in the treatment of an autoimmune disease or an inflammatory disease.

[0475] It also relates to the use of a lipid-based nanoparticle or a pharmaceutical composition comprising of the invention for treating an autoimmune disease or an inflammatory disease, in a subject. It also concerns the use a lipid-based nanoparticle or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating an autoimmune disease or an inflammatory disease, in a subject.

[0476] In some aspects, the autoimmune disease or an inflammatory disease is an acute or chronic disease. In some aspects, the disease is an acute inflammatory disease or inflammation, including serous inflammation, fibrinous inflammation, purulent inflammation and hemorrhagic inflammation.

[0477] Finally, it relates to a method for treating an autoimmune disease or an inflammatory disease, in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition or a lipid-based nanoparticle such as disclosed herein.

[0478] In one embodiment, the invention relates to a method of treatment of a disease and / or disorder selected from the group consisting of a disease wherein the resolution of inflammation is delayed or disrupted, and / or a disease selected from the group of inflammatory diseases, autoimmune diseases in a subject in need thereof, comprising administering to said subject an effective amount of the lipid-based nanoparticle or pharmaceutical composition as defined above. Examples of such diseases and disorders are more particularly described hereafter.

[0479] Particularly, the invention concerns a treatment method that comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of the lipid-based nanoparticle or pharmaceutical composition described herein.

[0480] “An effective amount” or a “therapeutic effective amount” as used herein refers to the amount of active agent (i.e., the lipid-based nanoparticle disclosed herein) required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The “effective amount” will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art. In some aspects, the disease to be treated by the lipid-based nanoparticle or a pharmaceutical composition of the invention is selected from the group comprising Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoathetosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti -N-Methyl-D- Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, membranous nephropathy, allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune urticaria, Behcet's disease, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune disfunction syndrome, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barre syndrome, graft-versus-host disease (GVHD), hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgM polyneuropathies, juvenile arthritis, Kawasaki's disease, lichen plantus, lichen sclerosus, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes mellitus, Multifocal motor neuropathy (MMN), pemphigoid gestationis, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynauld's phenomenon, Reiter's syndrome, sarcoidosis, solid organ transplant rejection, Takayasu arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteristis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, anti-neutrophil cytoplasmic antibody-associated vasculitides, vitiligo, asthma, autoimmune pancreatitis, IgA nephropathy and Wegner's granulomatosis; optionally selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoathetosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti-N-Methyl-D-Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, and membranous nephropathy, preferably selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, antiphospholipid syndrome, Hashimoto's thyroiditis, Graves' disease and graft rejection.

[0481] In some aspects, the disease to be treated by the lipid-based nanoparticle or a pharmaceutical composition of the invention is an autoimmune disease. Preferably, the invention concerns a pharmaceutical composition comprising a lipid-based nanoparticle (LNP) comprising one or more mRNA molecule(s) encoding a membrane IL-35 molecule as described herein, for use in the treatment of an auto-immune disease.

[0482] As used herein, the term "autoimmune disease" refers to a physiological condition in a subject that is resultant from the subject’s own body producing an inappropriate immune response that targets and damages the subject’s own cells. In autoimmune diseases, this immune tolerance breaks down, and the immune system identifies the body's own cells and / or tissues as foreign. As a result, the immune system mounts an immune response, producing antibodies and activating immune cells, leading to inflammation and damage to organs and tissues.

[0483] Examples of auto-immune diseases to be treated according to the invention are diabetes, in particular type 1 diabetes, NASH, psoriasis, lupus, in particular Systemic Lupus Erythematosus (SLE), rheumatoid arthritis, myasthenia gravis, multiple sclerosis, Sjogren’s syndrome, Hashimoto's Thyroiditis, celiac disease and vasculitis.

[0484] In some aspects, the disease to be treated by the lipid-based nanoparticle or a pharmaceutical composition of the invention is an inflammatory disease. Preferably, the invention concerns a pharmaceutical composition comprising a lipid-based nanoparticle (LNP) comprising one or more mRNA molecule(s) encoding a membrane IL-35 molecule as described herein, for use in the treatment of an inflammatory disease. As used herein, the term "inflammatory disease" or "inflammatory condition" encompasses any disease or condition characterized by inflammation. Inflammation is a basic physiological response to a variety of external or internal insults, such as infectious agents, physical injury, hypoxia, or disease processes. Therefore, diseases or conditions falling within "inflammatory disease" do not have to share a common genetic or physiological basis, so long as the disease or condition results in inflammation. Representative conditions that are encompassed in the term inflammatory disease include allergies, alopecia areata, aphthous ulcer, arthritis (such as rheumatoid arthritis, inflammatory arthritis, juvenile rheumatoid, gouty arthritis, and osteoarthritis), asthma, celiac disease, Crohn’s disease, Inflammatory Bowel Disease (IBD), dermatitis (including atopic dermatitis, contact dermatitis, seborrheic dermatitis, discoid dermatitis, lichen simplex chronicus and eczematous dermatitis), diabetes, diabetes mellitus, Hashimoto’s thyroiditis, irritable bowel disease, cutaneous and systemic lupus erythematosus, Periodontal Disease, multiple sclerosis, myasthenia gravis, cystic fibrosis, osteoporosis, psoriasis, scleroderma, Sjogren’s syndrome (including keratoconjunctivitis sicca secondary to Sjogren's syndrome), Type 2 Diabetes, Chronic Obstructive Pulmonary Disease (COPD), ulcerative colitis, vaginitis and Wegener's granulomatosis.

[0485] In some aspects, the autoimmune or inflammatory disease is an autoimmune or inflammatory disease of the skin. In such aspects, the pharmaceutical composition according to the invention is preferably formulated for any suitable route of administration preferably a topical, intradermal, transdermal, epi cutaneous, subcutaneous route of administration.

[0486] Particularly, the LNPs described herein have optimized formulations adapted for specific skin biodistribution. Such formulations allow the carrying of RNA encoding membrane IL-35. In particular, some formulations of the present invention are suitable for a topical administration, typically with better localized and controlled effect than a systemic administration.

[0487] Accordingly, the present invention relates to a lipid-based nanoparticle or a pharmaceutical composition comprising the LNP of the invention for use in the treatment of an autoimmune skin disease or an inflammatory skin disease and wherein the lipid-based nanoparticle or pharmaceutical composition comprising the LNP is or is to be administered by topical, intradermal, transdermal, epicutaneous or subcutaneous route, preferably topical route. It also relates to the use of a lipid-based nanoparticle or a pharmaceutical composition comprising the LNP of the invention for treating an autoimmune skin disease or an inflammatory skin disease in a subject and wherein the lipid-based nanoparticle or pharmaceutical composition comprising the LNP is or is to be administered by topical, intradermal, transdermal, epicutaneous or subcutaneous route, preferably topical route. It further concerns the use a lipid-based nanoparticle or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating an autoimmune skin disease or an inflammatory skin disease, in a subject, wherein the lipid-based nanoparticle or pharmaceutical composition comprising the LNP is or is to be administered by topical, intradermal, transdermal, epicutaneous or subcutaneous route, preferably topical route. Finally, it relates to a method for treating an autoimmune skin disease or an inflammatory skin disease, in a subject in need thereof comprising administering by topical, intradermal, transdermal, epicutaneous or subcutaneous route, preferably topical route to said subject a therapeutically effective amount of a pharmaceutical composition or a lipid-based nanoparticle such as disclosed herein.

[0488] In some aspects, the disease to be treated by the lipid-based nanoparticle or a pharmaceutical composition of the invention is from the group consisting eczema (atopic dermatitis), contact dermatitis, discoid dermatitis, psoriasis, seborrheic dermatitis, lichen simplex chronicus, pityriasis rosea, rosacea, urticaria, erythema multiforme, erythroderma, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, dermatitis herpetiformis, epidermolysis bullosa acquisita, cutaneous lupus erythematosus, discoid lupus erythematosus, dermatomyositis, scleroderma Henoch-Schbnlein purpura, vitiligo, alopecia areata, lichen planus, lichen sclerosis, morphea, and psoriasis.

[0489] In some specific aspects, the pharmaceutical composition of the invention is a topical pharmaceutical composition, the skin disease is psoriasis or atopic dermatitis; and the lipid composition of the lipid-based nanoparticle consists of about 48%-52% ALC-0315, 8%-12% DOPE, 38%-42% cholesterol and about 0.5% and 2% DMG-PEG of the total lipids of the lipid- based nanoparticle.

[0490] In some aspects, the disease is selected from the group consisting of Type 1 or 2 diabetes, Inflammatory Bowel Disease (IBD), rheumatoid arthritis, multiple sclerosis, Systemic lupus erythematosus, Inflammatory bowel disease, Atherosclerosis, Primary Sjogren syndrome, asthma and hepatitis.

[0491] In some aspects, the disease is an inflammatory and / or auto-immune disease of the liver. Preferably, the disease is selected from the group consisting of hepatitis, Autoimmune hepatitis (AH), Primary biliary cholangitis (PBC), Primary sclerosing cholangitis (PSC) and nonalcoholic fatty liver disease. Preferably, the disease is Autoimmune hepatitis (AH4). The lipid-based nanoparticle or pharmaceutical compositions of the invention can finally be used for the treatment of patients affected by infection diseases such as sepsis, severe viral infections with severe inflammatory conditions, such as coronavirus (e.g. COVID-19 or H1N1).

[0492] In some embodiment, the lipid-based nanoparticle or the pharmaceutical composition according to the invention may be used in combination with another therapeutic agent or therapy, in particular for the treatment of autoimmune diseases or autoimmune diseases.

[0493] The present invention also relates to a method for treating a disease, such as an autoimmune disease or a disease wherein the resolution of inflammation is delayed or disrupted, and / or an inflammatory disease, in a subject, comprising administering to said subject a therapeutically effective amount of the lipid-based nanoparticle or the pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent or therapy.

[0494] Examples of additional or second therapeutic agents include Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) such as ibuprofen and naproxen, Corticosteroids, Disease-Modifying Antirheumatic Drugs (DMARDs) such as methotrexate, hydroxychloroquine, azathioprine and sulfasalazine, immunosuppressive drugs, such as azathioprine, mycophenolate mofetil and cyclosporine, TNF inhibitors such as infliximab, adalimumab and etanercept, interleukin inhibitors such as tocilizumab and anakinra, rituximab, immune checkpoint inhibitors, interferons, Janus Kinase (JAK) Inhibitors, Plasmapheresis, Pain relief medications such as acetaminophen.

[0495] The additional or second therapeutic agent can be administered by the same or different route of administration than the lipid-based nanoparticle, the pharmaceutical composition of the invention.

[0496] Preferably, the additional or second therapeutic agent or the combined therapy is administered to the subject orally, via subcutaneous, intra-cutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-tumoral, intra-sternal, intra-thecal, intra-lesion, and intracranial injection or infusion techniques.

[0497] Subject, regimen and administration

[0498] A subject in need of a treatment may be a human having, at risk for, or suspected of having a disease such as an autoimmune disease or an inflammatory disease. Such a patient can be identified by routine medical examination. In particular, diagnosis of inflammatory and / or auto-immune diseases can include symptom assessment, examination of the family history, physical examination, imaging, laboratory tests such as Complete Blood Count (CBC), C-Reactive Protein (CRP) and Erythrocyte Sedimentation Rate (ESR) determination, auto-antibodies or immunoglobulin levels, and the like.

[0499] As used herein, the term “subject”, “host”, “individual,” or “patient” refers to human, including adult and child. The subject to treat may particularly be a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old, even more preferably an adult of at least 70 years old.

[0500] The form of the pharmaceutical compositions, the route of administration and the dose of administration can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, size, sex, and / or general physical condition. The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.

[0501] In some embodiments, the subject has already received at least one line of treatment, preferably several lines of treatment, prior to the administration of the lipid-based nanoparticle, the pharmaceutical composition or the combined therapy of the invention.

[0502] Methods

[0503] The invention also relates to a method of inhibiting immune cell activity, comprising the step of contacting immune cells with a lipid-based nanoparticle or a pharmaceutical composition according to the invention. Such methods aim to inhibit the inflammatory potential of T cells and / or B cells, especially Treg and / or Breg cells, and provide powerful tools to reduce the immune response, in particular for the treatment of a disease such as an autoimmune disease or an inflammatory disease.

[0504] In a particular aspect, the lipid-based nanoparticle disclosed herein can be administered to a subject, e.g., in vivo, to reduce immunity or to reduce the immune response, preferably in order to treat a disorder and / or disease. Accordingly, in one aspect, the invention provides a method of inhibiting an immune response in a subject comprising administering to the subject a lipid-based nanoparticle or pharmaceutical composition of the invention such that the immune response in the subject is inhibited. The lipid-based nanoparticle or pharmaceutical composition is preferably used to inhibit immune responses such as immune cell activation in a subject in need of a treatment. In a particular embodiment, the lipid-based nanoparticle or pharmaceutical composition according to the invention is used to increase T cells exhaustion or to deactivate activated T cells.

