Method for synthesizing targeted lipid nanoparticles and their use

Abstract

The present invention relates to a method for producing lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules using a mixing device, to lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules that can be obtained by such a method, and to the use thereof.

Description

[Technical Field]

[0001] This invention relates to the field of immunotherapy. It relates to a method for producing lipid nanoparticles comprising at least one nucleic acid molecule, and to their use in conditions such as treating cancer and infectious diseases. [Background technology]

[0002] As the COVID-19 pandemic clearly demonstrates, there have been significant advancements in related technologies in the field of RNA molecules, and mRNA vaccines have shown some success in treating various infectious diseases such as influenza, Ebola, and cottage virus. mRNA vaccines deliver mRNA to cells, causing them to express the proteins they produce, thereby providing immune defense to the body. Following mRNA vaccines, other applications using mRNA, such as antibody expression through mRNA transfection, have been explored.

[0003] 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.

[0004] To reduce the risk of off-target effects, the insertion of targeting agents (e.g., antibodies) into LNPs is being developed, particularly to target specific cell populations. Among targeting agents, antibodies are preferred because they exhibit high specificity and affinity in lipid-receptor binding.

[0005] However, even if prior art has shown that the use of targeted ligands enhances the delivery and therapeutic efficiency of lipid nanoparticles (LNPs), it is recognized that binding such moieties presents a significant challenge. Antibodies offer a very good alternative due to their high specificity and broad selectivity for targets, but in the case of LNPs, antibody production, conjugation, and subsequent nanoparticle purification can be extremely costly, time-consuming, and result in very low production yields. Applying known prior art processes to industrial-scale production results in very high production complexity and cost. This is all the more important in the case of RNA-containing LNP particles, considering that the production process for RNA-containing LNPs, including the mixing of RNA and lipid components under specific conditions, must be suitable for coupling the targeted entity (e.g., antibody) with the LNP.

[0006] Furthermore, obtaining targeted ligands on the surface of lipid nanoparticles, and maintaining their good binding specificity for targeting, remains a supplementary and advanced technical challenge. In fact, the specific binding ability of ligands to their biological targets may be lost when the produced lipid nanoparticles are exposed to the targets. In particular, due to steric hindrance and potential conformational modifications of the antibody, the accessibility of the antigen-binding portion of the antibody to the recognition portion of the target protein can be greatly reduced, or even lost.

[0007] For both reasons i) to enable coupling of the antibody to the lipid surface of the LNP, and ii) to create distance between the antibody and the LNP membrane, in the prior art, the antibody is coupled by the addition of a coupling moiety (generally a linked PEG-type group), the coupling moiety comprising a lipophilic portion for anchoring the complex [PEG group-antibody] to the lipid membrane. And therefore, lipid nanoparticles of the prior art contain such coupling groups for anchoring the complex [PEG group-antibody] to the lipid membrane.

[0008] More precisely, targeting agents, such as antibodies, can be conjugated to lipid nanoparticles by two approaches, including 1) a “one-pot” assembly of ionizable / cationic lipids, helper lipids, sterols, and PEG-lipids, the latter of which must be pre-decorated with the targeting agent, and 2) “post-inserting” the targeting ligand into pre-existing simple lipid nanoparticles (which have functionalized PEG-lipids ready to be conjugated with the targeting agent at any time).

[0009] These methods require an additional step in the targeted LNP synthesis process, either before or after LNP formation. This additional step complicates the production of targeted LNPs, as it induces not only additional costs but also supplementary burdens for implementing the method. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] WO 2016 / 021683 [Patent Document 2] WO 2015 / 011633 [Patent Document 3] WO 2011 / 153493 [Patent Document 4] WO 2013 / 126803 [Patent Document 5] WO 2010 / 054401 [Patent Document 6] WO 2010 / 042877 [Patent Document 7] WO 2016 / 104580 [Patent Document 8] WO 2015 / 005253 [Patent Document 9] WO 2014 / 007398 [Patent Document 10] WO 2017 / 117528 [Patent Document 11] WO 2017 / 075531 [Patent Document 12] WO 2017 / 00414 [Patent Document 13] WO 2015 / 199952 [Patent Document 14] U.S. Patent Application Publication No. 2015 / 0239834 [Patent Document 15] WO2019 / 131839 [Patent Document 16] WO2022 / 261101 [Patent Document 17] U.S. Patent No. 5,023,243 [Patent Document 18] WO21159130 [Patent Document 19] WO2013052523 [Patent Document 20] WO2014093924 [Patent Document 21] WO2015051173 [Patent Document 22] WO2015051169 [Patent Document 23] WO2015089511 [Patent Document 24] WO2015196130 [Patent Document 25] WO2015196118 [Patent Document 26] WO2015196128 [Patent Document 27] WO2017153936 [Patent Document 28] WO2014 / 186334 [Patent Document 29] WO2022 / 261490 [Patent Document 30] WO 2009 / 080253 [Patent Document 31] WO2020 / 127366 [Patent Document 32] WO 2014 / 194302 [Patent Document 33] WO 2017 / 040790 [Patent Document 34] WO 2017 / 19846 [Patent Document 35] WO 2017 / 024465 [Patent Document 36] WO 2017 / 025016 [Patent Document 37] WO 2017 / 132825 [Patent Document 38] WO 2017 / 133540 [Patent Document 39] WO 2006 / 121168 [Patent Document 40] WO2013006490 [Patent Document 41] WO2016 / 161270 [Patent Document 42] WO 2018 / 085469 [Patent Document 43] WO 2018 / 129553 [Patent Document 44] WO 2011 / 155607 [Patent Document 45] U.S. Patent No. 8,552,156 [Patent Document 46] EP 2581113 [Patent Document 47] U.S. Patent Application Publication No. 2014 / 044728 [Patent Document 48] WO18025178

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[0012] Therefore, there is room to improve the production of targeted LNPs (t-LNPs) without sacrificing their targeting or transfection capabilities. [Means for solving the problem]

[0013] The inventors have unexpectedly discovered a simple and efficient method for producing targeted lipid-based nanoparticles by a mixing system, which involves adding an antigen-binding domain to either a lipid-based composition or a nucleic acid-based composition before mixing the two compositions using a mixing system. This method can be performed in a single step and is cost-effective.

[0014] Surprisingly, mixing the antigen-binding domain with either a lipid-based composition or a nucleic acid composition does not hinder LNP formation, and the LNPs obtained by this process exhibit excellent targeting ability. Furthermore, the majority of the antigen-binding domain in t-LNPs is available for antigen recognition. In addition, this method produces a composition without any free antibodies, meaning that, other than the mixing and subsequent solvent exchange steps, this method generally does not require any supplemental purification steps.

[0015] Compared to the method of the present invention, the preparation of t-LNPs using "one-pot" assemblies such as those described above has several drawbacks. The synthesis yield of antibody-lipid conjugates is variable, and the purification step often requires separating the antibody-lipid conjugate from the unconjugated antibody. The resulting antibody-lipid is a large molecule that can interfere with LNP formation. Antibody-lipid conjugates are fragile and can be modified during the production process (organic solvents, mechanical forces), and may become trapped within the LNP rather than on its surface, thereby ultimately rendering its targeting properties inactive.

[0016] Compared to the method of the present invention, the "post-insertion" method requires the use of a fifth component in LNPs having a functionalized PEG lipid (e.g., DMG-PEG-maleimide). Antibody reduction is often required before coupling, which can alter the functionality of the antibody. The number of steps required to carry out this method is also greater than that of the present invention. Finally, this method does not enable coupling of all antibodies, and a purification step is often required to remove unbound antibodies.

[0017] The method described herein can be applied to the generation of multiple LNPs having various antigen-binding domains without the need to prepare the LNP and / or the nucleic acid molecule being carried.

[0018] As described in detail in this application, the applicant now has a novel LNP particle, in particular a revolutionary RNA-containing LNP that exhibits highly satisfactory specific targeting properties but lacks coupling / anchor groups and / or is not covalently bound to any of the lipids of the LNP, and contains at least one targeting entity (preferably at least one antibody or antigen-binding moiety).

[0019] In a first aspect, the present invention relates to a method for producing lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules, - A step of supplying a first composition comprising a lipid-based composition and a polar organic solvent through a first inlet of a mixing device. - A step of supplying a second composition comprising one or more nucleic acid molecules through a second inlet of the mixing device, wherein the second composition is an acidic aqueous composition. - A step of mixing the first composition and the second composition in a mixing device to generate lipid-based nanoparticles. - A step of removing polar organic solvents from the mixed composition and adjusting the pH of the composition to a neutral pH, and - A step to recover lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes; Prior to the mixing step of the first and second compositions, the antigen-binding domain is mixed with the first or second composition. Regarding the method.

[0020] The present invention relates to a method for producing lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules, - A step of supplying a first composition comprising a lipid-based composition and a polar organic solvent through a first inlet of a mixing device. - A step of supplying a second composition comprising one or more nucleic acid molecules through a second inlet of the mixing device, wherein the second composition is an acidic aqueous composition. - A step of mixing the first composition and the second composition in a mixing device to generate lipid-based nanoparticles. - A step of removing polar organic solvents from the mixed composition and adjusting the pH of the composition to a neutral pH, and - A step to recover lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes; Prior to the step of mixing the first and second compositions in a mixing device, the antigen-binding domain is mixed with the first or second composition; The antigen-binding domain is an antibody or its antigen-binding fragment. It also depends on the method.

[0021] Preferably, the step of removing the polar solvent is carried out by dialysis or buffer exchange.

[0022] Preferably, the pH of the second composition is between about 3 and about 6, preferably between about 4 and about 5, and more preferably between about 4 and about 4.5.

[0023] Preferably, pH adjustment to a neutral pH is an adjustment to a pH in the range of about 6.5 to about 7.5, preferably to 7.

[0024] Lipid-based compositions include, in particular, cationic or ionizable lipids, helper lipids, sterols, and PEG-lipids.

[0025] The ionizable lipids are preferably [(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 Selected from the group consisting of dodecane-2-ol (lipidoid C12-200), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) or bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide (SS-OP), 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.

[0026] 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.

[0027] Preferably, the helper lipid is selected from the group consisting of DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DEPC, DOPC, and DSPE, and is preferably DOPE or DSPC.

[0028] Preferably, the PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG, and PEG-c-DMA, ALC-0159, and any mixture thereof, and is preferably PEG-DMG, PEG-DSPE, or a mixture thereof.

[0029] The PEG is generally between 2000 Daltons and 5000 Daltons, preferably DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000, or a mixture thereof. The first composition or lipid-based composition of lipid-based nanoparticles is particularly, 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; and 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.

[0030] Preferably, the lipid-based composition contains about 10 mol% to about 70 mol% of cationic or ionizable lipids, about 5 mol% to about 70 mol% of helper lipids, about 10 mol% to about 70 mol% of sterols, and about 0.5 mol% to about 4 mol% of PEG-lipids.

[0031] More preferably, the lipid-based composition contains or comprises 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.

[0032] The lipid concentration in the first composition is, in particular, about 1 to about 100 mM.

[0033] The concentration of nucleic acid molecules in the second composition is preferably about 0.01 mg / mL to about 100 mg / mL.

[0034] In some embodiments, the antigen-binding domain is mixed with the first composition before the mixing step of the first and second compositions, and the concentration of the antigen-binding domain in the first composition is about 0.01 μg / μL to about 0.5 μg / μL.

[0035] In some other embodiments, the antigen-binding domain is mixed with the second composition before the mixing step of the first and second compositions, and the concentration of the antigen-binding domain in the second composition is about 0.005 μg / μL to about 0.25 μg / μL.

[0036] In some embodiments, the first composition comprises an ionizable lipid, the second composition has a pH lower than the pKa of the ionizable lipid, and / or the second composition has a pH between about 3 and about 6.

[0037] In some embodiments, the flow rate ratio of the first composition to the second composition is between 1:10 and 1:1, preferably between 1:7 and 1:1, and more preferably between 1:4 and 1:1.

[0038] The antigen-binding domain preferably includes an Fc domain, and more preferably the antigen-binding domain is IgG, and more preferably monoclonal IgG.

[0039] Preferably, the antigen-binding domain is 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 / T NFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, 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, C D39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 It binds to a target selected from the group consisting of / 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, CD127, SIRPa, and CD160.

[0040] Preferably、antigen binding domainは、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 / T NFSF6、GITR / TNFRSF18、GPR32、TIM3 / HAVCR2、ICOS、IL18R1 / CXCR1 / CD218a、ITGAE、LAG3、TRAILR、OX40L、LY108 / SlamF6、NKG2D、OX40 / TNFRSF4、PD-1、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 / TNFRSF18 11 / CD120B、CDCR3 / TNFRSF6B、TNFRSF12A / FN14 / TWEAKR、BAFFR / TNFRSF13C / CD268、WHEM / 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;The target is selected from the group consisting of the following, preferably a target selected from the group including CD137 / 41BB / TNFRSF9, PD-1, CRTAM, CTLA4, FasL / TNFSF6, TIM-3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4, and TIGIT.

[0041] In particular, the antigen-binding domain binds to a target selected from the group consisting of PD-1, CD127, SIRPa, and CLEC-1A. Preferably, the antigen-binding domain binds to human PD-1.

[0042] In particular, the antigen-binding domain is (i) VH including HCDR1, HCDR2 and HCDR3, (ii) VL including LCDR1, LCDR2 and LCDR3 An anti-PD-1 antigen binding domain comprising, a) - 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) - 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 c) - 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 - Light chain CDR3 (LCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 36. This is the anti-PD-1 antigen binding domain.

[0043] In some embodiments, the antigen-binding domain includes an Fc domain, preferably an IgG Fc domain.

[0044] Preferably, the antigen-binding domain is not covalently bound to any of the lipids in the lipid-based nanoparticles, and does not involve any modifications for coupling or grafting the antigen-binding domain to the lipids. Alternatively, the lipid-based nanoparticles do not contain an anchor moiety that includes a lipidized peptide or motif.

[0045] In some embodiments, the lipid-based nanoparticles include additional antigen-binding domains.

[0046] In particular, the first or second composition is mixed with a second antigen-binding domain before the mixing step of the first and second compositions, wherein the second antigen-binding domain is an antibody or an antigen-binding fragment thereof.

[0047] Preferably, the nucleic acid molecule is mRNA.

[0048] In particular, nucleic acid molecules, a) Immune cell-enhancing or inhibitory compounds, in particular, those described herein, preferably selected from the following: - TCF1, LEF1, WNT, FRIZZLED, Beta-catenin, BCL2, BCLXL, BIRC3, MCL1, PGC1a, TCF7, NFAT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glut1, Glut3, HK2, Arginine resynthesis enzyme, Argininosuccinate synthase (ASS) ), ornithine transcarbamylase (OTC), GYS, OXPHOS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD107a, lymphotoxin (LT) α1β2, granzyme B, perforin, POU2F1, BBS10, BBS12, TCP1, HSP, integrin alpha 1, integrin alpha 2, integrin alpha 2b, integrin alpha 11, integrin alpha 3, integrin alpha 6, Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, LFA-1, LFA-2, LFA-3, Integrin Beta 1, Integrin Beta 7, CD103, Integrin Alpha V, ITGAE, CD11C, CRTAM, SELL, GAL3, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Rab Protein, NLRP3, TAP, LAMP, Ubiquitin Ligase, CD74, Peptid -se, calreticulin, aurora, LGR6, HAT, KDM1, TGD, TET1, C-FOS, JUN, EGR-2, EGR-3, phosphatidylinositol 3-kinase (PI3K), BCLXL, GPR35, interferon regulators, e.g., IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, NFκB, T-bet, AhR, STING, MAVS, MyD88, IRAK1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKα, IKKβ, TRAM, TRIF, TBK1, D3-phosphoinositide, phosphatidylinositol derivatives, DECTIN, CD122, CD132, CD25, CD215, -1, CLEC-9A, CLEC-2, DECTIN-2, MCL, MINCLE, BDCA-2, ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, OX40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAM Compounds or molecules selected from the group consisting of the CD2 family, CD27, TL1A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, CD160, and any combination thereof, in particular, those described herein, for example; - PUMA, BIMS, 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-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, 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, Lamini 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, SUV420H2, TGFR, TNFR, GCSFR, CD72, DCIR, MICL, CLEC-1, TGFB, IFNa Compounds or molecules selected from the group consisting of R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM4, 2B4, 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, A2AR, SOCS, RIPK1, and any member of the STAT family;Furthermore, any combination thereof, preferably compounds or molecules 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, in particular, those described herein, for example; b) Cytokines, preferably selected from the group consisting of IL12A, IL12B, IFNG, IFNa, IL21, IL7, IL2, IL15, IL13, IL4, IL-10, IL35, IL37, IL38, and IL18, and any combination thereof, in particular, as described herein; c) Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-17R, IL-18R, IL-21R, IL-23R, and IL-35R; d) Chemokines, preferably selected from the group consisting of CXCL9 or CXCL10; e) 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; f) Antigen fragments derived from viral pathogens, bacterial pathogens and / or parasitic pathogens, in particular, those described herein, for example; g) Antibodies or fragments or derivatives thereof, preferably antibodies or fragments or derivatives thereof against targets selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, LAG3, BTLA, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, CD39, CD30, NKG2D, NKG2A, 2B4, DR3, CD101, CD44, SIRPG, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122; and any combination thereof; in particular, for example, those described herein, and h) Chimeric antigen receptors (CARs), in particular those described herein, for example. This is an mRNA that codes for a molecule selected from the group consisting of the following:

[0049] In particular, the mRNA encodes BCL2, IL7, IL7R, CXCL9, and / or CXCL10.

[0050] In some embodiments, the second composition comprises at least two different mRNA molecules. In particular, the second composition comprises an mRNA molecule encoding IL-7 and an mRNA molecule encoding IL-7R.

[0051] In certain embodiments, the mixing device is a microfluidic device.

[0052] The present invention also relates to lipid-based nanoparticles obtained or obtainable by the method of the present invention. The present invention also relates to lipid-based nanoparticles obtained or obtainable by the method of the present invention for use as pharmaceuticals or vaccines.

[0053] In some embodiments, the lipid-based nanoparticles obtained or achievable by the method of the present invention include cationic or ionizable lipids, helper lipids, sterols, PEG-modified lipids, one or more nucleic acid molecules, preferably mRNA molecules, and one or more antigen-binding domains.

