Lipid-based nanoparticles targeted to activated immune cells for the expression of immune cell inhibitory molecules and their use

Abstract

The present invention relates to lipid-based nanoparticles comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an inhibitory protein of the activated immune cells, and to the use thereof.

Description

[Technical Field]

[0001] This invention relates to the field of immunotherapy. It concerns lipid-based nanoparticles containing mRNA molecules, and their use for treating conditions such as autoimmune diseases and inflammatory diseases. [Background technology]

[0002] Both autoimmune diseases and inflammatory conditions stem from an inappropriate response of the immune system to either the body's own cells or byproducts of inflammatory processes, resulting in excessive activation of immune cells.

[0003] One strategy to inhibit or reduce immune cell activation is through direct activation of dissipation pathways or through suppression of inflammatory cells. However, this strategy can result in acute and serious adverse effects due to the nonspecific suppression of immune cells.

[0004] To avoid such nonspecific inactivation of immune cells, several solutions have been developed in previous research.

[0005] One of these solutions attempts to reduce immune cell activation by using molecules that inhibit the activity of immune cells, the most common of which are immunosuppressants. However, due to serious side effects resulting in severe toxicity and limited effectiveness, strategies were needed to overcome these drawbacks.

[0006] Solutions explored in the prior art to inhibit the immune response have focused on targeting specific subsets of immune cells for the delivery of inhibitory compounds.

[0007] Since immune cells are notoriously difficult to transfect, there is a need for solutions to efficiently reduce immune cell activation by targeting and delivering highly influential molecules, particularly within specific localizations.

[0008] Vaccines based on mRNA-containing lipid nanoparticles (LNPs) represent a promising new delivery platform. LNPs are used to deliver mRNA to cells, leading to the expression of encoded proteins and ultimately, the provision of immune defense to the body. Expressing proteins by delivering coding mRNA offers numerous advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to the cell, thus avoiding all side effects associated with plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA-based and viral-based vectors, mRNA does not carry the risk of integration into the genome, and protein production begins immediately after mRNA delivery.

[0009] Although LNPs have been successfully used in leading COVID-19 vaccines, many issues remain regarding the risk of off-target expression of systemic mRNA-LNP distribution and immunogens, which may generate systemic cytokines, activate complement, increase the frequency or severity of adverse events (as observed in recent clinical trials), and / or impair immune response development. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] WO2017049245 [Patent Document 2] WO2017112865 [Patent Document 3] WO2017218704 [Patent Document 4] WO2015164674 [Patent Document 5] WO2017031232 [Patent Document 6] WO2017099823 [Patent Document 7] WO2016118724 [Patent Document 8] WO2017223135 [Patent Document 9] WO2014152211 [Patent Document 10] WO2015038892 [Patent Document 11] WO2017049074 [Patent Document 12] WO2013090648 [Patent Document 13] WO2017180917 [Patent Document 14] WO 2017 / 075531 [Patent Document 15] WO 2017 / 117528 [Patent Document 16] US Patent Application Publication No. 20120276209 [Patent Document 17] PCT / EP2024 / 058775 [Patent Document 18] WO 2016 / 021683 [Patent Document 19] WO 2015 / 011633 [Patent Document 20] WO 2011 / 153493 [Patent Document 21] WO 2013 / 126803 [Patent Document 22] WO 2010 / 054401 [Patent Document 23] WO 2010 / 042877 [Patent Document 24] WO 2016 / 104580 [Patent Document 25] WO 2015 / 005253 [Patent Document 26] WO 2014 / 007398 [Patent Document 27] WO 2017 / 00414 [Patent Document 28] WO 2015 / 199952 [Patent Document 29] U.S. Patent Application Publication No. 2015 / 0239834 [Patent Document 30] WO2019 / 131839 [Patent Document 31] WO2022 / 261101 [Patent Document 32] WO2021194672 [Patent Document 33] WO 2009 / 080253 [Patent Document 34] WO2020 / 127366 [Patent Document 35] WO 2014 / 194302 [Patent Document 36] WO 2017 / 040790 [Patent Document 37] WO 2017 / 19846 [Patent Document 38] WO 2017 / 024465 [Patent Document 39] WO 2017 / 025016 [Patent Document 40] WO 2017 / 132825 [Patent Document 41] WO 2017 / 133540 [Patent Document 42] WO 2006 / 121168 [Patent Document 43] WO 2017 / 024465 [Patent Document 44] WO 2017 / 025016 [Patent Document 45] WO 2017 / 132825 [Patent Document 46] WO 2006 / 121168 [Patent Document 47] WO2013006490 [Patent Document 48] WO2016 / 161270 [Patent Document 49] WO 2018 / 085469 [Patent Document 50] WO 2018 / 129553 [Patent Document 51] WO 2011 / 155607 [Patent Document 52] U.S. Patent No. 8,552,156 [Patent Document 53] EP 2581113 [Patent Document 54] U.S. Patent Application Publication No. 2014 / 044728 [Patent Document 55] WO18025178 [Patent Document 56] WO19179388 [Patent Document 57] WO19179391 [Patent Document 58] WO19174603 [Patent Document 59] WO19148444 [Patent Document 60] WO19120232 [Patent Document 61] WO19056281 [Patent Document 62] WO19023482 [Patent Document 63] WO18209701 [Patent Document 64] WO18165895 [Patent Document 65] WO18160536 [Patent 66] WO18156250 [Patent Document 67] WO18106862 [Patent Document 68] WO18106864 [Patent Document 69] WO18068182 [Patent Document 70] WO18035710

Patent document 71

Patent document 72

Patent document 73

Patent document 74

Patent document 75

Patent document 76

Patent document 77

Patent document 78

Patent document 79

Patent document 80

Patent document 81

Patent document 82

Patent document 83

Patent document 84

Patent document 85

Patent document 86

Patent document 87

Patent document 88

Patent document 89

Patent document 90

Patent document 91

Patent document 92

Patent document 93

Patent document 94

Patent document 95

Patent document 96

Patent document 97

Patent document 98

Patent document 99

Patent document 100

Patent Document 101

Patent document 102

Patent document 103

Patent document 104

Patent document 105

Patent document 106

Patent document 107

Patent document 108

Patent document 109

Patent document 110

Patent document 111

Patent document 112

Patent document 113

Patent document 114

Patent document 115

Patent document 116

Patent document 117

Patent document 118

Patent document 119

Patent document 120

Patent document 121

Patent document 122

Patent document 123

Patent document 124

Patent document 125

Patent document 126

Patent document 127

Patent document 128

Patent document 129

Patent document 130

Patent document 131

Patent document 132

Patent document 133

Patent document 134

Patent document 135

Patent document 136

Patent document 137

[0011] [Non-licensed Document 1] Wardら、1989 Nature 341:544~546 pages [Non-licensed Document 2] Birdら、1988 Science 242:423~426 pages [Non-licensed Document 3] Huston, 1988, Proc. Natl. Acad. Sci. 85: 5879~5883. [Non-licensed Document 4] E. Meyers and W. Miller (Comput. Appl. Biosci., pp. 4:11~17, 1988) [Non-licensed Document 5] Needleman and Wunsch (J. Mol, Biol. 48:444~453 pages, 1970) [Non-licensed Document 6] Soding, J. (2005) "Protein homology detection by HMM-HMM comparison", Bioinformatics 21, pages 951~960 [Non-licensed Document 7] Edelman, 1969, Proc Natl Acad Sci USA 63: 78-85 [Non-licensed Document 8] Kabatら, 1991, Sequences of Proteins of Immunological Interest, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda [Non-licensed Document 9] Sempleら, 2010, Nat Biotechnol., 28(2): pages 172~176 [Non-licensed Document 10] Bashaら, 2011, Mol Ther, 19(12): 2186~2200 pages [Non-licensed Document 11] Leung, 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440~18450 [Non-licensed Document 12] Leeら, 2012, Int J Cancer., 131(5): E781-90 [Non-licensed Document 13] Belliveauら, 2012, Mol Ther nucleic Acids, 1: e37 [Non-licensed Document 14] Jayaramanら, 2012, Angew Chem Int Ed Engl., 51(34): 8529~8533 pages [Non-licensed Document 15] Muiら, 2013, Mol Ther Nucleic Acids. 2, el39 [Non-licensed Document 16] Maier, 2013, Mol Ther., 21(8): 1570-1578 [Non-licensed Document 17] Tamら, 2013, Nanomedicine, 9(5): 665-74 [Non-licensed Document 18] Dongら, Proc Natl Acad Sci US A. April 15, 2014; 111(15):5753 [Non-licensed Document 19] Love KTら, Proc Natl Acad Sci US A. May 25, 2010; 107(21):9915 [Non-licensed Document 20] Gao SH, Huang K, Tu H, Adler AS, BMC Biotechnology. 2013: 13:55 [Non-licensed Document 21] Kedmi, Ranit, "A modular platform for targeted RNAi therapeutics", Nature nanotechnology, Volume 13, 3 (2018): Pages 214~219, doi:10.1038 / s41565-017-0043-5 [Non-licensed Document 22] Qin J, Xue L, Gong N, Zhang H, Shepherd SJ, Haley RM, Swingle KL, Mitchell MJ., RGD peptide-based lipids for targeted mRNA delivery and gene editing applications. RSC Adv. 2022 Sep 7;12(39):25397~25404, doi: 10.1039 / d2ra02771b. PMID: 36199352; PMCID: PMC9450108 [Non-licensed Document 23] Schaefer, W.ら, PNAS, 108 (2011) pages 11187~1191 [Non-licensed Document 24] Si-Yang Liuら, J. Hematol. Oncol.10:136 (2017) [Non-licensed Document 25] Koreら、Bioorganic & Medicinal Chemistry 20132: pages 4570~4574 [Non-licensed Document 26] Sionov R., Vogt, Vlahopoulos SA, Granot Z., Regulation of Bim in Health and Disease. Oncotarget. 2015; 6: 23058~23134 [Non-Patent Document 27] Cao, H., Chen, X. (2012). General Consideration on Sialic Acid Chemistry. In: Chevolot, Y. (ed.) Carbohydrate Microarrays. Methods in Molecular Biology, Volume 808. Humana Press. https: / / doi.org / 10.1007 / 978-1-61779-373-8_3 [Non-Patent Document 28] Leo, O, Sachs, DH, Samelson, L, et al. J. Immunol. 137:3874 (1986) [Non-Patent Document 29] Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005) [Non-Patent Document 30] Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980) [Non-Patent Document 31] Samuelson, LE, Germain, RN, Schwartz, RH, PNAS(USA) 80:6972 (1983) [Overview of the project] [Problems that the invention aims to solve]

[0012] Therefore, it is necessary to improve the technology to overcome these shortcomings. [Means for solving the problem]

[0013] To this end, the inventors have developed targeted lipid-based nanoparticles that target activated immune cells, containing mRNA molecules encoding proteins that inhibit the activity of immune cells.

[0014] The present invention ensures that mRNA encoding an activity-inhibiting protein is specifically and locally delivered to a particular immune cell population, such as an immune cell population located in chronically inflammatory tissue, in order to maximize efficiency with little to no systemic side effects. Therefore, immune cell inhibitory proteins can be selected even from among proteins that exhibit severe side effects, because their actions are limited to a precise location, and the proteins are not expressed systemically, thus avoiding off-target side effects.

[0015] With the present invention, those skilled in the art will also be able to easily modify the mRNA cargo to replace or combine the effects of different immune cell inhibitory proteins in order to adapt the invention to the needs of the patient, without the need to evaluate and prove the safety of delivery of lipid-based nanoparticles.

[0016] The inventors herein provide a method for specifically and selectively targeting and inhibiting a selected subset of immune cells, rather than using LNPs to deliver inhibitory compounds to cells located in inflammatory tissue, or targeting any given type of cell and indiscriminately inactivating them.

[0017] Furthermore, targets can be selected based on the location of the immune cells that possess them. For example, CD80 and CD86 are expressed in pro-inflammatory M1 macrophages. By targeting CD80 and / or CD86-positive immune cells, mRNA can be delivered to the immune cells where it is most needed, thus ensuring a highly effective and specific effect. In contrast, targeting antigens expressed on a wide range of cell types, including naive or inactive cells, such as CD3 or CD5, does not yield the same level of precision.

[0018] Since the mRNA-encoded proteins envisioned in this specification can be selected from among the most potent effectors, it is therefore extremely important for patient safety that the risk of off-target effects be kept as low as possible, or even completely suppressed.

[0019] In addition, a further aspect of the present invention is to provide LNPs that are taken up very little by a capturing organ, particularly the liver, but also, if necessary, by other organs, such as the spleen and / or lungs. In this aspect, an increase in the free distribution of LNPs throughout the body, particularly in the blood or other bodily fluids, may be achieved, thereby potentially enabling the delivery of LNPs to an area of ​​interest (e.g., a site of inflammation).

[0020] In a first aspect, the present invention relates to lipid-based nanoparticles comprising an antigen-binding domain that can specifically bind to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an inhibitory protein of the activated immune cells, wherein the mRNA molecules encode i) an intracellular protein having an intracellular effect on activated immune cells, and / or ii) a transmembrane protein that is preferably not a chimeric-associated receptor (CAR).

[0021] In particular, the present invention relates to lipid-based nanoparticles comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an inhibitory protein of the activated immune cells, wherein the inhibitory protein is i) an intracellular protein having an intracellular effect on activated immune cells, and / or ii) a transmembrane protein, and the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

[0022] In one embodiment, the lipid-based nanoparticles comprise at least two mRNA molecules, one of which encodes a transmembrane protein that is a receptor, and the other of which encodes a secreted protein that is a ligand for the receptor.

[0023] In one embodiment, preferably, the activated immune cells are selected from the group consisting of activated T cells, activated B cells, activated myeloid cells, such as activated macrophages and activated dendritic cells.

[0024] In one embodiment, the targets expressed on the surface of activated immune cells are PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAV CR2, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138(SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD 120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFR The selection is made from the group consisting of SF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, CD160, CD127, and SIRPa.

[0025] In one embodiment, the targets expressed on the surface of activated immune cells are BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, C D83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, IC OS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVE The selection is made from the group consisting of M / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, and CD160.

[0026] Preferably, the target expressed on the surface of activated immune cells is selected from the group consisting of PD-1, CD127, SIRPa, and CLEC-1A.

[0027] In one embodiment, the target expressed on the surface of activated immune cells is PD-1.

[0028] In one embodiment, the antigen-binding domain is a) (i) VH including HCDR1, HCDR2 and HCDR3, and (ii) VL including LCDR1, LCDR2 and LCDR3, - Heavy chain CDR1 (HCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 1; - Heavy chain CDR2 (HCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 2; - Heavy chain CDR3 (HCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 3; - The light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 4; - Light chain CDR2 (LCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 5; and - The light chain CDR3 (LCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 6; or b)i) VH containing or consisting of the amino acid sequence of SEQ ID NO: 15, and ii) VL containing or consisting of the amino acid sequence of SEQ ID NO: 16, It is an anti-PD-1 binding domain.

[0029] In one embodiment, the antigen-binding domain is a) (i) VH including HCDR1, HCDR2 and HCDR3, and (ii) VL including LCDR1, LCDR2 and LCDR3, - Heavy chain CDR1 (HCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 31; - Heavy chain CDR2 (HCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 32; - Heavy chain CDR3 (HCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 33; - The light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 34; - Light chain CDR2 (LCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 35; and - The light chain CDR3 (LCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 36; or b)i) VH comprising the amino acid sequence of SEQ ID NO: 37 or consisting thereof, and ii) VL comprising the amino acid sequence of SEQ ID NO: 38 or consisting thereof, It is an anti-PD-1 binding domain.

[0030] In one embodiment, the antigen-binding domain is a) (i) VH including HCDR1, HCDR2 and HCDR3, and (ii) VL including LCDR1, LCDR2 and LCDR3, - Heavy chain CDR1 (HCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 23; - Heavy chain CDR2 (HCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 24; - Heavy chain CDR3 (HCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 25; - The light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 26; - Light chain CDR2 (LCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 27; and - The light chain CDR3 (LCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 28; or b)i) VH containing or consisting of the amino acid sequence of SEQ ID NO: 29, and ii) VL containing or consisting of the amino acid sequence of SEQ ID NO: 30, It is an anti-PD-1 binding domain.

[0031] Generally, the antigen-binding domains disclosed herein include Fc domains, preferably IgG Fc domains.

[0032] Preferably, the antigen-binding domain is not covalently bound to any of the lipids in the lipid-based nanoparticles, and does not include any modifications for coupling or grafting the antigen-binding domain onto the lipids. In particular, the lipid-based nanoparticles do not contain an anchor moiety containing a lipidized peptide or motif.

[0033] In one embodiment, the lipid-based nanoparticles include additional antigen-binding domains that can specifically bind to another target expressed on the surface of activated immune cells.

[0034] Preferably, the additional antigen-binding domain is i) not covalently bound to any of the lipids in the lipid-based nanoparticles, ii) does not include any modifications for coupling or grafting the antigen-binding domain to the lipids, and / or iii) is not covalently bound to either the lipidized peptide or the lipidized motif.

[0035] In one embodiment, the mRNA molecule encodes an intracellular protein that has an intracellular effect on activated immune cells, selected from the group consisting of cytoplasmic proteins, intracellular signaling proteins, enzymes, transcription factors, intrabodies, dominant-negative receptors, or engineered antagonist proteins, such as engineered blocking proteins.

[0036] Preferably, one or more mRNA molecules encode molecules selected from the following group: a) TIM3, ENTPD1, ​​LAG3, PD-1, PD-L1, TIGIT, FOXO1, MLH1, MSH2, MSH6, APC, CDKN2A, Caspase, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, BIM, BIM-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, P53, FOXP3, TOX, EOMES, BCL6 and BACH2, HIFa, PKCq and VHL, IDO-1, IDO-2, ARG1; TDO, mTOR / DAPTOR / RAPTOR, SHP, SMAD, SHIP-1, SHP-1 / 2, PTEN, PTP1B, Ikaros, EGR2 / 3, CREM, P27(KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS10, BBS12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Tapasin, LMP7, Er Proteins selected from the group consisting of p57, Cbl-b, CHEMR23, GPR37, GPR32, GPR18, FPR2, GPR35, HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3, or SUV420H2, preferably proteins selected from the group consisting of TIM3, LAG3, PD-L1, TIGIT, FAS, TRAIL, PUMA, BIM-S, FOXP3, TOX, IDO-1, IDO-2, ARG1, CHEMR23, and FPR2, and any combination thereof; b) Cytokines, preferably selected from the group consisting of IFNG, IFNa, IL2, IL13, IL4, and IL-10, and any combination thereof; c) Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-8R, IL-10R, IL-11R, IL-17R, IL-18R, IL-21R, IL-23R, IL-35R and any combination thereof; and d) Chemokine receptors, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and XCR1, and any combination thereof.

[0037] In one embodiment, one or more mRNA molecules are CCR4, CCR10, CXCR3, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR5, IL10R, TGFR, TNFR, ILR1A, GCSFR, IL4R, CXCR1, CXCR2, CXCR6, CCR1, CCR3, CCR6, CCR8, CCR9, CCR11, XCR1, CD72, DCIR, MICL, CLEC-1, TGFB, IL13, IL4, IL-10, IL35, IL37, IL38, IL-6R, IL17R, IL23R, IL35R, IL21R, IFNa It codes for R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, Galectin 9, CD48, HVEM, B7DC, CD155, CD113, CD112, ILT2, ILT3, NKG2A, LILRB1, IRp60, KIR, CD22, CD5, CD66a, PIR-B, KIR2DL1, KIR2DL2 / 3, ILT4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, TIGIT, LAG3 or A2AR, SOCS, RIPK1, and any member of the STAT family, or any combination thereof.

[0038] Preferably, one or more mRNA molecules encode BIM-S and / or PUMA.

[0039] In some embodiments, the lipid-based nanoparticles comprise at least two mRNA molecules, one of which encodes a transmembrane protein that is a receptor, and the other of which encodes a secreted protein that is a ligand for the receptor.

[0040] In some embodiments, the lipid-based composition of the lipid-based nanoparticles includes or comprises cationic or ionizable lipids, helper lipids, sterols, and PEG-lipids.

[0041] Preferably, the ionizable lipids are [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and dimethyl dio Dioctadecylammonium (DDAB); 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-Diacyloxy-3-dimethylammoniumpropane; 1,2-Dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradeoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1, 2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-Dimyristoyl-3-trimethylammoniumpropane (DMTAP), 1,2-Dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-Dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyl Norenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-Dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-DMA) , 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)-N-(3- (Aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy) C)-1-propanaminonium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (aminiiim) (DOBAQ), 2-({8-[(3P)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane N-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), di((Z)-nona-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propane-1-amine (DLDMA), N,N-dimethyl-2,3-Bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-nona-2-en-1-yl)-9-((4-(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]piperazine-1-yl]ethyl]amino]dodecane-2-ethyl A solution is selected from the group consisting of (lipidoid C12-200), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide (SS-OP; for example, CAS number 2377474-67-2)bis{2-[4-(α-D-tocopherol hemisuccinate ethyl)piperidyl]ethyl}disulfide (SS-EC) and any mixture thereof, preferably ALC-0315, SM-102, Dlin-MC3-DMA or SS-OP, more preferably ALC-0315 or SS-OP.

[0042] Preferably, the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixture thereof, and is preferably cholesterol.

[0043] Preferably, the helper lipid is selected from DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DOPC, DEPC, and DSPE, and any mixture thereof, preferably DOPE or DSPC.

[0044] Preferably, the PEG-lipid is selected from PEG-DMG, PEG-DSPE, PEG-c-DOMG, DMG-PEG-2000, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG, and PEG-c-DMA, ALC-0159, and any mixture thereof, and is preferably PEG-DMG, PEG-DSPE, or a mixture thereof.

[0045] In particular, the PEG has a size between approximately 2000 Daltons and approximately 5000 Daltons, and is preferably DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000, or a mixture thereof.

[0046] In some cases, lipid-based compositions of lipid-based nanoparticles, 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, i) ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG, j) SS-OP, DOPE, cholesterol, and DMG-PEG; k) SS-OP, DSPC, cholesterol and DSPE-PEG; and l) SS-OP, DOPC, cholesterol and DMG-PEG It is selected from the group consisting of the following.

[0047] In some embodiments, the lipid-based nanoparticles contain approximately 35 mol% to 55 mol% of cationic or ionizable lipids, approximately 5 mol% to 20 mol% of helper lipids, approximately 30 mol% to 60 mol% of sterols, and approximately 0.5 mol% to 4 mol% of PEG-lipids. Preferably, the lipid-based nanoparticles contain or consist of approximately 45 mol% to 55 mol% of cationic or ionizable lipids, approximately 5 mol% to 15 mol% of helper lipids, approximately 35 mol% to 45 mol% of sterols, and approximately 0.5 mol% to 2.5 mol% of PEG-lipids.

[0048] In one embodiment, the mRNA molecule is selected from the group comprising modified nucleotides, preferably alternative uracil, alternative cytosine, alternative guanine, and alternative adenine, preferably pseudouridine, 1-methylpsoidouridine, 1-ethylpsoidouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-psoidouridine, 2-thio-1-methylpsoidouridine, 2-thio-5-aza-uri The modified nucleotides include din, 2-thio-dihydropsoiduridine, 2-thio-dihydropsoiduridine, 2-thiopsoiduridine, 4-methoxy-2-thiopsoiduridine, 4-methoxypsoiduridine, 4-thio-1-methylpsoiduridine, 4-thiopsoiduridine, 5-aza-uridine, dihydropsoiduridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0049] In one embodiment, the mRNA molecule includes a 5'UTR, a 5' cap structure, a Kozak sequence, an IRES sequence, a chain termination nucleotide, a stem-loop, a 3'UTR, a poly(A) sequence, and / or a polyadenylation signal.

[0050] The present invention also relates to a pharmaceutical composition comprising at least one lipid-based nanoparticle according to the present invention and, optionally, a pharmaceutically acceptable carrier or excipient.

[0051] In some embodiments, the pharmaceutical composition further comprises additional lipid-based nanoparticles containing an antigen-binding domain that can specifically bind to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitory protein.

[0052] The present invention also relates to lipid-based nanoparticles or pharmaceutical compositions according to the present invention for use as pharmaceuticals.

[0053] In some embodiments, lipid-based nanoparticles or pharmaceutical compositions are for use in the treatment of diseases in subjects requiring them, diseases being selected from the group of diseases in which the resolution of inflammation is delayed or impaired, and / or inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases, e.g., chronic inflammatory lung disease (e.g., asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, skin inflammation; autoimmune diseases, e.g., diabetes, NASH, in particular type 1 diabetes, psoriasis, lupus, arthritis The following conditions are selected from the group consisting of: malarthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases, e.g., sepsis; severe viral indications with severe inflammatory states, e.g., coronavirus (e.g., COVID-19); peritonitis; degenerative diseases; impaired wound healing; dry eye syndrome; cancer, especially solid and humoral cancers; metastatic cancers, especially carcinomas, especially breast or colon cancer, or colorectal cancer, lung cancer, or mesothelioma, or myeloid cancers, especially leukemia; fibrosis (especially pulmonary and hepatic fibrosis); and ANCA (anti-neutrophil cytoplasmic autoantibody) conditions (vasculitis).

[0054] The present invention also relates to the use of lipid-based nanoparticles or pharmaceutical compositions according to the present invention for the manufacture of pharmaceuticals for the treatment of diseases listed herein in subjects requiring such treatment. The present invention further relates to a method for treating diseases listed herein in subjects requiring such treatment, comprising the step of administering a therapeutically effective amount of the lipid-based nanoparticles or pharmaceutical composition according to the present invention to the patient.

