Lipid-based nanoparticles comprising non-glycosylated FC domains and uses thereof

Abstract

The invention relates to a lipid-based nanoparticle comprising an antigen-binding domain covalently linked to a nonglycolsylated Fc domain, and uses thereof.

Description

[0001] LIPID-BASED NANOPARTICLES COMPRISING NON-GLYCOSYLATED FC

[0002] DOMAINS AND USES THEREOF

[0003] FIELD OF THE INVENTION

[0004] The invention pertains to the field of medicine, in particular immunotherapy.

[0005] BACKGROUND OF THE INVENTION

[0006] Over the past decade, the therapeutic potential of lipid-based nanoparticles (LNPs) as novel drug delivery systems complementing conventional pharmacology has been widely acknowledged. LNPs have particularly shown remarkable pharmacological performance and promising therapeutic outcomes, thus gaining substantial interest in preclinical and clinical research. They can encapsulate and / or expose various therapeutic agents including small molecules, nucleic acids and monoclonal antibodies for a diverse range of applications.

[0007] LNPs are particularly used to deliver nucleic acids molecules such as mRNA to cells. LNPs need to reach target tissues, followed by internalization by target cells, for the efficient delivery of the nucleic acids molecules. In particular, the delivery of mRNA leads 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] However, the lack of precise control over the biodistribution of LNPs also complicates the delivery of nucleic acids such as mRNA, siRNA, and DNA, which require accurate targeting to specific cells or tissues to achieve therapeutic benefit. In addition, upon systemic administration, LNPs are distributed throughout the body, leading to substantial accumulation in non-target tissues such as the liver, spleen, and lungs. This off-target accumulation not only reduces the therapeutic efficacy of the encapsulated drug but also increases the risk of adverse side effects due to unintended interactions with healthy tissues.

[0009] Therefore, there is a pressing need to develop novel strategies that can enhance the targeted delivery of LNPs. The invention seeks to meet this and other needs.

[0010] SUMMARY OF THE INVENTION

[0011] The invention described herein aims to address these challenges by providing an improved targeted LNP that enhances specificity for target tissues, thereby maximizing therapeutic efficacy and minimizing the risk of adverse side effects.

[0012] To this end, the inventors have developed a targeted lipid-based nanoparticle comprising an antigen binding domain of an antibody covalently linked to a nonglycosylated Fc domain.

[0013] Glycosylation of proteins is a complex and versatile posttranslational modification that influences biological activity, protein conformation, stability, solubility, secretion, susceptibility to aggregation and proteolysis, pharmacokinetics, and antigenicity. Typically, immunoglobulins such as IgG are composed of three globular domain structures, two of which are the fragments for antigen binding (Fab) and the other is the fragment crystalizable (Fc) that activates Fey receptors (FcyRs) on leukocytes and Cl component of complement. In particular, all human IgG antibodies contain an invariant N-linked glycosylation site at the asparagine at position 297 (numbered according to the EU index as in Kabat et al., 1991) of the heavy chain CH2 domain within the Fc region.

[0014] The inventors show herein that a t-LNP comprising an antibody with an unglycosyated Fc domain allow a precise and efficient delivery of mRNA in the target site of interest. This advantage is not bound to a specific method of synthesis of a t-LNP, as it was obtained with several methods of production of t-LNPs. The inventors have shown that the deglycosylation may be obtained through different means, allowing the deglycosylation process to be adaptable for multiple types of process.

[0015] The inventors particularly demonstrate the ability of t-LNP comprising an anti-PDl antibody, an anti-CD127 antibody or an anti-CLECl antibody with an unglycosylated Fc domain to significantly improve targeting and internalization of the t-LNPs and the delivery of mRNA molecules. As shown herein, the invention is not limited to a specific target. LNPs comprising bivalent, monovalent and sc-Fv-Fc format have been tested, all showing an improvement in targeting and internalization, showing the usefulness of deglycosylation of the Fc domain in different antibody formats.

[0016] Additionally, the effect of deglycosylation of the Fc domain is not tied to a specific composition of LNP. In a first aspect, the invention concerns a lipid-based nanoparticle comprising an antigen binding domain of an antibody covalently linked to a N-nonglycosylated Fc domain and optionally one or more nucleic acid molecules.

[0017] Preferably, the Fc domain is an IgGl, IgG2, IgG3 or IgG4 Fc domain, preferably an IgGl or IgG4 Fc domain, even more preferably an IgGl Fc domain.

[0018] The N-nonglycosylated Fc domain is typically a N-deglycosylated Fc domain. Preferably, the N- deglycosylated Fc domain is also O-deglycosylated.

[0019] Alternatively, the N-nonglycosylated Fc domain is an N-aglycosylated Fc domain.

[0020] In particular, the Fc domain of the invention i) does not have an Asparagine (N) at position 297 according to Eu numbering and / or ii) has an amino acid residue at position 298 and / or 299 according to Eu numbering that reduces or eliminates glycosylation at position 297. Preferably, the N-aglycosylated Fc domain comprises an amino acid substitution in the CH2 domain selected from the group consisting of N297A, N297D, N297Q, N297G, N297F, N297K, N297R, N297H, N297V, N297L, N297I, N297G, N297P, N297E, from the group consisting of N297A, N297Q and N297G, according to Eu numbering.

[0021] Preferably, the N-aglycosylated Fc domain comprises an amino acid substitution N297A in the CH2 domain, according to Eu numbering. More preferably, the N-aglycosylated Fc domain comprises an amino acid sequence comprising or consisting of SEQ ID NO: 59 or 63.

[0022] In some instances, the Fc domain i) is not covalently bound to any of the lipids of the lipid-based nanoparticle, and / or ii) does not comprise any modification for coupling or grafting the antigen binding domain to a lipid.

[0023] In some instances, the Fc domain further comprises a knob-into-hole modification, preferably wherein the Fc domain comprise a first domain comprising or consisting of SEQ ID NO: 66 and a second domain comprising or consisting of SEQ ID NO: 67.

[0024] Preferably, the antigen binding domain is selected from the group consisting of a Fab, a Fab’, a F(ab')2, a Fv, a crossMAb Fab, a crossMAb Fab’, a crossMAb F(ab')2, a single-chain variable fragment (scFV) and a VHH.

[0025] In particular, the antigen binding domain binds to a target selected from the group consisting of PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, 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, CD160, CD127 and SIRPg.

[0026] Preferably, the antigen binding domain binds to a target selected from the group consisting of PD- 1, CLEC-lA and CD127.

[0027] Particularly, the antigen binding domain is an anti-PD-1 binding domain comprising:

[0028] (i) a VH comprising a heavy chain CDR1 (HCDR1), CDR2 (HCDR2) and CDR3 (HCDR3), and

[0029] (ii) a VL comprising a light chain CDR1 (LCDR1), CDR2 (LCDR2) and CDR3 (LCDR3), wherein:

[0030] - the HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 1;

[0031] - the HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 2;

[0032] - the HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 3;

[0033] - the LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 4;

[0034] - the LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 5, and

[0035] - the LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO:6.

[0036] Particularly, the antigen binding domain and the N-nonglycosylated Fc domain form an antibody, said antibody being an anti-PD-1 antibody comprising a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 70 or 71 and a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 20.

[0037] In particular, the lipid-based nanoparticle comprises targeting moieties comprising the antigen binding domain covalently linked to the N-nonglycosylated Fc domain, said targeting moieties being antibodies; wherein in said lipid based nanoparticles at least 50% of the targeting moieties comprises a N-nonglycosylated Fc domain.

[0038] The lipid-based composition of the lipid based nanoparticle typically comprises or consists of i) a cationic or ionizable lipid, ii) a helper lipid, iii) a sterol and iv) a PEG-lipid or a combination of PEG-lipids, said combination preferably comprising a first PEG-lipid that does not comprise a reactive functional group for conjugation to the non-glycosylated Fc domain and a second PEG- lipid that comprises a reactive functional group for conjugation to the non-glycosylated Fc domain. In particular: 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), 1,2-di-O-octadecenyl -3 -trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-di oleoyl -3-dimethylammonium-propane (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), 1 ,2-dimyristoyl-sn-glycero-3 - ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl 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' -di oleylcarbamyl -3- dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen- 19-yl-4-(dimethylamino)butanoate (DLin-MC3 -DMA), N-(2 -Hydroxy ethyl)-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- (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), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12- 200), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide (SS-OP) and any mixtures thereof, preferably is ALC-0315, SM-102, Dlin-MC3- DMA or SS-OP and any mixture thereof, more preferably is ALC-0315 or SS-OP;

[0039] - 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;

[0040] - the helper lipid is selected from DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DEPC, DOPC and DSPE and any mixture thereof, preferably is DOPE or DSPC or a mixture thereof; and / or

[0041] - the PEG-lipid is selected from 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-DSG, PEG-DSPE or ALC-0159 and any mixture thereof.

[0042] Preferably, the lipid-based nanoparticle comprises from about 40 mol % to about 60 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 25 mol% to about 50 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid, preferably 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 30 mol% to about 45 mol% of a sterol, and from about 1 mol% to about 2.5 mol% of a PEG-lipid.

[0043] In some instances, the LNP comprises one or more nucleic acid molecules that is / are mRNA.

[0044] In some instances, the LNP of the invention comprises or consists essentially of: a) a combination of lipids comprising or consisting of:

[0045] - an ionizable lipid;

[0046] - a helper lipid;

[0047] -a sterol;

[0048] - a mixture of PEG lipids, said mixture comprising a first PEG-lipid that is not linked to a targeting moiety and a second PEG-lipid that is covalently linked to a targeting moiety, said targeting moiety comprising an antigen binding domain of an antibody, preferably of an anti-PD-1 antibody, covalently linked to a N-nonglycosylated Fc domain such as disclosed herein; and b) optionally a nucleic acid molecule, preferably one or more mRNA molecule(s).

[0049] Preferably, the LNP of the invention comprises a combination of lipids comprising or consisting of:

[0050] - the ionizable lipid is selected from the group consisting of ALC-0315, SM-102, Dlin- MC3-DMA and SS-OP;

[0051] - the helper lipid is DOPE or DSPC;

[0052] -the sterol is cholesterol;

[0053] - the mixture of PEG lipids is PEG-DSPE and / or PEG-DMG.

[0054] In a second aspect, the invention concerns a pharmaceutical composition comprising at least one lipid-based nanoparticle according to the invention and optionally a pharmaceutically acceptable carrier or excipient.

[0055] In a third aspect, the invention concerns the lipid-based nanoparticle according to the invention or the pharmaceutical composition according to the invention for use as a medicament, particularly for use in the treatment of a cancer, an infectious disease, an inflammatory disease or an autoimmune disease.

[0056] The invention also concerns the use of the lipid-based nanoparticle or of the pharmaceutical composition disclosed herein, in the manufacture or a medicament for treating a disease, preferably selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an auto-immune disease.

[0057] The invention also concerns a method for treating a disease, preferably selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an auto-immune disease, in a subject in need thereof, wherein the method comprises administering to said subject a therapeutic amount of the lipid-based nanoparticle according of the pharmaceutical composition disclosed herein.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] Definitions

[0060] 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.

[0061] 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.

[0062] 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. The percent identity between two amino acid sequences or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences or nucleotide sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as the BLASTN program for nucleic acid or amino acid sequences using as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. Alternatively, sequence identity can also be typically determined using sequence analysis software Clustal Omega using the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960, with the default settings.

[0063] By "position" as used herein is meant a location in the sequence of a protein or a number of a residue in the sequence of a protein, preferably such as a Fc domain or Fc chain. Positions may be numbered sequentially, or according to an established format, for example the EU index as in Kabat. For heavy chain constant region, such as CH2 and CH3 domains, amino acid positions discussed in the invention, numbering is according to the Eu index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1): 78-85 describing the amino acid sequence of the myeloma protein Eu, which reportedly was the first human IgGl sequenced. The Eu index of Edelman is also set forth in Kabat. The terms "Eu index as set forth in Kabat", "Eu index of Kabat", "Eu index", "Eu numbering" or “Eu nomenclature” 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). For example, position 297 is position in human IgGl.

[0064] 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. The preferred amino acid modification herein is a substitution. 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. For example, the substitution N297A refers to a variant polypeptide, in this case a Fc variant, in which the asparagine at position 297 is replaced with an alanine. 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. 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:

[0065] Table A - Amino Acid Residue

[0066] Table B - Alternative Conservative Amino Acid Residue Substitution Groups

[0067] Table C - Further Alternative Physical and Functional Classifications of Amino Acid Residues

[0068] As used herein, the term "isolated" indicates that the recited material (e.g., antibody or fragment thereof) 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.

[0069] 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.

[0070] 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.

[0071] 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%, +1-9.5%). 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%).

[0072] 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.

[0073] The term “at least one” means “one or more” or “one or several”. For instance, it refers to one, two, three or more.

[0074] Targeting moieties, Fc domains and antigen binding domains

[0075] The invention disclosed herein relates to a lipid-based nanoparticle comprising a targeting molecule, wherein said targeting molecule comprises or consists of an antigen binding domain of an antibody covalently linked to a nonglycosylated Fc domain.

[0076] By “targeting molecule” or “targeting moiety” it is meant a molecule, in particular a protein that is designed to bind or interact with a particular target or antigen. The targeting moiety envisioned herein typically comprises a) an antigen binding domain of an antibody, particularly such as described below, b) optionally a hinge domain and / or a peptide linker, particularly such as described below, c) a nonglycosylated Fc domain particularly such as described below.

[0077] Therefore, the LNP of the invention is preferably a targeted lipid nanoparticle (t-LNP). Each of the component of the targeting moiety are further described here below.

[0078] Fc domain

[0079] In a first aspect the invention concerns a lipid-based nanoparticle comprising an antigen binding domain of an antibody covalently linked to a nonglycosylated Fc domain.