[0505] The invention particularly provides a method of inhibiting an immune response in a subject, comprising administering to the subject a therapeutic effective amount of any of the lipid-based nanoparticles or pharmaceutical compositions comprising such described herein, such that an immune response in the subject is decreased. In a particular embodiment, the lipid-based nanoparticle or pharmaceutical composition can be used to inhibit the cytotoxic T-lymphocyte (CTL) response, to increase T cells exhaustion or to deactivate activated T cells.

[0506] The term "immune response" refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complements) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0507] In some embodiments, the method is an ex vivo or an in vitro method.

[0508] The invention particularly concerns an in vitro method for inhibiting immune cells activity, comprising the step of contacting activated immune cells with a lipid-based nanoparticle (LNP) of the invention.

[0509] The method can particularly comprise the step of: i) providing T cells and / or B cells from a subject, in particular of a subject suffering of an inflammatory and / or auto-immune disease ii) contacting said T cells and / or B cells with a lipid-based nanoparticle (LNP) of the invention, thereby inhibiting or lowering their activity, especially their inflammatory and / or auto-immune activity.

[0510] Especially, the inflammatory and / or auto-immune activity is reduced in comparison to a reference level, which is preferably the inflammatory and / or auto-immune activity of said cells in the absence of contact with the LNP.

[0511] Optionally, the method further comprises a step of administering the T cells and / or B cells having an inhibited or reduced inflammatory and / or auto-immune activity to the subject. Kit

[0512] Any of the lipid-based nanoparticle or compositions described herein may be included in a kit provided by the present invention. The present disclosure particularly provides kits for use in treating an autoimmune disease and / or inflammatory disease or for inhibiting immune cells activation.

[0513] In the context of the present invention, the term “kit” means two or more components (one of which corresponding to the lipid-based nanoparticle) packaged in a container, recipient or otherwise. A kit can hence be described as a set of products and / or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit. The kits of this invention are in suitable packaging.

[0514] The kit may include, in suitable container means, the pharmaceutical composition or lipid-based nanoparticle of the present invention.

[0515] The components comprised in the kit according to the invention may particularly be formulated into a syringe compatible composition.

[0516] In some embodiments, in particular when the kit is for use in the treatment of a disease, the kit further includes an additional agent for treating autoimmune diseases or inflammatory diseases, and the additional agent may be combined with the pharmaceutical composition and / or lipid- based nanoparticle of the present invention, or other components of the kit of the present invention or may be provided separately in the kit. Particularly, the kit described herein may include one or more additional therapeutic agents such as those described in the “Combined Therapy” described hereabove. The kit(s) may be tailored to a particular autoimmune and / or inflammatory disease for an individual and comprise respective second autoimmune and / or inflammatory disease therapies for the individual as described hereabove.

[0517] When the kit is for use in the method for inhibiting immune cell activation such as disclosed herein, the kit may further comprise an immune cell culture medium. For instance, the lipid- based nanoparticles according to the invention may be comprised in such immune cell culture medium.

[0518] The instructions related to the use of the lipid-based nanoparticle or pharmaceutical composition described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, means for reconstituting the lipid-based nanoparticle and / or means for diluting the lipid-based nanoparticle of the invention. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual).

[0519] All the references cited in this description are incorporated by reference in the present application. Others features and advantages of the invention will become clearer in the following figures and examples which are given for purposes of illustration and not by way of limitation.

[0520] Items

[0521] The present patent application also concerns a pharmaceutical composition comprising a lipid- based nanoparticle (LNP) comprising one or more mRNA molecule(s) encoding a membrane interleukin 35 (IL-35) molecule and optionally a pharmaceutically acceptable carrier, for use in the treatment of an inflammatory and / or auto-immune disease.

[0522] Particularly, the IL-35 molecule is a mammalian IL-35 molecule, preferably a human IL-35 molecule.

[0523] The LNP typically comprises a mRNA molecule encoding a single-chain polypeptide comprising an EBB subunit, a p35 subunit and, optionally a linker therebetween.

[0524] The membrane IL-35 molecule particularly comprises or consists of from N-terminus to C- terminus:

[0525] - optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a membrane anchoring domain; or

[0526] - optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, a membrane anchoring domain.

[0527] The membrane anchoring domain can be a transmembrane domain and / or a Glycosylphosphatidylinositol (GPI)-anchor domain.

[0528] The IL-35 molecule can comprise a hinge domain, a transmembrane domain or a fragment thereof and optionally a cytosolic domain or a fragment thereof.

[0529] Typically, the membrane IL-35 molecule comprises or consists of, from N-terminus to C- terminus:

[0530] - optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a transmembrane domain and optionally a cytosolic domain; or

[0531] - optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, a transmembrane domain and optionally a cytosolic domain; or

[0532] - optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a GPI-anchor domain; or - optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, and a GPI-anchor domain.

[0533] In some aspects, the membrane IL-35 comprises a peptide linker, said peptide linker being selected from the group consisting of i) a cleavable linker, preferably a 2A self-cleaving peptide, ii) a flexible linker, preferably selected from the group consisting of Glycine-Serine linkers, Glycine-Proline linkers and Proline-Rich linkers, and iii) a rigid linker, preferably selected from the group consisting of a-Helical Linkers, coiled-coil linkers and P-Sheet linkers.

[0534] Additionally or alternatively, the membrane IL-35 comprises a peptide signal, said peptide signal being p35 or IL-2 peptide signal, preferably comprising or consisting of a sequence of SEQ ID NO: 42 or 44.

[0535] In some aspects, the membrane IL-35 further comprises a hinge domain, said hinge domain being selected from the group consisting of a CD8 hinge domain, a CD28 hinge domain, an IgGl hinge domain, an IgG4 hinge domain, a CH2-CH3 domain of an IgGl, a CH2-CH3 domain of an IgG4, a hinge-CH2-CH3 of an IgGl, a hinge-CH2-CH3 domain of an IgG4 and a peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:22-35, 50 and 54. In some aspects wherein the membrane IL-35 comprises a transmembrane domain, said transmembrane domain being selected from the group consisting of a cytokine receptor transmembrane domain, a chemokine receptor transmembrane domain, a type I transmembrane protein transmembrane domain, a type II transmembrane protein transmembrane domain. Preferably, the transmembrane domain is selected from the group consisting of a CD80 transmembrane domain, a MHC1 transmembrane domain, a FAS transmembrane domain and a TNF receptor transmembrane domain, preferably selected from the group consisting of CD80 transmembrane domain, a MHC1 transmembrane domain, a FAS transmembrane domain and a TNF receptor transmembrane domain.

[0536] Alternatively, the membrane IL-35 comprises a Glycosylphosphatidylinositol (GPI)-anchor domain of a protein selected from the group consisting of ACHE, ALPG, ALPI, ALPL, ALPP, ART3, ART4, BST1, BST2, CD109, CD14, CD160, CD48, CD52, CD55, CD59, CDH13, CEACAM5, CEACAM7, CNTN3, CNTN4, CNTN5, CPM, CPO, CRIPTO, DPEP1, EFNA4, EFNA5, FCGR3B, FOLR1, FOLR2, GP2, GPC1, GPIHBP1, HYAL2, IZUMO1R, LSAMP, LY6D, LY6E, LY6G6C, LY6G6D, LY6H, LY6K, LYPD1, LYPD2, LYPD3, LYPD4, LYPD5, LYPD6B, LYPD8, MDGA1, MDGA2, MELTF, MMP17, MMP25, MSLN, NEGRI, NRN1, NRN1L, NTM, NTNG1, NTNG2, OPCML, OTO A, PLAUR, PLET1, PRND, PRNP, PRSS21, PRSS41, PRSS55, PSCA, RAET1G, RAET1L, RECK, RGMB, RTN4RL1, RTN4RL2, SEMA7A, SPACA4, SPRN, TECTA, TECTB, TEX101, THY1, ULBP2, VNN1, VNN2, XPNPEP2, GPCR3, more preferably from the group consisting of CD48, CD52, CD55, CD59 and PLET1, even more preferably comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NO: 57-145.

[0537] In some aspects, the membrane IL-35 further comprises a cytosolic domain, said cytosolic domain being selected from the group consisting of a cytokine receptor cytosolic domain, a type I transmembrane protein cytosolic domain and a type II transmembrane protein cytosolic domain. Typically, the mRNA molecule comprises a 5’UTR, a 3’UTR, 5' cap structure, a Kozak sequence, a chain terminating nucleotide, a stem loop, a poly-A sequence and / or a polyadenylation signal. Particularly, the mRNA molecule comprises or consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 46-49 or a variant thereof having at least 85% sequence identity thereto. The lipid-based nanoparticle may comprise a cationic or ionizable lipid, a helper lipid, a sterol and a PEG-lipid. Typically, the lipid-based nanoparticle comprises from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid.

[0538] The lipid composition of the lipid-based nanoparticle may comprise or consist of:

[0539] ALC-0315, DDAB, MC3-DLin-DMA or any mixture thereof, preferably from about 20 mol% to about 60 mol% of the total lipids of the lipid-based nanoparticle;

[0540] DSPC and / or DOPE, preferably from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle;

[0541] Cholesterol, preferably from about 30 mol% to about 70 mol% of the total lipids of the lipid-based nanoparticle; and

[0542] DMG-PEG, preferably from about 0.1 mol% to about 10 mol% of the total lipids of the lipid-based nanoparticle.

[0543] The disease may be selected from the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoath etosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti-N-Methyl-D-Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, membranous nephropathy, allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune urticaria, Behcet's disease, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune disfunction syndrome, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barre syndrome, graft-versus-host disease (GVHD), hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgM polyneuropathies, juvenile arthritis, Kawasaki's disease, lichen plantus, lichen sclerosus, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes mellitus, Multifocal motor neuropathy (MMN), pemphigoid gestationis, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynauld's phenomenon, Reiter's syndrome, sarcoidosis, solid organ transplant rejection, Takayasu arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteristis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, anti-neutrophil cytoplasmic antibody-associated vasculitides, vitiligo, asthma, autoimmune pancreatitis, IgA nephropathy and Wegner's granulomatosis; optionally selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoathetosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti -N-Methyl-D- Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, and membranous nephropathy, preferably selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, antiphospholipid syndrome, Hashimoto's thyroiditis, Graves' disease and graft rejection.

[0544] Typically, the disease is an inflammatory and / or auto-immune disease of the liver, preferably selected from the group consisting of Autoimmune hepatitis (AIH), Primary biliary cholangitis (PBC), Primary sclerosing cholangitis (PSC) and non-alcoholic fatty liver disease.

[0545] The LNP may further comprise a targeting moiety or an antigen-binding domain of an antibody capable of specifically binding to a target expressed on activated immune cells surface, preferably a target expressed on activated T cells, activated B cells, activated myeloid cells including activated macrophages, activated dendritic cells and / or activated neutrophils, preferably the target being selected from the group consisting of CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD154, CD183, CD3, CD4, CD8, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL / TNFSF6, PD1 / PDCD1, GITR / TNFRSF18, GPR32, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LY108 / SlamF6, OX40 / TNFRSF4, RGS1, LTBR / CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3 / TNFRSF6B, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, ICOSL, CD 160, CD 19, CD20, CD24, CD38 TIM-1, TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R, CLEC-1A, CDl lb, CD14, CD66b, CD163, CD206, SIGLEC 6, BCMA / TNFRSF17, CD150, CD86, OX40L, LOX1, TACVTNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74 and / or CD 165.

[0546] Disclosed herein is also a lipid-based nanoparticle comprising mRNA molecule(s) encoding membrane interleukin 35 (IL-35) molecule as defined above and a pharmaceutical composition comprising said lipid-based nanoparticle.

[0547] BRIEF DESCRIPTION OF THE DRAWINGS

[0548] Figure 1: Down-regulation of proinflammatory cytokines after LNP-mRNA-mIL-35 treatment in mouse Con A model compared to a control group. Eight-week-old female Balb / c mice were IV injected with Concavalin A at 15 mg / kg on Day 0. LNP without mRNA, LNP containing mRNA encoding mIL-35 PGE, mRNA-mIL-35 EGP or mRNA-mIL-35 PPE, were intravenously injected at 2pg / mouse 3 times (T-24h / T-12h / T0) and mice were sacrificed 6h post Con A. Pro-inflammatory cytokines (mIL-2, mIL-6, mlENy, mIL-12p40) were quantified by ELISA in the sera. Statistical significance was calculated using Mann-Whitney test: * : p < 0,05 ;** : p < 0,005 ;*** : p < 0,0005 ;**** : p < 0.0001 (Graphpad prism). 3 independent experiments were performed and each group were composed of 11 to 13 mice.