[0055] The present invention relates to an in vitro method for reducing immune cell activity, the method comprising the step of contacting activated immune cells with lipid-based nanoparticles or a pharmaceutical composition according to the present invention. [Brief explanation of the drawing]

[0056] [Figure 1] This figure shows the flow cytometry analysis of U937 (WT compared to transduced cells), Jurkat (WT compared to transduced cells), and HPB-ALL. Flow cytometry was used to measure the percentage of PD-1+ cells. Dashed line: Cells stained with PE-Cy7-labeled control isotype mouse IgG (#557646 batch: 8155598, BD Biosciences). Dark gray: Cells stained with PE-Cy7-labeled anti-human PD-1 antibody (#561272 batch: 1319137, BD Biosciences). Dead cells were stained with LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from the analysis. [Figure 2] This figure shows that the addition of anti-PD-1 mAb OSE-279 before LNP formation enables the preparation of targeted LNPs with improved transfection efficacy in PD-1+ cells. Different modalities for adding mAb OSE-279 during the LNP production process were tested. The transfection efficiency of LNPs containing fluc-mRNA was evaluated by comparing OSE-279-added modalities #1A, #1B, and #1C in WT and PD-1+ U937 cells (Figure 2A) and in WT and PD-1+ Jurkat cells (Figure 2B). Untargeted LNPs (nt-LNPs = LNPs produced without mAb addition) were used as negative controls. [Figure 3]This figure shows that transfection was improved by increasing the dose of mAb in targeted LNPs. Targeted LNPs were prepared using gradually increasing amounts (1, 10, and 50 μL) of mAb OSE-279 added during LNP preparation. The transfection efficiency of t-LNPs containing fluc-mRNA was evaluated in WT and PD-1+ U937 cells (Figure 3A), WT and PD-1+ Jurkat cells (Figure 3B), and HPB-ALL cells (Figure 3C). Untargeted LNPs were used as a negative control. [Figure 4] This figure shows that different lipid ratios can be used for the efficient preparation of targeted LNPs. OSE-279 t-LNPs were prepared by changing the relative ratio of the lipids constituting the LNPs (Ratio 1 and Ratio 2 as described in Table 2 (Table 9)). The transfection efficiency of targeted LNPs containing Fluc-mRNA was compared in WT and PD-1+U937 cells (Figure 4A), WT and PD-1+Jurkat cells (Figure 4B), and HPB-ALL cells (Figure 4C). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 5] This figure shows that blocking the PD-1 receptor on PD-1+ cells reduced the efficacy of the enhanced transfection by targeted LNPs. Targeted LNPs were prepared using gradually increasing amounts (1, 10, and 50 μL) of mAb OSE-279 added during the LNP preparation process. Pre-incubation experiments were performed by adding mAb OSE-279 at a concentration that allowed for complete occupancy of the cellular PD-1 receptor before adding the targeted LNPs. The transfection efficiency of LNPs containing fluc-mRNA was compared in WT and PD-1+ U937 cells (Figure 5A), WT and PD-1+ Jurkat cells (Figure 5B), and HPB-ALL cells (Figure 5C), with and without the pre-incubation step. Untargeted LNPs were used as a negative control. [Figure 6]This figure shows that targeted LNPs exhibit efficient binding to the PD-1 receptor. An ELISA binding assay was performed to characterize the binding efficiency of OSE-279 targeted LNPs to the PD-1 receptor compared to OSE-279 mAb "alone" (meaning the mAb was not complexed within the LNP). [Figure 7] This figure shows that the type of anti-PD-1 mAb used for targeted LNPs can be changed while maintaining improved transfection in PD-1-positive cells. Targeted LNPs were prepared using either pembrolizumab, nivolumab, or OSE-279 anti-PD-1 mAb. The transfection efficiency of LNPs containing fluc-mRNA was compared for pembrolizumab t-LNP, nivolumab t-LNP, and OSE-279 t-LNP (with or without anti-PD-1 pre-incubation) in WT and PD-1+ U937 cells (Figure 7A), WT and PD-1+ Jurkat cells (Figure 7B), and HPB-ALL cells (Figure 7C). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 8]This figure shows that targeted LNPs can efficiently transfect activated T cells expressing PD-1 compared to untargeted LNPs. Activated T cells (isolated from PBMCs and stimulated with the agonist CD3 / CD28 mAb) were analyzed by flow cytometry to determine the percentage of PD-1-positive cells (Figure 8A). Dashed line: Cells were stained with PE-Cy7-labeled control isotype mouse IgG (#557646 batch: 8155598, BD Biosciences). Dark gray: Cells were stained with PE-Cy7-labeled anti-human PD-1 antibody (#561272 batch: 1319137, BD Biosciences). Dead cells were stained with LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from the analysis. The transfection efficiency of OSE-279-targeted LNPs into activated T cells was compared with that of untargeted LNPs under different experimental conditions: 60,000 / 100,000 / 300,000 / 500,000 cells and 25 / 35 / 50 μL of LNPs added to each well (Figures 8B and 8C). [Figure 9]This figure shows that the receptor targeted by t-LNP can be changed by altering the mAb introduced into the t-LNP. U937 cells were transduced to express both the PD-1 receptor and the Target 2 receptor, while HPB-ALL cells spontaneously express both PD-1 and Target 2. Flow cytometry was used to measure the percentage of PD-1+ cells and cells expressing Target 2 (Figure 9A). Dashed line: Cells were stained with PE-Cy7 labeled control isotype mouse IgG (#557646 batch: 8155598, BD Biosciences). Dark gray: Cells were stained using PE-Cy7-labeled anti-human PD-1 antibody (#561272 batch: 1319137, BD Biosciences) to measure PD-1+ cells, or using PE-Cy7-labeled anti-human CD-127 antibody (#351320 batch: B251081, BioLegend) to measure cells expressing target 2. Dead cells were stained using LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from analysis. Targeted LNPs were prepared using either an anti-target 2 mAb or an anti-PD-1 mAb, and their relative transfection efficiencies were evaluated in U937 cells (Figures 9B-9C) and HPB-ALL cells (Figures 9D-9E), which express both target 2 receptors and PD-1 receptors. Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. Target 2 was CD127. [Figure 10]This figure shows that improved apoptosis was observed in PD-1 expressing cells transfected with targeted LNPs containing mRNA encoding BIM-S or PUMA proteins. mRNA encoding the apoptosis-inducing proteins BIM-S (Figures 10A, 10C, and 10E) and PUMA (Figures 10B, 10D, and 10F) was produced in-house using IVT transcription and encapsulated in targeted LNPs obtained using OSE-279 mAb. Transfection experiments were performed in PD-1 expressing cell lines: U937 (Figures 10A-10B), Jurkat (Figures 10C-10D), and HPB ALL (Figures 10E-10F). Transfection experiments were compared with and without a pre-incubation step with anti-PD-1 mAb. Cell viability was quantified 48 hours after treatment using a flow cytometer, CytoFLEX (Beckman Coulter). Non-targeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls in the targeting experiments. [Figure 11]This figure shows that LNPs targeted with an anti-CD127 antagonist mAb can target CD-127-expressing cells. U937 and Jurkat cells were transduced to express the CD-127 receptor. The percentage of CD127+ cells was measured using flow cytometry (Figure 11A). Dashed line: Cells were stained with PE-Cy7-labeled control isotype mouse IgG1 (#557646 batch: 8155598, BD Biosciences). Dark gray: Cells were stained with PE-Cy7-labeled anti-human CD-127 antibody (#351320 batch: B251081, BioLegend) to measure CD-127-positive cells. Dead cells were stained with LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from analysis. The transfection efficiency of anti-human CD-127 targeted LNPs was evaluated in U937 cells (Figure 11B) and HPB-ALL cells (Figure 11C), which express the CD-127 receptor. Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 12]This figure shows that LNPs targeted with an anti-CLEC antagonist mAb can target CLEC-1 expressing cells. U937 and THP-1 cells were transduced to express the CLEC-1 receptor. The percentage of CLEC-1 positive cells was measured using flow cytometry (Figure 12A). Dashed line: Cells were stained with 10 μg / mL purified control isotype human IgG1 (OSE Immunotherapeutics). Dark gray: Cells were stained with 10 μg / mL purified anti-human CLEC-1 antibody (OSE Immunotherapeutics) to measure CLEC-1 positive cells. PE-labeled anti-human IgG antibody (clone: ​​QA19A42 #366904 batch: B359783, BioLegend) was used to detect the purified primary antibody. Dead cells were stained using the LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from analysis. Human Fc receptors were then saturated using Human FcBlock (#564220 batch: 2122225, BD Biosciences). The transfection efficiency of anti-CLEC targeted LNPs was evaluated in U937 (Figure 12B) and THP-1 (Figure 12C), cells expressing the CLEC-1 receptor. Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 13]This figure shows that LNPs targeted with an anti-SIRPα antagonist mAb can target SIRPα-expressing cells. U937 cells were transduced to express the SIRPα receptor. The percentage of SIRPα-positive cells was measured using flow cytometry (Figure 13A). Dashed line: Cells were stained with 10 μg / mL purified control isotype human IgG4m (MOTA hIgG4m #PI08898, EVITRIA). Dark gray: Cells were stained with 10 μg / mL purified anti-human SIRPα antibody to measure SIRPα-positive cells. PE-labeled anti-human IgG antibody (clone: ​​QA19A42 #366904 batch: B359783, BioLegend) was used to detect the purified primary antibody. Dead cells were stained using the LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2622316, Life Technologies) and excluded from analysis. Human Fc receptors were then saturated using Human FcBlock (#564220, 2122225, BD Biosciences). The transfection efficiency of anti-human SIRPα-targeted LNPs was evaluated in U937 cells, which express the SIRPα receptor (Figure 13B). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 14]This figure shows that LNPs targeted with both anti-PD-1 antagonist mAbs and anti-CD-127 antagonist mAbs can target PD-1 and CD-127 expressing cells. U937 cells were transduced to express both PD-1 receptors and CD-127 receptors. The percentage of PD-1 and CD127-positive cells was measured using flow cytometry (Figure 14A). Dashed line: Cells were stained with PE-Cy7 labeled control isotype mouse IgG1 (#557646 batch: 8155598, BD Biosciences). Dark gray: Cells were stained using PE-Cy7-labeled anti-human PD-1 antibody (#561272 batch: 1319137, BD Biosciences) to measure PD-1-positive cells, or using PE-Cy7-labeled anti-human CD-127 antibody (#351320 batch: B251081, BioLegend) to measure CD-127-positive cells. Dead cells were stained using LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from analysis. The transfection efficiency of dual (anti-CD172 + OSE-279) targeted LNPs was evaluated in U937 cells (Figure 14B), which express both PD-1 receptors and CD-127 receptors. Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 15] This figure shows that different lipid ratios can be used for the efficient preparation of targeted LNPs. OSE-279 targeted LNPs were prepared by varying the relative ratio of DOPE and cholesterol that constitute the LNPs. The transfection efficiency of the targeted LNPs was compared in PD-1 positive cells: U937 cells (Figure 15A) and in HPB-ALL cells (Figure 15B). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 16]This figure shows that different types of helper lipids can be used for the efficient preparation of targeted LNPs. OSE-279 targeted LNPs were prepared by varying the type of helper lipids constituting the LNP (phospholipids DOPE and POPE or cationic lipid DDAB). The transfection efficiency of the targeted LNPs was compared in U937 PD-1 positive cells. Untargeted LNPs and targeted LNPs prepared using a control isotype mAb were used as negative controls. [Figure 17] This figure shows that different lipid lengths of PEG-lipids can be used for the efficient preparation of targeted LNPs. OSE-279 targeted LNPs were prepared by varying the length of the lipid tail of the PEG-lipid constituting the LNP (DMG-PEG2000 with a C14 length and DSG-PEG2000 with a C18 length). The transfection efficiency of the targeted LNPs was compared in PD-1 positive cells: U937 cells (Figure 17A) and Jurkat cells (Figure 17B). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 18] This figure shows that different lipid compositions can be used for the efficient preparation of targeted LNPs. OSE-279 targeted LNPs were prepared using the same lipid components as several FDA and EMA approved mRNA-based LNPs (Cominarty®, SpikeVax®, and Onpattro®). The transfection efficiency of untargeted LNPs compared to targeted LNPs was compared in PD-1 positive cells: U937 cells (Figures 18A-18D), Jurkat cells (Figures 18E-18H), and HPB-ALL (Figures 18I-18K). [Figure 19]This figure compares the transfection efficiency and binding of two types of OSE-279-targeted LNPs: LNPs containing OSE-279 mAbs grafted onto lipids, and LNPs containing OSE-279 mAbs (free OSE-279) not grafted onto lipids. Experiments were conducted to compare LNPs containing free OSE279 with LNPs obtained using a classical method for grafting mAbs onto the LNP surface (by thiol-Michael addition). First, the thiol-maleimide reaction process was optimized by testing different experimental conditions by changing the amount of DSPE-PEG-maleimide in the LNP (Figures 19A-19C) and the amount of OSE-279 mAb added to the reaction (Figures 19D-19F). Next, using optimized reaction conditions, the transfection capabilities of OSE-279-targeted LNPs prepared either via the thiol-maleimide pathway or by an alternative method were compared in Jurkat (Figure 19G) and HPB-ALL (Figure 19H) cells. Binding studies of the two types of targeted LNPs were also evaluated (Figure 19I). Figure 19J shows the prior art synthesis process for targeted LNPs, while Figure 19K presents the alternative synthesis process for targeted LNPs. [Figure 20] This figure shows that PD-1-positive cells can be targeted by targeted LNPs using different forms of OSE-279 mAbs. OSE-279 targeted LNPs were prepared using various forms (IgG vs. monovalent IgG) of the anti-PD-1 mAb OSE-279, and their transfection efficiency was evaluated in different PD-1 receptor-expressing cell lines: U937 (Figure 20A), Jurkat (Figure 20B), and HPB-ALL (Figure 20C). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 21]This figure shows that PD-1-positive cells can be targeted by targeted LNPs using different forms of OSE-279 mAbs. OSE-279 targeted LNPs were prepared using various forms (monovalent IgG form vs. ScFv-Fc form) of the anti-PD-1 mAb OSE-279, and their transfection efficiency was evaluated in different PD-1 receptor-expressing cell lines: U937 (Figure 21A), Jurkat (Figure 21B), and HPB-ALL (Figure 21C). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Modes for carrying out the invention]

[0057] Detailed description of the invention definition To facilitate understanding of this invention, certain terms are defined below. Additional definitions are provided throughout the detailed description.

[0058] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings that are generally understood by those skilled in the art to which the present invention relates.

[0059] As used herein, the term “antibody” is used in its broadest sense to describe a type of immunoglobulin molecule. In particular, an antibody includes an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site, and an immunologically active fragment of such an immunoglobulin molecule. An immunoglobulin molecule can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass of immunoglobulin molecules. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The term “antibody” specifically refers to an immunoglobulin molecule, and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing an antigen-binding domain that specifically binds to an antigen. Therefore, the term antibody encompasses the entire antibody molecule, for example, a four-chain antibody containing two heavy chains and two light chains, such as a polyclonal antibody, monoclonal antibody, or recombinant antibody, but also any antibody fragments that contain an antigen-binding domain.

[0060] The terms “antigen-binding fragment,” “antigen fragment,” or “antigen-binding domain” of an antibody, as used herein, refer to one or more fragments or derivatives of an antibody that possess the ability to specifically bind to an antigen (e.g., PD-1).

[0061] Examples of binding fragments that fall within the scope of the term "antigen-binding fragment" of an antibody include the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; the F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region; the Fd fragment, consisting of VH and CH1 domains; the Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; the dAb fragment, consisting of the VH domain (Ward et al., 1989 Nature 341: pp. 544-546); or any fusion protein containing such antigen-binding fragments. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked using recombination with a synthetic linker that enables the production of a single-chain protein (known as single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242: pp. 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85: pp. 5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be included within the scope of the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional methods known to those skilled in the art, and the fragments are screened for usefulness as they would be for intact antibodies.

[0062] As used herein, the term “isolated” means that the material described (e.g., antibodies, polypeptides, nucleic acids, etc.) has been substantially separated from other materials that coexist with it in nature, or has been concentrated compared to such other materials. In particular, an “isolated” antibody is an antibody that has been identified, isolated, and / or recovered from components of its natural environment.

[0063] As used herein, the terms “treatment,” “to treat,” or “to treat” refer to any action aimed at improving a patient’s health condition, such as treating, preventing, preventing onset, or delaying a disease. In certain embodiments, the terms refer to the improvement or elimination of a disease or symptoms associated with a disease, for example, interruption or delay of the resolution of inflammation leading to an inflammation-related disease, as per this disclosure. In other embodiments, the terms refer to minimizing the progression or worsening of a disease that results from the administration of one or more therapeutic agents to a subject having the disease.

[0064] Where used herein, “sequence identity” between two sequences is described by the parameters “sequence identity,” “sequence similarity,” or “sequence homology.” In this invention, the “identity percentage” between two sequences (A) and (B) is determined by comparing two optimally aligned sequences across a comparison region. More specifically, the identity percentage between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100). The alignment of sequences can be performed by methods well known in the art, for example, using the Needleman-Unsch algorithm for global alignment. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. Once the full alignment is obtained, the identity percentage can be obtained by dividing the total number of identical amino acid residues in the alignment by the total number of residues contained in the longest sequence between sequence (A) and sequence (B). Sequence identity is generally determined using sequence analysis software. To compare two amino acid sequences, for example, the "Emboss needle" tool for pairwise sequence alignment of proteins, provided by EMBL-EBI and available at www.ebi.ac.uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, can be used with default settings, for example: (I) Matrix: BLOSUM62, (ii) Gap start: 10, (iii) Gap extension: 0.5, (iv) Output format: Pair, (v) End gap penalty: False, (vi) End gap start: 10, (vii) End gap extension: 0.5.

[0065] The percentage of identity between two amino acid or nucleotide sequences can also be determined using the algorithm by E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988), which is incorporated into the ALIGN program (version 2.0), using a PAM120 residue weighting table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage of identity between two amino acid or nucleotide sequences can be determined using the algorithm by Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970), which is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using algorithms such as the BLASTN program for nucleic acids or amino acid sequences, for example, using a word length (W) of 11, an expected value (E) of 10, and M=5, N=4 as defaults, as well as comparing both strands.

[0066] Alternatively, sequence identity can generally be determined using the Clustal Omega sequence analysis software, with the HHalign algorithm and its default settings as its core alignment engine. This algorithm, along with its default settings, is described in Soding, J. (2005) "Protein homology detection by HMM-HMM comparison," Bioinformatics 21, pp. 951-960.

[0067] "Eu numbering" (also known as Eu indexing) refers to an antibody numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, United States Public Health Service, National Institutes of Health, Bethesda) based on the sequential numbering of the first sequenced human IgG1 (Eu antibody; Edelman et al., 1969, Proc Natl Acad Sci USA 63: pp. 78-85).

[0068] "Amino acid change" or "amino acid modification" as used herein means a change in the amino acid sequence of a polypeptide. "Amino acid modification" includes substitutions, insertions, and / or deletions in the polypeptide sequence. "Amino acid substitution" or "substitution" as used herein means the replacement of one amino acid with another at a specific position in the parent polypeptide sequence. "Amino acid insertion" or "insertion" means the addition of an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" as used herein means the removal of an amino acid at a specific position in the parent polypeptide sequence. Amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") that has similar chemical properties (e.g., charge, bulk, and / or hydrophobicity). As used herein, "amino acid position" or "amino acid position number" are used synonymously and refer to the position of a particular amino acid in an amino acid sequence, which is generally identified by a single-letter abbreviation of the amino acid. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) is considered to be at position 1.

[0069] A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") with similar chemical properties (e.g., charge, bulk, and / or hydrophobicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of a protein. Conservative substitutions and their corresponding rules are well-documented in the current state of the art. For example, a conservative substitution can be defined by substitutions within the group of amino acids shown in the table below.

[0070] [Table 1]

[0071] [Table 2]

[0072] [Table 3]

[0073] The term "and / or" as used herein should be interpreted as a specific disclosure of each of two specified features or components, with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as each of them is shown individually.

[0074] The terms "a" or "an" can refer to one or more of the elements they modify, unless the context makes it clear that either one or more of the elements are being described (for example, "a reagent" means one or more reagents).

[0075] When the term "about" is used herein in relation to any and all values ​​(including the lower and upper limits of a numerical range), it means any value that has a tolerance of deviation of up to + / -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 range of values ​​modifies each of the values ​​(i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). Furthermore, if an enumeration of values ​​(e.g., about 50%, 60%, 70%, 80%, 85%, or 86%) is described herein, the enumeration includes all of its intermediate and decimal values ​​(e.g., 54%, 85.4%).

[0076] When the term “essentially” is used herein in relation to any given biological sequence, it means that the biological sequence differs from the reference sequence included in the sequence listing by up to 10% in biological sequence length. In particular, “essentially consisting of” is intended to mean that a biological sequence consists of that sequence, but may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, additions, deletions, or mixtures thereof, preferably 1, 2, 3, 4, or 5 substitutions, additions, deletions, or mixtures thereof, provided that the biological sequence differs from the reference sequence included in the sequence listing by up to 10% in biological sequence length.

[0077] Lipid-based nanoparticles This disclosure relates to lipid-based nanoparticles comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an inhibitory protein of the activated immune cells.

[0078] The present invention relates in particular to lipid-based nanoparticles comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an inhibitory protein of the activated immune cells, wherein the inhibitory protein is i) an intracellular protein having an intracellular effect on activated immune cells, and / or ii) a transmembrane protein, and the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

[0079] The lipid-based nanoparticles according to the present invention are formulated, in particular, as either liposomes or lipid nanoparticles (LNPs), and especially as lipid nanoparticles containing a mixture of lipids.

[0080] Lipid-based nanoparticles also include similar nanoparticles, such as micelles and nanoemulsions, for example, but not limited to these. Lipid-based nanoparticles also include polymer-lipid hybrid compounds, such as polamines-polaxamers, and in particular, hybrid nanoparticles containing those described herein.

[0081] The lipid-based nanoparticles according to the present invention are preferably t-LNPs. As used herein, the term "t-LNP" refers to targeted lipid nanoparticles, i.e., lipid nanoparticles containing an antigen-binding domain. Alternatively, "nt-LNP" refers to lipid nanoparticles lacking an antigen-binding domain.

[0082] The lipid-based nanoparticles of the present invention generally contain helper lipids, sterols, and / or PEG lipid components together with the target mRNA. The elements of the LNP may be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more elements. Similarly, specific formulations of lipid-based nanoparticles may be selected for a specific application or target, for example, according to the efficacy and toxicity of a particular combination of elements.

[0083] The lipid-based nanoparticles of this disclosure are, in particular, components, compositions and methods generally known in the art, such as WO2017049245, WO2017112865, WO2017218704, WO2015164674, WO2017031232, WO2017099823, WO2016118724, WO2016118724, and WO2017223135. These can be produced using the components, compositions and methods disclosed in WO2014152211, WO2015038892, WO2017049074, WO2013090648, WO2017180917, WO2017075531 and WO2017117528, all of which are incorporated herein by reference in their entirety.

[0084] The production of LNPs in this art is described in, for example, U.S. Patent Application Publication No. 20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): pp. 172-176; Akinc et al., 2010, Mol Ther., 18(7): pp. 1357-1364; Basha et al., 2011, Mol Ther, 19(12): pp. 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): pp. 18440-18450; Lee et al., 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. This is fully explained in Engl., 51(34): pp. 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): pp. 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, and each of these references is incorporated herein by reference in its entirety.

[0085] In some embodiments, the method for obtaining lipid-based nanoparticles of the present invention is as described in the "Examples" section below, particularly in Examples 1 to 3, or as shown in Figures 19J to 19K. In particular, the method for obtaining lipid-based nanoparticles of the present invention is described in PCT / EP2024 / 058775.

[0086] In certain embodiments, the lipid-based nanoparticles comprise one or more ionizable or cationic lipids, one or more helper lipids, one or more sterols, and / or one or more polyethylene glycol (PEG)-modified lipids.

[0087] In a very specific embodiment, the lipid-based nanoparticles comprise one or more ionizable or cationic lipids (also known as component number 1), one or more helper lipids (also known as component number 2), one or more sterols (also known as component number 3), and / or one or more polyethylene glycol (PEG)-modified lipids (also known as component number 4).

[0088] In some embodiments, the lipid-based nanoparticles according to the present invention comprise one or more ionizable or cationic lipids. As used herein, the term “ionizable or cationic lipid” refers to a lipid molecule that has a positive charge in an acidic environment.

[0089] Ionizable or cationic lipids enhance the delivery and transfection efficiency of nucleic acid molecules. Their mechanism of action is based on complexing nucleic acids through electrostatic interactions. Several properties, such as charge, lipid shape, and protein corona formation, are considered important factors to consider when studying the structure-activity relationship and, consequently, when interpreting the design of new ionizable lipids.

[0090] In one embodiment, the ionizable or cationic lipid includes a head group having a positive charge or containing at least one nitrogen atom (N) that can be protonated.

[0091] In some embodiments, the ionizable or cationic lipids are [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Ch ol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradeoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2- Dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl Lenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-Dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-D MA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)- N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecenyloxy) Tradecyloxy)-1-propanaminonium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2-({8-[(3P)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane N-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), di((Z)-nona-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propane-1-amine (DLDMA), N,N-dimethyl-2,3-Bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-nona-2-en-1-yl)-9-((4-(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]piperazine-1-yl]ethyl]amino]dodecane-2-ol (Lipidoid C12-200), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide (SS-OP; e.g., CAS number 2377474-67-2), bis{2-[4-(α-D-tocopherol hemisuccinate ethyl)piperidyl]ethyl}disulfide (SS-EC), and any mixture thereof are selected from the group, preferably ALC-0315, SM-102, Dlin-MC3-DMA, or SS-OP, more preferably ALC-0315 or SS-OP.

[0092] Ionizable or cationic lipids include 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy C-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradeoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-di Railoxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3- Beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]- Dioxolane (DLin-KC2-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminonium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N -Dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2-( {8-[(3P)-Cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane(DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane(DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amonium bromide (DMORIE), di((Z)-non-2-e N-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-nona-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L 319), 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 [Lu]piperazin-1-yl]ethyl]amino]dodecane-2-ol (lipidoid C12-200), [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), and any mixture thereof may be selected from the group.

[0093] Examples of the addition of ionizable or cationic lipids include 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 These are described in U.S. Patent Application Publication No. 2015 / 199952, U.S. Patent Application Publication No. 2015 / 0239834, and WO2019 / 131839, all of which are incorporated herein by reference in their entirety.

[0094] In addition, synthetic ionizable or cationic lipids described by Dong et al. (Proc Natl Acad Sci US A. April 15, 2014; 111(15):5753, this disclosure is incorporated herein by reference) (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, K K-A12, PK-4K-E12, cWK-E12, PK500-012, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-O12, cIK-E12, cKK-E10, cKK-E14, and cKK-E16 (preferably cKK-E12, cKK-E14), and Love Synthetically ionizable or cationic lipids described by KT et al. (Proc Natl Acad Sci US A. May 25, 2010; 107(21):9915, this disclosure is incorporated herein by reference) (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) may also be considered.

[0095] In a preferred embodiment, the lipid-based nanoparticles according to the present invention include ionizable or cationic lipids, for example, ionizable or cationic lipids described in WO 2016 / 021683 or WO 2019 / 131839, which are incorporated herein by reference in their entirety.

[0096] In particular, the lipid composition of the lipid-based nanoparticles according to the present invention comprises ionizable or cationic lipids selected from the group consisting of [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 9-heptadecanyl8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), and bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide (SS-OP), as well as any mixture thereof.

[0097] In some embodiments, the ionizable or cationic lipid is selected from the group consisting of ALC-0315, SM-102, Dlin-MC3-DMA, and SS-OP.

[0098] Preferably, the ionizable or cationic lipid contained in the lipid-based nanoparticles is ALC-0315. Alternatively, the ionizable or cationic lipid contained in the lipid-based nanoparticles is SS-OP. In some embodiments, the ionizable or cationic lipids correspond to 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 nanoparticles according to the present invention.

[0099] In some embodiments, the ionizable or cationic lipids, preferably ALC-0315 or SS-OP, constitute about 45 mol% to about 55 mol% of the total lipids present in the lipid composition of the lipid-based nanoparticles according to the present invention. In particular, the ionizable or cationic lipids, preferably ALC-0315 or SS-OP, constitute about 48 mol% to about 52 mol% of the total lipids present in the lipid composition of the lipid-based nanoparticles according to the present invention.

[0100] Furthermore, in particular, ionizable or cationic lipids, preferably ALC-0315 or SS-OP, account for approximately 50 mol% of the total lipids present in the lipid composition of the lipid-based nanoparticles according to the present invention.

[0101] In some embodiments, the lipid-based nanoparticles according to the present invention include helper lipids. As used herein, the term “helper lipid” refers to a class of lipid molecules that increase the particle stability, fluidity, tolerance and / or biodistribution of lipid-based nanoparticles.

[0102] Helper lipids are also components of LNPs and play an important role in terms of stability and fusionability. Primarily, these lipids are phospholipids that form the main backbone of LNPs (e.g., DOPE, DSPC, DEPC, DSPE). Helper lipids modulate the fluidity of nanoparticles and enhance their efficacy by promoting lipid phase transitions that facilitate membrane fusion with endosomes. Generally, helper lipids can be saturated phospholipids that can increase the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayers, and stabilize their structural arrangement.