[0080] As used herein, the terms “fragment crystallizable region” “Fc region” or “Fc domain” are interchangeable and refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors. The Fc region or domain is typically composed of two domains derived from the second and third constant domains of antibody's heavy chains. A Fc chain typically comprises a CH2 domain and a CH3 domain, preferably covalently linked to each other. Portion of the Fc domain refers to the CH2 or the CH3 domain.

[0081] The Fc domain of the invention is typically dimeric. Accordingly, the Fc domain comprises or consists of two Fc chains, the Fc chains being able to form a dimeric Fc domain. The dimeric Fc domain can be a homodimer, each Fc chain being identical or essentially identical. Alternatively, the dimeric Fc domain can be a heterodimer, each Fc chain being different and complementary in order to promote the formation of the heterodimeric Fc domain. Heterodimeric Fc domains are made by altering the amino acid sequence of each Fc chain. The heterodimeric Fc domains rely on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and / or allow for ease of purification of heterodimers over the homodimers. Typically, amino acid variants that lead to the production of heterodimers are referred to as "heterodimerization variants". Heterodimerization variants can include steric variants (e.g. the "knobs and holes" or "skew" variants described below and the "charge pairs" variants described below) as well as "pi variants", which allows purification of homodimers away from heterodimers. WO2014 / 145806, hereby incorporated by reference in its entirety, discloses useful mechanisms for heterodimerization include "knobs and holes", "electrostatic steering" or "charge pairs", pi variants, and general additional Fc variants.

[0082] In some aspects, the Fc domain is an Fc domain of a monoclonal antibody.

[0083] In some aspects, the Fc domain is an IgG Fc domain, preferably a human or humanized IgG Fc domain.

[0084] By "IgG" as used herein is meant a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class typically comprises IgGl, lgG2, lgG3 and lgG4. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, "CH" domains in the context of IgG are as follows: "CHI" refers to positions 118-215 according to the EU index as in Kabat. "Hinge" refers to positions 216-230 according to the EU index as in Kabat. "CH2" refers to positions 231-340 according to the EU index as in Kabat, and "CH3" refers to positions 341-447 according to the EU index as in Kabat.

[0085] Preferably, the Fc domain is an IgGl, IgG2, IgG3 or IgG4 Fc domain, preferably an IgGl or IgG4 Fc domain, even more preferably an IgGl Fc domain.

[0086] Preferably, the Fc domain comprises or consists of CH2 and CH3 domains of an IgGl, IgG2, IgG3 or IgG4, preferably of an IgGl or IgG4, even more preferably of an IgGl .

[0087] The Fc domain of the invention is a nonglycosylated Fc domain, preferably a N-nonglycosylated Fc domain.

[0088] As used herein the term “ nonglycosylated”, “non-glycosylated” or “ unglycosylated “ refers to proteins, particularly antibodies, more particularly Fc domains, that do not have or are not linked to a glycan (sugar). This can occur naturally or be achieved through genetic engineering or production in systems that lack glycosylation machinery. This term encompasses aglycosylated and deglycosylated protein, such as aglycosylated or deglycosylated Fc domains. Preferably, the Fc domain of the invention does not comprise a N-glycosylation site or are not N- glycosylated

[0089] Preferably, the nonglycosylated Fc domain according to the invention, in particular the N- nonglycosylated Fc domain, does not comprise N-Acetylglucosamine (GlcNAc) and / or N-glycan.

[0090] Preferably, the nonglycosylated Fc domains, in particular the N-nonglycosylated Fc domain, to the invention have a low glycan site occupancy.

[0091] As used herein, “glycan site occupancy” refers to the proportion of a specific glycosylation site on a protein, such as an antibody or Fc domain, that is glycosylated. Preferably, the nonglycosylated Fc domains have a glycan site occupancy of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 4%, 3%, 2% or 1%. Preferably, the nonglycosylated Fc domains have a glycan site occupancy of less than 20%, preferably less than 10%, even more preferably less than 5%.

[0092] Glycan occupancy can be determined by methods known to the man skilled in the art, for example Mass Spectrometry (MS)-based methods, such as LC-MS / MS. Further suitable methods are described in Zhu et al., J Am Soc Mass Spectrom. 2014 Jun;25(6): 1012-7. Glycan site occupancy can alternatively be determined by calculating a ratio between nonglycosylated Fc domains and glycosylated Fc domains. In some aspects, the Fc domain is aglycosylated, preferably N- aglycosylated. Preferably, the Fc domain is an aglycosylated IgGl Fc domain, preferably a N- aglycosylated IgGl Fc domain.

[0093] As used herein, "aglycosylated" means a protein, such as a Fc domain, that is expressed or produced without carbohydrate, for example by mutation of one or more residues that encode the glycosylation pattern or by expression in an organism that does not attach carbohydrates to proteins, for example bacteria.

[0094] In some aspects, the Fc domain comprise one or more mutation(s) of one or more residues that encode the glycosylation pattern in the CH2 and / or CH3 domain, preferably in the CH2, even more preferably at position 297, 298 and / or 299 of the CH2 domain according to Eu numbering.

[0095] Typically, the aglycosylated Fc domain of the invention does not have the carbohydrate chains attached at position N297 of standard IgG Fc domains. Preferably, the Fc domain of the invention does not comprise a N-glycosylation site or are not N-glycosylated.

[0096] In particular, the N297 mutation envisioned herein does not necessarily occur at position 297 of a specific antibody. Although the Fc domains are highly conserved, small sequence differences can alter the residue numbering. For example, the position of the glycosylation site in the CH2 domain may depend on the size of the CDRs of the antibody. The man skilled in the art is familiar with this concept: the glycosylation site is indeed on an asparagine (N), but glycosylation occurs on the N of an NxS or NxT motif, where x is any residue except proline (P). For example, the glycosylation site may be reported as N297 in IgGl according to EU numbering, but in other immunoglobulin subclasses or according to other numbering methods (e.g., Clothia or Kabat numbering), it could be N293, N295 or N300, for example. The exact amino acid position of the glycosylation site can easily be determined by the man skilled in the art, for example by identifying the NxS or NxT motif described above in the sequence of said antibody, in particular in the vicinity of amino acid position 297, and / or by aligning the sequence of a specific antibody of interest with an IgGl Fc domain (where N is at position 297), particularly thanks to the NxS or NxT motif described above.

[0097] In particular, the aglycosylated Fc domain of the invention, preferably an IgGl or IgG4 Fc domain, i) does not comprise an Asparagine (N) at position 297 and / or ii) has an amino acid residue at position 298 and / or 299 that reduces or eliminates glycosylation at position 297. Additionally or alternatively, the aglycosylated Fc domain of the invention i) does not comprise a Serine (S) at position 298 and / or ii) does not comprise a Serine (S) or a Threonine (T) at position 299.

[0098] In another embodiment, the aglycosylated Fc domain of the invention, preferably an IgGl or IgG4 Fc domain, i) does not comprise a Serine (S) or a Threonine (T) at position 299 and / or ii) has an amino acid residue at position 297 and / or 298 that reduces or eliminates glycosylation at position

[0099] 297. Additionally or alternatively, the aglycosylated Fc domain of the invention i) does not comprise an Asparagine (N) at position 297 and / or ii) does not comprise a Serine (S) at position

[0100] 298.

[0101] In another embodiment, the aglycosylated Fc domain of the invention, preferably an IgGl or IgG4 Fc domain, i) comprises a Proline (P) at position 298 and / or ii) does not comprises an amino acid residue at position 297 and / or 298 that reduces or eliminates glycosylation at position 297. Additionally or alternatively, the aglycosylated Fc domain of the invention i) does not comprise an Asparagine (N) at position 297 and / or ii) does not comprise a Serine (S) or a Threonine (T) at position 299.

[0102] By “does not have a (N, T or S) residue” or “does not comprise a (N, T or S) residue” at a particular position in an amino acid sequence, it means that such amino acid is absent at this position, for example by amino acid mutation, such as substitution.

[0103] In some aspects, the aglycosylated Fc domain of the invention does not comprise an Asparagine (N) at position 297 (i.e., in the CH2 domain according to Eu nomenclature). Typically, such Fc domain comprises an amino acid mutation, preferably a substitution at position 297 according to Eu nomenclature. Aglycosylated antibodies can be produced by different methods, including the modification of the amino acid sequence at the positions 297-299 to prevent the attachment of glycans. The man skilled in the art is well aware of biomolecular techniques that allows to introduce mutations in a parent sequence, typically starting with introducing mutation in the polynucleotides parent sequence.

[0104] Preferably, the aglycosylated Fc domain comprises an amino acid substitution, preferably in the CH2 domain selected from the group consisting of N297A, N297D, N297Q, N297G, N297F, N297K, N297R, N297H, N297V, N297L, N297I, N297G, N297P, N297E, according to Eu nomenclature.

[0105] In some preferred aspects, the aglycosylated Fc domain comprises an amino acid substitution, preferably in the CH2 domain, selected from the group consisting of from the group consisting of N297A, N297Q, N297D and N297G, according to Eu nomenclature.

[0106] Preferably, the Fc domain of the invention is an IgGl Fc domain comprising an amino acid substitution, preferably in the CH2 domain, selected from the group consisting of N297A, N297D, N297Q, N297G, N297F, N297K, N297R, N297H, N297V, N297L, N297I, N297G, N297P, N297E according to Eu nomenclature, preferably from the group consisting of N297A, N297Q and N297G, preferably a N297A substitution.

[0107] In some aspects, the nonglycosylated Fc domain comprises a CH2 domain that comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 54 or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and a N297A or N297D substitution, respectively. Preferably, said CH2 domain is covalently linked to a CH3 domain that comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 57 or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0108] In some aspects, the nonglycosylated Fc domain comprises a CH2 domain that comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 61, or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and the N297A or N297D substitution, respectively. Preferably, said CH2 domain is covalently linked to a CH3 domain that comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 62 or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0109] In some aspects, the nonglycosylated Fc domain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 59 or 63, or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and the N297A or N297D substitution, respectively.

[0110] In some aspects, the aglycosylated Fc domain does not comprise a serine at position 298 according to Eu numbering.

[0111] In some aspects, the aglycosylated Fc domain comprises an amino acid substitution in the CH2 domain selected from the group consisting of S298A, S298G, S298D, S298E, S298Q, S298K, S298R, S298I, S298F, S298M, S298Y, S298W, S298H, preferably S298A or S298G. Any of these substitutions can be combined with any of the N297 substitutions described above.

[0112] In some aspects, the aglycosylated Fc domain does not comprise a threonine at position 299 according to Eu numbering.

[0113] In some aspects, the aglycosylated Fc domain comprises an amino acid substitution in the CH2 domain selected from the group consisting of T299D, T299E, T299N, T299Q, T299K, T299R, T299L, T299F, T299M, T299Y, T299W, T299P, T299G, preferably T299A or T299G. Any of these substitutions can be combined with any of the S298 and / or N297, preferably S298 substitutions described above.

[0114] In some aspects, the aglycosylated Fc domain comprises an amino acid substitution S298G / T299A or S298A / T299G according to Eu nomenclature. Preferably, said Fc domain has an asparagine at position 297. Alternatively, said Fc domain does not have an asparagine at position 297 (i.e., typically comprise an amino acid substitution at position 297, preferably as described above).

[0115] In some aspects, if the aglycosylated Fc domain comprises an amino acid mutation at position 298 and / or 299 according to Eu numbering, said Fc domain does not comprise an amino acid mutation at position 297. Typically, if said aglycosylated Fc domain does not comprise a serine at position 298 and / or a threonine at position 299, said Fc domain comprises an asparagine at position 297. Alternatively, said Fc domain may not have an asparagine at position 297.

[0116] In some aspects, preferably wherein the aglycosylated Fc domain is an IgG2 or IgG3, the Fc domain comprises a N392 mutation, preferably a N392 substitution. Preferably, said amino acid substitution, preferably in the CH2 domain, is selected from the group consisting of N392A, N392D, N392Q, N392G, N392F, N392K, N392R, N392H, N392V, N392L, N392I, N392G, N392P, N392E according to Eu nomenclature, preferably from the group consisting of N392A, N392Q and N392G, preferably is a N392A substitution. Any of these substitutions can be combined with any of the N297, S298 and / or T299 substitutions described above. Preferably, the N-aglycosylated Fc domain has a level of glycosylation that is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in comparison to the level of glycosylation of a wild-type Fc domain, preferably a Fc domain that has an asparagine in position 297 in the amino acid sequence of the CH2 domain according to EU numbering, more preferably that does not have one or more of the above-mentioned amino acids substitutions.

[0117] Typically, the N-aglycosylated Fc domain has a level of glycosylation of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. Preferably, the N- aglycosylated Fc domain has a level of glycosylation of less than 10%, preferably of less than 5%, more preferably of less than 2%.

[0118] The man skilled in the art is aware of methods for measuring glycosylation levels, especially in antibodies. For example, suitable methods include Mass Spectrometry (MS)-based methods, such as liquid chromatography (HPLC), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), nuclear magnetic resonance (NMR), matrix- assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), ultraperformance liquid chromatography (UPLC), and liquid chromatography (LC)-electrospray ionization (ESI)-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS / MS), or Glycan Profiling Assay Reagent Kit with detection on LabChip GXII Touch HT (protein analyser), or ELISA.

[0119] In some aspects, the Fc domain is deglycosylated, preferably N-deglycosylated. Preferably, the Fc domain is deglycosylated IgGl Fc domain, preferably a N-deglycosylated IgGl Fc domain.

