Method of synthesis of targeted lipid nanoparticle and uses thereof

Abstract

The invention relates to a method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s) using a mixing device, to a lipid-based nanoparticle comprising an antigen-binding domain and one or several nucleic acid molecule(s) obtainable trough such method and to uses thereof.

Description

[0001] METHOD OF SYNTHESIS OF TARGETED LIPID NANOPARTICLE AND USES

[0002] THEREOF

[0003] FIELD OF THE INVENTION

[0004] The invention pertains to the field of immunotherapy. The invention relates to a method of producing a lipid nanoparticle comprising at least one nucleic acid molecule, and its use to treat conditions such as cancer and infections.

[0005] BACKGROUND OF THE INVENTION

[0006] As evidenced by the COVID19 epidemic, there has been a breakthrough in relevant techniques in the field of RNA molecules, and mRNA vaccines have achieved some results in a variety of infectious diseases such as influenza virus, Ebola virus, and cottage virus. mRNA vaccines have delivered mRNA to cells and expressed producing proteins, thus providing immunoprotection to the body. In the wake of mRNA vaccines, other applications using mRNA were explored, such as the expression of antibodies through mRNA transfection.

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

[0008] To reduce the risk of off-target, insertion of targeting agents (such as antibodies) to LNPs has been developed, in particular to target particular cells populations. Among targeting agents, antibodies are the preferred molecules as they have a high specificity and a high affinity in ligand-receptor binding.

[0009] However, even if in the prior art, the use of targeting ligands has been shown to enhance the delivery and therapeutic efficiency of lipid nanoparticles (LNP), it is recognized that attaching such moieties represent a very strong challenge. While antibodies present a very good option, due to their high specificity and broad selection of target, the antibody production, conjugation, and subsequent nanoparticle purification can be extremely costly and time consuming for LNP, with a very low yield of production. The production complexity and cost are highly increased with the known process of the prior art for a production at an industrial scale. This is even more critical with LNP particles that contain RNA, considering that the manufacturing process of LNPs comprising RNA, including a mix of the RNA and the lipid components in specific conditions, has to be compatible with the coupling of the targeting entity (e.g., an antibody) to the LNP.

[0010] Further, obtaining a targeting ligand at the surface of a lipid nanoparticle, while maintaining its good binding property to target said ligand, is still a supplementary high technical problem. Indeed the specific binding capacity of ligands for their biological target can be lost when the lipid nanoparticles produced are exposed to said target. In particular, due to steric encumbrance and potential modification of conformation of the antibody, the accessibility of the antigen binding part of the antibody can be much lowered and even lost towards the recognizing moiety of a targeted protein of interest.

[0011] For the reasons of both i) allowing the coupling of the antibody to the lipidic surface of the LNP and ii) putting a distance between the antibody to the LNP membrane, the antibody has been coupled in the prior art thanks to the addition of a coupling part (typically a linking PEG-type group), said coupling part comprising a lipophilic part for the anchoring of the complex [PEG group - antibody] to the lipidic membrane. And thus the lipid nanoparticles of the prior art contain such a coupling group for the anchoring of the complex [PEG group - antibody] to the lipid membrane.

[0012] To be more precise, targeting agents such as antibodies can be conjugated to lipid nanoparticles via two approaches including 1) “one-pot” assembly of ionizable / cationic lipid, helper lipid, sterol and PEG-lipid; the latter needing to be beforehand decorated with the targeting agent and 2) “post-insertion” of targeting ligands into preformed plain lipid nanoparticles (with functionalized PEG-lipid ready for conjugation with the targeting agent).

[0013] These methods require additional steps in the process of synthesis of a targeted LNP, either before or after the formation of the LNP. This additional step complicates the production of targeted LNPs, as it induces an additional cost as well a supplementary burden to carry out the method.

[0014] There is therefore room for improving the production of targeted LNPs (t-LNPs), without sacrificing their targeting or their transfection abilities. SUMMARY OF THE INVENTION

[0015] The inventors unexpectedly found a way to simply and efficiently produce a targeted lipid- based nanoparticle through a mixing system, by mixing the antigen binding domain to either a composition comprising a lipid-based composition or a composition comprising nucleic acids, before mixing the two compositions together through the mixing system. This method can be carried in a single step and is cost-effective.

[0016] Unexpectedly, the mixing of antigen binding domain with either the lipid-based composition or the nucleic acids composition does not impede the formation of a LNP, and the LNP obtained through the process exhibits excellent targeting capacity. Moreover, in t-LNPs most of the antigen binding domain is accessible for antigen recognition. Additionally, the method yields a composition without any free antibody, which means that apart from the mixing and the subsequent solvent exchange steps, the method generally does not require any supplementary purification step.

[0017] Compared to the method of the invention, preparing a t-LNP with a “one-pot” assembly such as described hereabove has several drawbacks. The yield to synthesize antibody-lipid conjugates is variable and a purification step is often needed to separate antibody-lipid conjugate from unconjugated antibodies. The resulting antibody-lipid is a large molecule that can interfere with the formation of the LNP. The antibody-lipid conjugate is fragile and can be altered during the production process (organic solvents, mechanical forces) and may even get trapped within the LNP and not at its surface, rendering it ultimately inactive for targeting properties.

[0018] When compared to the method of the invention, the “post-insertion” method necessitates to use a 5thcomponent in the LNP with a functionalized PEG lipid (ex: DMG-PEG-maleimide). Before the coupling, reduction of the antibodies is often needed, which may alter their functionality. The number of steps required to carry out this method is also higher than the method of the invention. Finally, the method does not permit the coupling of all antibodies and a purification step is often needed to remove unbound antibodies.

[0019] The method described herein can be applied to generate multiple LNPs with different antigen binding domains, without the need for calibrating the LNP and / or the carried nucleic acid molecules.

[0020] As explained in detail in the present application, the applicant has now obtained new LNP particles, and in particular disruptive RNA-containing LNPs comprising at least a targeting entity (advantageously at least an antibody or antigen binding part) exhibiting very satisfying specific targeting property, but without coupling / anchoring group, and / or without a covalent bound to any of the lipids of the LNP.

[0021] In a first aspect, the invention relates to a method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s), the method comprising the steps of:

[0022] - feeding through a first entry of a mixing device a first composition comprising a lipid- based composition and a polar organic solvent,

[0023] - feeding through a second entry of said mixing device a second composition comprising one or several nucleic acid molecule(s), wherein said second composition is an acidic aqueous composition,

[0024] - mixing the first and second compositions in the mixing device so as to generate lipid- based nanoparticles,

[0025] - removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH; and, recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s); wherein the antigen binding domain is mixed with the first or second composition before the mixing step of the first and second compositions.

[0026] The invention also concerns a method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s), the method comprising the steps of:

[0027] - feeding through a first entry of a mixing device a first composition comprising a lipid- based composition and a polar organic solvent,

[0028] - feeding through a second entry of said mixing device a second composition comprising one or several nucleic acid molecule(s), wherein said second composition is an acidic aqueous composition,

[0029] - mixing the first and second compositions in the mixing device so as to generate lipid- based nanoparticles,

[0030] - removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH; and - recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s); wherein the antigen binding domain is mixed with the first or second composition before the mixing step of the first and second compositions into the mixing device; and wherein the antigen binding domain is an antibody or an antigen binding fragment thereof.

[0031] Preferably, the step of removing the polar solvent is performed by dialysis or buffer exchange.

[0032] Preferably, the pH of the second composition is comprised between about 3 to about 6, preferably between about 4 and about 5, even more preferably between about 4 and about 4.5.

[0033] Preferably, the adjustment of the pH to a neutral pH is an adjustment of the pH to a range from about 6.5 to about 7.5, preferably to 7.

[0034] The lipid-based composition particularly comprises a cationic or ionizable lipid, a helper lipid, a sterol and a PEG-lipid.

[0035] The ionizable lipid is preferably selected from the group consisting of [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 1,2-dioleoyl- 3 -trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine

[0036] (DODMA), l,2-di-O-octadecenyl-3 -trimethylammoniumpropane (DOTMA), 3-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammoniumpropanes; l,2-dialkyloxy-3-dimethylammoniumpropanes; dioctadecyldimethylammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5- en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di oleyloxybenzylamine (DMOBA), 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19- yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N- (3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP- DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminiiim (DOBAQ), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn- glycero-3 -ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-

[0037] (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2- dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98Niz-5), 1- [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200), 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-(a-D-tocopherolhemisuccinateethyl)piperidyl]ethyl} disulfide (SS-EC) and any mixtures thereof, preferably is ALC-0315, SM-102, Dlin-MC3-DMA or SS-OP, more preferably ALC-0315 or SS-OP. Preferably, the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alphatocopherol, and any mixtures thereof, preferably is cholesterol.

[0038] Preferably, the helper lipid is selected from the group consisting of DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DEPC, DOPC and DSPE, preferably is DOPE or DSPC.

[0039] 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, preferably is PEG-DMG, PEG-DSPE or a mixture thereof.

[0040] The PEG is typically of between 2000 Daltons and 5000 Daltons, preferably is DSPE-PEG- 2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000 or a mixture thereof.

[0041] The first composition or the lipid-based composition of the lipid based nanoparticles is particularly 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; and k) SS-OP, DSPC, cholesterol and DSPE-PEG; and l) SS-OP, DOPC, cholesterol and DMG-PEG.

[0042] Preferably, the lipid-based composition comprises from about 10 mol % to about 70 mol % of a cationic or ionizable lipid, from about 5 mol% to about 70 mol % of a helper lipid, from about 10 mol% to about 70 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG- lipid.

[0043] Even more preferably, the lipid-based composition comprises or consists of from about 45 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 15 mol % of a helper lipid from about 35 mol% to about 45 mol% of a sterol, and from about 0.5 mol% to about 2,5 mol% of a PEG-lipid.

[0044] The concentration of lipids in the first composition is particularly from about 1 to about 100 mM.

[0045] The concentration of nucleic acid molecules in the second composition is preferably from about 0.01 mg / mL to about 100 mg / mL.

[0046] In some aspects, the antigen binding domain is mixed with the first composition before the mixing step of the first and second compositions, and wherein the concentration of the antigen binding domain in the first composition is from about 0.01 pg / pL to about 0.5 pg / pL.

[0047] In some other aspects, the antigen binding domain is mixed with the second composition before the mixing step of the first and second compositions, and wherein the concentration of the antigen binding domain in the second composition is from about 0.005 pg / pL to about 0.25 Pg / pL.

[0048] In some aspects, the first composition comprises a ionizable lipid and the second composition has a pH inferior to the pKa of the ionizable lipid and / or the second composition has pH between about 3 and about 6.

[0049] In some aspects, the flow rate ratio between the first and the second compositions is comprised between 1 : 10 and 1 : 1, preferably between 1 :7 and 1 : 1, even more preferably between 1 :4 and 1 :1.

[0050] The antigen binding domain preferably comprises a Fc domain, preferably wherein the antigen binding domain is an IgG, even more preferably a monoclonal IgG.

[0051] Preferably, the antigen binding domain binds to a target selected from the group consisting of BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD 163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, 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.

[0052] Preferably, the antigen binding domain binds to a target selected from the group consisting of BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PD-1, PTPN22, RGS1, LOX1, SIGLEC 6, TACVTNFRSF13B, TIGIT, CD 163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, 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 and CD160; preferably from the group comprising CD137 / 41BB / TNFRSF9, PD-1, CRTAM, CTLA4, FasL / TNFSF6, TIM-3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4 and TIGIT.

[0053] Particularly, the antigen binding domain binds to a target selected from the group consisting of PD-1, CD 127, SIRPa and CLEC-1A. Preferably, the antigen binding domain binds to human PD-1.

[0054] Particularly, the antigen binding domain is an anti-PD-1 antigen binding domain comprising:

[0055] (i) a VH comprising HCDR1, HCDR2 and HCDR3, and

[0056] (ii) a VL comprising LCDR1, LCDR2 and LCDR3, wherein: a) - the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1;

[0057] - the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2;

[0058] - the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3;

[0059] - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 4;

[0060] - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 5, and

[0061] - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 6; or b) the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 23;

[0062] - the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 24;

[0063] - the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 25;

[0064] - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 26;

[0065] - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 27, and

[0066] - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 28; or c) the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 31;

[0067] - the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 32;

[0068] - the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 33; - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 34;

[0069] - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 35, and

[0070] - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 36.

[0071] In some aspects, the antigen binding domain comprises a Fc domain, preferably an IgG Fc domain.

[0072] Preferably, the antigen binding domain is not covalently bound to any of the lipids of the lipid- based nanoparticle or does not comprise any modification for coupling or grafting the antigen binding domain to a lipid. Alternatively or additionally, the lipid-based nanoparticle does not comprise an anchoring moiety comprising a lipidation peptide or motif.

[0073] In some aspects, the lipid-based nanoparticle comprises an additional antigen binding domain.

[0074] Particularly, the first or second composition is mixed with a second antigen binding domain before the mixing step of the first and second compositions, said second antigen binding domain being an antibody or an antigen binding fragment thereof.

[0075] Preferably, the nucleic acid molecule is a mRNA.

[0076] In particular, the nucleic acid molecule is a mRNA encoding for a molecule selected from the group consisting of: a) an immune cell enhancing or inhibiting compound, in particular such as described herein, and preferably selected from: a compound or molecule selected from the group consisting of TCF1, LEF1, WNT, FRIZZLED, Beta catenin, BCL2, BCLXL, BIRC3, MCL1, PGCla, TCF7, NF AT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCTI, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glutl, Glut3, HK2, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS, OXPHOS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD 107a, Lymphotoxin (LT) aip2, granzyme B, perforin, POU2F1, BBS 10, BBS 12, TCP1, HSP, Integrin alpha 1, Integrin alpha 2, Integrin alpha 2b, Integrin alpha 11, Integrin alpha 3, Integrin alpha 6, Integrin alpha7, 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, DEFI, 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 Regulatory Factors such as IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, NFKB, T-bet, AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKa, IKKp, TRAM, TRIF, TBK1, D3 -phosphoinositides, 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, 0X40, 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, CD160, and any combination thereof, in particular such as described herein; a compound or molecule selected from the group consisting of 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, IKAROS, EGR2 / 3, CREM, P27 (KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS 10, BBS 12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEFI, 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, galectin9, 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, preferably form 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 such as described herein; b) a cytokine, 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 such as described herein; c) a cytokine receptor, 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, IL-35R ; d) a chemokine, preferably selected from the group consisting of CXCL9 or CXCL10; e) a chemokine receptor, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, XCR1; f) an antigen fragment derived from viral pathogens, bacterial pathogens and / or parasitic pathogens, in particular such as described herein; g) an antibody or a fragment or a derivative thereof, preferably directed against a target selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, LAG3, BTLA, CD160, CD40L, ICOS, CD27, 0X40, 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 such as described herein and h) a Chimeric Antigen Receptor (CAR), in particular such as described herein.

[0077] Particularly, the mRNA encodes for BCL2, IL7, IL7R, CXCL9 and / or CXCL10.

[0078] In some aspects, the second composition comprises at least two different mRNA molecules. Particularly, the second composition comprises a mRNA molecule encoding for IL-7 and a mRNA molecule encoding for IL-7R.

[0079] In a particular aspect, the mixing device is a microfluidic device.

[0080] The invention also concerns a lipid-based nanoparticle obtained or obtainable through the method of the invention. The invention also relates to a lipid-based nanoparticle obtained or obtainable through the method of the invention for use as a medicament or vaccine.

[0081] In some aspects, the lipid-based nanoparticle obtained or obtainable by the methods of the invention comprises a cationic or ionizable lipid, a helper lipid, a sterol, a PEG-modified lipid, one or more nucleic acid molecule(s), preferably mRNA molecules, and one or more antigen biding domain(s). Preferably, said lipid-based nanoparticle comprises an antigen binding domain that is not covalently bound to any of the lipids of the lipid-based nanoparticle or an antigen binding domain that does not comprise any modification for coupling or grafting the antigen binding domain to a lipid.

[0082] In particular, the lipid-based nanoparticle does not comprise a) an antigen binding domain that is covalently bound to a lipid; ii) an antigen binding domain that comprises a modification for coupling or grafting the antigen binding domain to a lipid and / or iii) an anchoring moiety comprising a lipidation peptide or motif.

[0083] Preferably, the lipid-based nanoparticle comprises 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 an antibody against a checkpoint inhibitor.

[0084] The invention is also related to the lipid-based nanoparticle obtained or obtainable through the method of the invention for use as a medicament or vaccine. The invention further relates to a pharmaceutical composition comprising the lipid-based nanoparticle obtained through the method of the invention and optionally a pharmaceutically acceptable carrier.

[0085] The invention also concerns the lipid-based nanoparticle or the pharmaceutical composition comprising said lipid-based nanoparticle for use in the treatment of cancer, of an infectious disease, of an autoimmune disease or of an inflammatory disease.

[0086] The invention also relates to the use of the lipid-based nanoparticle the lipid-based nanoparticle obtained or obtainable through the method of the invention or of the pharmaceutical composition comprising it, for the manufacture of a medicament for the treatment of cancer, of an infectious disease, of an autoimmune disease or of an inflammatory disease.

[0087] Finally, the invention concerns a method for treating a cancer, an infectious disease, an autoimmune disease or an inflammatory disease in a subject, wherein the method comprises administering to said subject the lipid-based nanoparticle obtained or obtainable through the method of the invention or a pharmaceutical composition comprising it.

[0088] DETAILED DESCRIPTION OF THE INVENTION

[0089] The present disclosure relates to methods of production of a lipid nanoparticle one or several antigen binding domain(s) and one or several nucleic acid molecule(s). The disclosure also relates to a lipid nanoparticle produced by said methods, and its use as a medicament, in particular to treat diseases such as cancer.

[0090] Definitions

[0091] In order that the present invention may be more readily understood, certain terms are defined hereafter. Additional definitions are set forth throughout the detailed description.

[0092] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.

[0093] As used herein, the term “t-LNP” refers to a targeted lipid nanoparticle, i.e. a lipid nanoparticle having an antigen binding domain. The term “nt-LNP” refers to a lipid nanoparticle devoid of antigen binding domain.

[0094] As used herein, the term “antibody” describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The heavychain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The term “antibody” particularly refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses whole antibody molecules such as four-chain antibodies comprising 2 heavy chains and 2 light chains, such as polyclonal antibodies, monoclonal antibodies or recombinant antibodies.

[0095] The term "antigen-binding fragment” or “antigen binding domain” of an antibody (or simply “antibody fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1).

[0096] Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain, or any fusion proteins comprising such antigen-binding fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0097] As used herein, the term “isolated” indicates that the recited material (e.g., antibody, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature. Particularly, an “isolated” antibody is one which has been identified and separated and / or recovered from a component of its natural environment.

[0098] As used herein, the term “treatment”, “treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain aspects, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease, such as according to the present disclosure, the disruption or the delay in the resolution of the inflammation leading to inflammation associated disease. In other aspects, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0099] As used herein, the “sequence identity” between two sequences is described by the parameter "sequence identity", “sequence similarity” or “sequence homology”. For purposes of the present invention, the "percentage identity" between two sequences (A) and (B) is determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. The percent identity between the two sequences is particularly a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Said alignment of sequences can be carried out by well-known methods in the art, for example, using the algorithm for global alignment of Needleman-Wunsch. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. Once the total alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two amino acid sequences, one can use, for example, the tool “Emboss needle” for pairwise sequence alignment of proteins providing by EMBL-EBI and available on: www.ebi.ac. uk / Tools / services / web / toolform.ebi?tool=emboss_needle&context=protein, for example using default settings: (I) Matrix: BLOSUM62, (ii) Gap open: 10, (iii) gap extend: 0.5, (iv) output format: pair, (v) end gap penalty: false, (vi) end gap open: 10, (vii) end gap extend: 0.5.

[0100] The percent identity between two amino acid sequences or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences or nucleotide sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as the BLASTN program for nucleic acid or amino acid sequences using as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0101] Alternatively, Sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with the default settings.

[0102] "Eu numbering" (also known as Eu index) refers to the antibody numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda), which is based on the sequential numbering of the first human IgGl sequenced (the Eu antibody; Edelman, et al., 1969, Proc Natl Acad Sci USA 63: 78-85). By "amino acid change" or “amino acid modification” is meant herein a change in the amino acid sequence of a polypeptide. "Amino acid modifications" include substitution, insertion and / or deletion in a polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By "amino acid insertion" or "insertion" is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By "amino acid deletion" or "deletion" is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. The amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). As used herein, “amino acid position” or “amino acid position number” are used interchangeably and refer to the position of a particular amino acid in an amino acids sequence, generally specified with the one letter codes for the amino acids. The first amino acid in the amino acids sequence (i.e., starting from the N terminus) should be considered as having position 1.

[0103] A conservative substitution is the replacement of a given amino acid residue by another residue having a side chain (“R-group”) with similar chemical properties (e.g., charge, bulk and / or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Conservative substitutions and the corresponding rules are well-described in the state of the art. For instance, conservative substitutions can be defined by substitutions within the groups of amino acids reflected in the following tables:

[0104] Table A - Amino Acid Residue

[0105] Table B - Alternative Conservative Amino Acid Residue Substitution Groups

[0106] Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues

[0107] The term “and / or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually.

[0108] The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.

[0109] The term “about” as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means any value having an acceptable range of deviation of up to + / - 10% (e.g., + / - 0.5%, + / -1 %, + / -1 .5%, + / - 2%, + / - 2.5%, + / - 3%, + / - 3.5%, + / - 4%, + / - 4.5%, + / - 5%, + / - 5.5%, + / - 6%, + / - 6.5%, + / - 7%, + / - 7.5%, + / - 8%, + / - 8.5%, + / - 9%, + / - 9.5%). The use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3). Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%) the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%).

[0110] The term “essentially” as used herein in connection with any given biological sequence means said biological sequence varies from the reference sequence contained in the sequence listing by up to 10% of the biological sequence length. In particular, by “consists essentially of’ is intended that the biological sequence consists of that sequence, but it may also include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, additions, deletions or a mixture thereof, preferably 1, 2, 3, 4, or 5 substitutions, additions, deletions or a mixture thereof, with the proviso that said biological sequence varies from the reference sequence contained in the sequence listing by up to 10% of the biological sequence length.

[0111] Synthesis method In a first aspect, the invention relates to a method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s).

[0112] The method for producing such lipid-based nanoparticles comprises the steps of

[0113] - feeding through a first entry of a mixing device a first composition comprising a lipid- based composition and a polar organic solvent,

[0114] - feeding through a second entry of said mixing device a second composition comprising one or several nucleic acid molecule(s), wherein said second composition is an acidic aqueous composition,

[0115] - mixing the first and second compositions in the mixing device so as to generate lipid- based nanoparticles,

[0116] - removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH; and recovering the lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s); wherein the antigen binding domain is mixed with the first or second composition before the mixing step of the first and second compositions into the mixing device.

[0117] Any type of mixing device known or used for the generation of LNPs may be used to perform the method according to the invention. In particular, mixing devices suitable for an industrialscale production are preferred.

[0118] Examples of suitable mixing devices include, but are not limited to, T-mixer, U-mixer, V-mixer, NanoAssembler (Precision Nanosystems), Impigement Jet Mixing (Knauer), Microfluidizer M700 or M805 (Microfluidics International Corporation).

[0119] Other suitable mixing devices may be contemplated, as long as they allow for a production compatible with industrial scale needs.