[0054] Preferably, the lipid-based nanoparticles include an antigen-binding domain that is not covalently bound to any of the lipids in the lipid-based nanoparticles, or an antigen-binding domain that does not include any modifications for coupling or grafting the antigen-binding domain to the lipids.

[0055] In particular, lipid-based nanoparticles do not contain a) an antigen-binding domain covalently bound to a lipid, ii) an antigen-binding domain including modifications for coupling or grafting the antigen-binding domain to a lipid, and / or iii) an anchor portion including a lipidized peptide or motif.

[0056] Preferably, the lipid-based nanoparticles include one or more mRNA molecules encoding an immune cell-enhancing compound, an antigen fragment, a chimeric antigen receptor (CAR), an antigen-binding domain, or an antibody, such as an antigen-binding domain or antibody against a checkpoint inhibitor.

[0057] The present invention also relates to lipid-based nanoparticles obtained or obtainable by the method of the present invention for use as pharmaceuticals or vaccines. The present invention further relates to pharmaceutical compositions comprising lipid-based nanoparticles obtained by the method of the present invention and, optionally, a pharmaceutically acceptable carrier.

[0058] The present invention also relates to lipid-based nanoparticles, or pharmaceutical compositions comprising the lipid-based nanoparticles, for use in the treatment of cancer, infectious diseases, autoimmune diseases, or inflammatory diseases.

[0059] The present invention also relates to the use of lipid-based nanoparticles, which are lipid-based nanoparticles obtained or obtainable by the method of the present invention, or pharmaceutical compositions containing them, for the manufacture of pharmaceuticals for the treatment of cancer, infectious diseases, autoimmune diseases, or inflammatory diseases.

[0060] Finally, the present invention relates to a method for treating cancer, infectious disease, autoimmune disease or inflammatory disease in a subject, comprising the step of administering to the subject a lipid-based nanoparticle or a pharmaceutical composition containing the same obtained or obtainable by the method of the present invention.

[0061] The present invention will be better understood in consideration of the drawings and the following detailed description thereof. [Brief explanation of the drawing]

[0062] [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 the anti-PD-1 mAb OSE-279 prior to LNP formation enables the preparation of targeted LNPs with improved transfection efficacy in PD-1-positive cells. Different modalities for the addition of mAb OSE-279 during the LNP production process were tested. The transfection efficiency of LNPs encapsulating FLuc-mRNA was evaluated by comparing modalities #1A, #2, and #3 in WT and PD-1+U937 cells (Figure 2A) or in WT and PD-1+Jurkat cells (Figure 2B). Similarly, transfection efficiency was compared for addition modalities #1A, #1B, and #1C in WT and PD-1+U937 cells (Figure 2C) and in WT and PD-1+Jurkat cells (Figure 2D). 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 10)). 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-positive 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 gel filtration chromatography of the t-LNP solution demonstrated the absence of free mAb OSE-279. Size exclusion chromatograms were recorded and compared for i) an immediate mixture of nt-LNP and OSE-279 (top figure) and ii) a solution of t-LNP prepared as previously described using 15 μL of OSE-279 (bottom figure). [Figure 8]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 FLuc-mRNA-encapsulating LNPs was compared for pembrolizumab t-LNPs, nivolumab t-LNPs, and OSE-279 t-LNPs (with or without the anti-PD-1 pre-incubation step) in WT and PD-1+ U937 cells (Figure 8A), WT and PD-1+ Jurkat cells (Figure 8B), and HPB-ALL cells (Figure 8C). Untargeted LNPs and targeted LNPs prepared with a control isotype mAb were used as negative controls. [Figure 9] This figure shows that targeted LNPs can more efficiently transfect PD-1-expressing activated T cells 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 9A). 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 9B and 9C). [Figure 10] This figure shows that the receptor targeted by a targeted LNP can be changed by altering the mAb introduced into the targeted 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. The percentage of PD-1+ and Target 2+ cells was measured using flow cytometry (Figure 10A). 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) to measure PD-1+ cells, or with PE-Cy7-labeled anti-human Target 2 antibody to measure Target 2+ cells. Dead cells were stained using the LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from the 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 10B-10C) and HPB-ALL cells (Figures 10D-10E), which express both target 2 and PD-1 receptors. Untargeted LNPs and targeted LNPs prepared using a control isotype mAb were used as negative controls. Target 2 is CD127. [Figure 11A] In vivo and ex vivo distribution of LNPs: This figure shows the in vivo distribution of LNP#1 and LNP#2. [Figure 11B] The figure shows the in vivo and ex vivo distribution of LNPs: the ex vivo distribution of LNP#1 and LNP#2. [Figure 11C] In vivo and ex vivo distribution of LNPs: This figure shows the in vivo distribution of LNP#3 and LNP#4. [Figure 11D] This figure shows the in vivo and ex vivo distribution of LNPs: LNP#3 and LNP#4. [Figure 12A] This figure shows the ex vivo in vivo distribution of LNPs in the liver, lungs, and spleen. [Figure 12B] This figure shows the ex vivo in vivo distribution of LNPs in the liver, lungs, and spleen. [Figure 13] This figure shows the in vivo and ex vivo distribution of untargeted and targeted LNPs. [Figure 14] 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 14A). 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 using the LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and excluded from the analysis. The transfection efficiency of anti-human CD-127 targeted LNPs was evaluated in U937 cells (Figure 14B) and HPB-ALL cells (Figure 14C), which express the CD-127 receptor. Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 15]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 15A). 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 15B) and THP-1 (Figure 15C), cells expressing the CLEC-1 receptor. Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 16]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 16A). 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 16B). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 17]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 17A). 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 17B), which express both PD-1 receptors and CD-127 receptors. Untargeted LNPs and targeted LNPs prepared using a control isotype mAb were used as negative controls. [Figure 18] 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 18A) and HPB-ALL cells (Figure 18B). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 19]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 20] This figure shows that different lipid tail 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 20A) and Jurkat cells (Figure 20B). Untargeted LNPs and targeted LNPs prepared using control isotype mAbs were used as negative controls. [Figure 21] 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 21A-21D), Jurkat cells (Figures 21E-21H), and HPB-ALL (Figures 21I-21K). [Figure 22]This figure shows a comparison of the transfection efficiency and binding of two types of OSE-279-targeted LNPs prepared by either the method of the present invention or thiol-Michael addition. To evaluate the effectiveness of the method of the present invention, experiments were conducted comparing this novel process with classical methods used to graft mAbs (by thiol-Michael addition) onto the surface of LNPs. First, the thiol-maleimide reaction process was optimized by testing different experimental conditions by changing the amount of DSPE-PEG-maleimide in the LNPs (Figures 22A-22C) and the amount of OSE-279 mAb added to the reaction (Figures 22D-22F). Next, using the optimized reaction conditions, the transfection ability of OSE-279-targeted LNPs prepared either via the thiol-maleimide pathway or by the method of the present invention was compared in Jurkat (Figure 22G) and HPB-ALL (Figure 22H) cells. Binding studies of the two types of targeted LNPs were also evaluated (Figure 22I). Figure 22J shows the prior art synthesis process for targeted LNPs, while Figure 22K shows the synthesis process for targeted LNPs according to one embodiment of the present invention. [Figure 23] 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 23A), Jurkat (Figure 23B), and HPB-ALL (Figure 23C). Untargeted LNPs and targeted LNPs prepared using a control isotype mAb were used as negative controls. [Figure 24]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 24A), Jurkat (Figure 24B), and HPB-ALL (Figure 24C). Untargeted LNPs and targeted LNPs prepared using a control isotype mAb were used as negative controls. [Figure 25] 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 25A, 25C, and 25E) and PUMA (Figures 25B, 25D, and 25F) 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 25A-25B), Jurkat (Figures 25C-25D), and HPB ALL (Figures 25E-25F). 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 26]This figure shows that increased expression of the anti-apoptotic BCL-2 protein is observed in PD-1 expressing cells transfected with targeted LNPs containing mRNA encoding the BCL-2 protein. mRNA encoding the anti-apoptotic protein BCL-2 (B-cell lymphoma-2) was produced by IVT and encapsulated in untargeted LNPs and OSE-279 targeted LNPs. Transfection experiments were performed in wild-type cells (WT) and in PD-1 expressing cell lines U937 (Figures 26A-26B) and Jurkat (Figures 26C-26D). [Figure 27] This figure shows that increased expression of the CXCL-9 chemokine is observed in PD-1 expressing cells transfected with targeted LNPs containing mRNA encoding CXCL-9. mRNA encoding chemokine ligand-9 (CXCL-9) was produced by IVT and encapsulated in untargeted LNPs and OSE-279 targeted LNPs. Transfection experiments were performed in WT cells and PD-1 expressing Jurkat cells (Figures 27A-27B). [Figure 28] This figure shows that increased expression of the CXCL-10 chemokine is observed in PD-1 expressing cells transfected with targeted LNPs containing mRNA encoding CXCL-10. mRNA encoding chemokine ligand-10 (CXCL-10) was produced by IVT and encapsulated in untargeted LNPs and OSE-279 targeted LNPs. Transfection experiments were performed in wild-type cells and PD-1 expressing U937 cells (Figures 28A-28B). [Figure 29] This figure shows that increased luciferase expression was observed ex vivo in mice treated with PD-1-targeted LNPs in an EL-4 tumor model. Mice were treated with EL-4 cells transduced using human PD-1, and then treated with OSE-279-targeted and untargeted LNPs on day 16. Bioluminescence was measured ex vivo in the tumors (Figures 29A-29D). [Figure 30A]This figure shows that sequential transfection with LNPs containing IL-7Ra mRNA and IL-7 mRNA induces a stronger pSTAT5 signal for OSE-279-targeted LNPs in PD-1-positive cells compared to untargeted LNPs or WT cells. Cell phenotypes regarding spontaneous CD-127 and CD-132 expression were evaluated at the start of the experiment: U937 cells spontaneously express CD-132 but not CD-127, while Jurkat cells spontaneously express low levels of CD-132 but not CD-127. WT or PD-1-positive cells were first transfected with OSE-279-targeted LNPs or untargeted LNPs containing IL-7Ra mRNA. After 24 hours, the percentage of CD-127-positive cells was measured, and a second round of transfection was performed using the same cells, this time with OSE-279-targeted or untargeted LNPs containing IL-7 mRNA. After 4 hours, a pSTAT5 assay was performed to evaluate specific cell activation via the IL-7 pathway. [Figure 30B] This figure shows that sequential transfection with LNPs containing IL-7Ra mRNA and IL-7 mRNA induces a stronger pSTAT5 signal for OSE-279-targeted LNPs in PD-1-positive cells compared to untargeted LNPs or WT cells. Cell phenotypes regarding spontaneous CD-127 and CD-132 expression were evaluated at the start of the experiment: U937 cells spontaneously express CD-132 but not CD-127, while Jurkat cells spontaneously express low levels of CD-132 but not CD-127. WT or PD-1-positive cells were first transfected with OSE-279-targeted LNPs or untargeted LNPs containing IL-7Ra mRNA. After 24 hours, the percentage of CD-127-positive cells was measured, and a second round of transfection was performed using the same cells, this time with OSE-279-targeted or untargeted LNPs containing IL-7 mRNA. After 4 hours, a pSTAT5 assay was performed to evaluate specific cell activation via the IL-7 pathway. [Figure 30C] This figure shows that sequential transfection with LNPs containing IL-7Ra mRNA and IL-7 mRNA induces a stronger pSTAT5 signal for OSE-279-targeted LNPs in PD-1-positive cells compared to untargeted LNPs or WT cells. Cell phenotypes regarding spontaneous CD-127 and CD-132 expression were evaluated at the start of the experiment: U937 cells spontaneously express CD-132 but not CD-127, while Jurkat cells spontaneously express low levels of CD-132 but not CD-127. WT or PD-1-positive cells were first transfected with OSE-279-targeted LNPs or untargeted LNPs containing IL-7Ra mRNA. After 24 hours, the percentage of CD-127-positive cells was measured, and a second round of transfection was performed using the same cells, this time with OSE-279-targeted or untargeted LNPs containing IL-7 mRNA. After 4 hours, a pSTAT5 assay was performed to evaluate specific cell activation via the IL-7 pathway. [Figure 30D] This figure shows that sequential transfection with LNPs containing IL-7Ra mRNA and IL-7 mRNA induces a stronger pSTAT5 signal for OSE-279-targeted LNPs in PD-1-positive cells compared to untargeted LNPs or WT cells. Cell phenotypes regarding spontaneous CD-127 and CD-132 expression were evaluated at the start of the experiment: U937 cells spontaneously express CD-132 but not CD-127, while Jurkat cells spontaneously express low levels of CD-132 but not CD-127. WT or PD-1-positive cells were first transfected with OSE-279-targeted LNPs or untargeted LNPs containing IL-7Ra mRNA. After 24 hours, the percentage of CD-127-positive cells was measured, and a second round of transfection was performed using the same cells, this time with OSE-279-targeted or untargeted LNPs containing IL-7 mRNA. After 4 hours, a pSTAT5 assay was performed to evaluate specific cell activation via the IL-7 pathway. [Figure 30E] This figure shows that sequential transfection with LNPs containing IL-7Ra mRNA and IL-7 mRNA induces a stronger pSTAT5 signal for OSE-279-targeted LNPs in PD-1-positive cells compared to untargeted LNPs or WT cells. Cell phenotypes regarding spontaneous CD-127 and CD-132 expression were evaluated at the start of the experiment: U937 cells spontaneously express CD-132 but not CD-127, while Jurkat cells spontaneously express low levels of CD-132 but not CD-127. WT or PD-1-positive cells were first transfected with OSE-279-targeted LNPs or untargeted LNPs containing IL-7Ra mRNA. After 24 hours, the percentage of CD-127-positive cells was measured, and a second round of transfection was performed using the same cells, this time with OSE-279-targeted or untargeted LNPs containing IL-7 mRNA. After 4 hours, a pSTAT5 assay was performed to evaluate specific cell activation via the IL-7 pathway. [Figure 30F] This figure shows that sequential transfection with LNPs containing IL-7Ra mRNA and IL-7 mRNA induces a stronger pSTAT5 signal for OSE-279-targeted LNPs in PD-1-positive cells compared to untargeted LNPs or WT cells. Cell phenotypes regarding spontaneous CD-127 and CD-132 expression were evaluated at the start of the experiment: U937 cells spontaneously express CD-132 but not CD-127, while Jurkat cells spontaneously express low levels of CD-132 but not CD-127. WT or PD-1-positive cells were first transfected with OSE-279-targeted LNPs or untargeted LNPs containing IL-7Ra mRNA. After 24 hours, the percentage of CD-127-positive cells was measured, and a second round of transfection was performed using the same cells, this time with OSE-279-targeted or untargeted LNPs containing IL-7 mRNA. After 4 hours, a pSTAT5 assay was performed to evaluate specific cell activation via the IL-7 pathway. [Figure 31] This figure shows that OSE-279-targeted LNPs result in higher transfection and luciferase expression in activated T cells expressing the PD-1 receptor compared to untargeted LNPs. The percentage of PD-1-positive cells was determined by flow cytometry analysis of activated human PBMCs (stimulated with PHA / IL-2) (Figure 31A). Dashed line: Live cells were unstained. Dark gray: Cells were stained using PE-labeled anti-human PD-1 antibody (#130-117-384 batch: 5230902507, Miltenyi). Dead cells were stained using LIVE / DEAD® Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2622316, Life Technologies) and excluded from the analysis. The transfection efficiency of OSE-279-targeted LNPs to activated T cells was compared with that of untargeted LNPs (Figure 31B). [Modes for carrying out the invention]

[0063] Detailed description of the invention This disclosure relates to a method for producing lipid nanoparticles comprising one or more antigen-binding domains and one or more nucleic acid molecules. This disclosure also relates to the lipid nanoparticles produced by the said method and their use as pharmaceuticals, in particular for treating diseases such as cancer.

[0064] definition To facilitate understanding of this invention, certain terms are defined below. Additional definitions are provided throughout the detailed description.

[0065] 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.

[0066] As used herein, the term "t-LNP" refers to targeted lipid nanoparticles, i.e., lipid nanoparticles having an antigen-binding domain. The term "nt-LNP" refers to lipid nanoparticles lacking an antigen-binding domain.

[0067] 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 molecule. The heavy chain constant domains corresponding to different classes of immunoglobulin are called alpha, delta, epsilon, gamma, and mu, respectively. The term “antibody” specifically refers to an immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that binds immunospecifically to an antigen, and an immunologically active portion of such an immunoglobulin molecule. 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.

[0068] The terms “antigen-binding fragment” or “antigen-binding domain” of an antibody (or simply “antibody fragment”), as used herein, refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., PD-1).

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] "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).

[0076] "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.

[0077] 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.

[0078] [Table 1]

[0079] [Table 2]

[0080] [Table 3]

[0081] 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.

[0082] 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).

[0083] 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%).

[0084] 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.

[0085] Synthesis method In a first aspect, the present invention relates to a method for producing lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules.

[0086] Methods for producing such lipid-based nanoparticles are: - A step of supplying a first composition comprising a lipid-based composition and a polar organic solvent through a first inlet of a mixing device. - A step of supplying a second composition comprising one or more nucleic acid molecules through a second inlet of the mixing device, wherein the second composition is an acidic aqueous composition. - A step of mixing the first composition and the second composition in a mixing device to generate lipid-based nanoparticles. - A step of removing polar organic solvents from the mixed composition and adjusting the pH of the composition to a neutral pH, and - A step to recover lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes; The antigen-binding domain is mixed with the first or second composition before the step of mixing the first and second compositions in a mixing device.

[0087] The method according to the present invention can be carried out using any type of known mixing device for LNP generation or any type used for LNP generation. Mixing devices suitable for industrial-scale production are particularly preferred.

[0088] Suitable mixing devices include, but are not limited to, T-type mixers, U-type mixers, V-type mixers, NanoAssembler (Precision Nanosystems), Impigement Jet Mixing (Knauer), and Microfluidizer M700 or M805 (Microfluidics International).

[0089] Other suitable mixing devices may be considered if they enable production that meets industrial-scale needs.