[0549] Figure 2: Down-regulation of transaminase levels (Aspartate aminotransferase and Alanine aminotransferase) after LNP-mRNA-mIL-35 treatment in mouse Con A model compared to control group. Eight-weeks-old female Balb / c mice were injected with Concavalin A at 15 mg / kg by intravenous way at Day 0. LNP without mRNA, LNP containing mRNA-mlL- 35 PGE, mRNA-mIL-35 EGP, mRNA-mIL-35 PPE, were injected at 2pg / mouse by intravenous way 3 times (T-24h / T-12h / T0) and mice were sacrificed 6h post Con A. Transaminases were measured using ALTPM / ASTPM kits and read using Roche / Hitachi cobas ®c 503 analyser. Statistical significance was calculated using Mann-Whitney test: * : p < 0,05 ;** : p < 0,01 ;*** : p < 0,0005 (Graphpad prism). 3 independent experiments were performed, and each group was composed of 11 to 13 mice.

[0550] Figure 3: Down regulation of transaminase levels (Aspartate aminotransferase and Alanine aminotransferase) and pro-inflammatory cytokines after LNP-mRNA-mIL-35 treatment in mouse Con A model compared to control group at multiple dose. Eight-weeks-old female Balb / c mice were injected with Concavalin A at 15 mg / kg by intravenous way at Day 0. LNP without mRNA, LNP containing mRNA-mIL-35 PGE, mRNA-mIL-35 EGP, mRNA-mIL-35 PPE, were injected at 0,5, 2 or 4pg / mouse by intravenous way 3 times (T-24h / T-12h / T0). Pro- inflammatory cytokines (mIL-2, mIL-6, mlENy, mIL-12p40) were quantified by ELISA in the sera (A). Transaminase levels were measured using ALTPM / ASTPM kits and read using Roche / Hitachi cobas ®c 503 analyser (B). Statistical significance was calculated using Mann- Whitney test: * : p < 0,05 (Graphpad prism). 1 independent experiment was performed, and each group were composed of 2 to 5 mice.

[0551] Figure 4: Down-regulation of mouse anti-hCYP2D6 antibody titer mean after 6 treatment with LNP mRNA encoding IL-35 PGE in CYP2D6 mice model compared to control group. Eight-week-old female Balb / c mice were IP injected with emulsion consisting by hCYP2D6 at 0.1 mg / kg and CFA (200uL) on Day 0 and D14. PBS / LNP without mRNA or LNP containing mRNA encoding mIL-35 PGE, mRNA-mIL-35 EGP or mRNA-mIL-35 PPE were IV injected at 2 pg / mouse 3 times per week for 2 weeks starting at day 44. At day 60, ELISA to detect mouse anti-hCYP2D6 antibody was performed. Statistical significance was calculated using Mann- Whitney test: * : p < 0,05 (Graphpad prism). 1 independent experiment was performed, and each group was composed of 6 to 8 mice. Figure 5: Concentration of mIL-35 after transfection of HuH7 cell line with LNP-mRNA encoding mIL-35 PGE is higher than mIL-35 EGP / PPE formats. HuH7 cells were incubated with media, LNP mRNA encoding mRNA IL-35 PGE, LNP mRNA encoding mRNA IL-35 EGP and LNP mRNA encoding mRNA IL-35 PPE during 24h at 37°C. Supernatant was recuperated after centrifugation at 2500 T / min and ELISA LEGEND MAX™ Mouse IL-35 Heterodimer ELISA kit was used to quantified mouse IL-35 heterodimer. 1 independent experiment was performed.

[0552] Figure 6: Down regulation of proinflammatory cytokines after LNP-mRNA encoding transmembrane mIL-35 or hIL-35 treatment in mouse Con A model compared to control group. Eight- week-old Balb / c mice were IV injected with Concavalin A at 15 mg / kg on Day 0. Mice without LNP and with LNP containing mRNA encoding transmembrane human and mouse IL-35, were intravenously injected at 2pg / mouse 3 times (mIL-35) (T-24h / T-18h / T0) or 6pg / mouse 3 times (hIL-35) and mice were sacrificed 6h post Con A. Pro-inflammatory cytokines (mIL-2, mIL-6 and mIL-12p40) were quantified by ELISA in the sera. 1 independent experiment was performed, and each group was composed of 8 mice.

[0553] Figure 7: Down regulation of proinflammatory cytokines after LNP-mRNA encoding transmembrane mIL-35 treatment in mouse Con A model compared to LNP mRNA encoding Luciferase group. Eight- week-old Balb / c mice were IV injected with Concavalin A at 15 mg / kg on Day 0. Mice without LNP and with LNP containing mRNA encoding transmembrane mouse IL-35, were intravenously injected at 2pg / mouse 3 times (mIL-35) (T- 24h / T-18h / T0) or 2 pg / 6pg / mouse 3 times (Luciferase) and mice were sacrificed 6h post Con A. Pro-inflammatory cytokines (mIL-6 and IFNy) were quantified by ELISA in the sera. 2 independent experiments were performed, and each group was composed of 7 to 24 mice.

[0554] Figure 8: Downregulation of transaminases after treatment with LNP-mRNA encoding human IL-35 compared to no LNP in mouse Con A model. Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution without LNPs or a solution with LNPs containing mRNA encoding hIL-35 were intravenously injected at 0.5pg / 2pg / 4pg per mouse 3 times (T-24h / T-18h / T0). Mice were sacrificed 6h post Concavalin A injection. Transaminases were quantified by ELISA in the sera. Statistical significance was calculated using Kruskal -Wallis : **** : p < 0.0001 and Mann-Whitney test: * : p = 0,01 for ALAT and p = 0.0181 (Graphpad prism). 6 independent experiments were performed, and each group were composed of 23 to 26 mice. Figure 9: Downregulation of proinflammatory cytokines after treatment with LNP-mRNA encoding human IL-35 compared to empty LNP in mouse Con A model. Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution with LNPs containing mRNA encoding hIL-35 or a solution containing no LNPs were intravenously injected at 0.5pg Mpg Mpg and 6pg per mouse 3 times (T-24h / T-18h / T0) and mice were sacrificed 6h post Con A injection. Pro-inflammatory cytokines (mIL-2, mIL-6, mlL- 12p40) were quantified by ELISA in the sera. Statistical significance was calculated using Kruskal-Wallis test (mIL-2 : **** : p < 0.0001 and ** : p = 0.0021 ; mIL-6 :**** ; p < 0.0001 and ** : p = 0.0020 ; mIL-12p40 : **** : p < 0.0001 and Mann-Whitney test: * : p = 0,0196 (Graphpad prism). 10 independent experiments were performed, and each group were composed of 56 to 66 mice.

[0555] Figure 10: Downregulation of proinflammatory cytokines after treatment with LNP- mRNA encoding human IL-35 compared to treatment with LNP-mRNA encoding Luciferase in mouse Con A model. Eight- week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution with LNPs containing mRNA encoding luciferase or a solution with LNP containing mRNA encoding hIL-35 were intravenously injected at 6pg per mouse 3 times (T-24h / T-18h / T0) and mice were sacrificed 6h post Con A injection. Pro-inflammatory cytokines (mlENy) were quantified by ELISA in the sera. Each group were composed of 6 to 8 mice.

[0556] Figure 11: Downregulation of transaminases after treatment with LNP-mRNA encoding mIL-35 compared to no LNP in mouse Con A model. Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution without LNPs and a solution with LNPs containing mRNA encoding mIL-35 were intravenously injected at 0.5pg / 2pg / 4pg per mouse 3 times (T-24h / T-18h / T0) and mice were sacrificed 6h post Con A injection. Transaminases were quantified by ELISA in the sera. Statistical significance was calculated using Kruskal -Wallis: **** : p < 0.0001 and : *** : p < 0.0005 ; Mann-Whitney test; ** : p < 0,01 (Graphpad prism).6 independent experiments were performed, and each group were composed of 26 to 34 mice.

[0557] Figure 12: Downregulation of proinflammatory cytokines after treatment with LNP- mRNA encoding mIL-35 in mouse Con A model compared to no LNP. Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution without LNPs or a solution with LNPs containing mRNA encoding mIL-35 were intravenously injected at 0.5 pg / 2pg pg and 6pg per mouse 3 times (T-24h / T-18h / T0) and mice were sacrificed 6h post Con A injection. Pro-inflammatory cytokines (mIL-2, mIL-6, mlL-

[0558] 12p40, mZFNy) were quantified by ELISA in the sera. Statistical significance was calculated using Kruskal Wallis (mIL-2 : **** : p < 0.0001 ; mIL-6 ■**** ; p < 0.0001 and ** : p = 0.0076 ; mIL-12p40 : **** : p < 0.0001 ; mfFNy : **** : p = 0.0001 and Mann-Whitney test :*** : p = 0.0009 (Graphpad prism). 10 independent experiments were performed, and each group were composed of 56 to 66 mice.

[0559] Figure 13: Downregulation of proinflammatory cytokines after treatment with LNP- mRNA encoding mouse IL-35 compared to LNP-mRNA encoding Luciferase in mouse Con A model. Five / Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution with LNPs containing mRNA encoding luciferase or a solution with LNPs containing mRNA encoding mIL-35 PGE was intravenously injected at 2pg per mouse 3 times (T-24h / T-18h / T0) and mice were sacrificed 6h post Con A injection. Pro- inflammatory cytokines (mIL-2, mfFNy, mIL-12p40) were quantified by ELISA in the sera. Statistical significance was calculated using Mann-Whitney test: mIL-2 : *** : p = 0.0002 ; mfFNy : ** : p = 0.0070 ; mIL-12p40 : *** : p = 0.007). Each group were composed of 8 mice.

[0560] Figure 14: Down regulation of proinflammatory cytokines after treatment with LNP- mRNA encoding mouse IL-35 compared to LNP-mRNA encoding Luciferase in the liver (Figure A) and in the sera (Figure B)in mouse Con A model. Five / Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. A solution with LNP containing mRNA encoding luciferase or a solution with LNP containing mRNA encoding mIL-35 PGE were intravenously injected at 2pg per mouse 1 / 2 / 3 times (T-24h, T-24h + T-18h, T-24h + T-18h + TO) and mice were sacrificed 6h post Con A injection. Pro- inflammatory cytokines (mIL-2, mIL-6, mIL-12p40) were quantified by ELISA in the liver and in the sera (mIL-2, L-12p40). Each group were composed of 3 mice.

[0561] Figure 15: Downregulation of proinflammatory cytokines after treatment with LNP- mRNA encoding mouse IL-35 compared to LNP-mRNA encoding Luciferase in the liver (Figure A) and in the sera (Figure B)in mouse Con A model. Five / Eight-week-old female Balb / c mice were injected intravenously with Concavalin A at 15 mg / kg on Day 0. Solutions comprising LNPs ((ALC-315 / DOPE / Chol / DMG-PEG), (DDAB / DSPCE / Chol / DMG-PEG), (MC3-DLin-DMA / DSPC / Chol / DMG-PEG), (MC3-DLin-DMA / DOPE / Chol / DMG-PEG) containing mRNA encoding luciferase or mIL-35 were intravenously injected at 2pg per mouse 3 times (T-24h, T-18h, TO) and mice were sacrificed 6h post Con A injection. Pro-inflammatory cytokines (mIL-2, mIL-6, mIL-12p40, mZFNy) were quantified by ELISA in the liver and in the sera. Each group were composed of 3 mice.

[0562] Figure 16: Improved expression of IL-35 is observed in PD-1 expressing cells transfected with targeted-LNPs encapsulating mRNA encoding for IL-35. Jurkat human PD1+ cells were transfected with PD1 -targeted, isotype-targeted or not targeted LNP (lOOng of encapsulated RNA / well) containing mRNA encoding soluble human or mouse IL-35 RNA in presence of antagonistic anti human PD1 antibody (black bar) or without antagonistic anti human PD1 antibody (grey bar). Expression of soluble IL-35 was quantified by measure of AlexaFluor647 fluorescence in live cells. Results are shown in geometric mean of Al exaFluor 647 in live transfected cells (A647+) normalized to the condition with not targeted LNP.

[0563] Jurkat human PD1+ cells were transfected in the presence of PD1 -targeted or not targeted LNP containing mRNA encoding mIL-35 or hIL-35 in presence of antagonistic anti human PD1 antibody (black bar) or without antagonistic anti human PD1 antibody (grey bar). Expression of soluble IL-35 was quantified by measure of AlexaFluor647 fluorescence in live cells. Results are shown in geometric mean of AlexaFluor 647 in live transfected cells (A647+) normalized to the condition with not targeted LNP.

[0564] Figure 17: Comparison of human membrane IL-35 cytokine expression after transfection of HuH7 cells at day 1 and day 3. The Fold of cells expressing human IL-12p35 and human EBB after transfection of HuH7 cells with Messenger Max and mRNA encoding soluble and membrane human IL-35 constructions was assessed by flow cytometry. Immunostaining was performed using anti-human EBB and anti-human IL-12p35 antibodies.