[0120] Such suitable mixing devices comprises one, several or all the following features: a flow rate capacity greater than about 500 mL / min, preferably greater than about 1000 mL / min, preferably greater than about 1500 mL / min, preferably greater than about 2000 mL / min, preferably greater than about 2500 mL / min, preferably greater than about 3000 mL / min, preferably greater than about 3500 mL / min, preferably greater than about 4000 mL / min, an inlet tubing internal diameter greater than about 0.1 mm, preferably greater than about 0.2 mm, preferably greater than about 0.3 mm, preferably greater than about 0.4 mm, preferably greater than about 0.5 mm, preferably greater than about 1 mm, preferably greater than about 1.5 mm, an outlet tubing internal diameter greater than about 0.1 mm, preferably greater than about 0.2mm, preferably greater than about 0.3 mm, preferably greater than about 0.4 mm, preferably greater than about 0.5 mm, preferably greater than about 1 mm, preferably greater than about 1.5 mm, preferably greater than about 2 mm, preferably greater than about 2.5 mm, preferably greater than about 3 mm, preferably greater than about 3.5 mm, preferably greater than about 4 mm, preferably greater than about 4.5 mm, preferably greater than about 5 mm, preferably greater than about 5.5 mm, preferably greater than about 6 mm, preferably greater than about 6.5 mm, preferably greater than about 7 mm, preferably greater than about 7.5 mm, preferably greater than about 8 mm, preferably greater than about 8.5 mm, preferably greater than about 9 mm, preferably greater than about 9.5 mm, preferably greater than about 10 mm. an inlet back pressure greater than about 10 psi, preferably greater than 20 psi, preferably greater than 30 psi, preferably greater than 40 psi, preferably greater than 50 psi, a Reynold number greater than about 2000.

[0121] Alternatively, the process can be carried out with a microfluidic system. In such a case, the mixing device is a microfluidic device. Examples of suitable microfluidic devices include microfluidic mixer system (NanoAssemblr Spark, Ignite or Blaze from Precision NanoSystems, Vancouver, BC, Canada), microfluidic micro mixture (Precision NanoSystems) as described by Cohen et.al., 2015 ACS nano 9(2): 1581-1591, or any device using mixing technology to generate lipid-based nanoparticles, such as, but not limited to, crossflow injection, microfluidic bifurcating mixing, staggered herringbone mixing or microfluidic hydrodynamic focusing mixing. Preferably, the microfluidic device is a microfluidic mixer such as the LNP Pack provided by Inside Therapeutics.

[0122] Alternatively, the process can be carried out with a paralleled microfluidic device, which is an array of microfluidic mixing devices that operate simultaneously.

[0123] In another aspect of the invention, the method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s), comprises the steps of: feeding through a first entry of a mixing device a first composition comprising a lipid- based composition, feeding through a second entry of said mixing device a second composition comprising one or several nucleic acid molecule(s), mixing the first and second compositions in the mixing device so as to generate lipid- based nanoparticles, and recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s); wherein the antigen binding domain is mixed with the first or second composition before the mixing step of the first and second compositions into the mixing device.

[0124] In some aspects, the method for obtaining the lipid-based nanoparticles of the invention is as described under the “Examples” section below, in particular in Examples 1-3, or as described in Figure 22J-K.

[0125] First composition

[0126] In the method according to the invention, the first composition comprising the lipid-based composition comprises a polar organic solvent, preferably a polar protic organic solvent.

[0127] Examples of polar organic solvents include but are not limited to ethanol, methanol, / / -propanol, isopropyl alcohol, / / -butanol, acetic acid, formic acid, acetone, dimethylformamide, acetonitrile, dimethylsulfoxide, and propylene carbonate, preferably ethanol, methanol, n- propanol, isopropyl alcohol, / / -butanol, acetic acid, formic acid. The polar organic solvent is miscible in water.

[0128] Preferably, the polar organic solvent is an alcohol, preferably ethanol.

[0129] In some aspects, the first composition comprising the lipid based composition includes a volume of about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 97% or about 100% polar organic solvent, preferably ethanol.

[0130] In some aspects, the polar organic solvent is isopropyl alcohol. For example, the first composition comprising the lipid-based composition includes a volume of 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.

[0131] In some aspects, the polar organic solvent is dimethyl sulfoxide. For example, the first composition comprising the lipid-based composition comprises a volume of 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.

[0132] In some aspects, the concentration of lipids in the lipid-based composition is from about 1 mM to about 100 mM, from 2 mM about to about 95 mM, from about 3 mM to about 90 mM, from about 4 mM to about 85 mM, from about 5 mM to about 80 mM, from about 10 mM to about 75 mM, from about 15 mM to about 70 mM, from about 20 mM to about 60 mM, from about 30 mM to about 50 mM.

[0133] The lipid-based composition is more particularly defined hereafter under the section “Lipid- based composition”.

[0134] The elements of the lipid-based composition or of the first composition may be selected based on a particular application or target of the final LNP to be obtained, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements.

[0135] In a particular aspect, the lipid-based composition or the first composition comprises one or more ionizable or cationic lipid(s), one or more helper lipid(s), one or more sterol(s), and / or one or more polyethylene glycol (PEG)-modified lipid(s).

[0136] In some aspects, the lipid-based composition or the first composition comprises one or more ionizable or cationic lipid. As used herein, the term “ionizable or cationic lipid” refers to a lipid molecule positively charged in an acidic environment.

[0137] The lipid-based composition or the first composition typically comprises ionizable or cationic lipid, helper lipid, sterol and / or PEG lipid components. In one aspect, the ionizable or cationic lipid comprises a head group which includes at least one nitrogen atom (N) which is positively charged or capable of being protonated.

[0138] In some aspects, the ionizable or cationic lipid is selected from the group consisting of [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 1,2-dioleoyl- 3 -trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3 -trimethylammoniumpropane (DOTMA), 3-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammoniumpropanes; l,2-dialkyloxy-3-dimethylammoniumpropanes; dioctadecyldimethylammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5- en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di oleyloxybenzylamine (DMOBA), 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19- yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N- (3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP- DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminiiim (DOBAQ), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn- glycero-3 -ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-

[0139] (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2- dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98Niz-5), 1- [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200), 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., CASnumber2377474-67-2) bis{2-[4-(a-D- tocopherolhemisuccinateethyl)piperidyl]ethyl} disulfide (SS-EC) and any mixtures thereof, preferably is ALC-0315, SM-102, Dlin-MC3-DMA or SS-OP, more preferably ALC-0315 or SS-OP.

[0140] Ionizable or cationic lipids included in the lipid-based composition can be selected from the group consisting of l,2-dioleoyl-3 -trimethylammonium propane (DOTAP); N,N-dimethyl-2,3- di oleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3 -trimethylammoniumpropane

[0141] (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3 -dimethylammonium -propane

[0142] (DODAP); l,2-diacyloxy-3 -dimethylammoniumpropanes; l,2-dialkyloxy-3- dimethylammoniumpropanes; dioctadecyldimethylammonium chloride (DODAC), 1,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3- dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- di oleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2, 2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2, 2-dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-di oxolane (DLin-KC2-DMA), heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2- Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l- propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminiiim (DOBAQ), 2-({8-[(3P)- cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]propan-l-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3- bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-l-amonium bromide (DLRIE), N- (2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-

[0143] (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2- dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98Niz-5), 1- [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200), [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315), 9-

[0144] Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) and any mixtures thereof.

[0145] Additional examples of ionizable or cationic lipids are described in WO 2016 / 021683, WO 2015 / 011633, WO 2011 / 153493, WO 2013 / 126803, WO 2010 / 054401, WO 2010 / 042877, WO 2016 / 104580, WO 2015 / 005253, WO 2014 / 007398, WO 2017 / 117528, WO 2017 / 075531, WO 2017 / 00414, WO 2015 / 199952, US 2015 / 0239834, WO2019 / 131839 all of which are incorporated by reference herein in their entirety.

[0146] In addition, synthetic ionizable or cationic lipids (e.g., K-E12, H-A12, Y-E12, G-O12, K-A12, R-A12, CKK-E12, cPK-E12, PK1K-E12, PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K- E12, CWK-E12, PK500-012, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-012, CIK-E12, cKK-ElO, cKK-E14, and cKK-E16, preferably, cKK-E12, cKK-E14) described in Dong et al. (Proc Natl Acad Sci U S A. 2014 Apr 15; 111 (15): 5753 , the disclosure thereof being incorporated herein by reference), and the synthetic ionizable or cationic lipid (e.g., C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96 and C12-200, preferably C14-110, C16-96 and C12-200) described in Love KT et al. (Proc Natl Acad Sci U S A. 2010 May 25; 107(21):9915, the disclosure thereof being incorporated herein by reference) can be also envisioned.

[0147] In a preferred aspect, the lipid-based composition or the first composition comprises an ionizable or cationic lipid such as described in WO 2016 / 021683 or WO 2019 / 131839 which are incorporated herein by reference in their entirety.

[0148] Particularly, the lipide-based nanoparticle or the first composition comprises an ionizable or cationic lipid selected from the group consisting of [(4-hydroxybutyl)azanediyl]di(hexane-6,l- 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) and any mixtures thereof.

[0149] In some aspects, the ionizable or cationic lipid is selected from the group consisting of ALC- 0315, SM-102, Dlin-MC3-DMA and SS-OP.

[0150] Preferably, the ionizable or cationic lipid comprised in the lipid-based nanoparticle or first composition is ALC-0315. Alternatively, the ionizable or cationic lipid comprised in the lipid- based nanoparticle or first composition is SS-OP.

[0151] Ionizable or cationic lipids particularly promotes nucleic acid molecule(s) delivery and transfection efficiency. Their mechanism of action is based on complexing the nucleic acid by electrostatic interactions. Several properties, such as the charge or lipid shape, as well as the protein corona formation, have been described as important factors to consider when understanding structure-activity relationship studies, and thus, the design of new ionizable lipids.

[0152] In some aspects, the ionizable or cationic lipid represents from about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the lipid- based nanoparticle or first composition. In some aspects, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, represents from about 45 mol % to about 55 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle or first composition. Particularly, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, represents from about 48 mol % to about 52 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle or first composition.

[0153] More particularly, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, represents about 50 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle or first composition.

[0154] In some aspects, the lipid-based nanoparticle or the first composition comprises a helper lipid. As used herein, the term “helper lipid” refers to a class of lipid molecules that increases particle stability, fluidity tolerability and / or biodistribution of lipid-based nanoparticles.

[0155] Helper lipids are also constituents of LNPs, playing an important role in terms of stability and fusogenicity. These lipids are mainly phospholipids (such as DOPE, DSPC, DEPC, DSPE), which forms the main skeleton of LNPs. Helper lipids modulate nanoparticle fluidity and enhance efficacy by promoting lipid phase transitions that aid membrane fusion with the endosome. Helper lipids are generally saturated phospholipids, which can increase the phase transition temperature of cationic liposomes, support the formation of lamellar lipid bilayers and stabilize their structural arrangement.

[0156] For instance, the helper lipid can be selected from the group consisting of 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di- 0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,l,2- diarachidonoyl-sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 - phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine (SOPC), ethyl phosphatidylcholine (EPC), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphocholine (18 : 1 Lyso PC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16:0 PE), l-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (C16— 18: 1), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME), l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), l,2-dielaidoylsn-glycero-3- phosphoethanolamine (DEPE), N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)- phosphatidylethanolamine (NBD-PE), N-(lisamineRhodamine B sulfonyl)- phosphatidylethanolamine (Rh-PE), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphoethanolamine (18 : 1 Lyso PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (18 : 1 Monomethyl PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl (18 : 1 Dimethyl PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(hexanoylamine) (18 : 1 Caproylamine PE), l,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-l-phospho-sn-3'-(l'-(9Z- octadecenoyl)-2'-hydroxy)-glycerol (BMP-S,R), l,2-dioleoyl-sn-glycero-3-phospho-rac-(l- glycerol) sodium salt (DOPG), l,2-Diacyl-sn-glycero-3-phospho-L-serine (DSPS), L-a- phosphatidylserine (PS), l,2-dioleoyl-sn-glycero-3 -phosphate (PA), l,2-dioleoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (PG), l,2-dioleoyl-sn-glycero-3 -phosphomethanol (18 : 1 Phosphatidymethanol), l,2-dioleoyl-sn-glycero-3 -phosphoethanol (18 : 1 Phosphatidyethanol), l,2-dioleoyl-sn-glycero-3 -phosphopropanol (18 : 1 Phosphatidypropanol), 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (18: 1 PS, DOPS), l,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), N-oleoyl-D-erythro-sphingosine (Ceramide), Sphingomyelin (SM), Phosphatidylinositol (PI), 9A1P9, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), Dimethyl dioctadecylammonium (18:0 DDAB), and any mixtures thereof.

[0157] Particularly, the helper lipid can be selected from the group consisting of 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC), l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-di- 0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,l,2- diarachidonoyl-sn-glycero-3 -phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3 - phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3 -phosphocholine (SOPC), ethyl phosphatidylcholine (EPC), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphocholine (18 : 1 Lyso PC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16:0 PE), l-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (C16— 18: 1), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4ME), l-stearoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), l,2-dielaidoylsn-glycero-3- phosphoethanolamine (DEPE), N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)- phosphatidylethanolamine (NBD-PE), N-(lisamineRhodamine B sulfonyl)- phosphatidylethanolamine (Rh-PE), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphoethanolamine (18 : 1 Lyso PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (18 : 1 Monomethyl PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl (18 : 1 Dimethyl PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(hexanoylamine) (18 : 1 Caproylamine PE), l,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-l-phospho-sn-3'-(l '-(9Z- octadecenoyl)-2'-hydroxy)-glycerol (BMP-S,R), l,2-dioleoyl-sn-glycero-3-phospho-rac-(l- glycerol) sodium salt (DOPG), l,2-Diacyl-sn-glycero-3-phospho-L-serine (DSPS), L-a- phosphatidylserine (PS), l,2-dioleoyl-sn-glycero-3 -phosphate (PA), l,2-dioleoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (PG), l,2-dioleoyl-sn-glycero-3 -phosphomethanol (18 : 1 Phosphatidymethanol), l,2-dioleoyl-sn-glycero-3 -phosphoethanol (18 : 1 Phosphatidyethanol),

[0158] 1.2-dioleoyl-sn-glycero-3 -phosphopropanol (18 : 1 Phosphatidypropanol), 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (18: 1 PS, DOPS), l,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), N-oleoyl-D-erythro-sphingosine (Ceramide), Sphingomyelin (SM), Phosphatidylinositol (PI), 9A1P9, l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), Dimethyl dioctadecylammonium (18:0 DDAB), l,2-dioleyloxy-3 -dimethylaminopropane (DODMA), l,2-dioleoyl-3- dimethylammonium-propane (DODAP), l,2-dierucoyl-sn-glycero-3 -phosphocholine (DEPC),

[0159] 1.2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and any mixtures or combinations thereof. Preferably, the helper lipid is selected from the group consisting of DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DOPC, DEPC and DSPE, and any combinations thereof.

[0160] Preferably, the helper lipid is selected from the group consisting of DOPE, DOPC, DDAB, POPE and DSPC, and any combinations thereof..

[0161] In some embodiments, the helper lipid is DOPE. Alternatively, the helper lipid is DSPC.

[0162] In some aspects, the helper lipid represents from about 5 mol% to about 100 mol%, from about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the lipid-based composition of the invention.

[0163] In some aspects, the helper lipid, preferably DOPE or DSPC, represents from about 5 mol% to about 15 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0164] In some aspects, the helper lipid, preferably DOPE or DSPC, represents from about 8 mol% to about 12 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0165] In some aspects, the helper lipid, preferably DOPE or DSPC, represents about 10 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0166] In some aspects, the lipid of the lipid-based composition comprises one or more molecules comprising polyethylene glycol. Accordingly, the lipid-based composition may comprise PEG or PEG-modified lipids.

[0167] PEG lipid stabilizes lipid nanoparticles, regulates nanoparticle size by limiting lipid fusion and increases nanoparticle half-life by reducing nonspecific interactions with macrophages, improving colloidal stability and preventing the formation of the protein corona. PEG lipid phospholipids that are located on the surface of nanoparticles, improve their hydrophilicity, avoid rapid clearance by the immune system, prevent particle aggregation, and increase stability.

[0168] As used herein, the term “PEG lipid” may refer to polyethylene glycol (PEG) -modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerCI4 or PEG-CerC20), PEG- modified dialkylamines and PEG- modified l,2-diacyloxypropan-3 -amines. Such lipids are also referred to as PEGylated lipids. In some aspects, a PEG lipid can be PEG-c-DOMG, PEG- DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some aspects, the PEG- modified lipids are a modified form of PEG DMG.. A PEG lipid may particularly be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG- modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some aspects, a PEG lipid is selected from the group consisting of PEG-c- DOMG, PEG-DMG, DMG-PEG-2000, PEG-DLPE, PEG- DMPE, PEG-DPPC, and PEG-DSPE lipid.

[0169] In some aspects, the PEG-lipid includes, but is not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

[0170] In some aspects, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA) and ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG), and any mixture thereof.

[0171] In one aspect, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

[0172] For example, such PEG is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG- dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c-DMA), PEG-c-DOMG, PEG-DMG, DMG-PEG-2000, PEG-DLPE, PEG- DMPE, PEG-DPPC, and PEG-DSPE.

[0173] 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-dimyristylamide of 2-hydroxyacetic acid, O- pegylated to a PEG), and any mixture thereof; particularly from the group consisting of PEG- DMG, PEG-DSPE, ALC-0159 and any mixture thereof.

[0174] In a particular aspect, the LNP comprises a PEG that is not functionalized, which is a PEG that does not comprises any reactive specie or group at its end, said reactive specie or group being usable to conjugate a target moiety such as an antibody or a fragment thereof to the PEG.

[0175] In some aspects, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16.

[0176] Whereas the abundant prior art focuses on a high variety of structures and ratio of the cationic lipid and of the helper lipids in the LNP, the applicant has tested different structures and lengths of PEG-lipid (component n°4) to determine their impact on the biodistribution of the LNP in different areas of the body. A typical biodistribution of LNP particles that has been observed so far was with a high ratio in the liver, and much less in other organs that contain immune cells such as the spleen. Due to said liver uptake, the circulation of LNP towards various other tissues, and in particular towards tumoral tissues, is variable. An increased targeting of liver cells is for example described in WO2022 / 261101. However, in other clinical situations, a high or excessive uptake of LNP by capturing organs (mostly the liver, and potentially also the spleen and lungs notably) is not advantageous. Therefore, there is a need to provide LNP formulations that can escape the liver and reach other organs such as the spleen, or even to escape the spleen to favor tumor areas..

[0177] The inventors observed that the PEG-lipid has an impact on the biodistribution of the LNPs. The choice of the PEG-lipid can modify the uptake of the LNPs by the capturing organs (mostly liver, and optionally spleen and / or lungs). For instance, a PEG-C14 lipid seems to favour liver uptake whereas a PEG-C18 lipid decreases liver uptake and may optionally favour spleen targeting. Optionally, the PEG-lipid and the amount thereof in the LNP can be selected so as favouring free distribution of the LNP throughout the circulation of the body, in particular in direction of an area of interest for a specific targeted therapeutic treatment. More specifically, the PEG-lipid and the amount thereof in the LNP can be selected so as to be sufficiently free to target immune cells and tumoral cells, especially in the tumor microenvironment... As an illustrative advantage, targeted LNP carrying targeting ligand such as antibodies, are designed to escape the uptake by organs and target activated immune cells (such as PD1+ T lymphocytes) located within the tumor micro-environment.). The mRNA carried by the LNP and transfected into said activated immune cells allows to reinforce their anti-tumoral activity locally and specifically.

[0178] In a specific aspect, the lipid, in particular the lipid moiety of the PEG-lipids, includes those having a lengths of from about C16 to about C22 (Cl 6, Cl 7, Cl 8, Cl 9, C20, C21 or C22), preferably C16 to C20 (C16, C17, C18, C19 or C20), especially Cl 8.

[0179] Such PEG lipid can be selected in the group consisting of l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG), and PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), optionally in the group consisting of l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), and PEG-dioleyl, PEG-distearyl. In a very particular aspect, the PEG-lipid is PEG-disteryl glycerol (PEG-DSG).

[0180] Optionally, the PEG may present a molecular weight within the range from 0.5 to 50 kD, more preferably from 1 to 20 kD In some aspects, a PEG moiety, for example a mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In a particular aspect, the PEG has a size of about 2000. In a very particular aspect, the PEG has a size of between about 2000 Daltons and 5000 Daltons. In some instances, the PEG is selected from the group consisting of PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500 and PEG- 5000.

[0181] In one aspect, the PEG-lipid is PEG 2000-DMG. In another very particular aspect, the PEG- lipid is PEG 2000-DSG. In another very particular aspect, the PEG-lipid is ALC-0159.

[0182] In a particular aspect, the PEG has a size of about 2000. Alternatively, the PEG has a size of about 5000 Daltons.

[0183] In some instances, the PEG lipid is selected from the group consisting of DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG-PEG-5000 or a mixture thereof.

[0184] In a very particular aspect, the PEG-lipid is PEG 5000-DMG. In another very particular aspect, the PEG-lipid is PEG 5000-DSG.

[0185] In some aspects, the PEG lipid represents from about 1 mol% to about 100 mol%, from about 2 mol% to about 100 mol%, about 3 mol% to about 100 mol%, about 4 mol% to about 100 mol%, about 5 mol% to about 100 mol%, about 10 mol% to about 100 mol%, or about 15 mol% to about 100 mol% of the total lipids present in the lipid-based composition or the first composition. Optionally, the PEG lipid in the lipid-based nanoparticle or first composition is within the range from about 0.5 mol% to about 2 mol% of the total lipids present in the nanoparticle or first composition, for instance 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%, especially 1.5 mol%.

[0186] In addition, the inventors have surprisingly observed that the amount of PEG lipid has an impact of the biodistribution of the LNPs. It appears that a lower amount of PEG lipid may decrease the uptake of LNPs by capturing organs, especially liver but also spleen.

[0187] Accordingly, the PEG lipid could be less than 1.5 mol% of the total lipids present in the nanoparticle or first composition, 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 nanoparticle or first composition.

[0188] In some aspects, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 0,5 mol% to about 5 mol% or from about 0,5 mol% to about 2,5 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0189] Particularly, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 1 mol% to about 2 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0190] Preferably, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents about 1,5 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0191] In some aspects, the lipid-based composition or the first composition comprises one or more sterol. The sterol can particularly be selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and any mixtures thereof. Preferably, the sterol is cholesterol.

[0192] Sterol, more so cholesterol, enhances the stability of the nanoparticles by filling gaps between lipids, and aids fusion with the endosomal membrane during uptake into the cell.

[0193] In some aspects, the sterol comprises from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, about 50 mol% to about 100 mol%, about 60 mol% to about 100 mol%, or about 70 mol% to about 100 mol% of the total lipid present in the lipid-based composition or the first composition.

[0194] In some aspects, the sterol, preferably cholesterol, represents from about 30 mol% to about 50 mol% or from about 35 mol% to about 45 mol% of the total lipids present in the lipid-based nanoparticle or first composition. Particularly, the sterol, preferably cholesterol, represents from about 35 mol% to about 40 mol% of the total lipids present in the lipid-based nanoparticle or first composition. Such sterol is preferably cholesterol.

[0195] Particularly, the sterol, preferably cholesterol, represents about 38.5 mol% of the total lipids present in the lipid-based nanoparticle or first composition. Such sterol is preferably cholesterol.

[0196] In a preferred aspect, the lipid-based composition or first composition comprises a ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid.

[0197] In a preferred aspect, the first composition or the lipid-based nanoparticle consists of an ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid, these lipids being preferably as described above.

[0198] In some aspect, the ionizable or cationic lipid is from about 35 mol % to about 55 mol % of the total lipid present in the lipid-based composition or the first composition, the helper lipid is from about 5 mol% to about 20 mol % of the total lipid present in the lipid-based composition or the first composition, the sterol is from about 30 mol% to about 60 mol% of the total lipid present in the lipid-based composition or the first composition, and the PEG lipid is from about 0.5 mol% to about 4 mol% of the total lipid present in the lipid-based composition or the first composition.

[0199] Preferably, the lipid-based nanoparticle or first composition comprises or consists of from about 45 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 15 mol % of a helper lipid from about 35 mol% to about 45 mol% of a sterol, and from about 0.5 mol% to about 2. ,5 mol% of a PEG-lipid.

[0200] Optionally, the PEG lipid is within the range from about 0.5 mol% to about 2 mol%, for instance 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%, especially 1.5 mol% of the total lipids present in the nanoparticle or the first composition.

[0201] Optionally, the PEG lipid in said lipid-based nanoparticle could be less than 1.5 mol% of the total lipids present in the nanoparticle, 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 nanoparticle or the first composition.