[0090] Such a suitable mixing device includes one, some, or all of the following features: - Flow rate capacity greater than approximately 500 mL / min, preferably greater than approximately 1000 mL / min, preferably greater than approximately 1500 mL / min, preferably greater than approximately 2000 mL / min, preferably greater than approximately 2500 mL / min, preferably greater than approximately 3000 mL / min, preferably greater than approximately 3500 mL / min, preferably greater than approximately 4000 mL / min, - Injection tube inner diameter greater than approximately 0.1 mm, preferably greater than approximately 0.2 mm, preferably greater than approximately 0.3 mm, preferably greater than approximately 0.4 mm, preferably greater than approximately 0.5 mm, preferably greater than approximately 1 mm, preferably greater than approximately 1.5 mm, - Outlet pipe inner diameter, preferably greater than approximately 0.1 mm, preferably greater than approximately 0.2 mm, preferably greater than approximately 0.3 mm, preferably greater than approximately 0.4 mm, preferably greater than approximately 0.5 mm, preferably greater than approximately 1 mm, preferably greater than approximately 1.5 mm, preferably greater than approximately 2 mm, preferably greater than approximately 2.5 mm, preferably greater than approximately 3 mm, preferably greater than approximately 3.5 mm, preferably greater than approximately 4 mm, preferably greater than approximately 4.5 mm, preferably greater than approximately 5 mm, preferably greater than approximately 5.5 mm, preferably greater than approximately 6 mm, preferably greater than approximately 6.5 mm, preferably greater than approximately 7 mm, preferably greater than approximately 7.5 mm, preferably greater than approximately 8 mm, preferably greater than approximately 8.5 mm, preferably greater than approximately 9 mm, preferably greater than approximately 9.5 mm, preferably greater than approximately 10 mm - Entrance back force, greater than approximately 10 psi, preferably greater than 20 psi, preferably greater than 30 psi, preferably greater than 40 psi, preferably greater than 50 psi, - A Reynolds number greater than approximately 2000.

[0091] Alternatively, the process can be carried out using a microfluidic system. In such cases, the mixing device is a microfluidic device. Examples of suitable microfluidic devices include microfluidic mixer systems (NanoAssemblr Spark, Ignite, or Blaze from Precision NanoSystems, Vancouver, BC, Canada), microfluidic micromixtures (Precision NanoSystems) as described by Cohen et al., 2015 ACS nano 9(2): pp. 1581-1591, or any device that uses mixing techniques for producing lipid-based nanoparticles, such as cross-flow injection, microfluidic bifurcated mixing, staggered herringbone mixing, or hydrodynamic convergent mixing of microfluidics, but is not limited to these. Preferably, the microfluidic device is a microfluidic mixer, for example, the LNP Pack offered by Inside Therapeutics.

[0092] Alternatively, the process can be carried out using a parallel microfluidic device, which is a series of microfluidic mixing devices operating simultaneously.

[0093] In another aspect of the present invention, a method for producing lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules is: - A step of supplying a first composition, including a lipid-based composition, through a first inlet of a mixing device. - A step of supplying a second composition comprising one or more nucleic acid molecules through a second inlet of the mixing device, - A step of mixing the first composition and the second composition in a mixing device to generate lipid-based nanoparticles, and - A step to recover lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes; The antigen-binding domain is mixed with the first or second composition before the step of mixing the first and second compositions in a mixing device.

[0094] 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 22J to 22K.

[0095] First composition In the method according to the present invention, the first composition, which includes a lipid-based composition, includes a polar organic solvent, preferably a polar protic organic solvent.

[0096] Examples of polar organic solvents include, but are not limited to, ethanol, methanol, n-propanol, isopropyl alcohol, n-butanol, acetic acid, formic acid, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, and propylene carbonate, preferably ethanol, methanol, n-propanol, isopropyl alcohol, n-butanol, acetic acid, and formic acid. Polar organic solvents are miscible with water.

[0097] Preferably, the polar organic solvent is an alcohol, preferably ethanol.

[0098] In some embodiments, the first composition comprising a lipid-based composition comprises about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 97%, or about 100% by volume of a polar organic solvent, preferably ethanol.

[0099] In some embodiments, the polar organic solvent is isopropyl alcohol. For example, a first composition comprising a lipid-based composition contains, by volume, about 10% isopropyl alcohol, about 15% isopropyl alcohol, about 20% isopropyl alcohol, about 25% isopropyl alcohol, about 30% isopropyl alcohol, about 35% isopropyl alcohol, or about 40% isopropyl alcohol.

[0100] In some embodiments, the polar organic solvent is dimethyl sulfoxide. For example, a first composition comprising a lipid-based composition contains, by volume, about 10% dimethyl sulfoxide, about 15% dimethyl sulfoxide, about 20% dimethyl sulfoxide, about 25% dimethyl sulfoxide, about 30% dimethyl sulfoxide, about 35% dimethyl sulfoxide, or about 40% dimethyl sulfoxide.

[0101] In some embodiments, the lipid concentrations in the lipid-based composition are approximately 1 mM to 100 mM, 2 mM to 95 mM, 3 mM to 90 mM, 4 mM to 85 mM, 5 mM to 80 mM, 10 mM to 75 mM, 15 mM to 70 mM, 20 mM to 60 mM, and 30 mM to 50 mM.

[0102] Lipid-based compositions are defined in more detail below under the section "Lipid-based compositions".

[0103] The components of the lipid-based composition or the first composition may be selected based on the specific application or target of the final LNP to be obtained, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components.

[0104] In certain embodiments, the lipid-based composition or the first composition comprises 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.

[0105] In some embodiments, the lipid-based composition or the first composition comprises 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.

[0106] The lipid-based composition or the first composition generally comprises ionizable or cationic lipids, helper lipids, sterols and / or PEG lipid components. In one embodiment, the ionizable or cationic lipid comprises a head group having a positive charge or being protonable, containing at least one nitrogen atom (N).

[0107] 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.

[0108] The ionizable or cationic lipids contained in the lipid-based composition are 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 (DOD AP); 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-Dimiristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-Dimiristoyl-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 (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-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-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-D MA), 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-propanaminonium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide Umbromide (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-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)propane-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-[ The following may be selected from the group consisting of 4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-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.

[0109] 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.

[0110] 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.

[0111] In a preferred embodiment, the lipid-based composition or the first composition comprises ionizable or cationic lipids, such as those described in WO 2016 / 021683 or WO 2019 / 131839, these reference patent documents are incorporated herein by reference in their entirety.

[0112] In particular, the lipid-based nanoparticles or the first composition include 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-heptadecanyl 8-{(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 combination thereof.

[0113] 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.

[0114] Preferably, the lipid-based nanoparticles or the ionizable or cationic lipid contained in the first composition is ALC-0315. Alternatively, the lipid-based nanoparticles or the ionizable or cationic lipid contained in the first composition is SS-OP.

[0115] 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.

[0116] In some embodiments, the ionizable or cationic lipid corresponds 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 or the first composition.

[0117] In some embodiments, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, corresponds to about 45 mol% to about 55 mol% of the total lipids present in the lipid composition of the lipid-based nanoparticles or the first composition. In particular, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, corresponds to about 48 mol% to about 52 mol% of the total lipids present in the lipid composition of the lipid-based nanoparticles or the first composition.

[0118] Even more particularly, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, corresponds to about 50 mol% of the total lipids present in the lipid composition of the lipid-based nanoparticles or the first composition.

[0119] In some embodiments, the lipid-based nanoparticles or the first composition contain 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.

[0120] Helper lipids are also components of LNPs and play an important role in terms of stability and fusibility. Primarily, these lipids are phospholipids (e.g., DOPE, DSPC, DEPC, DSPE) that form the main backbone of the LNP. Helper lipids modulate the fluidity of the nanoparticles and enhance efficacy by promoting lipid phase transitions that assist in membrane fusion with endosomes. Generally, helper lipids can be saturated phospholipids that can raise the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayers, and stabilize their structural arrangement.

[0121] 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,It 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-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (18:0 DDAB), and any combination thereof.,

[0122] 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 It 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 or combination thereof.

[0123] 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 combination thereof.

[0124] Preferably, the helper lipid is selected from the group consisting of DOPE, DOPC, DDAB, POPE, and DSPC, and any combination thereof.

[0125] In some embodiments, the helper lipid is DOPE. Alternatively, the helper lipid is DSPC.

[0126] 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 composition of the present invention.

[0127] In some embodiments, the helper lipids, preferably DOPE or DSPC, constitute about 5 mol% to about 15 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0128] In some embodiments, the helper lipids, preferably DOPE or DSPC, constitute about 8 mol% to about 12 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0129] In some embodiments, the helper lipids, preferably DOPE or DSPC, constitute about 10 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0130] In some embodiments, the lipids in the lipid-based composition include one or more molecules containing polyethylene glycol. Therefore, the lipid-based composition may contain PEG or PEG-modified lipids.

[0131] 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.

[0132] 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.

[0133] 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).

[0134] 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.

[0135] 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.

[0136] 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.

[0137] Preferably, the PEG-lipid is selected from the group consisting of 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 (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.

[0138] In certain embodiments, the LNP comprises unfunctionalized PEG, which is PEG that does not contain any reactive species or any reactive group at its terminus, and the reactive species or group can be used to conjugate a target portion, such as an antibody or a fragment thereof, to the PEG.

[0139] 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.

[0140] 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, or even bypass the spleen and prioritize tumor areas.

[0141] The inventors observed that PEG-lipids affect the in vivo distribution of LNPs. By selecting PEG-lipids, the uptake of LNPs by capture organs (mainly the liver, and optionally the spleen and / or lungs) can be altered. For example, PEG-C14 lipids appear to promote liver uptake, while PEG-C18 lipids can reduce liver uptake and, optionally, promote spleen targeting. Optionally, the PEG-lipids and their amounts in the LNP can be selected to promote the free distribution of the LNP throughout the body's blood circulation, particularly in the direction of an area targeted for a specific therapeutic treatment. More specifically, the PEG-lipids and their amounts in the LNP can be selected to be free enough to target immune cells and tumor cells, particularly in the tumor microenvironment. As an illustrative advantage, targeted LNPs having targeting ligands such as antibodies are designed to avoid organ uptake and target activated immune cells (e.g., PD1+ T lymphocytes) located within the tumor microenvironment. The mRNA carried by the LNP and transfected into the activated immune cells can locally and specifically enhance the anti-tumor activity of those immune cells.

[0142] In a specific embodiment, the lipid moiety of the lipid, particularly the PEG-lipid, comprises 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.

[0143] 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).

[0144] If necessary, PEG may exhibit 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 certain embodiments, PEG has a size of about 2,000. In very specific embodiments, PEG has a size between about 2,000 and 5,000 daltons. In some examples, PEG is selected from the group consisting of PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000.

[0145] 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.

[0146] In certain embodiments, the PEG has a size of approximately 2000. Alternatively, the PEG has a size of approximately 5000 Daltons.

[0147] 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.

[0148] In a very specific embodiment, the PEG-lipid is PEG 5000-DMG. In another very specific embodiment, the PEG-lipid is PEG 5000-DSG.

[0149] In some embodiments, the PEG lipids constitute approximately 1 mol% to approximately 100 mol%, approximately 2 mol% to approximately 100 mol%, approximately 3 mol% to approximately 100 mol%, approximately 4 mol% to approximately 100 mol%, approximately 5 mol% to approximately 100 mol%, approximately 10 mol% to approximately 100 mol%, or approximately 15 mol% to approximately 100 mol% of the total lipids present in the lipid-based composition or the first composition.

[0150] If necessary, the PEG lipids in the lipid-based nanoparticles or the first composition 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%.

[0151] 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.

[0152] Therefore, the amount of PEG lipids may be less than 1.5 mol% of the total lipids present in the nanoparticles or the first composition, and in particular, 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 or the first composition.

[0153] In some embodiments, the PEG lipid, preferably PEG-DMG or PEG-DSPE, constitutes 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 or the first composition.

[0154] In particular, PEG lipids, preferably PEG-DMG or PEG-DSPE, constitute about 1 mol% to about 2 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0155] Preferably, PEG lipids, preferably PEG-DMG or PEG-DSPE, constitute about 1.5 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0156] In some embodiments, the lipid-based composition or the first composition comprises one or more sterols. The sterols may be 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.

[0157] Sterols, and more specifically cholesterol, enhance the stability of nanoparticles by filling the gaps between lipids, and aid in their fusion with the endosomal membrane during cellular uptake.

[0158] In some embodiments, the sterols comprise 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%, about 50 mol% to about 100 mol%, about 60 mol% to about 100 mol%, or about 70 mol% to about 100 mol% of the total lipids present in the lipid-based composition or the first composition.

[0159] 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 or the first composition.

[0160] In particular, sterols, preferably cholesterol, constitute about 35 mol% to about 40 mol% of the total lipids present in the lipid-based nanoparticles or the first composition. Such sterols are preferably cholesterol.

[0161] In particular, sterols, preferably cholesterol, account for about 38.5 mol% of the total lipids present in the lipid-based nanoparticles or the first composition. Such sterols are preferably cholesterol.

[0162] In a preferred embodiment, the lipid-based composition or the first composition comprises ionizable or cationic lipids, helper lipids, sterols, and PEG lipids.

[0163] In a preferred embodiment, the first composition or lipid-based nanoparticles consist of ionizable or cationic lipids, helper lipids, sterols, and PEG lipids, which are preferably the lipids described above.

[0164] In some embodiments, ionizable or cationic lipids constitute about 35 mol% to about 55 mol% of the total lipids present in the lipid-based composition or the first composition; helper lipids constitute about 5 mol% to about 20 mol% of the total lipids present in the lipid-based composition or the first composition; sterols constitute about 30 mol% to about 60 mol% of the total lipids present in the lipid-based composition or the first composition; and PEG lipids constitute about 0.5 mol% to about 4 mol% of the total lipids present in the lipid-based composition or the first composition.

[0165] Preferably, the lipid-based nanoparticles or the first composition 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.

[0166] If necessary, the PEG lipids 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%.

[0167] If necessary, the PEG lipids in the lipid-based nanoparticles may be less than 1.5 mol% of the total lipids present in the nanoparticles, and in particular 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 or the first composition.

[0168] In some embodiments, the PEG lipid, preferably PEG-DMG or PEG-DSPE, constitutes 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 or the first composition.

[0169] In particular, PEG lipids, preferably PEG-DMG or PEG-DSPE, constitute about 1 mol% to about 2 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0170] Preferably, PEG lipids, preferably PEG-DMG or PEG-DSPE, constitute about 1.5 mol% of the total lipids present in the lipid-based nanoparticles or the first composition.

[0171] In specific embodiments, the lipid-based composition or the first composition comprises [(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.

[0172] In a more specific embodiment, ALC-0315 constitutes about 35 mol% to about 55 mol% of the total lipids present in the nanoparticles or the first composition, DOPE constitutes about 5 mol% to about 20 mol% of the total lipids present in the nanoparticles or the first composition, cholesterol constitutes about 30 mol% to about 60 mol% of the total lipids present in the nanoparticles or the first composition, and the one or more polyethylene glycol (PEG) modified lipids constitute about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticles or the first composition. If necessary, the one or more polyethylene glycol (PEG)-modified lipids are present in the nanoparticles or the first composition in an amount of about 0.5 mol% to about 2 mol%, 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%. If necessary, the one or more polyethylene glycol (PEG)-modified lipids are present in the nanoparticles or the first composition in an amount of about 0.5 mol% to about 1.5 mol%, for example, about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9, or 1.0 mol%, for the total lipids present in the nanoparticles or the first composition.

[0173] In a very specific embodiment, the lipid-based composition or the first composition includes a lipid mixture.

[0174] 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.

[0175] Preferably, the lipid mixture comprises [(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 1-monomethoxypolyethylene glycol-2,3-dimyristylglycerol (PEG2000 DMG) having polyethylene glycol with an average molecular weight of 2000 as a PEG-modified lipid.

[0176] In a very specific embodiment, ALC-0315 constitutes about 35 mol% to about 55 mol% of the total lipids present in the lipid-based composition or the first composition; DOPE constitutes about 5 mol% to about 20 mol% of the total lipids present in the lipid-based composition or the first composition; cholesterol constitutes about 30 mol% to about 60 mol% of the total lipids present in the lipid-based composition or the first composition; and PEG2000 DMG constitutes about 0.5 mol% to about 4 mol% of the total lipids present in the lipid-based composition or the first composition.

[0177] If necessary, PEG2000-DMG is present in the nanoparticles or the first composition in an amount of about 0.5 mol% to about 2 mol%, 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%. If necessary, PEG2000-DMG is present in the nanoparticles or the first composition in an amount of about 0.5 mol% to about 1.5 mol%, for example, about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9, or 1.0 mol%, preferably about 0.5 mol%, for the total lipids present in the nanoparticles or the first composition.

[0178] In another very specific embodiment, the lipid-based composition or the first composition comprises [(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.

[0179] In very specific embodiments, ALC-0315 constitutes about 35 mol% to about 55 mol% of the total lipids present in the nanoparticles or the first composition; DOPE constitutes about 5 mol% to about 20 mol% of the total lipids present in the nanoparticles or the first composition; cholesterol constitutes about 30 mol% to about 60 mol% of the total lipids present in the nanoparticles or the first composition; and PEG2000-DSG constitutes about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticles or the first composition. If necessary, PEG2000-DSG may be 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%. If necessary, PEG2000-DSG is present in the nanoparticles or the first composition in an amount of about 0.5 mol% to about 1.5 mol% of the total lipids present in the nanoparticles or the first composition. If necessary, PEG2000-DSG is present in an amount of about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9, or 1.0 mol%, preferably about 0.5 mol%, of the total lipids present in the nanoparticles or the first composition.

[0180] In a very specific embodiment, the lipid-based composition or the first composition includes the following: - ALC-0315 in nanoparticles or approximately 35 mol% to approximately 55 mol% of the total lipids present in the first composition. - DOPE comprising approximately 5 mol% to approximately 20 mol% of the total lipids present in the nanoparticles or the first composition. - Cholesterol comprising approximately 30 mol% to approximately 60 mol% of the total lipids present in the nanoparticles or the first composition, and - PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG, in an amount of about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticles or the first composition, preferably about 0.5 mol% to about 1.5 or 2 mol%, and optionally about 0.5 mol% to about 1.0 mol% of the total lipids present in the nanoparticles or the first composition.