[0103] For example, helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-Oleoyl-2-Cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1-Stearoyl-2-Oleoyl-sn-glycero-3-phosphocholine (SOPC), Ethylphosphatidylcholine (EPC), 1-Oleoyl-2-Hydroxy-sn-glycero-3-phosphocholine (18:1 LysoPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-Hexadecyl-2-(9Z-Octadecenoyl)-sn-Glycero-3-Phosphoethanolamine (C16-18:1), 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Dilinoleoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Dilinolenoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Diarachidonoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-Glycero-3-Phosphoethanolamine, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (DEPE), N-(7-nitrobenzo-2-oxa-1,3-diazole-4-yl)-phosphatidylethanolamine (NBD-PE), N-(lisamineRhodamine)B-sulfonyl)-phosphatidylethanolamine (Rh-PE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 LysoPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (18:1 Monomethyl PE), 1,2-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 BiotinylPE), sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-1'-(3'-oleoyl-2'-hydroxy)-glycerol(BMP-S,S), sn-(3-(9Z-octadecenoyl)-2-hydroxy)-glycerol-1-phospho-sn-3'-(1'-(9Z-octadecenoyl)-2'-hydroxy)-glycerol(BMP-S,R), 1,2-dioleoyl-sn-glycerol -3-Phospho-rac-(1-glycerol) sodium salt (DOPG), 1,2-Diacyl-sn-glycero-3-phospho-L-serine (DSPS), L-α-phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphate (PA), 1,2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PG), 1,2-Dioleoyl-sn-glycero-3-phosphomethanol (18:1 phosphatidylmethanol), 1,2-Dioleoyl-sn-glycero-3-phosphoethanol (18:1 phosphatidylethanol), 1,The following may be selected from the group consisting of 2-dioleoyl-sn-glycero-3-phosphopropanol (18:1 phosphatidylpropanol), 1,2-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, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), dimethyldioctadecylammonium (18:0 DDAB), and any mixture thereof.

[0104] In particular, the helper lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-Oleoyl-2-Cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1-Stearoyl-2-Oleoyl-sn-glycero-3-phosphocholine (SOPC), Ethylphosphatidylcholine (EPC), 1-Oleoyl-2-Hydroxy-sn-glycero-3-phosphocholine (18:1 LysoPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0 PE), 1-Hexadecyl-2-(9Z-Octadecenoyl)-sn-Glycero-3-Phosphoethanolamine (C16-18:1), 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Dilinoleoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Dilinolenoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Diarachidonoyl-sn-Glycero-3-Phosphoethanolamine, 1,2-Didocosahexaenoyl-sn-Glycero-3-Phosphoethanolamine, 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dierydoyl-sn-glycero-3-phosphoethanolamine (DEPE), N-(7-nitrobenzo-2-oxa-1,3-diazole-4-yl)-phosphatidylethanolamine (NBD-PE), N-(lysamin rhodamine B sulfonyl)-phosphatidylethanolamine (Rh-PE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (18:1 lysoPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (18:1 monomethylPE), 1,2-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-1-phospho-sn-1'-(3'-oleoyl-2'-hydroxy)-glycerol(BMP-S,S), sn-(3-(9Z-octadecenoyl)-2-hydroxy)-glycerol-1-phospho-sn-3'-(1'-(9Z-octadecenoyl)-2'-hydroxy)-glycerol(BMP-S,R), 1,2-Dioleoyl-sn-glycerol -3-Phospho-rac-(1-glycerol) sodium salt (DOPG), 1,2-Diacyl-sn-glycero-3-phospho-L-serine (DSPS), L-α-phosphatidylserine (PS), 1,2-Dioleoyl-sn-glycero-3-phosphate (PA), 1,2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PG), 1,2-Dioleoyl-sn-glycero-3-phosphomethanol (18:1 phosphatidylmethanol), 1,2-Dioleoyl-sn-glycero-3-phosphoethanol (18:1 phosphatidylethanol), 1,2-Dioleoyl-sn-glycero-3-phosphopropanol (18:1 phosphatidylpropanol), 1,2-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, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), dimethyldioctadecylammonium (18:0 The following may be selected from the group consisting of DDAB, 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dielcoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and any mixture thereof.

[0105] 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 mixture thereof, and more preferably from the group consisting of DOPE, DOPC, DDAB, POPE, and DSPC and any mixture thereof.

[0106] In some embodiments, the helper lipid is DOPE. Alternatively, the helper lipid is DSPC.

[0107] In some embodiments, the helper lipids correspond to about 5 mol% to about 100 mol%, 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 nanoparticles of the present invention.

[0108] In some embodiments, helper lipids, preferably DOPE or DSPC, constitute about 5 mol% to about 15 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0109] In some embodiments, helper lipids, preferably DOPE or DSPC, constitute about 8 mol% to about 12 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0110] In some embodiments, helper lipids, preferably DOPE or DSPC, constitute about 10 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0111] In some embodiments, the lipids in the lipid-based nanoparticles disclosed herein include one or more molecules containing polyethylene glycol (PEG). Therefore, the lipid-based nanoparticles may contain PEG or PEG-modified lipids.

[0112] As used herein, the term "PEG lipid" may refer to polyethylene glycol (PEG)-modified lipids.

[0113] PEG lipids stabilize lipid nanoparticles, regulate nanoparticle size by limiting lipid fusion, reduce nonspecific interactions with macrophages, improve colloidal stability, and increase the half-life of nanoparticles by preventing protein corona formation. PEG lipid phospholipids located on the surface of nanoparticles enhance their hydrophilicity, avoid rapid clearance by the immune system, prevent particle aggregation, and increase stability.

[0114] As used herein, the term “PEG lipid” may refer to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates (e.g., PEG-CerCI4 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also called PEGylated lipids. In some embodiments, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. In some embodiments, PEG-modified lipids are modified forms of PEG-DMG. PEG lipids may be particularly selected from a 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 embodiments, the 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 lipids.

[0115] In some embodiments, the PEG-lipid includes, but is not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0116] In some embodiments, the PEG-lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitrail, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA) and ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid with the O PEGylated to PEG), and any mixture thereof.

[0117] In one embodiment, the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

[0118] For example, such PEGs are selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitrail, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), PEG-c-DOMG, PEG-DMG, DMG-PEG-2000, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0119] Preferably, the PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, PEG-c-DOMG, DMG-PEG-2000, DSPE-PEG-2000, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG, and PEG-c-DMA, ALC-0159 (N,N-dimyristilamide of 2-hydroxyacetic acid in which O is PEGylated), and any mixture thereof, and in particular from the group consisting of PEG-DMG, PEG-DSPE, ALC-0159, and any mixture thereof.

[0120] Preferably, the PEG is contained in the range of 2000 to 5000 daltons (i.e., PEG-2000 to PEG-5000), and is preferably DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000, or any mixture thereof.

[0121] In certain embodiments, the LNP comprises unfunctionalized PEG, which does not contain any reactive species at its terminus, and the reactive species can be used to conjugate a target portion, such as an antibody or a fragment thereof, to the PEG.

[0122] In some embodiments, the lipid portion of the PEG-lipid includes a lipid portion having a length of about C14 to about C22, preferably about C14 to about C16.

[0123] While numerous prior art studies have focused on the diverse structures and ratios of cationic and helper lipids in LNPs, the applicant tested different structures and lengths of PEG lipids (component count 4) to determine their effects on the biodistribution of LNPs in different areas of the body. Typical biodistribution of LNP particles observed to date shows a high proportion in the liver and a much lower proportion in other organs containing immune cells, such as the spleen. Due to the aforementioned hepatic uptake, the circulation of LNPs to various other tissues, particularly tumor tissue, is variable. Increased targeting of hepatocytes is described, for example, in WO2022 / 261101. However, in other clinical situations, high or excessive uptake of LNPs by capturing organs (primarily the liver, and potentially especially the spleen and lungs) is unfavorable. Therefore, there is a need to provide LNP formulations that can bypass the liver and reach other organs such as the spleen.

[0124] The inventors have observed that PEG-lipids affect the in vivo distribution of LNPs. By selecting PEG-lipids, the uptake of LNPs by capture organs (primarily the liver, and optionally the spleen and / or lungs) can be altered. For example, PEG-C14 lipids appear to promote hepatic uptake, while PEG-C18 lipids may decrease hepatic uptake and, optionally, promote splenic targeting. If necessary, the PEG-lipids in LNPs and their amounts can be selected to promote the free distribution of LNPs throughout the blood circulation in the body, particularly towards the target area of ​​a specific targeted therapeutic treatment. As an advantage that helps to illustrate this, targeted LNPs with targeting ligands such as antibodies are designed to evade organ uptake and target activated immune cells (e.g., PD1+ T lymphocytes). mRNA delivered by the LNPs and transfected into the activated immune cells makes it possible to locally and specifically enhance the antitumor activity of those immune cells.

[0125] Therefore, in a more specific embodiment, the lipids, particularly the lipid portion of PEG-lipids, include lipids having a length of about C16 to about C22 (C16, C17, C18, C19, C20, C21, or C22), preferably C16 to C20 (C16, C17, C18, C19, or C20), particularly C18.

[0126] Such PEG lipids may be selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitrail, PEG-dioleil, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), and PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), and, if necessary, from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dioleil, and PEG-distearyl. In a very specific embodiment, the PEG-lipid is PEG-disterylglycerol (PEG-DSG).

[0127] If necessary, PEG may have a molecular weight in the range of 0.5 to 50 kD, more preferably 1 to 20 kD. In some embodiments, the PEG portion, e.g., mPEG-NH2, has a size of about 1,000, 2,000, 5,000, 10,000, 15,000, or 20,000 daltons. In a very specific embodiment, PEG has a size of about 2,000. In a very specific embodiment, the PEG-lipid is PEG 2000-DMG.

[0128] In very specific embodiments, PEGs have sizes between approximately 2000 Daltons and 5000 Daltons (i.e., PEG-2000 to PEG-5000). In some examples, PEGs are selected from the group consisting of PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000.

[0129] In one embodiment, the PEG-lipid is PEG 2000-DMG. In another very specific embodiment, the PEG-lipid is PEG 2000-DSG. In yet another very specific embodiment, the PEG-lipid is ALC-0159.

[0130] In certain embodiments, the PEG has a size of approximately 2000. Alternatively, the PEG has a size of approximately 5000 Daltons.

[0131] In some cases, the PEG lipid is selected from the group consisting of DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000, or mixtures thereof.

[0132] In a very specific embodiment, the PEG-lipid is PEG 5000-DMG. In another very specific embodiment, the PEG-lipid is PEG 5000-DSG. In some embodiments, the PEG-lipid corresponds to about 1 mol% to about 100 mol%, 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 nanoparticles of the present invention.

[0133] If necessary, the PEG lipids in the lipid-based nanoparticles are in the range of about 0.5 mol% to about 2 mol% of the total lipids present in the nanoparticles or the first composition, for example, about 0.5, 0.6, 0.7, 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%, particularly 1.5 mol%.

[0134] Furthermore, the inventors surprisingly observed that the amount of PEG lipids affects the in vivo distribution of LNPs. Lower amounts of PEG lipids appear to reduce LNP uptake by capture organs, particularly the liver, but also by the spleen.

[0135] Thus, the PEG lipid in the lipid-based nanoparticles may be less than 1.5 mol% of the total lipids present in the nanoparticles, particularly, it may be less than 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6 or 0.5 mol% of the total lipids present in the nanoparticles.

[0136] In some embodiments, the PEG lipid, preferably PEG-DMG or PEG DSPE, corresponds to about 0.5 mol% to about 5 mol%, or about 0.5 mol% to about 2.5 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0137] Particularly, the PEG lipid, preferably PEG-DMG or PEG DSPE, corresponds to about 1 mol% to about 2 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0138] Preferably, the PEG lipid, preferably PEG-DMG or PEG DSPE, corresponds to about 1.5 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0139] In some embodiments, the lipid-based nanoparticles of the present invention contain one or more sterols. The sterol may be particularly selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixture thereof. Preferably, the sterol is cholesterol.

[0140] The sterol, and more particularly cholesterol, enhances the stability of the nanoparticles by filling the gaps between the lipids and assists in the fusion with the endosomal membrane during cellular uptake.

[0141] In some embodiments, sterols constitute approximately 10 mol% to approximately 100 mol%, approximately 20 mol% to approximately 100 mol%, approximately 30 mol% to approximately 100 mol%, approximately 40 mol% to approximately 100 mol%, approximately 50 mol% to approximately 100 mol%, approximately 60 mol% to approximately 100 mol%, or approximately 70 mol% to approximately 100 mol% of the total lipids present in the lipid-based nanoparticles of the present invention.

[0142] In some embodiments, sterols, preferably cholesterol, constitute about 30 mol% to about 50 mol%, or about 35 mol% to about 45 mol%, of the total lipids present in the lipid-based nanoparticles of the present invention.

[0143] In particular, sterols, preferably cholesterol, account for about 35 mol% to about 40 mol% of the total lipids present in the lipid-based nanoparticles of the present invention. Such sterols are preferably cholesterol.

[0144] In particular, sterols, preferably cholesterol, account for about 38.5 mol% of the total lipids present in the lipid-based nanoparticles of the present invention. Such sterols are preferably cholesterol.

[0145] In a preferred embodiment, the lipid-based nanoparticles of the present invention include ionizable or cationic lipids, helper lipids, sterols, and PEG lipids.

[0146] In a preferred embodiment, the lipid composition of the lipid-based nanoparticles of the present invention comprises ionizable or cationic lipids, helper lipids, sterols, and PEG lipids, wherein these lipids are preferably the lipids described above.

[0147] Preferably, in the lipid-based nanoparticles, ionizable or cationic lipids constitute about 10 mol% to about 70 mol% of the total lipids present in the nanoparticles, helper lipids constitute about 5 mol% to about 70 mol% of the total lipids present in the nanoparticles, sterols constitute about 10 mol% to about 70 mol% of the total lipids present in the nanoparticles, and PEG lipids constitute about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticles.

[0148] In particular, lipid-based nanoparticles contain or consist of approximately 35 mol% to 55 mol% cationic or ionizable lipids, approximately 5 mol% to 20 mol% helper lipids, approximately 30 mol% to 60 mol% sterols, and approximately 0.5 mol% to 4 mol% PEG-lipids.

[0149] Preferably, the lipid-based nanoparticles contain or consist of approximately 45 mol% to 55 mol% cationic or ionizable lipids, approximately 5 mol% to 15 mol% helper lipids, approximately 35 mol% to 45 mol% sterols, and approximately 0.5 mol% to 2.5 mol% PEG-lipids.

[0150] In some embodiments, the polymer may be included in and / or used to encapsulate or partially encapsulate the lipid-based nanoparticles according to the present invention. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acid), polyacid anhydrides, polyorthoesters, poly(esteramides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes, e.g., polyethylene and polypropylene, polyalkylene glycols, e.g., poly(ethylene glycol) (PEG), polyalkylene oxides (PEO ), polyalkylene terephthalates, e.g., poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters, e.g., poly(vinyl acetate), polyhalogenated vinyls, e.g., poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses, e.g., alkylcelluloses, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropylcellulose Acrylic acid polymers, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), This may include poly(dioxanone) and its copolymers and mixtures, poly(dioxanone) and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0151] In some embodiments, the lipid-based particles contain poloxamine and / or poloxamer.

[0152] In some embodiments, the lipid-based particles include polyethyleneimine, protamine (rotamine), and / or polyaspartamide.

[0153] In some embodiments, surface modifiers may be positioned within and / or on the surface of lipid-based nanoparticles of the present invention (e.g., by coating, adsorption, covalent linking, or other processes). Examples of surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, e.g., 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 trichotomum, bromhexine, carbocysteine, eprazinon, mesna, ambroxol, sobrelol, domiodol, letosteine, stepronin, thiopronin, gelzolin, thymosin b4, dorunase alpha, neltenexin, and erdosteine), and DNases (e.g., rhDNase).

[0154] In a very specific embodiment, lipid-based nanoparticles include a lipid mixture.

[0155] Preferably, the lipid mixture includes or consists of ionizable or cationic lipids, helper lipids, sterols, and PEG lipids, and these lipids are preferably the lipids described above.

[0156] In specific embodiments, the lipid-based nanoparticles include [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) as an ionizable or cationic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a neutral lipid, cholesterol as a sterol, and one or more polyethylene glycol (PEG)-modified lipids.

[0157] In a more specific embodiment, ALC-0315 constitutes approximately 35 mol% to approximately 55 mol% of the total lipids present in the LNP, DOPE constitutes approximately 5 mol% to approximately 20 mol% of the total lipids present in the LNP, cholesterol constitutes approximately 30 mol% to approximately 60 mol% of the total lipids present in the LNP, and the one or more polyethylene glycol (PEG) modified lipids constitute approximately 0.5 mol% to approximately 4 mol% of the total lipids present in the LNP. If necessary, the one or more polyethylene glycol (PEG) modified lipids constitute approximately 0.5 mol% to approximately 2 mol%, for example, approximately 0.5, 0.6, 0.7, 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%, particularly 1.5 mol%. If necessary, the one or more polyethylene glycol (PEG) modified lipids constitute approximately 0.5 mol% to approximately 1.5 mol% of the total lipids present in the LNP. If necessary, the one or more polyethylene glycol (PEG) modified lipids constitute approximately 0.5 mol% to approximately 0.6, 0.7, 0.8, 0.9, or 1.0 mol% of the total lipids present in the LNP.

[0158] In one aspect, the lipid-based nanoparticles comprise [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) as an ionizable or cationic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a neutral lipid, cholesterol as a sterol, and 1-methoxypolyethylene glycol-2,3-dimyristyl glycerol (PEG2000-DMG) having a polyethylene glycol with an average molecular weight of 2000 as a PEG-modified lipid.

[0159] In a very specific aspect, ALC-0315 is from about 35 mol% to about 55 mol% of the total lipids present in the LNP, DOPE is from about 5 mol% to about 20 mol% of the total lipids present in the LNP, cholesterol is from about 30 mol% to about 60 mol% of the total lipids present in the LNP, and PEG2000 DMG is from about 0.5 mol% to about 4 mol% of the total lipids present in the LNP.

[0160] Optionally, PEG2000-DMG is from about 0.5 mol% to about 2 mol% of the total lipids present in the LNP, such as about 0.5, 0.6, 0.7, 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%, particularly 1.5 mol%. Optionally, PEG2000-DMG is from about 0.5 mol% to about 1.5 mol% of the total lipids present in the LNP. Optionally, PEG2000-DMG is from about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9 or 1.0 mol% of the total lipids present in the LNP, preferably about 0.5 mol%.

[0161] In another very specific embodiment, the lipid-based nanoparticles include [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) as an ionizable or cationic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a neutral lipid, cholesterol as a sterol, and disterylglycerol (DSG) (PEG2000-DSG) having polyethylene glycol with an average molecular weight of 2000 as a PEG-modified lipid.

[0162] In very specific embodiments, ALC-0315 constitutes approximately 35 mol% to 55 mol% of the total lipids present in the LNP, DOPE constitutes approximately 5 mol% to 20 mol% of the total lipids present in the LNP, cholesterol constitutes approximately 30 mol% to 60 mol% of the total lipids present in the LNP, and PEG2000-DSG constitutes approximately 0.5 mol% to 4 mol% of the total lipids present in the LNP. If necessary, PEG2000-DSG may be present in approximately 0.5 mol% to 2 mol% of the total lipids present in the LNP, for example, approximately 0.5, 0.6, 0.7, 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%, particularly 1.5 mol%. If necessary, PEG2000-DSG is present in an amount of approximately 0.5 mol% to approximately 1.5 mol% of the total lipids in the LNP. If necessary, PEG2000-DSG is present in an amount of approximately 0.5 mol% to approximately 0.6, 0.7, 0.8, 0.9, or 1.0 mol%, preferably approximately 0.5 mol%, of the total lipids in the LNP.

[0163] In a very specific embodiment, the lipid composition of LNP according to the present invention includes the following: - ALC-0315 accounts for approximately 35 mol% to 55 mol% of the total lipids present in LNP. - Approximately 5 mol% to 20 mol% of the total lipids present in LNPs are DOPE. - Cholesterol, which accounts for approximately 30 mol% to 60 mol% of the total lipids present in LNP, - Approximately 0.5 mol% to approximately 4 mol% of the total lipids present in the LNP, preferably approximately 0.5 mol% to approximately 1.5 or 2 mol%, and optionally approximately 0.5 mol% to approximately 1.0 mol%, of the total lipids present in the LNP, consisting of PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG.

[0164] In some embodiments, the lipid-based nanoparticles include or consist 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, i) ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG, j) SS-OP, DOPE, cholesterol and DMG-PEG, k) SS-OP, DSPC, cholesterol and DSPE-PEG, and l) SS-OP, DOPC, cholesterol, and DMG-PEG.

[0165] In some embodiments, the lipid-based composition comprises or consists of a lipid mixture selected from the group consisting of: a) SS-OP, POPE, cholesterol, and DMG-PEG 2000; b) SS-OP, DEPC, cholesterol, and DMG-PEG 2000; c) SS-OP, DOPC, cholesterol, and DMG-PEG 2000; d) SS-OP, DOPC, cholesterol and DSG-PEG 5000 or 2000; and e) SS-OP, DSPC, cholesterol, and DSPE-PEG 2000.

[0166] Preferably, the lipid-based composition comprises a lipid mixture SS-OP, DSPC, cholesterol, and DSPE-PEG, preferably DSG-PEG 2000 or DSG-PEG 5000, or a mixture thereof.

[0167] Alternatively, the lipid-based composition may contain or consist of a lipid mixture selected from the group consisting of the following: 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) ALC-0315, DOPE, cholesterol, DMG-PEG, and DSPE-PEG;

[0168] Preferably, in such a specific embodiment, the PEG has a size of 2000 Daltons (i.e., PEG-2000).

[0169] Preferably, in such embodiments, the lipid composition of the lipid-based nanoparticles comprises or consists of about 35 mol% to about 55 mol% cationic or ionizable lipids, about 5 mol% to about 20 mol% helper lipids, about 30 mol% to about 60 mol% sterols, and about 0.5 mol% to about 4 mol% PEG-lipids.

[0170] Preferably, in such embodiments, the lipid composition of the lipid-based nanoparticles comprises or consists of about 45 mol% to about 55 mol% of cationic or ionizable lipids, about 5 mol% to about 15 mol% of helper lipids, about 35 mol% to about 45 mol% of sterols, and about 0.5 mol% to about 2.5 mol% of PEG-lipids.

[0171] In a very specific embodiment, the lipid composition of LNP according to the present invention includes the following: - Approximately 45 mol% to approximately 55 mol% of the total lipids present in the LNP, preferably approximately 48 mol% to approximately 52 mol%, more preferably approximately 50 mol%, of ALC-00315, SM-102, Dlin-MC3-DMA, or SS-OP, or any mixture thereof. - Approximately 5 mol% to approximately 15 mol%, preferably approximately 8 mol% to approximately 12 mol%, more preferably approximately 10 mol%, of the total lipids present in the LNP, DOPE, DDAB, DOPC, POPE, or DSPC, or any mixture thereof. - Cholesterol and more preferably about 37 mol% to about 40 mol%, more preferably about 38.5 mol%, of the total lipids present in the LNP. - Approximately 0.5 mol% to approximately 2.5 mol%, preferably approximately 1 mol% to approximately 2 mol%, more preferably approximately 1.5 mol%, of the total lipids present in the LNP, PEG 2000-DSG, PEG 2000-DMG, PEG 5000-DSG, PEG 5000-DMG, or ALC-0159, or any mixture thereof.

[0172] In a very specific embodiment, the lipid-based composition includes or consists of the following: - SS-OP, which is about 35 mol% to about 55 mol% of the total lipids present in the LNP, preferably about 48 mol% to about 52 mol%, more preferably about 50 mol% of the total lipids present in the LNP. - DSPC, which is about 5 mol% to about 20 mol% of the total lipids present in the LNP, preferably about 8 mol% to about 12 mol%, more preferably about 10 mol%, of the total lipids present in the LNP. - Cholesterol and more preferably about 37 mol% to about 40 mol%, more preferably about 38.5 mol%, of the total lipids present in the LNP. - PEG 2000-DSPE in an amount of approximately 0.5 mol% to approximately 2.5 mol%, preferably approximately 1 mol% to approximately 2 mol%, more preferably approximately 1.5 mol%, of the total lipids present in the LNP.

[0173] In a very specific embodiment, the lipid-based composition includes or consists of the following: - ALC-0315, which is present in the LNP at a concentration of approximately 35 mol% to approximately 55 mol%, preferably approximately 48 mol% to approximately 52 mol%, and more preferably approximately 50 mol%, of the total lipids present in the LNP. - Approximately 5 mol% to approximately 20 mol% of the total lipids present in the LNP, preferably approximately 8 mol% to approximately 12 mol%, more preferably approximately 10 mol%, of DOPE. - Cholesterol and more preferably cholesterol, making up about 30 mol% to about 60 mol% of the total lipids present in the LNP, preferably about 37 mol% to about 40 mol%, more preferably about 38.5 mol% of the total lipids present in the LNP. - Approximately 0.5 mol% to approximately 4 mol% of the total lipids present in the LNP, preferably approximately 0.5 mol% to approximately 1.5 or 2 mol% of the total lipids present in the LNP, and optionally approximately 0.5 mol% to approximately 1.5 mol% of the total lipids present in the LNP, consisting of PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG.

[0174] The lipid-based nanoparticles according to the present invention may also include one or more functionalized lipids. For example, the lipids may be functionalized with alkyne groups that can undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, the lipid bilayer may be functionalized in this way with one or more groups that are useful for facilitating membrane permeability, cell recognition, imaging, or for conjugation of antigen-binding domains to LNPs.

[0175] The LNP of the present invention may optionally include one or more coatings. For example, the LNP may be formulated as a capsule, film, or tablet having a coating. A capsule, film, or tablet containing the LNP, for example, the LNP described herein, may have any useful size, tensile strength, hardness, or density.

[0176] Lipid-based nanoparticles or compositions containing LNPs can be characterized in various ways. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to investigate the morphology and size distribution of LNPs or compositions containing LNPs. Zeta potential can be measured using dynamic light scattering or potentiometric measurements (e.g., potentiometric titration). Particle size can also be determined using dynamic light scattering. Multiple characteristics of LNPs or compositions containing LNPs, such as particle size, polydispersity index, and zeta potential, can also be measured using instruments such as the Zetasizer Nano ZS (Malvern Instruments, Malvern, Worcestershire, UK).

[0177] The physiological properties of lipid-based nanoparticles can be modified to increase their selectivity for specific bodily targets. For example, particle size can be adjusted based on the window sizes of various organs.

[0178] The average size of the lipid-based nanoparticles of the present invention may be between 10 nm and 200 nm, as measured, for example, by dynamic light scattering (DLS). For example, the average size may be between approximately 40 nm and approximately 200 nm, for example, approximately 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 200 nm. In some embodiments, the average size of LNPs is approximately 50nm to 200nm, 50nm to 190nm, 50nm to 180nm, 50nm to 170nm, 50nm to 160nm, 60nm to 200nm, 60nm to 190nm, 60nm to 180nm, 60nm to 170nm, 70nm to 200nm, 70nm to 190nm, 70nm to 180nm, 80nm to 200nm, 80nm to 190nm, or 90nm to 200nm. In some embodiments, the average size of LNPs is approximately 50 nm to 200 nm, 50 nm to 190 nm, 50 nm to 180 nm, 50 nm to 170 nm, 50 nm to 160 nm, 60 nm to 190 nm, 60 nm to 180 nm, 60 nm to 170 nm, 60 nm to 160 nm, 70 nm to 180 nm, 70 nm to 170 nm, 70 nm to 160 nm, 80 nm to 170 nm, 80 nm to 160 nm, or 90 nm to 160 nm. In certain embodiments, the average size of LNPs may be approximately 70 nm to 150 nm. In certain embodiments, the average size is approximately 120 nm. In other embodiments, the average size of lipid-based nanoparticles is approximately 150 nm.