[0202] In some aspects, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 0.5 mol% to about 5 mol% or from about 0.5 mol% to about 2.5 mol% of the total lipids present in the lipid-based nanoparticle or first composition. Particularly, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 1 mol% to about 2 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0203] Preferably, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents about 1.5 mol% of the total lipids present in the lipid-based nanoparticle or first composition.

[0204] In a specific aspect, the lipid-based composition or the first composition comprises [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315) as ionizable or cationic lipid, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and one or more polyethylene glycol (PEG)-modified lipid(s).

[0205] In a more specific aspect, ALC-0315 is from about 35 mol % to about 55 mol % of the total lipids present in the nanoparticle or the first composition, DOPE is from about 5 mol% to about 20 mol % of the total lipids present in the nanoparticle or the first composition, cholesterol is from about 30 mol% to about 60 mol% of the total lipids present in the nanoparticle or the first composition, and said one or more polyethylene glycol (PEG)-modified lipid(s)is from about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticle or the first composition. Optionally, said one or more polyethylene glycol (PEG)-modified lipid(s) is from about 0.5 mol% to about 2 mol% of the total lipids present in the nanoparticle or the first composition, for instance 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%, especially 1.5 mol%. Optionally, said one or more polyethylene glycol (PEG)-modified lipid(s)is from about 0.5 mol% to about 1.5 mol% of the total lipids present in the nanoparticle or the first composition. Optionally, said one or more polyethylene glycol (PEG)-modified lipid(s) is from about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9 or 1.0 mol% of the total lipids present in the nanoparticle or the first composition.

[0206] In a very specific aspect, the lipid-based composition or the first composition comprises a lipid mixture.

[0207] Preferably, the lipid mixture comprises or consists of an ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid, these lipids being preferably as described here above.

[0208] Preferably, the lipid mixture comprises [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyl decanoate) (ALC-0315) as ionizable or cationic lipid, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG) as PEG-modified lipid. In a very specific aspect, ALC-0315 is from about 35 mol % to about 55 mol % of the total lipid present in the lipid-based composition or the first composition, DOPE is from about 5 mol% to about 20 mol % of the total lipid present in the lipid-based composition or the first composition, cholesterol is from about 30 mol% to about 60 mol% of the total lipid present in the lipid-based composition or the first composition, and the PEG2000 DMG is from about 0.5 mol% to about 4 mol% of the total lipid present in the lipid-based composition or the first composition.

[0209] Optionally, PEG2000-DMG is from about 0.5 mol% to about 2 mol% of the total lipids present in the nanoparticle or the first composition, for instance 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%, especially 1.5 mol%. Optionally, PEG2000- DMG is from about 0.5 mol% to about 1.5 mol% of the total lipids present in the nanoparticle or the first composition. Optionally, PEG2000-DMG is from about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9 or 1.0 mol% of the total lipids present in the nanoparticle or the first composition, preferably about 0.5 mol%.

[0210] In another very specific aspect, the lipid-based composition or the first composition comprises [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315) as ionizable or cationic lipid, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and a disteryl glycerol (DSG) with polyethylene glycol of average molecular weight 2000 (PEG2000-DSG) as PEG-modified lipid.

[0211] In a very specific aspect, ALC-0315 is from about 35 mol % to about 55 mol % of the total lipids present in the nanoparticle or the first composition, DOPE is from about 5 mol% to about 20 mol % of the total lipids present in the nanoparticle or the first composition, cholesterol is from about 30 mol% to about 60 mol% of the total lipids present in the nanoparticle or the first composition, and the PEG2000-DSG is from about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticle or the first composition. Optionally, PEG2000-DSG is from about 0.5 mol% to about 2 mol% of the total lipids present in the nanoparticle or the first composition, for instance 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%, especially 1.5 mol%. Optionally, PEG2000-DSG is from about 0.5 mol% to about 1.5 mol% of the total lipids present in the nanoparticle or the first composition. Optionally, PEG2000-DSG is from about 0.5 mol% to about 0.6, 0.7, 0.8, 0.9 or 1.0 mol% of the total lipids present in the nanoparticle or the first composition, preferably about 0.5 mol%.

[0212] In a very specific aspect, the lipid-based composition or the first composition comprises: ALC-0315 from about 35 mol % to about 55 mol % of the total lipids present in the nanoparticle or the first composition,

[0213] - DOPE from about 5 mol% to about 20 mol % of the total lipids present in the nanoparticle or the first composition,

[0214] Cholesterol from about 30 mol% to about 60 mol% of the total lipids present in the nanoparticle or the first composition, and

[0215] - PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG, from about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticle or the first composition, preferably from about 0.5 mol% to about 1.5 or 2 mol% of the total lipids present in the nanoparticle or the first composition, optionally from about 0.5 mol% to about 1.0 mol% of the total lipids present in the nanoparticle or the first composition.

[0216] In some aspects, 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.

[0217] In some aspects, 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.

[0218] Preferably, the lipid-based composition or the first composition comprises or consists of the following lipid mixture SS-OP, DSPC, cholesterol and DSPE-PEG, preferably DSG-PEG 2000 or DSG-PEG 5000.

[0219] Alternatively, 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) ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG;

[0220] Preferably, in such specific aspects, the PEG has a size of 2000 Daltons (i.e., PEG-2000).

[0221] Preferably, in such specific aspects, the first composition or the lipid-based nanoparticle comprises or consists of from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid.

[0222] Preferably, in such specific aspect, the first composition or the lipid-based nanoparticle comprises or consists of from about 45 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 15 mol % of a helper lipid from about 35 mol% to about 45 mol% of a sterol, and from about 0.5 mol% to about 2,5 mol% of a PEG-lipid.

[0223] In a very specific aspect, the lipid-based composition or the first composition comprises or consists of a lipid mixture comprising or consisting of:

[0224] ALC-00315, SM-102, Dlin-MC3-DMA or SS-OP or any mixture thereof from about 45 mol % to about 55 mol 0%, preferably from about 48 mol % to about 52 mol %, more preferably of about 50 mol% of the total lipids present in the LNP,

[0225] DOPE, DDAB, DOPC, POPE or DSPC or any mixture thereof from about 5 mol% to about 15 mol %, preferably from about 8 mol% to about 12 mol %, more preferably of about 10 mol% of the total lipids present in the LNP, Cholesterol from about 35 mol% to about 45 mol%, preferably from about 37 mol% to about 40 mol %, more preferably of about 38.5 mol% of the total lipids present in the LNP, and

[0226] - PEG 2000-DSG, PEG 2000-DMG, PEG 5000-DSG, PEG 5000-DMG or ALC-0159 or any mixture thereof, from about 0.5 mol% to about 2.5 mol%, preferably from about 1 mol% to about 2 mol%, more preferably of about 1.5 mol% of the total lipids present in the LNP.

[0227] In a very specific aspect, the lipid-based composition or the first composition comprises or consists of:

[0228] SS-OP from about 35 mol % to about 55 mol % of the total lipids present in the LNP, preferably from about 48 mol % to about 52 mol %, more preferably of about 50 mol% of the total lipids present in the LNP

[0229] - DSPC from about 5 mol% to about 20 mol % of the total lipids present in the LNP, preferably from about 8 mol% to about 12 mol %, more preferably of about 10 mol% of the total lipids present in the LNP,

[0230] - Cholesterol from about 35 mol% to about 45 mol%, preferably from about 37 mol% to about 40 mol %, more preferably of about 38.5 mol% of the total lipids present in the LNP, and

[0231] PEG 2000-DSPE from about 0.5 mol% to about 2.5 mol%, preferably from about 1 mol% to about 2 mol%, more preferably of about 1.5 mol% of the total lipids present in the LNP.

[0232] In a very specific aspect, the lipid-based composition or the first composition comprises or consists of:

[0233] ALC-0315 from about 35 mol % to about 55 mol % of the total lipids present in the LNP, preferably from about 48 mol % to about 52 mol %, more preferably of about 50 mol% of the total lipids present in the LNP;

[0234] - DOPE from about 5 mol% to about 20 mol % of the total lipids present in the LNP, preferably from about 8 mol% to about 12 mol %, more preferably of about 10 mol% of the total lipids present in the LNP, Cholesterol from about 30 mol% to about 60 mol% of the total lipids present in the LNP, preferably from about 37 mol% to about 40 mol %, more preferably of about 38.5 mol% of the total lipids present in the LNP, and

[0235] - and PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DSG, from about 0.5 mol% to about 4 mol% of the total lipids present in the LNP, preferably from about 0.5 mol% to about 1.5 or 2 mol% of the total lipids present in the LNP, optionally from about 0.5 mol% to about 1.5 mol% of the total lipids present in the LNP.

[0236] The lipid-based composition or the first composition according to the invention may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition or imaging.

[0237] In some aspects, a polymer may be included in and / or used to encapsulate or partially encapsulate the lipid-based nanoparticle according to the invention. The polymer may be biodegradable and / or biocompatible. The polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, the polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(gly colic acid) (PGA), poly(lactic acid-co-gly colic acid) (PLGA), poly(L-lactic acid-co-gly colic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L- lactide) (PLLA), poly(D,L-lactide-co- caprolactone), poly(D,L-lactide-co-caprolactone-co- glycolide), poly(D,L-lactide-co-PEO-co- D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L- lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, 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) and copolymers and mixtures thereof, polydioxanone 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.

[0238] In some aspects, the lipid based particle comprises a poloxamine and / or a poloxamer.

[0239] In some aspects, the lipid based particle comprises a plyethyleneimine, rotamine and / or polyaspartamide.

[0240] Second

[0241] In the method of the invention, the second composition is an aqueous solution that comprises one or more nucleic acid molecule(s). Preferably, the nucleic acid molecule(s) is / are isolated nucleic acid molecule(s). Optionally, said aqueous solution contains a buffering agent or salt. Exemplary buffering agent may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate. Exemplary salt may include sodium chloride, magnesium chloride, and potassium chloride.

[0242] In an aspect, the one or several nucleic acid molecule(s) are dissolved in appropriate buffer solutions, for example such as an acetate buffer or a sodium acetate buffer.

[0243] Preferably, the pH of the second composition is comprised between about 3 to about 6, preferably between about 4 and about 5, even more preferably between about 4 and about 4.5.

[0244] In an aspect, when the lipid-based composition comprises an ionizable lipid, the pH of the aqueous composition comprising one or several nucleic acid molecule(s) (i.e., the second composition) is inferior to the pKa of the ionizable lipid in the lipid-based composition (i.e., the first composition).

[0245] In an aspect, the concentration of nucleic acid molecules in the composition comprising one or several nucleic acid molecule(s) is from about 0.01 mg / mL to about 100 mg / mL, from about 0.1 mg / mL to about 50 mg / mL, from about 0.5 mg / mL to about 25 mg / mL, from about 1 mg / mL to about 20 mg / mL, from about 2 mg / mL to about 15 mg / mL, from about 3 mg / mL to about 10 mg / mL.

[0246] The second composition comprises one or several nucleic acid molecule(s). The nucleic acid molecule(s) can be DNA molecule(s) and / or RNA molecule(s).

[0247] In particular, the nucleic acid molecule(s) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 DNA or mRNA molecule(s) encoding 1, 2, 3, 4, 5, 6,7 8, 9 or 10 different protein(s), respectively.

[0248] More specifically, the nucleic acid molecules are selected from the list consisting of mRNA, siRNA, cDNA, saRNA (self-amplifying RNA), taRNA (trans-amplifying RNA), shRNA, miRNA, antisense RNA, IncRNA, piRNA, gRNA, tsRNA. Preferably the one or several nucleic acid molecule(s) are one or several mRNA molecules.

[0249] In one aspect, the second composition or the lipid-based nanoparticle comprises an siRNA or an antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide.

[0250] In certain aspects, the second composition or the lipid-based nanoparticle comprises a short hairpin RNA (shRNA) agent. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain aspects, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleave the shRNA to form siRNA.

[0251] In some aspects, the nucleic acid molecules comprised in the second composition or the lipid- based nanoparticle are antisense molecules. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0252] In some aspects, the nucleic acid molecules comprised in the second composition or the lipid- based nanoparticle are one or more components of a CRISPR-Cas system, such as nucleic acid molecules encoding gRNA and a Cas protein, such as a Cas9 protein.

[0253] In some aspects, the nucleic acid molecule comprised in the second composition or the lipid- based nanoparticle is a miRNA or a mimic of a miRNA. MiRNAs are small non-coding RNA molecules that are capable of causing post-transcriptional silencing of specific genes in cells by the inhibition of translation or through degradation of the targeted mRNA. A miRNA can be completely complementary or can have a region of non-complementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity.

[0254] In order to assess the expression of the nucleic acid molecules the second composition or the lipid-based nanoparticle can also contain either a selectable marker gene or a reporter gene or both to facilitate identification of expressing cells from the population of cells sought to be transfected or infected using the LNP of the invention. In other aspects, the selectable marker may be carried on a separate piece of DNA and also be contained within the LNP. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[0255] The second composition or the lipid-based nanoparticle may also contain a selectable marker gene, which facilitates the selection of host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, P- galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0256] The second composition particularly includes an mRNA encoding a polypeptide of interest capable of being translated into a targeted cell of interest.

[0257] In a particular aspect, the lipid-based nanoparticle comprises a mRNA polynucleotide or a set of mRNA polynucleotides. Preferably, the lipid-based nanoparticle of the invention comprises one or more isolated mRNA molecule(s).

[0258] The technology of mRNA polynucleotide is now well-known by the person skilled in the art, as illustrated in WO21159130, the disclosure thereof being incorporated herein by reference.

[0259] The mRNA molecule of the invention particularly comprises structural elements that allows its encapsulation into the lipid-based nanoparticle and / or its expression into the targeted cell.

[0260] Preferably, the mRNA molecule contains stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5'-end (5'-UTR) and / or at their 3'-end (3'-UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly-A tail.

[0261] In some aspects, the mRNA of the invention includes a flanking region, a 5'-cap structure, a chain terminating nucleotide, a stem loop, a poly-A sequence and / or a polyadenylation signal. In some aspects, the mRNA of the invention comprises a flanking region. A 5'-UTR or a 3’- UTR may be provided as a flanking region to the mRNA of the invention. A 5’-UTR may be homologous or heterologous to the coding region of the mRNA. Multiple 5'- UTRs or 3’-UTRs may be included in the flanking region and may be of the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical alterations, before and / or after codon optimization. Variants of the 5'-UTRs and / or 3 ’-UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G. 5'-UTRs and / or 3 ’-UTRs may also be codon-optimized, or altered in any manner described herein.

[0262] In some aspects, the mRNA comprises an Internal Ribosome Entry Site (IRES) or a Kozak sequence in the 5’-UTR region. The Kozak consensus sequence (Kozak consensus or Kozak sequence) is a nucleic acid motif that functions as the protein translation initiation site. An internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in a cap-independent manner.

[0263] In some aspects, the mRNA of the invention comprises a 5 ’-capping region or structure. The 5'-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of an mRNA molecule.

[0264] The mRNA of the invention may particularly comprise a 5’-cap analog. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type, or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / linked to a polynucleotide.

[0265] In some aspects, the mRNA of the invention includes a stem loop such as, but not limited to, a histone stem loop. The histone stem loop may be before and / or after the poly-A region. The mRNA including the histone stem loop and a poly-A region sequence may include a chain terminating nucleoside. In other instances, the mRNA includes a histone stem loop and a 5'-cap structure such as described herein and / or known in the art. In some cases, the conserved stem loop region may include a miR sequence. As a non-limiting example, the stem loop region may include the seed sequence of a miR sequence. For example, the stem loop region may include a miR-122 seed sequence. Preferably, the stem loop is a nucleotide sequence of about 25 or about 26 nucleotides in length. In other cases, the mRNA, which includes the histone stem loop is stabilized by an alteration to the 3'-region of the polynucleotide that can prevent and / or inhibit the addition of oligo(U).

[0266] In yet other cases, the mRNA, which includes the histone stem loop is stabilized by the addition of an oligonucleotide that terminates in a 3 '-deoxynucleoside, 2', 3 '-dideoxynucleoside 3'-O- methylnucleosides, 3-O-ethylnucleosides, 3 '-arabinosides, and other alternative nucleosides known in the art and / or described herein.

[0267] The mRNA may particularly include at least one histone stem-loop and a poly-A region or polyadenylation signal.

[0268] In some aspects, the mRNA in the second composition or the t-LNP, includes a poly-A sequence and / or polyadenylation signal. A poly-A sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A poly-A sequence may be a tail located adjacent to a 3' untranslated region of a nucleic acid. The length of a poly-A region of the present disclosure is of 20, 40, 80, 100, 120, 140 or 160 nucleotides in length on an mRNA molecule described herein. The poly-A region may also be designed as a fraction of the mRNA to which it belongs. In this context, the poly-A region may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A region.

[0269] In some instances, the mRNA includes a poly-A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this aspect, the G-quartet is incorporated at the end of the poly-A region.

[0270] In some aspects, the mRNA molecule of the invention may include one or more naturally occurring components, including any of the canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside) which impart useful properties including increased stability and / or the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. For example, a modRNA may exhibit reduced degradation in a cell into which the modRNA is introduced, relative to a corresponding unaltered mRNA. These alternative species may enhance the efficiency of protein production, intracellular retention of the polynucleotides, and / or viability of contacted cells, as well as possess reduced immunogenicity. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published patent application Nos. WO2013052523; WO2014093924; W02015051173; W02015051169; W02015089511; W02015196130; WO2015196118; WO2015196128; or WO2017153936 all of which are incorporated by reference herein.

[0271] Different sugar alterations and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in a polynucleotide. One of ordinary skill in the art will appreciate that the nucleotide analogs or other alteration(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. An alteration may also be a 5'- or 3'-terminal alteration. In some aspects, the polynucleotide includes an alteration at the 3'-terminus. The mRNA may contain from about 1% to about 100% alternative nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. , any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of a canonical nucleotide (e.g., A, G, U, or C).

[0272] The mRNA may contain at a minimum zero and at maximum 100% alternative nucleotides, or any intervening percentage, such as at least 5% alternative nucleotides, at least 10% alternative nucleotides, at least 25% alternative nucleotides, at least 50% alternative nucleotides, at least 80% alternative nucleotides, or at least 90% alternative nucleotides. For example, polynucleotides may contain an alternative pyrimidine such as an alternative uracil or cytosine. In some aspects, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in a polynucleotide is replaced with an alternative uracil (e.g., a 5- substituted uracil). The alternative uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with an alternative cytosine (e.g., a 5-substituted cytosine). The alternative cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0273] In some aspects, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (y), pyridin-4- one ribonucleoside, 5-aza- uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio- uracil (s4U), 4-thio- pseudouridine, 2-thio-pseudouridine, 5 -hydroxy -uracil (ho5U), 5- aminoallyl-uracil, 5-halo- uracil (e.g., 5-iodo-uracil or 5 -bromo-uracil), 3-methyl-uracil (mU), 5 -meth oxy -uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5- carboxymethyl-uracil (cm5U), 1 -carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl- uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5- methoxycarbonylmethyl-uracil (mcm5U), 5- methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5-aminomethyl-2-thio-uracil (nmVu), 5-methylaminomethyl-uracil (mnm5U), 5- methylaminomethyl-2-thio-uracil (mnmVu), 5-methylaminomethyl-2-sel eno-uracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2 -thio-uracil (cmnmVu), 5 -propynyl -uracil, 1- propynyl-pseudouracil, 5-taurinomethyl-uracil (xm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uracil(xm5s2U), l-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., having the nucleobase deoxythymine), 1 -methyl-pseudouridine (mly), 5-methyl-

[0274] 2 -thio-uracil (m5s2U), l-methyl-4-thio-pseudouridine (m xy), 4-thio- 1 -methyl-pseudouridine,

[0275] 3-methyl-pseudouridine (m3y), 2 -thio- 1 -methyl-pseudouridine, 1 -methyl- 1 -deazapseudouridine, 2-thiol-methyl-l-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, N1 -methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uracil (acp U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp y), 5- (isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2 -thio-uracil (inm5s2U), 5,2'-0-dimethyl-uridine (m5Um), 2-thio-2'-0-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-0-methyl-uridine (mem Urn), 5-carbamoylmethyl-2'-0-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm5Um), 3,2'-0- dimethyl-uridine (mUrn), and 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm5Um), 1- thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)- uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2 -thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(l-E- propenylamino)]uracil. In some aspects, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine 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), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo- cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl- 1 -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5-ethyl-

[0276] 1-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5- methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine (k2C), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acetyl-2'-0-methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formyl-2'-0-methyl-cytidine (f5Cm), N4,N4,2'-0- trimethyl-cytidine (m42Cm), 1 -thio-cytosine, 5 -hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2- azidoethyl)-cytosine.

[0277] In some aspects, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine,2,6- diaminopurine, 2- amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6- chloro-purine), 2- amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methy 1 -adenine (ml A), 2-methyl-adenine (m2 A), 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,

[0278] 2 -methylthio-adenine, 2-methoxy-adenine, N6,2'-0-dimethyl-adenosine (m6Am), N6,N6,2'-0- trimethyl-adenosine (m62Am), l,2'-0-dimethyl-adenosine (ml Am), 2-amino-N6-methyl- purine, 1 -thio-adenine, 8-azido-adenine, N6-(19-amino- pentaoxanonadecyl)-adenine, 2,8- dimethyl-adenine, N6-formyl-adenine, and N6- hydroxymethyl-adenine.

[0279] In some aspects, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl- queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza- guanine (preQi), archaeosine (G+), 7-deaza-8-aza-guanine,

[0280] 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6- thio-7- methyl-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-6-thio-guanine, N2- methyl-2'-0-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-0-methyl-guanosine (m22Gm), 1- methyl-2'-0-methyl-guanosine (mIGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'- O-methyl-inosine (Im), l,2'-0-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl- guanine.The alternative nucleobase of a nucleotide can be independently a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine. In another aspect, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4- d]pyrimidines, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 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,

[0281] 7- deazaadenine, 3 -deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a] 1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimi dines, pyrazin-2-ones, 1,2,4- triazine, pyridazine; or 1,3,5 triazine. When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and / or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7- deaza adenine).

[0282] Optionally, the mRNA can be a circular RNA, in particular a circular mRNA, especially as described in WO2014 / 186334 and WO2022 / 261490. In a preferred aspect, the one or several nucleic acid molecule(s) are one or several mRNA molecule(s) encoding for a molecule selected from the group consisting of an immune cell enhancing compound, an antigen fragment, an antigen binding domain and a Chimeric Antigen Receptor (CAR).

[0283] In some embodiments, the second composition does not comprise nucleic acids but comprises an active ingredient such as drugs and therapeutic molecules.

[0284] Additionally or alternatively, the second composition may comprises an imaging agent. Such imaging agent may be for instance a fluorescent protein such as GFP or luciferase. In an embodiment, the imaging agent may be one or several fluorescent mRNA molecule.

[0285] In particular, the lipid-based nanoparticle to be obtained by the invention or the second composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mRNA molecule(s) encoding 1, 2, 3, 4, 5, 6,7 8, 9 or 10 different protein(s), respectively. In a particular aspect, the lipid-based nanoparticle or the second composition comprises mRNA molecules encoding 2 different immune cell proteins such as disclosed herein. The immune cell protein can be selected from a single or from different cell types. For example, the mRNA molecules may encode different T cells proteins. Alternatively, the mRNA molecules may encode different immune cell protein, for example for a T cell protein and for another immune cell inhibiting protein such as a Natural killer protein.

[0286] Immune cell compound or molecule

[0287] In some aspects, the nucleic acid molecule as described herein encodes for an immune cell protein, in particular an immune cell inhibiting or enhancing protein. Accordingly, the lipid- based nanoparticle of the invention comprises one or more different mRNA molecule encoding an immune cell inhibiting or activating protein.

[0288] The terms “immune cell compound”, “immune cell molecule” or “immune cell protein” refers to molecules and proteins that are specifically produced or expressed by immune cells, such as activated immune cells. These proteins play crucial roles in the functioning of the immune system, including antigen recognition, signaling, cell communication, and immune responses. Immune cell proteins are diverse and can include receptors, enzymes, cytokines, antibodies, and other molecules that are essential for the proper functioning of immune cells or immune system.