[0120] As used herein, “deglycosylated” proteins, such as antibodies or Fc domains, are proteins that originally had glycans attached but have had them enzymatically or chemically removed. Typically, deglycosylated Fc domains are Fc domains that have been submitted to an enzymatic deglycosylation, resulting in the cleavage of the glycosylation motif attached to the N297 residue.

[0121] Preferably, the Fc domain of the invention has gone through enzymatic deglycosylation. The man skilled in the art is well aware of deglycosylation methods for proteins and antibodies, such as using a PNGase F (peptide:N-glycosidase F) or a Protein Deglycosylation Mix (e.g., NEB, Catalog # P6044S).

[0122] Preferably, the N-deglycosylated Fc domain has a level of glycosylation that is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, in particular in comparison to the level of glycosylation of a wild-type Fc domain, preferably a Fc domain that has not gone through enzymatic deglycosylation, for example prior to the use of deglycosylation methods such as described above.

[0123] Preferably, the Fc domain of the invention does not comprise a N-glycosylation site or are not N- glycosylated. Preferably, the Fc domain of the invention does not comprise a O-glycosylation site or are not O-glycosylated.

[0124] Preferably, the Fc domain of the invention i) does not comprise a N-glycosylation site or are not N-glycosylated and ii) does not comprise a O-glycosylation site or are not O-glycosylated.

[0125] Typically, the Fc domain of the invention are N-deglycosylated and O-deglycosylated.

[0126] Preferably, the deglycosylated Fc domain, preferably N-deglycosylated and O-deglycosylated Fc domain, has a level of glycosylation that is reduced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in comparison to the level of glycosylation of a wild-type Fc domain, preferably a Fc domain that has not gone through enzymatic deglycosylation, for example prior to the use of deglycosylation methods such as described above.

[0127] Typically, the deglycosylated Fc domain has a level of glycosylation of less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. Typically, the deglycosylated Fc domain has a level of glycosylation of less than 20%, preferably less than 10%, even more preferably less than 5%.

[0128] In some particular aspects, the Fc domain is further modified to increase the binding to FcRn, thereby increasing the half-life of the bifunctional molecule. In another aspect or additional aspect, the Fc domain is modified to decrease the binding to FcyR, thereby reducing ADCC or CDC, or to increase the binding to FcyR, thereby increasing ADCC or CDC. The alteration of amino acids near the junction of the Fc portion and the non-Fc portion can dramatically increase the serum half-life of the Fc fusion protein as shown in WO 01 / 58957.

[0129] In some aspects, the nonglycosylated IgGl Fc domain further comprises a substitution or a combination of substitutions selected from the group consisting of T250Q / M428L; M252Y / S254T / T256E + H433K / N434F; E233P / L234V / L235A / G236A + A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257VQ311; K326W / E333S; S239D / I332E / G236A;

[0130] L234A / L235A; P329G; M252Y / S254T / T256E; K322A and K444A, preferably selected from the group consisting of M252Y / S254T / T256E, and L234A / L235A optionally with P329G.

[0131] In some aspects, the nonglycosylated IgG4 Fc domain further comprises a substitution or a combination of substitutions selected from the group consisting of S228P; L234A / L235A; L234A / L235A / P329G, P329G, S228P + M252Y / S254T / T256E, K444A K444E, K444D, K444G and K444A. Even more preferably, the bifunctional molecule, preferably the binding moiety, comprises an IgG4 Fc-region with a S228P substitution that stabilizes the IgG4. In some embodiments, the non-glycosylated Fc domain is IgG4 Fc-region with a S228P substitution.

[0132] As mentioned herein, when referring to amino acid mutations, the and “+” refer to mutations that are cumulative. Thus, by the mutation S228P + M252Y / S254T / T256E, it is meant the following mutations : S228P, M252Y, S254T and T256E.

[0133] In some aspects, the non- glycosylated Fc domain is a knob-into-hole Fc domain.

[0134] Preferably, the non- glycosylated Fc domain is a knob-into-hole heterodimeric Fc domain comprising i) a first Fc chain comprising the substitutions T366W / S354C and ii) a second Fc chain comprising the substitutionsT366S / L368A / Y407V / Y349C.

[0135] In some aspects, the non-glycosylated Fc domain comprises i) a first Fc chain that comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 66 or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and ii) a second Fc chain that comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 67 or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0136] In some aspects, wherein the antigen binding domain is a monovalent, the binding domain is covalently linked to only one chain of the non-glycosylated domain of the invention. Preferably, the first Fc chain is covalently linked to the antigen binding domain and the second Fc chain is devoid / not link to an antigen binding domain.

[0137] In some aspects, wherein the nonglycosylated Fc domain is a knob-into-hole and the antigen binding domain is a monovalent, the binding domain is preferably covalently linked in N-terminal of the hole Fc chain.

[0138] In some aspects, the nonglycosylated Fc domain of the invention i) is not covalently bound to any of the lipids of the lipid-based nanoparticle, ii) does not comprise any modification for coupling or grafting the nonglycosylated Fc domain to a lipid. Typically, the Fc domain does not comprise an additional cysteine, in particular in C-terminus of the Fc domain.

[0139] Alternatively, the nonglycosylated Fc domain i) is covalently bound to a lipid of the LNP, preferably a PEG-lipid, or ii) comprises a modification for coupling or grafting the nonglycosylated Fc domain to a lipid. For example, the Fc domain can be conjugated with a lipid by chemical reaction, for example a maleimide thiol reaction. Typically, the Fc domain comprises an additional cysteine, preferably in C-terminus of the Fc domain for covalent linkage with a lipid of the LNP, preferably a PEG-lipid. and linker

[0140] In some aspects, the antigen binding domain is covalently linked to the Fc domain by a hinge domain or a peptide linker.

[0141] In some aspects, the antigen binding domain is covalently linked to the Fc domain by a hinge domain.

[0142] The “hinge domain”, “hinge region” or “D domain” of an antibody is a flexible region that typically connects the Fab region to the Fc region. The hinge region typically contains a series of amino acids, including proline, cysteine, and glycine, which contribute to its flexibility and ability to form disulfide bonds. The “Hinge" preferably refers to amino acid positions 216-230 according to the EU index.

[0143] The hinge region can be derived from an immunoglobulin heavy chain, e.g., IgGl, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human or humanized IgGl, IgG2, IgG3, IgG4. More preferably, the hinge region is derived from a human or humanized IgGl or IgG4 heavy chain. The hinge may be of the same IgG class or different IgG class than the Fc domain. Preferably, the hinge is an IgGl hinge and the Fc domain is an IgGl Fc domain. Alternatively, the hinge is an IgG4 hinge and the Fc domain is an IgG4 Fc domain.

[0144] The IgGl hinge region has three cysteines, two of which are involved in disulfide bonds between the two heavy chains of the immunoglobulin. These same cysteines permit efficient and consistent disulfide bonding formation between Fc portions. Therefore, a preferred hinge region of the present invention is derived from IgGl, more preferably from human IgGl. In some aspects, the first cysteine within the human IgGl hinge region is mutated to another amino acid, preferably serine. Preferably, the IgGl hinge domain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 64, or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0145] The hinge region of IgG4 is known to form interchain disulfide bonds inefficiently. However, a suitable hinge region for the present invention can be derived from the IgG4 hinge region, preferably containing a mutation that enhances correct formation of disulfide bonds between heavy chain-derived moieties (Angal S, et al. (1993) Mol. Immunol., 30: 105-8). More preferably, the hinge region is derived from a human IgG4 heavy chain. Preferably, the IgG4 hinge domain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 65, or a variant thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some aspects, the hinge domain is an unglycosylated hinge, preferably a aglycosylated or deglycosylated hinge. Preferably, the hinge domain is not O-glycosylated or does not comprise a O-glycosylation site / residue. Preferably, the hinge domain is not N-glycosylated or does not comprise a N-glycosylation site / residue.

[0146] In some aspects, the antigen binding domain is covalently linked to the unglycosylated Fc domain, preferably by a peptide linker. For example, when the antigen binding domain is a scFV, said scFV can be directly linked to the a nonglycosylated Fc domain or can be linked to the a nonglycosylated Fc domain via a peptide linker.

[0147] As used herein, the term "linker" or “peptide linker” refers to a sequence of at least one amino acid. Such a linker may be useful to prevent steric hindrances. The linker is usually 3-44 amino acid residues in length. Preferably, the linker has 3-30 amino acid residues. In some aspects, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues.

[0148] One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Other examples are poly-alanine linker sequences. Preferably, the linker is a flexible linker. Examples of flexible linkers are Glycine-Serine linker, Gly cine-Proline linker, Proline-Rich linker, (GGGS)n (e.g., SEQ ID NO: 40, 47 or 48), linker and (GGGGS)n linker (e.g., SEQ ID NO: 49-52), wherein n indicates the number of repeats of the motif and is an integer selected from 1-10. In some embodiments, the linker is selected from the group consisting of (GGGGS)2(SEQ ID NO: 40), (GGGS)3(SEQ ID NO: 47), (GGGS)4(SEQ ID NO: 48), (GGGGS)4(SEQ ID NO: 49), (GGGGS)3(SEQ ID NO: 50), (GGGGS)2(SEQ ID NO: 51) and GGGGS (SEQ ID NO: 52) linker. Preferably, the linker between the antigen binding domain and the unglycosylated Fc domain of the invention is (GGGGS)3(SEQ ID NO: 50).

[0149] Antigen biding domain

[0150] The unglycosylated Fc domain of the invention is linked to an antigen binding domain of an antibody.

[0151] Preferably, the C-terminal end of the antigen biding domain is covalently linked to the N-terminal end of the Fc domain or Fc chain, typically via a hinge domain or a peptide linker.

[0152] Preferably the targeting moiety of the invention comprises or consists essentially of, from N- terminal to C-terminal : an antigen biding domain, optionally a hinge domain or a peptide linker and a nonglycosylated Fc domain such as described herein. As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. Preferably, the term "antibody" includes intact immunoglobulins and "antibody fragment" or "antigen binding fragment" (such as Fab, Fab', F(ab')2, Fv), single chain (scFv or scFab), CrossMAb, mutants thereof, molecules comprising an antibody portion, diabodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term antibody refers to a humanized antibody.

[0153] An "antibody heavy chain" as used herein, refers to the larger of the two types of polypeptide chains present in antibody conformations. The CDRs of the antibody heavy chain are typically referred to as “HCDR1”, “HCDR2” and “HCDR3”. The framework regions of the antibody heavy chain are typically referred to as “HFR1”, “HFR2”, “HFR3” and “HFR4”. These framework regions and complementary determining regions are preferably operably linked in the following order: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 (from amino terminus to carboxy terminus).

[0154] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in antibody conformations; K and A light chains refer to the two major antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as “LCDR1”, “LCDR2” and “LCDR3”. The framework regions of the antibody light chain are typically referred to as “LFR1”, “LFR2”, “LFR3” and “LFR4”. These framework regions and complementary determining regions are preferably operably linked in the following order: LFR1-LCDR1-LFR2- LCDR2-LFR3-LCDR3-LFR4 (from amino terminus to carboxy terminus).

[0155] Preferably, the antigen binding domain of the invention is or derived from a monoclonal antibody.

[0156] In some aspects, the antigen binding domain is or derived from a human or a humanized antibody. As used herein, the term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., chimeric antibodies that contain minimal sequence derived from a non-human antibody). 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.

[0157] In some aspects, the antigen binding domain is an unglycosylated antigen binding domain, preferably deglycosylated antigen binding domain. In particular, the framework regions of the antigen binding domain are unglycosylated, preferably deglycosylated. Alternatively or additionally, the CDR regions of the antigen binding domain are unglycosylated, preferably deglycosylated.

[0158] In some aspects, the antigen binding domain is antigen-binding fragment of an antibody, preferably of a human or humanized monoclonal antibody.

[0159] As used herein, an “antigen-binding fragment” or “antigen-binding domain” of an antibody means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody of the invention, that exhibits antigen-binding capacity for a particular antigen; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. Advantageously, the antigen-binding fragments have a similar binding affinity as the corresponding 4-chain antibodies. However, antigen-binding fragment that have a reduced antigen-binding affinity with respect to corresponding 4-chain antibodies are also encompassed within the invention. The antigen-binding capacity can be determined by measuring the affinity between the antibody and the target fragment. These antigen-binding fragments may also be designated as “functional fragments” or “function conservative fragments” of antibodies. Antigenbinding fragments of antibodies are preferably fragments which comprise their hypervariable domains designated CDRs (Complementary Determining Regions) or part(s) thereof.

[0160] Preferably, the antigen binding domain linked to the unglycosylated Fc domain of the invention is selected from the group consisting of a Fab, a Fab’, a F(ab')2, a Fv, a crossMAb Fab, a crossMAb Fab’, a crossMAb F(ab')2, a single-chain variable fragment (scFV) and a VHH. In some aspects, the antigen binding domain is a F(ab')2, so that when the antigen binding domain is covalently linked to a Fc domain of the invention, optionally via an hinge, it forms a full conventional bivalent antibody (i.e., 4 chain antibody).

[0161] In some aspects, the antigen binding domain is a Fab or a Fab’, so that when the antigen binding domain is covalently linked to a nonglycosylated Fc domain of the invention, optionally via an hinge or peptide linker, it forms a monovalent antibody. Preferably, the C-terminal end of the Fab or Fa’b is covalently linked to the N-terminal end of one chain of the Fc domain.

[0162] Preferably, the nonglycosylated Fc domain is a knob-into-hole and the Fab or Fa’b is covalently linked in N-terminal of the hole Fc chain.

[0163] In some aspects, the antigen binding domain is a scFv covalently linked to a nonglycosylated Fc domain of the invention, optionally via an hinge or peptide linker. Preferably, the C-terminal end of the scFV is covalently linked to the N-terminal end of one chain of the Fc domain.