[0179] In another embodiment, the average size of the lipid-based nanoparticles of the present invention may be between 10 nm and 400 nm, as measured, for example, by dynamic light scattering (DLS). For example, the average size may be between approximately 40 nm and approximately 350 nm, for example, approximately 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, 200 nm, 250 nm, 300 nm, or 350 nm.

[0180] The zeta potential of lipid-based nanoparticles can be used to describe the interfacial dynamic potential of a composition containing such lipid-based nanoparticles. For example, the zeta potential can describe the surface charge of lipid-based nanoparticles. Since species with higher charges may interact undesirably with cells, tissues, and other elements in the body, lipid-based nanoparticles with relatively low, positive or negative charges are generally preferred. In some embodiments, the zeta potential of lipid-based nanoparticles may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0181] In some embodiments, lipid-based nanoparticles include imaging agents, particularly those for MRI (magnetic resonance imaging), PET (positron emission tomography), SPECT (single-photon emission computed tomography), ultrasound, radiography, X-ray tomography, and optical imaging (fluorescence, bioluminescence, diffusion, etc.). These imaging agents may enable tracking of the location of lipid-based nanoparticles (and therefore, immune cell inhibitory proteins), particularly after their administration to a patient. Examples of imaging agents include paramagnetic gadolinium chelates, paramagnetic lanthanide chelates (DOTA, DO3A, DTPA, PCTA), in particular paramagnetic lanthanide chelates having a film lipophilic portion, and iron gluconate, as well as iron sulfide or iron oxide contained in the cavities of lipid-based nanoparticles, for example (or instance), magnetite (FesCU), magnethematite (y-Fe2O3), wustite (FeO), hematite (a-Fe2O3), or combinations thereof, for example, those described in WO2021194672, the disclosure of which is incorporated herein by reference, but which are not limited thereto.

[0182] The lipid-based nanoparticles of the present invention comprise i) an antigen-binding domain and ii) one or more mRNA molecules. Each of these two components is described more precisely below. Therefore, any of the embodiments described below apply to all of the lipid-based nanoparticles disclosed above.

[0183] antigen-binding domain The lipid-based nanoparticles according to the present invention include an antigen-binding domain that can specifically bind to a target expressed on the surface of activated immune cells.

[0184] The terms "specifically binds to," "specifically binds," "is specific for," or "selectively binds to" a particular target or epitope on a particular antigen mean that the antigen-binding domain recognizes and binds to the particular antigen or epitope but does not substantially recognize or bind to other molecules in the sample. For example, an antigen-binding domain that specifically (or preferentially) binds to an antigen is an antigen-binding domain that binds to the antigen with, for example, a greater affinity, binding strength, more readily, and / or for a longer duration than it binds to other / different antigens. Preferably, the terms "specifically binds to" or "specifically binds" refer to an affinity of at least about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M, or greater affinity for binding to the antigen, and / or a binding affinity for the target that is at least 2-fold higher than its affinity for non-specific antigens. Affinity can be determined by various methods well known to those skilled in the art. These methods include, but are not limited to, Biacore analysis, Blitz analysis, and Scatchard plots.

[0185] In one aspect, the antigen-binding domain contained in the lipid-based nanoparticles according to the invention has a KD value of 10 -8 M or less, preferably 10 -9 M or less, more preferably 1.10 -10 M or less, for a target expressed on activated immune cells, if determined by biosensor analysis, particularly by Biacore analysis.

[0186] As used herein, the term “target” of an antigen-binding domain means a carbohydrate, lipid, peptide, polypeptide, protein, antigen, or epitope that is specifically recognized or bound to by the antigen-binding domain according to the present invention and is expressed on the outer surface of an activated immune cell. With respect to the expression of a target on the surface of an immune cell, the term “expressed” means a target that is present on or presented on the outer surface of an immune cell, such as a carbohydrate, lipid, peptide, polypeptide, protein, antigen, or epitope.

[0187] In one embodiment, the target is specifically expressed by activated immune cells in healthy subjects or in subjects suffering from a disease, particularly, for example, an autoimmune disease or an inflammatory disease. This means that the target has a higher expression level in activated immune cells than in other cells, or that the ratio of activated immune cells expressing the target to all immune cells is higher than the ratio of other cells expressing the target to all other cells. Preferably, the expression level or ratio is 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher.

[0188] When used herein, "immune cells" include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells). Immune cells preferably refer to T cells, more specifically CD4+ T cells, CD8+ T cells, effector T cells, and / or exhausted T cells.

[0189] "Activated immune cells" refer to immune cells that are involved in an immune response to the presence of non-self cells such as pathogens or cancer cells, or to self-cells in the case of autoimmune diseases, or that have been activated during such an immune response. Activated immune cells are particularly recruited to localized sites where inflammation occurs due to the presence of non-self cells. Specific activation markers of immune cells that can be targeted by antigen-binding domains are described in detail below.

[0190] Preferably, the activated immune cells are selected from the group consisting of activated T cells, activated B cells, and activated myeloid cells, and the activated myeloid cells include activated macrophages and activated dendritic cells. Preferably, the activated immune cells are activated T cells.

[0191] In one embodiment, the targets of the antigen-binding domain are PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, and LGR6. , CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, Fas L / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, LOX1, SIGLEC6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138(SDC1), CD38 , CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CD The group is selected from CR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, CD160, CD127, and SIRPa.

[0192] In one embodiment, the targets of the antigen-binding domain are BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD8 3, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICO S, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVE The selection is made from the group consisting of M / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, and CD160.As used herein, “T cell” or “T lymphocyte” includes CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, effector T cells, effector memory stem-like T cells, tumor-infiltrating lymphocytes (TILs), anerious T cells, and exhausted T cells. In very specific embodiments, T cells are effector T cells, exhausted T cells, tumor-infiltrating lymphocytes (TILs), or effector memory stem-like T cells. “Activated T cell” or “activated T lymphocyte” is a T cell activated by simultaneously receiving signal-1 from T cell recognition of an antigen via the T cell receptor and signal-2 from a costimulatory molecule. Markers expressed by activated T cells include, but are not limited to, CD137 / 41BB / TNFRSF9, PD-1, CTLA4, FasL / TNFSF6, ITGAE, and OX40 / TNFRSF4. Exhausted T cells may be characterized by the presence of the marker TNFRSF9.

[0193] Preferably, the term "T cell" does not include regulatory T cells (Treg), suppressive T cells, and / or senescent T cells. Regulatory T cells may be characterized by the presence of the marker CD25.

[0194] When used herein, “B cells” or “B lymphocytes” include, but are not limited to, B-1 B cells, follicular B cells, and marginal zone (MZ) B cells. “Activated B cells” or “activated B lymphocytes” are B cells that are activated when they bind to an antigen via their B cell receptor. Activated B cells can, in particular, secrete immunoglobulins. Markers expressed by activated B cells include, but are not limited to, BCMA / TNFRSF17, CD150, and CD86.

[0195] Neutrophils are a type of white blood cell called granulocytes, produced from stem cells in the bone marrow. Neutrophils play a crucial role in innate immunity. Activated neutrophils are neutrophils that are activated in response to inflammatory stimuli when they invade an inflammatory or infected tissue site. Neutrophil activation is characterized by the release of granule proteins, acquisition of phagocytic activity, and production of NETs, ​​all of which are designed to enhance the cell's ability to destroy pathogens. Markers of activated neutrophils include, but are not limited to, CD11b, CD18, CD66b, CD177, and PRTN3.

[0196] Eosinophils are a type of white blood cell and a component of the immune system responsible for eliminating multicellular parasites and causing certain infections in vertebrates. Eosinophils are granulocytes that originate in the bone marrow during hematopoietic cell regeneration and then migrate into the bloodstream. Activated eosinophils are eosinophils that have been recruited from the blood to the tissues at the site of inflammation or infection and have received activation signals mediated by cytokines. When activated, eosinophils can release a wide variety of inflammatory mediators. Markers of activated eosinophils include, but are not limited to, CD69, L-selectin, ICAM-1, CD44, and PSGL-1.

[0197] Basophils are a type of polymorphonuclear leukocyte characterized by having a nucleus that is divided into two or three lobes and the presence of cytoplasmic granules. Activated basophils are activated by crosslinking of FceRI receptor-binding IgE with antigens, leading to rapid degranulation and release of their cellular contents. In addition, basophils can be activated without IgE crosslinking by inflammatory mediators, such as complement factors C5a and C3a, MBP, PAF, and chemokines. Markers of activated basophils include, but are not limited to, CD63, CD203c, and CD164.

[0198] Mast cells, also known as mast cells or labrocytes, are commensal cells of connective tissue containing many granules rich in histamine and heparin. Activated mast cells are those stimulated by allergens via cross-linking with immunoglobulin E receptors (e.g., FcεRI), by physical injury via pattern recognition receptors for damage-associated molecular patterns (DAMPs), by microbial pathogens via pattern recognition receptors for pathogen-associated molecular patterns (PAMPs), and by various compounds via their G protein-coupled or ligand-gated ion channels. Upon activation, mast cells can selectively and rapidly release mediators or inflammation-inducing components, such as histamine, heparin, cytokines, and growth factors. Markers of activated mast cells include, but are not limited to, CD63, CD203, c-Kit, IL-3Rα, and FcεRI.

[0199] A macrophage is a type of white blood cell that helps eliminate foreign substances by engulfing them 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) up to giant cells, microglia, Hofbayer cells, glomerular mesangial cells, osteoclasts, Langerhans cells, epithelioid cells, red pulp macrophages (sinusoidal parietal cells), peritoneal macrophages, and LysoMac. An activated macrophage is a macrophage activated by either IFNg-mediated priming signaling followed by encounter with appropriate stimuli such as bacterial LPS, or by direct stimulation with IL4 and / or IL13. Activated macrophages can kill non-self cells by phagocytosis. Markers of activated macrophages include, but are not limited to, TNFRSF12A / FN14 / TWEAKR.

[0200] "Dendritic cells" or "DCs" are antigen-presenting cells of the immune system. Their primary function is to process antigenic substances and present them to T cells of the immune system on their cell surface. As used herein, this term includes, for example, plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs). "Activated dendritic cells" are dendritic cells directly activated by conserved pathogen molecules, and dendritic cells indirectly activated by inflammatory mediators produced by other cell types that recognize such molecules. A marker for activated dendritic cells is, but is not limited to, TRAF4.

[0201] "Natural killer cells," "NK cells," or "large granular lymphocytes (LGLs)" are a type of cytotoxic lymphocyte that is extremely important to the innate immune system. These lymphocytes contain small granules containing proteins, such as perforin and proteases known as granzymes, in their cytoplasm. As used herein, this term includes natural killer cells, adaptive natural killer cells, memory natural killer cells, and memory-like natural killer cells. "Activated natural killer cells" are natural killer cells activated by a dominance of activating receptor stimulation over inhibitory receptor stimulation. Markers for activated natural killer cells include, but are not limited to, NKG2A and TRAF3IP1.

[0202] The antigen-binding domain of the present invention particularly targets markers of immune cell activation, such as the markers described above.

[0203] The targets expressed on activated immune cells may be selected from among the targets listed in Table D (Table 4) below.

[0204] [Table 4A]

[0205] [Table 4B]

[0206] [Table 4C]

[0207] [Table 4D]

[0208] [Table 4E]

[0209] [Table 4F]

[0210] [Table 4G]

[0211] [Table 4H]

[0212] [Table 4I]

[0213] If necessary, targets expressed on the surface of activated immune cells are further expressed specifically on the surface of activated immune cells.

[0214] "Specifically expressed" on the surface of activated immune cells means that the antigen-binding domain target is expressed on the outer surface of immune cells but is substantially not expressed by other cell types, such as tumor cells. This specifically means that the expression of the target is higher in immune cells than in other cells (i.e., non-immune cells).

[0215] Targets specifically expressed on the surface of activated immune cells include BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD30, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, I COS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVE The selection is made from the group consisting of M / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, and CD160.

[0216] For example, the targets expressed on the surface of activated B cells are selected from the group consisting of BCMA / TNFRSF17, CD150, CD86, OX40L, LOX1, TACI / TNFRSF13B, CD138(SDC1), FCRL4, CD78, FRAF3 / CD40BP, TRAP1, BAFFR / TNFRSF13C / CD268, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, and CD165.

[0217] For example, targets expressed on the surface of activated myeloid cells are selected from the group consisting of CD163, CD206, Siglec 6, TRAF1, TRAF4, TRAF7, TRAP100 / MED24, TNFRSF12A / FN14 / TWEAKR, CD301, IL4R, and CLEC-1A.

[0218] For example, the targets expressed on the surface of activated natural killer cells are selected from the group consisting of CST7, CXCR4, NKG2A, TRAF3IP1, and CMKLR1.

[0219] Preferably, the target specifically expressed on the surface of activated immune cells is not an antigen of the TCR pathway (interaction between antigen-presenting cells and T cells).

[0220] For example, targets expressed on the surface of activated T cells include CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD154, CD183, CD25, CD254, CD26, CD275, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, CD80, CD95, CTLA4, CXCR3, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, and LY108. The selection is made from the group consisting of / 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, and CD160.

[0221] Preferably, the target expressed on the surface of activated immune cells is selected from the group consisting of PD-1, CD127, SIRPa, and CLEC-1A.

[0222] In a preferred embodiment, the target is PD-1.

[0223] In certain embodiments, the immune cells are T cells, and therefore the lipid-based nanoparticles according to the present invention include antigen-binding domains that can specifically bind to targets expressed on the surface of activated T cells. Preferably, the targets expressed on the surface of activated T cells are selected from the group consisting of CD137 / 41BB / TNFRSF9, PD-1, CRTAM, CD39, CXCR5, CD70, CTLA-4, TIM-3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4, and TIGIT.

[0224] Preferably, the target specifically expressed on the surface of activated immune cells is not a pan-T cell marker, i.e., a marker expressed on multiple subtypes of T cells. In one embodiment, the pan-T antigen is CD2, CD3, CD5, or CD7. Therefore, if the lipid-based nanoparticles are intended to target immune cells such as T cells, the target of the antigen-binding domain is not CD2, CD3, CD5, and / or CD7.

[0225] In certain embodiments, the activated T cells are effector memory stem-like T cells, and the target is a factor expressed on the surface of effector memory stem-like T cells, preferably specifically expressed on the surface of effector memory stem-like T cells. Preferably, the target expressed on the surface of effector memory stem-like T cells is selected from the group consisting of CXCR5, SLAMF7, and CRTAM.

[0226] In certain embodiments, the activated T cells are tumor-infiltrating lymphocytes (TILs), and the target is a factor expressed on the surface of tumor-infiltrating lymphocytes, preferably specifically expressed on the surface of TILs. Preferably, the target expressed on the surface of TILs is selected from the group consisting of CD101, CD137 (Tnfrsf9 / 4-1BBL), CRTAM, CST7, CTLA4, CXCR3, FAS, IL18R1 / CXCR1 / CD218A, LAG-3 PTPN22, RGS1, TNFSF14, and PD1.

[0227] In certain embodiments, the activated T cells are cytotoxic T cells, and the target is a factor expressed on the surface of the cytotoxic T cells, preferably specifically expressed on the surface of the cytotoxic T cells. Preferably, the target expressed on the surface of the cytotoxic T cells is selected from the group consisting of CD25, CD38, CD69, PD1, TIM-3, LAG-3, and TIGIT.

[0228] In one embodiment, if the target is a receptor that has an inhibitory effect on cells, the antigen-binding domain has antagonist activity against the target.

[0229] The term "antagonist," as used herein, refers to a substance that blocks or reduces the activity or functionality of another substance. In particular, the term refers to a binding domain that binds to a cell receptor (e.g., PD-1) as a reference substance (e.g., PD-L1 and / or PD-L2), thereby preventing the receptor from producing all or part of its useful biological effect (e.g., creation of an immunosuppressive microenvironment). Antagonist activity can be evaluated by competitive ELISA.

[0230] In an alternative embodiment, if the target is a receptor that has an inhibitory effect on cells, the antigen-binding domain has agonist activity against the target.

[0231] As used herein, the term "agonist" refers to a substance that activates or enhances the activity or functionality of another substance. In particular, the term refers to a binding domain that binds to a cell receptor as a reference substance, thereby causing all or part of its useful biological effect (e.g., creation of an immunosuppressive microenvironment) to occur in that receptor.

[0232] In the alternative embodiment, the antigen-binding domain does not have activity against the target.

[0233] In some embodiments, the antigen-binding domain does not interfere with the binding of its target to its native ligand. In other words, the antigen-binding domain according to the present invention is neither an agonist nor an antagonist of the interaction between its target and its native ligand. The absence of such agonist and / or antagonistic ability can be assessed using methods generally known to those skilled in the art.

[0234] In a preferred embodiment, the antigen-binding domain of the lipid-based nanoparticle does not compete with the intrinsic ligand for binding to a target specifically expressed on activated immune cells. The absence of competition between the antigen-binding domain of the present invention and the intrinsic ligand of a target specifically expressed on activated immune cells can be determined when the binding of the intrinsic ligand to the target specifically expressed on activated immune cells in the presence of the antigen-binding domain of the present invention is at least 50%, more preferably at least 80%, even more preferably at least 90%, and most preferably the same as the binding of the intrinsic ligand to the target specifically expressed on activated immune cells under the same experimental conditions but without the presence of the antigen-binding domain of the present invention.

[0235] In some embodiments, the antigen-binding domain is an antibody, a fragment thereof, or a derivative thereof. Preferably, the antigen-binding domain is derived from a form selected from the group consisting of IgA, IgM, IgE, IgD, and IgG, or a variant thereof.

[0236] When used herein, the terms "derive from" and "derived from" refer to a compound having a structure derived from the structure of a parent compound or protein, whose structure is sufficiently similar to those disclosed herein, and which, based on that similarity, is expected by those skilled in the art to exhibit the same or similar properties, activities, and utility as the claimed compound.

[0237] The antigen-binding domain is preferably a monoclonal antibody, preferably a human, humanized, chimeric, or recombinant antibody, or an antigen-binding fragment thereof.

[0238] In some embodiments, the antigen-binding domain is a monoclonal antibody or an antigen-binding fragment thereof.

[0239] In some embodiments, the antigen-binding domain is a monoclonal antibody or its antigen-binding fragment. The term "monoclonal antibody," as used herein, refers to a preparation of a single-specific antibody molecule. Monoclonal antibodies exhibit single-binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to a single-binding specific antibody having variable and constant regions derived from, based on, or derived from a human germline immunoglobulin sequence or a fully synthetic sequence. The method of preparing the monoclonal antibody is irrelevant to the binding specificity. In some embodiments, the antibody of this disclosure is a monoclonal antibody.

[0240] In some embodiments, the antigen-binding domain is a recombinant antibody or its antigen-binding fragment. As used herein, the term “recombinant antibody” means an antibody produced, expressed, generated or isolated by recombinant means, for example, an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; an antibody isolated from an animal (e.g., mouse) that is transgenic due to a human immunoglobulin gene; or an antibody produced, expressed, generated or isolated by any other method in which a particular immunoglobulin gene sequence (e.g., a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0241] In some embodiments, the antigen-binding domain is a chimeric antibody or its antigen-binding fragment. As used herein, "chimeric antibody" refers to an antibody in which a variable domain sequence derived from the germline of a mammalian species such as mouse is grafted onto a constant domain sequence derived from the germline of another mammalian species such as human. Chimeric antibodies generally contain a constant domain from human and a variable domain from another mammalian species, thereby reducing the risk of reaction of such chimeric antibodies with exogenous antibodies from non-human animals when used in therapeutic procedures.

[0242] In some embodiments, the antigen-binding domain is a humanized antibody or its antigen-binding fragment. As used herein, “humanized antibody” refers to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. “Humanized form” of antibody, e.g., non-human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) that maintains the desired specificity, affinity, and efficacy of the original antibody despite the replacement of one or more residues from CDRs with residues from at least one CDR of a non-human antibody (donor antibody). Additional framework region modifications may also be added within the human framework sequence. Preferably, the humanized antibody has a T20 human-ness score greater than 80%, 85%, or 90%. The "human-likeness" of an antibody can be measured using a T20 score analyzer to quantify the human-likeness of the variable region of an antibody, as described, for example, by Gao SH, Huang K, Tu H, Adler AS, BMC Biotechnology. 2013: 13:55, or by a web-based tool for calculating the T20 score of an antibody sequence using T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com.

[0243] In another embodiment, the antigen-binding domain according to this disclosure is a modified antibody or its antigen-binding fragment. As used herein, “modified antibody” is a molecule comprising an antibody or its antigen-binding fragment, where the antibody or its functional fragment is accompanied by a functionally different molecule. The modified antibody of the present invention may be either a fusion chimeric protein or a conjugate obtained as a result of any preferred form of binding, including covalent bonding, grafting, chemical bonding, or coupling, with a chemical or biological group, or with a molecule, such as a PEG polymer or another protecting group or protecting molecule suitable for protection against protease cleavage in vivo, for improved stability and / or half-life of the antibody or functional fragment. PEGylation of an antibody or its functional fragment is a particularly interesting embodiment because it improves the delivery conditions of the active substance to the host, especially for therapeutic applications. PEGylation may be site-specific to prevent interference with the recognition site of the antibody or functional fragment and may be carried out using high molecular weight PEG. PEGylation can be achieved by free cysteine ​​residues present in the sequence of the antibody or functional fragment, or by additional free cysteine ​​residues in the amino sequence of the antibody or functional fragment. In some embodiments, the antigen-binding domain includes a hydrophobic domain. The hydrophobic domain may include or consist of one or more alpha-helix regions. The hydrophobic region is configured to interact with the hydrophobic lipids of the lipid-based nanoparticles of the present invention in particular.

[0244] In some embodiments, the antigen-binding domain includes, or is modified to include, a group that can react with a group on the lipid of the nanoparticle, such as PEG-maleimide, for conjugating the antigen-binding domain to the lipid of the lipid-based nanoparticle, such as a thiol group. Suitable reactive groups and conjugation chemistry are well known to those skilled in the art. For example, conjugation can be carried out by click chemistry, or by enzymes.

[0245] In one embodiment, the antigen-binding domain is conjugated to the lipids of the lipid-based nanoparticle by a maleimide moiety that is conjugated, bound, or linked to, for example, a PEG derivative. Preferably, the antigen-binding domain may include a thiol group that can react with PEG-maleimide to conjugate the antigen-binding domain to the lipid-based nanoparticle. The antigen-binding domain can be conjugated to the lipids of the lipid-based nanoparticle, optionally via a linker, using other methods known to those skilled in the art, for example, the method disclosed in Kedmi, Ranit, et al., "A modular platform for targeted RNAi therapeutics," Nature nanotechnology, vol. 13, 3 (2018): pp. 214-219, doi:10.1038 / s41565-017-0043-5, whose disclosure is incorporated herein by reference.

[0246] In certain embodiments, the antigen-binding domain is not covalently bound to any lipid of the LNP, nor does it include any modifications for coupling or grafting the antigen-binding domain onto a lipid. In particular, the antigen-binding domain does not include a lipophilic moiety or a graft moiety, such as a free cysteine ​​or thiol group.

[0247] In certain embodiments, the LNP does not include any antigen-specific antigen-binding domains located on an antigen-binding domain that can specifically bind to a target expressed on the surface of activated immune cells, and in particular does not include an antigen-binding domain that is directed toward the Fc domain of an antibody.

[0248] In certain embodiments, antigen-binding domains capable of specifically binding to targets expressed on the surface of activated immune cells are not bound to LNPs by "antigen-antibody" type interactions. More specifically, antigen-binding domains are not restricted to antigen-specific antigen-binding domains present on antigen-binding domains capable of specifically binding to targets expressed on the surface of activated immune cells, particularly antigen-binding domains that are directional to the Fc domain of an antibody.

[0249] Preferably, the lipid-based nanoparticles do not contain a secondary antibody (e.g., an anti-Fc antibody or its antigen-binding fragment or derivative) that enables the binding of an antigen-binding domain to the lipid-based nanoparticles.

[0250] In particular, lipid-based nanoparticles do not contain antibodies, fragments thereof, or derivatives thereof that are lipid-modified or covalently bound to lipid-modified peptides or motifs. Preferably, lipid-based nanoparticles do not contain lipid-modified secondary antibodies that enable the binding of antigen-binding domains to lipid-based nanoparticles.

[0251] In some embodiments, the lipid-based nanoparticles do not contain a portion comprising a lipidized peptide or motif.

[0252] In some embodiments, the antigen-binding domain does not contain an anchor portion containing a lipidized peptide or motif, nor is it covalently bound to such anchor portion.

[0253] As used herein, the terms “anchor moiety,” “anchor molecule,” or “anchor entity” refer to a component that anchors or binds an antigen-binding domain to a lipid-based nanoparticle. Preferably, the anchor moiety is a protein.

[0254] As used herein, the term “lipidized peptide or motif” refers to a specific sequence pattern in a protein or protein entity (e.g., an antibody or a fragment thereof) that is related to the binding or anchoring of a lipid moiety.

[0255] With respect to the present invention, entities comprising lipidized peptides, motifs, or patterns are entities (e.g., antigen-binding domains, scFvs, or antibodies) that will be anchored to or bound to lipid molecules, particularly to lipids in lipid-based nanoparticles.

[0256] Lipidized peptides or motifs may include different types of lipid modifications, such as cysteine ​​prenylation (e.g., attachment of hydrophobic isoprene polymers, such as farnesyl or geranylgeranyl, to cysteine ​​residues of proteins), N-terminal glycine myristoylation, cysteine ​​palmitoylation, serine and lysine lipoacylation (e.g., addition of lipoacyl groups to serine and lysine residues of proteins), palmitoylation, and GPI anchoring, or peptides derived from parts of endometrial bacterial lipoproteins. One common example of a lipidized motif is the CAAX box, which serves as a recognition motif for isoprenylation.

[0257] In some embodiments, the lipid-based nanoparticles do not contain bacterial anchor polypeptides, lipoproteins, such as bacterial lipoproteins, or recombinant membrane-anchored lipoproteins. Preferably, the antigen-binding domain does not contain bacterial anchor polypeptides, lipoproteins, such as bacterial lipoproteins, or recombinant membrane-anchored lipoproteins, and is not covalently bound to bacterial anchor polypeptides, lipoproteins, such as bacterial lipoproteins, or recombinant membrane-anchored lipoproteins.

[0258] Preferably, the lipid-based nanoparticles of the present invention do not contain NipA lipoprotein or any fragment thereof. Preferably, the antigen-binding domain does not contain NipA lipoprotein or any fragment thereof, and is not covalently bound to NipA lipoprotein or any fragment thereof.

[0259] Preferably, the lipid-based nanoparticles or antigen-binding domains do not contain a portion containing the amino acid sequence: CDNSSS (SEQ ID NO: 41) or CDQSSS (SEQ ID NO: 42), or a lipidized peptide or motif consisting thereof.

[0260] In some embodiments, the antigen-binding domain to be contained in the lipid-based nanoparticles according to the present invention has binding activity similar to that of the antigen-binding domain in its free form. As used herein, “antigen-binding domain in its free form” refers to an antigen-binding domain that is not linked, grafted, or conjugated to an LNP. In preferred embodiments, the antigen-binding domain to be contained in the lipid-based nanoparticles according to the present invention has binding activity equal to about 70%, more preferably about 75%, and even more preferably about 80%, of the binding activity of the antigen-binding domain in its free form.