[0289] The immune cell protein encoded by a mRNA molecule included in the lipid-based nanoparticle of the invention may particularly be selected based on the type of immune cell targeted by the antigen binding domain comprised in the lipid-based nanoparticle. In particular, when the immune cell targeted is a T-cell (i.e., so that the lipid-based nanoparticle comprises an antigen binding domain that binds to a target expressed on T cells such as PD-1), the mRNA encodes T cell protein.

[0290] Additionally, the immune cell protein encoded by a mRNA molecule to be included in the lipid- based nanoparticle of the invention may particularly be selected based on the desired effect. For example, the immune cell protein may be selected for a particular indication, condition, disease, or disorder.

[0291] For example, the immune cell protein may be selected for its effect on the targeted immune cell itself. Typically, the immune cell protein can be an immune cell inhibiting protein or an immune cell enhancing protein.

[0292] In some aspects, the mRNA molecule encodes for an immune cell inhibiting protein.

[0293] The term “immune cell inhibiting protein” refers to a protein that decreases, suppresses or dampens the activity of immune cells, in particular in a subject. These proteins typically play a role in modulating the immune response to prevent excessive inflammation or inappropriate immune reactions. Such protein can typically 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. Immune cell inhibiting proteins are known to be important for maintaining immune homeostasis and preventing autoimmunity.

[0294] In particular, such immune cell inhibiting protein exhibits an effect on activated immune cells selected from the group consisting of:

[0295] Inducing or increasing the exhaustion of immune cells

[0296] Inhibiting or decreasing the proliferation or renewal of immune cells

[0297] Inducing or increasing the apoptosis or cell death of immune cells

[0298] Increasing the production or concentration of mitochondrial enzymes and / or transporters of immune cells

[0299] Increasing the production or concentration of transcription factors of immune cells Inducing or increasing hypoxia of the microenvironment or tissue

[0300] Increasing the production or concentration of metabolism enzymes of immune cells Inducing or Increasing the production of anti-inflammatory signalling molecule of immune cells

[0301] Inhibiting or decreasing the production of cytotoxic compounds by immune cells Inducing or increasing the internalization and exocytosis of immune cells Increasing the production or concentration of chaperone protein of immune cells Inducing or increasing the production of cytoskeleton regulation protein of immune cells Inhibiting or decreasing the degradation of protein through immuno-proteasome and ubiquitination, as well as the antigen presentation of immune cells Inducing or increasing the migration and / or motility of immune cells Inducing or increasing the inflammation resolution of the tissue

[0302] Inducing or increasing the active membrane transport of immune cells Increasing the production or concentration of carrier protein of immune cells

[0303] - Modifying the epigenetic of immune cells

[0304] Increasing the production or concentration of tRNA of immune cells, Inducing or increasing the production and / or secretion of checkpoint inhibitors, Inducing a phenotype switch of the immune cell, from a pro-inflammatory to an antiinflammatory immune cell type,

[0305] Inhibiting a compound, protein or molecule that inhibits the exhaustion of immune cells, the apoptosis 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 metabolism enzymes of immune cells, the hypoxia of the microenvironment or tissue, the efficiency of signalling pathways of immune cells, the secretion of immune cells, the internalization of endosome of immune cells, the production or concentration of chaperone protein of immune cells, the production of cytoskeleton regulation protein of immune cells, the migration and / or motility of immune cells, the active membrane transport of immune cells, the production or concentration of carrier protein of immune cells or the production or concentration of tRNA of immune cells, the production and / or secretion of checkpoint inhibitors, or the production of inflammation resolution factors ; or that induces or increases the proliferation of immune cells, the production of cytotoxic compounds by immune cells or the degradation of protein through immuno-proteasome and ubiquitination, as well as the antigen presentation of immune cells.

[0306] Alternatively, the mRNA molecule encodes for an immune cell activating protein.

[0307] The terms “immune cell activating protein” or “immune cell enhancing protein” refer to a protein that induces, increases, enhances or boosts the activity of immune cells or activates immune cells. These proteins typically play critical roles in augmenting the immune response, improving the ability of the immune system to detect and eliminate pathogens, infected cells, or abnormal cells, such as cancer cells. Immune cell enhancing 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. They are known to be crucial for mounting effective immune responses against infections and tumors.

[0308] In particular, such immune cell enhancing protein exhibits an effect on activated immune cells selected from the group consisting of:

[0309] Inducing or increasing the sternness of immune cells

[0310] Inducing or increasing the proliferation or renewal of immune cells

[0311] Inhibiting or decreasing the apoptosis of immune cells

[0312] Increasing the production or concentration of mitochondrial enzymes and / or transporters of immune cells

[0313] Increasing the production or concentration of transcription factors of immune cells Inhibiting or decreasing hypoxia of the microenvironment or tissue

[0314] Increasing the production or concentration of metabolism enzymes of immune cells Increasing the efficiency of signaling pathways of immune cells

[0315] Inducing or increasing the production of cytotoxic compounds by immune cells Inducing or increasing the internalization of endosome of immune cells

[0316] Increasing the production or concentration of chaperone protein of immune cells Inducing or increasing the production of cytoskeleton regulation protein of immune cells Inducing or increasing the degradation of protein through immuno-proteasome and ubiquitination, as well as the antigen presentation of immune cells

[0317] Inducing or increasing the migration and / or motility of immune cells

[0318] Inducing or increasing the inflammation of the tumor microenvironment (TME)

[0319] Increasing the anergy resistance of immune cells

[0320] Inducing or increasing the active membrane transport of immune cells

[0321] Increasing the production or concentration of carrier protein of immune cells

[0322] - Modifying the epigenetic of immune cells

[0323] Increasing the production or concentration of tRNA of immune cells,

[0324] Inhibiting or decreasing the production and / or secretion of checkpoint inhibitors, Inducing a phenotype switch of the immune cell, from a pro-tumoral to an anti-tumoral immune cell type,

[0325] Inhibiting a compound, protein or molecule that inhibits the sternness of immune cells, the proliferation or renewal 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 metabolism enzymes of immune cells, the efficiency of signaling pathways of immune cells, the secretion of immune cells, the production of cytotoxic proteins by immune cells, the internalization of endosome of immune cells, the production or concentration of chaperone protein of immune cells, the production of cytoskeleton regulation protein of immune cells, the degradation of protein through immuno-proteasome and ubiquitination, as well as the antigen presentation of immune cells, the migration and / or motility of immune cells, autophagy of immune cells, the inflammation of TME, the active membrane transport of immune cells, the production or concentration of carrier protein of immune cells or the production or concentration of tRNA of immune cells; or that induces or increases the apoptosis of immune cells, hypoxia of the microenvironment or tissue or the production and / or secretion of checkpoint inhibitors.

[0326] In particular, the immune cell inhibiting protein inducing or increasing the exhaustion of immune cells is selected from the group comprising or consisting of TIM3, ENTPD1, LAG3, PD-1 and TIGIT.

[0327] In particular, the immune cell enhancing protein inducing or increasing the sternness of immune cells is selected from the group comprising or consisting of TCF1, LEF1, WNT, FRIZZLED and Beta catenin.

[0328] In particular, the immune cell inhibiting protein inhibiting or decreasing the proliferation or renewal of immune cells is selected from the group comprising or consisting of FOXO1, MLH1, MSH2, MSH6, APC and CDKN2A.

[0329] In particular, the immune cell enhancing protein inducing or increasing the proliferation or renewal of immune cells is selected from the group comprising or consisting of LRP6, CYCLIN, TOP2A, MUCL1 and MDM2.

[0330] In particular, the immune cell inhibiting protein inducing or increasing the apoptosis or cell death of immune cells is selected from the group comprising or consisting of CASPASE, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid et Bim, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GAB ARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA and P53.

[0331] In particular, the immune cell enhancing protein inhibiting or decreasing the apoptosis of immune cells is selected from the group comprising or consisting of BCL2, BCLXL, BIRC3 and MCLl . In particular, the immune cell inhibiting protein increasing the production or concentration of mitochondrial enzymes and / or transporters of immune cells is from the SLC25 family.

[0332] In particular, the immune cell enhancing protein increasing the production or concentration of mitochondrial enzymes and / or transporters of immune cells is PGCla.

[0333] In particular, the immune cell inhibiting protein increasing the production or concentration of transcription factors of immune cells is selected from the group comprising or consisting of FOXP3, TOX, EOMES, BCL6, and BACH2.In particular, the immune cell inhibiting protein increasing the production or concentration of transcription factors of immune cells is selected from the group comprising or consisting of FOXP3, TOX, EOMES, BCL6, BACH2 SOCS, RIPK1 and any member of the STAT family.

[0334] In particular, the immune cell enhancing protein increasing the production or concentration of transcription factors of immune cells is selected from the group comprising or consisting of TCF7, NF AT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GAT A3, JUNB, POU2AF1, OCTI, BLIMP- 1, XBP-1 and FOXO1.

[0335] In particular, the immune cell enhancing protein increasing the production or concentration of transcription factors of immune cells is selected from the group comprising or consisting of TCF7, NF AT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCTI, BLIMP- 1, XBP-1 and FOXO1.

[0336] In particular, the immune cell inhibiting protein inducing or increasing hypoxia of the microenvironment or tissue is selected from the group comprising or consisting of HIFa, PKCq and VHL.

[0337] In particular, the immune cell enhancing protein inhibiting or decreasing hypoxia of the microenvironment or tissue is PTGS2.

[0338] In particular, the immune cell inhibiting protein increasing the production or concentration of metabolism enzymes of immune cells is selected from the group comprising or consisting of IDO-1, IDO-2, ARG1 and TDO.

[0339] In particular, the immune cell enhancing protein increasing the production or concentration of metabolism enzymes of immune cells is selected from the group comprising or consisting of CSE, Glutl, Glut3, HK2, FOXO1, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS.

[0340] In particular, the immune cell enhancing protein increasing the production or concentration of metabolism enzymes of immune cells is selected from the group comprising or consisting of CSE, Glutl, Glut3, HK2, FOXO1, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS and OXPHOS.

[0341] In particular, the immune cell inhibiting protein inducing or increasing the production of antiinflammatory signaling molecule of immune cells is selected from the group comprising or consisting of mTOR / DAPTOR / RAPTOR, SHP and SMAD.

[0342] In particular, the immune cell enhancing protein increasing the efficiency of signaling pathways of immune cells is selected from the group comprising or consisting of AKT, PLC, STAT, SMAD, Blys, BTK and BLK.

[0343] In particular, the immune cell inhibiting protein inhibiting or decreasing the production of cytotoxic compounds by immune cells is selected from the group comprising or consisting of SHIP-1, SHP-1 / 2, PTEN, PTP1B, IKAROS, EGR2 / 3, CREM and P27 (KIP1).

[0344] In particular, the immune cell enhancing protein inducing or increasing the production of cytotoxic proteins by immune cells is selected from the group comprising or consisting of CD 107a, Lymphotoxin (LT) aip2, granzyme B, perforin.

[0345] In particular, the immune cell inhibiting protein inducing or increasing the internalization and exocytosis of immune cells is selected from the group comprising or consisting of CD 107a, RAC1, AP2, RB7, M6P and MPR.

[0346] In particular, the immune cell enhancing protein inducing or increasing internalization of endosome is POU2F1.

[0347] In particular, the immune cell protein increasing the production or concentration of chaperone protein of immune cells is selected from the group comprising or consisting of BBS10, BBS12, TCP1 and HSP.

[0348] In particular, the immune cell inhibiting protein inducing or increasing the production of cytoskeleton regulation protein, the migration and / or motility of immune cells is selected from the group comprising or consisting of APC, Laminin, Actin, Vimentin, DEFI, Dynein, Kinesin and Rab protein. In particular, the immune cell enhancing protein inducing or increasing the production of cytoskeleton regulation protein, the migration and / or motility of immune cells is selected from the group comprising or consisting of Integrin alpha 1, Integrin alpha 2, Integrin alpha 2b, Integrin alpha 11, Integrin alpha 3, Integrin alpha 6, Integrin alpha?, 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, DEFI, Dynein, Kinesin, Rab protein.

[0349] In particular, the immune cell inhibiting protein inhibiting or decreasing the degradation of protein through immune-proteasome and ubiquitination, as well as the antigen presentation of immune cells is selected from the group comprising or consisting of TAPASIN, LMP7, Erp57 and Cbl-b.

[0350] In particular, the immune cell enhancing protein inducing or increasing the degradation of protein through immuno-proteasome and ubiquitination, as well as the antigen presentation of immune cells is selected from the group comprising or consisting of NLRP3, TAP, LAMP, Ubiquitin ligase, CD74, Peptidase, Calreticulin and Aurora.

[0351] In particular, the immune cell inhibiting protein inducing or increasing the inflammation resolution of the tissue is selected from the group comprising or consisting of CHEMR23, GPR37, GPR32, GPR18, FPR2 and GPR35.

[0352] In particular, the immune cell enhancing protein inducing or increasing the inflammation of TME is LGR6.

[0353] In particular, the immune cell enhancing protein modifying the epigenetic of immune cells is selected from the group comprising or consisting of HAT, KDM1, TGD, TET1.

[0354] In particular, the immune cell inhibiting protein modifying the epigenetic of immune cells is selected from the group comprising or consisting of HDAC, HMT, SIRT1, DOT1L, PRDME, PRMT1, PRMT3, PRMT5, PRMT6, SETD7, SETD8, SMYD2, SMYD3 and SUV420H2.

[0355] In particular, the immune cell enhancing protein increasing the anergy resistance of immune cells is selected from the group comprising or consisting of C-FOS, JUN, EGR-2, EGR-3.

[0356] These proteins are detailed in Table D here below. In some aspects, the mRNA encodes for a protein selected in Table D.

[0357] Table D.

[0358] In some aspects, the one or several mRNA molecule(s) encode(s) for TIM3, ENTPD1, LAG3, PD-1, PD-L1, TIGIT, FOXO1, MLH1, MSH2, MSH6, APC, CDKN2A, CASPASE, FAS, TRAIL, Bax, Bak, Bok, Bad, Bid etBim, BIM, BIM-S, FADD, FASL, TRAILR, TNFR, ATG5, LC3, GABARAP, GATE16, ATG 5 / 7 / 10 / 12, NOXA, PUMA, P53, FOXP3, TOX, EOMES, BCL6 and BACH2, HIFa, PKCq and VHL, IDO-1, IDO-2, ARG1; TDO, mTOR / DAPTOR / RAPTOR, SHP, SMAD, SHIP-1, SHP-1 / 2, PTEN, PTP1B, IKAROS, EGR2 / 3, CREM, P27 (KIP1), CD 107a, RAC1, AP2, RB7, M6P, MPR, BBS 10, BBS 12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEFI, 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.

[0359] Preferably, the one or several mRNA molecule(s) encode(s) for 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.

[0360] Preferably, the one or several mRNA molecule(s) encode(s) for 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.

[0361] In some aspects, the mRNA molecule encodes for BIM-S and / or PUMA.

[0362] Preferably, the mRNA molecule encoding for PUMA comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 48, or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0363] In some aspects, the mRNA molecule encodes for a BIM protein. As used herein, the term “BIM protein” encompasses the three Bim isoforms (i.e., Bim-EL, Bim-L and Bim-S) that are generated by alternative splicing. Preferably, the mRNA molecule encoding for BIM-S comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 49 or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0364] The effect of an immune cell inhibiting protein on the activated immune cells may be determined when, in presence of the immune cell inhibiting protein of the invention, activated immune cells exhibits a lower activity compared to activated immune cells under the same experimental conditions but without the presence of the immune cell inhibiting protein. Said 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 in need of a treatment as detailed hereunder. Immune cell activity can be measured by any method known to the person skilled in the art. In particular, the inhibition of immune cell activity may be measured by comparing the immune cell activity of a population of immune cells obtained from a sample, without the immune cell inhibiting protein to be assessed, to the immune cell activity of a population of immune cells obtained from a sample and treated with the immune cell inhibiting protein to be assessed.

[0365] In some aspects, the activity-enhancing protein is selected from the group consisting of: TCF1, LEF1, WNT, FRIZZLED, Beta catenin, LRP6, CYCLIN, TOP2A, MUCL1, MDM2, BCL2, BCLXL, BIRC3, MCL1, PGCla, TCF7, NF AT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCTI, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glutl, Glut3, HK2, FOXO1, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD 107a, Lymphotoxin (LT) aip2, 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 alpha7, 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, DEFI, 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 Regulatory Factors such as IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, SOCS, NFKB, STAT3, AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKK a, IKKp, TRAM, TRIF, RIPK1, TBK1, PI3K, D3 -phosphoinositides, derivatives of phosphatidylinositol, IL7R, CD122, CD132, CD25, CD215, IL12R, IL17R, IL8R, IL21R, IL11R, IL18R, IL10R, IL1R, IL6R, CXCR3, CXCR5, CXCR4, CXCR1, CXCR2, CXCR6, 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, 0X40, 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, 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.

[0366] In some aspects, the activity-enhancing protein is selected from the group consisting of: TCF1, LEF1, WNT, FRIZZLED, Beta catenin, LRP6, CYCLIN, TOP2A, MUCL1, MDM2, BCL2, BCLXL, BIRC3, MCL1, PGCla, TCF7, NF AT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCTI, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glutl, Glut3, HK2, FOXO1, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS, AKT, PLC, SMAD, Blys, BTK, BLK, CD107a, Lymphotoxin (LT) al 2, 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 alpha7, Integrin alpha E, Integrin beta 2, Integrin beta 4, LFA-1, LFA-2, LFA-3, Integrin beta

[0367] I, Integrin beta 7, CD103, Integrin alpha V, ITGAE, CD11C, CRTAM, CXCR5, CXCR3, CCR7, SELL, GAL3, Laminin, Actin, Vimentin, DEFI, 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 Regulatory Factors such as IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, TRAM, TBK1, NFKB, AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKa, IKK0, TRIF, PI3K, D3 -phosphoinositides, derivatives of phosphatidylinositol, IL7R, CD 122, CD132, CD25, CD215, IL12R, IL17R, IL8R, IL21R, IL11R, IL18R, IL10R, IL1R, IL6R, CXCR3, CXCR5, CXCR4, CXCR1, CXCR2, CXCR6, 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, 0X40, OX40L, CD 155, 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, 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.

[0368] Preferably, the activity-enhancing protein is selected from the group consisting of TCF1, WNT, BCL2, BCLXL, TBET, Glutl, LGR6, ICOS, CD28, CD40L, 4- IBB, Perforin, CXCL9, CXCL10, GrB, OXPHOS, Integrin alpha 1, Integrin alpha 2, Integrin alpha 2b, Integrin alpha

[0369] I I, Integrin alpha 3, Integrin alpha 6, Integrin alpha7, Integrin alpha E, Integrin beta 2, Integrin beta 4, Integrin beta 1, Integrin beta 7 and Integrin alpha V.

[0370] 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 alpha7, 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. Particularly, the activity-enhancing protein is selected from the group consisting of: BCL2, IL7, IL7R, CXCL9 and CXCL10.

[0371] Preferably, the mRNA molecule encoding for BLC2 comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 43 or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0372] Preferably, the mRNA molecule encoding for IL7 comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 44 or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0373] Preferably, the mRNA molecule encoding for IL7R comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 45 or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0374] Preferably, the mRNA molecule encoding for CXCL9 comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 46 or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0375] Preferably, the mRNA molecule encoding for CXCL10 comprises or consists of a nucleic acid sequence as set forth in SEQ ID NO: 47 or of a nucleic acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity thereto.

[0376] The effect of an immune cell enhancing protein on the immune cells may be determined when, in presence of the immune cell enhancing protein of the invention, immune cells exhibits a 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 the person skilled in the art. In particular, the enhancement of immune cell activity may 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 assessed, to the immune cell activity of a population of immune cells obtained from a sample and treated with the immune cell enhancing compound or molecule to be assessed.

[0377] In an aspect, the immune cell inhibiting protein is an intracellular protein having an intracellular effect on the activated immune cell or a transmembrane

[0378] In an aspect, the immune cell enhancing protein is selected from the group comprising intracellular proteins or proteins having an intracellular effect on the immune cell, transmembrane proteins and secreted proteins. In an aspect, the immune cell protein is an intracellular protein or a protein having an intracellular effect on the immune cell.

[0379] Use of intracellular proteins, in combination with the targeting of a specific subset of immune cells, preferentially T cells, even more preferably TILs, results in a very specific and efficient inhibition or enhancement of said immune cells. Potent intracellular proteins may be used, with an elevated activation and / or proliferation of immune cells in a specific environment.

[0380] As used herein, a “protein that have an intracellular effect on the immune cell” or an “intracellular protein” refers to a protein which is produced / expressed inside a cell and which does not enter the extracellular medium, either alone or in a vesicle, nor that is expressed in the cellular membrane. The intracellular protein is thus contained in the boundaries of the cellular membrane, and acts in one of the cellular compartments (e.g. cytosol, endoplasmic reticulum, mitochondria, nucleus, etc.). Such protein can be present in any of the cellular compartments, such as nucleus, interci sternal space, organelles or cytosol. Accordingly, the protein can be a cytoplasmic protein, a nuclear protein or a mitochondrial protein, or an intercistemal protein, preferably nuclear or cytoplasmic protein.

[0381] Optionally, the immune cell intracellular protein can be an enzyme, an intracellular signalling protein or a transcription factor, preferably a transcription factor.

[0382] In a particular aspect, the transmembrane protein is selected from the group comprising FRIZZLED, BCLXL, CCR4, CCR10, CXCR3, CCR10, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR3, CXCR5, CRTAM, CCR7, CXCR5, GPR35, GPR37 and TAP.

[0383] In a particular aspect, the immune cell protein is a transcription factor. As used herein, a “transcription factor” refers to a DNA-binding protein that regulates the transcription of a gene. Preferably, the transcription factor is selected from the group consisting of RORgt, SOCS, NFKB, STAT, TRAM, RIPK1, and TBK1 and a variant thereof having at least 80% of identity with the wildtype protein or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof. Preferably, the transcription factor is selected from the group consisting of RORgt, NFKB, TRAM and TBK1 and a variant thereof having at least 80% of identity with the wildtype protein or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0384] Preferably, the transcription factor is selected from the group consisting TCF7, NF AT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCTI, BLIMP- 1, XBP-1 and FOXO1 and any combinations thereof. Preferably, the transcription factor is selected from the group consisting of Interferon Regulatory Factors (IRFs, including IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9), CREB, RORg, RORgt, SOCS, NFKB, T-bet, STAT3, AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKa, IKKP, TRAM, TRIF, RIPK1, and TBK1 and a variant thereof having at least 80% of identity with the wildtype protein or having 1 to 10 modifications selected from the group consisting of addition, deletion, substitution and combinations thereof.

[0385] In a specific aspect, the immune cell inhibiting protein is not a chimeric antigen receptor (CAR). In an aspect, the immune cell activating protein is not a CAR, a T cell receptor (TcR) (i.e., including TCR alpha, TCR beta, CD3 and CD247) or a B cell receptor (BcR).

[0386] In some aspects, the one or more mRNA molecule(s) does not encode a cytokine and / or a chemokine. In a specific aspect, the immune cell inhibiting protein is not a cytokine and / or a chemokine.

[0387] In a particular aspect, the transmembrane protein is selected from the group comprising CCR4, CCR10, CXCR3, CCR10, CCR5, CCR2, CX3CR1, CCR7, CXCR4, CXCR3, CXCR5, CCR7, CXCR5.

[0388] Alternatively, the immune cell protein is an enzyme. For example, such enzyme can be phosphatidylinositol 3-kinase (PI3K).

[0389] Alternatively, the immune cell protein is an intracellular signalling protein. For example, such intracellular signalling molecules can be D3 -phosphoinositides and derivatives of phosphatidylinositol such as phosphorylated at the D-3 position of the inositol ring and encompasses the compounds phosphatidylinositol-(3)-monophosphate (PtdIns(3)P), phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol(3,4,5)- trisphosphate (PtdIns(3,4,5)P3). Therefore, the intracellular signalling protein can be those involved in the synthesis of these molecules.

[0390] In a specific aspect, the immune cell inhibiting protein is not a chimeric antigen receptor (CAR). Preferably, the immune cell inhibiting protein is not a CAR, a T cell receptor (TcR) (i.e., including TCR alpha, TCR beta, CD3 and CD247) or a B cell receptor (BcR).