[0181] In some embodiments, the lipid-based composition or the first composition comprises or consists of a lipid mixture selected from the group consisting of: a) ALC-0315, DOPE, cholesterol, and DMG-PEG; b) ALC-0315, DDAB, cholesterol, and DMG-PEG; c) ALC-0315, POPE, cholesterol, and DMG-PEG; d) ALC-0315, DOPE, cholesterol, and DSPE-PEG; e) ALC-0315, DSPC, cholesterol, and DMG-PEG; f) ALC-0315, DSPC, cholesterol, and ALC-0159; g) SM-102, DSPC, cholesterol, and DMG-PEG; h) Dlin-MC3-DMA, DSPC, cholesterol, and DMG-PEG; 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.

[0182] In some embodiments, the lipid-based composition or the first 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 2000; e) SS-OP, DOPC, cholesterol and DSG-PEG 5000; and f) SS-OP, DSPC, cholesterol, and DSPE-PEG 2000.

[0183] Preferably, the lipid-based composition or the first composition comprises or consists of a lipid mixture SS-OP, DSPC, cholesterol, and DSPE-PEG, preferably DSG-PEG 2000 or DSG-PEG 5000.

[0184] Alternatively, the lipid-based composition or the first composition may contain 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) ALC-0315, DOPE, cholesterol, DMG-PEG, and DSPE-PEG;

[0185] Preferably, in such a specific embodiment, the PEG has a size of 2000 Daltons (i.e., PEG-2000).

[0186] Preferably, in such specific embodiments, the first composition or lipid-based nanoparticles contain or consist 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.

[0187] Preferably, in such specific embodiments, the first composition or lipid-based nanoparticles contain or consist of about 45 mol% to about 55 mol% cationic or ionizable lipids, about 5 mol% to about 15 mol% helper lipids, about 35 mol% to about 45 mol% sterols, and about 0.5 mol% to about 2.5 mol% PEG-lipids.

[0188] In a very specific embodiment, the lipid-based composition or the first composition includes or consists of a lipid mixture comprising 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 cholesterol, making up about 35 mol% to about 45 mol% of the total lipids present in the LNP, preferably about 37 mol% to about 40 mol%, and more preferably about 38.5 mol%. - 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.

[0189] In a very specific embodiment, the lipid-based composition or the first 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 cholesterol, making up about 35 mol% to about 45 mol% of the total lipids present in the LNP, preferably about 37 mol% to about 40 mol%, and more preferably about 38.5 mol%. - 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.

[0190] In a very specific embodiment, the lipid-based composition or the first 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.

[0191] The lipid-based composition or the first composition 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, lipid bilayers may be functionalized in this way with one or more groups useful for facilitating membrane permeation, cell recognition, or imaging.

[0192] 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.

[0193] In some embodiments, the lipid-based particles contain poloxamine and / or poloxamer.

[0194] In some embodiments, the lipid-based particles include polyethyleneimine, protamine (rotamine), and / or polyaspartamide.

[0195] Second composition In the method of the present invention, the second composition is an aqueous solution containing one or more nucleic acid molecules. Preferably, the nucleic acid molecules are isolated nucleic acid molecules. If necessary, the aqueous solution contains a buffer or a salt. Exemplary buffers include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. Exemplary salts include sodium chloride, magnesium chloride, and potassium chloride.

[0196] In one embodiment, one or more nucleic acid molecules are dissolved in a suitable buffer solution, such as an acetate buffer or a sodium acetate buffer.

[0197] Preferably, the pH of the second composition is between about 3 and about 6, preferably between about 4 and about 5, and more preferably between about 4 and about 4.5.

[0198] In one embodiment, if the lipid-based composition contains ionizable lipids, the pH of an aqueous composition containing one or more nucleic acid molecules (i.e., the second composition) is lower than the pKa of the ionizable lipids in the lipid-based composition (i.e., the first composition).

[0199] In one embodiment, the concentrations of nucleic acid molecules in a composition containing one or more nucleic acid molecules are approximately 0.01 mg / mL to approximately 100 mg / mL, approximately 0.1 mg / mL to approximately 50 mg / mL, approximately 0.5 mg / mL to approximately 25 mg / mL, approximately 1 mg / mL to approximately 20 mg / mL, approximately 2 mg / mL to approximately 15 mg / mL, and approximately 3 mg / mL to approximately 10 mg / mL.

[0200] The second composition comprises one or more nucleic acid molecules. The nucleic acid molecules may be DNA molecules and / or RNA molecules.

[0201] In particular, nucleic acid molecules can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 DNA or mRNA molecules that encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different proteins, respectively.

[0202] More specifically, the nucleic acid molecule is selected from a list consisting of mRNA, siRNA, cDNA, saRNA (self-amplified RNA), taRNA (trans-amplified RNA), shRNA, miRNA, antisense RNA, lncRNA, piRNA, gRNA, and tsRNA. Preferably, one or more nucleic acid molecules are one or more mRNA molecules.

[0203] In one embodiment, the second composition or lipid-based nanoparticles include siRNA or antisense polynucleotides. Preferably, the siRNA or antisense polynucleotides can inhibit the expression of a target polypeptide.

[0204] In certain embodiments, the second composition or lipid-based nanoparticles contain a short hairpin RNA (shRNA) agent. ShRNA molecules are well known in the art and are directed toward target mRNA, thereby reducing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell and then processed into siRNA. For example, in certain cases, the cell has a native enzyme (e.g., a dicer) that cleaves the shRNA to form siRNA.

[0205] In some embodiments, the nucleic acid molecules contained in the second composition or lipid-based nanoparticles are antisense molecules. Antisense oligomers of about 10 to about 30, more preferably about 15 nucleotides, are preferred. The synthetic antisense molecules intended by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0206] In some embodiments, the nucleic acid molecules contained in the second composition or lipid-based nanoparticles are one or more components of the CRISPR-Cas system, such as a nucleic acid molecule encoding a gRNA and a Cas protein, for example, a Cas9 protein.

[0207] In some embodiments, the nucleic acid molecules contained in the second composition or lipid-based nanoparticles are miRNAs or mimetic miRNAs. miRNAs are small, non-coding RNA molecules that can induce post-translational silencing of specific genes within a cell by inhibiting translation or by degrading targeted mRNA. miRNAs may be perfectly complementary to the target nucleic acid or may have regions that are incomplementary to the target nucleic acid, which may result in the formation of a "bulge" in the incomplementary region.

[0208] To evaluate the expression of nucleic acid molecules, the second composition or lipid-based nanoparticles may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification of cell expression from a population of cells that are being sought to be transfected or infected using the LNP of the present invention. In other embodiments, the selectable marker may be supported on another DNA fragment and may also be contained within the LNP. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes such as neomycin resistance.

[0209] The second composition or lipid-based nanoparticles may also contain selectable marker genes to facilitate the selection of host cells. Suitable selectable marker genes are proteins that confer resistance to a particular drug, such as G418 and hygromycin, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, such as genes encoding the Fc portion of immunoglobulins, preferably IgG. The selectable marker may also be introduced using a vector separate from the nucleic acid of interest.

[0210] The second composition includes, in particular, mRNA encoding a target polypeptide, which, upon entering a target cell, can translate the polypeptide.

[0211] In certain embodiments, the lipid-based nanoparticles comprise mRNA polynucleotides or a set of mRNA polynucleotides. Preferably, the lipid-based nanoparticles of the present invention comprise one or more isolated mRNA molecules.

[0212] The mRNA polynucleotide technology is 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.

[0213] 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 target cells.

[0214] 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.

[0215] 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.

[0216] In some embodiments, the mRNA of the present invention includes an adjoining region. A 5'-UTR or 3'-UTR may be provided as the adjoining 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 adjoining region, and these 5'-UTRs or 3'-UTRs may have the same sequence or different sequences. Any portion of the adjoining 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. 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 at 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.

[0217] 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.

[0218] In some embodiments, the mRNA of the present invention includes a 5' capping region or structure. The 5' cap structure of a polynucleotide is involved in nuclear export and increased polynucleotide stability, binding to mRNA cap-binding proteins (CBPs), and the association of CBPs and poly(A)-binding proteins to form a mature circular mRNA species, thereby contributing to intracellular polynucleotide stability and translational competency. Multiple distinctly different 5' cap structures can be used to generate the 5' cap of an mRNA molecule.

[0219] The mRNA of the present invention particularly includes a 5' cap analog. In this specification, synthetic cap analogs, chemical caps, chemical cap analogs, structural or functional cap analogs are also used. Cap analogs retain cap function but differ in their chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5' caps. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or linked to polynucleotides.

[0220] In some embodiments, the mRNA of the present invention includes a stem-loop, for example, a histone stem-loop, but is not limited to these. The histone stem-loop may be before and / or after the poly(A) region. The mRNA, including the histone stem-loop and poly(A) region sequence, may include a strand termination nucleotide. 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 non-limiting examples, the stem-loop region may include a seed sequence for the miR sequence. For example, the stem-loop region may include the miR-122 seed sequence. Preferably, the stem-loop is a nucleotide sequence of about 25 or about 26 nucleotides in length. In other cases, the mRNA including the histone stem-loop is stabilized by modification of the 3' region of the polynucleotide, which can prevent and / or inhibit the addition of oligo(U).

[0221] 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.

[0222] The mRNA, in particular, includes at least one histone stem-loop and a poly-A region or polyadenylation signal.

[0223] In some embodiments, the mRNA in the second composition or t-LNP includes a polyA sequence and / or a polyadenylation signal. The polyA sequence may consist entirely or largely of adenine nucleotides or their analogs or derivatives. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the nucleic acid. The length of the polyA region in this disclosure is 20, 40, 80, 100, 120, 140, or 160 nucleotides on the mRNA molecule described herein. The polyA region may also be designed as part of the mRNA to which it belongs. In this regard, the polyA region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct with the polyA region subtracted.

[0224] In some cases, 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.

[0225] 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.

[0226] Different sugar modifications and / or internucleoside linkages (e.g., skeletal structures) can be located at various positions within a 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).

[0227] 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).

[0228] 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.

[0229] 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.

[0230] In some embodiments, the nucleic acid base is an alternative adenine. Examples of 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-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, and 1-methyl-1-adenine (ml A) 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)adenine (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-hydroxynorvalylcarbamoyl-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 Examples include 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.

[0231] 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, 8 -This includes 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.

[0232] If necessary, the mRNA should be circular RNA, in particular circular mRNA, as described in WO2014 / 186334 and WO2022 / 261490.

[0233] In a preferred embodiment, one or more nucleic acid molecules are one or more mRNA molecules encoding molecules selected from the group consisting of immune cell-enhancing compounds, antigen fragments, antigen-binding domains, and chimeric antigen receptors (CARs).

[0234] In some embodiments, the second composition does not contain nucleic acids but contains active ingredients, such as drugs and therapeutic molecules.

[0235] In addition, or alternatively, the second composition may include an imaging agent. Such an imaging agent may be, for example, a fluorescent protein, such as GFP or luciferase. In some embodiments, the imaging agent may be one or more fluorescent mRNA molecules.

[0236] In particular, the lipid-based nanoparticles or second composition obtained by the present invention may comprise mRNA molecules 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, each encoding a different protein of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. In certain embodiments, the lipid-based nanoparticles or second composition comprise mRNA molecules encoding two different immune cell proteins, such as those disclosed herein. The immune cell proteins may be selected from a single cell type or from different cell types. For example, the mRNA molecules may encode different T cell proteins. Alternatively, the mRNA molecules may encode different immune cell proteins, such as T cell proteins, and another immune cell inhibitory protein, such as a natural killer protein.

[0237] - Immune cell compounds or molecules In some embodiments, the nucleic acid molecules described herein encode immune cell proteins, particularly immune cell inhibitory or enhancing proteins. Accordingly, the lipid-based nanoparticles of the present invention comprise one or more different mRNA molecules encoding immune cell inhibitory or activating proteins.

[0238] The terms "immune cell compound," "immune cell molecule," or "immune cell protein" refer to molecules or proteins specifically produced or expressed by immune cells, such as activated immune cells. These proteins play crucial roles in immune system functions, including antigen recognition, signal transduction, cell communication, and immune responses. Immune cell proteins are diverse and may include receptors, enzymes, cytokines, antibodies, and other molecules essential for the proper functioning of immune cells or the immune system.

[0239] The immune cell proteins encoded by the 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 a T cell (i.e., the lipid-based nanoparticles contain an antigen-binding domain that binds to a target expressed on T cells, such as PD-1), the mRNA encodes a T cell protein.

[0240] In addition, the immune cell proteins encoded by mRNA molecules that will be contained in the lipid-based nanoparticles of the present invention may be selected based on the desired effect. For example, immune cell proteins may be selected for specific indications, conditions, diseases, or disorders.

[0241] For example, immune cell proteins may be selected for their effect on the target immune cells themselves. Generally, immune cell proteins can be either immune cell inhibitory proteins or immune cell enhancing proteins.

[0242] In some embodiments, mRNA molecules encode immune cell inhibitory proteins.

[0243] 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.

[0244] In particular, such 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.

[0245] Alternatively, mRNA molecules encode immune cell-activating proteins.

[0246] The terms "immune cell activation carrier protein" or "immune cell enhancement protein" refer to proteins that induce, increase, enhance, or elevate the activity of immune cells, or activate them. These proteins generally play a crucial role in enhancing the immune response and, consequently, improving the immune system's ability to detect and eliminate pathogens, infected cells, or abnormal cells, such as cancer cells. Immune cell enhancement proteins can act by promoting the proliferation, activation, or function of various types of immune cells, including T cells, B cells, natural killer cells, macrophages, dendritic cells, and others. It is well known that they are critical to initiating effective immune responses against infectious diseases and tumors.

[0247] In particular, such immune cell-enhancing proteins exhibit effects on activated immune cells, selected from the following group: - Inducing or increasing the stem cell properties of immune cells - To induce or increase the proliferation or regeneration of immune cells. - Inhibiting or reducing apoptosis 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. - To inhibit or reduce the hypoxic state of the microenvironment or tissues. - To increase the production or concentration of metabolic enzymes in immune cells. - To increase the efficiency of signaling pathways in immune cells. - Inducing or increasing the production of cytotoxic compounds by immune cells. - Inducing or increasing the internalization of endosomes in 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. - Inducing or increasing 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. - Inducing or increasing inflammation in the tumor microenvironment (TME) - To increase the anergy resistance of immune cells - 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. - Inhibiting or reducing the production and / or secretion of checkpoint inhibitors. - Inducing a phenotypic switch in immune cells from tumor-promoting immune cell type to anti-tumor immune cell type. - Inhibiting the stem cell properties of immune cells, the proliferation or regeneration of immune cells, the production or concentration of mitochondrial enzymes and / or transporters of immune cells, the production or concentration of transcription factors of immune cells, the production or concentration of metabolic enzymes of immune cells, the efficiency of signaling pathways of immune cells, secretion of immune cells, production of cytotoxic proteins by immune cells, internalization of endosomes by immune cells, production or concentration of chaperone proteins of immune cells, production of cytoskeletal regulatory proteins of immune cells, degradation of proteins by immunoproteasomes and ubiquitination, and antigen presentation of immune cells, migration and / or mobility of immune cells, autophagy of immune cells, inflammation of the TME, active membrane transport of immune cells, production or concentration of carrier proteins of immune cells, or production or concentration of tRNA of immune cells; or inducing or increasing apoptosis of immune cells, hypoxia of the microenvironment or tissue, or production and / or secretion of checkpoint inhibitors, by compounds, proteins or molecules.

[0248] 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.

[0249] In particular, immune cell-enhancing proteins that induce or increase the stem cell properties of immune cells are selected from the group including or comprising TCF1, LEF1, WNT, FRIZZLED, and beta-catenin.

[0250] 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.

[0251] In particular, immune cell-enhancing proteins that induce or increase the proliferation or regeneration of immune cells are selected from the group including or comprising LRP6, CYCLIN, TOP2A, MUCL1, and MDM2.

[0252] 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.

[0253] In particular, immune cell-enhancing proteins that inhibit or reduce apoptosis of immune cells are selected from the group including or comprising BCL2, BCLXL, BIRC3, and MCL1.

[0254] 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.

[0255] In particular, PGC1a is an immune cell-enhancing protein that increases the production or concentration of mitochondrial enzymes and / or transporters in immune cells.

[0256] 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.

[0257] In particular, immune cell-enhancing proteins that increase the production or concentration of transcription factors in immune cells are selected from the group including or consisting of TCF7, NFAT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, and FOXO1.

[0258] In particular, immune cell-enhancing proteins that increase the production or concentration of transcription factors in immune cells are selected from the group including or comprising TCF7, NFAT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, and FOXO1.

[0259] 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.

[0260] In particular, PTGS2 is an immune cell-enhancing protein that inhibits or reduces hypoxic conditions in the microenvironment or tissues.

[0261] 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.

[0262] In particular, immune cell-enhancing proteins that increase the production or concentration of metabolic enzymes in immune cells are selected from the group including or consisting of CSE, Glut1, Glut3, HK2, FOXO1, arginine resynthesis enzyme, argininosuccinate synthase (ASS), ornithine transcarbamylase (OTC), and GYS.

[0263] In particular, immune cell-enhancing proteins that increase the production or concentration of metabolic enzymes in immune cells are selected from the group including or comprising CSE, Glut1, Glut3, HK2, FOXO1, arginine resynthesis enzyme, argininosuccinate synthase (ASS), ornithine transcarbamylase (OTC), GYS, and OXPHOS.

[0264] 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.

[0265] In particular, immune cell-enhancing proteins that increase the efficiency of immune cell signaling pathways are selected from the group including or comprising AKT, PLC, STAT, SMAD, Blys, BTK, and BLK.

[0266] 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).

[0267] In particular, immune cell-enhancing proteins that induce or increase the production of cytotoxic proteins by immune cells are selected from the group including or comprising CD107a, lymphotoxin (LT) α1β2, granzyme B, and perforin.

[0268] 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.

[0269] In particular, POU2F1 is an immune cell-enhancing protein that induces or increases endosome internalization.

[0270] In particular, immune cell 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.

[0271] 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, kinesin, and Rab proteins.