[0261] In one other aspect, the antigen-binding domain according to this disclosure is an antigen-binding antibody mimetic. As used herein, the term “antigen-binding antibody mimetic” means an artificial protein, peptide, and any chemical substance that has the ability to bind to an antigen in a manner that mimics the ability of an antibody to bind to an antigen. Such mimics include afitins and anticarins, as well as aptamers (peptide aptamers and oligonucleotide aptamers).

[0262] Alternatively, the antigen-binding domain according to this disclosure is a targeted peptide. Preferably, the peptide is not covalently linked to a lipid or a lipid-forming motif. For example, the peptide is an RGD peptide, e.g., Qin J, Xue L, Gong N, Zhang H, Shepherd SJ, Haley RM, Swingle KL, Mitchell MJ., RGD peptide-based lipids for targeted mRNA delivery and gene editing applications. RSC Adv. 7 Sept. 2022;12(39):25397~25404, doi: 10.1039 / d2ra02771b. PMID: 36199352; PMCID: PMC9450108.

[0263] In one embodiment, the antigen-binding domain is an aptamer. Aptamers are a class of molecules that can serve as an alternative to antibodies for molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences that have the ability to recognize virtually any class of target molecules with high affinity and specificity. Such ligands can be isolated by phylogenetic evolution of ligands by exponential enrichment of a random sequence library (SELEX). A random sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer of a unique sequence that will eventually be chemically modified. The peptide aptamer consists of a structure-constrained antibody-variable region exhibited by a platform protein, such as thioredoxin A of E. coli, selected from the combinatorial library by a two-hybrid method.

[0264] The antigen-binding domain of the lipid-based nanoparticle may be of any form known in the art.

[0265] In a preferred embodiment, the antigen-binding domain is an antibody, a fragment thereof, or a derivative thereof, such as Fab, F(ab)2, Fab', F(ab')2, Fd, Fv, crossMAb, or a single-stranded variable fragment (scFV), VHH, or a single-stranded Fab fragment.

[0266] Examples of included binding fragments include the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; the F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region; the Fd fragment, consisting of VH and CH1 domains; the Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; the dAb fragment, consisting of a VH domain (Ward et al., 1989 Nature 341: pp. 544-546); or any fusion protein containing such antigen-binding fragments. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked using recombination with a synthetic linker that enables the production of a single-chain protein (known as single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242: pp. 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85: pp. 5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be included within the scope of the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional methods known to those skilled in the art, and the fragments are screened for usefulness in the same way as intact antibodies.

[0267] As used herein, the term “CrossMAb” refers to an antigen-binding domain in which the CL and CH1 domains are inverted, particularly in one binding arm of an antibody. Thus, such a binding domain includes a VH domain linked to the CL domain and a VL domain linked to the CH1 domain. Such a configuration reduces byproduct formation (compared to approaches without such CL-CH1 domain exchange) caused by a mismatch between the light chain of the first binding domain, which specifically binds to the first antigen, and the incorrect heavy chain of the second binding domain, which specifically binds to the second antigen. CrossMAb is described, for example, in WO 2009 / 080253 and Schaefer, W. et al., PNAS, 108 (2011) pp. 11187–1191, and the disclosures of these references are incorporated herein by reference.

[0268] In some examples, the antigen-binding domain is an antibody, or comprises or consists of an Fc domain, preferably an IgG Fc domain, such as those described herein, covalently linked to a Fab, Fv, Fab', scFV, or VHH.

[0269] In some cases, the antigen-binding domain is an aptamer or a short peptide sequence, such as an RGD peptide.

[0270] Aptamers are short ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) sequences that are generated in vitro to bind to a given target with high affinity and specificity.

[0271] In a preferred embodiment, the antigen-binding domain is an antibody, fragment thereof, or derivative specific to PD-1, CTLA-4, BTLA, TIGIT, CD160, LAG-3, or TIM-3, such as F(ab')2, Fab, Fab', F(ab)2, Fd, Fv, crossMAb, single-strand variable fragment (scFV), VHH, or single-strand Fab fragment. A great many antibodies against PD-1, TIM-3, CTLA-4, LAG-3, BTLA, TIGIT, and CD160 have already been described in the art.

[0272] 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 synonymously and refer to the programmed death-1 receptor, also known as CD279, and include variants and isoforms of human PD-1, as well as analogs having at least one common epitope with PD-1. PD-1 is a major regulator of immune response and peripheral immune tolerance thresholds. PD-1 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 human PD-1 is disclosed in GenBank accession number NP_005009. PD-1 has four splice variants expressed on human peripheral blood mononuclear cells (PBMCs). Therefore, the PD-1 protein includes not only full-length PD-1 but also alternative splice variants of PD-1, such as PD-1Aex2, PD-1Aex3, PD-1Aex2,3 and PD-1Aex2,3,4. Unless otherwise specified, these terms include any variant and isoforms of human PD-1 expressed spontaneously by PBMCs or by cells transfected with the PD-1 gene.

[0273] Some anti-PD-1 antibodies are already clinically approved, while others are still in clinical development. For example, anti-PD-1 antibodies include pembrolizumab (also known as keytruda lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), OSE-279 (see WO2020 / 127366), pizilizumab (CT-011), semiprimab (ribtayo), camrelizumab, AUNP12, AMP-224, and AGEN-203. 4. BGB-A317 (tislerizumab), PDR001 (spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, genolimuzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), JS001 (Si-Yang See Liu et al., J. Hematol. Oncol. 10:136 (2017), BI-754091, 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 See 2017 / 133540 (disclosures in these reference documents are incorporated herein by reference), and the monoclonal antibodies may be selected from the group consisting of 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168 (disclosures in this reference document are incorporated herein by reference).

[0274] Antibodies against TIM-3, such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, and LY3321367, are also known. In some embodiments, TFM-3 antibodies are disclosed in International Patent Application Publication Nos. WO2013006490, WO2016 / 161270, WO 2018 / 085469, or WO 2018 / 129553, WO 2011 / 155607, U.S. Patent No. 8,552,156, EP 2581113, and U.S. Patent Application Publication No. 2014 / 044728, the disclosures of these references are incorporated herein by reference.

[0275] Antibodies against CTLA-4, such as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), and MEDI5752 (AstraZeneca), are also known. Anti-CTLA-4 antibodies include WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232, WO19056281, WO19023482, WO18209701, WO18165895, WO18160536, WO18156250, WO18106862, WO18106864, WO18068182, Disclosures are also made in WO18035710, WO18025178, WO17194265, WO17106372, WO17084078, WO17087588, WO16196237, WO16130898, WO16015675, WO12120125, WO09100140 and WO07008463, and these disclosures are incorporated herein by reference.

[0276] Antibodies against LAG-3, such as BMS-986016, IMP701, or MGD012, are also known. Anti-LAG-3 antibodies are also disclosed in WO2008132601, EP2320940, and WO19152574, and the disclosures in these reference documents are incorporated herein by reference.

[0277] Antibodies against BTLA, such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mab19A7, or hu Mab4C7, are also known in the art. The antibody against BTLA, TAB004, is currently undergoing clinical trials in subjects with advanced malignant disease. Anti-BTLA antibodies are also disclosed in WO08076560, WO10106051 (e.g., BTLA8.2), WO11014438 (e.g., 4C7), WO17096017, and WO17144668 (e.g., 629.3), and the disclosures in these reference documents are incorporated herein by reference.

[0278] Antibodies against TIGIT, e.g., BMS-986207 or AB154, BMS-986207 CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536.1 CH A.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8 are also known in the art, as disclosed in WO19232484. The anti-TIGIT antibodies are WO16028656, WO16106302, WO16191643, WO17030823, WO17037707, WO17053748, WO17152088, WO18033798, WO18102536, WO18102746, WO18160704, WO18200430, and WO1820 Disclosures are also made in 4363, WO19023504, WO19062832, WO19129221, WO19129261, WO19137548, WO19152574, WO19154415, WO19168382 and WO19215728, and these disclosures are incorporated herein by reference.

[0279] In some embodiments, the target is CD127 or IL-7R, and the antigen-binding domain is specific to CD127, preferably human CD127. Preferably, the antigen-binding domain is a CD127 antagonist.

[0280] As used herein, the term “IL-7R” refers to any form of IL-7R and its variants that retain at least some of the activity of IL-7R. One exemplary human IL-7R can be found as Uniprot accession number P16871. Antagonist IL-7R antibodies include antibodies that block, weaken, suppress or reduce (to any extent, including significantly blockade) the biological activity of IL-7R, including downstream pathways mediated by IL-7R signaling, such as interaction with IL-7 and / or induction of a cellular response to IL-7.

[0281] Antibodies against CD127 or IL7-R, such as GSK2618960, RN168, AbD11590, MAB306-100, R34.34, A019D5, eBioRDR5, 40131, 1A12, M21, 47H4, HIL-7R-M21, eBioYL8, and RDR5, are also known in the art. Anti-CD127 antibodies are also disclosed in WO14102430, WO20077190, WO04000238, WO11104687, WO16059512, and WO17062748, the disclosures of these reference documents are incorporated herein by reference.

[0282] In some embodiments, the target is SIRPa, and the antigen-binding domain is specific to SIRPa, preferably human SIRPa. Preferably, the antigen-binding domain is an antagonist of SIRPa.

[0283] As used herein, the terms “signal-regulating protein alpha,” “SIRPα,” and “SIRPa” refer to the receptor transmembrane glycoprotein that is the mammalian immunoglobulin-like cell surface receptor for CD47. The term “anti-SIRP” refers to an antibody of this disclosure intended for use as a therapeutic or diagnostic agent that specifically binds to SIRPa, and more particularly to human SIRPa, to one or both of the two commonly identified variants, SIRPaV1 and SIRPaV2. For example, the amino acid sequence of human SIRPa is approximately 504 amino acids and has the Genbank accession numbers NP_001035111.1, NP_001035112.1, NP_001317657.1, or NP_542970.1.

[0284] Antibodies against SIRPa, such as OSE-172, CC-95251, BI 765063, HPA054437, maglorimab, TTI-621, TTI-622, and evolpacept (ALX148), are also known in the art. Anti-SIRPa antibodies are also disclosed in WO17178653, WO19073080, WO22254379, WO20102422, WO23202672, WO21222746, WO18008470, WO16205042, WO22121980, WO22110922, WO19226973, WO22254379, WO23020459 and WO18107058, and the disclosures in these reference documents are incorporated herein by reference.

[0285] In some embodiments, the antigen-binding domain comprises or consists of an anti-SIRPa antibody, for example, an anti-SIRPa antibody disclosed in WO19073080. In particular, the antigen-binding domain is an anti-SIRPa domain comprising or consisting of a VH domain comprising or consisting of the sequence shown in SEQ ID NO: 39, and a VL domain comprising or consisting of the sequence shown in SEQ ID NO: 40, or comprising the VH domain and the VL domain. Preferably, the antigen-binding domain further comprises or covalently ligated an Fc domain, preferably an IgG Fc domain, for example, one described herein.

[0286] In some embodiments, the antigen-binding domain includes a heavy chain containing or comprising the sequence shown in SEQ ID NO: 51, and a light chain containing or comprising the sequence shown in SEQ ID NO: 52, or is an anti-SIRPa antibody comprising the heavy chain and the light chain.

[0287] In some embodiments, the target is CLEC-1A, and the antigen-binding domain is specific to CLEC-1A, preferably human CLEC-1A. Preferably, the antigen-binding domain is an antagonist of CLEC-1A.

[0288] As used herein, the term "CLEC-1 A" refers to the C-type lectin-like receptor-1 A from a mammalian species, preferably human CLEC-1 A. The reference sequence of human CLEC-1 A corresponds to the sequence linked to accession number Q8NC01 Uniprot. As used herein, the term "CLEC-1 antagonist" has its general meaning in the art and refers to any compound, e.g., an antibody or a fragment thereof, that blocks, inhibits, or reduces the biological activity of CLEC-1. In particular, CLEC-1 antagonists inhibit the interaction of CLEC-1 with at least one of its ligands.

[0289] Antibodies against CLEC-1A, such as MAB1704, ABIN526589, AF1704, and ABIN526590, are also known in the art.

[0290] In some embodiments, the target is PD-1, and the antigen-binding domain is specific to PD-1. Preferably, the antigen-binding domain is a PD-1 antagonist. More preferably, the anti-PD-1 antibody is pembrolizumab (also known as keytruda lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), or OSE-279 (e.g., as described in WO2020 / 127366 (the disclosure of this reference document is incorporated herein by reference)).

[0291] Accordingly, in some embodiments, the present invention relates to a t-LNP comprising an anti-PD-1 antigen-binding domain that can specifically bind to PD-1 expressed on the surface of activated T cells, for example, the anti-PD-1 antigen-binding domain disclosed herein, and one or more mRNA molecules encoding an inhibitory protein of the activated T cells.

[0292] Preferably, the antigen-binding domain contained in the lipid-based nanoparticles according to the present invention is an anti-PD-1 antibody, for example, the above-mentioned anti-PD-1 antibody, or an antigen-binding fragment thereof, preferably a human, humanized, or chimeric anti-PD-1 antibody or an antigen-binding fragment thereof. In particular, the antigen-binding domain is F(ab')2, Fab, crossMAb, or scFv, which are specific to PD-1. Preferably, the antigen-binding domain further comprises or is covalently linked to an Fc domain, preferably an IgG Fc domain, for example, those described herein.

[0293] In a very specific aspect of this disclosure, the antigen-binding domain is derived from an antibody that targets PD-1 and is disclosed in WO2020 / 127366, the disclosure of which reference document is incorporated herein by reference in its entirety.

[0294] Therefore, in some embodiments, the antigen-binding domain is (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, - The heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, wherein it optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, particularly at any position other than position 3 of SEQ ID NO: 1; - The heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of Sequence ID No. 2, wherein it optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, particularly at any position other than positions 13, 14, and 16 of Sequence ID No. 2; - The heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 3, wherein it optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, particularly at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 3; - The light chain CDR1 (LCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 4, wherein it optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, particularly at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 4; - The light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 5, which may optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; and - The light chain CDR3 (LCDR3) optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 6, comprising or consisting of the amino acid sequence of SEQ ID NO: 6. This is the anti-PD-1 antigen binding domain.

[0295] In another embodiment, the anti-PD-1 antigen-binding domain comprises (i) a heavy chain variable region (VH) including CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; and (ii) a light chain variable region (VL) including CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6, or is essentially composed of the VH and VL.

[0296] In one embodiment, the anti-PD1 antibody or antigen-binding fragment according to the present invention includes framework regions, in particular heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3 and HFR4 and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3 and LFR4.

[0297] Preferably, the anti-PD-1 antigen binding domain is (i) A heavy chain variable region (VH) comprising HFR1 of SEQ ID NO: 7, HCDR1 of SEQ ID NO: 1, HFR2 of SEQ ID NO: 8, HCDR2 of SEQ ID NO: 2, HFR3 of SEQ ID NO: 9, HCDR3 of SEQ ID NO: 3, HFR4 of SEQ ID NO: 10, HFR4 of SEQ ID NO: 10, HFR4 of SEQ ID NO: 10, HFR3 of SEQ ID NO: 9, HCDR3 of SEQ ID NO: 3, HFR4 of SEQ ID NO: 10, HFR4 of SEQ ID NO: 10, HFR34 of SEQ ID NO: 10, HFR3 of SEQ ID NO: 10, HFR1 of SEQ ID NO: 10, HFR1 of SEQ ID NO: 10, HFR1 of SEQ ID NO: 10, HFR1 of SEQ ID NO: 10, HFR1 of SEQ ID NO: 10, HFR1 of SEQ ID NO: 10, HFR1 of SEQ ID NO (ii) Light chain variable region (VL) comprising LFR1 of SEQ ID NO: 11, LCDR1 of SEQ ID NO: 4, LFR2 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 5, LFR3 of SEQ ID NO: 13, LCDR3 of SEQ ID NO: 6, LFR4 of SEQ ID NO: 14, LFR4 of SEQ ID NO: 14, LFR4 of SEQ ID NO: 14, LFR34 of SEQ ID NO: 14, LFR3 of SEQ ID NO: 14, LFR4 of SEQ ID NO: 14, LFR This includes, or is essentially derived from (i) and (ii).

[0298] In one embodiment, the anti-PD-1 antigen binding domain is (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 15, which may include one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, particularly at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 15; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 16, which optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, particularly at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 16. It includes or is essentially derived from (a) and (b).

[0299] In another embodiment, the anti-PD-1 antigen binding domain is (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 15, which may optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, outside the CDR (i.e., only in the framework region); (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 16, which may optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, outside the CDR (i.e., only in the framework region). It includes or is essentially derived from (a) and (b).

[0300] Preferably, the anti-PD-1 antigen-binding domain includes (a) a heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 16, or essentially consisting of (a) and (b).

[0301] In one embodiment, the anti-PD-1 antigen-binding domain includes VH, VL, CH1, and CL domains such that the antigen-binding domain becomes Fab.

[0302] In such embodiments, the heavy chain constant domain (CH1) comprises or is essentially derived from Sequence ID No. 17, which may optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof.

[0303] Preferably, the light chain constant domain (CL) comprises or is essentially derived from SEQ ID NO: 18, which may optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof.

[0304] In one embodiment, the anti-PD-1 antigen-binding domain is Fab or Fab', Fab or F(ab')2, and includes: i) a VH domain and a CH1 domain, each having the amino acid sequences shown in SEQ ID NOs: 15 and 17, respectively, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof; and ii) a VL domain and a CL domain, each having the amino acid sequences shown in SEQ ID NOs: 16 and 18, respectively, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof.

[0305] Preferably, the antigen-binding domain is an anti-PD-1 Fab or F(ab')2 comprising i) a chain containing or consisting of a VH domain and a CH1 domain, wherein the VH and CH1 domains each have the amino acid sequences shown in SEQ ID NOs. 15 and 17, respectively, and ii) a chain containing or consisting of VL and CL domains, wherein the domains each have the amino acid sequences shown in SEQ ID NOs. 16 and 18, respectively, or comprising i) and ii).

[0306] In one embodiment, the antigen-binding domain comprises i) a chain comprising or having the amino acid sequence shown in SEQ ID NO: 19, which optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; and ii) a chain comprising or having the amino acid sequence shown in SEQ ID NO: 20, which optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; or is an anti-PD-1 Fab or F(ab')2 comprising i) and ii).

[0307] In one embodiment, the antigen-binding domain includes i) a chain comprising or consisting of a VH domain and a CL domain, wherein the domains each have the amino acid sequences shown in SEQ ID NOs: 15 and 18, wherein the domains each have the amino acid sequences shown in SEQ ID NOs: 16 and 17, wherein the domains each have the amino acid sequences shown in SEQ ID NOs: 16 and 17, wherein the domains each have the amino acid sequences shown in SEQ ID NOs: 16 and 17, wherein the domains each have the amino acid sequences shown in SEQ ID NOs: 16 and 17, wherein the anti-PD-1 CrossMAb comprises i) and ii).

[0308] Preferably, the antigen-binding domain includes i) a chain comprising or consisting of a VH domain and a CL domain, wherein the domains each have the amino acid sequences shown in SEQ ID NOs. 15 and 18, and ii) a chain comprising or consisting of a VL and CH1 domain, wherein the domains each have the amino acid sequences shown in SEQ ID NOs. 16 and 17, or an anti-PD-1 CrossMAb comprising i) and ii).

[0309] In one embodiment, the antigen-binding domain comprises i) a chain comprising or having the amino acid sequence shown in SEQ ID NO: 21, which optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; and ii) a chain comprising or having the amino acid sequence shown in SEQ ID NO: 22, which optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof; or an anti-PD-1 CrossMAb comprising i) and ii).

[0310] In some embodiments, where the antigen-binding domain is described as having one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof, such modifications are outside the CDR.

[0311] Alternatively, the antigen-binding domain is, (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 23. HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 24. HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 25. LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 26. LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 27, and LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 28. This is the anti-PD-1 antigen binding domain.

[0312] Preferably, the antigen-binding domain is (a) Heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 29; and (b) Light chain variable domain (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 30 (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 31. HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 32. HCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 33. LCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 34. LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 35, and LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 36. This is the anti-PD-1 antigen binding domain.

[0313] Preferably, the antigen-binding domain is (a) Heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 37; and (b) A light chain variable domain (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 38 It is an anti-PD-1 antigen binding domain that includes [the specified element].

[0314] In some embodiments, the antigen-binding domain is - A heavy chain containing or consisting of the sequence shown in Sequence ID No. 45, and a light chain containing or consisting of the sequence shown in Sequence ID No. 46; - A heavy chain containing or consisting of the sequence shown in Sequence ID No. 47, and a light chain containing or consisting of the sequence shown in Sequence ID No. 48; or - A heavy chain containing or consisting of the sequence shown in Sequence ID No. 49, and a light chain containing or consisting of the sequence shown in Sequence ID No. 50. It is an anti-PD-1 antibody that contains or consists of the heavy chain and light chain.

[0315] Preferably, the antigen-binding domain is an antagonist anti-PD-1 antibody comprising a heavy chain containing or consisting of the sequence shown in SEQ ID NO: 45, and a light chain containing or consisting of the sequence shown in SEQ ID NO: 46, or comprising the heavy chain and the light chain.

[0316] In certain aspects of this disclosure, the variable region of the antigen-binding domain may associate with an antibody constant region, particularly an antibody constant region from IgA, IgM, IgE, IgD, or IgG, such as IgG1, IgG2, IgG3, or IgG4, preferably IgG1, IgG2, or IgG4. Preferably, the antigen-binding domain includes an IgG Fc region, preferably IgG1, IgG2, or IgG4 Fc region. Preferably, the Fc domain includes all or part of a hinge region. The hinge region may originate from an immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region originates from human IgG1, IgG2, IgG3, or IgG4. Preferably, the hinge region originates from human or humanized IgG1, IgG2, or IgG4.

[0317] Preferably, the antigen-binding domain includes an Fc domain, preferably an IgG Fc domain, or is covalently linked to it. In particular, the Fc region is derived from human or humanized IgG1, IgG2, or IgG4. Preferably, the antigen-binding domain includes an Fc domain and a hinge region derived from human or humanized IgG1, IgG2, or IgG4.

[0318] Preferably, the antigen-binding domain includes an Fc domain, preferably an IgG Fc domain, or is covalently linked to it. In particular, the Fc region is derived from human or humanized IgG1, IgG2, or IgG4. Preferably, the antigen-binding domain includes an Fc domain and a hinge region derived from human or humanized IgG1, IgG2, or IgG4.

[0319] Preferably, the antigen-binding domain includes an Fc domain, preferably an IgG Fc domain. For example, this means that the antigen-binding domain is an antibody containing an IgG Fc domain, and / or the antigen-binding domain includes an antigen-binding fragment of an antibody (e.g., Fab or scFv) covalently linked to the IgG Fc domain.

[0320] As used herein, the terms “IgG Fc region” or “IgG Fc domain” are used to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as containing amino acid residues from position C226 or P230 of the IgG antibody to the carboxyl terminus. The numbering of residues in the Fc region is based on the Kabat EU index numbering. The constant region may be further mutated or modified by methods known in the art to alter their binding ability to Fc receptors.

[0321] Generally, the Fc domain includes two heavy chain constant domains known as the CH2 and CH3 domains. Where necessary, the Fc domain as envisioned herein also includes a hinge region.

[0322] In one embodiment, the antigen-binding domain includes a shortened Fc region or a fragment of the Fc region. In one embodiment, the Fc region includes a CH2 domain. In another embodiment, the Fc region includes CH2 and CH3 domains, or includes a hinge-CH2-CH3 domain. Alternatively, the Fc domain may include all or part of the hinge region, the CH2 domain, and / or the CH3 domain.

[0323] An antibody or an antigen-binding fragment thereof having an amino acid sequence that has at least 90% identity, for example, at least 95%, 96%, 97%, 98%, or 99% identity with any one of the amino acid sequences defined above is also part of this disclosure.

[0324] In some embodiments, the difference in amino acids is a conservative substitution, i.e., the substitution of one amino acid with another amino acid having similar chemical or physical properties (size, charge, or polarity), and such substitution generally does not adversely affect the biochemical, biophysical, and / or biological properties of the antibody or its antigen-binding fragment. In particular, the substitution does not interfere with the interaction between the antibody or its antigen-binding fragment and its target. The conservative substitution is advantageously selected from one of the following five groups: Group 1 - small aliphatic, nonpolar, or slightly polar residues (A, S, T, P, G); Group 2 - polar, charged residues and their amides (D, N, E, Q); Group 3 - polar, positively charged residues (H, R, K); Group 4 - large aliphatic, nonpolar residues (M, L, I, V, C); and Group 5 - large, aromatic residues (F, Y, W).

[0325] Those skilled in the art can determine the location of various regions / domains of an antibody by reference to the standard definitions shown in this regard, including reference numbering systems, by reference to the KABAT numbering system, or by applying the IMGT algorithm. In this regard, it should be noted that the definition of regions / domains in the sequence of the present invention may differ depending on the reference system. Therefore, the regions / domains defined in the present invention encompass sequences that show approximately + / - 10% differences in length or localization within the full-length sequence of the variable domain of the antibody.

[0326] In one embodiment, the lipid-based nanoparticles include an additional antigen-binding domain (i.e., in addition to the first antigen-binding domain) that binds to a target specifically expressed on the surface of activated immune cells. In this embodiment, the lipid-based nanoparticles include, in particular, a first antigen-binding domain that binds to a first target specifically expressed on the surface of activated immune cells, and a second antigen-binding domain that binds to a second target specifically expressed on the surface of activated immune cells.

[0327] In particular, the additional or second antigen-binding domain is i) not covalently bound to any lipids of the lipid-based nanoparticles, ii) does not involve any modifications for coupling or grafting the antigen-binding domain to lipids, and / or iii) not covalently bound to lipidized peptides or motifs, in particular those described herein.

[0328] Preferably, the additional or second antigen-binding domain is not a secondary antibody, generally a secondary antibody (e.g., an anti-Fc antibody or its antigen-binding fragment or derivative) that enables the binding of the first antigen-binding domain to lipid-based nanoparticles.

[0329] Preferably, the additional or second antigen-binding domain is not a secondary antibody containing a lipidized peptide or motif, such as those described herein.

[0330] In a preferred embodiment, the first antigen-binding domain and the additional (second) antigen-binding domain bind to a target expressed on the surface of the same activated immune cell. The target may be the same or different. For example, if the immune cell is a T cell, the first antigen-binding domain binds to, for example, PD-1, and the second antigen-binding domain binds to an antigen selected from the group consisting of CD137 / 41BB / TNFRSF9, CRTAM, CTLA4, FasL / TNFSF6, TIM-3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4SIRPg, and TIGIT. Alternatively, if the immune cell is a T cell, the first and second antigen-binding domains may bind to PD-1. Preferably, the first and second antigen-binding domains are not antibodies competing for PD-1, meaning that the first and second antigen-binding domains recognize different (non-overlapping) epitopes of PD-1.

[0331] In some embodiments, each of the first and second targets specifically expressed on the surface of activated immune cells (i.e., the targets recognized by the first and second antigen-binding domains, respectively) is CD4, CD8, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, L GR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR3 2, TIM3 / HAVCR2, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138(SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HV The group is selected from the following: EM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, and CD160.

[0332] By adding additional antigen-binding domains, the specificity of lipid-based nanoparticles to specific populations of activated immune cells is enhanced based on their type and / or localization. By enhancing the already high specificity of the lipid-based nanoparticles of the present invention, the risk of off-target or toxicity in healthy tissues and / or organs is limited or completely eliminated.