[0391] In some aspects, the immune cell compound or molecule is a secreted protein.

[0392] As used herein, “secreted compound” or “secreted protein” or “secreted molecule” refers to a compound which exits the cell into the extracellular medium, is addressed to the membrane of the immune cell or is directed to an adjacent cell. In an aspect, the secreted compound or molecule does not act on the immune cell which secreted it.

[0393] In an aspect, the secreted compound or molecule acts on an additional cell as well as on the immune cell which secreted it.

[0394] In some aspects, the one or more mRNA molecule(s) does not encode a cytokine and / or a chemokine.

[0395] In a particular aspect, the immune cell inhibiting protein is a cytokine receptor. Preferably, the cytokine receptor is selected from the group consisting of IL10R, TGFR, TNFR, ILR1A, GCSFR and IL4R.

[0396] In a particular aspect, 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.

[0397] In a particular aspect, the immune cell inhibiting 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.

[0398] In a particular aspect, the immune cell inhibiting protein is a lectin receptor. Preferably, the lectin receptor is selected from the group consisting of CD72, DCIR, MICL and CLEC-1.

[0399] In a particular aspect, 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.

[0400] In a particular aspect, the immune cell inhibiting protein is an engineered surface receptor or anchored membrane cytokine. Preferably, the engineered surface receptor or anchored membrane cytokine is selected from the group consisting of Membrane engineered cytokine (TGFB, IL13, IL4, IL-10), Dominant negative TLR, Dominant negative receptor (IL-6R, TNFR, IL17R, IL23R, IL35R, IL21R, IFNa R).

[0401] In a particular aspect, the immune cell enhancing protein is an engineered surface receptor or anchored membrane cytokine. Preferably, the engineered surface receptor or anchored membrane cytokine is selected from the group consisting of IL12A, IL12B, IFNG, IFNa, IL21, IL7, IL2, IL15 and IL18. In a particular aspect, the immune cell inhibiting protein is a coinhibitory receptor or ligand. Preferably, the coinhibitory 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, galectin9, 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

[0402] In a particular aspect, the immune cell enhancing protein is a costimulation receptor or ligand. Preferably, the costimulation receptor or ligand is selected from the group consisting of ICOS, ICOSL, CD28, CD80, CD86, CD70, CD40L, CD226, GITR, GITRL, 4-1BB, 4-1BBL, 0X40, 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.

[0403] In a particular aspect, the one or several mRNA molecule(s) encode(s) for 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 R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, galectin9, 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.

[0404] In a particular aspect, the one or several mRNA molecule(s) encode(s) for 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 R, BTLA, CTLA4, B7H1, B7H3, B7H4, PD1H, LAIR1, TIM1, TIM3, TIM4, 2B4, PD-1, PDL1, PDL2, galectin9, 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.

[0405] These proteins are detailed in Table E here below. In some aspects, the mRNa molecule encodes for a protein selected in Table E. Table E

[0406] In a particular aspect, the immune cell inhibiting protein is a protein inhibiting a compound, protein or molecule that inhibits the exhaustion of immune cells, the apoptosis 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 metabolism enzymes of immune cells, the hypoxia of the microenvironment or tissue, the efficiency of signalling pathways of immune cells, the secretion of immune cells, the internalization of endosome of immune cells, the production or concentration of chaperone protein of immune cells, the production of cytoskeleton regulation protein of immune cells, the migration and / or motility of immune cells, the active membrane transport of immune cells, the production or concentration of carrier protein of immune cells or the production or concentration of tRNA of immune cells, the production and / or secretion of checkpoint inhibitors, or the production of inflammation resolution factors ; or that induces or increases the proliferation of immune cells, the production of cytotoxic compounds by immune cells or the degradation of protein through immuno-proteasome and ubiquitination, as well as the antigen presentation of immune cells. In some aspects, the invention concerns a lipid-based nanoparticle comprising an antigenbinding domain capable of specifically binding to a target expressed on activated T cells surface and one or several mRNA molecule(s) encoding an activity-inhibiting protein of said T cells.

[0407] Preferably, the invention concerns a lipid-based nanoparticle comprising an anti-PD-1 antigenbinding domain and one or several mRNA molecule(s) encoding an activity-inhibiting protein of said T cells.

[0408] Even more preferably, the invention concerns a lipid-based nanoparticle comprising an anti- PD-1 antigen-binding domain and one or several mRNA molecule(s) encoding T cells transcription factors or T cell factors inhibiting growth.

[0409] Additionally, the lipid-based nanoparticle of the invention may comprise several mRNA molecules encoding different immune cell activity inhibiting proteins. In this aspect, the lipid- based nanoparticle comprises a first mRNA molecule encoding a T cell activity inhibiting protein and a second mRNA molecule encoding a second immune cell activity inhibiting protein. The second immune cell activity inhibiting protein may be another T cell activity inhibiting protein (i.e., different form the first T cell activity inhibiting component) or can be an activity inhibiting component of another type of immune cell, such as a NK or a macrophage.

[0410] In a particular aspect, the immune cell enhancing protein is specific of T cells, even more preferably of activated T cells. Preferably, the immune cell modulating compound or molecule is selected from the group comprising T cell growth factors, in particular growth factors to increase number and repertoire of naive immune cells, agonists to activate and stimulate immune cells, inhibitors of T cell checkpoint blockade, T cell growth factors to increase the growth and survival of immune T cells, immune-stimulating cytokines, immune-stimulating membrane protein.

[0411] In some aspects, the one or more mRNA molecule(s) does not encode a cytokine and / or a chemokine.

[0412] In an aspect, the lipid-based nanoparticle comprises at least two mRNA molecule(s), wherein one of said at least two mRNA molecules encodes a transmembrane protein that is a receptor and another of said at least two mRNA molecules encodes a secreted protein that is a ligand of said receptor. For example, the lipid-based nanoparticle comprises a mRNA encoding an interleukin receptor (e.g., IL10-R) and a mRNA encoding the associated interleukin (e.g., IL- 10). This allows the inhibition at the level of the same / single immune cell (i.e., cis-activation). Other examples are the followings: - TGFB and TGFBR,

[0413] TNF and TNFR,

[0414] - IL13 and IL13R,

[0415] - GCF and GCFR, - IL4 and IL4R,

[0416] - CD72 and CD100,

[0417] CD22 and a2-6Sia,

[0418] Siglec-10 and a2-3Sia, and

[0419] LILRB and HLA. Receptors and associated ligand are detailed in Table F hereunder. In some aspects, the mRNa molecule encodes for a protein selected in Table F.

[0420] Table F: Receptors and associated ligands

[0421]

[0422] In an aspect, the lipid-based nanoparticle comprises at least two mRNA molecule(s), wherein one of said at least two mRNA molecules encodes a transmembrane protein that is a receptor and another of said at least two mRNA molecules encodes a secreted protein that is a ligand of said receptor. For example, the lipid-based nanoparticle comprises a mRNA encoding an interleukin receptor (e.g., IL7-R) and a mRNA encoding the associated interleukin (e.g., IL-7). This allows the activation at the level of the same / single immune cell (i.e., cis-activation). Other examples are the followings:

[0423] - BAFFR and BAFF IL6RA and / or IL6RB and IL-6;

[0424] - IL-1R1 and IL-1;

[0425] - IL15RA (CD215) and IL15;

[0426] - IL21R and IL-21 - IL2Ralpha, IL2Rbeta, IL2Rgamma and IL2

[0427] - IL8RA, IL8RB and IL8

[0428] IL9R, IL2gamma and IL9

[0429] - ILlOR and lLlO

[0430] - ILl lR and lLl l - IL12R beta 1, IL12R beta 2 and IL12

[0431] - IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE and IL 17; and

[0432] - IL18R and IL18.

[0433] In some aspects, the lipid-based nanoparticle of the invention comprises a mRNA encoding IL7-R and a mRNA encoding IL-7. Interleukin receptors and ligand are detailed in Table G hereunder. In some aspects, the mRNa molecule encodes for a protein selected in Table G.

[0434] Table G: Receptors and associated ligands

[0435] In some aspects, the mRNa molecule encodes for a protein as described in Tables E, F and G.

[0436] Additionally, a2-3Sia and a2-6Sia refers to sialic acids linked together, either with an a2-3 linkage or an a2-6 linkage, respectively. Linkage of sialic acids residues is known in the art, see for example Fig. 23 and 24 of Cao, H., Chen, X. (2012). General Consideration on Sialic

[0437] Acid Chemistry. In: Chevolot, Y. (eds) Carbohydrate Microarrays. Methods in Molecular Biology, vol 808. Humana Press, https: / / doi.org / 10.1007 / 978-l-61779-373-8_3.

[0438] In some embodiments, the lipid-based nanoparticle comprises at least two mRNA molecule(s), wherein one of said at least two mRNA molecules encodes a costimulatory molecule, preferably selected from the group consisting of CD28, CD80, CD86, ICOS, ICOSL, 0X40, OX40L,

[0439] CD40, CD40L, GITRL, CD 137 and CD137L and another of said at least two mRNA molecules encodes another immune cell enhancing compound, such as an intracellular protein or another transmembrane protein. In some embodiments, the lipid-based nanoparticle comprises one or more mRNA molecule selected from the group consisting of: a mRNA encoding for BCL2, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 43 or having at least 80%, 90%, 95% or 99% sequence identity thereto; a mRNA encoding for IL7, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 44 or having at least 80%, 90%, 95% or 99% sequence identity thereto; a mRNA encoding for IL7R, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 45 or having at least 80%, 90%, 95% or 99% sequence identity thereto; a mRNA encoding for CXCL9, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 46 or having at least 80%, 90%, 95% or 99% sequence identity thereto; a mRNA encoding for CXCL10, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 47 or having at least 80%, 90%, 95% or 99% sequence identity thereto; a mRNA molecule encoding for PUMA, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 48 or having at least 80%, 90%, 95% or 99% sequence identity thereto; a mRNA encoding for BIM-S, preferably a mRNA molecule comprising a nucleic acid sequence such as described in SEQ ID NO: 49 or having at least 80%, 90%, 95% or 99% sequence identity thereto.

[0440] Antigen fragment

[0441] In some aspects, the nucleic acid molecule(s) of the invention, preferably the mRNA molecule(s), encodes for an antigen or an antigen fragment.

[0442] The antigen or fragment thereof is preferably selected among any molecule that is expressed by any viral, bacterial, or parasitic pathogen prior to or during entry into, colonization of, or replication in their host. These pathogens can be infectious in humans, domestic animals or wild animal hosts. In particular, the antigen or fragment thereof encoded by the nucleic acid molecule(s) of the invention promote protective immunity. Preferably, the antigen is a protective antigen or a fragment thereof. As used herein, any viral, bacterial, or parasitic molecule that elicits an immunological response that results in long-term acquired immune resistance in an host is called a "protective antigen".

[0443] In some aspects, the nucleic acid molecule encodes for a viral antigen or a fragment thereof. The viral pathogens, from which the viral antigens are derived, include, but are not limited to: Orthomyxoviruses, such as influenza virus; Retroviruses, such as RSV, HTLV-1, and HTLV- II, Herpesviruses such as EBV; CMV or herpes simplex virus; Lentiviruses, such as HIV-1 and HIV-2; Rhabdoviruses, such as rabies virus; Picornaviruses, such as Poliovirus; Poxviruses, such as vaccinia virus; Rotavirus; and Parvoviruses, such as Adeno- Associated Viruses (AAV); Betacoronaviruses, such as SARS-CoV, SARS-CoV 2 and MERS-CoV.

[0444] Examples of protective antigens of viral pathogens include the Human Immunodeficiency Virus (HIV) antigens Rev, Pol, Nef, Gag, Env, Tat, mutant derivatives of Tat, such as Tat-A31-45, T- and B-cell epitopes of gpl20, chimeric derivatives of HIV-1 Env and gpl20, such as a fusion between gpl20 and CD4, a truncated or modified HIV-1 Env, such as gpl40 or derivatives of HIV-1 Env and / or gpl40. Other examples are the hepatitis B surface antigen, rotavirus antigens, such as VP4 and VP7, influenza virus antigens such as hemagglutinin, neuraminidase, or nucleoprotein, and herpes simplex virus antigens such as thymidine kinase.

[0445] In some aspects, the nucleic acid molecule encodes for a bacterial antigen or a fragment thereof. Examples of bacterial pathogens, from which the bacterial antigens may be derived, include but are not limited to, Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsia spp., Listeria spp., Legionella pneumoniae, Fansicella spp., Pseudomonas spp., Vibrio spp., and Boreilia burgdorferi.

[0446] Examples of protective antigens of bacterial pathogens include the somatic antigens of enterotoxigenic E. coli, such as the CFA / I fimbrial antigen and the nontoxic B-subunit of the heat-labile toxin; pertactin of Bordetella pertussis, adenylate cyclase-hemolysin of B. pertussis, fragment C of tetanus toxin of Clostridium tetani, OspA of Boreilia burgdorferi, protective paracrystalline-surface-layer proteins of Rickettsia prowazekii and Rickettsia typhi, the listeriolysin (also known as "Lio" and "Hly") and / or the superoxide dismutase (also known as "SOD" and "p60") of Listeria monocytogenes, urease of Helicobacter pylori, and the receptorbinding domain of lethal toxin and / or the protective antigen of Bacillus anthrax. In some aspects, the nucleic acid molecule encodes for a parasitic antigen or a fragment thereof. The parasitic pathogens, from which the parasitic antigens are derived, include but are not limited to: Plasmodium spp. such as Plasmodium falciparum, Trypanosome spp. such as Trypanosoma cruzi, Giardia spp. such as Giardia intestinalis, Boophilus spp., Babesia spp. such as Babesia microti, Entamoeba spp. such as Entamoeba histolytica, Eimeria spp. such as Eimeria maxima, Leishmania spp., Schistosome spp., Brugia spp., Fascida spp., Dirofilaria spp., Wuchereria spp., and Onchocerea spp.

[0447] Examples of protective antigens of parasitic pathogens include the circumsporozoite (CS) or Liver Stage Specific (LSA) antigens LSA-1 and LSA-3 of Plasmodium spp. such as those of P. bergerii or P. falciparum, or immunogenic mutants thereof; the merozoite surface antigen of Plasmodium spp., the galactose specific lectin of Entamoeba histolytica, gp63 of Leishmania spp., gp46 of Leishmania major, paramyosin of Brugia malayi, the triose-phosphate isomerase of Schistosoma mansoni, the secreted globin-like protein of Trichostrongylus colubriformis, the glutathione-S-transferase of Frasciola hepatica, Schistosoma bovis and S. japonicum, and KLH of Schistosoma bovis and S. japonicum.

[0448] The antigen or fragment thereof may be encoded by a codon-optimized, synthetic gene and may be constructed using conventional recombinant DNA methods.

[0449] Antigen binding domain

[0450] In some aspects, the nucleic acid molecule(s) of the invention, preferably the mRNA molecule(s), encodes for an antigen binding domain.

[0451] In a preferred aspect, the antigen binding domain is an antibody, a fragment or a derivative thereof such as a F(ab')2, a Fab, a Fab’, a crossMAB or a single-chain variable fragment (scFV) or a VHH.

[0452] Exemplary antigen binding domain include, but are not limited to, antigen binding domain directed against a target selected from the group consisting of PD-1, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, 0X40, 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.

[0453] In some embodiments, the nucleic acid molecule(s) of the invention, preferably the mRNA molecule(s), encodes for an antigen biding domain, preferably an antibody; against a checkpoint inhibitor. In that way, the targeted cell, in particular in case of targeted immune cell, will be able to produce antigen biding domain, preferably an antibody, against checkpoint inhibitor(s). According to the present invention, a heavy chain and light chain of an antibody may be encoded and delivered by a single mRNA molecule or separate mRNA molecules. It is contemplated that it may be advantageous to deliver heavy chain encoding mRNA and light chain encoding mRNA at varying ratios in order to optimize production of fully assembled functional antibodies. Thus, in some aspects, the heavy chain encoding mRNA (also referred to as the first mRNA) and the light chain encoding mRNA (also referred to as the second mRNA) are used at a ratio ranging between approximately 10: 1 to 1 : 10 (e.g., between 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 aspects the heavy chain encoding mRNA (also referred to as the first mRNA) and the light chain encoding mRNA (also referred to as the second mRNA) are used at a ratio of or greater than approximately 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1 or 1 : 1. In some aspects, the heavy chain encoding mRNA (also referred to as the first mRNA) and the light chain encoding mRNA (also referred to as the second mRNA) are used at a ratio of approximately 1 : 1 (i.e., equal molar). In some aspects, the heavy chain encoding mRNA (also referred to as the first mRNA) and the light chain encoding mRNA (also referred to as the second mRNA) are used at a ratio other than 1 : 1 (equal molar). For example, the heavy chain encoding mRNA (also referred to as the first mRNA) and the light chain encoding mRNA (also referred to as the second mRNA) are used at a ratio greater than 1 (e.g., ranging between 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 referred to as the first mRNA) and the light chain encoding mRNA (also referred to as the second mRNA) are used at a ratio less than 1 (e.g., ranging between 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).

[0454] In some aspects, a nucleic acid molecule, preferably an mRNA, encoding a heavy chain and / or light chain incorporates a nucleotide sequence encoding a signal peptide. As used herein, the term "signal peptide" refers to a peptide present at a newly synthesized protein that can target the protein towards the secretory pathway. Typically, the signal peptide is cleaved after translocation into the endoplasmic reticulum following translation of the mRNA. Signal peptide is also referred to as signal sequence, leader sequence or leader peptide. Typically, a signal peptide is a short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids long) peptide. A signal peptide may be present at the N-terminus of a newly synthesized protein, such as a light or heavy chain of an antibody. Without wishing to be bound by any particular theory, the incorporation of a signal peptide encoding sequence on a heavy chain and / or light chain encoding mRNA may facilitate the secretion and / or production of the antibody produced from the mRNA in vivo.

[0455] A suitable signal peptide for the present invention can be a heterogeneous sequence derived from various eukaryotic and prokaryotic proteins, in particular secreted proteins. In some aspects, a suitable signal peptide is a leucine-rich sequence (see Yamamoto Y et al. (1989), Biochemistry, 28:2728-2732, which is incorporated herein by reference). A suitable signal peptide may be derived from a human growth hormone (hGH), serum albumin preproprotein, Ig kappa light chain precursor, Azurocidin preproprotein, cystatin-S precursor, trypsinogen 2 precursor, potassium channel blocker, alpha conotoxin lpl.3, alpha conotoxin, alfa- galactosidase, cellulose, aspartic proteinase nepenthesin- 1, acid chitinase, K28 prepro-toxin, killer toxin zygocin precursor, and Cholera toxin. Exemplary signal peptide sequences are also described in Kober, et al, Biotechnol. Bioeng., 110: 1164-73, 2012, which is incorporated herein by reference.

[0456] In some aspects, a heavy chain and / or light chain encoding mRNA may incorporate a sequence encoding a signal peptide derived from human growth hormone (hGH), or a fragment thereof.

[0457] CAR

[0458] In some aspects, the nucleic acid molecule(s) encodes a chimeric antigen receptor (CAR). In one aspect, the nucleic acid molecule is an mRNA molecule encoding a CAR. In one aspect, the nucleic acid molecule is a modified nucleoside mRNA molecule encoding a CAR.

[0459] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial T cell receptor that is engineered to be expressed on an immune effector cell and specifically bind an antigen. CARs may be used as a therapy with adoptive cell transfer. T cells are removed from a patient and modified so that they express the receptors specific to a particular form of antigen. In some aspects, the CARs have specificity to a selected target, for example a fibroblast cell surface receptor. CARs typically comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising an antigen binding region that specifically binds to a selected target, e.g., cell surface receptor.

[0460] In various aspects, the CARs contemplated herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element otherwise referred to as an antigen binding domain. In some aspects, the extracellular domain also comprises a hinge domain. In certain aspects, the intracellular domain or otherwise the cytoplasmic domain comprises, a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen.

[0461] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term "spacer domain" generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise 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.

[0462] The extracellular domain, transmembrane domain, and intracellular domain can be derived from any desired source.

[0463] CAR antigen binding domain

[0464] The antigen binding domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. In one aspect, the antigen binding domain may consist of an Ig heavy chain which may in turn be covalently associated with Ig light chain by virtue of the presence of CHI and hinge regions, or may become covalently associated with other Ig heavy / light chain complexes by virtue of the presence of hinge, CH2 and CH3 domains. In the latter case, the heavy / light chain complex that becomes joined to the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct. Depending on the function of the antibody, the desired structure and the signal transduction, the entire chain may be used or a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed. In various aspects, the CAR antigen binding domain may be humanized or comprise a fully human sequence. In one aspect, the antigen binding domain is a targeting domain, wherein the targeting domain directs the T cell expressing the CAR to a specific cell or tissue of interest. For example, in one aspect, the targeting domain comprises an antibody, antibody fragment, or peptide that specifically binds to an antigen (e.g., a salef-antigen or a foreign antigen) thereby directing the T cell expressing the CAR to a cell or tissue expressing the antigen.

[0465] The antigen binding domain of the CAR molecule of the invention can be generated to be reactive to any desirable antigen of interest, or fragment thereof, including, but not limited to a tumor antigen or a foreign antigen (e.g, a bacterial antigen, or a viral antigen).

[0466] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response. The selection of the antigen binding domain of the VM-domain containing fusion molecule of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostatecarcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF -II, IGF-I receptor and mesothelin. Another exemplary tumor antigen is chondroitin sulfate proteoglycan 4 (CSPG4) (also referred to as melanoma-associated chondroitin sulfate proteoglycan (MCSP), high- molecular-weight melanoma-associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2)).

[0467] In one aspect, the antigen binding of the CAR specifically binds to a tumor antigen that comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an 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 onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0468] The type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are 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 at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0469] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumorspecific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumorsuppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EB VA and the 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 27.29VBCAA, CA 195, CA 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\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0470] CAR transmembrane domain

[0471] With respect to the transmembrane domain, a CAR of the disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one aspect, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0472] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, for example, but not limited to between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In another aspect, the linker comprises a glycine-serine doublet.

[0473] CAR intracellular domain

[0474] In various aspects, the cytoplasmic domain or otherwise the intracellular domain of a CAR may be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines. The term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0475] Preferred examples of intracellular domains for use in the CARs of the disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

[0476] Primary intracellular signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[0477] Examples of IT AMs containing primary intracellular signaling sequences that are of particular use in the 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 aspect, the intracellular signaling molecule in the CAR of the invention comprises an intracellular signaling sequence derived from CD3 zeta.

[0478] In another aspect, the intracellular domain of the CAR can be designed to comprise the CD3- zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include 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 a ligand that specifically binds with CD83, and the like.

[0479] The intracellular signaling sequences within the intracellular domain of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a suitable linker in some aspects.

[0480] In one aspect, the intracellular domain is designed to comprise the signaling domain of CD3- zeta and the signaling domain of CD28. In yet another aspect, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB. In various aspects, the CAR can be a “first generation,” “second generation,” “third generation,” “fourth generation” or “fifth generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257: 127-133 (2014); Sharpe et al., Dis. Model Meeh. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32: 169-180 (2009)), each of which are incorporated by reference in its entirety).

[0481] “First generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3A-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3Achain signaling domain in a single fusion molecule, independent of HLA- mediated antigen presentation.

[0482] “Second-generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to augment T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388-398 (2013)). CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory molecules, for example, CD28, 4- IBB, ICOS, 0X40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

[0483] “Second generation” CARs provide both co- stimulation, for example, by CD28 or 4- IBB domains, and activation, for example, by a CD3Asignaling domain. Preclinical studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9): 1577-1583 (2012)). “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4- IBB domains, and activation, for example, by comprising a CD3Aactivation domain.

[0484] “Fourth generation” CARs provide co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3Asignaling domain in addition to a constitutive or inducible chemokine component.

[0485] “Fifth generation” CARs provide co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3Asignaling domain, a constitutive or inducible chemokine component, and an intracellular domain of a cytokine receptor, for example, IL-2Rp.