[0272] In particular, immune cell-enhancing 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 integrin alpha 1, integrin alpha 2, integrin alpha 2b, integrin alpha 11, integrin alpha 3, integrin alpha 6, integrin alpha 7, integrin alpha E, integrin beta 2, integrin beta 4, LFA-1, LFA-2, LFA-3, integrin beta 1, integrin beta 7, CD103, integrin alpha V, ITGAE, CD11C, CRTAM, CXCR5, CXCR3, CCR7, SELL, GAL3, laminin, actin, vimentin, DEF1, dynein, kinesin, and Rab proteins.

[0273] 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.

[0274] In particular, immunoproteasome and ubiquitination-mediated protein degradation, as well as immune cell antigen presentation, are selected from the group including or comprising NLRP3, TAP, LAMP, ubiquitin ligase, CD74, peptidase, calreticulin, and Aurora.

[0275] 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.

[0276] In particular, LGR6 is an immune cell-enhancing protein that induces or increases inflammation in TMEs.

[0277] In particular, immune cell-enhancing proteins that modify the epigenetics of immune cells are selected from the group including or comprising HAT, KDM1, TGD, and TET1.

[0278] 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.

[0279] In particular, immune cell-enhancing proteins that increase the anergy resistance of immune cells are selected from the group including or comprising C-FOS, JUN, EGR-2, and EGR-3.

[0280] These proteins are described in detail in Table D (Table 4) below. In some embodiments, the mRNA encodes a protein selected from Table D (Table 4).

[0281] [Table 4A]

[0282] [Table 4B]

[0283] [Table 4C]

[0284] [Table 4D]

[0285] [Table 4E]

[0286] [Table 4F]

[0287] [Table 4G]

[0288] [Table 4H]

[0289] [Table 4I]

[0290]

Table 4J

[0291]

Table 4K

[0292]

Table 4L

[0293]

Table 4M

[0294]

Table 4N

[0295] Table 4O

[0296] [Table 4P]

[0297]

Table 4Q

[0298]

Table 4R

[0299] 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.

[0300] 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.

[0301] Preferably, one or more mRNA molecules encode 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, and FPR2, and any combination thereof.

[0302] In some embodiments, the mRNA molecule encodes BIM-S and / or PUMA.

[0303] Preferably, the mRNA molecule encoding PUMA contains, or consists of, the nucleic acid sequence shown in Sequence ID No. 48, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0304] In some embodiments, the mRNA molecule encodes a BIM protein. As used herein, the term “BIM protein” encompasses the three Bim isoforms (i.e., Bim-EL, Bim-L, and Bim-S) produced by alternative splicing. Preferably, the mRNA molecule encoding BIM-S contains, or consists of, the nucleic acid sequence shown in SEQ ID NO: 49, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0305] 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.

[0306] In some embodiments, the activity-enhancing proteins include TCF1, LEF1, WNT, FRIZZLED, betacatenin, LRP6, CYCLIN, TOP2A, MUCL1, MDM2, BCL2, BCLXL, BIRC3, MCL1, PGC1a, TCF7, NFAT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glut1, Glut3, HK2, FO XO1, Arginine Resynthesis Enzyme, Argininosuccinate Synthase (ASS), Ornithine Transcarbamylase (OTC), GYS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD107a, Lymphotoxin (LT) α1β2, Granzyme B, Perforin, POU2F1, BBS10, BBS12, TCP1, HSP, Integrin Alpha 1, Integrin Alpha 2, Integrin Alpha 2b, Integrin Alpha 11, Integrin Alpha 3, Integrin Alpha 6, Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, LFA-1, LFA-2, LFA-3, Integrin Beta 1, Integrin Beta 7, CD103, Integrin Alpha V, ITGAE, CD11C, CRTAM, CXCR5, CXCR3, CCR7, SELL, GAL3, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Rab protein, NLRP3, TAP, LAMP, Ubiquitin ligase, CD74, peptidase, calreticulin, Aurora, LGR6, HAT, KDM1, TGD, TET1, C-FOS, JUN, EGR-2, EGR-3, phosphatidylinositol 3-kinase (PI3K), interferon regulators, e.g., IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, SOCS, NFκB, STAT3, AhR, STING, MAVS, MyD88, IRAK1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKα, IKKβ, TRAM, TRIF, RIPK1, TBK1, PI3K, D3-phosphoinositide, derivatives of phosphatidylinositol, IL7R, CD122, CD132, CD25, CD215, IL12R, IL17R, IL8R, IL21R, IL11R, IL18R, IL10R, IL1R, IL6R, CXCR3, CXCR5, CXCR4, CXCR1, C XCR2, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, DECTIN-1, CLEC-9A, CLEC-2, DECTIN-2, MCL, MIN CLE, BDCA-2, ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, OX40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAM The group is selected from the CD2 family, CD27, TL1A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, CD160, Perforin, CXCL9, CXCL10, GrB, OXPHOS, FRIZZLED, BCLXL, CCR4, CCR10, CXCR3, CCR10, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR3, CXCR5, CRTAM, CCR7, CXCR5, GPR35, GPR37, and TAP.

[0307] In some embodiments, the activity-enhancing proteins are TCF1, LEF1, WNT, FRIZZLED, betacatenin, LRP6, CYCLIN, TOP2A, MUCL1, MDM2, BCL2, BCLXL, BIRC3, MCL1, PGC1a, TCF7, NFAT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glut1, Glut3, HK2, F OXO1, Arginine Resynthesis Enzyme, Argininosuccinate Synthase (ASS), Ornithine Transcarbamylase (OTC), GYS, AKT, PLC, SMAD, Blys, BTK, BLK, CD107a, Lymphotoxin (LT) α1β2, Granzyme B, Perforin, POU2F1, BBS10, BBS12, TCP1, HSP, Integrin Alpha 1, Integrin Alpha 2, Integrin Alpha 2b, Integrin Alpha 11, Integrin Alpha 3, Integrin Alpha 6, Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, LFA-1, LFA-2, LFA-3, Integrin Beta 1, Integrin Beta 7, CD103, Integrin Alpha V, ITGAE, CD11C, CRTAM, CXCR5, CXCR3, CCR7, SELL, GAL3, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Rab protein, NLRP3, TAP, LAMP, Ubiquitin ligase, CD74, peptidase, calreticulin, Aurora, LGR6, HAT, KDM1, TGD, TET1, C-FOS, JUN, EGR-2, EGR-3, phosphatidylinositol 3-kinase (PI3K), interferon regulators, e.g., IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, TRAM, TBK1, NFκB, AhR, STING, MAVS, MyD88, IRAK1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKα, IKKβ, TRIF, PI3K, D3-phosphoinositide, derivatives of phosphatidylinositol, IL7R, CD122, CD132, CD25, CD215, IL12R, IL17R, IL8R, IL21R, IL11R, IL18R, IL10R, IL1R, IL6R, CXCR3, CXCR5, CXCR4, CXCR1, CXCR2, CXC R6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX3CR1, XCR1, DECTIN-1, CLEC-9A, CLEC-2, DECTIN-2, MCL, MINCLE, BDCA-2, ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, OX40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAM The group is selected from the CD2 family, CD27, TL1A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, CD160, Perforin, CXCL9, CXCL10, GrB, OXPHOS, FRIZZLED, BCLXL, CCR4, CCR10, CXCR3, CCR10, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR3, CXCR5, CRTAM, CCR7, CXCR5, GPR35, GPR37, and TAP.

[0308] Preferably, the activity-enhancing protein is selected from the group consisting of TCF1, WNT, BCL2, BCLXL, TBET, Glut1, LGR6, ICOS, CD28, CD40L, 4-1BB, perforin, CXCL9, CXCL10, GrB, OXPHOS, integrin alpha 1, integrin alpha 2, integrin alpha 2b, integrin alpha 11, integrin alpha 3, integrin alpha 6, integrin alpha 7, integrin alpha E, integrin beta 2, integrin beta 4, integrin beta 1, integrin beta 7, and integrin alpha V.

[0309] Preferably, the activity-enhancing protein is selected from the group consisting of BCL2, BCLXL, CD28, 4-1BB, ICOS, CD40L, TBET, TCF1, Glut-1, OXPHOS, IL7, IL12, IL-15, IL-21, IL7R, IL12R, IL-15R, IL-21R, integrin alpha 1, integrin alpha 2, integrin alpha 2b, integrin alpha 11, integrin alpha 3, integrin alpha 6, integrin alpha 7, integrin alpha E, integrin beta 2, integrin beta 4, integrin beta 1, integrin beta 7, and integrin alpha V, GrB, perforin, TCF1, Wnt, Rec or LGR6, and any combination thereof.

[0310] In particular, the activity-enhancing proteins are selected from the group consisting of BCL2, IL7, IL7R, CXCL9, and CXCL10.

[0311] Preferably, the mRNA molecule encoding BLC2 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.

[0312] Preferably, the mRNA molecule encoding IL7 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.

[0313] Preferably, the mRNA molecule encoding IL7R contains, or consists of, the nucleic acid sequence shown in SEQ ID NO: 45, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0314] Preferably, the mRNA molecule encoding CXCL9 contains, or consists of, the nucleic acid sequence shown in SEQ ID NO: 46, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0315] Preferably, the mRNA molecule encoding CXCL10 contains, or consists of, the nucleic acid sequence shown in SEQ ID NO: 47, or a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity thereto.

[0316] The effect of the immune cell-enhancing protein on immune cells can be determined when, in the presence of the immune cell-enhancing protein of the present invention, immune cells exhibit greater activity compared to immune cells under the same experimental conditions but without the presence of the immune cell-enhancing protein. Immune cell activity can be measured by any method known to those skilled in the art. In particular, enhancement of immune cell activity can be measured by comparing the immune cell activity of a population of immune cells obtained from a sample, without the immune cell-enhancing protein to be evaluated, with the immune cell activity of a population of immune cells treated with the immune cell-enhancing compound or molecule obtained from the sample and to be evaluated.

[0317] In one embodiment, an immune cell inhibitory protein is an intracellular protein that has an intracellular effect on activated immune cells or transmembrane cells.

[0318] In one embodiment, the immune cell-enhancing protein is selected from the group including intracellular proteins, proteins that have intracellular effects on immune cells, transmembrane proteins, and secreted proteins.

[0319] In some embodiments, immune cell proteins are intracellular proteins or proteins that have intracellular effects on immune cells.

[0320] By using intracellular proteins in combination with targeting immune cells, preferably T cells, and more preferably a specific subset of TILs, highly specific and efficient inhibition or enhancement of said immune cells can be achieved. Potent intracellular proteins that enhance the activation and / or proliferation of immune cells in specific environments may be used.

[0321] As used herein, “proteins having intracellular effects on 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.

[0322] If necessary, the intracellular proteins of immune cells may be enzymes, intracellular signaling proteins, or transcription factors, preferably transcription factors.

[0323] In certain embodiments, the transmembrane protein is selected from the group including FRIZZLED, BCLXL, CCR4, CCR10, CXCR3, CCR10, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR3, CXCR5, CRTAM, CCR7, CXCR5, GPR35, GPR37, and TAP.

[0324] In certain embodiments, immune cell proteins are transcription factors. As used herein, “transcription factor” refers to DNA-binding proteins that regulate gene transcription. Preferably, the transcription factor is selected from the group consisting of RORgt, SOCS, NFκB, STAT, TRAM, RIPK1, and TBK1 and its variants, wherein the variant has at least 80% identity with the wild-type protein or has 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions, and combinations thereof. Preferably, the transcription factor is selected from the group consisting of RORgt, NFκB, TRAM, and TBK1 and its variants, wherein the variant has at least 80% identity with the wild-type protein or has 1 to 10 modifications selected from the group consisting of additions, deletions, substitutions, and combinations thereof.

[0325] Preferably, the transcription factor is selected from the group consisting of TCF7, NFAT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, and FOXO1, and any combination thereof.

[0326] Preferably, the transcription factor is selected from the group consisting of interferon regulators (IRF, which includes IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9), CREB, RORg, RORgt, SOCS, NFκB, T-bet, STAT3, AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKα, IKKβ, TRAM, TRIF, RIPK1, and TBK1 and its variants, wherein the variant has at least 80% identity with the wild-type protein or has 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0327] 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).

[0328] 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.

[0329] 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.

[0330] Alternatively, immune cell proteins are enzymes. For example, such an enzyme could be phosphatidylinositol 3-kinase (PI3K).

[0331] Alternatively, immune cell 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.

[0332] 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).

[0333] In some embodiments, the immune cell compound or molecule is a secreted protein.

[0334] 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.

[0335] In some embodiments, the secreted compound or molecule does not act on the immune cells that secrete it.

[0336] In some embodiments, secreted compounds or molecules act not only on the immune cells that secrete them, but also on additional cells.

[0337] In some embodiments, one or more mRNA molecules do not encode cytokines and / or chemokines.

[0338] 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.

[0339] In certain embodiments, the immune cell-enhancing protein is a cytokine receptor. Preferably, the cytokine receptor is selected from the group consisting of IL7R, CD122, CD132, CD25, CD215, IL12R, IL17R, IL8R, IL21R, IL11R, IL18R, IL10R, IL1R, and IL6R.

[0340] In certain embodiments, the immune cell inhibitory or enhancing 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.

[0341] 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.

[0342] In certain embodiments, the immune cell-enhancing protein is a lectin receptor. Preferably, the lectin receptor is selected from the group consisting of DECTIN-1, CLEC-9A, CLEC-2, DECTIN-2, MCL, MINCLE, and BDCA-2.

[0343] 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).

[0344] In certain embodiments, the immune cell-enhancing 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 IL12A, IL12B, IFNG, IFNa, IL21, IL7, IL2, IL15, and IL18.

[0345] 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.

[0346] In certain embodiments, the immune cell-enhancing protein is a costimulatory receptor or ligand. Preferably, the costimulatory receptor or ligand is selected from the group consisting of ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, OX40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAM CD2 family, CD27, TL1A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, and CD160.

[0347] 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.

[0348] In certain embodiments, one or more mRNA molecules are CXCL9, CXCL10, CCR4, CCR10, CXCR3, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR5, PI3K, 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.

[0349] These proteins are described in detail in Table E (Table 5) below. In some embodiments, the mRNA molecule encodes a protein selected from Table E (Table 5).

[0350] [Table 5A]

[0351] [Table 5B]

[0352] [Table 5C]

[0353] [Table 5D]

[0354] [Table 5E]

[0355] [Table 5F]

[0356] [Table 5G]

[0357] [Table 5H]

[0358] [Table 5I]

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] In certain embodiments, the immune cell-enhancing protein is specific to T cells, and more preferably to activated T cells. Preferably, the immune cell-modulating compound or molecule is selected from the group including T cell growth factors, particularly growth factors that increase the number and repertoire of naive immune cells, agonists that activate and stimulate immune cells, inhibitors of T cell checkpoint blockade, T cell growth factors that increase the growth and survival of immune T cells, immunostimulatory cytokines, and immunostimulatory membrane proteins.

[0365] In some embodiments, one or more mRNA molecules do not encode cytokines and / or chemokines.

[0366] 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, and - LILRB and HLA.

[0367] The receptors and associated ligands 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).

[0368] [Table 6A]

[0369] [Table 6B]

[0370] [Table 6C]

[0371] 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., IL7-R) and mRNA encoding a related interleukin (e.g., IL-7). This allows for activation at the same / single immune cell (i.e., cis-activation) level. Other examples are as follows: - BAFFR and BAFF - IL6RA and / or IL6RB and IL-6; - IL-1R1 and IL-1; - IL15RA(CD215) and IL15; - IL-21R and IL-21 - IL2R Alpha, IL2R Beta, IL2R Gamma and IL2 - IL8RA, IL8RB and IL8 - IL9R, IL2 Gamma and IL9 - IL10R and IL10 - IL11R and IL11 - IL12R Beta 1, IL12R Beta 2 and IL12 - IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE and IL-17; and - IL18R and IL18.

[0372] In some embodiments, the lipid-based nanoparticles of the present invention include mRNA encoding IL7-R and mRNA encoding IL-7.

[0373] Interleukin 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).

[0374] [Table 7A]

[0375] [Table 7B]

[0376] In some embodiments, the mRNA molecule encodes the proteins listed in Tables E (Table 5), F (Table 6), and G (Table 7).

[0377] 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.

[0378] In some embodiments, the lipid-based nanoparticles comprise at least two mRNA molecules, one of which preferably encodes a co-stimulatory molecule selected from the group consisting of CD28, CD80, CD86, ICOS, ICOSL, OX40, OX40L, CD40, CD40L, GITRL, CD137, and CD137L, and the other of which encodes another immune cell-enhancing compound, such as an intracellular protein or another transmembrane protein.

[0379] In some embodiments, the lipid-based nanoparticles include one or more mRNA molecules selected from the group consisting of: - mRNA encoding BCL2, 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 IL7, 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; - mRNA encoding IL7R, preferably an mRNA molecule containing a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 45 or a nucleic acid sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto; - mRNA encoding CXCL9, preferably an mRNA molecule containing a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 46 or a nucleic acid sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto; - mRNA encoding CXCL10, preferably an mRNA molecule containing a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 47 or a nucleic acid sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto; - mRNA molecules encoding PUMA, preferably including a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 48 or an mRNA molecule having at least 80%, 90%, 95%, or 99% sequence identity thereto; - mRNA encoding BIM-S, preferably an mRNA molecule containing a nucleic acid sequence, for example, the nucleic acid sequence described in Sequence ID No. 49 or a nucleic acid sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto.

[0380] - antigen fragment In some embodiments, the nucleic acid molecule of the present invention, preferably an mRNA molecule, encodes an antigen or an antigenic fragment.

[0381] The antigen or fragment thereof is preferably selected from among molecules expressed by any virus, bacterium, or parasitic pathogen before or during invasion of their host, before or during colonization of their host, or before or during replication in their host. These pathogens can infect human, domesticated, or wild animal hosts.

[0382] In particular, antigens or fragments thereof encoded by the nucleic acid molecules of the present invention promote protective immunity. Preferably, the antigen is a protective antigen or fragment thereof. As used herein, any virus, bacterial, or parasitic molecule that elicits an immune response resulting in long-term acquired immune resistance in the host is referred to as a “protective antigen.”