[0333] In certain embodiments, the first and second antigen-binding domains are derived from Fab, Fab', F(ab')2, Fv, single-stranded (scFv), CrossMAb, or nanobody (VHH), preferably F(ab')2, Fab, CrossMAb, or scFV, and the first and second antigen-binding domains may be the same or different in form. For example, both the first and second antigen-binding domains may be Fab or scFv. Alternatively, neither the first nor the second antigen-binding domains may be scFv. Preferably, the first and second antigen-binding domains include or are covalently linked to an Fc domain, preferably an IgG Fc domain, such as those described herein.

[0334] The first antigen-binding domain and the second antigen-binding domain may be the same or different in form. For example, both the first and second antigen-binding domains may be antibodies, Fabs, or scFvs. Alternatively, neither the first nor the second antigen-binding domain may be scFvs.

[0335] In some embodiments, the lipid-based nanoparticles include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different antigen-binding domains. Such antigen-binding domains may be two distinct antigen-binding domains that recognize the same target (e.g., pembrolizumab and nivolumab, both of which bind to PD-1), or two distinct antigen-binding domains that recognize two different targets (e.g., PD-1 and CTLA-4).

[0336] In some embodiments, additional targeting molecules may be used in the lipid-based nanoparticles of the present invention, in addition to the antigen-binding domain. For example, modified sugars or modified lipids may be used in combination with the antigen-binding domain of the present invention.

[0337] mRNA The lipid-based nanoparticles of the present invention contain mRNA molecules encoding immune cell activity inhibitory proteins.

[0338] Preferably, the lipid-based nanoparticles of the present invention comprise one or more isolated mRNA molecules.

[0339] The lipid-based nanoparticles of the present invention particularly include mRNA encoding a target polypeptide, which can be translated by immune cells to produce the target polypeptide.

[0340] In certain embodiments, lipid-based nanoparticles comprise mRNA polynucleotides or sets of mRNA polynucleotides. The techniques of mRNA polynucleotides are now well known to those skilled in the art, as shown in WO21159130, and the disclosures of that reference document are incorporated herein by reference.

[0341] The amount of mRNA molecules in lipid-based nanoparticles may depend on the size, composition, desired target and / or application, or other properties of the lipid-based nanoparticles. For example, the amount of useful mRNA in lipid-based nanoparticles may also depend on the size, sequence, and other characteristics of the mRNA. The relative amounts of mRNA molecules to other elements (e.g., lipids) in lipid-based nanoparticles can also vary. In some embodiments, the wt / wt ratio of lipid components to mRNA molecules in lipid-based nanoparticles may be about 5:1 to about 60:1, for example, 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 lipid components to mRNA molecules can range from approximately 10:1 to approximately 40:1. In certain embodiments, the wt / wt ratio is approximately 20:1. The amount of mRNA molecules in lipid-based nanoparticles can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0342] Alternatively, the amounts of lipids and mRNA may be selected to obtain a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the mRNA. Generally, a lower N:P ratio is preferred. One or more mRNAs, lipids, and their amounts may be selected to obtain an N:P ratio of about 2:1 to about 30:1, for example, 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 embodiments, the N:P ratio may be about 2:1 to about 8:1. In other embodiments, the N:P ratio may be about 5:1 to about 8:1. For example, the N:P ratio can be approximately 5.0:1, 5.5:1, 5.67:1, 6.0:1, 6.5:1, or 7.0:1. For example, the N:P ratio can be approximately 5.67:1. Preferably, the N:P ratio is between approximately 5:1 and 7:1. Preferably, the N:P ratio is approximately 6:1.

[0343] The encapsulation efficiency of mRNA molecules is described as the amount of mRNA encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. The encapsulation efficiency is preferably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of mRNA in a solution containing lipid-based nanoparticles before and after disruption of the lipid-based nanoparticles with one or more organic solvents or surfactants. The amount of free mRNA in the solution can be measured using fluorescence. For the lipid-based nanoparticles described herein, the encapsulation efficiency can be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency is at least 70%. In a particular embodiment, the internalization efficiency is at least 80%, preferably 90%.

[0344] The mRNA molecule of the present invention includes, in particular, structural elements that enable its encapsulation in lipid-based nanoparticles and / or its expression upon entering targeted immune cells.

[0345] Preferably, the mRNA molecule contains stabilizing elements, which include, but are not limited to, untranslated regions (UTRs) at their 5' end (5'-UTR) and / or untranslated regions (UTRs) at their 3' end (3'-UTR), in addition to other structural features such as a 5' cap structure or a 3' poly-A tail. Preferably, the mRNA polynucleotide includes at least one base modification and at least one 5' end cap. The mRNA envisioned herein generally includes a region of linked nucleosides encoding the polypeptide of interest (e.g., coding region), a first adjacent region located at the 5' end of the linked nucleosides region (e.g., 5'-UTR), a second adjacent region located at the 3' end of the linked nucleosides region (e.g., 3'-UTR), at least one 5' cap region, and a 3' stabilizing region.

[0346] In some embodiments, the mRNA of the present invention includes a polyA region or a Kozak sequence (for example, in the 5'-UTR). In some cases, the mRNA of the present invention includes one or more intronic nucleotide sequences that can be excised from the polynucleotide.

[0347] In some embodiments, the mRNA of the present invention comprises an adjacent region, a 5' cap structure, a chain termination nucleotide, a stem-loop, a poly(A) sequence, and / or a polyadenylation signal.

[0348] In some embodiments, the mRNA of the present invention includes an adjacency region. A 5'-UTR or 3'-UTR may be provided as the adjacency region of the mRNA of the present invention. The 5'-UTR may be homologous or non-homologous to the coding region of the mRNA. Multiple 5'-UTRs or 3'-UTRs may be included in the adjacency region, and these 5'-UTRs or 3'-UTRs may have the same sequence or different sequences. Any portion of the adjacency region that contains nothing may be codon-optimized, and any of these may independently contain one or more different structural or chemical modifications before and / or after codon optimization.

[0349] To modify one or more properties of mRNA, a 5'-UTR or 3'-UTR that is non-homologous to the mRNA coding region may be manipulated. The mRNA may then be administered to cells, tissues, or organs, and results such as protein levels, localization, and / or half-life may be measured to evaluate the beneficial effects of the non-homologous 5'-UTR and / or 3'-UTR on the mRNA. Variants of the 5'-UTR and / or 3'-UTR in which one or more nucleotides, including A, T, C, or G, are added or removed from the terminal may also be utilized. The 5'-UTR and / or 3'-UTR may also be codon-optimized or modified in any of the manner described herein.

[0350] In some embodiments, mRNA contains an internal ribosome entry site (IRES) or Kozak sequence in the 5'-UTR region. The Kozak consensus sequence (Kozak consensus or Kozak sequence) is a nucleic acid motif that functions as a protein translation initiation site. The internal ribosome entry site (IRES) is an RNA element that enables cap-dependent translation initiation.

[0351] In some embodiments, the mRNA of the present invention includes a 5' capping region or structure. The 5' cap structure of the polynucleotide is involved in nuclear export and increased polynucleotide stability, and binds to mRNA cap-binding proteins (CBPs). The association of CBPs and poly(A)-binding proteins forms a mature circular mRNA species, thereby contributing to intracellular polynucleotide stability and translational competency.

[0352] The cap further aids in the removal of the 5' proximal intron during mRNA splicing. By capping the 5' end of an endogenous polynucleotide molecule, a 5'-ppp-5'-triphosphate linkage can be created between the terminal guanosine cap residue and the 5' terminal transcription sense nucleotide of the polynucleotide. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or immediately preceding (anteterminal) transcription nucleotides at the 5' end of the polynucleotide can also be 2'-O-methylated as needed. Removal of the 5' cap by hydrolysis and cleavage of the guanylate cap structure can target polynucleotide molecules, such as mRNA molecules, for degradation.

[0353] By modifying polynucleotides, it is possible to create a cap structure that cannot be hydrolyzed, thereby preventing cap removal and increasing the half-life of the polynucleotide. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester linkage, alternative nucleotides can be used during the capping reaction. For example, a vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotide according to the manufacturer's instructions to create a phosphorothioate linkage within the 5'-ppp-5' cap.

[0354] Additional alternative guanosine nucleotides, such as α-methyl-phosphate nucleotides and seleno-phosphate nucleotides, may also be used. Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar at the 5' end and / or the nucleotide immediately preceding the 5' end of the (as described above) polynucleotides using the 2'-hydroxyl group of the sugar. Multiple distinctly different 5' cap structures can be used to generate the 5' cap of the mRNA molecule.

[0355] To generate a more reliable 5' cap structure, mRNA can also be capped post-transcriptionally using enzymes. As used herein, the phrase “more reliable” refers to a feature element that faithfully reflects or mimics, either structurally or functionally, an endogenous or wild-type feature element. In other words, a “more reliable” feature element better represents endogenous, wild-type, natural or physiological cellular function and / or structure compared to a synthetic feature element or analogue of the prior art, or surpasses the corresponding endogenous, wild-type, natural or physiological feature element in one or more respects. Non-limiting examples of more reliable 5' cap structures useful in the polynucleotides of this disclosure include, among many others, those having enhanced binding of cap-binding proteins, increased half-life, reduced sensitivity to 5'-endonucleases, and / or reduced 5' cap removal compared to synthetic 5' cap structures known in the art (or wild-type, natural or physiological 5' cap structures). For example, recombinant vaccinia virus capping enzyme or Faust virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5' terminal nucleotide of a polynucleotide and the guanosine cap nucleotide if the cap guanosine contains N7-methylation and the 5' terminal nucleotide of the polynucleotide contains 2'-O-methylation. Such a structure is called a Capl structure. This cap results in higher translational capacity, cell stability, and reduced activation of pro-inflammatory cytokines compared to, for example, other 5' cap analog structures known in the art. Other 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).

[0356] Because mRNA can be capped after transcription, and this process is more efficient, nearly 100% of mRNA can be capped. This is significantly different from the 80% that occurs when a capping analog is ligated to a polynucleotide during the in vitro transcription reaction. The 5' end cap may contain an endogenous cap or a capping analog. The 5' end cap may contain a guanosine analog. Useful guanosine analogs include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, polynucleotides contain a modified 5' cap. Modifications to the 5' cap may increase the stability of the polynucleotide, increase its half-life, and improve the efficiency of polynucleotide translation. Modified 5' caps may include, but are not limited to, one or more of the following modifications: modification at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP); substitution of a sugar ring oxygen (which creates a carbocyclic ring) by a methylene moiety (CH2); modification of the triphosphate crosslinking portion of the cap structure; or modification of the nucleic acid base (G) portion.

[0357] 5' capping of polynucleotides can be completed during in vitro transcription reactions using the following chemical RNA capping analogs according to the manufacturer's protocol to generate a 5'-guanosine capped structure: 3'-O-Me-m7G(5')ppp(5')G[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 can be completed after transcription using vaccinia virus capping enzyme or Faust virus capping enzyme (New England BioLabs, Ipswich, MA) to generate a "capped 0" structure: m7G(5')ppp(5')G. Cap 1 can be generated using both vaccinia virus capping enzyme or Faust virus capping enzyme and 2'-O-methyltransferase to produce m7G(5')ppp(5')G-2'-O-methyl. Cap 2 can be generated from Cap 1, and then 2'-O-methylation of the third nucleotide from the 5' end can be performed using 2'-O-methyltransferase. Cap 3 can be generated from Cap 2, and then 2'-O-methylation of the fourth nucleotide from the 5' end can be performed using 2'-O-methyltransferase. The enzymes may be derived from recombinant sources. Alternatively, other caps, such as the CleanCap structure (Trilink), can be used. Cleancap is a trinucleotide in which 5'-m7G is linked to the AG sequence by a 5'-5' triphosphate linkage.

[0358] The mRNA of the present invention particularly includes a 5' cap analog. In this specification, synthetic cap analogs, chemical caps, chemical cap analogs, and structural or functional cap analogs are also referred to. Cap analogs retain cap function but differ from natural (i.e., endogenous, wild-type, or physiological) 5' caps in terms of their chemical structure. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to polynucleotides. For example, an anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, one of which contains both an N7-methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7G-3'mppp-G, which may be equivalently referred to as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanosine is ligated to the 5' terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7-methylated and 3'-O-methylated guanosine becomes the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0359] The 5' cap of mRNA may also be a dinucleotide cap analog. In non-limiting examples, the dinucleotide cap analog may be modified by a boranophosphate group or a phosphoroselenoate group at a different phosphate position, for example, the dinucleotide cap analog described in U.S. Patent No. 8,519,110, the cap structure of which is incorporated herein by reference. Alternatively, the cap analog may be an N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5)ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5)ppp(5')G cap analogs (see, for example, Kore et al., Bioorganic & Medicinal Chemistry 20132: pp. 4570-4574, where the cap structures are incorporated herein by reference). In other examples, useful cap analogs in the polynucleotides of this disclosure are 4-chloro / bromophenoxyethyl analogs.

[0360] In some embodiments, the mRNA of the present invention includes a stem-loop, for example, a histone stem-loop, but is not limited to the histone stem-loop. The histone stem-loop may be before and / or after a polyA region. The mRNA, including the histone stem-loop and polyA region sequence, may include the strand termination nucleotides described herein. In other examples, the mRNA includes a histone stem-loop and a 5' cap structure, for example, those described herein and / or known in the art. In some cases, the conserved stem-loop region may include a miR sequence. In a non-limiting example, the stem-loop region may include a seed sequence for a miR sequence. For example, the stem-loop region may include the miR-122 seed sequence.

[0361] Preferably, the stem-loop is a nucleotide sequence approximately 25 or 26 nucleotides in length. The histone stem-loop may be located 3' to the coding region (e.g., at the 3' end of the coding region). In non-limiting examples, the stem-loop may be located at the 3' end of the mRNA described herein. In some cases, the mRNA may contain more than one stem-loop (e.g., two stem-loops). The stem-loop may be located at the second terminal region of the polynucleotide. In non-limiting examples, the stem-loop may be located within the untranslated region (e.g., the 3'-UTR) of the second terminal region. In some cases, mRNA containing a histone stem-loop can be stabilized by the addition of a 3'-stabilizing region (e.g., a 3'-stabilizing region containing at least one strand termination nucleoside). Although we do not wish to be constrained by theory, the addition of at least one strand termination nucleoside can slow the degradation of the polynucleotide and, consequently, increase its half-life.

[0362] In other cases, mRNA containing histone stem loops is stabilized by modifications to the 3' region of the polynucleotide, which can prevent and / or inhibit the addition of oligo(U).

[0363] In other cases, mRNA containing a histone stem loop is stabilized by the addition of oligonucleotides terminated with 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3'-O-methylnucleosides, 3-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0364] The mRNA, in particular, includes at least one histone stem-loop and a poly-A region or polyadenylation signal.

[0365] In some embodiments, the mRNA contained in the lipid-based nanoparticles of the present invention includes a poly(A) sequence and / or a polyadenylation signal. The poly(A) sequence may consist entirely or largely of adenine nucleotides or their analogs or derivatives. The poly(A) sequence may be a tail located adjacent to the 3'-untranslated region of the nucleic acid. During RNA processing, a long chain of adenosine nucleotides (poly(A) region) is typically added to the messenger RNA (mRNA) molecule to increase its stability. Immediately after transcription, the 3' end of the transcript is cleaved, releasing a 3'-hydroxyl group. Poly(A) polymerase then adds the adenosine nucleotide chain to the RNA. This process, called polyadenylation, adds a poly(A) region that is between 100 and 250 residues in length. Specific poly(A) region lengths can bring certain advantages to the mRNA of the present disclosure. Generally, the length of the poly(A) region of the present disclosure is at least 30 nucleotides. In another embodiment, the poly(A) region is at least 35 nucleotides. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 70 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides.In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1700 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 1900 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In some examples, the polyA region may be 80, 120, or 160 nucleotides long on the mRNA molecule described herein. In other examples, the polyA region is a polyA region of 20, 40, 80, 100, 120, 140, or 160 nucleotides long on the mRNA molecule described herein. In some cases, the polyA region is designed relative to the total mRNA length. This design may be based on the length of the coding region of the mRNA, the length of a specific feature element or region, or the length of the final product expressed from the mRNA. Compared to any feature element of the mRNA (e.g., the mRNA portion containing the polyA region), the polyA region may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the additional feature element. The polyA region may also be designed as part of the mRNA to which it belongs.In this regard, the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the structure, or the total length of the structure minus the poly-A region.

[0366] In some examples, mRNA contains a poly-AG quadruplex. The G quadruplex can be a cyclic hydrogen-bonded array of 4 guanosine nucleotides, which can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G quadruplex is incorporated at the end of the poly-A region. The resulting mRNA can be assayed at various time points for other parameters, including stability, protein production, and half-life. The poly-AG quadruplex has been shown to result in protein production equivalent to at least 75% of the protein production seen when using a 120-nucleotide poly-A region alone. mRNA with a poly-AG quadruplex may further contain a 5' cap structure. In some cases, the 3' stabilizing region, which can be used to stabilize mRNA, includes either the poly-A region or the poly-AG quadruplex. In other cases, the 3' stabilizing regions that may be used in this disclosure include strand termination nucleosides, e.g., 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, e.g., 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleosides, or O-methylnucleosides. In other cases, mRNA containing a polyA region or polyAG quadruplex may be stabilized by modification of the 3' region of the polynucleotide, which can prevent and / or inhibit the addition of oligo(U). In further examples, mRNA containing a poly-A region or poly-AG quadruplex can be stabilized by the addition of oligonucleotides terminated with 3'-deoxynucleosides, 2',3'-dideoxynucleosides, 3-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0367] In certain cases, engineered binding sites and / or conjugations of mRNA to poly(A)-binding proteins are used to enhance expression. The engineered binding site can be a sensor sequence that can act as a binding site for a ligand in the mRNA's local microenvironment. As a non-limiting example, mRNA may contain at least one engineered binding site to modify the binding affinity of poly(A)-binding proteins (PABPs) and their analogues. In addition, by using alternative nucleotides at the 3' end of the poly(A) region, multiple distinctly different mRNA molecules can be ligated together to PABP (poly(A)-binding proteins) via the 3' end. While we do not wish to be constrained by theory, the poly(A) region may be useful in protein synthesis because it recruits PABPs, which in turn can interact with the translation initiation complex.

[0368] In some embodiments, the mRNA molecule of the present invention may contain one or more naturally occurring components, including any of the standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). Any one of the nucleic acid regions may contain one or more alternative components (e.g., alternative nucleosides) that confer useful properties, including increased stability of cells into which the polynucleotide is introduced and / or the absence of substantial induction of an innate immune response. For example, modRNA may exhibit reduced degradation in cells into which it is introduced compared with the corresponding unmodified mRNA. These alternative species may also have reduced immunogenicity, as well as improved protein production efficiency, intracellular retention of polynucleotides, and / or viability of contacted cells. Non-limiting examples of such naturally occurring modified nucleotides and nucleosides can be found, among others, in published patent applications WO2013052523;WO2014093924;WO2015051173;WO2015051169;WO2015089511;WO2015196130;WO2015196118;WO2015196128;or WO2017153936, all of which are incorporated herein by reference.

[0369] Different sugar modifications and / or internucleoside linkages (e.g., skeletal structures) can be located at various positions within the polynucleotide. Those skilled in the art will understand that the nucleotide analog or other modification may be located at any position within the polynucleotide such that the function of the polynucleotide is not substantially impaired. The modification may also be a 5'-terminus modification or a 3'-terminus modification. In some embodiments, the polynucleotide includes a modification at the 3' end. mRNA is composed of approximately 1% to 100% of alternative nucleotides (with respect to total nucleotide content, or with respect to one or more types of nucleotides, i.e., A, G, U, or C) or any intervening percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 10% It may contain 90%, 10%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80%, 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~95%, 80%~100%, 90%~95%, 90%~100%, and 95%~100%. It will be understood that the remaining percentage is occupied by the presence of standard nucleotides (e.g., A, G, U, or C).

[0370] mRNA may contain at least zero and at most 100% of alternative nucleotides, or any intervening percentage, for example, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, or at least 90% of alternative nucleotides. For example, a polynucleotide may contain alternative pyrimidines, such as alternative uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the polynucleotide is replaced with alternative uracil (e.g., 5-substituted uracil). The alternative uracil may be replaced by a single compound having a unique structure, or by multiple compounds having different structures (e.g., two, three, four or more unique structures). In some cases, at least 5%, 10%, 25%, 50%, 80%, 90%, or 100% of the cytosines in a polynucleotide are replaced with alternative cytosines (e.g., 5-substituted cytosines). These alternative cytosines may be replaced by a single compound having a unique structure, or by multiple compounds having different structures (e.g., two, three, four or more unique structures).

[0371] In some embodiments, the nucleic acid base is a uracil substitute. Exemplary nucleic acid bases and nucleosides having a uracil substitute include pseudouridine (ψ), pyridine-4-onyribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio-uracil (s4U), 4-thio-psoidouridine, 2-thio-psoidouridine, 5-hydroxyuracil (ho5U), 5-aminoallyl-uracil, and 5-halo-uracil (e.g., 5-iodouracil or 5-bromouracil). ), 3-methyl-uracil (mU), 5-methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-psoidouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonyl Methyl-2-thiouracil (mcm5s2U), 5-aminomethyl-2-thiouracil (nmVu), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnmVu), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyluracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnmVu) 5-propynyl-uracil, 1-propynyl-psoidouracil, 5-taurinomethyl-uracil (xm5U), 1-taurinomethyl-psoidouridine, 5-taurinomethyl-2-thio-uracil (xm5s2U), 1-taurinomethyl-4-thio-psoidouridine, 5-methyl-uracil (m5U, i.e., having the nucleic acid base deoxythymine), 1-methyl-psoidouridine (m1ψ), 5-methyl-2-thio-uracil (m5s2U), l-methyl-4-thio-psoidouridine (m(xψ), 4-thio-1-methyl-psoidouridine, 3-methyl-psoidouridine (m3ψ), 2-thio-1-methyl-psoidouridine, 1-methyl-1-deaza-psoidouridine, 2-thiol-methyl-1-deaza-psoidouridine, dihydrouracil (D), dihydropsoidouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropsoidouridine, 2-methoxyuracil, 2-methoxy-4-thiouracil, 4-methoxy-psoidouridine, 4-methoxy-2-thiopsoidouridine, N1-methyl-psoidouridine, 3-(3-amino-3-carboxypropyl)uracil (acp U), 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine (acp ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thiouracil (inm5s2U), 5,2'-O-dimethyluridine (m5Um), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mem Examples include 5-(Urn), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (mUrn), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyluracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propenylamino)]uracil.

[0372] In some embodiments, the nucleic acid base is a cytosine substitute. Exemplary nucleic acid bases and nucleosides having a cytosine substitute include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodocytosine), and 5 -Hydroxymethylcytosine (hm5C), 1-methylpsoidisocytidine, pyrrolocytosine, pyrrolocypsoidisocytidine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thiopsoidisocytidine, 4-thio-1-methyl-1-psoidisocytidine, 4-thio-1-methyl-1-deazapse-psoidisocytidine, 1-methyl-1-deazapse Idoisocytidine, Zebralin, 5-Aza-Zebralin, 5-Ethyl-1-Zebralin, 5-Aza-2-Thio-Zebralin, 2-Thio-Zebralin, 2-Methoxycytosine, 2-Methoxy-5-Methylcytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethylcytidine (m5Cm), N4 Examples include acetyl-2'-O-methylcytidine (ac4Cm), N4,2'-O-dimethylcytidine (m4Cm), 5-formyl-2'-O-methylcytidine (f5Cm), N4,N4,2'-O-trimethylcytidine (m42Cm), 1-thiocytosine, 5-hydroxycytosine, 5-(3-azidopropyl)cytosine, and 5-(2-azidoethyl)cytosine.

[0373] In some embodiments, the nucleic acid base is an alternative adenine. Exemplary nucleic acid bases and nucleosides having an alternative adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, and 7-deaza-8-aza-2-amino -purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-1-adenine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl) Denine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-H Examples include droxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (ml Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0374] In some embodiments, the nucleic acid base is a guanine substitute. Exemplary nucleic acid bases and nucleosides having a guanine substitute include inosine (I), 1-methyl-inosine (mil), waiosine (imG), methylwaiosine (mimG), 4-demethylwaiosine (imG-14), isowyosine (imG2), waibutosine (yW), peroxywaibutosine (o2yW), hydroxywaibutosine (OHyW), intermediate hydroxywaibutosine (OHyW*), 7-deaza-guanine, and quosine (Q). Epoxy cuosin (oQ), galactosyl cuosin (galQ), mannosyl cuosin (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQI), alkaeosin (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl Thiol-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (mIG), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-guanine Examples include O-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (mIGm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl-guanine. Nucleic acid bases that substitute for nucleotides can independently be purines, pyrimidines, or purine or pyrimidine analogs. For example, nucleotide bases can be substitutes for adenine, cytosine, guanine, uracil, or hypoxanthine.In another embodiment, nucleic acid bases may include, for example, naturally occurring and synthetic derivatives of bases, which include pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, This includes 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halos, especially 5-bromos, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazine-2-ones, 1,2,4-triazines, pyridazines; or 1,3,5-triazines. When nucleotides are described using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives, for example, A includes adenine or an adenine analog, such as 7-deazaadenine.

[0375] If necessary, the mRNA should be circular RNA, in particular circular mRNA, as described in WO2014 / 186334 and WO2022 / 261490.

[0376] Immune cell inhibitory proteins The mRNA molecules described above encode immune cell inhibitory proteins. Therefore, the lipid-based nanoparticles of the present invention contain one or more different mRNA molecules encoding immune cell inhibitory proteins.

[0377] The term "immune cell inhibitory protein" refers to proteins that reduce, suppress, or weaken the activity of immune cells, particularly in a given area. These proteins are generally involved in the modulation of the immune response to prevent excessive inflammation or inappropriate immune reactions. Such proteins can generally act by inhibiting the proliferation, activation, or function of various types of immune cells, such as T cells, B cells, natural killer cells, or antigen-presenting cells. It is well known that immune cell inhibitory proteins are important for maintaining immune homeostasis and preventing autoimmunity.

[0378] In particular, lipid-based nanoparticles may comprise mRNA molecules 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each encoding a different immune cell inhibitory protein, respectively. In certain embodiments, lipid-based nanoparticles may comprise mRNA molecules encoding two different immune cell inhibitory proteins, such as those disclosed herein. The immune cell inhibitory proteins may be selected from a single cell type or from different cell types. For example, the mRNA molecules may encode different T cell inhibitory proteins. Alternatively, the mRNA molecules may encode different immune cell inhibitory proteins, such as a T cell inhibitory protein and another immune cell inhibitory protein, such as a natural killer inhibitory protein.

[0379] The immune cell inhibitory proteins encoded by mRNA molecules contained in the lipid-based nanoparticles of the present invention can be selected, in particular, based on the type of immune cell targeted by the antigen-binding domain contained in the lipid-based nanoparticles. Specifically, if the targeted immune cell is an activated T cell (i.e., the lipid-based nanoparticle contains an antigen-binding domain that binds to a target expressed on activated T cells, such as PD-1), the mRNA encodes an activated T cell inhibitory protein.

[0380] In addition, the immunosuppressant proteins encoded by mRNA molecules contained in the lipid-based nanoparticles of the present invention may be selected based on the desired effect. For example, immunosuppressant proteins may be selected for specific indications, conditions, diseases, or disorders.