[0565] Figure 18: Down regulation of pro-inflammatory IFNy cytokine secreted by human PBMCh in presence of HuH7 cells transfected with several human membrane IL-35 cytokine. HuH7 cells were plated and transfected with several human membrane IL-35 constructions during one day and co-cultured with human PBMC a day before adding concavalin A at 0.5 pg / ml (Figure 18A / B) or 1 pg / ml for 5 days (Figure 18C). At day 6, the supernatant was isolated to evaluate pro inflammatory cytokine by ELISA.

[0566] Figure 19: Gene expression of 3 subunits of IL-35 receptor (IL12RB2, IL27RA and IL6ST / gpl30) in skin diseases. ACLE: Acute cutaneous lupus; CLE : cutaneous lupus erythematosus ; DLE : discoid lupus erythematosus ; SCLE: subacute CLE; pSo: psoriasis; AD: atopic dermatitis; CD: contact dermatitis; LP: lichen planus: EDB: epidermolysis bullosa; DD: discoid dermatitis. EXAMPLES

[0567] Advantages of the invention will become clearer in the following examples which are given for purposes of illustration and not by way of limitation.

[0568] Materials and Methods

[0569] Lipids: ALC-0315 was purchased from Avanti Lipids (8909000), Cholesterol was purchased from Merck (1046720001), DMG-PEG-2000 was purchased from Avanti Lipids (880151P), DOPE was purchased from Corden Pharma (LP-R4-069).

[0570] Preparation of lipid-based nanoparticles entrapping IL-35 mRNA

[0571] LNPs were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG- 2000: 1.5 %, at a total lipid concentration of 9.1 mM.

[0572] 11-35 mRNA (50 pg, 1 mg / mL) was diluted with 25 mM acetate buffer (550pL, pH 4.3) to a final concentration of 0.083 mg / mL. The selected experimental conditions allow for a N / P ratio of 6.

[0573] The lipid mixture (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNPs were stored in a final volume of 1 mL in PBS at 4°C and characterized.

[0574] LNP characterization

[0575] The mean hydrodynamic diameter of the mRNA-LNPs was measured by dynamic light scattering using a Malvern NanoZS (Malvern Instruments, UK). mRNA encapsulation efficiency was determined by mRNA accessibility to Ribogreen using the Quant-iT Ribogreen RNA assay (Thermo Fisher).

[0576] RNA design

[0577] Full length mouse IL-12 alpha (p35) cDNA sequence NM 008351.3 and full length mouse Ebi3 cDNA sequence ENSMUST00000003274.8 were optimized separately using GenSmart™ sequence optimization algorithm, in order to reduce uridine occurrence and generate the most linear mRNA structure. The nucleotide sequences obtained were joined by either a P2A peptide sequence (PPE design: Ms_IL35 p35-P2A-Ebi3) or a (G4S)3 linker sequence (EGP design: Ms_IL35 Ebi3-G4S3-p35, PGE design: Ms_IL35 p35-G4S3-Ebi3). The full-length sequence was further optimized to generate the most linear structured RNA, assessed by the lowest free energy on mRNA-fold tool. The non translatable msIL35 control RNA (non-coding IL-35) was based on the PGE design, were all ATG were replaced by different codons, mostly stop codons. The designed constructs were cloned by Gibson assembly into a plasmid that incorporate a T7 promoter, an hHbB 5’UTR, an hHbB 3’UTR and a poly- A tail. The final DNA matrices were prepared by linearization by BspQl restriction enzyme.

[0578] LNP PPE comprises mRNA molecules encoding from N-terminus to C-terminus : a p35 peptide signal, a p35 subunit, a P2A linker, an Ebi3 peptide signal and an Ebi3 subunit. In particular, the PPE mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 10 or 16.

[0579] LNP EGP comprises mRNA molecules encoding a fusion protein comprising from N-terminus to C-terminus : an Ebi3 peptide signal, an Ebi3 subunit, a (GGGGS)s linker and a p35 subunit. In particular, the EGP mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 12 or 20.

[0580] LNP PGE comprises mRNA molecules encoding a fusion protein comprising from N-terminus to C-terminus : a p35 peptide signal, a-p35 subunit, a (GGGGS)s linker and an Ebi3 subunit. In particular, the PGE mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 14 or 18.

[0581] RNA design for membrane mouse IL-35

[0582] Full length mouse IL-12 alpha (p35) cDNA sequence NM 008351.3 and full length mouse Ebi3 cDNA sequence ENSMUST00000003274.8 were optimized separately using GenSmartTM sequence optimization algorithm, in order to reduce uridine occurrence and generate the most linear mRNA structure. The nucleotide sequences obtained were joined by either a P2A peptide sequence (PPE design: Ms_IL35 p35-P2A-Ebi3) or a (G4S)3 linker sequence (EGP design: Ms_IL35 Ebi3-G4S3-p35, PGE design: Ms_IL35 p35-G4S3-Ebi3). The full-length sequence was further optimized to generate the most linear structured RNA, assessed by the lowest free energy on mRNA-fold tool. The ORF sequence was then continued by the in frame 3’ addition of a nucleotide sequence encoding three Alanine residues followed by a (G4S)3 linker and the CD80 transmembrane domain (SEQ ID NO: 38), or the CD80 transmembrane and intracytoplasmic domains (SEQ ID 38 and 40, respectively). The non translatable msIL35 control RNA (non-coding IL-35) was based on the PGE design, were all ATG were replaced by different codons, mostly stop codons. The designed constructs were cloned by Gibson assembly into a plasmid that incorporate a T7 promoter, an hHbB SARS Cov2 derived 5’UTR, an hHbB 3’UTR and a poly-A tail. The final DNA matrices were prepared by linearization by BspQl restriction enzyme.

[0583] In vitro transcription (IVT)

[0584] IVT reactions were performed using T7-Flashscribe™ transcription kit C-ASF3507 (Cellscript, USA) or NEB HiScribe High-yield kit E-2040S with the following modifications: CleanCap-AG reagent (N-7113, Trilink) was added for co-transcriptional Cap-1 capping; UTP was replaced by N1 -methyl -pseudo UTP (N-1081, Trilink). DNAse treated mRNAs were purified by LiCl precipitation and quality controlled by agarose gel electrophoresis.

[0585] Mouse Concavalin A model for soluble IL-35

[0586] Eight-week-old female Balb / c mice were injected intravascularly with Concavalin A (Sigma, #C2010) at 15 mg / kg (50uL) on Day 0. LNP without mRNA or LNP containing mRNA encoding mIL-35 PGE, mIL-35 EGP or mIL-35 PPE were injected in IV at 0.5, 2 or 4pg / mouse 3 times (T-24h / T-12h / T0). Blood retroorbital sampling was realized and centrifuged at 2500 t / min during 10 min and serum was stocked at -20°C prior cytokine dosing.

[0587] Mouse IL-2 (BD Biosciences, #555148 ; Biotechne, #DY402), mouse IFN-y (BD Biosciences, #555138 ; Biotechne, #DY485), mouse IL-6 (BD Biosciences, #555240 ; Biotechne, #DY406) and mouse mIL-12p40 (BD Biosciences, #555165 ; Biotechne, #DY499) cytokines were measured in the serum by ELISA according to the manufacturer’s instructions and samples were read using Spark®TECAN.

[0588] Capture Antibodies to each well were incubated overnight at 4°C. After aspiration and washing procedures, plate was block with the assay diluent and incubated 1 hour at Room Temperature. After aspiration and washing procedures, samples were added and incubated 2 hours at room temperature. After aspiration and washing procedures, working detector (Detection Ab + SAv- HRP) were added and incubated Ihour at room temperature. After aspiration and washing procedures substrate solution to each well were added and incubated 30 min at Room temperature. Stop solution were added to each well and plate was analyzed at 450 nm within 30 min with correction 570 nm.

[0589] Alanine Aminotransferase level in the sera was measured using Alanine Aminotransferase acc. To IFCC with pyridoxal phosphate activation kit (Roche, #08056773 190) and Aspartate Aminotransferase level was measured using Aspartate Aminotransferase acc. To IFCC with pyridoxal phosphate activation kit (Roche, #08056838 190). Samples were read using Roche / Hitachi cobas ®c 503 analyser. I l l

[0590] Mouse Concavalin A model for transmembrane IL-35

[0591] Eight-week-old female Balb / c mice were IV injected with Concavalin A (Sigma, #C2010) at 15 mg / kg (50uL) on Day 0. PBS or LNP containing mRNA encoding transmembrane mIL-35 (2pg) or hIL-35 PGE (6pg) were injected in IV at 3 times (T-24h / T-12h / T0). Blood retroorbital sampling was realized and centrifuged at 2500 t / min during 10 min and serum was stocked at - 20°C prior cytokine dosing.

[0592] Mouse IL-2 (Biotechne, #DY402 ; BD Biosciences, #555148), mouse IL-6 (Biotechne, #DY406 ; BD Biosciences, #555240) and mouse mIL-12p40 (Biotechne, #DY499 ; BD Biosciences, #555165) cytokines were measured in the serum by ELISA according to the manufacturer’s instructions and samples were read using Spark®TECAN.

[0593] Capture antibodies to each well were incubated overnight at 4°C. After aspiration and washing procedures, the plate was block with the assay diluent and incubated 1 hour at Room Temperature. After aspiration and washing procedures, samples were added and incubated 2 hours at room temperature. After aspiration and washing procedures, working detectors (Detection Ab + SAv-HRP) were added and incubated Ihour at room temperature. After aspiration and washing procedures substrate solution to each well were added and incubated 30 min at Room temperature. Stop solutions were added to each well and plates were analyzed at 450 nm within 30 min with correction 570 nm.

[0594] Chronic inflammatory CYP2D6 mouse model:

[0595] Eight-week-old female Balb / c mice were IP injected with emulsion consisting by human CYP2D6 (Sigma, #APrEST83904) at 0.1 mg / kg and CFA (Adjuvant complet de Freund , Sigma, #F5881) (200uL) on Day 0 and D22 . LNP without mRNA or LNP containing mRNA encoding mIL-35 PGE, mRNA-mIL-35 EGP or mRNA-mIL-35 PPE were IV injected at 2 pg / mouse 3 times per week for 2 weeks starting at day 44. Blood retroorbital sampling was realized and centrifuged at 2500 t / min during 10 min and serum was stocked at -20°C prior antibody dosing at day 60.

[0596] Capture protein hCYP2D6 (Sigma, APrEST83904, 0.5 ug / ml) to each well was incubated overnight at 4°C. After aspiration and washing procedures, plate was blocked with the PBS Tween 0,1% BSA 1% and incubated 2 hours at 37°C. After aspiration and washing procedures, samples were added and incubated 2 hours at 37°C. After aspiration and washing procedures, working detector (Peroxidase AffiniPure™ F(ab')2 Fragment Donkey Anti-Mouse IgG (H+L),#715-036- 151) were added and incubated Ihour at 37°C. After aspiration and washing procedures substrate solution (Sigma, TMB, T8665) to each well were added and incubated 30 min at Room temperature. Stop solution (H2SO4) were added to each well and plate was analyzed at 450 nm within 30 min with X correction 570 nm.

[0597] Cell culture:

[0598] HuH7 cells were cultured in T75 flasks using DMEM medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS) , 100 U / mL of penicillin, 0,1 mg / mL of streptomycin, and 2mM of L-Glutamin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0599] Transfection of HuH7 cells (Example 4):

[0600] The day before, 7500 HuH7 cells were plated in a 96-well transparent flat bottom culture plate and the day of transfection, medium was replaced by lOOpl of fresh DMEM medium supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U / mL of penicillin, 0,1 mg / mL of streptomycin, and 2mM of L-Glutamine. LNPs were diluted to 4.3 ng / pL with culture medium and added on cells previously prepared in their culture medium (addition of 25 pL LNP / well corresponding to 108 ng RNA / well). 24h post-treatment, supernatants were harvested and stored at -20°C until quantitative analysis of mIL35 production.

[0601] Then, to measure mouse IL-35 secretion in supernatants, an Enzyme-Linked ImmunoSorbent Assay (ELISA) was performed (#440507, BioLegend). Briefly, 96 well strip plate is pre-coated with a rat monoclonal anti -mouse IL-35 antibody. The Detection Antibody is a biotinylated goat polyclonal anti-mouse IL-12 antibody. This kit is specifically designed for the accurate quantitation of mouse IL-35 heterodimer from cell culture supernatant, serum, plasma, and other biological fluids. This kit is analytically validated with ready-to-use reagents. TECAN Spark plate reader was used to quantify the absorbance (405nm).

[0602] ELISA:

[0603] Mouse IL-2 (Biotechne, #DY406; BD Biosciences, #555148), mouse IFN-y (Biotechne, #DY485, BD Biosciences, #555138), mouse IL-6 (Biotechne, #DY406 ; BD Biosciences, #555240) and mouse mIL-12p40 (DY499 Mouse IL-12 / IL-23 p40 ; BD Biosciences, #555165) cytokines were measured in the serum by ELISA according to the manufacturer’s instructions and samples were read using Spark®TECAN.