[0383] In some embodiments, the nucleic acid molecule encodes a viral antigen or a fragment thereof. Viral pathogens from which the viral antigen originates include, but are not limited to, orthomyxoviruses, e.g., influenza virus; retroviruses, e.g., RSV, HTLV-1, and HTLV-II; herpesviruses, e.g., EBV; CMV, or herpes simplex virus; lentiviruses, e.g., HIV-1 and HIV-2; rhabdoviruses, e.g., rabies virus; piconaviruses, e.g., poliovirus; poxviruses, e.g., vaccinia virus; rotavirus; and parvoviruses, e.g., adeno-associated virus (AAV); and betacoronaviruses, e.g., SARS-CoV, SARS-CoV-2, and MERS-CoV.

[0384] Examples of protective antigens for viral pathogens include human immunodeficiency virus (HIV) antigens Rev, Pol, Nef, Gag, Env, Tat, variant derivatives of Tat, e.g., Tat-Δ31-45, T and B cell epitopes of gp120, chimeric derivatives of HIV-1 Env and gp120, e.g., fusions of gp120 and CD4, shortened or modified HIV-1 Env, e.g., gp140, or derivatives of HIV-1 Env and / or gp140. Other examples include hepatitis B surface antigens, rotavirus antigens, e.g., VP4 and VP7, influenza virus antigens, e.g., hemagglutinin, neuraminidase, or nucleoproteins, and herpes simplex virus antigens, e.g., thymidine kinase.

[0385] In some embodiments, nucleic acid molecules encode bacterial antigens or fragments thereof. Examples of bacterial pathogens from which bacterial antigens may originate include, but are not limited to, species of the genera Mycobacterium, Helicobacter pylori, Salmonella, Shigella, Escherichia coli, Rickettsia, Listeria, Legionella pneumoniae, Fansicella, Pseudomonas, Vibrio, and Borellia burgdorferi.

[0386] Examples of protective antigens of bacterial pathogens include the cell antigens of enterotoxigenic Escherichia coli, e.g., CFA / I ciliary antigen and the non-toxic B subunit of the heat-unstable toxin; pertactin of Bordetella pertussis; adenylyl cyclase-lysin of Bordetella pertussis; fragment C of tetanus toxin of Clostridium tetani; OspA of Borrelia burgdorferi; protective paracrystalline surface protein of Rickettsia prowazekii and Rickettsia typhi; listeriorin (also known as "Llo" and "Hly") and / or superoxide dismutase (also known as "SOD" and "p60") of Listeria monocytogenes; urease of Helicobacter pylori; and Bacillus anthrax Examples include the receptor-binding domains of lethal toxins and / or protective antigens of anthrax.

[0387] In some embodiments, the nucleic acid molecule encodes a parasitic antigen or a fragment thereof. Parasitic pathogens from which parasitic antigens originate include species of the genus Plasmodium (e.g., Plasmodium falciparum), species of Trypanosomes (e.g., Trypanosoma cruzi), species of Giardia (e.g., Giardia intestinalis), species of Boophilus (e.g., Boswellian tick), species of Babesia (e.g., Babesia microti), species of Entamoeba (e.g., Entamoeba histolytica), and species of Eimeria (e.g., Eimeria maxima). This includes, but is not limited to, species of the genera *Lyshmania maxima*, *Leishmania* spp., *Schistosome* spp., *Brugia* spp., *Fascida* spp., *Dirofilaria* spp., *Wuchereria* spp., and *Onchocerea* spp.

[0388] Examples of parasitic pathogen defense antigens include perisporozoite proteins (CS) or liver stage-specific (LSA) antigens LSA-1 and LSA-3 of Plasmodium species, such as those of Plasmodium bergerii or Plasmodium falciparum, or their immunogenic variants; merozoite surface antigens of Plasmodium species; galactose-specific lectin of Entamoeba histolytica; gp63 of Leishmania species; gp46 of Leishmania major; paramyosin of Brugia malayi; triose phosphate isomerase of Schistosoma mansoni; secretory globin-like protein of Trichostrongylus colubriformis; liver fluke (Frasciola hepatica); and bovine schistosomiasis (Schistosoma Examples include glutathione-S-transferase from Schistosoma bovis and Schistosoma japonicum, as well as KLH from Schistosoma bovine and Schistosoma japonicum.

[0389] Antigens or fragments thereof may be encoded by codon-optimized synthetic genes and can be constructed using conventional recombinant DNA methods.

[0390] - Antigen-binding domain In some embodiments, the nucleic acid molecule, preferably an mRNA molecule, of the present invention encodes an antigen-binding domain.

[0391] In a preferred embodiment, the antigen-binding domain is an antibody, a fragment or derivative thereof, such as F(ab')2, Fab, Fab', crossMAB, or a single-stranded variable fragment (scFV) or VHH.

[0392] Examples of antigen-binding domains include, but are not limited to, antigen-binding domains that are directional to targets selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, NKG2A, LAG3, 2B4, DR3, CD101, CD44, SIRPG, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM, and CD122.

[0393] In some embodiments, the nucleic acid molecule of the present invention, preferably an mRNA molecule, encodes an antigen-binding domain for a checkpoint inhibitor, preferably an antibody. Therefore, the target cell, in particular the case of a targeted immune cell, will produce an antigen-binding domain for a checkpoint inhibitor, preferably an antibody.

[0394] According to the present invention, the heavy and light chains of an antibody can be encoded and delivered by a single mRNA molecule or separate mRNA molecules. To optimize the production of a fully assembled functional antibody, it is considered advantageous to deliver the mRNA-encoding heavy chain and mRNA-encoding light chain in various ratios. Therefore, in some embodiments, the heavy chain encoding mRNA (also called the first mRNA) and the light chain encoding mRNA (also called the second mRNA) are used in ratios ranging from approximately 10:1 to 1:10 (for example, approximately 9:1 to 1:9, 8:1 to 1:8, 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, or 2:1 to 1:2). In some embodiments, the heavy chain encoding mRNA (also called the first mRNA) and the light chain encoding mRNA (also called the second mRNA) are used in a ratio of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1 or greater. In some embodiments, the heavy chain encoding mRNA (also called the first mRNA) and the light chain encoding mRNA (also called the second mRNA) are used in a ratio of approximately 1:1 (i.e., equimolar). In some embodiments, the heavy chain encoding mRNA (also called the first mRNA) and the light chain encoding mRNA (also called the second mRNA) are used in a ratio other than 1:1 (equomolar). For example, the heavy chain encoding mRNA (also called the first mRNA) and the light chain encoding mRNA (also called the second mRNA) are used in a ratio greater than 1 (for example, in the range of approximately 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1). Alternatively, the heavy chain encoding mRNA (also called the first mRNA) and the light chain encoding mRNA (also called the second mRNA) are used in a ratio less than 1 (for example, in the range of approximately 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:7, 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2).

[0395] In some embodiments, nucleic acid molecules encoding heavy and / or light chains, preferably mRNA, contain a nucleotide sequence encoding a signal peptide. As used herein, the term “signal peptide” refers to a peptide present in a newly synthesized protein that can target the protein to the secretory pathway. Generally, signal peptides are cleaved post-translation of mRNA after translocation to the endoplasmic reticulum. Signal peptides are also called signal sequences, leader sequences, or leader peptides. Generally, signal peptides are short peptides (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acid lengths). Signal peptides may be present at the N-terminus of the light or heavy chain of a newly synthesized protein, such as an antibody. While we do not wish to be constrained by any particular theory, the incorporation of a signal peptide encoding sequence onto the heavy and / or light chain encoding mRNA may facilitate the secretion and / or production of antibodies produced from mRNA in vivo.

[0396] Suitable signal peptides for the present invention may be heterologous sequences derived from various eukaryotic and prokaryotic proteins, particularly secreted proteins. In some embodiments, suitable signal peptides are leucine-rich sequences (see Yamamoto Y et al., (1989), Biochemistry, 28: pp. 2728-2732, incorporated herein by reference). Suitable signal peptides may be derived from human growth hormone (hGH), serum albumin preproprotein, Ig kappa light chain precursor, azulosidine preproprotein, cystatin-S precursor, trypsinogen 2 precursor, potassium channel blockers, alpha-conotoxin lpl.3, alpha-conotoxin, alpha-galactosidase, cellulose, aspartate proteinase nepenthesin-1, acid chitinase, K28 preprotoxin, killer toxin digosine precursor, and cholera toxin. An exemplary signal peptide sequence is also described in Kober et al., Biotechnol. Bioeng., 110: 1164-73, 2012, which is incorporated herein by reference.

[0397] In some embodiments, the heavy and / or light chain encoding mRNA may include sequences encoding a signal peptide or fragment thereof derived from human growth hormone (hGH).

[0398] - CAR In some embodiments, the nucleic acid molecule encodes a chimeric antigen receptor (CAR). In one embodiment, the nucleic acid molecule is an mRNA molecule encoding a CAR. In another embodiment, the nucleic acid molecule is a modified nucleoside mRNA molecule encoding a CAR.

[0399] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial T cell receptor that is expressed on immune effector cells and engineered to specifically bind to an antigen. CARs may be used as a therapeutic agent in conjunction with adoptive cell transfer. T cells are removed from the patient and modified to express receptors specific to a particular form of antigen. In some embodiments, CARs have specificity to a selected target, e.g., fibroblast surface receptors. A CAR generally comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain containing an antigen-binding region that specifically binds to a selected target, e.g., a cell surface receptor.

[0400] In various embodiments, the CARs contemplated herein include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element, also called an antigen-binding domain. In some embodiments, the extracellular domain also includes a hinge domain. In certain embodiments, the intracellular domain, or otherwise the cytoplasmic domain, includes a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. The costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand required for an efficient lymphocyte response to an antigen.

[0401] A spacer domain may be incorporated between the extracellular domain and the transmembrane domain of a CAR, or between the cytoplasmic domain and the transmembrane domain of a CAR. As used herein, the term “spacer domain” generally means any oligopeptide or polypeptide that functions to ligate a transmembrane domain to either the extracellular domain or the cytoplasmic domain of a polypeptide chain. The spacer domain may contain up to 5 amino acids, or 10 amino acids, or 20 amino acids, or 30 amino acids, or 40 amino acids, or 50 amino acids, or 60 amino acids, or 70 amino acids, or 80 amino acids, or 90 amino acids, or 100 amino acids, or 110 amino acids, or 120 amino acids, or 130 amino acids, or 140 amino acids, or 150 amino acids, or 160 amino acids, or 170 amino acids, or 180 amino acids, or 190 amino acids, or 200 amino acids, or 210 amino acids, or 220 amino acids, or 230 amino acids, or 240 amino acids, or 250 amino acids, or 260 amino acids, or 270 amino acids, or 280 amino acids, or 290 amino acids, or 300 amino acids.

[0402] The extracellular domain, transmembrane domain, and intracellular domain may be derived from any desired source.

[0403] CAR antigen-binding domain The antigen-binding domain can be derived from a wide variety of extracellular domains or secreted proteins involved in ligand binding and / or signal transduction. In one embodiment, the antigen-binding domain may consist of an Ig heavy chain, which may be covalently associated with an Ig light chain by the presence of CHI and hinge regions, or covalently associated with other Ig heavy / light chain complexes by the presence of hinge, CH2, and CH3 domains. In the latter case, the heavy / light chain complex that will bind to the chimeric construct may constitute an antibody with specificity distinctly different from that of the chimeric construct. Depending on the antibody function, desired structure, and signal transduction, the entire chain may be used, or a shortened chain may be used. In the case of a shortened chain, all or part of the CHI, CH2, or CH3 domains may be removed, or all or part of the hinge region may be removed. In various embodiments, the CAR antigen-binding domain may be humanized or may contain a complete human sequence.

[0404] In one embodiment, the antigen-binding domain is a targeting domain, which gives CAR-expressing T cells a targeting of specific cells or tissues. For example, in one embodiment, the targeting domain includes an antibody, an antibody fragment, or a peptide that specifically binds to an antigen (e.g., a self-antigen or an exogenous antigen), thereby giving CAR-expressing T cells a targeting of cells or tissues that express the antigen.

[0405] The antigen-binding domain of the CAR molecule of the present invention can be generated to react to any desired antigen or fragment thereof, including but not limited to tumor antigens or exogenous antigens (e.g., bacterial or viral antigens).

[0406] Tumor antigens are proteins produced by tumor cells that trigger an immune response. The selection of the antigen-binding domain in the VM domain-containing fusion molecule of the present invention will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostain, PSMA, Her2 / neu, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. Another exemplary tumor antigen is chondroitin sulfate proteoglycan 4 (CSPG4) (also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP), high molecular weight melanoma-associated antigen (HMW-MAA), or neuron-glia antigen 2 (NG2)).

[0407] In one embodiment, CAR antigen binding specifically binds to tumor antigens, including one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can act as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncoemetic antigens, such as carcinoembryonic antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, and have shown some success.

[0408] The types of tumor antigens referred to in this invention may also be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present on other cells in the body. TAA-associated antigens are not specific to tumor cells; on the contrary, they are expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present on normal cells at very low levels but expressed on tumor cells at much higher levels.

[0409] Non-limiting examples of TSA or TAA antigens include: differentiation antigens, e.g., MART-I / Melan A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multisystem antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor suppressor genes, e.g., p53, Ras, HER-2 / neu; specific tumor antigens resulting from chromosomal translocations, e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, VBCAA, CA 195, CA Examples include 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0410] CAR transmembrane domain With respect to the transmembrane domain, the CARs of this disclosure can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of the same or different surface membrane proteins to the transmembrane domain of the domain, thereby minimizing interaction with other members of the receptor complex.

[0411] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present invention may originate from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 (i.e., may include at least these transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it will mainly consist of hydrophobic residues, such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. If necessary, short oligopeptides or polypeptide linkers, for example but not limited to those between 2 and 10 amino acids in length, may form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In another embodiment, the linker comprises a glycine-serine doublet.

[0412] CAR intracellular domain In various aspects, the cytoplasmic domain or otherwise intracellular domain of a CAR may be involved in the activation of at least one of the normal effector functions of the immune cell on which the CAR is expressed. The term “effector function” refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. The term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and instructs the cell to perform a specialized function. Usually, the entire intracellular domain may be used, but often it is not necessary to use the entire chain. When a truncated portion of the intracellular domain is used, such a truncated portion can be used in place of the intact chain if it transmits effector function signals. Therefore, the term intracellular domain is intended to include any truncated portion of the intracellular domain that is sufficient to transmit effector function signals.

[0413] Preferred examples of intracellular domains for use in CARs of this disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in coordination to initiate signal transduction after antigen receptor ligation, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0414] It is well known that signals generated by TCRs alone are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation by TCRs (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0415] Primary intracellular signaling sequences regulate the primary activation of the TCR complex either stimulatingly or inhibitorily. Stimulating primary intracellular signaling sequences may contain signaling motifs known as immune receptor-activated tyrosine motifs or ITAMs.

[0416] Examples of ITAM-containing primary intracellular signaling sequences particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling molecule in the CAR of the present invention includes an intracellular signaling sequence derived from CD3 zeta.

[0417] In another embodiment, the intracellular domain of the CAR can be designed to include only the CD3-zeta signaling domain, or to include the CD3-zeta signaling domain in combination with any other desired cytoplasmic domain useful for the CAR of the present invention. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. The costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand required for an efficient lymphocyte response to an antigen. Examples of such molecules include ligands that specifically bind to CD2, CD27, CD28, 4-1BB (CD137), 0x40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0418] The intracellular signaling sequences within the intracellular domain of the CAR of the present invention may be linked randomly or in a specified order. If necessary, short oligopeptides or polypeptide linkers, for example, linkers between 2 and 10 amino acids in length, may form the linkage. Glycine-serine doublets are suitable linkers in some embodiments.

[0419] In one embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a CD28 signaling domain. In yet another embodiment, the intracellular domain is designed to include a CD3-zeta signaling domain and a 4-1BB signaling domain.

[0420] In various forms, CARs can be "first-generation," "second-generation," "third-generation," "fourth-generation," or "fifth-generation" CARs (for example, Sadelain et al., Cancer Discov. 3(4): pp. 388-398 (2013); Jensen et al., Immunol. Rev. 257: pp. 127-133 (2014); Sharpe et al., Dis. Model Meeh. 8(4): pp. 337-350 (2015); Brentjens et al., Clin. Cancer Res. 13: pp. 5426-5435 (2007); Gade et al., Cancer Res. 65: pp. 9080-9088 (2005); Maher et al., Nat. Biotechnol. 20: pp. 70-75 (2002); Kershaw et al., J. Immunol. 173: pp. 2143-2150 (2004); See Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32: pp. 169-180 (2009), each of these references is incorporated herein by reference in its entirety.

[0421] The “first-generation” CARs for use in this invention include an antigen-binding domain, e.g., a single-stranded variable fragment (scFv), fused to the cytoplasmic / intracellular domain of the T cell receptor chain, fused to the transmembrane domain, etc. The “first-generation” CARs generally have an intracellular domain from the CD3^ chain, which is the primary signaling molecule from the endogenous T cell receptor (TCR). The “first-generation” CARs can induce de novo antigen recognition and can cause activation of both CD4+ T cells and CD8+ T cells through their CD3^ chain signaling domains within a single fusion molecule, independently of HLA-mediated antigen presentation.

[0422] The “second-generation” CAR for use in this invention includes an antigen-binding domain, such as a single-stranded variable fragment (scFv), which is fused with an intracellular signaling domain capable of activating T cells and a costimulatory domain designed to enhance the potency and persistence of T cells (Sadelain et al., Cancer Discov. 3: pp. 388-398 (2013)). Thus, the CAR design can combine two functions physiologically carried out by two separate complexes: antigen recognition and signal transduction, namely the TCR heterodimer and the CD3 complex. The “second-generation” CAR includes intracellular domains from various costimulatory molecules, such as CD28, 4-1BB, ICOS, OX40, etc., in the cytoplasmic tail of the CAR to provide additional signaling to the cell.

[0423] "Second-generation" CARs provide both co-stimulation, for example, via the CD28 or 4-IBB domain, and activation, for example, via the CD3^ signaling domain. Preclinical studies have shown that "second-generation" CARs can enhance the antitumor activity of T cells. For example, the robust efficacy of "second-generation" CAR-modified T cells has been demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9): pp. 1577-1583 (2012)).