[0164] Preferably, the nonglycosylated Fc domain is a knob-into-hole and the scFv is covalently linked in N-terminal of the hole Fc chain.

[0165] In some aspects, the antigen binding domain is a CrossMAb covalently linked to a nonglycosylated Fc domain of the invention, optionally via an hinge or a peptide linker. Preferably, the C-terminal end of the FcrossMab is covalently linked to the N-terminal end of the Fc domain.

[0166] Preferably, the nonglycosylated Fc domain is a knob-into-hole and the crossMab is covalently linked in N-terminal of the hole Fc chain.

[0167] 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 binding 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.

[0168] In some aspects, the antigen binding domain specifically binds to a target expressed on immune cells surface, a tumor cell, an infected cell or on pathogens of the human body, particularly targets that are only or specifically expressed on immune cells. As used herein, the term “target” refers to a carbohydrate, lipid, peptide, polypeptide, protein, antigen or epitope that is specifically recognized or targeted 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 a cell. The term “specifically expressed” mean that the target is expressed on immune cells, but is not substantially expressed by other cell type, particularly such as tumoral cells.

[0169] With regard to the binding of an antibody to a target molecule, the terms "bind" or "binding" refer to peptides, polypeptides, proteins, fusion proteins and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it refers to an antigen-antibody type interaction. The terms "specific binding", "specifically binds to," "specific for," "selectively binds" and "selective for" a particular antigen (e.g., PD-1) or an epitope on a particular antigen (e.g., PD-1) mean that the antibody recognizes and binds a specific antigen, but does not substantially recognize or bind other molecules in a sample. Preferably, the term “specific binding” means the contact between an antibody and an antigen with a binding affinity equal or lower than 10-7 M. In certain aspects, antibodies bind with affinities equal or lower than 10-8 M, 10-9 M or 10-10 M.

[0170] 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).

[0171] 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 E6ZE7 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. M16789.1, M16790.1, M16791.1, M16792.1), 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 AccessionNos. 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.

[0172] Examples of antibodies targeting such TAAs are for example Rituximab and Trastuzumab.

[0173] 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.

[0174] Examples of antibodies targeting such pathogens are for example Duvratoxumab, Palivizumab, Raxibacumab, Bezlotoxumab and Ansuvimab

[0175] In an embodiment, the target of the antigen binding domain is selected from the group comprising or consisting of PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4- 1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, L0X1, SIGLEC 6, TACVTNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, CD160, CD127 and SIRPg.

[0176] The target expressed on activated immune cells can particularly be selected among the target described in Table D below.

[0177] Table D: Examples of targets of interest.

[0178]

[0179]

[0180] Preferably, the antigen biding domain specifically binds to a target selected from the group consisting of PD-1, TIM-3, CTLA-4, LAG-3, BTLA, TIGIT, CD160, CD127 and CLEC-1A, preferably from the group consisting of PD-1, CD127 and CLEC-1A. In some aspects, the antigen binding domain is or derives from an anti-TIM-3 antibody. Antibodies directed against TIM-3 targeting TIM-3 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.

[0181] In some aspects, the antigen binding domain is or derives from an anti-CTLA-4 antibody.

[0182] Antibodies directed against CTLA-4 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,

[0183] WO16196237, WO16130898, WO16015675, WO12120125, W009100140 and W007008463, the disclosure thereof being incorporated herein by reference.

[0184] In some aspects, the antigen binding domain is or derives from an anti-LAG-3 antibody.

[0185] Antibodies directed against LAG-3 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.

[0186] In some aspects, the antigen binding domain is or derives from an anti-BTLA antibody.

[0187] 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.

[0188] In some aspects, the antigen binding domain is or derives from an anti-TIGIT antibody.

[0189] 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, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8,

[0190] CHAN.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5, CHA.9.560.6, CHA.9.560.7,

[0191] CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4,

[0192] CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8 as disclosed in WO 19232484. Anti-TIGIT antibodies are also disclosed in WO 16028656,

[0193] W016106302, WO16191643, W017030823, W017037707, WO17053748, WO17152088,

[0194] WO18033798, WO18102536, WO18102746, W018160704, W018200430, WO18204363,

[0195] W019023504, WO19062832, WO19129221, WO19129261, WO19137548, WO19152574,

[0196] WO19154415, WO19168382 and WO19215728, the disclosure thereof being incorporated herein by reference.

[0197] In some aspects, the antigen binding domain is or derives from an anti-CD127 antibody. In some aspects, the target is CD127 or IL-7R and the antigen binding domain is specific to CD127, preferably human CD 127. Preferably, the antigen binding domain is an antagonist of CD 127.

[0198] 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 signalling, such interaction with IL-7 and / or elicitation of a cellular response to IL-7.

[0199] Antibodies directed against CD127 or IL7-R are also known in the art, such as GSK2618960, RN168, AbDl 1590, MAB306-100, R34.34, A019D5, eBioRDR5, 40131, 1 A12, M21, 47H4, HIL- 7R-M21, eBioYL8, RDR5. Anti-CD127 antibodies are also disclosed in W014102430, W020077190, W004000238, WO11104687, WO16059512 and WO17062748, the disclosure thereof being incorporated herein by reference.

[0200] In some aspects, the antigen binding domain is or derives from an anti-CLEC-lA antibody.

[0201] In some aspects, the antigen binding domain is specific to CLEC-1A, preferably human CLEC- 1 A. Preferably, the antigen binding domain is an antagonist of CLEC-1 A.

[0202] 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. Antibodies directed against CLEC-1 A are also known in the art, such as MAB 1704, ABIN526589, AF1704 and ABIN526590.

[0203] In some aspects, the antigen binding domain is or derives from an anti-PD-1 antibody.

[0204] As used herein, the terms "Programmed Death 1 ", "Programmed Cell Death 1 ", “PD-1”, "PDCD1 ", “PD-1 antigen”, “human PD-1”, "hPD-1" and "hPD-1" are used interchangeably and refer to the Programmed Death-1 receptor, also known as CD279, and include variants and isoforms of human PD-1, and analogs having at least one common epitope with PD-1. PD-1 is a key regulator of the threshold of immune response and peripheral immune tolerance. It is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. As an example, the amino acid sequence of a human PD-1 is disclosed under GenBank accession number NP 005009. PD-1 has four splice variants expressed on human Peripheral blood mononuclear cells (PBMC). Accordingly, PD-1 proteins include full-length PD-1, as well as alternative splice variants of PD-1, such as PD-lAex2, PD- 1 Aex3, PD-1 Aex2,3 and PD-1 Aex2,3,4. 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.

[0205] Several anti-PD-1 are already clinically approved, and others are still in clinical developments. For instance, the anti-PD-1 antibody can be selected from the group consisting of Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS- 936558, ONO-4538), OSE-279 (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 (Anno), 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.

[0206] In some aspects, the target is PD-1 and 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-PD-1 antibody is Pembrolizumab (also known as Keytruda lambrolizumab, MK-3475), Nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538) or OSE-279 (such as described in WO2020 / 127366, the disclosure thereof being incorporated herein by reference).

[0207] 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. Preferably, said antigen binding domain further comprises or is covalently linked to a Fc domain, preferably an IgG Fc domain such as described herein.

[0208] 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.

[0209] Then, in some aspects, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0210] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0211] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein:

[0212] - 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;

[0213] - 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;

[0214] - 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;

[0215] - the light chain CDR1 (LCDR1) comprises or consists of an amino acid sequence of SEQ ID NO:

[0216] 4, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof,;

[0217] - the light chain CDR2 (LCDR2) comprises or consists of an amino acid sequence of SEQ ID NO:

[0218] 5, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; and - 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.

[0219] 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.

[0220] In one embodiment, 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.

[0221] Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0222] (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

[0223] (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.

[0224] In some aspects, the anti-PD-1 antigen-binding domain comprises or consists essentially of: (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence SEQ ID NO: 15, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof;

[0225] (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.

[0226] Particularly, the modifications are outside of the CDRs (i.e., are in the FR).

[0227] Preferably, the anti-PD-1 antigen-binding domain comprises or consists essentially of:

[0228] (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.

[0229] In some aspects, the antigen-binding domain, preferably the anti-PDl antigen binding domain comprises or consists of VH, VL, CHI and a CL domains, so that the antigen binding domain is typically a Fab, a Fab’ or a scFV.

[0230] In some aspects, the heavy chain constant domain (CHI) of the antigen binding domain linked to a non-glycosylated IgGl Fc domain of the invention 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.

[0231] In some aspects, the heavy chain constant domain (CHI) of the antigen binding domain linked to a non-glycosylated IgGl Fc domain of the invention comprises or consists essentially of SEQ ID NO: 17 or a variant thereof having at least 80%, 85%, 90%, 95%, 97% or 99% sequence identity thereto.

[0232] In some aspects, the heavy chain constant domain (CHI) of the antigen binding domain linked to a aglycosylated IgG4 Fc domain comprises or consists essentially of SEQ ID NO: 68, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0233] In some aspects, the heavy chain constant domain (CHI) of the antigen binding domain linked to a non-glycosylated IgG4 Fc domain of the invention comprises or consists essentially of SEQ ID NO: 68 or a variant thereof having at least 80%, 85%, 90%, 95%, 97% or 99% sequence identity thereto. 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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 68 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.

[0238] In some aspects, the antigen binding domain is an anti-PD-1 Fab or F(ab’)2, comprising or consisting of i) a heavy 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 and of ii) a light 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.

[0239] In some aspects, the antigen binding domain is an anti-PD-1 Fab or F(ab’)2, comprising or consisting of i) a heavy chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 69 optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof and of ii) a light 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.

[0240] In some aspects, the antigen binding domain comprises an anti-PD-1 CrossMAb comprising or consisting of i) a chain comprising or consisting of VH and CL domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 16 and 17, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0241] In some aspects, the antigen binding domain comprises an anti-PD-1 CrossMAb comprising or consisting of i) a chain comprising or consisting of VH and CL domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 15 and 18, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof; ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 16 and 68, respectively, optionally with one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0242] In some aspects, the antigen binding domain comprises an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID NOs: 15 and 18, respectively, and ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 16 and 17, respectively.

[0243] In some aspects, the antigen binding domain comprises an anti-PD-1 CrossMAb, comprising or consisting of i) a chain comprising or consisting of a VH domain and a CL domain, said domains having the amino acid sequence as set forth in SEQ ID NOs: 15 and 18, respectively, and ii) a chain comprising or consisting of VL and CHI domains, said domains having the amino acid sequence as set forth in SEQ ID NOs: 16 and 68, respectively.

[0244] In some aspects, 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 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.

[0245] In some aspects, the antigen binding domain is an anti-PD-1 scFV, preferably comprising or consisting of a sequence as set forth in SEQ ID NO: 39, or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, or having one, two or three modification(s) selected from substitution(s), addition(s), deletion(s) and any combination thereof.

[0246] Preferably, 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.

[0247] Alternatively, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0248] (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0249] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein

[0250] HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 23, HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 24, HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 25, LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 26, LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 28,

[0251] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0252] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 29;

[0253] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 30.

[0254] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising: (i) a heavy chain variable domain comprising HCDR1, HCDR2 and HCDR3, and

[0255] (ii) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein

[0256] HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 31, HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 32, HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 33, LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 34, LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 35, and

[0257] LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 36,

[0258] Preferably, the antigen binding domain is an anti-PD-1 antigen-binding domain comprising:

[0259] (a) a heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO: 37;

[0260] (b) a light chain variable region (VL) comprising or consisting of an amino acid sequence of SEQ ID NO: 38.

[0261] In some aspects, the antigen binding domain is or is derived from 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: 19 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 20; an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 69 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 20; an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 43 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 44; or an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 45 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 46.

[0262] Preferably, the antigen binding domain is an antagonist of PD-1 comprising or consisting of an heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 19 and a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0263] Typically, the covalent linkage between the antigen binding domain and the nonglycosylated Fc domain, preferably N-nonglycosylated Fc domain, form a targeting moiety, typically an antibody, especially a full length antibody. Particularly, any one of the antigen binding domain disclosed herein can be linked to any one of the nonglycosylated Fc domain, preferably N-nonglycosylated Fc domain, to form a targeting moiety, typically an antibody, especially a full length antibody.

[0264] In some instances, the targeting moiety comprising the antigen binding domain disclosed herein covalently linked to the nonglycosylated Fc domain disclosed herein is an anti-PD-1 antibody comprising a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 70 and a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 20.

[0265] In some instances, the targeting moiety comprising the antigen binding domain disclosed herein covalently linked to the nonglycosylated Fc domain disclosed herein is an anti-PD-1 antibody comprising a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 71 and a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 20.

[0266] Lipid-based nanoparticles

[0267] The lipid-based nanoparticle according to the invention is particularly formulated either as a liposome or a lipid nanoparticle (LNP), especially a lipid nanoparticle comprising a mixture of lipids.

[0268] 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.

[0269] The elements of a LNP may be selected based on a particular application or target, and / or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a lipid-based nanoparticle may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combination of elements.

[0270] The lipid-based nanoparticles of the disclosure can particularly be generated using components, compositions, and methods as generally known in the art, for example such as disclosed in WO2017049245; WO2017112865; WO2017218704; WO2015164674; WO2017031232; WO2017099823; WO2016118724; WO2016118724; WO2017223135; WO2014152211; WO2015038892; W02017049074; W02013090648; W02017180917; WO2017075531 and WO2017117528 all of which are incorporated by reference herein in their entirety.

[0271] The manufacture of LNP s is well described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Then, 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[0272] In some aspects, the method for obtaining the lipid-based nanoparticles of the invention is as described under the “Examples” section below. In particular, the method for obtaining the lipid- based nanoparticles of the invention is as described in PCTZEP2024 / 058775.