[0486] In some aspects, the one or more mRNA molecule(s) encode(s) for a molecule selected from the group consisting of: a) an immune cell enhancing or inhibiting compound, in particular such as described herein, and preferably selected from: a compound or molecule selected from the group consisting of TCF 1 , LEF 1 , WNT, FRIZZLED, Beta catenin, BCL2, BCLXL, BIRC3, MCL1, PGCla, TCF7, NF AT, NFKB, RORgt, TBET, EOMES, RUNX3, GATA3, JUNB, POU2AF1, OCTI, BLIMP-1, XBP-1, FOXO1, PTGS2, CSE, Glutl, Glut3, HK2, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS, OXPHOS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD 107a, Lymphotoxin (LT) aip2, granzyme B, perforin, POU2F1, BBS 10, BBS 12, TCP1, HSP, Integrin alpha 1, Integrin alpha 2, Integrin alpha 2b, Integrin alpha 11, Integrin alpha 3, Integrin alpha 6, Integrin alpha7, 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, DEFI, 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 Regulatory Factors such as IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, NFKB, T-bet, AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK-TAB1, MKK3, MKK4, MKK6, MKK7, IKKa, IKKp, TRAM, TRIF, TBK1, D3 -phosphoinositides, 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- IBB, 4-1BBL, 0X40, 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, CD160, and any combination thereof, preferably 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 alpha7, 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, preferably 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 alpha7, 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 such as described herein; a compound or molecule selected from the group consisting of 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, IKAROS, EGR2 / 3, CREM, P27 (KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS 10, BBS 12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEFI, 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, galectin9, 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, 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 such as described herein; b) a cytokine, 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 such as described herein; c) a cytokine receptor, 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, IL-35R; d) a chemokine, preferably selected from the group consisting of CXCL9 or CXCL10; e) a chemokine receptor, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, XCR1; f) an antigen fragment derived from viral pathogens, bacterial pathogens and / or parasitic pathogens, in particular such as described herein; g) an antibody or a fragment or a derivative thereof, preferably directed against a target selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, LAG3, BTLA, CD160, CD40L, ICOS, CD27, 0X40, 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 such as described herein and h) a Chimeric Antigen Receptor (CAR), in particular such as described herein.

[0487] In some aspects, the one or more mRNA molecule(s) encode(s) for a molecule selected from the group consisting of: a) an immune cell molecule selected from the group consisting of:

[0488] - BCL2, BCLXL, CD28, 4- IBB, 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 alpha7, 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 such as described herein; or

[0489] - 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 such as described herein.

[0490] Antigen binding domain

[0491] The methods of the invention finally involve one or more antigen binding domain(s).

[0492] In the methods of the invention, an antigen binding domain is mixed with the first or second composition before the first and second compositions are mixed together. In some aspects, the antigen binding domain is mixed with the first or second composition before the feeding of the first and second compositions into the mixing device. It means that in certain aspects, the antigen binding domain is mixed with the first or second composition outside of the mixing device.

[0493] Then, in an aspect, the method of the invention comprises the steps of:

[0494] - mixing a composition comprising one or more antigen biding domain(s) with a first composition, said first composition comprising a lipid-based composition and a polar organic solvent,

[0495] - feeding said mixed composition through a first entry of a mixing device,

[0496] - feeding through a second entry of said mixing device a second composition, wherein said second composition is an acidic aqueous composition comprising one or several nucleic acid molecule(s),- mixing the compositions from the first and second entry in the mixing device so as to generate lipid-based nanoparticles,

[0497] - removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH; and b) recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s).

[0498] Alternatively, the method of the invention comprises the steps of:

[0499] - mixing a composition comprising one or more antigen biding domain(s) with the second composition, wherein said second composition is an acidic aqueous composition comprising one or several nucleic acid molecule(s),

[0500] - feeding said mixed composition through a second entry of a mixing device, - feeding through a first entry of a mixing device the first composition, said first composition comprising a lipid-based composition and a polar organic solvent,

[0501] - mixing the compositions from the first and second entry in the mixing device so as to generate lipid-based nanoparticles,

[0502] - removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH; and c) recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s).

[0503] In an aspect, the composition comprising the antigen binding domain comprises an appropriate buffer, typically PBS.

[0504] Preferably, the antigen binding domain is not covalently bound to any of the lipids of the LNP or does not comprise any modification for coupling or grafting the antigen binding domain to a lipid. In particular, the antigen binding domain does not comprise a lipophilic moiety or a grafting moiety such as free cysteine(s) or a thiol group.

[0505] In an aspect, the antigen binding domain is mixed with the first composition and the concentration of antigen binding domain in the first composition is from about 0.01 pg / pL to about 0.5 pg / pL, from about 0.02 pg / pL to about 0.45 pg / pL, from about 0.03 pg / pL to about 0.4 pg / pL, from about 0.04 pg / pL to about 0.35 pg / pL, from about 0.05 pg / pL to about 0.3 pg / pL, from about 0.1 pg / pL to about 0.2 pg / pL, from about 0.125 pg / pL to about 0.175 pg / pL. Particularly, when 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 from about 0.01 pg / pL to about 0.5 pg / pL.

[0506] In an alternative aspect, the antigen binding domain is mixed with the second composition and the concentration of antigen binding domain in the second composition is from about 0.005 pg / pL to about 0.5 pg / pL, from about 0.01 pg / pL to about 0.45 pg / pL, from about 0.02 pg / pL to about 0.4 pg / pL, from about 0.03 pg / pL to about 0.35 pg / pL, from about 0.04 pg / pL to about 0.3 pg / pL, from about 0.05 pg / pL to about 0.25 pg / pL, from about 0.1 pg / pL to about 0.2 pg / pL, from about 0.125 pg / pL to about 0.175 pg / pL. Particularly, when 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 from about 0.005 pg / pL to about 0.25 pg / pL.

[0507] The antigen binding domain used for mixing with the first or second composition is capable to specifically bind to a target. Not wishing to be bound by theory, the inventors believe that the I l l presence of the antigen binding domain will help to address or target the lipid-based nanoparticle obtained by the methods of the invention in a particular localization, tissue or cell, for the specific delivery of the nucleic acid molecule(s) comprised in the lipid nanoparticle.

[0508] The terms "specific binding", "specifically binds to," "specific for" or "selectively binds" a particular target or an epitope on a particular antigen mean that the antigen binding domain recognizes and binds a specific antigen or epitope, but does not substantially recognize or bind other molecules in a sample. For example, an antigen binding domain that specifically (or preferentially) binds to an antigen is an antigen binding domain that binds said antigen for example with greater affinity, avidity, more readily, and / or with greater duration than it binds to other / different antigens. Preferably, the term "specifically binds to" or "binds specifically" refers to the ability of an antigen receptor to bind to an antigen with an affinity of at least about 1 x IO’6M, 1 x IO’7M,1 x IO’8M, 1 x IO’9M, 1 x IO’10M, 1 x 10’11M, 1 x IO’12M, or more, and / or bind to a target with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen. The affinity can be determined by various methods well known from one skilled in the art. These methods include, but are not limited to, Biacore Analysis, Blitz analysis and Scatchard plot.

[0509] In an aspect, the antigen-binding domain to be comprised in the lipid-based nanoparticle according to the invention has a KD value inferior or equal to 10'8M, preferably inferior or equal to 10'9M for the target expressed on immune cells, more preferably inferior or equal to 1.1 O’10M, as may be determined by biosensor analysis, particularly by Biacore Analysis.

[0510] As used herein, the term “target” of the antigen binding domain refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or bound by the antigen binding domain according to the invention and expressed on the external surface of immune cells. With regards to the expression of a target on the surface of immune cells, the term “expressed” refers to a target, such as carbohydrates, lipids, peptides, polypeptides, proteins, antigens or epitopes that are present or presented at the outer surface of an immune cell.

[0511] In an aspect, the antigen-binding domain to be comprised in the lipid-based nanoparticle according to the invention has a binding activity which is similar to the same antigen-binding domain in a free form. As used herein, a “free form antigen binding domain” refers to an antigen-binding domain which is not linked, grafted or conjugated to an LNP. In a preferred embodiment, the antigen-binding domain to be comprised in the lipid-based nanoparticle according to the invention has a binding activity equal to about 70% of the binding activity of the free form antigen-binding domain, even preferably to about 75%, even more preferably to about 80%.

[0512] The antigen binding domain to be incorporated in the lipid-based nanoparticle by the methods of the invention is particularly capable of specific binding to a target that is expressed on an immune cell, a tumor cell, an infected cell or on pathogens of the human body.

[0513] In some aspects, the antigen binding domain is capable of specific binding to a target that is expressed on a tumor cell. Preferably, the antigen binding domain is capable of specific binding to Tumor Associated Antigens (TAAs), such as carcinoembryonic antigen (CEA), p53, human epidermal receptor-2 / neurological (HER-2 / neu), melanoma antigen 2 and 3 (MAGE-2 / 3), Human papillomavirus protein 6 (HPVE6), alphafetoprotein (AFP) and prostate specific antigen (PSA).

[0514] Tumor antigens targeted by the antigen binding domain of the present invention include, but are not limited to, any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE 1 (e.g., GenBank Accession No. M77481), MAGE 2 (e.g., GenBank Accession No. U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); and p97 melanoma antigen (e.g., GenBank Accession No. M12154). Other tumor-specific antigens include the Ras peptide and p53 peptide associated with advanced cancers, the HPV 16 / 18 and E6 / E7 antigens associated with cervical cancers, MUC1-KLH antigen associated with breast carcinoma (e.g., GenBank Accession No. J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession No. X98311), gplOO (e.g., GenBank Accession No. S73003) or MARTI antigens associated with melanoma, and the PSA antigen associated with prostate cancer (e.g., GenBank Accession No. X14810). The p53 gene sequence is known (See e.g., Harris et al. (1986) Mol. Cell. Biol., 6:4650-4656) and is deposited with GenBank under Accession No. M14694. Tumor antigens encompassed by the present invention further include, but are not limited to, Her- 2 / Neu (e.g. GenBank Accession Nos. Ml 6789.1, Ml 6790.1, M16791.1, Ml 6792.1), NY-ESO- 1 (e.g. GenBank Accession No. U87459), hTERT (aka telomerase) (GenBank Accession. Nos. NM003219 (variant 1), NM198255 (variant 2), NM 198253 (variant 3), and NM 198254 (variant 4), proteinase 3 (e.g. GenBank Accession Nos. M29142, M75154, M96839, X55668, NM 00277, M96628 and X56606) HPV E6 and E7 (e.g. GenBank Accession No. NC 001526) and WT-1 (e.g. GenBank Accession Nos. NM000378 (variant A), NM024424 (variant B), NM 024425 (variant C), and NM024426 (variant D)). Thus, the present invention can be used as immunotherapeutics for cancers including, but not limited to, cervical, breast, colorectal, prostate, lung cancers, and for melanomas.

[0515] Examples of antibodies targeting such TAAs are for example Rituximab and Trastuzumab.

[0516] In some aspects, the antigen binding domain is capable of specific binding to a target that is expressed on a pathogen, such as a bacterium, virus or fungus. The present invention further includes, antigen binding domains that target antigens from the following infectious diseases; measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, and HIV (e.g., GenBank Accession No. U18552). Bacterial and parasitic antigens will be derived from known causative agents responsible for diseases including, but not limited to, diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonias (e.g., Haemophilus influenzae, Pneumocystis carinii, etc.), cholera, typhoid, plague, shigellosis, salmonellosis (e.g., GenBank Accession No. L03833), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), malaria (e.g., GenBank Accession No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis, leshmaniasis, giardiasis (e.g., GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis.

[0517] Examples of antibodies targeting such pathogens are for example Duvratoxumab, Palivizumab, Raxibacumab, Bezlotoxumab and Ansuvimab

[0518] The antigen binding domain to be incorporated in the lipid-based nanoparticle by the methods of the invention is particularly capable of specific binding to a target that is expressed on an immune cell (i.e., at the outer surface of an immune cell), preferably, to a target that is expressed on an activated immune cell. Particularly, the target is expressed by immune cells in a healthy subject or in a subject suffering from a disease, preferably in a subject suffering from a disease such as a cancer, an infectious disease an autoimmune disease or an inflammatory disease. 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 by the total immune cells is higher than the ratio of other cells expressing the target by the total other cells. Preferably the expression level or ratio is higher by a factor 2, 5, 10, 20, 50 or 100. “Immune cell” as used herein includes neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells). It preferably refers to T cells, more specifically CD4+ T cells, CD8+ T cells, effector T cells and / or exhausted T cells. By “activated immune cells” it is meant immune cells that are involve have been activated during an immune response towards the presence of non-self cells such as pathogens or cancer cells. Activated immune cells are particularly recruited in the localization wherein the inflammation, triggered by the presence of non-self cells, occurs. Particular markers of immune cells of activation that can be targeted by the antigen binding domain are particularly described here after.

[0519] “T cell” or “T lymphocytes” as used herein includes CD4 + T cells, CD8 + T cells, T helper 1 type T cells, T helper 2 type T cells, T helper 17 type T cells, effector T cells, effector memory stem like T cells, Tumor Infiltrating Lymphocyte (TIL), anergic T cells, inhibitory T cells and / or senescent T cells and exhausted T cells . In a very particular aspect, the T cell is an effector T cell, an exhausted T cell, a Tumor Infiltrating Lymphocyte (TIL) or an effector memory stem like T cell. “Activated T cell” or “Activated T lymphocytes” are T cells activated by simultaneously receiving signal-1 from T-cell recognition of antigen via the T cell receptor and signal-2 from costimulatory molecule. Markers expressed by activated T cells include but are not limited to CD137 / 41BB / TNFRSF9, PD-1, CRTAM, CTLA4, FasL / TNFSF6, TIM- 3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4, SIRPg and TIGIT .

[0520] Preferably, the term “T cell” does not include regulatory T cells (Treg), inhibitory T cells and / or senescent T cells.

[0521] Preferably, the immune cell is an activated T cell.

[0522] The target expressed on immune cells can particularly be selected among the target described in Table G below.

[0523] Table G: Examples of targets of interest.

[0524] In an embodiment, the target of the antigen binding domain is selected from the group consisting of PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4- 1BB / TNFRSF9, CD 150, CD 153, CD 154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF 1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L,

[0525] TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC- 1A, CD21, CLEC-9A, CD 180, CD59, CD54, CD71, CD35, CD218a, CD74, CD 165, 4- 1BBL / CD137L, ICOSL, CD160, CD127 and SIRPa.

[0526] The target of the antigen binding domain expressed on cells surface is preferably selected from the group consisting of PD-1, CTLA-4, GITR, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, 0X40, 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; preferably of PD-1, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, 0X40, 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. Particularly, the target expressed of the antigen binding domain is selected from the group consisting of PD-1, CD127, SIRPa and CLEC-1A.

[0527] Preferably, the target expressed of the antigen binding domain is selected from the group consisting of PD-1, CTLA4, 4-1BB, ICOS, LAG3, TIM3, TIGIT, BTLA or CLEC-1, preferably to PD-1;

[0528] Preferably, the target of the antigen binding domain specifically expressed on immune cells surface is selected from the group consisting of PD-1, BTLA, TIGIT, CD 160, LAG3 and TIM3. In a preferred aspect, the target is PD-1. These targets are, in particular, not antigens of the TCR pathway (interaction between antigen presenting cells and T cells).

[0529] Preferably, the antigen binding domain to be mixed with the first or second composition is an anti -PD-1 antigen binding domain so that the lipid-based nanoparticle to be obtained by the method of the invention aims to target immune cells that are present in a tumoral environment (e.g., that are recruited on tumoral site), for example PD-1 positive immune cells.

[0530] In a particular aspect, the T cell is a Tumor Infiltrating lymphocytes (TILs) and the target is a factor expressed on the surface of Tumor Infiltrating lymphocytes, preferably specifically expressed on the surface of TILs. Preferably, the target expressed on the surface of TILS is selected from the group consisting of CD101, CD137 (Tnfrsf9 / 4-lBBL), CRTAM, CST7, CTLA4, CXCR3, FAS, IL18R1 / CXCR1 / CD218A, LAG-3 PTPN22, RGS1, TNFSF14 and PD1.

[0531] In an aspect, the antigen binding domain has an antagonist activity on the target when the target is a receptor having an inhibitory effect on the cell, in particular for checkpoint inhibitors between tumor cells and T cells (for example PD-1).

[0532] The term “antagonist” as used herein, refers to a substance that block or reduces the activity or functionality of another substance. Particularly, this term refers to a binding domain that binds to a cellular receptor (e.g., PD-1) as a reference substance (e.g., PD-L1 and / or PD-L2), preventing it from producing all or part of its usual biological effects (e.g., the creation of an immune suppressive microenvironment). The antagonist activity may be assessed by competitive ELISA.

[0533] In an alternative aspect, the antigen binding domain has an agonist activity on the target when the target is a receptor having an activator effect on the cell (e.g CD137).The term “agonist” as used herein, refers to a substance that activates or increases the activity or functionality of another substance. Particularly, this term refers to a binding domain that binds to a cellular receptor as a reference substance, causing it to produce all or part of its usual biological effects (e.g., the creation of an immune suppressive microenvironment).

[0534] In an aspect, the antigen binding domain does not interfere nor compete with the binding between its target and its natural ligand. Particularly, the antigen binding domain has no activity on the target, in particular for checkpoint inhibitors between tumor cells and T cells (for example PD-1).

[0535] The antigen binding domain to be used in the methods of the invention can be of any format known in the art.

[0536] Particularly, the antigen binding domain is an antibody, a fragment or a derivative thereof such as a Fab, a F(ab)2, a Fab’, a F(ab')2, a Fd, a Fv, a crossMAb or a single-chain variable fragment (scFV) a VHH or a single-chain Fab fragment.

[0537] Examples of antigen binding fragments encompassed typically include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 : 544-546), which consists of a VH domain, or any fusion proteins comprising such antigenbinding fragments. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0538] As used herein, the term “CrossMAb” refers to antigen binding domains with an inversion of CL and CHI domains, in particular in one binding arm of antibodies. Thus, such binding domain comprises a VH domain linked to a CL domain and a VL domain linked to a CHI domain. Such format reduces the byproduct formation caused by a mismatch of a light chain of a first binding domain that specifically binds to a first antigen with the wrong heavy chain of a second biding domain that specifically binds to a second antigen (when compared to approaches without such CL-CH1 domain exchanges). CrossMAb are for example described in WO 2009 / 080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191, the disclosure of which being incorporated herein by reference.

[0539] In some instances, the antigen binding domain is an antibody or comprises or consists of a Fab, a Fv, a Fab’, a scFV, a CrossMab or a VHH covalently linked to a Fc domain, preferably an IgG Fc domain such as described herein.

[0540] In one another aspect, the antigen binding domain according to the present disclosure is an antigen-binding antibody mimetic. As used herein the term “antigen-binding antibody mimetic” refers to artificial proteins, peptides and any chemical compounds with the capacity to bind antigens mimicking that of antibodies. Such mimetics comprise affitins and anticalins as well as aptamers (peptide aptamers and oligonucleotide aptamers).

[0541] In one aspect, the antigen binding domain is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

[0542] Alternatively, the antigen binding domain according to the present disclosure is a targeting peptide. Preferably, the peptide is not covalently linked to a lipid or to a lipidation motif. For example, the peptide is the RGD peptide such as described in Qin J, Xue L, Gong N, Zhang H, Shepherd SJ, Haley RM, Swingle KL, Mitchell MJ. RGD peptide-based lipids for targeted mRNA delivery and gene editing applications. RSC Adv. 2022 Sep 7;12(39):25397-25404. doi: 10.1039 / d2ra02771b. PMID: 36199352; PMCID: PMC9450108.

[0543] In some instances, the antigen binding domain is an aptamer or a short peptide sequence such as RGD peptide.

[0544] Aptamers are short ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) sequences generated in vitro to bind with high affinity and specificity to a given target. In a preferred aspect, the antigen binding domain of the lipid-based nanoparticle does not compete with the natural ligand for the binding to the target specifically expressed on activated immune cells. The absence of competition between the antigen binding domain of the invention and the natural ligand of the target specifically expressed on the activated immune cells may be determined when, in presence of the antigen binding domain of the invention, the binding of the natural ligand to the target specifically expressed on the activated immune cells is at least 50%, more preferably at least 80%, still more preferably at least 90% and most preferably similar, to the binding of the natural ligand to the target specifically expressed on the activated immune cells, under the same experimental conditions but without the presence of the antigen binding domain of the invention.

[0545] Preferably, the activated immune cells are selected from the group consisting of activated T cells, activated B cells, activated myeloid cells including activated macrophages and activated dendritic cells.

[0546] In a preferred aspect, the antigen binding domain of the lipid-based nanoparticle does not compete with an antigen binding domain which binds to the same target.

[0547] In an aspect, the antigen binding domain is an antibody or is derived from an antibody. Preferably, the antigen binding domain is preferably derived from an IgA, IgM, IgE, IgD and IgG, preferably an IgG.

[0548] The terms “derive from” and “derived from” as used herein refers to a compound or molecule having a structure derived from the structure of a parent compound, molecule or protein and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar properties, activities and utilities as the claimed compounds.

[0549] In some aspects, the antigen binding domain is a monoclonal antibody or an antigen binding fragment thereof. The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single specificity. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant for the binding specificity. In an aspect, the antibodies of the disclosure are monoclonal antibodies. In some aspects, the antigen binding domain is a recombinant antibody or an antigen binding fragment thereof. As used herein, the term "recombinant antibody" refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0550] In some aspects, the antigen binding domain is a chimeric antibody or an antigen binding fragment thereof. As used herein, a “chimeric antibody” refers to an antibody in which the sequence of the variable domain derived from the germline of a mammalian species, such as a mouse, have been grafted onto the sequence of the constant domain derived from the germline of another mammalian species, such as a human. Chimeric antibodies generally comprise constant domains from human and variable domains from another mammalian species, reducing the risk of a reaction to foreign antibodies from a non-human animal when they are used in therapeutic treatments.

[0551] In some aspects, the antigen binding domain is a humanized antibody or an antigen binding fragment thereof. As used herein “humanized antibody” refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences”. A "humanized form" of an antibody, e.g., a non- human antibody, also refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from at least one CDR of a non-human antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. Additional framework region modifications may be made within the human framework sequences. Preferably humanized antibody has a T20 humanness score greater than 80%, 85% or 90%. “Humanness” of an antibody can for example be measured using the T20 score analyzer to quantify the humanness of the variable region of antibodies as described in Gao S H, Huang K, Tu H, Adler A S. BMC Biotechnology. 2013: 13:55 or via a web-based tool to calculate the T20 score of antibody sequences using the T20 Cutoff Human Databases: http: / / abAnalyzer.lakepharma.com. In a particular aspect, the antigen binding domain is not covalently bound to any of the lipids of the LNP or does not comprise any modification for coupling or grafting the antigen binding domain to a lipid. In particular, the antigen binding domain does not comprise a lipophilic moiety or a grafting moiety such as free cysteine(s) or a thiol group.

[0552] In a particular aspect, the LNP does not include any antigen binding domain specific for an antigen present on the antigen binding domain of the LNP, in particular an antigen binding domain directed against the Fc domain of an antibody.

[0553] In a particular aspect, the antigen binding domain of the LNP is not bound to the LNP by a type of interaction “antigen-antibody”. More specifically, the antigen binding domain is not bound by an antigen binding domain specific for an antigen present on the antigen binding domain of the LNP, in particular an antigen binding domain directed against the Fc domain of an antibody.

[0554] Preferably, the lipid-based nanoparticle does not comprise a secondary antibody that allows the attachment of the antigen binding domain to the lipid-based nanoparticle (e.g., such as an anti- Fc antibody or antigen binding fragment or derivative thereof).

[0555] Particularly, the lipid-based nanoparticle does not comprise an antibody or any fragment or derivative thereof that is lipidated or that is covalently bound to a peptide or motif that is lipidated. Preferably, the lipid-based nanoparticle does not comprise a lipidated secondary antibody that allows the attachment of the antigen binding domain to the lipid-based nanoparticle.

[0556] In some aspects, the lipid-based nanoparticle does not comprise a moiety comprising a lipidation peptide or motif.

[0557] In some aspects, the antigen binding domain does not comprise or is not covalently bound to an anchoring moiety comprising a lipidation peptide or motif.