[0604] Capture antibodies were added to each well and incubated overnight at 4°C. After aspiration and washing procedures, the ELISA plate was blocked with the assay diluent and incubated 1 hour at Room Temperature. After aspiration and washing procedures, samples were added and incubated 2 hours at room temperature. After aspiration and washing procedures, working detectors (Detection antibodies and-streptavidin HRP) were added and incubated 1 hour at room temperature. After aspiration and washing procedures substrate solution was added to each well and incubated 30 min at Room temperature. Stop solution was added to each well and the ELISA plate was analyzed at 450 nm within 30 min with X correction 570 nm.

[0605] Transaminases:

[0606] Alanine Aminotransferase level was measured in the sera using Alanine Aminotransferase according to The International Federation of Clinical Chemistry (IFCC) standards with pyridoxal phosphate activation kit (Roche, #08056773 190) and Aspartate Aminotransferase level was measured using Aspartate Aminotransferase according to IFCC standards with pyridoxal phosphate activation kit (Roche, #08056838 190). Samples were read using Roche / Hitachi cobas ®c 503 analyser.

[0607] Preparation of LNP :

[0608] LNPs were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios, at a total lipid concentration of 9.1 mM. :

[0609] • Formulation A : ALC-0315 : 50% / DOPE : 10% / Cholesterol : 38.5% / DMG-PEG- 2000 : 1.5%,

[0610] • Formulation B : DDAB : 29% / DSPC : 9% / Cholesterol: 63% / DMG-PEG-2000 : 5%,

[0611] • Formulation C : MC3-DLin-DMA: 50% / DSPC : 10% / Cholesterol: 38.5% / DMG- PEG-2000 : 1.5%,

[0612] • Formulation D : MC3-DLin-DMA: 50% / DOPE : 10% / Cholesterol: 38.5% / DMG- PEG-2000 : 1.5%, mRNAs encoding either Luciferase or IL35 (75 pL, 1 mg / mL) were diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL.

[0613] The selected experimental conditions allow for a N / P ratio of 6.

[0614] The lipids (300 pL) and the mRNA solutions (900 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) overnight using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were filtered on 0.22 pm filter (Acrodisc, Supor Strl, Pall Corporation) and stored at 4°C at a final concentration of 22.2 pg / mL . Table 1. LNPs formulation

[0615] LNP characterization:

[0616] The mean hydrodynamic diameter of the mRNA-LNPs was measured by dynamic light scattering using a Malvern NanoZS (Malvern Instruments, UK). mRNA encapsulation efficiency was determined by mRNA accessibility to Ribogreen using the Quant-iT Ribogreen RNA assay (Thermo Fisher).

[0617] Preparation of targeted LNP

[0618] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 4.55 mM. ms-IL-35-PGE or h-IL-35-PGE mRNA (25 pL, 1 mg / mL) were diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.041 mg / mL. For the preparation of targeted LNP, the monoclonal antibody (anti-PDl mAb) (0.95 pg / pL in PBS, 1.3x10-6 pmol mAb / pg mRNA) was added to the mRNA solution prior to microfluidic mixing.

[0619] The selected experimental conditions allow for a N / P ratio of 6.

[0620] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) overnight using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored at 4°C at a final concentration of 13 pg / mL. Table 2. LNPs formulation

[0621] The anti-human PD-1 antibody is OSE-279, in particular such as described in WO2020 / 127366.

[0622] Results

[0623] Example 1: Preparation and characterization of LNPs encapsulating IL-35-mRNA.

[0624] The LNPs were prepared as detailed above using microfluidic mixing system (Inside Tx).

[0625] This method resulted in particles with Z-Ave = 122 nm (number-weighed diameter = 79 nm) and PDI = 0.160 and with good encapsulation of mRNA (93 % EE). For the LNP encapsulating the mRNA IL-35 with the different constructions PGE, EGP and PPE, the following characteristics average are obtained as followed Table 3.

[0626] Table 3. Characteristics of LNP constructions

[0627] Example 2: LNP encapsulating mRNA encoding IL-35 therapy suppress autoimmune inflammation in vivo in acute inflammatory model

[0628] The therapeutic efficacy of LNPs comprising mRNA molecules encoding IL-35 was evaluated in the concanavalin A (Con A)-induced liver injury mouse model, which is a typical animal model in the field of autoimmune hepatitis (AIH) (Lieu et al., Open Life Sci 2022 Feb 28;17(l):91-101, PMID#35291566). Briefly, mice were injected with Concavalin A that triggers acute liver injury characterized by liver necrosis (increase of AS AT and / or AL AT transaminase levels), activation of T lymphocytes and overexpression of proinflammatory cytokines (e.g., IL- 12, IL-6 and IFNg). To evaluate efficacy of LNP / mRNA IL-35 treatment, 3 doses were injected into the mice, and secretion of pro-inflammatory cytokines as well as transaminase levels were quantified in the sera 6 hours post Con-A injection. These data are represented in Figure 1 (cytokine secretion) and Figure 2 (ASAT ALAT transaminase levels). Data represent pool of 3 independent experiments (n = 11 / 13 mice per group). A dose response is represented in Figure 3 (cytokine secretion and transaminases levels). Data represent 1 independent experiment (n = 2 / 5 mice per group).

[0629] Pro-inflammatory cytokines (mIL-2 (**** : p < 0,0001), mIL-6 (**** : p < 0,0001), mlFNy (**** ; p < 0,0001), mIL-12p40 (**** : p < 0,0001) (Mann-Whitney test) as well as transaminase levels (ASAT and ALAT : ** : p < 0,01) (Mann-Whitney test) were increased after Con A injection compared to mice injected with PBS without Con A, validating the inflammatory mouse model (Figure 6 and Figure 7). Moreover, in ConA group, pro-inflammatory cytokines (mIL-6 (* : p < 0,05 ) and mlFNy (** : p < 0,01), were significantly increased after injection of LNP without mRNA, compared to mice injected with No LNP. An increase of IL-12, IL-2 and IFNy (proinflammatory cytokines) are also observed in the “LNP without mRNA” group versus the “No LNP” group, demonstrating that lipid nanoparticles alone are capable of inducing inflammation, a therapeutic concern well-described (Moghimi et al., Mol Ther 2022 Jun l;30(6):2109-2110, PMID#35487214).

[0630] The inventors have also tested the efficacy of IL-35 therapy in single or double chain format (constructions PGE, EGP and PPE) as different formats. Mice treated with LNP-mRNA-mIL-35 EGP suppress inflammation with a significant and strong decrease of proinflammatory cytokines: mIL-2 (**** : p < 0.0001), mIFNy (*(*** : p < 0,05001 ), mIL-12p40 (**** : p < 0.0001) and mIL-6 (* : p < 0,05 ) (Mann Whitney test). Similar results were observed with the other IL-35 constructions. Mice treated with LNP -mRNA-mIL-35 PPE presents a decrease of mIL-2 (**** : p < 0.0001) and mIL-12p40 (**** : p < 0.0001) and mIL-6 (*(** : p < 0,05005) (Mann Whitney test) and a trend was observed for mIFNy. Mice treated with LNP-mRNA-mIL-35 PGE presents a decrease of mIL-2 (**** : p < 0.0001), mJFNy (**** : p < 0.0001), mIL-12p40 (**** : p < 0.0001 ) and for mIL-6 (**** : p < 0.0001).

[0631] Transaminase levels (ASAT / ALAT), manifesting hepatic damages and inflammation, were increased after Con A injection compared to mice injected with PBS (ASAT and ALAT: ** : p

[0632] < 0,01) (Mann-Whitney test), manifesting hepatic damages and inflammation. Interestingly, transaminases (ASAT / ALAT) were increased after injection of LNP without mRNA in Con A group compared to mice injected with Con A without LNP. ASAT was significantly decreased with LNP-mRNA-mIL-35 EGP, LNP-mRNA-mIL-35 PGE and LNP-mRNA-mIL-35 PPE (*: p

[0633] < 0,05) (Mann-Whitney test) compared to the group treated with LNP -without mRNA. ALAT was significantly decreased with LNP-mRNA-mIL-35 EGP, LNP-mRNA-mIL-35 PPE and LNP-mRNA-mIL-35 PGE (**: p < 0,01) compared to group treated with LNP -without mRNA. These data demonstrate the therapeutic efficacy of mRNA encoding IL-35 encapsulated in LNP therapy to suppress autoimmune hepatitis and induced liver injury.

[0634] Very surprisingly and interestingly, contrary to the prior art that dissuades to use LNP in case of inflammatory conditions, the effect of expression of IL35 is therefore greater than the inflammation induced by the LNP and is capable of reversing said inflammation. Pro- inflammatory mechanism associated to notably IL2, IL12p40, IL6, ASAT / ALAT expression are highly decreased.

[0635] Additionally, further experiments were performed with dose response of mRNA encoding IL-35 (0.5, 2 and 4ug ARN / per mice) (Figure 3A and B) and shows that even a very low dose 0.5pg of mRNA is sufficient to reverse inflammation (pro-inflammatory cytokines : * : p < 0,05 ) (Mann- Whitney test) and liver damage in vivo (transaminase levels : * : p < 0,05) (Mann-Whitney test) compared to the group treated with LNP -without mRNA. Such a dose of RNA is low in the field of LNP therapeutic approaches in vivo (Wu et al., 2022 PMID#3506990; Liu et al., 2022 PMID#35301053; Li et al., 2021 PMID 34447945) demonstrating that the present invention is highly efficient.

[0636] Altogether, these results prove that IL-35 delivered by LNPs, is able to decrease the inflammation caused by Con A and is therefore a pertinent candidate for treating conditions linked with inflammation, such as auto-immune diseases, allergies and / or inflammatory diseases.

[0637] Example 3: LNP encapsulating mRNA encoding IL-35 PGE therapy suppress chronic autoimmune inflammation in vivo

[0638] The therapeutic efficacy of LNPs comprising mRNA molecules encoding mIL-35 was evaluated in a chronic type 2 Auto Immune Hepatitis (AIH) mouse model characterized by induction of autoreactive B cell and T cell response and chronic liver damage, a typical animal model in the field of autoimmune hepatitis (AIH). Briefly, mice were IP injected with recombinant human cytochrome P450 (CYP) 2D6 emulsified with complete Freund’s adjuvant (CFA). This is a chronic inflammatory condition of the liver characterized by parenchymal destruction, hypergammaglobulinemia, specific autoantibody production, and hepatic fibrosis and necrosis. Immunization with CFA, in combination with a natural human autoantigen like CYP2D6, was demonstrated to break tolerance, resulting in a chronic form of autoimmune-related liver damage. This murine model of type 2 AIH is expected to be instrumental in understanding the immunologic mechanisms of the pathogenesis of this autoimmune liver disease. (Am J Pathol 2022, 192: 21e30; https: / / doi.org / 10.1016 / j.ajpath.2021.10.006). To evaluate efficacy of LNP / mRNA mIL-35 treatment, 6 doses of mRNA IL-35 / LNP therapy (2pg / inj ection) were injected into the mice every 2 / 3 days, and secretion of auto immune mouse anti hCYP2D6 antibody was evaluated by ELISA assay. These data are represented in Figure 4. Data represent 1 independent experiment (n = 6 / 8 mice per group). Mouse anti hCYP2D6 antibody were increased after two injections of CYP2D6 / CFA emulsion compared to mice injected with CFA / PBS, validating the inflammatory mouse model (Data not shown).

[0639] The inventors have also tested the efficacy of IL-35 therapy in single or double chain format (constructions PGE, EGP and PPE) and compared effect to LNP mRNA control therapy, a noncoding IL-35 mRNA (mock mRNA). Mice treated with LNP-mRNA-mIL-35 PGE suppress inflammation with a significant and strong decrease of autoimmune mouse anti hCYP2D- antibodies (* : p < 0,05 ) (Mann Whitney test) after 6 injections. This treatment is able to reverses the inflammation observed in no LNP no mRNA group and LNP non coding mRNA group (* : p < 0,05 ) (Mann Whitney test)(Figure 4). . Surprisingly, these results were not observed with the other LNP mRNA mIL-35 constructions (PPE and EGP). A statistical significance difference was obtained between the different constructions showing higher therapeutic effect of the IL-35 PGE constructions vs PPE and EGP IL-35 construction (* : p < 0,05 ) (Mann Whitney test)(Figure 4).

[0640] These data demonstrate the therapeutic efficacy of mRNA encoding mIL-35 encapsulated in LNP therapy to suppress autoimmune hepatitis.

[0641] Altogether, these results prove that IL-35 delivered by LNPs, is able to decrease the auto immune antibody caused by CYP2D6 / CFA and is therefore a pertinent candidate for treating conditions linked with chronic inflammation, such as auto-immune diseases, allergies and / or inflammatory diseases.