[0424] "Third-generation" CARs provide multiple co-stimulation, for example, by including both the CD28 domain and the 4-1BB domain, and provide activation, for example, by including the CD3^ activation domain.

[0425] "Fourth-generation" CARs provide co-stimulation, for example, through the CD28 or 4-1BB domain, and activation, for example, through the CD3^ signaling domain in addition to constitutive or inducible chemokine components.

[0426] "Fifth-generation" CARs provide co-stimulation, for example, through the CD28 or 4-1BB domain, and activation, for example, through the CD3^ signaling domain, constitutive or inducible chemokine components, and the intracellular domain of a cytokine receptor, for example, IL-2Rp.

[0427] In some embodiments, one or more mRNA molecules encode molecules selected from the following group: a) Immune cell-enhancing or inhibitory compounds, in particular, those described herein, preferably selected from the following: - TCF1, LEF1, WNT, FRIZZLED, Beta-catenin, BCL2, BCLXL, BIRC3, MCL1, PGC1a, TCF7, NFAT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glut1, Glut3, HK2, Arginine resynthesis enzyme, Argininosuccinate synthase (ASS), Ornithine Transcarbamylase (OTC), GYS, OXPHOS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD107a, Lymphotoxin (LT) α1β2, Granzyme B, Perforin, POU2F1, BBS10, BBS12, TCP1, HSP, Integrin Alpha 1, Integrin Alpha 2, Integrin Alpha 2b, Integrin Alpha 11, Integrin Alpha 3, Integrin Alpha 6, Integr Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, LFA-1, LFA-2, LFA-3, Integrin Beta 1, Integrin Beta 7, CD103, Integrin Alpha V, ITGAE, CD11C, CRTAM, SELL, GAL3, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Rab Protein, NLRP3, TAP, LAMP, Ubiquitin Ligase, CD74, Peptidase Calreticulin, Aurora, LGR6, HAT, KDM1, TGD, TET1, C-FOS, JUN, EGR-2, EGR-3, Phosphatidylinositol 3-kinase (PI3K), BCLXL, GPR35, Interferon regulators, e.g., IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, NFκB, T-bet, AhR, STING, MAVS, MyD88, IRAK1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKα, IKKβ, TRAM, TRIF, TBK1, D3-phosphoinositide, phosphatidylinositol derivatives, DECTIN, CD122, CD132, CD25, CD215, -1, CLEC-9A, CLEC-2, DECTIN-2, MCL, MINCLE, BDCA-2, ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, OX40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAMFrom the group consisting of the CD2 family, CD27, TL1A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, CD160, and any combination thereof, preferably BCL2, BCLXL, CD28, 4-1BB, ICOS, CD40L, TBET, TCF1, G lut-1, OXPHOS, IL7, IL12, IL-15, IL-21, IL7R, IL12R, IL-15R, IL-21R, Integrin Alpha 1, Integrin Alpha 2, Integrin Alpha 2b, Integrin Alpha 11, Integrin Alpha 3, Integrin Alpha 6, Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, Integrin Beta 1, Integrin Beta 7 and Compounds or molecules selected from the group consisting of integrin alpha V, GrB, perforin, TCF1, Wnt, Rec or LGR6 and any combination thereof, preferably from the group consisting of BCL2, BCLXL, CD28, 4-1BB, ICOS, CD40L, TBET, TCF1, Glut-1, OXPHOS, IL7, IL12, IL15, IL21, IL7R, IL12R, IL21R, IL12R, GrB, perforin, TCF1, Wnt, integrin alpha 1, integrin alpha 2, integrin alpha 2b, integrin alpha 11, integrin alpha 3, integrin alpha 6, integrin alpha 7, integrin alpha E, integrin beta 2, integrin beta 4, integrin beta 1, integrin beta 7 and integrin alpha V, Rec or LGR6 and any combination thereof; in particular, for example, those described herein; - PUMA, BIMS, 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-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, 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, Lamini 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, SUV420H2, TGFR, TNFR, GCSFR, CD72, DCIR, MICL, CLEC-1, TGFB, IFNa R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM4, 2B4, 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 A2AR, SOCS, RIPK1, and any member of the STAT family;and any combination thereof, compounds or molecules 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, in particular, those described herein, for example; b) Cytokines, preferably selected from the group consisting of IL12A, IL12B, IFNG, IFNa, IL21, IL7, IL2, IL15, IL13, IL4, IL-10, IL35, IL37, IL38, and IL18, and any combination thereof, in particular, those described herein, for example; c) Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-17R, IL-18R, IL-21R, IL-23R, and IL-35R; d) Chemokines, preferably selected from the group consisting of CXCL9 or CXCL10; e) 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; f) Antigen fragments derived from viral pathogens, bacterial pathogens and / or parasitic pathogens, in particular, those described herein, for example; g) Antibodies or fragments or derivatives thereof, preferably antibodies or fragments or derivatives thereof against targets selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, LAG3, BTLA, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, CD39, CD30, NKG2D, NKG2A, 2B4, DR3, CD101, CD44, SIRPG, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122; and any combination thereof; in particular, for example, those described herein, and h) Chimeric antigen receptors (CARs), in particular those described herein, for example.

[0428] In some embodiments, one or more mRNA molecules encode molecules selected from the following group: a) Immune cell molecules selected from the following group: - BCL2, BCLXL, CD28, 4-1BB, ICOS, CD40L, TBET, TCF1, Glut-1, OXPHOS, IL7, IL12, IL15, IL21, IL7R, IL12R, IL21R, IL12R, GrB, Perforin, TCF1, Wnt, Integrin Alpha 1, Integrin Alpha 2, Integrin Alpha 2b, Integrin Alpha 11, Integrin Alpha 3, Integrin Alpha 6, Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, Integrin Beta 1, Integrin Beta 7 and Integrin Alpha V, Rec or LGR6 and any combination thereof, in particular, for example, those described herein; or - 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, in particular, as described herein, for example.

[0429] antigen-binding domain The method of the present invention ultimately comprises one or more antigen-binding domains.

[0430] In the method of the present invention, the antigen-binding domain is mixed with the first or second composition before the first and second compositions are mixed. In some embodiments, the antigen-binding domain is mixed with the first or second composition before the step of supplying the first and second compositions to a mixing device. This means that in certain embodiments, the antigen-binding domain is mixed with the first or second composition outside of the mixing device.

[0431] Therefore, in a certain embodiment, the method of the present invention is - A step of mixing a composition comprising one or more antigen-binding domains with a first composition, wherein the first composition comprises a lipid-based composition and a polar organic solvent. - A step of supplying the mixed composition through the first inlet of the mixing device, - A step of supplying a second composition through a second inlet of the mixing device, wherein the second composition is an acidic aqueous composition comprising one or more nucleic acid molecules, - A step of mixing the composition from the first inlet and the composition from the second inlet in a mixing device to generate lipid-based nanoparticles. - A step of removing polar organic solvents from the mixed composition and adjusting the pH of the composition to a neutral pH, and b) A step of recovering lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes.

[0432] Alternatively, the method of the present invention is - A step of mixing a composition comprising one or more antigen-binding domains with a second composition, wherein the second composition is an acidic aqueous composition comprising one or more nucleic acid molecules. - A step of supplying the mixed composition through a second inlet of the mixing device, - A step of supplying a first composition through a first inlet of a mixing device, wherein the first composition comprises a lipid-based composition and a polar organic solvent. - A step of mixing the composition from the first inlet and the composition from the second inlet in a mixing device to generate lipid-based nanoparticles. - A step of removing polar organic solvents from the mixed composition and adjusting the pH of the composition to a neutral pH; and c) A step of recovering lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes.

[0433] In some embodiments, the composition comprising the antigen-binding domain comprises a suitable buffer, generally PBS.

[0434] Preferably, the antigen-binding domain is not covalently bound to any of the lipids of the LNP, and does not include any modifications for coupling or grafting the antigen-binding domain to the lipids. In particular, the antigen-binding domain does not include a lipophilic portion or a graft portion, such as a free cysteine ​​or thiol group.

[0435] In one embodiment, the antigen-binding domain is mixed with the first composition, and the concentration of the antigen-binding domain in the first composition is about 0.01 μg / μL to about 0.5 μg / μL, about 0.02 μg / μL to about 0.45 μg / μL, about 0.03 μg / μL to about 0.4 μg / μL, about 0.04 μg / μL to about 0.35 μg / μL, about 0.05 μg / μL to about 0.3 μg / μL, about 0.1 μg / μL to about 0.2 μg / μL, and about 0.125 μg / μL to about 0.175 μg / μL. In particular, if the antigen-binding domain is mixed with the first composition before the mixing step of the first and second compositions, the concentration of the antigen-binding domain in the first composition is preferably about 0.01 μg / μL to about 0.5 μg / μL.

[0436] In an alternative embodiment, the antigen-binding domain is mixed with a second composition, and the concentration of the antigen-binding domain in the second composition is approximately 0.005 μg / μL to approximately 0.5 μg / μL, approximately 0.01 μg / μL to approximately 0.45 μg / μL, approximately 0.02 μg / μL to approximately 0.4 μg / μL, approximately 0.03 μg / μL to approximately 0.35 μg / μL, approximately 0.04 μg / μL to approximately 0.3 μg / μL, approximately 0.05 μg / μL to approximately 0.25 μg / μL, approximately 0.1 μg / μL to approximately 0.2 μg / μL, and approximately 0.125 μg / μL to approximately 0.175 μg / μL. In particular, if the antigen-binding domain is mixed with the second composition before the mixing step of the first and second compositions, the concentration of the antigen-binding domain in the second composition is preferably about 0.005 μg / μL to about 0.25 μg / μL.

[0437] The antigen-binding domain used for mixing with the first or second composition can specifically bind to a target. While we do not wish to be constrained by theory, we believe that the presence of the antigen-binding domain would be useful for designating or targeting destinations of lipid-based nanoparticles obtained by the method of the present invention into specific localization sites, tissues, or cells for the specific delivery of nucleic acid molecules contained in the lipid nanoparticles.

[0438] The terms “specific binding,” “specifically binding to,” “specific to,” or “selectively binding” to a specific target or epitope on a particular antigen mean that the antigen-binding domain recognizes and binds to a particular antigen or epitope, but substantially does not 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 more easily and / or for a longer duration with greater affinity, binding strength, etc., than it would to bind to other / different antigens. Preferably, the terms “specifically binding to” or “specifically binding” mean at least about 1 × 10⁻⁶ -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 Refers to the ability of an antigen receptor to bind to an antigen with an affinity equal to or greater than M, and / or to bind to a target with an affinity 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.

[0439] In certain embodiments, the antigen-binding domain that will be included in the lipid-based nanoparticles according to the present 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, when determined by biosensor analysis, particularly by Biacore analysis, for targets expressed on immune cells.

[0440] As used herein, the term "target" of an antigen-binding domain refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen, or epitope that is specifically recognized or bound by an antigen-binding domain according to the present invention and that is expressed on the outer surface of an immune cell. With respect to the expression of a target on the surface of an immune cell, the term "expressed" refers to a target that is present or presented on the outer surface of the immune cell, such as a carbohydrate, lipid, peptide, polypeptide, protein, antigen, or epitope.

[0441] In certain embodiments, the antigen-binding domain that will be included in the lipid-based nanoparticles according to the present invention has a binding activity that is similar to that of the antigen-binding domain in its free form. As used herein, the "antigen-binding domain in free form" refers to an antigen-binding domain that is not linked, grafted, or conjugated to an LNP. In a preferred embodiment, the antigen-binding domain that will be included in the lipid-based nanoparticles according to the present invention has a binding activity equal to about 70%, more preferably equal to about 75%, even more preferably equal to about 80%, of the binding activity of the antigen-binding domain in free form.

[0442] The antigen-binding domain incorporated into lipid-based nanoparticles by the method of the present invention can specifically bind to targets expressed on immune cells, tumor cells, infected cells, or pathogens in the human body.

[0443] In some embodiments, the antigen-binding domain can specifically bind to targets expressed on tumor cells. Preferably, the antigen-binding domain can specifically bind to tumor-associated antigens (TAAs), such as carcinoembryonic antigen (CEA), p53, human epithelial receptor-2 / neurological (HER-2 / neu), melanoma antigens 2 and 3 (MAGE-2 / 3), human papillomavirus protein 6 (HPVE6), alpha-fetoprotein (AFP), and prostate-specific antigen (PSA).

[0444] Examples of tumor antigens targeted by the antigen-binding domain of the present invention include, but are not limited to, any of the various MAGE (melanoma-associated antigen E) including MAGE 1 (e.g., GenBank accession number M77481), MAGE 2 (e.g., GenBank accession number U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank accession numbers X54156 and AA494311); and p97 melanoma antigen (e.g., GenBank accession number M12154). Other tumor-specific antigens include Ras peptide and p53 peptide associated with advanced cancer, HPV 16 / 18 and E6 / E7 antigens associated with cervical cancer, MUC1-KLH antigen associated with breast cancer (e.g., GenBank accession number J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank accession number X98311), gp100 associated with melanoma (e.g., GenBank accession number S73003) or MART1 antigen, and PSA antigen associated with prostate cancer (e.g., GenBank accession number X14810). The p53 gene sequence is publicly known (see, for example, Harris et al., (1986) Mol. Cell. Biol., 6: pp. 4650-4656) and is deposited in GenBank under accession number M14694.The tumor antigens included in this invention are Her-2 / Neu (e.g., GenBank accession numbers M16789.1, M16790.1, M16791.1, M16792.1), NY-ESO-1 (e.g., GenBank accession number U87459), hTERT (also known as telomerase) (GenBank accession numbers NM003219 (variant 1), NM198255 (variant 2), NM 198253 (variant 3), and NM 198254 (variant 4)), proteinase 3 (e.g., GenBank accession numbers M29142, M75154, M96839, X55668, NM 00277, M96628, and X56606), HPV E6 and E7 (e.g., GenBank accession number NC Further examples include, but are not limited to, 001526) and WT-1 (e.g., GenBank accession numbers NM000378 (Variant A), NM024424 (Variant B), NM024425 (Variant C), and NM024426 (Variant D)). Accordingly, the present invention can be used as an immunotherapy for cancers including, but not limited to, cervical cancer, breast cancer, colorectal cancer, prostate cancer, and lung cancer, and for melanoma.

[0445] Examples of antibodies that target such TAAs include rituximab and tratuzumab.

[0446] In some embodiments, the antigen-binding domain can specifically bind to a target expressed on a pathogen such as bacteria, viruses, or fungi. The present invention further includes antigen-binding domains that target antigens from the following infectious diseases: measles, mumps, rubella, poliomyelitis, hepatitis A, hepatitis B (e.g., GenBank accession number E02707), and hepatitis C (e.g., GenBank accession number E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank accession number M34678), yellow fever, Japanese encephalitis (e.g., GenBank accession number E07883), dengue fever (e.g., GenBank accession number M24444), hantavirus, and HIV (e.g., GenBank accession number U18552). Bacterial and parasitic antigens include those for diphtheria, pertussis (e.g., GenBank accession number M35274), tetanus (e.g., GenBank accession number M64353), tuberculosis, bacterial and fungal pneumonia (e.g., Haemophilus influenzae, Pneumocystis jiroensis). The causes of these diseases may include, but are not limited to, known causative agents of diseases such as carinii, cholera, typhoid fever, plague, bacterial dysentery, salmonellosis (e.g., GenBank accession number L03833), legionellosis, Lyme disease (e.g., GenBank accession number U59487), malaria (e.g., GenBank accession number X53832), hookworms, onchocerciasis (e.g., GenBank accession number M27807), schistosomiasis (e.g., GenBank accession number L08198), trypanosomiasis, leishmaniasis, giardiasis (e.g., GenBank accession number M33641), amoebicosis, filariasis (e.g., GenBank accession number J03266), borreliosis, and trichinellosis.

[0447] Examples of antibodies that target such pathogens include dubratoxumab, palivizumab, laxibakumab, bezlotoxumab, and ansubimab.

[0448] The antigen-binding domain incorporated into lipid-based nanoparticles by the method of the present invention can specifically bind, in particular, to targets expressed on immune cells (i.e., on the outer surface of immune cells), preferably to targets expressed on activated immune cells. In particular, the targets are expressed by immune cells in healthy subjects or subjects suffering from disease, preferably in subjects suffering from diseases such as cancer, infectious diseases, autoimmune diseases, or inflammatory diseases. This means that the target has a higher expression level in immune cells than in other cells, or that the ratio of 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, 5, 10, 20, 50, or 100 times higher.

[0449] "Immune cells," as used herein, 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. "Activated immune cells" means immune cells that have been activated in the course of an immune response to the presence of non-self cells such as pathogens or cancer cells. Activated immune cells are particularly recruited to localized sites where inflammation occurs, triggered by 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.

[0450] When 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), anergistic T cells, suppressor T cells, and / or senescent 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, CRTAM, CTLA4, FasL / TNFRSF6, TIM-3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4, SIRPg, and TIGIT.

[0451] Preferably, the term "T cell" does not include regulatory T cells (Treg), suppressive T cells, and / or senescent T cells.

[0452] Preferably, the immune cells are activated T cells.

[0453] The targets expressed on immune cells may be selected from among the targets listed in Table G (Table 8) below.

[0454] [Table 8A]

[0455] [Table 8B]

[0456] [Table 8C]

[0457]

Table 8D

[0458] Table 8E

[0459]

Table 8F

[0460]

Table 8G

[0461]

Table 8H

[0462] Table 8I

[0463] 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.

[0464] The targets of the antigen-binding domain expressed on the cell surface are preferably PD-1, CTLA-4, GITR, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, HVEM, Tim-1, LFA-1, LAG3, TIM3, CD39, CD30, NKG2D, NKG2A, PD-1, 2B4, DR3, CD101, CD44, SIRPG, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, and CEACAM. From the group consisting of and CD122; preferably selected from the group consisting of PD-1, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, HVEM, Tim-1, LFA-1, LAG3, TIM3, CD39, CD30, NKG2D, NKG2A, PD-1, 2B4, DR3, CD101, CD44, SIRPG, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122.

[0465] In particular, the targets on which the antigen-binding domain is expressed are selected from the group consisting of PD-1, CD127, SIRPa, and CLEC-1A.