[0273] In particular, in the preparation of the LNP according to the invention, a first composition comprising the lipids and a second composition comprising the antigen binding domain of an antibody covalently linked to a N-nonglycosylated Fc domain are mixed, for example in a microfluidic device.

[0274] In said second composition, the proportion of antibodies comprising a N-nonglycosylated Fc domain can typically of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the total of antibodies.

[0275] Therefore, in some embodiments, the LNP comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of an antigen binding domain of an antibody covalently linked to a N-nonglycosylated Fc domain.

[0276] Preferably, the lipid-based nanoparticle comprises full length antibodies wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of said antibodies comprises a N- nonglycosylated Fc domain. More preferably, the lipid-based nanoparticle comprises full length antibodies wherein at least at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of said antibodies comprises a N-nonglycosylated Fc domain.

[0277] Preferably, the LNP comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of an antibody comprising a N-nonglycosylated Fc domain as described herein. Preferably, the LNP comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of antibodies comprising a N-nonglycosylated Fc domain as described herein.

[0278] In particular, said percentage is the number of the antibodies comprising the nonglycosylated Fc domain divided by the total number of antibodies comprised in the LNP. Kinetics of antibody glycosylation and antibodies concentrations (total, glycosylated and nonglycosylated forms) can be easily determined by the man skilled in the art, for example using Mass Spectrometry (MS)- based methods, such as UHPLC-MS and / or the automated photometric analyzer Gallery from Thermo Fisher Scientific.

[0279] In a particular aspect, the lipid-based nanoparticle 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). Such lipids can typically be comprised in various mole percent or molar ratio (mol%).

[0280] In some aspects, the lipid-based nanoparticle according to the invention comprises one or more ionizable or cationic lipid(s). As used herein, the term “ionizable or cationic lipid” refers to a lipid molecule positively charged in an acidic environment.

[0281] 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.

[0282] 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.

[0283] 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), l,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), l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-di oxolane (DLin-K-XTC2-DMA), 2, 2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-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- 1-amine (Octyl-CLinDMA), l,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2- 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), l-[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. 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, WO 2016 / 021683 or WO 2019 / 131839 all of which are incorporated by reference herein in their entirety.

[0284] 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., C 14-98, CIS- 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.

[0285] Particularly, the lipid composition of the lipide-based nanoparticle according to the invention comprises an ionizable or cationic lipid selected from the group consisting of [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2 -hexyl decanoate) (ALC-0315), heptatri aconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), and bis[2-(4-{2-[4-(cis- 9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide (SS-OP; e.g., CAS number 2377474-67-2) and any mixtures thereof.

[0286] 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. Preferably, the ionizable or cationic lipid comprised in the lipid-based nanoparticle is ALC-0315. Alternatively, the ionizable or cationic lipid comprised in the lipid-based nanoparticle is SS-OP.

[0287] In some aspects, the ionizable or cationic lipid represents from about 5 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle according to the invention.

[0288] In some aspects, the ionizable or cationic lipid, preferably ALC-0315 or SS-OP, represents from about 40 mol % to about 60 mol %, preferably rom about 45 mol % to about 55 mol %, even more preferably from about 48 mol % to about 52 mol % of the total lipids present in the lipid composition of the lipid-based nanoparticle according to the invention. More 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 according to the invention.

[0289] In some aspects, the lipid-based nanoparticle according to the invention comprises a helper lipid. As used herein, the term “helper lipid” refers to a class of lipid molecules that increases particle stability, fluidity tolerability and / or biodistribution of lipid-based nanoparticles. 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.

[0290] 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

[0291] (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), l,2-di-0- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn-glycero-3- phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,l,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), ethyl phosphatidylcholine (EPC), l-oleoyl-2-hydroxy-sn-glycero-3 -phosphocholine (18 : 1 Lyso PC), 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), 1- 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

[0292] (NBD-PE), N-(lisamineRhodamine B sulfonyl)-phosphatidylethanolamine (Rh-PE), l-oleoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine (18 :1 Lyso PE), 1.2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-methyl (18 : 1 Monomethyl PE), 1.2-dioleoyl-sn-glycero-3 - phosphoethanolamine-N,N-dimethyl (18 : 1 Dimethyl PE), 1 ,2-dioleoyl-sn-glycero-3 - phosphoethanolamine-N-(hexanoylamine) (18 : 1 Caproylamine PE), 1 ,2-dioleoyl-sn-glycero-3 - phosphoethanolamine-N-(biotinyl) (18 : 1 Biotinyl PE), sn-(3-oleoyl-2-hydroxy)-glycerol-l- phospho-sn-l'-(3'-oleoyl-2'-hydroxy)-glycerol (BMP-S,S), sn-(3-(9Z-octadecenoyl)-2-hydroxy)- glycerol- l-phospho-sn-3'-(l'-(9Z-octadecenoyl)-2'-hydroxy)-glycerol (BMP-S,R), 1,2-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), 1,2- 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),

[0293] 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), Dimethyldioctadecylammonium (18:0 DDAB), l,2-dioleyloxy-3 -dimethylaminopropane (DODMA), l,2-dioleoyl-3- dimethylammonium-propane (DODAP), l,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),

[0294] 1.2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and any mixtures or combinations thereof.

[0295] In some embodiments, 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. Preferably, the helper lipid is selected from the group consisting of from the group consisting of DOPE, DOPC, DDAB, POPE and DSPC, and any combinations thereof.

[0296] In some embodiments, the helper lipid is DOPE. Alternatively, the helper lipid is DSPC.

[0297] In some aspects, the helper lipid represents from about 5 mol% to about 100 mol%, from about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0298] In some aspects, the helper lipid, preferably DOPE or DSPC, represents from about 5 mol% to about 15 mol%, preferably from about 8 mol% to about 12 mol%, of the total lipids present in the lipid-based nanoparticle of the invention. Typically, the helper lipid, preferably DOPE or DSPC, represents about 10 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0299] In some aspects, the lipid-based nanoparticle disclosed herein comprises one or more molecules comprising polyethylene glycol (PEG). Accordingly, the lipid-based nanoparticle may comprise PEG or PEG-modified lipids.

[0300] 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.

[0301] In a particular aspect, the LNP comprises a PEG that is not functionalized, which is a PEG that does not comprises any reactive species at its end, said reactive species being usable to conjugate a target moiety such as an antibody or a fragment thereof to the PEG.

[0302] In some aspects, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22 (C14, C15, C16, C17, C18, C19, C20, C21 or C22), preferably from about C14 to about C18, even more preferably from about C14 to about C16. In some aspects, the lipid moiety of the PEG-lipids is a C14 or a C18.

[0303] In some aspects, 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.

[0304] In some aspects, the PEG-lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG- dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2-dimyristyloxlpropyl-3-amine (PEG-c- DMA) is ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG), PEG- c-DOMG, PEG-DMG, DMG-PEG-2000, PEG-DLPE, PEG- DMPE, PEG-DPPC and PEG-DSPE.

[0305] Preferably, the PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, PEG- c- DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG and PEG-c-DMA, ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG), and any mixture thereof; particularly from the group consisting of PEG-DMG, PEG-DSPE, ALC-0159 and any mixture thereof.

[0306] 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.

[0307] Preferably, the PEG has a size comprised between 2000 Daltons and 5000 Daltons (i.e., PEG-2000 to PEG-5000). In a particular aspect, the PEG has a size of about 2000. Alternatively, the PEG has a size of about 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, preferably is DSPE-PEG-2000, DMG- PEG-2000, DSPE-PEG-5000, DMG-PEG-5000 or any mixture thereof

[0308] In a very particular aspect, the PEG-lipid is PEG 5000-DMG. In another very particular aspect, the PEG-lipid is PEG 5000-DSG.

[0309] In a very particular 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.

[0310] In some aspects, the PEG lipid represents from about 0.5 mol% to about 50 mol%, from about 1 mol% to about 25 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 100 mol%, about 10 mol% to about 100 mol%, or about 15 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0311] Optionally, the PEG lipid in the lipid-based nanoparticle is within the range from about 0.5 mol% to about 2.5 mol% of the total lipids present in the nanoparticle, 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, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 mol%, especially 1.5 mol%.

[0312] In some aspects, the PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 0.5 mol% to about 5 mol%, preferably from about 0.5 mol% to about 2.5 mol%, even more preferably from about 1 mol% to about 2 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0313] 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 of the invention.

[0314] In some aspects, the PEG lipid comprises a reactive group, especially a functional reactive group, that allows for conjugation to the Fc domain of the invention. Preferably, said reactive group is selected from a thiol reagent, a maleimide reagent, or click chemistry reagent, e.g, a reagent selected from the group consisting of an alkyne reagent, such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent and an azide (AZ) reagent.

[0315] In some embodiments, the PEG lipid is selected from the group consisting of DSPE-PEG- maleimide, DSG-PEG-maleimide, DSPE-PEG-azide and DSG-PEG-azide.

[0316] Preferably, the PEG lipid is DSPE-PEG-maleimide. In some embodiments, the lipid base nanoparticle comprises a combination or mixture of PEG lipids, a first PEG-lipid comprising a reactive group for conjugation to a targeting moiety such as described above, and a second PEG lipid being devoid of it.

[0317] Therefore, in some of the LNP described herein the LNP may comprises a mixture of PEG lipids comprising or consisting of a non-conjugated PEG-lipid and a PEG lipid conjugated to a targeting moiety such as described herein.

[0318] In some embodiments, the PEG lipid comprised in the LNP of the invention is a combination of PEG lipids consisting of a) DSG-PEG or DMG-PEG, preferably DSG-PEG and b) DSG-PEG- maleimide or DSPE-PEG-maleimide, preferably DSPE-PEG-maleimide.

[0319] The man skilled in the art is aware that the maleimide group is a reactive group for conjugation with a targeting moiety, said maleimide group forming a stable covalent bond with thiol groups (- SH), which are present in the cysteine residues of Fc domains of antibodies. Therefore, when reference is made herein to PEG lipid comprising a reactive group such as maleimide, it should be understood that such PEG-lipid is intended to conjugation with a targeting moiety, said reactive group per se being transformed in a covalent bond upon coupling to the targeting moiety.

[0320] In some aspects, the lipid-based nanoparticle of the invention comprises one or more sterol. The sterol can particularly be selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixtures thereof. Preferably, the sterol is cholesterol.

[0321] In some aspects, the sterol represents from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, about 50 mol% to about 100 mol%, about 60 mol% to about 100 mol%, or about 70 mol% to about 100 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0322] In some aspects, the sterol, preferably cholesterol, represents from about 30 mol% to about 50 mol%, preferably from about 35 mol% to about 45 mol%, even more preferably from about 35 mol% to about 40 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0323] Particularly, the sterol, preferably cholesterol, represents about 38.5 mol% of the total lipids present in the lipid-based nanoparticle of the invention. Such sterol being preferably cholesterol.

[0324] In some aspects, the lipid-based nanoparticle comprises a lipid mixture. 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. In a preferred aspect, the lipid composition of the lipid-based nanoparticle of the invention comprises or consists of an ionizable or cationic lipid, a helper lipid, a sterol and a PEG lipid, these lipids being preferably as described above.

[0325] Preferably, in said lipid-based nanoparticle, the ionizable or cationic lipid is from about 10 mol % to about 70 mol % of the total lipids present in the nanoparticle, the helper lipid is from about 5 mol% to about 70 mol % of the total lipids present in the nanoparticle, the sterol is from about 10 mol% to about 70 mol% of the total lipids present in the nanoparticle, and the PEG lipid is from about 0.5 mol% to about 4 mol% of the total lipids present in the nanoparticle.

[0326] Particularly, the lipid-based nanoparticle comprises or consists of from about 35 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 30 mol% to about 60 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid.

[0327] Preferably, the lipid-based nanoparticle comprises or consists of from about 45 mol % to about 55 mol % of a cationic or ionizable lipid, from about 5 mol% to about 15 mol % of a helper lipid from about 35 mol% to about 45 mol% of a sterol, and from about 0.5 mol% to about 2.5 mol% of a PEG-lipid.

[0328] In a specific aspect, the lipid-based nanoparticle comprises [(4-hydroxybutyl)azanediyl]di(hexane- 6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) as ionizable or cationic lipid, 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE) as neutral lipid, cholesterol as sterol and one or more polyethylene glycol (PEG)-modified lipid(s).

[0329] In a specific aspect, the lipid-based nanoparticle comprises a cationic or ionizable lipid, a helper lipid, a structural lipid or sterol, a PEG-lipid that do not comprise a reactive group for conjugation to a targeting moiety and a PEG lipid comprising a reactive group for conjugation to a targeting moiety.

[0330] In a specific aspect, the lipid-based nanoparticle comprises a cationic or ionizable lipid, a helper lipid, a structural lipid or sterol, a non-conjugated PEG-lipid and a PEG lipid conjugated to a targeting moiety.

[0331] In order to have a biodistribution which favour the escape of capture organs in particular the liver, the non-conjugated PEG-lipid is preferably a C16-C22 PEG lipid, preferably a Cl 8 PEG (such as DSPE PEG, DSG PEG). In some embodiments, the PEG-lipid it is not a C14 PEG (such as DMG PEG). Said PEG lipid typically has stealth property allowing to escape said organs and this allow the LNP to reach blood circulation and targeted areas of interest, in particular tumoral tissues. The LNP of the invention is capable of very specifically targeting immune cells of interest locally in the target tissue, in particular thanks to the contribution of the PEG lipid conjugated to a targeting entity, in particular activated immune cells that are locally present in the tissue, such as PD1 positive immune cells (T cells) in tumoral tissue when using an anti-PDl antibody conjugated to the conjugated PEG lipid of the LNP.