[0642] Example 4: LNP encapsulating mRNA encoding IL-35 PGE construction demonstrated a higher IL-35 production than other IL-35 formats (EGP,PPE)

[0643] To evaluate whether IL-35 mRNA construction has an impact on production of IL-35 by cells, the inventors transfected Hepatocytes HuH7 cells with LNP encoding mRNA of IL-35 PGE, EGP and PPE construction (108ng of mRNA). Surprisingly and interestingly, a production of IL- 35 was higher when HuH7 cells were transfected with LNP mRNA encoding mouse IL-35 PGE construction compared to LNP mRNA encoding for mouse IL-35 PPE or mouse IL-35 EGP constructs. This higher production demonstrates the advantage of IL-35 PGE (Figure 5). Example 5: LNP encapsulating mRNA encoding transmembrane mIL-35 or hIL-35 suppress chronic autoimmune inflammation in vivo

[0644] The therapeutic efficacy of the present invention and the use of the mRNA encoding transmembrane mouse / human IL-35 encapsulated in LNP was evaluated in the concanavalin A (Con A)-induced liver injury mouse model, that is a well-known animal model in the field of autoimmune hepatitis (AIH) (Lieu et al., 2022 PMID#35291566). Briefly, mice were injected with Concavalin A that triggers acute liver injury characterized by liver necrosis (increase of ASAT, ALAT transaminase levels, activation of T lymphocytes and overexpression of proinflammatory cytokines (IL-12, IL-6, IFNg)). To evaluate the efficacy of LNP / mRNA IL-35 treatment, 3 doses were injected into the mice, and secretion of pro-inflammatory cytokines were quantified in the sera 6 hours post Con- A injection. Data are presented in Figure 6 (cytokine secretion). Data represents 1 independent experiment (n = 8 mice per group).

[0645] Pro-inflammatory cytokines (mIL-2, mIL-6 and mIL-12p40) were increased after Con A injection compared to mice injected with PBS without Con A, validating the inflammatory mouse model (Figure 6).

[0646] The inventors have tested the efficacy of LNP comprising mRNA molecules encoding transmembrane mouse / human IL-35 .

[0647] In particular, the human transmembrane IL-35 is a human PGE construct covalently linked to a CD80 transmembrane domain. The mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 170. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 162.

[0648] In particular, the mouse transmembrane IL-35 is a mouse PGE construct covalently linked to a CD80 transmembrane domain. The mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 172. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 173.

[0649] Mice treated with LNPs comprising mRNA molecules encoding transmembrane mouse or human IL-35 suppress inflammation with a significant and strong decrease of proinflammatory cytokines, such as: mIL-2, mIL-12p40 and mIL-6 (Kruskal -Wallis test, Multiple Comparisons) : mIL-6 : PBS vs. PBS wo ConA : **** : p < 0.0001 ; PBS vs. transmembrane mIL-35 : * : p = 0.0214). mIL-12p40 : PBS vs. PBS wo ConA : **** : p < 0.0001 ; PBS vs. transmembrane hIL-35 : ** : p = 0.0035 ; PBS vs. transmembrane mIL-35 : * : p = 0.0467). mIL-2 : PBS vs. PBS wo ConA : **** : p < 0.0001 ; PBS vs. transmembrane hIL-35 : *** : p = 0.0003).

[0650] The inventors have also compared the efficacy of LNPs comprising mRNA molecules encoding transmembrane mouse IL-35 to LNPs containing mRNA molecules encoding Luciferase. Mice treated with LNP-mRNA-transmembrane mouse IL-35 suppress inflammation compared to LNP containing mRNA molecules encoding Luciferase (control group). Pro-inflammatory cytokines (mIL-6 and IFNy) were decreased after LNP-mRNA-transmembrane mouse IL-35 injection compared to mice injected with LNP containing mRNA encoding Luciferase (Figure 7).

[0651] These results prove that the delivery of mRNA molecules by LNPs allows the expression of membrane IL35, regardless of the construction considered, and can limit the inflammation caused by Con A. They are therefore a pertinent candidate for treating conditions linked with inflammation, such as auto-immune diseases, allergies or inflammatory diseases.

[0652] Example 6: LNP encapsulating mRNA encoding human IL-35 therapy suppresses autoimmune inflammation in vivo

[0653] To evaluate efficacy of LNP / mRNA human IL-35 treatment, 0,5 pg, 2pg, 4pg per mouse were intravenously injected into the mice 3 times (-24h,-18h, Oh), and secretion of transaminases (ASAT / ALAT) were quantified in the sera 6h post Con-A injection. Data are presented in Figure 8. A significant increase was observed in mice treated with Con A and then with a solution without LNP (n = 27) compared to the condition without Con A and without LNP (n = 26). ASAT / ALAT were significantly decreased after treatment with ConA and then with LNPs comprising mRNAs encoding hIL-35 (n = 23) (Statistical significance was calculated using Kruskal-Wallis : **** : p < 0.0001 and Mann-Whitney test: * : p = 0,01 for ALAT and p = 0.0181 (Graphpad prism). Secretion of proinflammatory cytokine were quantified in the sera 6h post Con-A injection and data are presented in Figure 9. A significant increase was observed in mice treated with Con A without LNP (n = 66) compared to the condition without Con A and without LNP (n = 56). Proinflammatory cytokines were decreased after treatment with LNPs comprising mRNAs encoding hIL-35 (n = 63) (Statistical significance was calculated using Kruskal-Wallis test (mIL-2 : **** : p < 0.0001 and ** : p = 0.0021 ; mIL-6 :**** ; p < 0.0001 and ** : p = 0.0020 ; mIL-12p40 : **** : p < 0.0001 and Mann-Whitney test: * : p = 0,0196 (Graphpad prism)). A decrease was observed in mice treated with Con A and then with LNPs comprising mRNA encoding human IL-35 (n = 8) compared to group treated with Con A and then with LNPs comprising mRNAs encoding Luciferase (n = 8) (Figure 10). Example 7: LNPs encapsulating mRNA encoding IL-35 therapy suppress autoimmune inflammation in vivo

[0654] To evaluate efficacy of LNP / mRNA mouse IL-35 (mIL-35) treatment, the same experiment was performed with mIL-35. Secretion of transaminases (ASAT / ALAT) were quantified in the sera 6h post Con-A injection and data are presented in Figure 11. A significant increase was observed in mice treated with Con A and later with a solution without LNP (n = 27) compared to with the condition without Con A (n = 26) and without LNP. ASAT / ALAT induced by Con A injection were significantly decreased after treatment with LNPs comprising mRNAs encoding mIL-35 (n = 34). compared to LNPs comprising mRNA encoding Luciferase (n = 8) (Statistical significance was calculated using Kruskal-Wallis: **** : p < 0.0001 and : *** : p < 0.0005 ; Mann-Whitney test; ** : p < 0,01 (Graphpad prism)).

[0655] Secretion of proinflammatory cytokines was quantified in the sera 6h post Con-A injection and data are presented in Figure 12. A significant increase was observed in mice treated with Con A and later with a solution without LNPs (n = 66) compared to mice not treated with Con A (n = 56) and with a solution without LNP. Proinflammatory cytokines induced by Con A injection were decreased after treatment with LNPs comprising mRNA encoding mIL-35 (n = 54) (Statistical significance was calculated using Kruskal Wallis (mIL-2 : **** : p < 0.0001 ; mlL- 6 .**** . p < 0.0001 and ** : p = 0.0076 ; mIL-12p40 : **** : p < 0.0001 ; mlFNy : **** : p = 0.0001 and Mann-Whitney test :*** : p = 0.0009 (Graphpad prism)). A significant increase was observed in mice treated with Con A and later with LNPs comprising mRNA encoding Luciferase (n = 8) compared to the mice without Con A treatment (n = 8) and without LNP (Figure 13). Proinflammatory cytokines were significantly decreased after treatment with LNPs comprising mRNAs encoding mIL-35 (n = 8) (Statistical significance was calculated using Mann-Whitney test: mIL-2 : *** : p = 0.0002 ; mlFNy : ** : p = 0.0070 ; mIL-12p40 : *** : p = 0.007). Each group were composed by 8 mice).

[0656] Those experiments lead to the conclusion that mIL-35 and hIL-35 delivered by LNPs can decrease proinflammatory cytokine secretion and prevent liver injury by decreasing transaminases level (ASAT / ALAT).

[0657] Example 8: IL-35 mRNA can suppress inflammation induced by chronic LNPs injection and overall disease inflammation

[0658] Additional experiments were performed with a variable number of injections to decipher the impact of inflammation induced by LNPs in the liver (A) and in the sera (B). Pro-inflammatory cytokines (mIL-2, mIL-6 and mIL12-p40) were quantified by ELISA in the liver and were decreased after Con A injection in mice treated with LNPs comprising mRNAs encoding mlL- 35 compared to mice treated with LNPs comprising mRNA encoding Luciferase (Figure 14A). Interestingly, three injections compared to one or two injections, induced an increase of pro inflammatory cytokines secretion in the liver, which can be controlled by mIL-35 expression in the liver. In periphery, mIL-35 can decrease the secretion of proinflammatory cytokines (mlL- 12p40 and mIL-2 (Figure 14B)). This means that in autoimmune disease and chronic inflammation, where several injections could be necessary, mIL-35 is able to prevent inflammation induce by LNP and also by the disease.

[0659] Example 9: LNPs encapsulating mRNA encoding IL-35 can prevent inflammation induced by several pro-inflammatory LNP and by chronic injection in acute Con A mice model

[0660] The inventors have pursued their work and wondered if mIL-35 could control inflammation induced by other proinflammatory LNP of different compositions. The inventors then tested other inflammatory LNPs having the following compositions:

[0661] - (ALC-0315 / DOPE / Chol / DMG-PEG),

[0662] - (DDAB / DSPCE / Chol / DMG-PEG),

[0663] - (MC3-DLin-DMA / DSPC / Chol / DMG-PEG),

[0664] - (MC3-DLin-DMA / DOPE / Chol / DMG-PEG).

[0665] Pro-inflammatory cytokines (mIL-2, mIL-6 and mIL12-p40) were quantified by ELISA in the liver and were decreased after Con A injection with LNPs ((ALC-0315 / DOPE / Chol / DMG-PEG) ; (DDAB / DSPCE / Chol / DMG-PEG)) comprising mRNA encoding mIL-35 compared to mice treated with LNPs ((ALC-0315 / DOPE / Chol / DMG-PEG) ; (DDAB / DSPCE / Chol / DMG-PEG)) comprising mRNA encoding Luciferase (Figure 15 A). Proinflammatory cytokines (mIL-2 and mlENy) were quantified by ELISA in the sera and were decreased after Con A injection in mice treated with LNPs ((ALC-0315 / DOPE / Chol / DMG-PEG) ; (DDAB / DSPCE / Chol / DMG-PEG)) comprising mRNA encoding mIL-35 compared to mice treated with LNPs ((ALC- 0315 / DOPE / Chol / DMG-PEG) ; (DDAB / DSPCE / Chol / DMG-PEG)) comprising mRNA encoding Luciferase (Figure 15B).

[0666] Altogether, these results prove that the expression of IL35, regardless of the construction considered, through LNP delivery, is able to limit / decrease the inflammation caused by Con A. LNPs comprising mRNA encoding IL-35 are therefore a pertinent candidate for treating conditions linked with inflammation, such as auto-immune diseases, allergies or inflammatory diseases. Example 10: anti-PDl-targeted-LNPs encapsulating mRNA encoding for either mIL-35 or hIL-35 lead to higher IL-35 expression in PD-1 positive cells, compared to non-targeted-

[0667] LNPs.

[0668] The inventors have tested the specific delivery of mouse or human IL-35 into Jurkat human PD1+ cells.

[0669] Jurkat human PD1+cells were plated at 105cells / well in a Flat bottom 96 well-plate and transfected with PDl-targeted LNPs (i.e., comprising anti-PDl antibodies) or not targeted LNP ((i.e., comprising control isotope IgGl antibodies that do not target PD-1) formulated with mRNA (lOOng of encapsulated RNA / well) encoding soluble human or mouse IL-35. Antihuman PD1 antibody N297A or control isotope IgGl antibody was used to prepare the LNP to target PD1+cells. To evaluate the possibility to target PD1+and sparing PDF cells, antagonistic anti-human PD1 antibody N297A was added during 30 min at 37°C at 500 pg / ml final concentration before the incubation. Jurkat human PD1+were transfected with PDl-targeted or not targeted LNP mRNA encoding soluble mIL-35 or hIL-35 during 24h. After 24h, to block the cytokine secretion and keep soluble human or mouse IL-35 intracellular, cells were treated with Brefeldin A (BioLegend #420601,) at a final concentration of 5pg / ml. After 2 hours, cells were harvested, and dead cells were stained with LIVE / DEAD™ Fixable Yellow Dead Cell Stain Kit (Invitrogen #L34968A) for 30min at 4°C. Then, cells were fixed, permeabilized (BD biosciences #554714), saturated with human AB serum 5% 20 minutes at room temperature. Staining was performed with purified anti-human / mouse IL12p35 (Biotechne #MAB1570) at 5pg / ml for 30 minutes at Room Temperature. An AlexaFluor647 labeled anti-mouse (Invitrogen #A21236) was used at I pg / ml as secondary antibody for 30 minutes at room temperature. Cells were then assessed on the flow cytometer CytoFlex (Beckman Coulter). Expression of soluble IL-35 was quantified by measure of AlexaFluor647 fluorescence in live cells. Results are shown in % of positive cells of AlexaFluor 647 in live transfected cells (A647+) normalized to the condition with not targeted LNP.