[0466] Preferably, the target on which the antigen-binding domain is expressed is selected from the group consisting of PD-1, CTLA4, 4-1BB, ICOS, LAG3, TIM3, TIGIT, BTLA, or CLEC-1, and is preferably PD-1.

[0467] Preferably, the target of the antigen-binding domain specifically expressed on the surface of immune cells is selected from the group consisting of PD-1, BTLA, TIGIT, CD160, LAG3, and TIM3. In a preferred embodiment, the target is PD-1. These targets are not antigens of the TCR pathway (interaction between antigen-presenting cells and T cells).

[0468] Preferably, the antigen-binding domain to be mixed with the first or second composition is an anti-PD-1 antigen-binding domain, and therefore the lipid-based nanoparticles obtained by the method of the present invention aim to target immune cells present in the tumor environment (e.g., recruited to the tumor site), such as PD-1 positive immune cells.

[0469] In certain embodiments, the T cells are tumor-infiltrating lymphocytes (TILs), and the targets are factors expressed on the surface of tumor-infiltrating lymphocytes, preferably specifically expressed on the surface of TILs. Preferably, the targets expressed on the surface of TILs are 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.

[0470] In one embodiment, when the target is a receptor that has an inhibitory effect on cells, particularly a receptor for a checkpoint inhibitor between tumor cells and T cells (e.g., PD-1), the antigen-binding domain has antagonist activity against the target.

[0471] 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.

[0472] In an alternative embodiment, if the target is a receptor that has an activating effect on cells (e.g., CD137), the antigen-binding domain has agonist activity against the target. The term "agonist," as used herein, 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.

[0473] In some embodiments, the antigen-binding domain does not interfere with or compete for the binding of its target to its native ligand. In particular, the antigen-binding domain has no activity against the target, especially the target of checkpoint inhibitors between tumor cells and T cells (e.g., PD-1).

[0474] The antigen-binding domain used in the method of the present invention may be of any form known in the art.

[0475] In particular, the antigen-binding domain is an antibody, a fragment thereof, or a derivative, 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.

[0476] 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.

[0477] 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.

[0478] 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 Fab, Fv, Fab', scFV, CrossMab, or VHH covalently linked to one described herein.

[0479] 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).

[0480] 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.

[0481] 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.

[0482] In some cases, the antigen-binding domain is an aptamer or a short peptide sequence, such as an RGD peptide.

[0483] 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.

[0484] 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.

[0485] 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.

[0486] In a preferred embodiment, the antigen-binding domain of the lipid-based nanoparticle does not compete with other antigen-binding domains that bind to the same target.

[0487] In some embodiments, the antigen-binding domain is an antibody or derived from an antibody. Preferably, the antigen-binding domain is derived from IgA, IgM, IgE, IgD, and IgG, preferably IgG.

[0488] The terms "derive from" and "derived from," as used herein, refer to a compound or molecule having a structure derived from the structure of a parent compound, molecule, 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.

[0489] 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.

[0490] 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.

[0491] 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.

[0492] 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.

[0493] In certain embodiments, the antigen-binding domain is not covalently bound to any of the lipids of the LNP, and does not include any modifications for coupling or grafting the antigen-binding domain to the lipids. In particular, the antigen-binding domain does not include a lipophilic moiety or a graft moiety, such as a free cysteine ​​or thiol group.

[0494] In certain embodiments, the LNP does not contain any antigen-specific antigen-binding domains present on the antigen-binding domain of the LNP, and in particular does not contain an antigen-binding domain that is directed toward the Fc domain of an antibody.

[0495] In certain embodiments, the antigen-binding domain of an LNP is not bound to the LNP by an "antigen-antibody" type interaction. More specifically, the antigen-binding domain is not bound to antigen-specific antigen-binding domains present on the LNP's antigen-binding domain, particularly antigen-binding domains that are directed toward the Fc domain of an antibody.

[0496] 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.

[0497] 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.

[0498] In some embodiments, the lipid-based nanoparticles do not contain a portion comprising a lipidized peptide or motif.

[0499] 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.

[0500] As used herein, the term “anchor moiety” refers to a component that anchors or binds an antigen-binding domain to a lipid-based nanoparticle.

[0501] 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.

[0502] In relation 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.

[0503] 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.

[0504] 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.

[0505] 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.

[0506] 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.

[0507] In some preferred embodiments, the antigen-binding domain to be mixed with the first or second composition in the method of the present invention is an antibody known in the art or derived therefrom. A great many antibodies against PD-1, TIM3, CTLA-4, LAG-3, BTLA, and TIGIT have already been described.

[0508] In some embodiments, the antigen-binding domain specifically binds to a target selected from the group consisting of PD-1, CTLA4, 4-1BB, ICOS, LAG3, TIM3, TIGIT, BTLA, or CLEC-1, preferably to PD-1.

[0509] As used herein, the terms “programmed death 1,” “programmed cell death 1,” “PD1,” “PD-1,” “PDCD1,” “PD-1 antigen,” “human PD-1,” “hPD-1,” and “hPD1” 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. PD1 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.

[0510] 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), OSE279 (see WO2020 / 127366), pizilizumab (CT-011), semiplimab (ribtayo), camrelizumab, AUNP12, AMP-224, AGEN-2034, 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 (see Si-Yang 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 (WO See 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540 (disclosures in these reference documents are incorporated herein by reference), and may be selected from the group consisting of monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168 (disclosures in this reference document are incorporated herein by reference).

[0511] Antibodies targeting TIM3, 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 which are incorporated herein by reference.

[0512] Antibodies targeting CTLA-4, such as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), and MEDI5752 (AstraZeneca), are also known. The anti-CTLA-4 antibodies are 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.

[0513] Antibodies targeting 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.

[0514] 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.

[0515] 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 the disclosures in these reference documents are incorporated herein by reference.

[0516] 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.

[0517] 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.

[0518] 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.

[0519] 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.

[0520] 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.

[0521] Antibodies against SIRPa, such as 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.

[0522] 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.

[0523] In some embodiments, the antigen-binding domain is an anti-SIRPa antibody comprising a heavy chain containing or consisting of the sequence shown in SEQ ID NO: 56, and a light chain containing or consisting of the sequence shown in SEQ ID NO: 57, or comprising the heavy chain and the light chain.

[0524] 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.

[0525] 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.

[0526] Antibodies against CLEC-1A, such as MAB1704, ABIN526589, AF1704, and ABIN526590, are also known in the art.

[0527] In some embodiments, the antigen-binding domain is specific to PD-1. Preferably, the antigen-binding domain is a PD-1 antagonist. More preferably, the anti-PD1 antigen-binding domain is an anti-PD-1 antibody selected from the group consisting of pembrolizumab (also known as keytruda lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), and OSE279 (e.g., as described in WO2020 / 127366 (the disclosure of this reference document is incorporated herein by reference)).

[0528] Accordingly, in some embodiments, the present invention relates to a method for obtaining a t-LNP comprising an anti-PD-1 antigen-binding domain that can specifically bind to PD-1 expressed on the surface of immune cells such as activated T cells, for example, the anti-PD-1 antigen-binding domain disclosed herein, and one or more mRNA molecules encoding an activity-enhancing protein of the immune cells. Accordingly, the present invention also relates to a t-LNP that can be obtained by the method of the present invention, comprising an anti-PD-1 antigen-binding domain that can specifically bind to PD-1 expressed on immune cells such as 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 activity-enhancing protein of the activated immune cells.

[0529] 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.

[0530] 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.

[0531] 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.

[0532] 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.

[0533] 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.

[0534] 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).

[0535] 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 include, in particular, one, two, or three modifications selected from substitutions, additions, deletions, and any combination...

Claims

1. A method for producing lipid-based nanoparticles comprising an antigen-binding domain and one or more nucleic acid molecules, - A step of supplying a first composition comprising a lipid-based composition and a polar organic solvent through a first inlet of a mixing device. - A step of supplying a second composition comprising one or more nucleic acid molecules through a second inlet of the mixing device, wherein the second composition is an acidic aqueous composition. - A step of mixing the first composition and the second composition in the mixing device to generate lipid-based nanoparticles, - A step of removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH, and - A step of recovering the lipid-based nanoparticles containing an antigen-binding domain and one or more nucleic acid molecules. Includes; Prior to the step of mixing the first and second compositions in the mixing device, the antigen-binding domain is mixed with the first or second composition; A method wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

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

3. The method according to claim 1 or 2, wherein the antigen-binding domain is not covalently bound to any of the lipids of the lipid-based nanoparticles, or the method does not involve any modification 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. The method according to any one of claims 1 to 4, wherein the step of removing the polar solvent is performed by dialysis or buffer exchange.

6. The method according to any one of claims 1 to 5, wherein the lipid-based composition comprises or consists of cationic or ionizable lipids, helper lipids, sterols and PEG-lipids.

7. 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, The method according to claim 6, wherein a compound is 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) or bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl]disulfide (SS-OP), 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.

8. The method according to claim 6 or 7, 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.

9. The method according to any one of claims 6 to 8, wherein the helper lipid is selected from the group consisting of DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DEPC, DOPC, and DSPE, and is preferably DOPE or DSPC.

10. The method according to any one of claims 6 to 9, wherein the PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG, and PEG-c-DMA, ALC-0159, and any mixture thereof, and is preferably PEG-DMG, PEG-DSPE, or a mixture thereof.

11. Lipid-based nanoparticles as defined in claim 10, wherein the PEG is between 2000 daltons and 5000 daltons, and is preferably DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000, or a mixture thereof.

12. The lipid-based composition 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; and k) SS-OP, DSPC, cholesterol and DSPE-PEG; and l) SS-OP, DOPC, cholesterol and DMG-PEG A method according to any one of claims 1 to 11, selected from the group consisting of the following.

13. The method according to any one of claims 1 to 12, wherein the lipid-based composition comprises or consists of 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.

14. The method according to any one of claims 1 to 12, wherein the lipid-based composition 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.

15. The method according to any one of claims 1 to 14, wherein the concentration of lipids in the first composition is about 1 to about 100 mM, and / or the concentration of nucleic acid molecules in the second composition is about 0.01 mg / mL to about 100 mg / mL.

16. - The antigen-binding domain is mixed with the first composition before the mixing step of the first and second compositions, and the concentration of the antigen-binding domain in the first composition is about 0.01 μg / μL to about 0.5 μg / μL, or - The antigen-binding domain is mixed with the second composition before the mixing step of the first and second compositions, and the concentration of the antigen-binding domain in the second composition is about 0.005 μg / μL to about 0.25 μg / μL. The method according to any one of claims 1 to 15.

17. The method according to any one of claims 1 to 16, wherein the first composition comprises an ionizable lipid, and the second composition has a pH lower than the pKa of the ionizable lipid, preferably a pH between about 3 and about 6.

18. The method according to any one of claims 1 to 17, wherein the flow rate ratio of the first composition to the second composition is between 1:10 and 10:1, preferably between 1:5 and 5:1, and more preferably between 1:2 and 1:

4.

19. The antigen-binding domains 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, GP R18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TN FSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18R1 / CXCR1 / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, 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, F CRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, T The method according to any one of claims 1 to 18, wherein the target is conjugated to a target selected from the group consisting of NFRSF12A / 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, CD127, SIRPa, and CD160.

20. The method according to any one of claims 1 to 19, wherein the antigen-binding domain binds to a target selected from the group consisting of PD-1, CD127, SIRPa, and CLEC-1A.

21. The method according to any one of claims 1 to 20, wherein the antigen-binding domain binds to human PD-1.

22. The antigen-binding domain, (i) VH containing heavy chain CDR1 (HCDR1), CDR2 (HCDR2), and CDR3 (HCDR3), and (ii) VL containing light chain CDR1 (LCDR1), CDR2 (LCDR2), and CDR3 (LCDR3) It contains an anti-PD-1 antigen binding domain, a) The HCDR1 contains or consists of the amino acid sequence of SEQ ID NO: 1; The HCDR2 contains or is derived from 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 LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 6; or b) The HCDR1 contains or comprises the amino acid sequence of SEQ ID NO: 23; The HCDR2 contains or comprises the amino acid sequence of SEQ ID NO: 24; The HCDR3 contains or comprises the amino acid sequence of SEQ ID NO: 25; The aforementioned LCDR1 contains or comprises 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 LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 28; or c) The HCDR1 contains or comprises the amino acid sequence of SEQ ID NO: 31; The 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 comprises 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. The method according to claim 21.

23. 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. The method according to claim 21, comprising an anti-PD-1 antigen-binding domain.

24. The method according to any one of claims 1 to 23, wherein the first or second composition is mixed with a second antigen-binding domain before the mixing step of the first and second compositions, and the second antigen-binding domain is an antibody or an antigen-binding fragment thereof.

25. The method according to any one of claims 1 to 24, wherein the nucleic acid molecule is mRNA.

26. The aforementioned mRNA a) Immune cell-enhancing or inhibitory compounds, in particular, those described herein, preferably selected from the following: - TCF1, LEF1, WNT, FRIZZLED, Beta-catenin, BCL2, BCLXL, BIRC3, MCL1, PGC1a, TCF7, NFAT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCT1, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glut1, Glut3, HK2, Arginine resynthesis enzyme, Argininosuccinate synthase (ASS) ), ornithine transcarbamylase (OTC), GYS, OXPHOS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD107a, lymphotoxin (LT) α1β2, granzyme B, perforin, POU2F1, BBS10, BBS12, TCP1, HSP, integrin alpha 1, integrin alpha 2, integrin alpha 2b, integrin alpha 11, integrin alpha 3, integrin alpha 6, i Integrin Alpha 7, Integrin Alpha E, Integrin Beta 2, Integrin Beta 4, LFA-1, LFA-2, LFA-3, Integrin Beta 1, Integrin Beta 7, CD103, Integrin Alpha V, ITGAE, CD11C, CRTAM, SELL, GAL3, Laminin, Actin, Vimentin, DEF1, Dynein, Kinesin, Rab Protein, NLRP3, TAP, LAMP, Ubiquitin Ligase, CD74, Peptid Ze, Calreticulin, Aurora, LGR6, HAT, KDM1, TGD, TET1, C-FOS, JUN, EGR-2, EGR-3, Phosphatidylinositol 3-kinase (PI3K), BCLXL, GPR35, Interferon regulators, e.g., IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, NFκB, T-bet, AhR, STING, MAVS, MyD88, IRAK1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKα, IKKβ, TRIF, D3-phosphoinositide, derivatives of phosphatidylinositol, DECTIN, CD122, CD132, CD25, CD215, -1, CLEC-9A, CLEC-2, DECTIN-2, MCL, MINCLE, BDCA-2, ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, OX40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAM Molecules selected from the group consisting of the CD2 family, CD27, TL1A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, CD160, and any combination thereof, in particular those described herein, for example; - PUMA, BIMS, 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-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, 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, Lami Nin, 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, SUV420H2, TGFR, TNFR, GCSFR, CD72, DCIR, MICL, CLEC-1, TGFB, IFNa From R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM4, 2B4, 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, A2AR, SOCS, RIPK1, and any member of the STAT family, preferably FOXO1, Fas, TRAIL, PUMA, NOXA, BIM, LAG3, CTLA4, PDL1, FOXP3, TOX, IDO, ARG1 A molecule selected from the group consisting of IL35+IL35R, TGFB+TGFBR, TIM3, TIGIT, ChemR23, and FPR2;and any combination thereof, in particular, those described herein, for example; b) Cytokines, preferably selected from the group consisting of IL12A, IL12B, IFNG, IFNa, IL21, IL7, IL2, IL15, IL13, IL4, IL-10, IL35, IL37, IL38, and IL18, and any combination thereof, in particular those described herein; c) Cytokine receptors, preferably selected from the group consisting of IL-1R, IL-4R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-17R, IL-18R, IL-21R, IL-23R, and IL-35R; d) Chemokines, preferably selected from the group consisting of CXCL9 or CXCL10; e) 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; f) Antigen fragments derived from viral pathogens, bacterial pathogens and / or parasitic pathogens, in particular, those described herein, for example; g) Antibodies or fragments or derivatives thereof, preferably antibodies or fragments or derivatives thereof against targets selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, LAG3, BTLA, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, CD39, CD30, NKG2D, NKG2A, 2B4, DR3, CD101, CD44, SIRPG, CD38, CXCR3, CXCR5, CD4, CD8, CD25, CRTAM, CD96, CD226, CD112R, CD103, CEACAM and CD122; and any combination thereof; in particular, for example, those described herein, and h) Chimeric antigen receptors (CARs), in particular those described herein, for example. The method according to claim 25, which encodes a molecule selected from the group consisting of the following.

27. The method according to claim 26, wherein the mRNA encodes BCL2, IL7, IL7R, CXCL9 and / or CXCL10.

28. The method according to any one of claims 1 to 27, wherein the second composition comprises at least two different mRNA molecules.

29. The method according to claim 28, wherein the second composition comprises an mRNA molecule encoding IL-7 and an mRNA molecule encoding IL-7R.

30. The method according to any one of claims 1 to 29, wherein the mixing device is a microfluidic device.

31. Lipid-based nanoparticles that can be obtained by the method described in any one of claims 1 to 30.

32. a) an antigen-binding domain covalently bound to a lipid; ii) an antigen-binding domain including modifications for coupling or grafting the antigen-binding domain to a lipid and / or iii) an anchor portion including a lipidized peptide or motif, according to claim 31.

33. A pharmaceutical composition comprising lipid-based nanoparticles as described in claim 31 and, optionally, a pharmaceutically acceptable carrier.

34. Lipid-based nanoparticles according to claim 31 or 32, or a pharmaceutical composition according to claim 33, for use as a pharmaceutical or vaccine.

35. i) for use in the treatment of cancer or infectious diseases, or ii) for use in autoimmune diseases or inflammatory diseases, as described in claim 33.

36. Use of the lipid-based nanoparticles according to claim 31 or 32 or the pharmaceutical composition according to claim 33 for the manufacture of a pharmaceutical for the treatment of cancer or infectious diseases, autoimmune diseases or inflammatory diseases.

37. A method for treating cancer, infectious disease, autoimmune disease, or inflammatory disease in a subject, comprising the step of administering to the subject a lipid-based nanoparticle according to claim 31 or 32 or a pharmaceutical composition according to claim 33.

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