[0558] As used herein, the term “anchoring moiety” refers to a component that anchors or attaches the antigen binding domain into the lipid-based nanoparticle.

[0559] As used herein, the terms “lipidation peptide or motif’ refers to a specific sequence pattern in proteins or proteic entity (such as antibodies or fragment thereof) that is associated with the attachment or anchoring of lipid moi eties.

[0560] In the context of the invention, an entity that comprises a lipidation peptide, motif or pattern is an entity (e.g., an antigen binding domain, scFv or antibody) that will be anchored or attachment to lipid molecules, in particular to lipids of the lipid-based nanoparticle. Lipidation peptide or motif can involve different types of lipid modifications, including: cysteine prenylation (e.g., the attachment of hydrophobic isoprene polymers such as farnesyl or geranylgeranyl to cysteine residues of proteins), N-terminal Glycine Myristoylation, Cysteine Palmitoylation, Serine and Lysine Fatty Acylation (e.g., the addition of fatty acyl groups to serine and lysine residues of proteins), Palmitoylation, GPI-Anchor Addition, or peptides that derive from part of an inner membrane bacterial lipoprotein. One common example of a lipidation motif is the CAAX box, which serves as a recognition motif for isoprenylation.

[0561] In some aspects, the lipid based nanoparticle does not comprise a bacterial anchor polypeptide, a lipoprotein, such as a bacterial lipoprotein, or a recombinant membrane-anchored lipoprotein. Preferably, the antigen binding domain does not comprise or is not covalently bound to a bacterial anchor polypeptide, a lipoprotein, such as a bacterial lipoprotein, or a recombinant membrane-anchored lipoprotein.

[0562] Preferably, the lipid based nanoparticle of the invention does not comprise a NipA lipoprotein or any fragment thereof. Preferably, the antigen binding domain does not comprise or is not covalently bound to a NipA lipoprotein or any fragment thereof.

[0563] Preferably, the lipid based nanoparticle or antigen binding domain does not comprise a moiety comprising a lipidation peptide or motif that comprises or consists of the amino acid sequence : CDNSSS (SEQ ID NO : 41) or CDQSSS (SEQ ID NO: 42).

[0564] In some preferred aspects, the antigen binding domain to be mixed with the first or second compositions in the method of the invention is or derives from antibodies known in the art. Numerous antibodies directed against PD-1, TIM3, CTLA-4, LAG-3, BTLA and TIGIT, have already been described.

[0565] In some aspect, 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

[0566] 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 "hPDl" are used interchangeably and refer to the Programmed Death- 1 receptor, also known as CD279, and include variants and isoforms of human PD-1, and analogs having at least one common epitope with PD-1. PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. As an example, the amino acid sequence of a human PD-1 is disclosed under GenBank accession number NP 005009. PD1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC). Accordingly, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD-1, such as PD-lAex2, PD-lAex3, PD-lAex2,3 and PD-lAex2,3,4. Unless specified otherwise, the terms include any variant and, isoform of human PD-1 that are naturally expressed by PBMC, or that are expressed by cells transfected with a PD-1 gene.

[0567] Several anti-PD-1 are already clinically approved, and others are still in clinical developments. For instance, the anti-PD-1 antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (OPDIVO, MDX-1106, BMS-936558, ONO-4538), OSE279 (see WO2020 / 127366), Pidilizumab (CT- 011), Cemiplimab (Libtayo), Camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (Tisleizumab), PDR001 (spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ- 63723283, genolimzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), JS001 (see Si-Yang Liu et al., J. Hematol. Oncol.10: 136 (2017)), BL754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR- 042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI- 1110 (see WO 2014 / 194302), AGEN2034 (see WO 2017 / 040790), MGA012 (see WO 2017 / 19846), or IBI308 (see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, the disclosure thereof being incorporated herein by reference), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006 / 121168, the disclosure thereof being incorporated herein by reference.

[0568] Antibodies targeting TIM3 are also known such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367,. In some aspects, the TFM-3 antibody is as disclosed in International Patent Application Publication Nos. W02013006490, W02016 / 161270, WO 2018 / 085469, or WO 2018 / 129553, WO 2011 / 155607, U.S. 8,552,156, EP 2581113 and U.S 2014 / 044728, the disclosure thereof being incorporated herein by reference.

[0569] Antibodies targeting CTLA-4 are also known such as ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), MEDI5752 (AstraZeneca). Anti-CTLA-4 antibodies are also disclosed in WO18025178, WO19179388, WO19179391, WO19174603, WO19148444, WO19120232, WO19056281, WO19023482, W018209701, WO18165895, WO18160536, WO18156250, WO18106862, WO18106864, WO18068182, W018035710, WO18025178, WO17194265, WO17106372, W017084078, WO17087588, WO16196237, WO16130898, WO16015675, WO12120125, W009100140 and W007008463, the disclosure thereof being incorporated herein by reference.

[0570] Antibodies targeting LAG-3 are also known such as BMS- 986016, IMP701 or MGD012. Anti- LAG-3 antibodies are also disclosed in W02008132601, EP2320940, WO19152574, the disclosure thereof being incorporated herein by reference.

[0571] Antibodies directed against BTLA are also known in the art such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mabl9A7, or hu Mab4C7. The antibody TAB004 against BTLA are currently under clinical trial in subjects with advanced malignancies. Anti -BTLA antibodies are also disclosed in W008076560, W010106051 (e.g., BTLA8.2), WO11014438 (e.g., 4C7), W017096017 and WO17144668 (e.g., 629.3), the disclosure thereof being incorporated herein by reference.

[0572] Antibodies directed against TIGIT are also known in the art, such as BMS-986207 or AB 154, 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, CHAN.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8,

[0573] CHAN.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5, CHA.9.560.6, CHA.9.560.7,

[0574] CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

[0575] CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1,

[0576] CHAN.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8 as disclosed in WO19232484. Anti-TIGIT antibodies are also disclosed in WO16028656, W016106302, WO16191643, W017030823, W017037707, WO17053748, WO17152088, WO18033798, WO18102536, WO18102746, W018160704, W018200430, WO18204363,

[0577] W019023504, WO19062832, WO19129221, WO19129261, WO19137548, WO19152574, WO19154415, WO19168382 and WO 19215728, the disclosure thereof being incorporated herein by reference.

[0578] In some aspects, 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 an antagonist of CD127.

[0579] As used herein, the term "IL-7R" refers to any form of IL-7R and variants thereof that retain at least part of the activity of IL-7R. One exemplary human IL-7R is found as Uniprot Accession Number P16871. Antagonist IL-7R antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) IL-7R biological activity, including downstream pathways mediated by IL-7R signaling, such interaction with IL-7 and / or elicitation of a cellular response to IL-7.

[0580] Antibodies directed against CD127 or IL7-R are also known in the art, such as GSK2618960, RN168, AbD11590, MAB306-100, R34.34, A019D5, eBioRDR5, 40131, 1A12, M21, 47H4, HIL-7R-M21, eBioYL8, RDR5. Anti-CD127 antibodies are also disclosed in W014102430, W020077190, W004000238, WO11104687, WO 16059512 and WO 17062748, the disclosure thereof being incorporated herein by reference.

[0581] In some aspects, 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.

[0582] As used herein, the terms “Signal-regulatory protein alpha”, "SIRPa" and "SIRPa" refers to a receptor-type transmembrane glycoprotein that is mammalian Immunoglobulin-like cell surface receptor for CD47. The term "anti-SIRP" refer to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and specifically binds to SIRPa, in particular to a human SIRPa, to one or both of two common variants identified, SIRPaVl and SIRPaV2. For example, the human SIRPa amino acid sequence is about 504 amino acids and has a Genbank accession number of NP_001035111.1, NP_001035112.1, NP_001317657.1, or NP_542970.1.

[0583] Antibodies directed against SIRPa are also known in the art, such as CC-95251, BI 765063, HPA054437, Magrolimab, TTI-621, TTL622 and Evorpacept (ALX148). Anti-SIRPa antibodies are also disclosed in WO17178653, W019073080, WO22254379, W020102422, WO23202672, WO21222746, W018008470, W016205042, WO22121980, WO22110922, WO19226973, WO22254379, WO23020459 and W018107058 the disclosure thereof being incorporated herein by reference.

[0584] In some aspects, the antigen binding domain comprises or consists of an anti-SIRPa antibody such as disclosed in W019073080. Particularly, the antigen binding domain is an anti-SIRPa domain, comprising or consisting of a VH domain comprising or consisting of a sequence as set forth in SEQ ID NO: 39 and a VL domain comprising or consisting of a sequence as set forth in SEQ ID NO: 40. Preferably, said antigen binding domain further comprises or is covalently linked to a Fc domain, preferably an IgG Fc domain such as described herein. In some aspects, the antigen binding domain is an anti-SIRPa antibody, comprising or consisting of : an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 56 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 57.

[0585] In some aspects, 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.

[0586] As used herein, the term "CLEC-1 A" relates to a C- type lectin-like receptor- 1 A from a mammal species, preferably a human CLEC-1 A. A reference sequence of the human CLEC-1 A corresponds to the sequence associated to the Accession number Q8NC01 Uniprot. As used herein, the term "CLEC-1 antagonist" has its general meaning in the art and refers to any compound, such as an antibody or a fragment thereof, that blocks, suppresses, or reduces the biological activity of CLEC-1. In particular, the CLEC-1 antagonist inhibits the interactions between the CLEC-1 and at least one of its ligands.

[0587] Antibodies directed against CLEC-1A are also known in the art, such as MAB1704, ABIN526589, AF1704 and ABIN526590.

[0588] In some aspects, the antigen binding domain is specific to PD-1. Preferably, the antigen binding domain is an antagonist of PD-1. Even more preferably, the anti-PDl 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 (such as described in WO2020 / 127366, the disclosure thereof being incorporated herein by reference).

[0589] Accordingly, in some aspect, the invention concerns a method to obtain a t-LNP, comprising an anti -PD-1 antigen-binding domain such as disclosed herein, capable of specifically binding to PD-1 expressed on immune cells surface such as activated T cells, and one or several mRNA molecule(s) encoding an activity-enhancing protein of said immune cells. Accordingly, the invention also concerns a t-LNP obtainable by the method of the invention, comprising an anti- PD-1 antigen-binding domain such as disclosed herein, capable of specifically binding to PD- 1 expressed on immune cells such as activated T cells surface and one or several mRNA molecule(s) encoding an activity-enhancing protein of said activated immune cells.

[0590] Preferably, the antigen binding domain comprised in the lipid-based nanoparticle according to the invention is an anti -PD-1 antibody such as described above an or antigen binding fragment thereof, preferably a human, humanized or chimeric anti -PD-1 antibody or antigen binding fragment thereof. Particularly, the antigen binding domain is a F(ab')2, a Fab, a crossMaB or a scFv that is specific to PD-1.

[0591] In a very specific aspect of the present disclosure, the antigen binding domain targets PD-1 and is derived from the antibody disclosed in WO2020 / 127366, the disclosure thereof being incorporated herein by reference in its entirety.

[0592] Then, in an aspect, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0593] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0594] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

[0595] - the heavy chain CDR1 (HCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 1, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but position 3 of SEQ ID NO: 1;

[0596] - the heavy chain CDR2 (HCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 2, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 13, 14 and 16 of SEQ ID NO: 2;

[0597] - the heavy chain CDR3 (HCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 3; optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 2, 3, 7 and 8 of SEQ ID NO: 3;

[0598] - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO: 4, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular at any position but positions 5, 6, 10, 11 and 16 of SEQ ID NO: 4;

[0599] - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO: 5, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and

[0600] - the light chain CDR3 (LCDR3) comprises or consists of an amino acid sequence of SEQ ID NO: 6, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof at any position but positions 1, 4 and 6 of SEQ ID NO: 6.

[0601] In another aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of: (i) a heavy chain variable region (VH) comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3; and (ii) a light chain variable region (VL) comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5 and a CDR3 of SEQ ID NO: 6.

[0602] In one aspect, the anti-PDl antibody or antigen binding fragment according to the invention comprises 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.

[0603] Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0604] (i) a heavy chain variable region (VH) comprising a HFR1 of SEQ ID NO : 7, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR1 of SEQ ID NO: 1, a HFR2 of SEQ ID NO : 8, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR2 of SEQ ID NO: 2, a HFR3 of SEQ ID NO : 9, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a HCDR3 of SEQ ID NO: 3; and a HFR4 of SEQ ID NO : 10, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, and

[0605] (ii) a light chain variable region (VL) comprising a LFR1 of SEQ ID NO : 11, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR1 of SEQ ID NO: 4, a LFR2 of SEQ ID NO : 12, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR2 of SEQ ID NO: 5, a LFR3 of SEQ ID NO : 13, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, a LCDR3 of SEQ ID NO: 6 and a LFR4 of SEQ ID NO : 14, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0606] In another aspect, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0607] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 15 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular outside of the CDRs (i.e., in the framework region(s) only);

[0608] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 16 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular outside of the CDRs.

[0609] Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0610] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 15 and (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 16.

[0611] In an aspect, the anti-PD-1 antigen-binding domain comprises VH, VL, CHI and a CL domains, so that the antigen binding domain is a Fab.

[0612] In such aspect, the heavy chain constant domain (CHI) comprises or consists essentially of SEQ ID NO: 17, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0613] Preferably, the light chain constant domain (CL) comprises or consists essentially of SEQ ID NO: 18 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0614] In an embodiment, the anti-PD-1 antigen-binding domain is a Fab or a Fab’, a Fab or a F(ab’)2 and comprises i) a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID Nos: 15 and 17, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and ii) a VL domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID Nos: 16 and 18, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0615] Preferably, the antigen binding domain is an anti-PD-1 Fab or F(ab’)2, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CHI domain, said VH and CHI domains having the amino acid sequence as set forth in SEQ ID Nos: 15 and 17 respectively and ii) a chain comprising or consisting of VL and CL domains, said domains having the amino acid sequence as set forth in SEQ ID Nos: 16 and 18, respectively. In an aspect, the antigen binding domain is an anti-PD-1 Fab or F(ab’)2, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 19 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular outside of the CDRs and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 20 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular outside of the CDRs.

[0616] In an aspect, the antigen binding domain is an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 21 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular outside of the CDRs and of ii) a chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 22 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, in particular outside of the CDRs.

[0617] In some aspects, when the antigen binding domain is described as having one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof, said modifications are outside of the CDRs.

[0618] Alternatively, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0619] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0620] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein

[0621] HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 23,

[0622] HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 24,

[0623] HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 25,

[0624] LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 26,

[0625] LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 27, and

[0626] LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 28,

[0627] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0628] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 29; (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 30.

[0629] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0630] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein

[0631] HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 31,

[0632] HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 32,

[0633] HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 33,

[0634] LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 34,

[0635] LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 35, and

[0636] LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 36,

[0637] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0638] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 37;

[0639] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 38.

[0640] In some aspects, the antigen binding domain is an anti-PD-1 antibody, comprising or consisting of : an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 50 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 51; an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 52 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 53; or an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 54 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 55.

[0641] Preferably, the antigen binding domain is an antagonist anti-PD-1 antibody comprising or consisting of an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 50 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 51. In a preferred aspect, the antigen binding domain is designed to be specifically activable in the TME. In this instance, the antigen binding domain may be a pH sensitive antigen binding domain, having a lower affinity for its ligand at a physiological pH, and a higher affinity at a pH of the TME, typically more acidic than the physiological pH. In other instances, the antigen binding domain may comprise a masking group designed to bar the antigen binding domain from binding with its ligand, or at least limit its binding to its ligand, when out of the TME, while exposing the antigen binding domain once in the TME. The exposure of the antigen binding domain may be caused by the switch of pH, the presence of a protease, or any other mechanism known to the person skilled in the art.

[0642] In a particular aspect of the disclosure, the variable regions of the anti-PD-1 antigen binding domain as described above may be associated with antibody constant regions, in particular from IgA, IgM, IgE, IgD or IgG such as IgGl, IgG2, IgG3, IgG4, preferably IgGl, IgG2 , or IgG4. Preferably, the antigen binding domain comprises an IgG Fc region, preferably an IgGl, IgG2, or IgG4 Fc region. Preferably, the Fc domain includes all or a portion of a hinge region. Preferably, the hinge region is derived from a human or humanized IgGl, IgG2, or IgG4.

[0643] As used herein, the term “IgG Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl-terminus of the IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The constant regions may be further mutated or modified, by methods known in the art, for modifying their binding capability towards Fc receptor.

[0644] Preferably, the antigen binding domain comprises or is covalently linked to a Fc domain, preferably an IgG Fc domain. Particularly, the Fc region is derived from human or humanized IgGl, IgG2 or IgG4. Preferably, the antigen binding domain comprises a Fc domain and a hinge region derived from a human or humanized IgGl, IgG2 or IgG4.

[0645] Preferably, the antigen binding domain comprises or is covalently linked to a Fc domain, preferably an IgG Fc domain. Particularly, the Fc region is derived from human or humanized IgGl, IgG2 or IgG4. Preferably, the antigen binding domain comprises a Fc domain and a hinge region derived from a human or humanized IgGl, IgG2 or IgG4.

[0646] Preferably, the antigen binding domain comprises a Fc domain, preferably an IgG Fc domain. For example, this means that the antigen binding domain is an antibody that comprises a Fc domain and / or that the antigen binding domain comprises an antigen binding fragment of an antibody (such as a Fab or scFv) that is covalently linked to a Fc domain.

[0647] As used herein, the term “IgG Fc region” or “IgG Fc domain” is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl-terminus of the IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The constant regions may be further mutated or modified, by methods known in the art, for modifying their binding capability towards Fc receptor.

[0648] Typically a Fc domain comprises two heavy chain constant domain, known as CH2 and CH3 domains. Optionally, the Fc domain envisioned herein also comprises a hinge region.

[0649] In one embodiment, the antigen binding domain comprises a truncated Fc region or a fragment of the Fc domain. In one embodiment, the Fc domain includes a CH2 domain. In another embodiment, the Fc domain includes CH2 and CH3 domains or includes hinge-CH2-CH3. Alternatively, the Fc domain can include all or a portion of the hinge region, a CH2 domain and / or a CH3 domain.

[0650] Antibodies or antigen-binding fragments thereof with amino acid sequences having at least 90%, for example, at least 95%, 96%, 97%, 98%, or 99% identity to any one of the above defined amino acid sequences are also part of the present disclosure.

[0651] In an aspect, the lipid-based nanoparticle obtained or obtainable by the methods of the invention comprises several antigen binding domain.

[0652] Preferably the method of the invention involves 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different antigen binding domains. Such antigen binding domains can be two distinct antigen biding domains that recognize the same target (e.g., such as pembrolizumab and nivolumab that both binds to PD-1) or two distinct antigen biding domains that recognize two different target (e.g., such as PD-1 and CTLA-4).

[0653] In a particular aspect, two different antigen binding domains are used in the method of the invention, so that the lipid nanoparticle comprises two different antigen binding domains, the first antigen binding domain and the second antigen may have the same or different formats. For example, the first antigen binding domain and the second antigen binding domain can be both antibodies, Fab or scFV. Alternatively, the first antigen binding domain and the second antigen binding domain are not both scFv. In a preferred aspect, the first antigen binding domain and the additional (second) antigen binding domain bind to targets expressed on the surface of the same activated immune cells. Said targets can be the same or different. For example, when the immune cell is a T cell, the first antigen binding domain binds for example to PD-1, and the second antigen binding domain binds to an antigen selected from the group consisting of CD137 / 41BB / TNFRSF9, CRTAM, CTLA4, FasL / TNFSF6, TIM-3 / HAVCR2, ITGAE, LAG-3, OX40 / TNFRSF4 and TIGIT. Alternatively, when the immune cell is a T cell, both the first and second antigen binding domains may bind to PD-1. Preferably, the first and second antigen binding domains are not competing antibodies for PD-1, which means that the first and second antigen binding domains recognize a different (non-overlapping) epitope of PD-1.

[0654] In some aspects, each of the first and second targets specifically expressed on activated immune cell surface (i.e., said targets being recognized by the first and second antigen binding domains, respectively) are selected from the group comprising BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACI / TNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF 1811 / CD120B,

[0655] CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L,

[0656] TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC- 1A, CD21, CLEC-9A, CD 180, CD59, CD54, CD71, CD35, CD218a, CD74, CD 165, 4- 1BBL / CD137L, ICOSL and CD160.

[0657] In a particular aspect, the first antigen binding domain and the second antigen binding domain are derived from Fab, Fab', F(ab')2, Fv, single chain (scFv), CrossMAb or nanobody (VHH), preferably a F(ab’)2, a Fab, a CrossMAb or a scFV and the first and second antigen binding domains may have the same or different formats. For example, the first antigen binding domain and the second antigen binding domain can be both Fab or scFV. Alternatively, the first antigen binding domain and the second antigen binding domain are not both scFv. Preferably, the first antigen binding domain and the second antigen binding antigen binding domain comprise or are covalently linked to a Fc domain, preferably an IgG Fc domain such as described herein.

[0658] Particularly, the additional or second antigen binding domain is i) not covalently bound to any of the lipids of the lipid-based nanoparticle, ii) does not comprise any modification for coupling or grafting the antigen binding domain to a lipid and / or iii) is not covalently bound to a lipidation peptide or motif, in particular a lipidation peptide or motif such as described herein.

[0659] Preferably, the additional or second antigen binding domain is not a secondary antibody, typically a secondary antibody that allows the attachment of the first antigen binding domain to the lipid-based nanoparticle (e.g., such as an anti-Fc antibody or antigen binding fragment or derivative thereof).

[0660] Preferably, the additional or second antigen binding domain is not a secondary antibody comprising a lipidation peptide or motif such as described herein.

[0661] In some embodiments, in addition to the antigen binding domain, additional targeting molecules may be used in the method of the invention. For example, modified sugars or modified lipids can be used in combination with the antigen binding domain of the invention.

[0662] The method of the invention comprises a step of mixing the first and second compositions, once one of said composition has been mixed with the antigen binding domain.

[0663] In an aspect, the first composition is mixed with the second composition comprising with a volume:volume ratio from about between about 1 :1 and about 1 :25, from about 1 : 1 to about 1:20, from about 1 :2 to about 1: 10, from about 1 :1 to about 1 :5, from about 1 :2 to about 1 :5, from about 1 :2 to about 1 :4.

[0664] In an aspect, the first composition is mixed with the second composition to produce a mixed composition with an organic polar solvent (e.g., ethanol) to water ratio (volume:volume) between about 1 : 1 and about 1 :25, from about 1 :1 to about 1 :20, from about 1 :2 to about 1 : 10, from about 1 : 1 to about 1 :5, from about 1 :2 to about 1 :5, from about 1 :2 to about 1 :4. Preferably, the mixed composition comprises a ratio ethanol / water (volume:volume) of between 1 : 1 to 1 :10, preferably between 1 :2 and 1 :5. Suitable flow rates for mixing the first composition and the second composition may be determined based on the scales.

[0665] In some aspects, the second composition is mixed at a flow rate ranging from about 1-100 ml / minute, 10-200 ml / minute, 20-300 ml / minute, 40-400 ml / minute, 60-500 ml / minute, 70-600 ml / minute, 80-700 ml / minute, 90-800 ml / minute, 100-900 ml / minute, 110-1000 ml / minute, 120-1100 ml / minute, 130-1200 ml / minute, 140-1300 ml / minute, 150-1400 ml / minute, 160- 1500 ml / minute, 170-1600 ml / minute, 180-1700 ml / minute, 150-250 ml / minute, 250-500 ml / minute, 500-1000 ml / minute, 1000-2000 ml / minute, 2000-3000 ml / minute, 3000-4000 ml / minute, or 4000-5000 ml / minute. In some aspects, the second composition is mixed at a flow rate of about 200 ml / minute, about 500 ml / minute, about 1000 ml / minute, about 2000 ml / minute, about 3000 ml / minute, about 4000 ml / minute, or about 5000 ml / minute.