[0670] Interestingly, as shown in Figure 16, expressions of mIL-35 or hIL-35 were increased by threefold in Jurkat human PD1+cells with anti-PDl -targeted LNPs comprising mRNA molecules encoding mIL-35 or hIL-35 compared to not targeted LNP comprising mRNA molecules encoding mIL-35 or hIL-35. More interestingly, expressions of mIL-35 or hIL-35 were totally abrogated with anti-PDl -targeted LNPs comprising mRNA encoding mIL-35 or hIL-35 when Jurkat human PD1+were pretreated with the anti- human PD1 antagonistic antibody N297A alone. This leads to the conclusion that IL-35 can be delivered into Jurkat human PD1+cells and can spare PDL cells, the specific delivery of IL-35 into activated T and B cells (i.e., expressing PD- 1) will help to prevent inflammation without impact on naive T and B cells. Furthermore, these results suggest that other markers than PD1 could be used to deliver IL-35 in a cell population

[0671] Example 11: LNP / RNA encoding IL-35 cytokine expressed in membrane form allows surface expression of the cytokine on transfected cells and inhibition of inflammation

[0672] Preparation of LNP

[0673] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 9.1 mM. mRNA (100 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. The selected experimental conditions allow for a N / P ratio of 6.

[0674] The lipids (400 pL) and the mRNA solutions (1200 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) overnight using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were concentrated using Vivaspin Centrifugal Concentrator (10k MWCO, PES membrane, Sartorius), filtered on 0.22 pm filter (Acrodisc, Supor Strl, Pall Corporation) and stored at 4°C at a final concentration of 25 pg / mL.

[0675] RNA design

[0676] The human IL35 sequence was constructed as a PGE IL35 construct; by joining the signal peptide of human IL2 NP 000577.2 (amino acids 1-20) to the human IL-12 alpha (p35) amino acid sequence NP_000873.2 devoid of its natural signal peptide (amino acids 23-219), followed by a (G4S)3 sequence and finally the human P27 precursor (Ebi3) amino acid sequence NP 005746.2 (amino acids 21-229). This full amino acid construct sequence was submitted to GenSmartTM sequence optimization algorithm, in order to reduce uridine occurrence and generate the most linear mRNA structure, assessed by the lowest free energy on mRNA-fold tool.

[0677] The ORF sequence was then continued by the in frame 3’ addition of a nucleotide sequence encoding different linkers, i.e., El 5 (i.e., 15 consecutive E residues, SEQ ID NO: 153); GGI ; hinge FcGILALAPG (SEQ ID NO: 35); (EAAAK)3 (SEQ ID NO: 54); G4S6 (i.e., (GGGGS)6, SEQ ID NO: 32) and the CD80 transmembrane domain (SEQ ID NO: 38). This added sequence was further optimized in the context of the upstream unchanged human IL35 sequence. For some constructions, both the linker and the transmembrane domain were replaced (membrane hIL35-hinge; FcGILALAPG BCR TM; membrane hIL35-CD59 GPI anchor), and the new transmembrane encoding sequence was further optimized in the context of the whole sequence.

[0678] IL-35-CD59 GPI- Anchor construct comprises human IL35 sequence described above fused to a sequence encoding CD59 GPI anchor (SEQ ID NO : 145).

[0679] IL-35-hinge FcGILALAPG-BCRTM construct comprises human IL35 sequence described above fused to a sequence encoding a hinge IgGl Fc domain with a LALAPG mutation (SEQ ID NO : 56) and a BCR transmembrane domain (SEQ ID NO : 152).

[0680] The designed constructs were cloned by Gibson assembly into a plasmid that incorporates a T7 promoter, a SARS Cov2 derived 5’UTR, an hHbB 3’UTR and a poly- A tail. The final DNA matrices were prepared by linearization by BspQl restriction enzyme.

[0681] In particular, the hIL-35-hinge G1-CD80TM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 154. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 162.

[0682] In particular, the hIL-35-hinge FcGlLALAPG-CD80TM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 155. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 163.

[0683] In particular, the hIL-35-E15-CD80TM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 156. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 164.

[0684] In particular, the hIL-35-GGI-CD80TM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 157. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 165.

[0685] In particular, the hIL-35-(EAAAK)3-CD80TM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 158. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 166.

[0686] In particular, the hIL-35-G4S6-CD80TM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 159. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 167. In particular, the hIL-35-CD59 GPI- Anchor mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 160. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 168.

[0687] In particular, the hIL-35-hinge FcGILALAPG-BCRTM mRNA molecule comprises a nucleic acid sequence as described in SEQ ID NO: 161. This mRNA particularly encodes for a transmembrane IL-35 as set forth in SEQ ID NO: 169.

[0688] In vitro transcription (IVT)

[0689] IVT reactions were performed using T7-FlashscribeTM transcription kit C-ASF3507 (Cellscript, USA) or NEB HiScribe High-yield kit E-2040S with the following modifications: CleanCap-AG reagent (N-7113, Trilink) or CAP GAG reagent (Hzymes, RPC) was added for co-transcriptional Cap-1 capping; UTP was replaced by N1 -methyl-pseudo UTP (N-1081, Trilink; or HPB002500 Hzymes, RPC). DNAse treated mRNAs were purified by LiCl precipitation. Quality controls of the RNA included integrity control by agarose gel electrophoresis and signaling from doublestranded RNA contamination in IVT mRNA Samples by TLR3 bioassay (Promega).

[0690] Expression:

[0691] HuH7 cells (10000 cells / well) were incubated 24h in DMEM supplemented with 10% of SVF and a solution of messenger max and mRNA encoding different constructions of membrane form of IL-35 : mRNA encoding human membrane IL-35-hinge G1-CD80TM , human membrane IL- 35-hinge FcGlLALAPG-CD80TM, human membrane IL-35-E15-CD80TM, human membrane IL-35-GGI-CD80TM, human membrane IL-35-(EAAAK)3-CD80TM , human membrane IL- 35-CD59 GPI-Anchor, human membrane IL-35-hinge FcGILALAPG-BCRTM, human membrane IL-35-G4S6-CD80TM in a flat-bottom plate. Supernatants were harvested and cells were washed with Phosphate Buffer Saline (PBS). TrypLE Express was added and cells were incubated 5 min at 37°C. After 5 min, Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% of Fetal Bovine Serum (SVF) was added and cells were harvested to be transferred into a new plate to perform the immunostaining.

[0692] Cells were washed and Live / Dead Fixable Yellow Dead cell (Invitrogen, #L34968A) and human Fc block (BD, #564220) were added, then, cells were incubated 30 min at 4°C. Cells were washed and anti-human IL-12p35 or (MAB1570) anti-human EBB (NBP3-15735) antibodies at 5 ug / ml were added and incubated at 4°C during 30 min. Cells were washed and goat anti mouse IgG 647 (Biotechne,#A21236) or goat anti rabbit IgG A647 (Biotechne,#A21245) antibodies were incubated at 4°C during 30 min, respectively. Staining was quantifying using a Cytoflex ® reader. The expression of human IL12p35 or human EBI3 staining were expressed in live cell populations.

[0693] Functional assay: secretion of IFNg:

[0694] HuH7 cells (10000 cells / well) were incubated 24h in the presence of messenger max and mRNA encoding different constructions of membrane IL-35 : mRNA encoding human membrane IL- 35-hinge G1-CD80TM , mRNA encoding human membrane IL-35-hinge FcGlLALAPG- CD80TM, mRNA encoding human membrane IL-35-E15-CD80TM, mRNA encoding human membrane IL-35-GGI-CD80TM, mRNA encoding human membrane IL-35-(EAAAK)3- CD80TM, mRNA encoding human membrane IL-35-CD59GP I- Anchor, mRNA encoding human membrane IL-35-hinge FcGILALAPG-BCRTM, mRNA encoding human membrane IL-35-G4S6-CD80TM in a flat- bottom plate. Human PBMCs were co-cultured one day before adding concavalin A at 0,5 pg / ml (Figure 18A / B) or lug / ml (Figure 18C) during 5 days. At day 6, the supernatant was isolated to perform an ELISA. Human IFNy secretion was evaluated in the serum by ELISA according to the manufacturer’s instructions and samples were read using Spark®TECAN.

[0695] To evaluate membrane IL-35 expression, the inventors performed a transfection of HuH7 cells with several constructions of membrane human IL-35, especially in the form of a PGE construct. Several constructions were tested to evaluate the impact of the linker between the transmembrane domain CD80 or the CD59 GPI anchor protein and PGE IL-35 (i.e., E15, GGI, (EAAAK)3, G4S6 (i.e., (GGGGS)6 ), hinge Gl, hinge FcGILALAPG). The inventors have observed (Figure 17) a membrane expression of double positive cells (p35+EBI3+) after transfection of HuH7 cells with all constructions at day 1 : human membrane IL-35-E15-TM-CD80, human membrane IL-35-GGI-TM-CD80, human membrane IL-35-hinge G1-TM-CD80, human membrane IL-35- hinge FcGILALAPG BCR TM, human membrane IL-35 hinge FcGILALAPG- TM-CD80, human membrane IL-35-(EAAAK)3-TM-CD80 and human membrane IL-35-G4S6-TM-CD80 and human membrane IL-35 CD59 GPI anchor. Interestingly, membrane protein expression is maintained even at Day 3 for the different constructions despite the short half-life of RNA in the cells (~24h) showing potential long-term effect of the IL-35. A long-term expression of IL-35 is an important parameter to maintain a lasting inhibitory effect for the treatment of chronic inflammatory and auto-immune diseases.

[0696] The inventors next evaluated biological activity of the membrane IL-35 constructs to immune suppress inflammation and more particularly inhibition of pro-inflammatory cytokine by activated T cells (IFNg). In vitro, HuH7 cells were transfected with several constructs of membrane IL-35 to promote expression of IL-35 on the surface then co-cultured with hPBMC for one day before adding concavalin A (0.5 or lug / mL) stimulating agent for 5 days. Negative control of the experiment was performed with HuH7 transfected with control mRNA (non-coding IL-35 sequence). Pro-inflammatory cytokine IFNy were decreased for all IL-35 membrane constructions (Figure 18A / B / C).

[0697] Altogether, these results prove that membrane IL35 constructions are effective to inhibit T cell activation in vitro and inflammation.

[0698] Example 12: Gene expression analysis

[0699] The RNA-seq dataset was obtained from the Gene Expression Omnibus (GEO) database. The dataset includes gene expression profiles from both diseased and healthy tissue samples. After normalization and log2 transformation, log2 fold change (log2FC) and p-values for the difference in gene expression between diseased and healthy tissues were calculated for each dataset and represented in the same way in this graph. Diseases with higher expression levels are shown to the right of the dotted line, where a log2FC of 1 indicates that the gene is expressed twice as much in the diseased tissue compared to the healthy tissue.

[0700] Gene expression of 3 subunits of the IL-35 receptor, namely IL12RB2, IL27RA and IL6ST (also called gpl30) have been analysed in skin diseases. In lymphocytes B, the IL-35 receptor includes the two following subunits: IL12RB2 and IL27RA. In lymphocytes T, the IL-35 receptor can be made of the following combinations of subunits: gpl30 / gpl30; gpl30 / IL12RB2 and IL12RB2 / IL12RB2. The results are shown in Figure 19. An overexpression of IL12RB2 and IL27RA was observed in skin diseases, in particular CDLE, psoriasis, AD, DLE and LP. Therefore, this overexpression supports the efficiency of LNP IL-35 for the treatment of auto-immune or inflammatory skin diseases.

Claims

CLAIMS1. A pharmaceutical composition comprising a lipid-based nanoparticle (LNP) comprising one or more mRNA molecule(s) encoding a membrane interleukin 35 (IL-35) molecule comprising a membrane anchoring domain and optionally a pharmaceutically acceptable carrier, for use in the treatment of an inflammatory and / or auto-immune disease.

2. The pharmaceutical composition for use according to claim 1, wherein the IL-35 molecule is a mammalian IL-35 molecule, preferably a human IL-35 molecule.

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the membrane anchoring domain is a transmembrane domain and / or a Glycosylphosphatidylinositol (GPI)- anchor domain.