[0381] In particular, immune cell inhibitory proteins exhibit effects on activated immune cells, selected from the following group: - Inducing or increasing the exhaustion of immune cells - Inhibiting or reducing the proliferation or regeneration of immune cells. - Inducing or increasing apoptosis or cell death of immune cells. - To increase the production or concentration of mitochondrial enzymes and / or transporters in immune cells. - To increase the production or concentration of transcription factors in immune cells. - Inducing or increasing a hypoxic state in the microenvironment or tissues. - To increase the production or concentration of metabolic enzymes in immune cells. - Inducing or increasing the production of anti-inflammatory signaling molecules in immune cells. - Inhibiting or reducing the production of cytotoxic compounds by immune cells. - Inducing or increasing the internalization and exocytosis of immune cells. - To increase the production or concentration of chaperone proteins in immune cells. - To induce or increase the production of cytoskeletal regulatory proteins in immune cells. - Inhibiting or reducing the degradation of proteins by immunoproteasomes and ubiquitination, as well as antigen presentation by immune cells. - To induce or increase the migration and / or mobility of immune cells. - To induce or increase the reduction of inflammation in tissues. - Inducing or increasing active membrane transport of immune cells - To increase the production or concentration of carrier proteins in immune cells. - Modifying the epigenetics of immune cells - To increase the production or concentration of tRNA in immune cells. - Inducing or increasing the production and / or secretion of checkpoint inhibitors. - Inducing a phenotypic switch in immune cells from pro-inflammatory immune cell type to anti-inflammatory immune cell type. - Inhibiting immune cell exhaustion, immune cell apoptosis, production or concentration of mitochondrial enzymes and / or transporters in immune cells, production or concentration of transcription factors in immune cells, production or concentration of metabolic enzymes in immune cells, hypoxic state of the microenvironment or tissues, efficiency of immune cell signaling pathways, immune cell secretion, internalization of endosomes in immune cells, production or concentration of chaperone proteins in immune cells, production of cytoskeletal regulatory proteins in immune cells, immune cell migration and / or mobility, active membrane transport of immune cells, production or concentration of carrier proteins in immune cells, or production or concentration of tRNA in immune cells, production and / or secretion of checkpoint inhibitors, or production of anti-inflammatory factors; or inhibiting compounds, proteins or molecules that induce or increase / enhance immune cell proliferation, production of cytotoxic compounds by immune cells, or degradation of proteins by immunoproteasomes and ubiquitination, and antigen presentation by immune cells.

[0382] In particular, immune cell inhibitory proteins that induce or increase immune cell exhaustion are selected from the group including or comprising TIM3, ENTPD1, ​​LAG3, PD-1, and TIGIT.

[0383] In particular, immune cell inhibitory proteins that inhibit or reduce the proliferation or regeneration of immune cells are selected from the group including or consisting of FOXO1, MLH1, MSH2, MSH6, APC, and CDKN2A.

[0384] In particular, immune cell inhibitory proteins that induce or increase apoptosis or cell death of immune cells are selected from the group including or consisting of caspases, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, and P53.

[0385] In particular, immune cell inhibitory proteins that increase the production or concentration of mitochondrial enzymes and / or transporters in immune cells are derived from the SLC25 family.

[0386] In particular, immune cell inhibitory proteins that increase the production or concentration of transcription factors in immune cells are selected from the group including or comprising FOXP3, TOX, EOMES, BCL6, and BACH2.

[0387] In particular, immune cell inhibitory proteins that induce or increase hypoxic conditions in the microenvironment or tissues are selected from the group including or comprising HIFa, PKCq, and VHL.

[0388] In particular, immune cell inhibitory proteins that increase the production or concentration of metabolic enzymes in immune cells are selected from the group including or comprising IDO-1, IDO-2, ARG1, and TDO.

[0389] In particular, immune cell inhibitory proteins that induce or increase the production of anti-inflammatory signaling molecules by immune cells are selected from the group including or comprising mTOR / DAPTOR / RAPTOR, SHP, and SMAD.

[0390] In particular, immune cell inhibitory proteins that inhibit or reduce the production of cytotoxic compounds by immune cells are selected from the group including or consisting of SHIP-1, SHP-1 / 2, PTEN, PTP1B, Ikaros, EGR2 / 3, CREM, and P27(KIP1).

[0391] In particular, immune cell inhibitory proteins that induce or increase the internalization and exocytosis of immune cells are selected from the group including or comprising CD107a, RAC1, AP2, RB7, M6P, and MPR.

[0392] In particular, immune cell inhibitory proteins that increase the production or concentration of chaperone proteins in immune cells are selected from the group including or comprising BBS10, BBS12, TCP1, and HSP.

[0393] In particular, immune cell inhibitory proteins that induce or increase the production, migration, and / or mobility of cytoskeletal regulatory proteins of immune cells are selected from the group including or comprising APC, laminin, actin, vimentin, DEF1, dynein, and kinesin.

[0394] In particular, immunosuppressant proteins that inhibit or reduce the degradation of proteins by immunoproteasomes and ubiquitination, as well as antigen presentation by immune cells, are selected from the group including or comprising tapasin, LMP7, Erp57, and Cbl-b.

[0395] In particular, immune cell inhibitory proteins that induce or increase the resolution of inflammation in tissues are selected from the group including or comprising CHEMR23, GPR37, GPR32, GPR18, FPR2, and GPR35.

[0396] In particular, immune cell inhibitory proteins that modify the epigenetics of immune cells are selected from the group including or consisting of HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3, and SUV420H2.

[0397] These proteins are described in detail in Table D (Table 4) below. In some embodiments, the mRNA molecule encodes a protein selected from Table E (Table 5).

[0398] [Table 5A]

[0399] [Table 5B]

[0400] [Table 5C]

[0401] [Table 5D]

[0402] [Table 5E]

[0403] [Table 5F]

[0404] [Table 5G]

[0405] [Table 5H]

[0406] [Table 5I]

[0407] [Table 5J]

[0408] One or more mRNA molecules encode molecules selected from the following group: a) TIM3, ENTPD1, ​​LAG3, PD-1, PD-L1, TIGIT, FOXO1, MLH1, MSH2, MSH6, APC, CDKN2A, Caspase, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, BIM, BIM-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, P53, FOXP3, TOX, EOMES, BCL6 and BACH2, HIFa, PKCq and VHL, IDO-1, IDO-2, ARG1; TDO, mTOR / DAPTOR / RAPTOR, SHP, SMAD, SHIP-1, SHP-1 / 2, PTEN, PTP1B, Ikaros, EGR2 / 3, CREM, P27(KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS10, BBS12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Tapasin, LMP7, Er Proteins selected from the group consisting of p57, Cbl-b, CHEMR23, GPR37, GPR32, GPR18, FPR2, GPR35, HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3, or SUV420H2, preferably proteins selected from the group consisting of TIM3, LAG3, PD-L1, TIGIT, FAS, TRAIL, PUMA, BIM-S, FOXP3, TOX, IDO-1, IDO-2, ARG1, CHEMR23, and FPR2, and any combination thereof; b) Cytokines, preferably selected from the group consisting of IFNG, IFNa, IL2, IL13, IL4 and IL-10, IL35 (a dimeric protein containing IL-12α chain and IL-27β chain), IL37, IL38, and any combination thereof; c) Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-8R, IL-10R, IL-11R, IL-17R, IL-18R, IL-21R, IL-23R, IL-35R, and any combination thereof; and d) Chemokine receptors, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and XCR1, and any combination thereof.

[0409] In some embodiments, one or more mRNA molecules are TIM3, ENTPD1, ​​LAG3, PD-1, PD-L1, TIGIT, FOXO1, MLH1, MSH2, MSH6, APC, CDKN2A, caspase, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, BIM, BIM-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, P53, FOXP3, TOX, EOMES, BCL6 and BACH2, HIFa, PKCq and VHL, IDO-1, IDO-2, ARG1; TDO, mTOR / DAPTOR / RAPTOR, SHP, SMAD, SHIP-1, SHP-1 / 2, PTEN, PTP1B, Icarus, EGR2 / 3, CREM, P27(KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS10, BBS12, It codes for TCP1, HSP, APC, laminin, actin, vimentin, DEF1, dynein, kinesin, tapasin, LMP7, Erp57, Cbl-b, CHEMR23, GPR37, GPR32, GPR18, FPR2, GPR35, HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3, or SUV420H2, or any combination thereof.

[0410] Preferably, one or more mRNA molecules encode a protein selected from the group consisting of TIM3, LAG3, PD-L1, TIGIT, FAS, TRAIL, PUMA, FOXP3, TOX, IDO-1, IDO-2, ARG1, CHEMR23, FPR2, or BIM-S, or any combination thereof.

[0411] In some embodiments, the mRNA molecule encodes BIM-S and / or PUMA.

[0412] Preferably, the mRNA molecule encoding PUMA contains, or consists of, the nucleic acid sequence shown in Sequence ID No. 43, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0413] In some embodiments, the mRNA molecule encodes a BIM protein. As used herein, the term “BIM protein” encompasses the three Bim isoforms produced by alternative splicing (i.e., Bim-EL, Bim-L, and Bim-S, as described, for example, in Sionov R. Vogt, Vlahopoulos SA, Granot Z., Regulation of Bim in Health and Disease. Oncotarget. 2015; 6: pp. 23058-23134). Preferably, the mRNA molecule encoding BIM-S contains, or consists of, the nucleic acid sequence shown in Sequence ID No. 44, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0414] The effect of an immune cell inhibitory protein on activated immune cells can be determined when, in the presence of the immune cell inhibitory protein of the present invention, activated immune cells exhibit lower activity compared to activated immune cells under the same experimental conditions but without the presence of the immune cell inhibitory protein. The sample may be an immune cell culture from a sample of at least one healthy patient or from a sample of at least one patient requiring treatment, as detailed below. Immune cell activity can be measured by any method known to those skilled in the art. In particular, inhibition of immune cell activity can be measured by comparing the immune cell activity of a population of immune cells obtained from the sample, without the immune cell inhibitory protein to be evaluated, with the immune cell activity of a population of immune cells obtained from the sample and treated with the immune cell inhibitory protein to be evaluated.

[0415] In one embodiment, the immune cell inhibitory protein is an intracellular protein that has an intracellular effect on activated immune cells or transmembrane proteins, preferably intracellular proteins.

[0416] By using intracellular proteins in combination with targeting specific subsets of immune cells, highly specific and efficient inhibition of said immune cells can be achieved. Potent intracellular proteins that enhance the activation and / or proliferation of immune cells in specific environments can be used.

[0417] As used herein, “proteins having intracellular effects on activated immune cells” or “intracellular proteins” refers to proteins produced / expressed within a cell that do not enter the extracellular medium, whether alone or in a vesicle, nor are they expressed on the cell membrane. Therefore, intracellular proteins are contained within the cell membrane boundary and act in one of the intracellular compartments (e.g., cytosol, endoplasmic reticulum, mitochondria, nucleus, etc.). Such proteins may be present in any of the intracellular compartments, e.g., the nucleus, intervesical space, organelle, or cytosol. Thus, proteins may be cytoplasmic proteins, nuclear proteins or mitochondrial proteins, or intervesical proteins, preferably nuclear or cytoplasmic proteins.

[0418] If necessary, the intracellular proteins of immune cells may be enzymes, intracellular signaling proteins, or transcription factors, preferably transcription factors.

[0419] In certain embodiments, the immune cell inhibitory protein is a transcription factor. As used herein, “transcription factor” refers to a DNA-binding protein that regulates gene transcription. Preferably, the transcription factor is selected from the group consisting of SOCS family members, STAT family members, and RIPK1 and its variants, the variant having at least 80% identity with the wild-type protein or having 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions and combinations thereof.

[0420] In certain embodiments, immune cell inhibitory proteins are not chimeric antigen receptors (CARs). In certain embodiments, immune cell activating proteins are neither CARs, nor T cell receptors (TcRs) (i.e., including TCR alpha, TCR beta, CD3, and CD247), nor B cell receptors (BcRs).

[0421] In some embodiments, the immune cell compound or molecule is a secreted protein.

[0422] As used herein, “secretory compound,” “secretory protein,” or “secretory molecule” refers to a compound that leaves a cell, enters the extracellular culture medium, and is directed to the membrane of an immune cell or to an adjacent cell.

[0423] In some embodiments, the secreted compound or molecule does not act on the immune cells that secrete it. In other embodiments, the secreted compound or molecule acts not only on the immune cells that secrete it but also on additional cells.

[0424] In some embodiments, one or more mRNA molecules do not encode cytokines and / or chemokines. In specific embodiments, immune cell inhibitory proteins are not cytokines and / or chemokines.

[0425] In certain embodiments, the transmembrane protein is selected from the group including CCR4, CCR10, CXCR3, CCR10, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR3, CXCR5, CCR7, and CXCR5.

[0426] Alternatively, immune cell inhibitory proteins are intracellular signaling proteins. For example, such intracellular signaling molecules may be derivatives of D3-phosphoinositides and phosphatidylinositol, such as those in which the D-3 position of the inositol ring is phosphorylated, and include compounds such as phosphatidylinositol-(3)-monophosphate (PtdIns(3)P), phosphatidylinositol(3,4)-diphosphate (PtdIns(3,4)P2), and phosphatidylinositol(3,4,5)-triphosphate (PtdIns(3,4,5)P3). Therefore, intracellular signaling proteins may be involved in the synthesis of these molecules.

[0427] In specific embodiments, the immune cell inhibitory protein is not a chimeric antigen receptor (CAR). Preferably, the immune cell inhibitory protein is neither a CAR, nor a T cell receptor (TcR) (i.e., including TCR alpha, TCR beta, CD3 and CD247), nor a B cell receptor (BcR).

[0428] In some embodiments, one or more mRNA molecules do not encode cytokines and / or chemokines.

[0429] In certain embodiments, the immune cell inhibitory protein is a cytokine receptor. Preferably, the cytokine receptor is selected from the group consisting of IL10R, TGFR, TNFR, ILR1A, GCSFR, and IL4R.

[0430] In certain embodiments, the immune cell inhibitory protein is a chemokine receptor. Preferably, the chemokine receptor is selected from the group consisting of CXCR3, CXCR5, CXCR4, CXCR1, CXCR2, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, and XCR1.

[0431] In certain embodiments, the immune cell inhibitory protein is a lectin receptor. Preferably, the lectin receptor is selected from the group consisting of CD72, DCIR, MICL, and CLEC-1.

[0432] In certain embodiments, the immune cell inhibitory protein is an engineered surface receptor or membrane-anchored cytokine. Preferably, the engineered surface receptor or membrane-anchored cytokine is selected from the group consisting of engineered membrane cytokines (TGFB, IL13, IL4, IL-10), dominant-negative TLRs, and dominant-negative receptors (IL-6R, TNFR, IL17R, IL23R, IL35R, IL21R, IFNa R).

[0433] In certain embodiments, the immune cell inhibitory protein is a co-inhibitory receptor or ligand. Preferably, the co-inhibitory receptor or ligand is selected from the group consisting of BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, galectin 9, CD48, HVEM, B7DC, CD155, CD113, CD112, ILT2, ILT3, NKG2A, LILRB1, IRp60, KIR, CD22, CD5, CD66a, PIR-B, KIR2DL1, KIR2DL2 / 3, ILT4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, TIGIT, LAG3, and A2AR.

[0434] These proteins are described in detail in Table F (Table 6) below. In some embodiments, the mRNA molecule encodes a protein selected from Table F (Table 6).

[0435] [Table 6A]

[0436] [Table 6B]

[0437] [Table 6C]

[0438] [Table 6D]

[0439] [Table 6E]

[0440] In certain embodiments, one or more mRNA molecules are CCR4, CCR10, CXCR3, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR5, IL10R, TGFR, TNFR, ILR1A, GCSFR, IL4R, CXCR1, CXCR2, CXCR6, CCR1, CCR3, CCR6, CCR8, CCR9, CCR11, XCR1, CD72, DCIR, MICL, CLEC-1, TGFB, IL13, IL4, IL-10, IL-6R, IL17R, IL23R, IL35R, IL21R, IFNa This code encodes R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, Galectin 9, CD48, HVEM, B7DC, CD155, CD113, CD112, ILT2, ILT3, NKG2A, LILRB1, IRp60, KIR, CD22, CD5, CD66a, PIR-B, KIR2DL1, KIR2DL2 / 3, ILT4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, TIGIT, LAG3, or A2AR, or any combination thereof.

[0441] In certain embodiments, one or more mRNA molecules are CCR4, CCR10, CXCR3, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR5, IL10R, TGFR, TNFR, ILR1A, GCSFR, IL4R, CXCR1, CXCR2, CXCR6, CCR1, CCR3, CCR6, CCR8, CCR9, CCR11, XCR1, CD72, DCIR, MICL, CLEC-1, TGFB, IL13, IL4, IL-10, IL35, IL37, IL38, IL-6R, IL17R, IL23R, IL35R, IL21R, IFNa This code encodes R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, Galectin 9, CD48, HVEM, B7DC, CD155, CD113, CD112, ILT2, ILT3, NKG2A, LILRB1, IRp60, KIR, CD22, CD5, CD66a, PIR-B, KIR2DL1, KIR2DL2 / 3, ILT4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, TIGIT, LAG3, or A2AR, or any combination thereof.

[0442] In certain embodiments, an immune cell inhibitory protein is a protein that inhibits a compound, protein, or molecule that inhibits immune cell exhaustion, immune cell apoptosis, production or concentration of mitochondrial enzymes and / or transporters, production or concentration of transcription factors, production or concentration of metabolic enzymes, hypoxia of the microenvironment or tissues, efficiency of immune cell signaling pathways, immune cell secretion, internalization of endosomes by immune cells, production or concentration of chaperone proteins by immune cells, production of cytoskeletal regulatory proteins by immune cells, immune cell migration and / or mobility, active membrane transport by immune cells, production or concentration of carrier proteins or production or concentration of tRNA by immune cells, production and / or secretion of checkpoint inhibitors, or production of anti-inflammatory factors; or induces or increases immune cell proliferation, production of cytotoxic compounds by immune cells, or degradation of proteins by immunoproteasomes and ubiquitination, and antigen presentation by immune cells.

[0443] In some embodiments, the present invention relates to lipid-based nanoparticles comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated T cells, and one or more mRNA molecules encoding a protein that inhibits the activity of the T cells.

[0444] Preferably, the present invention relates to lipid-based nanoparticles comprising an anti-PD-1 antigen-binding domain and one or more mRNA molecules encoding a protein that inhibits the activity of the T cell.

[0445] More preferably, the present invention relates to lipid-based nanoparticles comprising an anti-PD-1 antigen-binding domain and one or more mRNA molecules encoding a T cell transcription factor or a growth-inhibiting T cell factor.

[0446] In addition, the lipid-based nanoparticles of the present invention may include several mRNA molecules encoding different immune cell activity inhibitory proteins. In this embodiment, the lipid-based nanoparticles include a first mRNA molecule encoding a T cell activity inhibitory protein and a second mRNA molecule encoding a second immune cell activity inhibitory protein. The second immune cell activity inhibitory protein may be another T cell activity inhibitory protein (i.e., different from the first T cell activity inhibitory component) or an inhibitory component of another type of immune cell, such as NK cells or macrophages.

[0447] In one embodiment, lipid-based nanoparticles comprise at least two mRNA molecules, one of which encodes a transmembrane protein that is a receptor, and the other of which encodes a secreted protein that is a ligand for the receptor. For example, lipid-based nanoparticles comprise mRNA encoding an interleukin receptor (e.g., IL10-R) and mRNA encoding a related interleukin (e.g., IL-10). This allows for inhibition at the same / single immune cell (i.e., cis-activated) level. Other examples are as follows: - TGFB and TGFBR, - TNF and TNFR, - IL13 and IL13R, - GCF and GCFR, - IL4 and IL4R, - CD72 and CD100, - CD22 and α2-6Sia, - Siglec-10 and α2-3Sia, - LILRB and HLA.

[0448] The receptors and ligands are described in detail in Table G (Table 7) below. In some embodiments, the mRNA molecule encodes a protein selected from Table G (Table 7).

[0449] [Table 7A]

[0450] [Table 7B]

[0451] [Table 7C]

[0452] In some embodiments, the mRNA molecule encodes a protein selected from Tables E (Table 5), F (Table 6), and G (Table 7).

[0453] In addition, α2-3Sia and α2-6Sia refer to sialic acids linked to each other by either α2-3 or α2-6 linkages, respectively. Linking of sialic acid residues is well known in the art; see, for example, Figures 23 and 24 in Cao, H., Chen, X. (2012). General Consideration on Sialic Acid Chemistry. In: Chevolot, Y. (ed.). Carbohydrate Microarrays. Methods in Molecular Biology, Vol. 808. Humana Press. https: / / doi.org / 10.1007 / 978-1-61779-373-8_3.

[0454] In some embodiments, the lipid-based nanoparticles include one or more mRNA molecules selected from the group consisting of: - mRNA encoding BIM, preferably an mRNA molecule containing a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 43 or a nucleic acid sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto; - mRNA encoding PUMA, preferably an mRNA molecule containing a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 44 or a nucleic acid sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto;

[0455] In some embodiments, the present invention relates in particular to lipid-based nanoparticles including the following: a) A lipid-based composition, - Approximately 45 mol% to approximately 55 mol% of the total lipids present in the LNP, preferably approximately 48 mol% to approximately 52 mol%, more preferably approximately 50 mol%, of ALC-00315, SM-102, Dlin-MC3-DMA, or SS-OP, or any mixture thereof. - Approximately 5 mol% to approximately 15 mol%, preferably approximately 8 mol% to approximately 12 mol%, more preferably approximately 10 mol%, of the total lipids present in the LNP, DOPE, DDAB, DOPC, POPE, or DSPC, or any mixture thereof. - Cholesterol and more preferably about 37 mol% to about 40 mol%, more preferably about 38.5 mol%, of the total lipids present in the LNP. - Approximately 0.5 mol% to approximately 2.5 mol%, preferably approximately 1 mol% to approximately 2 mol%, more preferably approximately 1.5 mol%, of the total lipids present in the LNP, PEG 2000-DSG, PEG 2000-DMG, PEG 5000-DSG, PEG 5000-DMG, or ALC-0159, or any mixture thereof. Lipid-based compositions containing; b) An antigen-binding domain that specifically binds to human PD-1 or human CLEC-1; c) One or more mRNA molecules encoding the following: - TIM3, ENTPD1, ​​LAG3, PD-1, PD-L1, TIGIT, FOXO1, MLH1, MSH2, MSH6, APC, CDKN2A, Caspase, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, BIM, BIM-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, P53, FOXP3, TOX, EOMES, BCL6 and BACH2, HIFa, PKCq and VHL, IDO-1, IDO-2, ARG1; TDO, mTOR / DAPTOR / RAPTOR, SHP, SMAD, SHIP-1, SHP-1 / 2, PTEN, PTP1B, Ikaros, EGR2 / 3, CREM, P27(KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS10, BBS12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Tapasin, LMP7, Er Proteins selected from the group consisting of p57, Cbl-b, CHEMR23, GPR37, GPR32, GPR18, FPR2, GPR35, HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3, or SUV420H2, preferably proteins selected from the group consisting of TIM3, LAG3, PD-L1, TIGIT, FAS, TRAIL, PUMA, BIM-S, FOXP3, TOX, IDO-1, IDO-2, ARG1, CHEMR23, and FPR2, and any combination thereof; - Cytokines, preferably selected from the group consisting of IFNG, IFNa, IL2, IL13, IL4, and IL-10, and any combination thereof; - Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-8R, IL-10R, IL-11R, IL-17R, IL-18R, IL-21R, IL-23R, IL-35R, and any combination thereof; and - Chemokine receptors, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and XCR1, and any combination thereof.

[0456] In some embodiments, the present invention relates in particular to lipid-based nanoparticles including the following: a) A lipid-based composition, - ALC-0315, DOPE, cholesterol and DMG-PEG, - ALC-0315, DDAB, cholesterol and DMG-PEG, - ALC-0315, POPE, cholesterol and DMG-PEG, - ALC-0315, DOPE, cholesterol and DSPE-PEG, - ALC-0315, DSPC, cholesterol and DMG-PEG, - ALC-0315, DSPC, cholesterol and ALC-0159, - SM-102, DSPC, cholesterol and DMG-PEG, - Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG, - ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG, - SS-OP, DOPE, cholesterol, and DMG-PEG; - SS-OP, DSPC, cholesterol and DSPE-PEG; and - SS-OP, DOPC, cholesterol and DMG-PEG Lipid-based compositions comprising, in particular, the lipid percentages described herein; b) An antigen-binding domain that specifically binds to human PD-1; c) One or more mRNA molecules encoding a protein selected from the group consisting of FOXO1, Fas, TRAIL, PUMA, NOXA, BIM, LAG3, CTLA4, PDL1, FOXP3, TOX, IDO, ARG1 IL35+IL35R, TGFB+TGFBR, TIM3, TIGIT, ChemR23, FPR2, and any combination thereof, in particular PUMA or BIMS.

[0457] Pharmaceutical composition The present invention also relates to pharmaceutical compositions comprising lipid-based nanoparticles, preferably the lipid-based nanoparticles described above as an active ingredient or compound, and optionally a pharmaceutically acceptable carrier or excipient.

[0458] As used herein, “pharmaceutical composition” refers to a preparation of one or more active agents, including those containing LNP according to the present invention, using physiologically suitable carriers and other chemical components as needed, such as excipients. The purpose of a pharmaceutical composition is to facilitate the administration of an active agent to a living organism. The compositions of the present invention may be in any conventional route of administration or in a form suitable for use. In one embodiment, “composition” generally refers to a combination of an active agent, such as a compound or composition, and an inactive or active, naturally occurring or non-naturally occurring carrier, such as an adjuvant, diluent, binder, stabilizer, buffer, or preservative, and includes a pharmaceutically acceptable carrier. “Acceptable vehicle” or “acceptable carrier” is, as referred herein, any known compound or combination of compounds that is known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0459] The pharmaceutical composition can be sterilized and, if desired, can be mixed with excipients that do not interact harmfully with the lipid-based nanoparticles of the present invention and do not impart any undesirable toxic effects, such as pharmaceutically acceptable carriers, excipients, salts, antioxidants, and / or stabilizers.

[0460] In particular, the pharmaceutical compositions according to the present invention can be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. To facilitate administration, the lipid-based nanoparticles described herein can be made into pharmaceutical compositions, in particular, for in vivo administration. Means for preparing such compositions are described in the Art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)). In yet another embodiment, the pharmaceutical composition is administered intranodal or intratumoral.

[0461] Pharmaceutical compositions can be prepared by mixing lipid-based nanoparticles of desired purity with pharmaceutically acceptable carriers, excipients, antioxidants, and / or stabilizers as needed in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, antioxidants, and / or stabilizers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0462] Preferably, pharmaceutical compositions containing lipid-based nanoparticles are relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the composition, for example, the particle size distribution of the lipid-based nanoparticles contained in the composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. Preferably, the pharmaceutical composition has a polydispersity index of about 0 to about 0.25, for example, 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 polydispersity index of the pharmaceutical composition is about 0.10 to about 0.25.

[0463] The pharmaceutical compositions according to the present invention can be formulated to release the active ingredient (e.g., lipid-based nanoparticles of the present invention) substantially immediately after administration, or after any predetermined time or period after administration. In some embodiments, the pharmaceutical compositions may utilize sustained-release, delayed-release, and gradual-release delivery systems, so that the composition is delivered before sensitization of the site to be treated, and with sufficient time to induce sensitization. By means known in the art, the release and absorption of the composition can be prevented or minimized until it reaches the target tissue or organ, or sustained release of the composition can be ensured. Such systems can increase convenience for subjects and physicians by avoiding repeated administration of the composition.

[0464] Those skilled in the art will understand that the formulations of the present invention may be isotonic with human blood, that is, that the formulations of the present invention may have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by vapor pressure or freezing point depression osmometer.

[0465] Pharmaceutical compositions must generally be sterile and stable under manufacturing and storage conditions. Prevention of microbial presence can be ensured by sterilization procedures (e.g., by microfiltration) and / or by the inclusion of various antimicrobial and antifungal agents.