[0666] In some aspects, the first composition is mixed at a flow rate ranging from about 1-100 ml / minute, 10-200 ml / minute, 20-300 ml / minute, 40-400 ml / minute, 60-500 ml / minute, 70-600 ml / minute, 80-700 ml / minute, 90-800 ml / minute, 100-900 ml / minute, 110-1000 ml / minute, 120-1100 ml / minute, 130-1200 ml / minute, 140-1300 ml / minute, 150-1400 ml / minute, 160- 1500 ml / minute, 170-1600 ml / minute, 180-1700 ml / minute, 150-250 ml / minute, 250-500 ml / minute, 500-1000 ml / minute, 1000-2000 ml / minute, 2000-3000 ml / minute, 3000-4000 ml / minute, or 4000-5000 ml / minute. In some aspects, the first composition is mixed at a flow rate of about 200 ml / minute, about 500 ml / minute, about 1000 ml / minute, about 2000 ml / minute, about 3000 ml / minute, about 4000 ml / minute, or about 5000 ml / minute.

[0667] In an aspect, the flow rate ratio of the first composition to the second composition is comprised between 1 to 10 and 1 to 1, between 1 to 9 and 1 to 1, between 1 to 8 and 1 to 1, between 1 to 7 and 1 to 1, between 1 to 6 and 1 to 1, between 1 to 5 and 1 to 1, between 1 to 4 and 1 to 1, between 1 to 3 and 1 to 1, between 1 to 2 and 1 to 1. Preferably, the flow rate ratio between the first composition and the second composition is comprised between 1 :1 and 1 :4, preferably is 1 :3.

[0668] Alternatively, the amount of lipid and mRNA may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an mRNA. In general, a lower N:P ratio is preferred. The one or more mRNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 30: 1, such as 2:1, 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20:1, 22:1, 24:1, 26: 1, 28: 1, or 30: 1. In certain aspects, the N:P ratio may be from about 2:1 to about 8: 1. In other aspects, the N:P ratio is from about 5: 1 to about 8: 1. For example, the N:P ratio may be about 5.0: 1, about 5.5:l, about 5.67: l, about 6.0: l, about 6.5: l, or about 7.0: 1. For example, the N:P ratio may be about 5.67: 1. Preferably, the N:P ratio is between about 5: 1 and about 7: 1. Preferably, the N:P ratio is about 6: 1.

[0669] The result of the mixing step is the generation of the lipid-based nanoparticles including the antigen binding domain and the nucleic acid molecule(s).

[0670] Removal of polar organic solvent and additional steps

[0671] After the mixing of the first and second compositions, the method of the invention comprises a step of removing the polar organic solvent and adjusting the pH of the composition to a neutral pH.

[0672] This step can be carried by any method known in the art, such as dialysis or buffer exchange.

[0673] In some aspects, the method may comprise a step of buffer exchange after the step of mixing the first and second compositions, for example using the Unagi’s buffer exchange.

[0674] Alternatively, the method may comprise a step of dialyzing the composition obtained after the step of mixing the first and second compositions. Preferably, the composition is dialyzed against an adequate buffer, such as phosphate buffered saline (PBS). Such method may further comprises a step of concentration of the LNPs after dialysis, in particular to reduce the sample volume.

[0675] In some aspects, the adjustment of the pH to a neutral pH is an adjustment of the pH to a range from about 6.5 to about 7.5, preferably to 7. The man skilled in the art knows how to adjust pH, for example by adding NaOH to the composition. Alternatively, the pH is adjusted by replacing the organic solvent with an aqueous solution of pH 7, for example by buffer exchange.

[0676] In some aspects, the lipid nanoparticles containing the nucleic acid molecule(s) and the antigen biding domain are concentrated after the removal of the polar organic solvent. Accordingly, the method of the invention may further comprise a step of purification and / or concentration of the lipid nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s).

[0677] In some aspects, the method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s), the method comprising the steps of:

[0678] - feeding through a first entry of a mixing device a first composition,

[0679] - feeding through a second entry of said mixing device a second composition, - mixing the first and second compositions in the mixing device so as to generate lipid-based nanoparticles,

[0680] - removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH,

[0681] - concentrating the lipid-based nanoparticles comprising the antigen binding domain and the one or several nucleic acid molecule(s),

[0682] - recovering the purified and / or concentrated lipid-based nanoparticles comprising the antigen binding domain and the one or several nucleic acid molecule(s).

[0683] Various purification methods may be used. In some aspects, lipid nanoparticles are purified using Tangential Flow Filtration. Tangential flow filtration (TFF), also referred to as cross-flow filtration, is a type of filtration wherein the material to be filtered is passed tangentially across a filter rather than through it. In TFF, undesired permeate passes through the filter, while the desired retentate passes along the filter and is collected downstream. It is important to note that the desired material is typically contained in the retentate in TFF, which is the opposite of what one normally encounters in traditional-dead end filtration.

[0684] Depending upon the material to be filtered, TFF is usually used for either microfiltration or ultrafiltration. Microfiltration is typically defined where the filter has a pore size of between 0.05 pm and 1.0 pm, inclusive, while ultrafiltration typically involves filters with a pore size of less than 0.05 pm. Pore size also determines the nominal molecular weight limits (NMWL), also referred to as the molecular weight cut off (MWCO) for a particular filter, with microfiltration membranes typically having NMWLs of greater than 1,000 kilodaltons (kDa) and ultrafiltration filters having NMWLs of between 1 kDa and 1,000 kDa.

[0685] A principal advantage of tangential flow filtration is that non-permeable particles that may aggregate in and block the filter (sometimes referred to as “filter cake”) during traditional “dead-end” filtration, are instead carried along the surface of the filter. This advantage allows tangential flow filtration to be widely used in industrial processes requiring continuous operation since down time is significantly reduced because filters do not generally need to be removed and cleaned.

[0686] Tangential flow filtration can be used for several purposes including concentration and diafiltration, among others. Concentration is a process whereby solvent is removed from a solution while solute molecules are retained. In order to effectively concentrate a sample, a membrane having a NMWL or MWCO that is substantially lower than the molecular weight of the solute molecules to be retained is used. Generally, one of skill may select a filter having a NMWL or MWCO of three to six times below the molecular weight of the target molecule(s).

[0687] In some aspects, lipid nanoparticles are purified using diafiltration. Diafiltration is a fractionation process whereby small undesired particles are passed through a filter while larger desired nanoparticles are maintained in the retentate without changing the concentration of those nanoparticles in solution. Diafiltration is often used to remove salts or reaction buffers from a solution. Diafiltration may be either continuous or discontinuous. In continuous diafiltration, a diafiltration solution is added to the sample feed at the same rate that filtrate is generated. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting concentration. Discontinuous diafiltration may be repeated until a desired concentration of nanoparticles is reached.

[0688] Purified and / or concentrated lipid nanoparticles comprising the antigen binding domain and the one or several nucleic acid molecule(s) may be finally formulated in a composition comprising a desired buffer such as, for example, PBS. Such composition may further comprise a preservative or stabilizing agent, preferably that allows good storage conditions. For examples, the t-LNP obtained by the method of the invention can be stably frozen in a 10% trehalose solution. Alternatively, the t-LNP obtained by the method of the invention can be lyophilized.

[0689] Lipid-based nanoparticles

[0690] The methods of the invention comprise a step of recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s).

[0691] The invention thus also refers to a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s) obtained or obtainable by any of the methods disclosed herein.

[0692] The lipid-based nanoparticle according to the invention is preferably formulated either as a liposome or a lipid nanoparticle (LNP), especially a lipid nanoparticle comprising a mixture of lipids.

[0693] The lipid-based nanoparticle also encompasses similar nanoparticles such as but not limited to micelles and nano-emulsions. Lipid based nanoparticles also include Hybrid nanoparticles comprising polymers-lipids hybrid compounds, such as polamines-polaxamers, in particular as described herein.

[0694] The lipid-based nanoparticle according to the invention is preferably a t-LNP. As used herein, the term “t-LNP” refers to a targeted lipid nanoparticle, i.e., a lipid nanoparticle comprising an antigen binding domain. Alternatively, a “nt-LNP” refers to a lipid nanoparticle devoid of antigen binding domain. Said LNP comprises any of the lipids, antigen binding domains and nucleic acid molecule(s) disclosed above.

[0695] In some aspects, the LNP obtained or obtainable by the methods of the invention comprises a cationic or ionizable lipid, a helper lipid, a sterol, a PEG-modified lipid, one or more nucleic acid molecule(s), preferably mRNA molecules, and one or more antigen biding domain(s).In some aspects, the LNP obtained or obtainable by the methods of the invention comprises [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315) as ionizable or cationic lipid, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol with polyethylene glycol of average molecular weight 2000 (PEG2000 DMG) as PEG-modified lipid.

[0696] In some aspects, the LNP obtained or obtainable by the methods of the invention comprises one or more antigen biding domain(s), said antigen binding domain(s) being not covalently bound to a lipid. Even more preferably, the antigen biding domain does not comprise any modification for coupling or grafting the antigen binding domain to a lipid, such as a PEG group or a lipophilic group.

[0697] Said antigen binding domain is preferably directed against PD-1, TIM3, CTLA-4, LAG-3, BTLA and TIGIT.

[0698] In some aspects, the LNP obtained or obtainable by the methods of the invention comprises 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 an antibody against a checkpoint inhibitor, such as described herein.

[0699] In some aspects, the t-LNPs (with antigen binding domain and nucleic acid molecule(s)) produced by the method of the present disclosure has an efficacy, intracellular delivery, and / or immunogenicity being higher than the efficacy, intracellular delivery, and / or immunogenicity of untargeted LNP (without antigen binding domain) by about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1 folds or more, about 2 folds or more, about 3 folds or more, about 4 folds or more, about 5 folds or more, about 10 folds or more, about 20 folds or more, about 30 folds or more, about 40 folds or more, about 50 folds or more, about 100 folds or more, about 200 folds or more, about 300 folds or more, about 400 folds or more, about 500 folds or more, about 1000 folds or more, about 2000 folds or more, about 3000 folds or more, about 4000 folds or more, about 5000 folds or more, or about 10000 folds or more.

[0700] In a particular aspect, the lipid-based nanoparticles with antigen binding domain are compared to the lipid-based nanoparticles without antigen binding domain are both prepared by the method of the invention.

[0701] In some aspects, the lipid-based nanoparticles (with antigen binding domain) produced by the method of the present disclosure exhibits a binding efficiency to their target (e.g., for example in terms of affinity) higher than the binding efficiency of an untargeted LNP (without antigen binding domain).

[0702] In some aspects, the LNPs produced by the method of the present disclosure exhibits a nucleic acid expression (e.g., mRNA expression) higher than the nucleic acid expression (e.g., mRNA expression) of untargeted LNP (without antigen binding domain).

[0703] In some aspects, the LNPs (with antigen binding domain) produced by the method of the present disclosure exhibits an good encapsulation of nucleic acid molecules. The efficiency of encapsulation of nucleic acid molecules describes the amount of nucleic acid molecules encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is preferably desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of nucleic acid molecules in a solution containing the lipid-based nanoparticle before and after breaking up the lipid-based nanoparticle with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free nucleic acid molecules in a solution. For the lipid- based nanoparticles obtainable by the method of the invention, the encapsulation efficiency may be of at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects, the encapsulation efficiency is of at least 70%. In certain aspects, the encapsulation efficiency is of at least 80%, preferably 90%.

[0704] Lipid-based nanoparticles or a composition comprising LNP obtainable by the method of the invention may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a LNP or of a composition comprising LNP. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a LNP or of a composition comprising LNP, such as particle size, poly dispersity index, and zeta potential. In one aspect, the mean size of the lipid-based nanoparticles of the invention may be between 10 of nm and 200 of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 200 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm or 200nm. In some aspects, the mean size of a LNP is from about 50 nm to about 200 nm, from about 50 nm to about 190 nm, from about 50 nm to about 180 nm, from about 50 nm to about 170 nm, from about 50 nm to about 160 nm, from about 60 nm to about 200 nm, from about 60 nm to about 190 nm, from about 60 nm to about 180 nm, from about 60 nm to about 170 nm, from about 70 nm to about 200 nm, from about 70 nm to about 190 nm, from about 70 nm to about 180 nm, from about 80 nm to about 200 nm, from about 80 nm to about 190 nm, or from about 90nm to about 200 nm. In some aspects, the mean size of a LNP is from about 50 nm to about 200 nm, from about 50 nm to about 190 nm, from about 50 nm to about 180 nm, from about 50 nm to about 170 nm, from about 50 nm to about 160 nm, from about 60 nm to about 190 nm, from about 60 nm to about 180 nm, from about 60 nm to about 170 nm, from about 60 nm to about 160 nm, from about 70 nm to about 180 nm, from about 70 nm to about 170 nm, from about 70 nm to about 160 nm, from about 80 nm to about 170 nm, from about 80 nm to about 160 nm, or from about 90nm to about 160 nm. In certain aspects, the mean size of a LNP may be from about 70 nm to about 150 nm. In a particular aspect, the mean size is about 120 nm. In other aspects, the mean size of the lipid-based nanoparticle is about 150 nm.

[0705] In another aspect, the mean size of the lipid-based nanoparticle of the invention may be between 10 nm and 400 nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 350 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 250 nm, 300 nm or 350 nm.

[0706] A composition comprising LNPs may be relatively homogenous. A poly dispersity index may be used to indicate the homogeneity of a composition comprising LNPs, e.g., the particle size distribution of the lipid-based nanoparticles. A small (e.g., less than 0.3) poly dispersity index generally indicates a narrow particle size distribution. A composition comprising LNPs may have a poly dispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some aspects, the poly dispersity index of a composition comprising LNPs is from about 0.10 to about 0.25.

[0707] The zeta potential of the lipid-based nanoparticle obtainable according to the method of the invention may be used to indicate the electrokinetic potential of a composition comprising said lipid-based nanoparticle. For example, the zeta potential may describe the surface charge of a lipid-based nanoparticle. Lipid-based nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some aspects, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about - 5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0708] In some embodiments, the methods disclosed herein comprise a step of LNP characterization. For example, the method of the invention can further comprise a step of: d) purity and / or size assessment, e) measurement of transfection of h...

Claims

CLAIMS1. A method for producing a lipid-based nanoparticle comprising an antigen binding domain and one or several nucleic acid molecule(s), the method comprising the steps of:- feeding through a first entry of a mixing device a first composition comprising a lipid- based composition and a polar organic solvent,- feeding through a second entry of said mixing device a second composition comprising one or several nucleic acid molecule(s), wherein said second composition is an acidic aqueous composition,- mixing the first and second compositions in the mixing device so as to generate lipid- based nanoparticles,- removing the polar organic solvent from the mixed composition and adjusting the pH of the composition to a neutral pH; and- recovering the lipid-based nanoparticles comprising an antigen binding domain and one or several nucleic acid molecule(s); wherein the antigen binding domain is mixed with the first or second composition before the mixing step of the first and second compositions into the mixing device; and wherein the antigen binding domain is an antibody or an antigen binding fragment thereof.

2. The method of claim 1, wherein the antigen binding domain comprises a Fc domain, preferably an IgG Fc domain.

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

4. The lipid-based nanoparticle of any one of claims 1-3, wherein the lipid-based nanoparticle does not comprise an anchoring moiety comprising a lipidation peptide or motif.

5. The method of any one of claims 1-4, wherein the step of removing the polar solvent is performed by dialysis or buffer exchange.

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

7. The method of claim 6, wherein the ionizable lipid is selected from the group consisting of [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 1,2- dioleoyl-3 -trimethylammonium propane (DOTAP); N,N-dimethyl-2, 3 -di oleyloxypropylamine (DODMA), l,2-di-O-octadecenyl-3 -trimethylammoniumpropane (DOTMA), 3-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); l,2-diacyloxy-3- dimethylammoniumpropanes; l,2-dialkyloxy-3-dimethylammoniumpropanes; dioctadecyldimethylammonium chloride (DODAC), l,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N- dimethyl-l-propanamium trifluoroacetate (DOSPA), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5- en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-di oleyloxybenzylamine (DMOBA), 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,3 l-tetraen-19- yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N- dimethyl-2,3-bis(cis-9-tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N- (3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-l-propanaminium bromide (GAP- DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (GAP -DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminiiim (DOBAQ), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), l,2-dipalmitoyl-3- dimethylammonium-propane (DPDAP), Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn- glycero-3 -ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)propan-l-aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3- bis(dodecyloxy)propan-l-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl- ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2- dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98Niz-5), 1- [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C 12-200), 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-(a-D-tocopherolhemisuccinateethyl)piperidyl]ethyl} disulfide (SS-EC) and any mixtures thereof, preferably is ALC-0315, SM-102, Dlin-MC3-DMA or SS-OP, more preferably ALC-0315 or SS-OP.

8. The method of 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 mixtures thereof, preferably is cholesterol.

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

10. The method of any one of claims 6-9, 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, preferably is PEG-DMG, PEG-DSPE or a mixture thereof.

11. The lipid-based nanoparticle of claim 10, wherein the PEG is of between 2000 Daltons and 5000 Daltons, preferably is DSPE-PEG-2000, DMG-PEG-2000, DSPE-PEG-5000, DMG- PEG-5000 or a mixture thereof.

12. The method of any one of claims 1 to 11, wherein the lipid-based composition is 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; and k) SS-OP, DSPC, cholesterol and DSPE-PEG; and l) SS-OP, DOPC, cholesterol and DMG-PEG.

13. The method of any one of claims 1-12, wherein the lipid-based composition comprises or consists of from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid.

14. The method of any one of claims 1-12, wherein the lipid-based composition comprises or consists of from about 45 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 15 mol % of a helper lipid from about 35 mol% to about 45 mol% of a sterol, and from about 0.5 mol% to about 2,5 mol% of a PEG-lipid.

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

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

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

18. The method of any one of claims 1-17, wherein the flow rate ratio between the first and the second compositions is comprised between 1 : 10 and 10: 1, preferably between 1 :5 and 5: 1, even more preferably between 1 :2 and 1 :4.

19. The method of any one of claims 1-18, wherein the antigen binding domain binds to a target selected from the group consisting of BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PDCD1, PTPN22, RGS1, LOX1, SIGLEC 6, TACVTNFRSF13B, TIGIT, CD 163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, 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 of any one of claims 1-19, wherein the antigen binding domain binds to a target selected from the group consisting of PD-1, CD 127, SIRPa and CLEC-1A.

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

22. The method of claim 21, wherein the antigen binding domain comprises an anti-PD-1 antigen binding domain comprising:(i) a VH comprising a heavy chain CDR1 (HCDR1), CDR2 (HCDR2) and CDR3 (HCDR3), and (ii) a VL comprising a light chain CDR1 (LCDR1), CDR2 (LCDR2) and CDR3 (LCDR3), wherein: a) the HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 5, and the LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO:6, or b) the HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 23, the HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 24, the HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 25, the LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 26, the LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 27, and the LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 28, or c) the HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 31, the HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 32, the HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 33, the LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 34, the LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 35, and the LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 36.

23. The method of claim 21, wherein the antigen binding domain comprises an anti-PD-1 antigen binding domain comprising: a) a VH comprising or consisting of an amino acid sequence of SEQ ID NO: 15; and a VL comprising or consisting of an amino acid sequence of SEQ ID NO: 16; b) a VH comprising or consisting of an amino acid sequence of SEQ ID NO: 29; and a VL comprising or consisting of an amino acid sequence of SEQ ID NO: 30; and c) VH comprising or consisting of an amino acid sequence of SEQ ID NO: 37; and a VL comprising or consisting of an amino acid sequence of SEQ ID NO: 38.

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

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

26. The method of claim 25, wherein the mRNA encodes for a molecule selected from the group consisting of: a) an immune cell enhancing or inhibiting compound, in particular such as described herein, and preferably selected from: a molecule selected from the group consisting of TCF 1 , LEF 1 , WNT, FRIZZLED, Beta catenin, BCL2, BCLXL, BIRC3, MCL1, PGCla, TCF7, NFAT, NFKB, RORgt, TRAM, TBK1, TBET, EOMES, RUNX3, GAT A3, JUNB, POU2AF1, OCTI, BLIMP- 1, XBP-1, FOXO1, PTGS2, CSE, Glutl, Glut3, HK2, arginine resynthesis enzymes, argininosuccinate synthase (ASS), Ornithine transcarbamylase (OTC), GYS, OXPHOS, AKT, PLC, STAT, SMAD, Blys, BTK, BLK, CD 107a, Lymphotoxin (LT) al 2, granzyme B, perforin, POU2F1, BBS 10, BBS 12, TCP1, HSP, Integrin alpha 1, Integrin alpha 2, Integrin alpha 2b, Integrin alpha 11, Integrin alpha 3, Integrin alpha 6, Integrin alpha7, Integrin alpha E, Integrin beta 2, Integrin beta 4, LFA-1, LFA-2, LFA-3, Integrin beta 1, Integrin beta 7, CD 103, Integrin alpha V, ITGAE, CD11C, CRTAM, SELL, GAL3, Laminin, Actin, Vimentin, DEFI, 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 Regulatory Factors such as IRF1, IRF-3, IRF-5, IRF-7, IRF-8 and IRF-9, CREB, RORg, RORgt, , NFKB, T-bet, , AhR, STING, MAVS, MyD88, IRAK 1, IRAK2, IRAK4, TRAF3, TRAF6, TAK1, TAB2, TAB3, TAK- TAB1, MKK3, MKK4, MKK6, MKK7, IKKa, IKKp, , TRIF, , , D3 -phosphoinositides, 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- IBB, 4-1BBL, 0X40, OX40L, CD155, LIGHT, HVEM, CD30, CD30L, SLAM CD2 family, CD27, TL1 A, DR3, TM1, TIM4, CD150, CD48, CD58, CD112, BAFFR, BCMA, TACI, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS1, CD160, and any combination thereof, in particular such as described herein; a molecule selected from the group consisting of 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, IKAROS, EGR2 / 3, CREM, P27 (KIP1), CD107a, RAC1, AP2, RB7, M6P, MPR, BBS 10, BBS 12, TCP1, HSP, APC, Laminin, Actin, Vimentin, DEFI, 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, galectin9, 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 from FOXO1, Fas, TRAIL, PUMA, NOXA, BIM, LAG3, CTLA4, PDL1, FOXP3, TOX, IDO, ARG1 IL35+IL35R, TGFB+TGFBR, TIM3, TIGIT, ChemR23, FPR2; and any combination thereof, in particular such as described herein;b) a cytokine, 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 such as described herein; c) a cytokine receptor, 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, IL-35R; d) a chemokine, preferably selected from the group consisting of CXCL9 or CXCL10; e) a chemokine receptor, preferably selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, XCR1; f) an antigen fragment derived from viral pathogens, bacterial pathogens and / or parasitic pathogens, in particular such as described herein; g) an antibody or a fragment or a derivative thereof, preferably directed against a target selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, LAG3, BTLA, CD160, CD40L, ICOS, CD27, 0X40, 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 such as described herein and h) a Chimeric Antigen Receptor (CAR), in particular such as described herein.

27. The method of claim 26, wherein the mRNA encodes forBCL2, IL7, IL7R, CXCL9 and / or CXCL10.

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

29. The method of claim 28, wherein the second composition comprises a mRNA molecule encoding for IL-7 and a mRNA molecule encoding for IL-7R.

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

31. A lipid-based nanoparticle obtainable through the method of any one of claims 1 to 30.

32. The lipid-based nanoparticle of claim 31, wherein said lipid-based nanoparticle does not comprise a) an antigen binding domain that is covalently bound to a lipid; ii) an antigen binding domain that comprises a modification for coupling or grafting the antigen binding domain to a lipid and / or iii) an anchoring moiety comprising a lipidation peptide or motif.

33. A pharmaceutical composition comprising the lipid-based nanoparticle of claim 31 and optionally a pharmaceutically acceptable carrier.

34. The lipid-based nanoparticle of claim 31 or 32 or the pharmaceutical composition of claim 33, for use as a medicament or vaccine.

35. The lipid-based nanoparticle or the pharmaceutical composition for use according to claim 33, for use in the treatment of i) a cancer or of an infectious disease or ii) an autoimmune disease or an inflammatory disease.

36. Use of the lipid-based nanoparticle of claim 31 or 32 or of the pharmaceutical composition of claim 33, for the manufacture of a medicament for the treatment of a cancer or of an infectious disease or an autoimmune disease or an inflammatory disease.

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

Images