[0332] In a very specific aspect, ALC-0315 is from about 35 mol % to about 55 mol % of the total lipids present in the LNP, DOPE is from about 5 mol% to about 20 mol % of the total lipids present in the LNP, cholesterol is from about 30 mol% to about 60 mol% of the total lipids present in the LNP, and the PEG2000-DSG is from about 0.5 mol% to about 4 mol% of the total lipids present in the LNP. Optionally, PEG2000-DSG is from about 0.5 mol% to about 2 mol% of the total lipids present in the LNP.

[0333] In some aspects, the lipid-based composition of the lipid-based nanoparticle comprises or consists of a lipid mixture selected from the group consisting of: a) ALC-0315, DOPE, cholesterol and DMG-PEG; b) ALC-0315, DDAB, cholesterol and DMG-PEG; c) ALC-0315, POPE, cholesterol and DMG-PEG; d) ALC-0315, DOPE, cholesterol and DSPE-PEG; e) ALC-0315, DSPC, cholesterol and DMG-PEG; f) ALC-0315, DSPC, cholesterol and ALC-0159; g) SM-102, DSPC, cholesterol and DMG-PEG; h) Dlin-MC3-DMA, DSPC, cholesterol and DMG-PEG; 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.

[0334] In some aspects, the lipid-based composition of the lipid-based nanoparticle comprises or consists of a lipid mixture comprising or consisting of: a) ALC-0315, DOPE, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; b) ALC-0315, DOPE, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; c) ALC-0315, DSPC, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; d) ALC-0315, DSPC, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; e) SS-OP, DOPE, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; f) SS-OP, DOPE, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; g) SS-OP, DSPC, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; h) SS-OP, DSPC, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; i) Dlin-MC3-DMA, DOPE, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; j) Dlin-MC3-DMA, DOPE, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; k) Dlin-MC3-DMA, DSPC, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; l) Dlin-MC3-DMA, DSPC, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; m) SM-102, DOPE, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; n) SM-102, DOPE, cholesterol, DMG-PEG and DSPE-PEG-Maleimide; o) SM-102, DSPC, cholesterol, DSG-PEG and DSPE-PEG-Maleimide; or p) SM-102, DSPC, cholesterol, DMG-PEG and DSPE-PEG-Maleimide;

[0335] In some aspects, the lipid composition of the LNP according to the invention comprises:

[0336] - An ionizable lipid selected from the group consisting of ALC-0315, SM-102, MC3-DLin- DMA and SS-OP, preferably between 40% and 55% of the total lipids present in the LNP;

[0337] A helper lipid which is DOPE or DSPC, preferably between 5% and 15% of the total lipids present in the LNP,;

[0338] Cholesterol, preferably between 35% and 45% of the total lipids present in the LNP,;

[0339] A PEG lipid which is DMG-PEG or DSG-PEG, preferably between 1% and 2% of the total lipids present in the LNP, optionally in combination with a DSPE-PEG-Maleimide, preferably between 0.05% and 0.2% of the total lipids present in the LNP.

[0340] In some aspects, the lipid composition of the LNP according to the invention comprises or consists of: a) ALC-0315: about 50% / DOPE: about 10% / Cholesterol: about 38.5 % / DMG- PEG: about 1.5 %; b) SM-102 : about 50% / DSPC : about 10% / Cholesterol : about 38.5 % / DMG- PEG : about 1.5%; c) MC3-DLin-DMA: about 50% / DSPC : about 10% / Cholesterol: about 38.5 % / DMG-PEG about 1.5%; d) ALC-0315 : about 46.3% / DSPC : about 9.4% / Cholesterol : about 42.7 % / ALC-0159 : about 1.6%; e) SS-OP: about 50% / DSPC : about 10% / Cholesterol: about 38.5 % / DSPE-PEG : about 1.5%; f) ALC-0315: about 50% / DOPE: about 10% / Cholesterol: about 38.5 % / DMG- PEG: about 1.4 % / DSPE-PEG-Maleimide : about 0.1 %, g) SS-OP: about 50% / DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.4 % / DSPE-PEG-Maleimide : about 0.1 %; or h) ALC-0315: about 50% / DOPE or DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.5 % / DSPE-PEG-Maleimide : about 0.1 %.

[0341] In some aspects, the lipid composition of the LNP according to the invention comprises or consists of ALC-0315: about 50% / DOPE and / or DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.5 % / DSPE-PEG-Maleimide : about 0.1 %.

[0342] In a very specific aspect, the lipid composition of the LNP according to the invention comprises:

[0343] ALC-00315, SM-102, Dlin-MC3-DMA or SS-OP or any mixture thereof, preferably 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,

[0344] DOPE, DDAB, DOPC, POPE or DSPC or any mixture thereof, preferably DSPC, preferably 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,

[0345] Cholesterol preferably 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

[0346] - PEG 2000-DSG, PEG 2000-DMG, PEG 5000-DSG, PEG 5000-DMG ALC-0159 or any mixture thereof, preferably 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.

[0347] In a very specific aspect, the lipid composition of the LNP according to the invention comprises:

[0348] A ionizable lipid selected from the group consisting of ALC-00315, SM-102, Dlin-MC3- DMA or SS-OP or any mixture thereof, preferably 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,

[0349] A helper lipid selected from the group consisting of DOPE, DDAB, DOPC, POPE or DSPC or any mixture thereof, preferably DOPE or DSPC, preferably 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, A sterol such as 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,

[0350] - A first PEG lipid selected from the group consisting of PEG-DSG, PEG-DSPE, PEG- DMG, and ALC-0159 or any mixture thereof, preferably PEG-DSG, preferably from about 0.5 mol% to about 2.5 mol%, preferably from about 1 mol% to about 2 mol%, more preferably of about 1.5 mol% of the total lipids present in the LNP; and

[0351] - A second PEG lipid selected from the group consisting of DSPE-PEG-maleimide, DSG- PEG-maleimide, DSPE-PEG-azide and DSG-PEG-azide, preferably DSPE-PEG- maleimide preferably from about 0.05 mol% to about 1 mol%, preferably from about 0,08 mol% to about 1,2 mol%, more preferably of about 0.1 mol% of the total lipids present in the LNP.

[0352] In a very specific aspect, the lipid-based composition comprises or consists of:

[0353] 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

[0354] 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,

[0355] 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

[0356] 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.

[0357] In a very specific aspect, the lipid-based composition comprises or consists of:

[0358] ALC-0315 from about 35 mol % to about 55 mol % of the total lipids present in the LNP, preferably from about 45 mol % to about 50 mol %, more preferably of about 50 mol% of the total lipids present in the LNP; ALC-0159 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,

[0359] 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 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.

[0360] In a very specific aspect, the lipid-based composition comprises or consists of:

[0361] ALC-0315 from about 35 mol % to about 55 mol % of the total lipids present in the LNP, preferably from about 45 mol % to about 50 mol %, more preferably of about 50 mol% of the total lipids present in the LNP;

[0362] 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,

[0363] 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 and PEG 2000-DSG and / or PEG 2000-DMG, preferably PEG 2000-DMG, 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.

[0364] In a very specific aspect, the lipid-based composition comprises or consists of:

[0365] SM-102 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;

[0366] 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, 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 and PEG 2000-DMG 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.

[0367] In a very specific aspect, the lipid-based composition comprises or consists of:

[0368] MC3-DLin-DMA 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;

[0369] 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,

[0370] 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 and PEG 2000-DMG 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.

[0371] The lipid-based nanoparticle according to the invention may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, imaging, or for the conjugation of the antigen binding domain to the LNP.

[0372] In some aspects, the functionalized lipids comprises polyethylene glycol (PEG). Accordingly, the lipid-based nanoparticle may comprise functionalized PEG or PEG-modified lipids.

[0373] In some aspects, the lipid moiety of the functionalized PEG-lipids includes those having lengths of from about C14 to about C22 (C14, C15, C16, C17, C18, C19, C20, C21 or C22), preferably from about C 14 to about C 18, even more preferably from about C 16 to about C 18. In some aspects, the lipid moiety of the functionalized PEG-lipids is a C18.

[0374] In some aspects, the functionalized PEG-lipid is selected from the group consisting of a functionalized PEG-modified phosphatidylethanolamine, a functionalized PEG-modified phosphatidic acid, a functionalized PEG-modified ceramide, a functionalized PEG-modified dialkylamine, a functionalized PEG-modified diacylglycerol, a functionalized PEG-modified dialkylglycerol, and mixtures thereof.

[0375] In some aspects, the PEG-lipid of functionalized PEG-lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-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) is ALC-0159 (N,N-dimyristylamide of 2- hydroxyacetic acid, O-pegylated to a PEG), PEG-c-DOMG, PEG-DMG, DMG-PEG-2000, PEG- DLPE, PEG- DMPE, PEG-DPPC and PEG-DSPE.

[0376] Preferably, the PEG-lipid of the functionalized PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, PEG-c- DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DPPE, PEG-DAG and PEG-c-DMA, ALC-0159 (N,N-dimyristylamide of 2-hydroxyacetic acid, O- pegylated to a PEG), and any mixture thereof; particularly from the group consisting of PEG- DMG, PEG-DSPE, ALC-0159 and any mixture thereof.

[0377] In some aspects, a functionalized 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.

[0378] Preferably, the PEG of the functionalized PEG lipid has a size comprised between 2000 Daltons and 5000 Daltons (i.e., PEG-2000 to PEG-5000). In a particular aspect, the PEG has a size of about 2000 Daltons. Alternatively, the PEG has a size of about 5000 Daltons.

[0379] 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, preferably is DSPE-PEG-2000, DMG- PEG-2000, DSPE-PEG-5000, DMG-PEG-5000 or any mixture thereof.

[0380] In a very particular aspect, the functionalized PEG-lipid is PEG 5000-DMG. In another very particular aspect, the functionalized PEG-lipid is PEG 5000-DSG. In a very particular aspect, the functionalized PEG-lipid is PEG 2000-DMG. In another very particular aspect, the functionalized PEG-lipid is PEG 2000-DSG. In another very particular aspect, the functionalized PEG-lipid is ALC-0159.

[0381] In some aspects, the functionalized PEG lipid represents from about 0.1 mol% to about 1.5 mol%, from about 0.2 mol% to about 1 mol%, about 0.4 mol% to about 0.6 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0382] Optionally, the functionalized PEG lipid in the lipid-based nanoparticle is within the range from about 0.1 mol% to about 1 mol% of the total lipids present in the nanoparticle, for instance about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mol%, especially 0.5 mol%.

[0383] In some aspects, the functionalized PEG lipid, preferably PEG-DMG or PEG DSPE, represents from about 0.1 mol% to about 1.5 mol%, from about 0.2 mol% to about 1 mol%, about 0.4 mol% to about 0.6 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0384] Preferably, the functionalized PEG lipid, preferably PEG-DMG or PEG DSPE, represents about 0.5 mol% of the total lipids present in the lipid-based nanoparticle of the invention.

[0385] In this embodiment, the lipid composition of the lipid-based nanoparticle of the invention comprises or consists of an ionizable or cationic lipid, a helper lipid, a sterol, a PEG lipid and a functionalized PEG lipid, these lipids being preferably as described above.

[0386] 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 poly(ethylene 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.

[0387] In some aspects, the lipid based particle comprises a poloxamine and / or a poloxamer.

[0388] In some aspects, the lipid based particle comprises a polyethyleneimine, rotamine and / or polyaspartamide.

[0389] In some aspects, a surface altering agent may be disposed within a lipid-based nanoparticle of the invention and / or on the surface of the lipid-based nanoparticle (e.g., by coating, adsorption, covalent linkage, or other process). Surface altering agents include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin b4, dornase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase).

[0390] The LNP of the invention may optionally comprise one or more coatings. For example, the LNP may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a LNP such as described herein may have any useful size, tensile strength, hardness, or density.

[0391] In an embodiment, 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. In some aspects, the lipid-based nanoparticle does not comprise a moiety comprising a lipidation peptide or motif.

[0392] 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.

[0393] As used herein, the term “anchoring moiety” or "anchoring molecule” or “anchoring entity” refers to a component that anchors or attaches the antigen binding domain into the lipid-based nanoparticle. Preferably, the anchoring moiety is a protein.

[0394] 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.

[0395] 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.

[0396] Lipidation peptide or motif can involve different types of lipid modifications, including: cysteine prenylation (e.g., the attachment of hydrophobic isoprene polymers such as famesyl 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, GP 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.

[0397] 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.

[0398] 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.

[0399] Preferably, the lipid based nanoparticle or antigen binding domain does not comprise a moiety comprising a lipidation peptide or motif, preferably that comprises or consists of the amino acid sequence : CDNSSS (SEQ ID NO: 41) or CDQSSS (SEQ ID NO: 42). In some aspects, 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 antigenbinding 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 antigenbinding domain, even preferably to about 75%, even more preferably to about 80%.

[0400] Lipid-based nanoparticles or a composition comprising LNPs may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a LNP or of a composition comprising LNPs. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a LNP or of a composition comprising LNPs, such as particle size, poly dispersity index, and zeta potential.

[0401] Physiochemical properties of lipid-based nanoparticles may be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs.

[0402] In one aspect, the mean size of the lipid-based nanoparticle of the invention may be between 10 of nm and 200 of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 200 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm or 200nm.

[0403] 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 embodiments, the mean size of the lipid- based nanoparticle is about 150 nm.

[0404] 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.

[0405] The zeta potential of a lipid-based nanoparticle 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 lipid-based nanoparticle 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.

[0406] Here below, are provided examples of specific targeting molecules comprising the antigen binding domain and Fc domain of the invention that are illustrative and non-limiting.

[0407] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein said targeting moiety comprises or consists of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 19; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and iii) a Fc domain heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 59, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0408] Preferably, the targeting moiety is bivalent, so that it comprises two of the above heavy chain and light chain.