4. The pharmaceutical composition for use according to any one of claims 1-3, wherein the membrane IL-35 is a single-chain polypeptide comprising an EBB subunit and a p35 subunit and, optionally a linker in between.

5. The pharmaceutical composition for use according to any one of claims 1-4, wherein the membrane IL-35 molecule comprises or consists of from N-terminus to C-terminus:- optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a membrane anchoring domain; or- optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, a membrane anchoring domain.

6. The pharmaceutical composition for use according to any one of claims 1-5, wherein the membrane IL-35 molecule comprises or consists of, from N-terminus to C-terminus:- optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, a transmembrane domain and optionally a cytosolic domain; or- optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, a transmembrane domain and optionally a cytosolic domain; or- optionally a peptide signal, a p35 subunit, optionally a peptide linker, an Ebi3 subunit, optionally a hinge domain, and a GPI-anchor domain; or- optionally a peptide signal, an Ebi3 subunit, optionally a peptide linker, a p35 subunit, optionally a hinge domain, and a GPI-anchor domain.

7. The pharmaceutical composition for use according to any one of claims 4-6, wherein the membrane IL-35 comprises a peptide linker, preferably between the EBB subunit and the p35subunit, said peptide linker being selected from the group consisting of i) a cleavable linker, preferably a 2A self-cleaving peptide, ii) a flexible linker, preferably selected from the group consisting of Glycine- Serine linkers, Glycine-Proline linkers and Proline-Rich linkers, and iii) a rigid linker, preferably selected from the group consisting of a-Helical Linkers, coiled-coil linkers and P-Sheet linkers.

8. The pharmaceutical composition for use according to any one of claims 1-7, wherein the membrane IL-35 comprises at least one peptide signal, said peptide signal being preferably p35 peptide signal or IL-2 peptide signal, more preferably comprising or consisting of an amino acid sequence of SEQ ID NO: 42 or 44.

9. The pharmaceutical composition for use according to any one of claims 1-8, wherein the membrane IL-35 comprises a hinge domain, said hinge domain being preferably selected from the group consisting of a CD8 hinge domain, a CD28 hinge domain, an IgGl hinge domain, an IgG4 hinge domain, a CH2-CH3 domain of an IgGl, a CH2-CH3 domain of an IgG4, a hinge-CH2-CH3 of an IgGl, a hinge-CH2-CH3 domain of an IgG4 and a peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:22-37, 50 and 54.

10. The pharmaceutical composition for use according to any one of claims 1-9, wherein the membrane anchoring domain is a transmembrane domain, said transmembrane domain being selected from the group consisting of a cytokine receptor transmembrane domain, a chemokine receptor transmembrane domain, a type I transmembrane protein transmembrane domain, a type II transmembrane protein transmembrane domain.

11. The pharmaceutical composition for use according to claim 10, wherein the transmembrane domain is selected from the group consisting of a CD80 transmembrane domain, a MHC1 transmembrane domain, a FAS transmembrane domain, a TNF receptor transmembrane domain or a B cell receptor (BCR) transmembrane domain, preferably is a CD80 transmembrane domain or a BCR transmembrane domain.

12. The pharmaceutical composition for use according to claim 10 or 11, wherein the membrane IL-35 further comprises a cytosolic domain, said cytosolic domain being selected from the group consisting of a cytokine receptor cytosolic domain, a type I transmembrane protein cytosolic domain and a type II transmembrane protein cytosolic domain.

13. The pharmaceutical composition for use according to any one of claims 1-9, wherein the membrane anchoring domain is a Glycosylphosphatidylinositol (GPI)-anchor domain of a protein selected from the group consisting of ACHE, ALPG, ALPI, ALPL, ALPP, ART3,ART4, BST1, BST2, CD109, CD14, CD160, CD48, CD52, CD55, CD59, CDH13, CEACAM5, CEACAM7, CNTN3, CNTN4, CNTN5, CPM, CPO, CRIPTO, DPEP1, EFNA4, EFNA5, FCGR3B, FOLR1, FOLR2, GP2, GPC1, GPIHBP1, HYAL2, IZUM01R, LSAMP, LY6D, LY6E, LY6G6C, LY6G6D, LY6H, LY6K, LYPD1, LYPD2, LYPD3, LYPD4, LYPD5, LYPD6B, LYPD8, MDGA1, MDGA2, MELTF, MMP17, MMP25, MSLN, NEGRI, NRN1, NRN1L, NTM, NTNG1, NTNG2, OPCML, OTO A, PLAUR, PLET1, PRND, PRNP, PRSS21, PRSS41, PRSS55, PSCA, RAET1G, RAET1L, RECK, RGMB, RTN4RL1, RTN4RL2, SEMA7A, SPACA4, SPRN, TECTA, TECTB, TEX101, THY1, ULBP2, VNN1, VNN2, XPNPEP2, GPCR3, more preferably from the group consisting of CD48, CD52, CD55, CD59, PRNP, FOLR1, LSAMP, MSLN, DPEP1, THY1 and PLET1, even more preferably comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NO: 57-151.

14. The pharmaceutical composition for use according to any one of claims 1-13, wherein the membrane IL-35 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 162-169 and 172-173, or a variant thereof having at least 85% sequence identity thereto.

15. The pharmaceutical composition for use according to any one of claims 1-13, wherein the mRNA molecule comprises or consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 46-49, 154-161 and 170-171 or a variant thereof having at least 85% sequence identity thereto.

16. The pharmaceutical composition for use according to any one of the preceding claims, wherein the mRNA molecule comprises a 5’UTR, a 3’UTR, 5' cap structure, a Kozak sequence, a chain terminating nucleotide, a stem loop, a poly-A sequence and / or a polyadenylation signal.

17. The pharmaceutical composition for use according to any one of the preceding claims, wherein the lipid-based nanoparticle comprises a cationic or ionizable lipid, a helper lipid, a sterol and a PEG-lipid.

18. The pharmaceutical composition for use according to claim 17, wherein the lipid-based nanoparticle comprises from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid.

19. The pharmaceutical composition for use according to claim 17 or 18, wherein the lipid composition of the lipid-based nanoparticle comprises or consists ofALC-0315, DDAB, MC3-DLin-DMA or any mixture thereof, preferably from about 20 mol% to about 60 mol% of the total lipids of the lipid-based nanoparticle;DSPC and / or DOPE, preferably from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle;Cholesterol, preferably from about 30 mol% to about 70 mol% of the total lipids of the lipid-based nanoparticle; andDMG-PEG, preferably from about 0.1 mol% to about 10 mol% of the total lipids of the lipid-based nanoparticle.

20. The pharmaceutical composition for use according to any of claims 17-19, wherein the lipid composition of the lipid-based nanoparticle comprises or consists of:ALC-0315 or a derivative thereof from about 45 mol% to about 55 mol% of the total lipids of the lipid-based nanoparticle;DOPE from about 1 mol% to about 20 mol% of the total lipids of the lipid-based nanoparticle;Cholesterol from about 30 mol% to about 70 mol% of the total lipids of the lipid- based nanoparticle; andDMG-PEG from about 0.1 mol% to about 10 mol% of the total lipids of the lipid- based nanoparticle.

21. The pharmaceutical composition for use according to any one of the preceding claims, wherein the LNP further comprises a targeting moiety or an antigen-binding domain of an antibody capable of specifically binding to a target expressed on activated immune cells surface, preferably a target expressed on activated T cells, activated B cells, activated myeloid cells including activated macrophages, activated dendritic cells and / or activated neutrophils, preferably the target being selected from the group consisting of CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD154, CD183, CD3, CD4, CD8, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL / TNFSF6, PD1 / PDCD1, GITR / TNFRSF18, GPR32, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LY108 / SlamF6, OX40 / TNFRSF4, RGS1, LTBR / CD70, TNFSF14, CD112R, CD28H, CD164, TRAF2, CDCR3 / TNFRSF6B, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, ICOSL, CD160, CD19, CD20, CD24, CD38 TIM-1, TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R, CLEC-1331A, CDl lb, CD14, CD66b, CD163, CD206, SIGLEC 6, BCMA / TNFRSF17, CD150, CD86, OX40L, L0X1, TACVTNFRSF13B, CD138 (SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74 and / or CD 165.

22. The pharmaceutical composition for use according to any one of the preceding claims, wherein the disease is selected from the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoath etosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti-N-Methyl-D- Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, membranous nephropathy, allogenic islet graft rejection, alopecia areata, ankylosing spondylitis, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune urticaria, Behcet's disease, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune disfunction syndrome, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, discoid lupus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Guillain-Barre syndrome, graft-versus-host disease (GVHD), hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (FTP), IgA neuropathy, IgM polyneuropathies, juvenile arthritis, Kawasaki's disease, lichen plantus, lichen sclerosus, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes mellitus, Multifocal motor neuropathy (MMN), pemphigoid gestationis, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynauld's phenomenon, Reiter's syndrome, sarcoidosis, solid organ transplant rejection, Takayasu arteritis, toxic epidermal necrolysis134(TEN), Stevens Johnson syndrome (SJS), temporal arteristis / giant cell arteritis, thrombotic thrombocytopenia purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitides, vitiligo, asthma, autoimmune pancreatitis, IgA nephropathy and Wegner's granulomatosis; optionally selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, immune thrombocytopenia (especially persistent or chronic immune thrombocytopenia), chronic inflammatory demyelinating polyneuropathy, scleroderma, CREST syndrome, inflammatory myopathy, primary biliary cirrhosis, coeliac disease, rheumatoid arthritis, granulomatosis, antiphospholipid syndrome, Goodpasture syndrome, chronic autoimmune hepatitis, polymyositis, small intestinal bacterial overgrowth, Hashimoto's thyroiditis, Graves' disease, paraneoplastic cerebellar degeneration, limbic encephalitis, encephalomyelitis, subacute sensory neuronopathy, choreoathetosis, opsoclonus myoclonus syndrome, Stiff-Person syndrome, diabetes mellitus type 1, Isaac's syndrome, optic neuropathy, anti-N-Methyl-D-Aspartate Receptor Encephalitis, neuromyelitis optica, Bullous pemphigoid, and membranous nephropathy, preferably selected in the group consisting of Myasthenia Gravis, Pemphigus vulgaris, systemic lupus erythematosus, Sjogren’s syndrome, antiphospholipid syndrome, Hashimoto's thyroiditis, Graves' disease and graft rejection.

23. The pharmaceutical composition for use according to anyone of claims 1-21, wherein the disease is an inflammatory and / or auto-immune disease of the liver, preferably selected from the group consisting of Autoimmune hepatitis (AIH), Primary biliary cholangitis (PBC), Primary sclerosing cholangitis (PSC) and non-alcoholic fatty liver disease.

24. The pharmaceutical composition for use according to any one of claims 1-21, wherein the inflammatory and / or auto-immune is a skin inflammatory and / or auto-immune disease, preferably selected from the group consisting of eczema (atopic dermatitis), contact dermatitis, discoid dermatitis, psoriasis, seborrheic dermatitis, lichen simplex chronicus, pityriasis rosea, rosacea, urticaria, erythema multiforme, erythroderma, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, dermatitis herpetiformis, epidermolysis bullosa acquisita, cutaneous lupus erythematosus, discoid lupus erythematosus, dermatomyositis, scleroderma Henoch-Schbnlein purpura, vitiligo, alopecia areata, lichen planus, lichen sclerosis, morphea, and psoriasis, preferably atopic dermatitis or psoriasis, even more preferably psoriasis.13525. The pharmaceutical composition for use according to claim 24, wherein pharmaceutical composition is formulated for topical, intradermal, transdermal, epi cutaneous or subcutaneous administration, preferably topical administration.

26. The pharmaceutical composition for use according to claim 24 or 25, wherein:- the pharmaceutical composition is a topical pharmaceutical composition;- the skin disease is psoriasis or atopic dermatitis; and- the lipid composition of the lipid-based nanoparticle consists of about 48%-52% ALC-0315, 8%-12% DOPE, 38%-42% cholesterol and about 0.5% and 2% DMG-PEG of the total lipids of the lipid-based nanoparticle.

27. A lipid-based nanoparticle comprising mRNA molecule(s) encoding a transmembrane interleukin 35 (IL-35) molecule as defined in any one of claims 1 to 21.

28. A pharmaceutical composition comprising the lipid-based nanoparticle of claim 27 and optionally a pharmaceutically acceptable carrier.

29. Use of the lipid-based nanoparticle of claim 27 or the pharmaceutical composition according to claim 28, for the manufacture of a medicament for the treatment of an autoimmune disease or an inflammatory disease, preferably a skin autoimmune or inflammatory disease.

30. A method for treating an autoimmune disease or an inflammatory disease, preferably a skin autoimmune or inflammatory disease, wherein the method comprises administering a therapeutic amount of the lipid-based nanoparticle of claim 27 or the pharmaceutical composition according to claim 28.

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