[0466] In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or auxiliary components, for example, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surfactants, buffers, and / or preservatives.

[0467] Surfactants and / or emulsifiers include natural emulsifiers (e.g., gum arabic, 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 Examples of polyoxyethylene ethers (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, sodium doxate, and / or combinations thereof.

[0468] Examples of preservatives include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol-based preservatives, and / or acidic preservatives.

[0469] Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite.

[0470] Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate.

[0471] 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, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid.

[0472] Examples of alcohol-based preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, 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 hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE®, KATHON®, and / or EUXYL®. Examples of free radical scavengers include butylated hydroxytoluene (BHT or butylated hydroxytoluene) or deferoxamine.

[0473] Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, 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, dicalcium phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, amino-sulfonic acid buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, and / or ethyl alcohol.

[0474] In some embodiments, the pharmaceutical composition containing lipid-based nanoparticles according to the present invention further comprises salts such as chloride salts. In some embodiments, the pharmaceutical composition containing lipid-based nanoparticles further comprises sugars such as disaccharides. In some embodiments, the pharmaceutical composition further comprises sugars but does not contain salts such as chloride salts. In some embodiments, the pharmaceutical composition further comprises one or more small hydrophobic molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0475] Formulations comprising amphiphilic polymers and lipid-based nanoparticles may be formulated, in whole or in part, as pharmaceutical compositions. A pharmaceutical composition may, in particular, comprise one or more amphiphilic polymers and one or more lipid-based nanoparticles. For example, a pharmaceutical composition may comprise one or more amphiphilic polymers and one or more lipid-based nanoparticles comprising one or more different mRNA therapeutic and / or prophylactic agents. In addition, excipients and auxiliary components may be used in the pharmaceutical composition, unless any conventional excipient or auxiliary component is incompatible with one or more components of the lipid-based nanoparticles or with the amphiphilic polymer.

[0476] In some embodiments, the pharmaceutical composition contains one or more amphiphilic polymers in an amount between 0.1% and 15% (wt / vol) (e.g., 0.5%, 1%, 2.5%, 5%, 10%, or 12.5% ​​w / vol).

[0477] The relative amounts of lipid-based nanoparticles, pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical compositions according to this disclosure may vary depending on the nature, size, and / or state of the object being treated, and further, on the route through which the composition will be administered. In some embodiments, the pharmaceutical composition contains one or more lipid-based nanoparticles, e.g., the lipid-based nanoparticles disclosed herein, in amounts between 0.1% and 100% (wt / wt). The amount of lipid-based nanoparticles that can be combined with a carrier material to produce a single dosage form generally corresponds to the amount of lipid-based nanoparticles that produce a therapeutic effect.

[0478] In some embodiments, one or more excipients or auxiliary components may constitute more than 50% of the total mass or volume of a pharmaceutical composition containing lipid-based nanoparticles, for example, the lipid-based nanoparticles disclosed herein. For example, one or more excipients or auxiliary components may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, pharmaceutically acceptable excipients are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for use in humans and / or veterinary use. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopoeia.

[0479] In certain embodiments, the pharmaceutical composition comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different lipid-based nanoparticles, each comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitor. "Different lipid-based nanoparticles" means lipid-based nanoparticles that i) have different lipid compositions, ii) have different antigen-binding domains (e.g., different sequences and / or targets), and / or iii) have different mRNA molecules.

[0480] Preferably, the pharmaceutical composition comprises at least two different lipid-based nanoparticles, each containing an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitor.

[0481] Preferably, the pharmaceutical composition comprises at least two different lipid-based nanoparticles, each comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitor, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof, and preferably comprises an Fc domain, preferably an IgG Fc domain.

[0482] In some specific embodiments, the pharmaceutical composition comprises at least two different lipid-based nanoparticles, none of which are: i) not covalently bonded to any lipid in the lipid-based nanoparticles; ii) not modified in any way to couple or graft an antigen-binding domain to a lipid; and / or iii) not covalently bonded to either a lipidized peptide or a lipidized motif, and thus not containing one or more antigen-binding domains.

[0483] Therefore, the pharmaceutical composition according to the present invention is - A first lipid-based nanoparticle comprising a first antigen-binding domain capable of specifically binding to a first target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitor; and - A second lipid-based nanoparticle comprising a second antigen-binding domain capable of specifically binding to a second (different) target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitor. It may include.

[0484] Preferably, one or more mRNA molecules encoding an immune cell activity inhibitory component may be the same or different in the first and second lipid-based nanoparticles.

[0485] In a preferred embodiment, the antigen-binding domain of a lipid-based nanoparticle and the antigen-binding domain of an additional lipid-based nanoparticle each have mRNA molecules encoding different immune cell activity inhibitors, but can specifically bind to the same target (e.g., PD-1).

[0486] Preferably, the antigen-binding domain of the first lipid-based nanoparticle and the antigen-binding domain of the additional (second) lipid-based nanoparticle can specifically bind to different / non-overlapping epitopes of the same target (e.g., different epitopes of PD-1).

[0487] Alternatively, the antigen-binding domain of the first lipid-based nanoparticle and the antigen-binding domain of the additional (second) lipid-based nanoparticle can specifically bind to the same epitope on the target (e.g., the same epitope on PD-1).

[0488] Therapeutic use The lipid-based nanoparticles and pharmaceutical compositions containing them, as defined above, have considerable in vitro and in vivo utility and applications. In particular, the lipid-based nanoparticles and pharmaceutical compositions provided herein can be used in therapeutic methods and / or for therapeutic purposes.

[0489] The present invention relates to lipid-based nanoparticles or pharmaceutical compositions containing them for use as a pharmaceutical or vaccine, and / or for use in the treatment of disorders or diseases, such as autoimmune diseases or inflammatory diseases.

[0490] The present invention relates to the use of one or more lipid-based nanoparticles or pharmaceutical compositions according to the present invention for the manufacture of pharmaceuticals for the treatment of autoimmune diseases or inflammatory diseases.

[0491] The present invention relates to a method for treating an autoimmune disease or inflammatory disease in a subject, comprising the step of administering one or more lipid-based nanoparticles or a pharmaceutical composition according to the present invention to the subject.

[0492] The present invention also relates to the use of lipid-based nanoparticles or pharmaceutical compositions containing them for treating diseases or disorders in a subject, such as autoimmune diseases or inflammatory diseases. The present invention also relates to the use of lipid-based nanoparticles or pharmaceutical compositions disclosed herein in the manufacture of pharmaceuticals for treating diseases or disorders in a subject, such as autoimmune diseases or inflammatory diseases.

[0493] Finally, the present invention relates to a method for treating a disease or disorder in a subject, for example, an autoimmune disease or an inflammatory disease, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition or lipid-based nanoparticles, for example, a pharmaceutical composition or lipid-based nanoparticles disclosed herein, to the subject.

[0494] In one embodiment, the present invention relates to a method for treating, in a subject requiring such treatment, a disease and / or disorder selected from the group consisting of diseases in which the resolution of inflammation is delayed or impaired, and / or a disease selected from the group consisting of inflammatory diseases and autoimmune diseases, comprising the step of administering an effective amount of the lipid-based nanoparticles or pharmaceutical composition defined above to the subject. Examples of such diseases and disorders are described in more detail below.

[0495] In particular, the present invention relates to a treatment method comprising the steps of (a) identifying a patient in need of treatment; and (b) administering a therapeutically effective amount of a lipid-based nanoparticle or pharmaceutical composition described herein to the patient.

[0496] "Effective dose" or "therapeutic dose," as used herein, refers to the amount of active agent (i.e., lipid-based nanoparticles disclosed herein) required to impart a therapeutic effect to a subject, either alone or in combination with one or more other active agents, for example, the amount of active agent required to treat a targeted disease or disorder or to produce a desired effect. The "effective dose" will vary depending on the agent, the disease and its severity, the characteristics of the subject to be treated, including age, health status, size, sex and weight, the duration of treatment, the type of concurrent therapy (if any), the specific route of administration, and similar factors within the scope of the knowledge and experience of the healthcare professional. These factors are well known to those skilled in the art.

[0497] - autoimmune diseases The lipid-based nanoparticles or pharmaceutical compositions of the present invention may be used in the treatment of patients suffering from autoimmune diseases, such as diabetes, NASH, particularly type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, celiac disease, vasculitis, myasthenia gravis; infectious diseases, such as sepsis; severe viral indications with severe inflammatory conditions, such as coronavirus (e.g., COVID-19), peritonitis; degenerative diseases; impaired wound healing, dry eye syndrome; cancer, particularly solid and humoral cancers, metastatic cancers, particularly carcinomas, particularly breast cancer or colon cancer, or colorectal cancer, lung cancer, or mesothelioma, or myeloid cancers, particularly leukemia.

[0498] Fibrosis (particularly pulmonary and hepatic fibrosis) and ANCA (anti-neutrophil cytoplasmic autoantibody) conditions (vasculitis) are particularly relevant.

[0499] - Inflammatory diseases The lipid-based nanoparticles or pharmaceutical compositions of the present invention may be used to treat patients suffering from diseases in which the resolution of inflammation is delayed or impaired, and / or inflammatory diseases, particularly acute inflammatory diseases, chronic inflammatory diseases, such as chronic inflammatory lung disease (e.g., asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, particularly Crohn's disease or colitis, particularly ulcerative colitis or spontaneous colitis, cystic fibrosis, and skin inflammations selected from the group.

[0500] Combination therapy In some embodiments, lipid-based nanoparticles or pharmaceutical compositions according to the present invention may be used in combination with other therapeutic agents or therapies, particularly for the treatment of autoimmune diseases or for other therapeutic agents or therapies for autoimmune diseases.

[0501] The present invention also relates to a method for treating a disease in a subject, such as an autoimmune disease or a disease in which the resolution of inflammation is delayed or impaired, and / or an inflammatory disease, the method comprising the step of administering to the subject a therapeutically effective amount of a lipid-based nanoparticle or pharmaceutical composition described herein, and a therapeutically effective amount of an additional or second therapeutic agent or therapy.

[0502] Specific examples of additional or second therapeutic agents are provided in WO2021 / 069709.

[0503] Accordingly, combination therapies for the treatment of a disease or disorder are also provided herein, using any of the lipid-based nanoparticles described herein or a pharmaceutical composition containing the same and a suitable second agent.

[0504] In some embodiments, lipid-based nanoparticles and a second agent may be present in a specific pharmaceutical composition. Alternatively, the terms “combination therapy” or “combination therapy,” as used herein, encompass sequential administration of these two agents (e.g., lipid-based nanoparticles as described herein and additional or second preferred therapeutic agents), i.e., administration in which each therapeutic agent is administered at different times, and substantially simultaneous administration of at least two of these therapeutic agents or agents. Sequential or substantially simultaneous administration of each agent may be influenced by any suitable route. The agents may be administered by the same route or by different routes. For example, the first agent (e.g., lipid-based nanoparticles) may be administered intramuscularly, and the additional therapeutic agent (e.g., an anti-inflammatory agent or immunomodulator) may be administered intravenously. Alternatively, a selected combination of agents may be administered by intravenous injection, while the other agents of that combination may be administered intramuscularly.

[0505] In some embodiments, additional therapeutic agents include alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), cell death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell engager) antibodies, antibody-drug conjugates, bioresponsive modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase 2 inhibitors, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, apoptosis inhibitor protein (IAP) inhibitors, intercausing antibiotics, and kinase inhibitors. The drugs may be selected from a non-exclusive list that includes kinesin inhibitors, Jak2 inhibitors, mammalian rapamycin target protein inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), poly(ADP)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids, plant alkaloids, small molecular weight inhibitory ribonucleic acid (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, epitopes or neoepitopes from tumor antigens, and combinations of one or more of these drugs.

[0506] Combination therapy may also rely on a combination of administration of lipid-based nanoparticles or pharmaceutical compositions containing them according to the present invention and surgical procedures.

[0507] Subjects, regimens, and administration Those requiring treatment may be individuals who have, are at risk of having, or are suspected of having a disease, such as an autoimmune disease or an inflammatory disease. Such patients can be identified through routine health checkups.

[0508] As used herein, the terms “subject,” “host,” “individual,” or “patient” refer to human beings, including adults and children. The subjects to be treated may be, in particular, human beings in the prenatal stage, neonates, children, infants, adolescents, or adults, in particular, adults at least 30 or 40 years of age, preferably at least 50 years of age, even more preferably at least 60 years of age, and even more preferably at least 70 years of age.

[0509] The form, route of administration, and dosage of the pharmaceutical composition may be adjusted by those skilled in the art according to the type and severity of the infection, and according to the patient, particularly their age, weight, size, sex, and / or health condition. The compositions of the present invention may be administered in a number of ways, depending on whether topical or systemic treatment is desired.

[0510] Using conventional methods known to those skilled in the medical field, the lipid-based nanoparticles, pharmaceutical compositions, or combination therapies disclosed herein can be administered to a target depending on the type or site of the disease to be treated, for example, by oral administration, parenteral administration, enteral administration, inhalation spray administration, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration, or implantable reservoir administration. Preferably, the lipid-based nanoparticles, pharmaceutical compositions, or combination therapies are administered by subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intratumoral, intrasternal, intrathecal, intrafocal, and intracranial injection or infusion techniques. Preferably, the lipid-based nanoparticles, pharmaceutical compositions, or combination therapies are administered intravenously. Alternatively, the lipid-based nanoparticles, pharmaceutical compositions, or combination therapies are administered intratumorally.

[0511] In some embodiments, the subject has already undergone at least one series of treatments, preferably several series of treatments, prior to administration of the lipid-based nanoparticles, pharmaceutical composition, or combination therapy of the present invention.

[0512] Methods to inhibit immune cells The present invention also relates to a method for inhibiting immune cell activity, comprising the step of contacting immune cells with lipid-based nanoparticles or pharmaceutical compositions according to the present invention. Such a method aims to inhibit the immune potential of T cells and serve as a powerful tool for reducing immune responses, particularly immune responses to the treatment of diseases, such as autoimmune diseases or inflammatory diseases.

[0513] In certain embodiments, the lipid-based nanoparticles disclosed herein may be administered to a subject, for example, in vivo, to reduce immunity or reduce the immune response, preferably to treat a disorder and / or disease. Accordingly, in one embodiment, the present invention provides a method for inhibiting an immune response in a subject, comprising the step of administering the lipid-based nanoparticles or pharmaceutical composition of the present invention to a subject to inhibit an immune response in the subject. The lipid-based nanoparticles or pharmaceutical composition are preferably used to inhibit an immune response, such as immune cell activation, in a subject requiring treatment.

[0514] In certain embodiments, the lipid-based nanoparticles or pharmaceutical compositions according to the present invention are used to increase T cell exhaustion or to inactivate activated T cells.

[0515] The present invention provides a method for inhibiting an immune response i...

Claims

1. Lipid-based nanoparticles comprising an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an inhibitory protein of the activated immune cells, wherein the inhibitory protein is i) an intracellular protein having an intracellular effect on the activated immune cells, and / or ii) a transmembrane protein, and the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

2. The lipid-based nanoparticle according to claim 1, wherein the antigen-binding domain comprises an Fc domain, preferably an IgG Fc domain.

3. The lipid-based nanoparticle according to claim 1 or 2, wherein the antigen-binding domain is not covalently bound to any of the lipids of the lipid-based nanoparticle, or does not include any modifications for coupling or grafting the antigen-binding domain to the lipids.

4. The lipid-based nanoparticles as defined in any one of claims 1 to 3, wherein the lipid-based nanoparticles do not contain an anchor moiety comprising a lipidized peptide or motif.

5. Lipid-based nanoparticles according to any one of claims 1 to 4, wherein the activated immune cells are selected from the group consisting of activated T cells, activated B cells, and activated myeloid cells, and the activated myeloid cells include activated macrophages and activated dendritic cells.

6. The targets expressed on the surface of activated immune cells are PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, ​​CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138(SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TR AP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GIT Lipid-based nanoparticles according to any one of claims 1 to 5, selected from the group consisting of R / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, CD160, CD127, and SIRPa.

7. Lipid-based nanoparticles according to any one of claims 1 to 5, wherein the target expressed on the surface of activated immune cells is selected from the group consisting of PD-1, CD127, SIRPa, and CLEC-1A.

8. The lipid-based nanoparticle according to any one of claims 1 to 5, wherein the target expressed on the surface of activated immune cells is PD-1.

9. The antigen-binding domain is an anti-PD-1 binding domain comprising (ii) a VH containing heavy chain CDR1 (HCDR1), CDR2 (HCDR2), and CDR3 (HCDR3), and (ii) a VL containing light chain CDR1 (LCDR1), CDR2 (LCDR2), and CDR3 (LCDR3), a) The HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 1, The aforementioned HCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 2, The aforementioned HCDR3 contains or comprises the amino acid sequence of SEQ ID NO: 3, The aforementioned LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:

4. The aforementioned LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 5, and The aforementioned LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 6, or b) The HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 23, The aforementioned HCDR2 contains or comprises the amino acid sequence of SEQ ID NO:

24. The aforementioned HCDR3 contains or comprises the amino acid sequence of SEQ ID NO:

25. The aforementioned LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:

26. The aforementioned LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 27; and The aforementioned LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 28, or c) The HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 31, The aforementioned HCDR2 contains or comprises the amino acid sequence of SEQ ID NO:

32. The aforementioned HCDR3 contains or comprises the amino acid sequence of SEQ ID NO: 33, The aforementioned LCDR1 contains or consists of the amino acid sequence of SEQ ID NO:

34. The aforementioned LCDR2 contains or consists of the amino acid sequence of SEQ ID NO: 35; and The aforementioned LCDR3 contains or consists of the amino acid sequence of SEQ ID NO:

36. Lipid-based nanoparticles according to claim 8, wherein the domain is an anti-PD-1 antigen binding domain.

10. The antigen-binding domain, a) VH containing or consisting of the amino acid sequence of SEQ ID NO: 15, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 16; b) VH containing or consisting of the amino acid sequence of SEQ ID NO: 29, and VL containing or consisting of the amino acid sequence of SEQ ID NO: 30; and c) VH containing or consisting of the amino acid sequence of SEQ ID NO: 37, and VL containing or consisting of the amino acid sequence of SEQ ID NO:

38. Lipid-based nanoparticles according to claim 8 or 9, comprising an anti-PD-1 binding domain.

11. Lipid-based nanoparticles according to any one of claims 1 to 10, comprising an additional antigen-binding domain capable of specifically binding to another target expressed on the surface of activated immune cells.

12. The lipid-based nanoparticle according to claim 11, wherein the additional antigen-binding domain is i) not covalently bound to any of the lipids of the lipid-based nanoparticle, ii) does not involve any modification for coupling or grafting the antigen-binding domain to a lipid, and / or iii) is not covalently bound to either a lipidized peptide or a lipidized motif.

13. Lipid-based nanoparticles according to any one of claims 1 to 12, wherein the mRNA molecule encodes an intracellular protein having an intracellular effect on activated immune cells, selected from the group consisting of cytoplasmic proteins, intracellular signaling proteins, enzymes, transcription factors, intrabodies, dominant-negative receptors, or engineered antagonist proteins, such as engineered blocking proteins.

14. The aforementioned one or more mRNA molecules a) TIM3, ENTPD1, ​​LAG3, PD-1, PD-L1, TIGIT, FOXO1, MLH1, MSH2, MSH6, APC, CDKN2A, Caspase, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, BIM, BIM-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, P53, FOXP3, TOX, EOMES, BCL6 and BACH2, HIFa, PKCq and VHL, IDO-1, IDO-2, ARG1; TDO, mTOR / DAPTOR / RAPTOR, SHP, SMAD, SHIP-1, SHP-1 / 2, PTEN, PTP1B, Icarus, EGR2 / 3, CREM, P27(KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS10, BBS12, TCP1, HSP, APC, Laminin, Actin, Vimentin, D Proteins selected from the group consisting of EF1, dynein, kinesin, tapasin, LMP7, Erp57, Cbl-b, CHEMR23, GPR37, GPR32, GPR18, FPR2, GPR35, HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3, or SUV420H2, preferably proteins selected from the group consisting of FOXO1, Fas, TRAIL, PUMA, NOXA, BIM, LAG3, CTLA4, PDL1, FOXP3, TOX, IDO, ARG1, IL35, IL35R, TGFB+TGFBR, TIM3, TIGIT, ChemR23, and FPR22, and any combination thereof; b) Cytokines, preferably selected from the group consisting of IFNG, IFNa, IL2, IL13, IL4, and IL-10, and any combination thereof; c) Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-8R, IL-10R, IL-11R, IL-17R, IL-18R, IL-21R, IL-23R, IL-35R and any combination thereof; and d) Chemokine receptors, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and XCR1, and any combination thereof. Lipid-based nanoparticles according to any one of claims 1 to 13, encoding a molecule selected from the group consisting of the following.

15. The aforementioned one or more mRNA molecules are CCR4, CCR10, CXCR3, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR5, IL10R, TGFR, TNFR, ILR1A, GCSFR, IL4R, CXCR1, CXCR2, CXCR6, CCR1, CCR3, CCR6, CCR8, CCR9, CCR11, XCR1, CD72, DCIR, MICL, CLEC-1, TGFB, IL13, IL4, IL-10, IL-6R, IL17R, IL23R, IL35R, IL21R, IFNa Lipid-based nanoparticles according to any one of claims 1 to 13, encoding R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, Galectin 9, CD48, HVEM, B7DC, CD155, CD113, CD112, ILT2, ILT3, NKG2A, LILRB1, IRp60, KIR, CD22, CD5, CD66a, PIR-B, KIR2DL1, KIR2DL2 / 3, ILT4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, TIGIT, LAG3 or A2AR, SOCS, RIPK1, and any member of the STAT family, or any combination thereof.

16. Lipid-based nanoparticles according to any one of claims 1 to 13, wherein one or more mRNA molecules encode BIM-S and / or PUMA.

17. The lipid-based nanoparticle according to any one of claims 1 to 16, wherein the lipid-based nanoparticle comprises at least two mRNA molecules, one of which encodes a transmembrane protein that is a receptor, and the other of which encodes a secreted protein that is a ligand for the receptor.

18. The lipid-based nanoparticles according to any one of claims 1 to 17, wherein the lipid-based composition of the lipid-based nanoparticles comprises or consists of cationic or ionizable lipids, helper lipids, sterols and PEG-lipids.

19. The ionizable lipids include [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and dimethyldioctadecylammonium Diaminenium (DDAB); 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-Diacyloxy-3-dimethylammoniumpropane; 1,2-Dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-Di(tetradeoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-Dimyristoyl-sn-glycero-3-ethyl Suchocolin (DMEPC), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLen DMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-Dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylamino Ethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy )-1-propanaminonium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2-({8-[(3P)-cholesta-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-Dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy) (C)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amonium bromide (DMORIE), di((Z)-nona-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (A TX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-nona-2-en-1-yl)-9-((4-(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]piperazine-1-yl, Lipid-based nanoparticles according to claim 18, selected from the group consisting of ethyl]amino]dodecane-2-ol (lipidoid C12-200), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide (SS-OP; e.g., CAS number 2377474-67-2), bis{2-[4-(α-D-tocopherol hemisuccinate ethyl)piperidyl]ethyl}disulfide (SS-EC) and any mixture thereof, preferably ALC-0315, SM-102, Dlin-MC3-DMA or SS-OP, more preferably ALC-0315 or SS-OP.

20. Lipid-based nanoparticles according to claim 18 or 19, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixture thereof, and is preferably cholesterol.

21. Lipid-based nanoparticles according to any one of claims 18 to 20, wherein the helper lipid is selected from DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DOPC, DEPC, and DSPE, and any mixture thereof, preferably DOPE or DSPC.

22. Lipid-based nanoparticles according to any one of claims 18 to 21, wherein the PEG-lipid is selected from PEG-DMG, PEG-DSPE, PEG-c-DOMG, DMG-PEG-2000, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG, and PEG-c-DMA, ALC-0159, and any mixture thereof, preferably PEG-DMG, PEG-DSPE, or a mixture thereof.

23. Lipid-based nanoparticles as defined in claim 24, wherein the PEG has a size between about 2000 daltons and about 5000 daltons, and is preferably DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000, or a mixture thereof.

24. The lipid-based composition of the lipid-based nanoparticles is 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, i) ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG, j) SS-OP, DOPE, cholesterol, and DMG-PEG; k) SS-OP, DSPC, cholesterol and DSPE-PEG; and l) SS-OP, DOPC, cholesterol and DMG-PEG Lipid-based nanoparticles according to claim 18, selected from the group consisting of the following.

25. The lipid-based nanoparticles according to any one of claims 18 to 24, wherein the lipid-based nanoparticles comprise about 35 mol% to about 55 mol% of cationic or ionizable lipids, about 5 mol% to about 20 mol% of helper lipids, about 30 mol% to about 60 mol% of sterols, and about 0.5 mol% to about 4 mol% of PEG-lipids.

26. The lipid-based nanoparticles according to any one of claims 18 to 24, wherein the lipid-based nanoparticles comprise about 45 mol% to about 55 mol% of cationic or ionizable lipids, about 5 mol% to about 15 mol% of helper lipids, about 35 mol% to about 45 mol% of sterols, and about 0.5 mol% to about 2.5 mol% of PEG-lipids.

27. A pharmaceutical composition comprising at least one lipid-based nanoparticle according to any one of claims 1 to 26, and optionally a pharmaceutically acceptable carrier or excipient.

28. The pharmaceutical composition according to claim 27, further comprising additional lipid-based nanoparticles, each containing an antigen-binding domain capable of specifically binding to a target expressed on the surface of activated immune cells, and one or more mRNA molecules encoding an immune cell activity inhibitory protein.

29. Lipid-based nanoparticles according to any one of claims 1 to 26 or a pharmaceutical composition according to claim 27 or 28, for use as a pharmaceutical.

30. The lipid-based nanoparticles or pharmaceutical composition are for use in the treatment of a disease in a subject requiring them, and the disease is selected from the group of diseases in which the resolution of inflammation is delayed or hindered, and / or inflammatory diseases, in particular acute inflammatory diseases, chronic inflammatory diseases, e.g., chronic inflammatory lung disease, asthma, keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn's disease or colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, skin inflammation; autoimmune diseases, e.g., diabetes, NASH, in particular type 1 diabetes, psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, etc. Lipid-based nanoparticles or pharmaceutical compositions for use according to claim 29, selected from the group consisting of Reac disease, vasculitis, myasthenia gravis; infectious diseases, e.g., sepsis; severe viral indications with severe inflammatory conditions, e.g., coronavirus (e.g., COVID-19); peritonitis; degenerative diseases; impaired wound healing; dry eye syndrome; cancer, in particular solid and humoral cancers; metastatic cancers, in particular carcinomas, in particular breast cancer or colon cancer, or colorectal cancer, lung cancer or mesothelioma, or myeloid cancers, in particular leukemia; fibrosis (in particular pulmonary and hepatic fibrosis); and ANCA (anti-neutrophil cytoplasmic autoantibody) conditions (vasculitis).

31. Use of lipid-based nanoparticles according to any one of claims 1 to 26 or the pharmaceutical composition according to claim 27 or 28 for the manufacture of a pharmaceutical for the treatment of an autoimmune disease or an inflammatory disease.

32. A method for treating an autoimmune disease or an inflammatory disease, comprising the step of administering a lipid-based nanoparticle according to any one of claims 1 to 26 or a pharmaceutical composition according to claim 27 or 28.

33. An in vitro method for inhibiting immune cell activity, comprising the step of contacting activated immune cells with lipid-based nanoparticles according to any one of claims 1 to 26 or with a pharmaceutical composition according to claim 27 or 28.

Images