[0409] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein said targeting moiety comprises or consists of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 69; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 65, and iii) a Fc domain comprising or consisting of a sequence as set forth in SEQ ID NO: 63, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0410] Preferably, the targeting moiety is bivalent, so that it comprises two of the above heavy chain and light chain.

[0411] Alternatively, the targeting moiety is monovalent.

[0412] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a monovalent targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein said targeting moiety comprises or consists of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 69; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 65, and iii) a first Fc chain comprising or consisting of a sequence as set forth in SEQ ID NO: 63, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20; c) a heavy chain comprising or consisting of i) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 65, and ii) a second Fc chain comprising or consisting of a sequence as set forth in SEQ ID NO: 63.

[0413] Typically, the first and second Fc chain form a homodimer. Preferably, the second Fc chain is devoid / not link to an antigen binding domain.

[0414] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a monovalent targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein said targeting moiety comprises or consists of: a) a first heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 19; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and iii) a first Fc chain comprising or consisting of a sequence as set forth in SEQ ID NO: 59, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20; c) a second heavy chain comprising or consisting of i) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and ii) a second Fc chain comprising or consisting of a sequence as set forth in SEQ ID NO: 59.

[0415] Typically, the first and second Fc chain form a homodimer.

[0416] Alternatively, the Fc domain is an heterodimeric Fc domain, in particular a knob into hole Fc domain, wherein the first Fc chain comprises or consists of a sequence as set forth in SEQ ID NO: 66 and wherein the second Fc chain comprises or consists of a sequence as set forth in SEQ ID NO: 67.

[0417] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a monovalent targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein said targeting moiety comprises or consists of: a) a first heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 19; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and iii) a first Fc chain comprising or consisting of a sequence as set forth in SEQ ID NO: 67, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20; c) a second heavy chain comprising or consisting of i) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and ii) a second Fc chain comprising or consisting of a sequence as set forth in SEQ ID NO: 66.

[0418] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of scFV covalently linked to an unglycosylated Fc domain, wherein said targeting moiety comprises or consists of i) a scFV comprising or consisting of a sequence as set forth in SEQ ID NO: 39, ii) optionally a peptide linker, preferably comprising or consisting of a sequence as set forth in SEQ ID NO: 50 and iii) a dimeric Fc domain comprising a first Fc chain and a second Fc chain, each chain comprising or consisting of a sequence as set forth in SEQ ID NO: 59 or 63, respectively. In some aspects, the first and second Fc chains share the same SEQ ID number. Alternatively, the Fc domain is knob into hole Fc domain, wherein the first Fc chain comprises or consists of a sequence as set forth in SEQ ID NO: 66 and wherein the second Fc chain comprises or consists of a sequence as set forth in SEQ ID NO: 67.

[0419] In some embodiments, where the targeting moiety is a scFV, the targeting moiety may be bivalent or monovalent (i.e., comprises one or two scFV)

[0420] In some aspects, the LNP of the invention comprises or consists essentially of: a) a combination of lipids comprising or consisting of:

[0421] - an ionizable lipid;

[0422] - a helper lipid;

[0423] -a sterol;

[0424] - a mixture of PEG lipids, said mixture comprising a first PEG-lipid that is not linked to a targeting moiety and a second PEG-lipid that is covalently linked to a targeting moiety, said targeting moiety comprising an antigen binding domain of an antibody, preferably of an anti-PD-1 antibody, covalently linked to a nonglycosylated Fc domain such as described herein; and b) optionally a nucleic acid molecule, preferably one or more mRNA molecule(s).

[0425] In some aspects, the LNP of the invention comprises or consists essentially of: a) a combination of lipids comprising or consisting of:

[0426] - a cationic or ionizable lipid, preferably between 5 and 55 mol% of the total lipids present in the LNP, preferably 50 mol%;

[0427] - a helper lipid, preferably between 5 and 15% mol% of the total lipids present in the LNP, preferably 10 mol%; -a structural lipid or sterol, preferably between 35% and 40% mol% of the total lipids present in the LNP, preferably 38.5 mol%;

[0428] - a non-conjugated PEG-lipid PEG, preferably between 0.5% and 2.5% mol% of the total lipids present in the LNP, preferably 1.5 mol%;

[0429] - a PEG lipid, preferably between 0.1 a 0.5 mol% of the total lipids present in the LNP, conjugated to c) a targeting moiety comprising an antigen binding domain of an antibody, preferably of an anti-PD-1 antibody, covalently linked to a nonglycosylated Fc domain such as described herein; and b) optionally a nucleic acid molecule, preferably a mRNA,

[0430] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein: i) the lipid based nanoparticle comprises or consists of the following combination of lipids: a) an ionizable lipid selected from the group consisting of ALC-0315, SM-102, MC3-DLin- DMA and SS-OP, preferably between 40 and 55 mol% of the total lipids present in the LNP; b) a helper lipid which is DOPE or DSPC, preferably between 5 and 15 mol% of the total lipids present in the LNP; c) cholesterol, preferably between 35 and 45 mol% of the total lipids present in the LNP; d) a PEG lipid which is DMG-PEG or DSG-PEG, preferably between 1 and 2 mol% of the total lipids present in the LNP, optionally in combination with a DSPE-PEG-Maleimide, preferably between 0.05 and 0.2 mol% of the total lipids present in the LNP; and ii) the targeting moiety comprises or consists of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 19; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and iii) a Fc domain heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 59 , and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0431] Preferably, the targeting moiety is bivalent, so that it comprises two of the above heavy chain and light chain. Preferably, when the LNP comprises a DSPE-PEG-Maleimide in the composition of lipids, the targeting moiety is conjugated / covalently linked to the DSPE-PEG.

[0432] Preferably, said LNP further comprises a nucleic acid molecule, such as one or more mRNA molecules.

[0433] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein: i) The lipid based nanoparticle comprises or consists of the following combination of lipids: a) an ionizable lipid selected from the group consisting of ALC-0315, SM-102, MC3-DLin- DMA and SS-OP, preferably between 40 and 55 mol% of the total lipids present in the LNP; b) a helper lipid which is DOPE or DSPC, preferably between 5 and 15 mol% of the total lipids present in the LNP; c) cholesterol, preferably between 35 and 45 mol% of the total lipids present in the LNP; d) a PEG lipid which is DMG-PEG or DSG-PEG, preferably between 1 and 2 mol% of the total lipids present in the LNP, optionally in combination with a DSPE-PEG-Maleimide, preferably between 0.05 and 0.2 mol% of the total lipids present in the LNP; and ii) the targeting moiety comprises or consists of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 69; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 65, and iii) a Fc domain heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 63, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0434] Preferably, the targeting moiety is bivalent, so that it comprises two of the above heavy chain and light chain.

[0435] Preferably, when the LNP comprises a DSPE-PEG-Maleimide in the composition of lipids, the targeting moiety is conjugated / covalently linked to the DSPE-PEG.

[0436] Preferably, said LNP further comprises a nucleic acid molecule, such as one or more mRNA molecules. In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein: i) the lipid based nanoparticle comprises or consists of the following combination of lipids: a) ALC-0315: about 50% / DOPE: about 10% / Cholesterol: about 38.5 % / DMG-PEG: about 1.5 %; b) SM-102 : about 50% / DSPC : about 10% / Cholesterol : about 38.5 % / DMG-PEG : about 1.5%; c) MC3-DLin-DMA: about 50% / DSPC : about 10% / Cholesterol: about 38.5 % / DMG-PEG about 1.5%; d) ALC-0315 : about 46.3% / DSPC : about 9.4% / Cholesterol : about 42.7 % / ALC- 0159 : about 1.6%; e) SS-OP: about 50% / DSPC : about 10% / Cholesterol: about 38.5 % / DSPE-PEG : about 1.5%; f) ALC-0315: about 50% / DOPE: about 10% / Cholesterol: about 38.5 % / DMG-PEG: about 1.4 % / DSPE-PEG-Maleimide : about 0.1 %, g) SS-OP: about 50% / DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.4 % / DSPE-PEG-Maleimide : about 0.1 %; or h) ALC-0315: about 50% / DOPE or DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.5 % / DSPE-PEG-Maleimide : about 0.1 %; and ii) the targeting moiety comprises or consists of an anti-PD-1 antigen binding domain of an antibody covalently linked to a nonglycosylated Fc domain such as described herein, said targeting moiety preferably comprising or consisting of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 19; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 64, and iii) a Fc domain heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 59, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0437] Preferably, the targeting moiety is bivalent, so that it comprises two of the above heavy chain and light chain. Preferably, when the LNP comprises a DSPE-PEG-Maleimide in the composition of lipids, the targeting moiety is conjugated / covalently linked to the DSPE-PEG.

[0438] Preferably, said LNP further comprises a nucleic acid molecule, such as one or more mRNA molecules.

[0439] In some embodiments, the invention concerns a lipid-based nanoparticle comprising a targeting moiety comprising or consisting of an antigen binding domain covalently linked to an unglycosylated Fc domain, wherein: i) the lipid based nanoparticle comprises or consists of the following combination of lipids: a) ALC-0315: about 50% / DOPE: about 10% / Cholesterol: about 38.5 % / DMG-PEG: about 1.5 %; b) SM-102 : about 50% / DSPC : about 10% / Cholesterol : about 38.5 % / DMG-PEG : about 1.5%; c) MC3-DLin-DMA: about 50% / DSPC : about 10% / Cholesterol: about 38.5 % / DMG-PEG about 1.5%; d) ALC-0315 : about 46.3% / DSPC : about 9.4% / Cholesterol : about 42.7 % / ALC- 0159 : about 1.6%; e) SS-OP: about 50% / DSPC : about 10% / Cholesterol: about 38.5 % / DSPE-PEG : about 1.5%; f) ALC-0315: about 50% / DOPE: about 10% / Cholesterol: about 38.5 % / DMG-PEG: about 1.4 % / DSPE-PEG-Maleimide : about 0.1 %, g) SS-OP: about 50% / DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.4 % / DSPE-PEG-Maleimide : about 0.1 %; or h) ALC-0315: about 50% / DOPE or DSPC: about 10% / Cholesterol: about 38.5 % / DSG-PEG: about 1.5 % / DSPE-PEG-Maleimide : about 0.1 %; and ii) the targeting moiety comprises or consists of an anti-PD-1 antigen binding domain of an antibody covalently linked to a nonglycosylated Fc domain such as described herein, said targeting moiety preferably comprising or consisting of: a) a heavy chain comprising or consisting of: i) a Fab heavy chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 69; ii) optionally a hinge domain comprising or consisting of a sequence as set forth in SEQ ID NO: 65, and iii) a Fc domain heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 63, and b) a light chain comprising or consisting of Fab light chain of an anti-PD-1 antibody comprising or consisting of a sequence as set forth in SEQ ID NO: 20.

[0440] Preferably, the targeting moiety is bivalent, so that it comprises two of the above heavy chain and light chain.

[0441] Preferably, when the LNP comprises a DSPE-PEG-Maleimide in the composition of lipids, the targeting moiety is conjugated / covalently linked to the DSPE-PEG.

[0442] Preferably, said LNP further comprises a nucleic acid molecule, such as one or more mRNA molecules.

[0443] Nucleic acid molecules

[0444] In some aspects, the lipid-based nanoparticle of the invention comprises one or more isolated nucleic acid molecule(s).

[0445] The nucleic acid molecule(s) can be DNA molecule(s) and / or RNA molecule(s).

[0446] In particular, the nucleic acid molecule(s) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 DNA or RNA molecule(s) encoding 1, 2, 3, 4, 5, 6,7 8, 9 or 10 different protein(s), respectively.

[0447] In some aspects, 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, circRNA.

[0448] In some aspects, the nucleic acid molecules 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.

[0449] In order to assess the expression of the nucleic acid molecules, 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.

[0450] 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.

[0451] Preferably the one or several nucleic acid molecule(s) are one or several mRNA molecules. Optionally, the mRNA can be a circular RNA, in particular a circular mRNA, especially as described in WO2014 / 186334 and WO2022 / 261490, incorporated herein by reference.

[0452] In some preferred aspects, 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).

[0453] The lipid-based nanoparticle of the invention particularly includes an mRNA encoding a polypeptide of interest capable of being translated by the immune cell to produce the polypeptide of interest. For example, the polypeptide encoded by the mRNA can be useful in the treatment of a disease or disorder such as cancer.

[0454] In a particular aspect, the lipid-based nanoparticle comprises a mRNA polynucleotide or a set of mRNA polynucleotides. The technology of mRNA polynucleotide is now well-known by the person skilled in the art, as illustrated in WO21159130, the disclosure thereof being incorporated herein by reference. Optionally, the mRNA can be a circular RNA, in particular a circular mRNA, especially as described in WO2014 / 186334 and WO2022 / 261490.

[0455] 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.

[0456] The amount of a mRNA molecule in a lipid-based nanoparticle may depend on the size, composition, desired target and / or application, or other properties of the lipid-based nanoparticle. For example, the amount of mRNA useful in the lipid-based nanoparticle may also depend on the size, sequence, and other characteristics of the mRNA. The relative amounts of a mRNA molecule and other elements (e.g., lipids) in a lipid-based nanoparticle may also vary. In some aspects, the wt / wt ratio of the lipid component to a mRNA molecule in a lipid-based nanoparticle may be from about 5: 1 to about 60: 1, such as 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18:1, 19: 1, 20: 1, 25: 1,30: 1,35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt / wt ratio of the lipid component to a mRNA molecule may be from about 10: 1 to about 40: 1. In certain aspects, the wt / wt ratio is about 20: 1. The amount of a mRNA molecule in a lipid-based nanoparticle may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy). 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: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio may be 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.

[0457] In particular, the lipid-based nanoparticle 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 immune cell proteins.

[0458] In some aspects, the mRNA molecule encodes for an immune cell inhibiting protein.

[0459] 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.

[0460] In some aspects, 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 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, preferably BIM or PUMA.

[0461] Alternatively, 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.

[0462] Alternatively, the mRNA molecule encodes for an immune cell activating protein.

[0463] 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. In some aspects, the activity-enhancing protein is selected from the group consisting of: TCF1, BCL2, BCLXL, TBET, Glutl, LGR6, ICOS, CD28, CD40L, 4- IBB, Perforin, CXCL9, CXCL10, GrB, 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, Integrin beta 1, Integrin beta 7 and Integrin alpha V. Particularly, the activity-enhancing protein is selected from the group consisting of: TCF1, BCL2, IL7, IL7R, CXCL9 and CXCL10, preferably from BCL2, IL7, IL7R, CXCL9 and CXCL10.

[0464] In some aspects, the mRNA encodes for a protein selected in Table E.

[0465] Table E. List of protein of interest.

[0466] Pharmaceutical compositions

[0467] The present invention also relates to pharmaceutical compositions comprising any of the lipid- based nanoparticles as described hereabove, preferably as the active ingredient or compound and optionally a pharmaceutically acceptable carrier or excipient.

[0468] As used herein, a “pharmaceutical composition” refers to a preparation of one or more of the active agents, such as comprising a LNP according to the invention, with optional other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism. Compositions of the present invention can be in a form suitable for any conventional route of administration or use. In one aspect, a “composition” typically intends a combination of the active agent, e.g., compound or composition, and a naturally-occurring or non-naturally-occurring carrier, inert or active, such as an adjuvant, diluent, binder, stabilizer, buffers, preservative or the like and include pharmaceutically acceptable carriers. An "acceptable vehicle" or “acceptable carrier” as referred to herein, is any known compound or combination of compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0469] The pharmaceutical compositions can be sterilized and, if desired, mixed with auxiliary agents such as pharmaceutically acceptable carriers, excipients, salts, antioxidant and / or stabilizers which do not deleteriously interact with the lipid-based nanoparticle of the invention and does not impart any undesired toxicological effects.

[0470] Particularly, the pharmaceutical composition according to the invention can be formulated for any conventional route of administration including a topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. Preferably, the pharmaceutical composition according to the invention is formulated for intravenous, intramuscular or subcutaneous administration. In some cases, LNPs may be administered directly to specific tissues or organs. For example, LNPs designed for targeted delivery to the lungs may be administered via inhalation or intratracheal injection for respiratory diseases.

[0471] To facilitate administration, the lipid-based nanoparticle as described herein can particularly be made into a pharmaceutical composition for in vivo administration. The means of making such a composition have been described in the art (see, for instance, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)). In yet another embodiment, the pharmaceutical composition is administered intranodally or intratum orally.

[0472] The pharmaceutical composition may be prepared by mixing a lipid-based nanoparticle having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, antioxidant, and / or stabilizers in the form of lyophilized formulations or aqueous solutions. Such suitable carriers, excipients, antioxidants, and / or stabilizers are well known in the art and have been for example described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0473] Preferably, the pharmaceutical composition comprising the lipid-based nanoparticle is relatively homogenous. A poly dispersity index may be used to indicate the homogeneity of the composition, e.g., the particle size distribution of the lipid-based nanoparticles comprised in the composition. A small (e.g., less than 0.3) poly dispersity index generally indicates a narrow particle size distribution. Preferably, the pharmaceutical composition has a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the pharmaceutical composition is from about 0.10 to about 0.25.

[0474] Pharmaceutical compositions according to the invention may be formulated to release the active ingredients (e.g. the lipid-based nanoparticle of the invention) substantially immediately upon administration or at any predetermined time or time period after administration. The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Means known in the art can be used to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.

[0475] It will be understood by one skilled in the art that the formulations of the invention may be isotonic with human blood that is the formulations of the invention have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity can be measured by, for example, a vapor pressure or ice-freezing type osmometer.

[0476] Pharmaceutical composition typically must be sterile and stable under the conditions of manufacture and storage. Prevention of presence of microorganisms may be ensured both by sterilization procedures (for example by microfiltration), and / or by the inclusion of various antibacterial and antifungal agents.

[0477] In some embodiments, the pharmaceutical composition includes one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, surface active agents, buffering agents and / or preservatives.

[0478] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, alginic acid, sodium alginate, cholesterol, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and / or combinations thereof.

[0479] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, free radical scavengers, antimicrobial preservatives, antifungal preservatives, alcohol preservatives and / or acidic preservatives.

[0480] Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite and / or sodium sulphite. Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, eidetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and / or trisodium edetate.

[0481] Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerine, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and / or sorbic acid.

[0482] Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulphite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMAB EN®II, NEOLONE™, KATHON™ and / or EUXYL®. An exemplary free radical scavenger includes butylated hydroxytoluene (BHT or butylhydroxytoluene) or deferoxamine.

[0483] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminium hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution and / or ethyl alcohol. In some embodiments, the pharmaceutical composition including a lipid-based nanoparticle according to the invention further includes a salt, such as a chloride salt. In some embodiments, the pharmaceutical composition including a lipid-based nanoparticle further includes a sugar such as a disaccharide. In some embodiments, the pharmaceutical composition further includes a sugar but not a salt, such as a chloride salt. In some embodiments, a pharmaceutical composition further includes one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E) or a sterol. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogues thereof).

[0484] The LNP of the invention can be formulated with amphiphilic polymers in a pharmaceutical composition. Common amphiphilic polymers used in the formulation of LNPs include polyethylene glycol (PEG) and polyvinyl alcohol (PVA).

[0485] Relative amounts of the lipid-based nanoparticles, pharmaceutically acceptable excipients and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. In some embodiments, the pharmaceutical composition comprises between 0.1% and 100% (wt / wt) of one or more lipid-based nanoparticles such as disclosed herein. The amount or number of lipid-based nanoparticles which can be combined with a carrier material to produce a single dosage form will generally be that amount of the lipid-based nanoparticles which produces a therapeutic effect.

[0486] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of the pharmaceutical composition including a lipid-based nanoparticle such as disclosed herein. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical composition. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and / or for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0487] In some aspects, the pharmaceutical composition comprises at least two different lipid-based nanoparticles comprising an antigen binding domain capable of specifically binding to a target expressed on activated immune cells surface and comprising one or several mRNA molecule(s) encoding an immune cell activity enhancing component.

[0488] Preferably, the pharmaceutical composition comprises at least two different lipid-based nanoparticles comprising an antigen binding domain capable of specifically binding to a target expressed on activated immune cells surface and comprising one or several mRNA molecule(s) encoding an immune cell activity inhibiting component, wherein the antigen binding domain is an antibody or an antigen binding fragment thereof and preferably comprises a Fc domain , preferably an IgG Fc domain.

[0489] In some particular embodiments, the pharmaceutical composition comprises at least two different lipid-based nanoparticles as described above, wherein none of the lipid-based nanoparticles comprises one or more antigen binding domain(s) that i) is / are not covalently bound to any of the lipids of the lipid-based nanoparticle, ii) does / do not comprise any modification for coupling or grafting the antigen binding domain to a lipid and / or iii) is / are not covalently bound to a lipidation peptide or motif.

[0490] Then, the pharmaceutical composition according to the invention may comprise:

[0491] A first lipid-based nanoparticle comprising a first antigen binding domain capable of specifically binding to a first target expressed on activated immune cells surface and one or several mRNA molecule(s) encoding an immune cell activity enhancing component; and

[0492] A second lipid-based nanoparticle comprising a second antigen binding domain capable of specifically binding to a second (different) target expressed on activated immune cells surface and one or several mRNA molecule(s) encoding an immune cell activity enhancing component.

[0493] Preferably, the one or several mRNA molecule(s) encoding an immune cell activity enhancing component may be the same or different in the first and second lipid-based nanoparticles.

[0494] In a preferred aspect, the antigen binding domain of the lipid-based nanoparticle and the antigen binding domain of the additional lipid-based nanoparticle are capable of specifically binding to the same target (e.g., PD-1) while each having mRNA molecule(s) encoding for a different immune cell activity enhancing component, respectively.

[0495] Preferably, the antigen binding domain of the first lipid-based nanoparticle and the antigen binding domain of the additional (second) lipid-based nanoparticle are capable of specifically binding to a different / non overlapping epitope of the same target (e.g., different epitopes of PD-1). Alternatively, the antigen binding domain of the first lipid-based nanoparticle and the antigen binding domain of the additional (second) lipid-based nanoparticle are capable of specifically binding to the same epitope of the target (e.g., the same epitope on PD-1).

[0496] Therapeutic uses

[0497] The lipid-based nanoparticle and the pharmaceutical composition comprising such as defined above have numerous in vitro and in vivo utilities and applications. Particularly, the lipid-based nanoparticles and the pharmaceutical compositions provided herein may be used in therapeutic methods and / or for therapeutic purposes.

[0498] The invention also relates to the lipid-based nanoparticle according to the present invention, or the pharmaceutical composition according to the present invention, for use as a medicament.

[0499] The invention also relates to the use of the lipid-based nanoparticle or of the pharmaceutical composition according to the invention, in the manufacture or a medicament for treating a disease, preferably selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an auto-immune disease.

[0500] The invention also relates to a method for treating a disease, preferably selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an auto-immune disease, in a subject in need thereof, wherein the method comprises administering to said subject a therapeutic amount of the lipid-based nanoparticle or of the pharmaceutical composition according to the invention.

[0501] 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.

[0502] As used herein, the terms “disorder” or “disease” refer to the incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease e.g., an illness that disrupts normal physical or mental functions. More preferably, the term disorder refers to immune and / or inflammatory diseases that affect animals and / or humans, such as cancer. Particularly, the invention concerns a treatment method that comprises: (a) identifying a patient in need of treatment; and (b) administering to the patient a therapeutically effective amount of the lipid-based nanoparticle or pharmaceutical composition described herein.

[0503] “An effective amount” or a “therapeutic effective amount” as used herein refers to the amount of active agent (i.e., the lipid-based nanoparticle disclosed herein) required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents, e.g., the amount of active agent that is needed to treat the targeted disease or disorder, or to produce the desired effect. The “effective amount” will vary depending on the agent(s), the disease and its severity, the characteristics of the subject to be treated including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art.

[0504] In a particular aspect, the invention provides a lipid-based nanoparticle or a pharmaceutical composition for use in the treatment of a subject having a cancer.

[0505] The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0506] In another embodiment, the invention provides the use a lipid-based nanoparticle or pharmaceutical composition as disclosed herein in the manufacture of a medicament.

[0507] In another embodiment, the invention provides the use a lipid-based nanoparticle or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a cancer.

[0508] Accordingly, in one aspect, the invention provides a method for treating a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the lipid-based nanoparticle or pharmaceutical composition of the invention, preferably such that the subject is treated from cancer. Particularly, the present invention relates to the treatment of a subject using a lipid-based nanoparticle or pharmaceutical composition of the invention such that immune response, especially the CTL response, is enhanced and growth of cancerous cells is inhibited.

[0509] In a specific aspect of the invention, the cancer is a PD-1 positive cancer or a PD-L1 negative cancer.

[0510] In a specific aspect of the invention, the cancer is a CD127 or CLEC-1 A positive cancer. In some aspects, the lipid-based nanoparticles or pharmaceutical compositions are for use in the treatment of an infectious disease or for use in the treatment of patients that have been exposed to toxins or pathogens.

[0511] The invention also provides the use of a lipid-based nanoparticle or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating an infectious disease.

[0512] Accordingly, some aspects of the invention provides a method for treating an infectious disease in a subject comprising administering to the subject a therapeutically effective amount of a lipid- based nanoparticle according to the present invention, or a pharmaceutical composition comprising such, preferably such that the subject is treated for the infectious disease.

[0513] Any suitable infection may be treated with a lipid-based nanoparticle or pharmaceutical composition according to the present invention.

[0514] Some examples of pathogenic viruses causing infections treatable by methods of the invention include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

[0515] Some examples of pathogenic bacteria causing infections treatable by methods of the invention include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.

[0516] Some examples of pathogenic fungi causing infections treatable by methods of the invention include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

[0517] Some examples of pathogenic parasites causing infections treatable by methods of the invention include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, and Nippostrongylus brasiliensis. In some embodiment, the lipid-based nanoparticle or the pharmaceutical composition according to the invention may be used in combination with another therapeutic agent or therapy, in particular for the treatment of cancer, of an infectious disease or of a chronic viral infection.

[0518] The present invention also relates to a lipid-based nanoparticle or a pharmaceutical composition comprising the LNP of the invention for use in the treatment of an autoimmune disease or an inflammatory disease.

[0519] It also relates to the use of a lipid-based nanoparticle or a pharmaceutical composition comprising of the invention for treating an autoimmune disease or an inflammatory disease, in a subject. It also concerns the use a lipid-based nanoparticle or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating an autoimmune disease or an inflammatory disease, in a subject.

[0520] Finally, it relates to a method for treating an autoimmune disease or an inflammatory disease, in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition or a lipid-based nanoparticle such as disclosed herein.

[0521] The inflammatory disease can typically be selected from the group consisting of acute inflammatory diseases, chronic inflammatory diseases such as chronic inflammatory pulmonary diseases (e.g., asthma), keratoconjunctivitis, periodontal disease, eczema, inflammatory bowel disease, in particular Crohn’s disease or colitis, in particular ulcerative colitis or spontaneous colitis, cystic fibrosis, cutaneous inflammation.

[0522] The auto-immune disease can typically be selected from the group consisting of psoriasis, lupus, rheumatoid arthritis, multiple sclerosis, Sjogren’s syndrome, celiac disease, vasculitis and myasthenia gravis.

[0523] Also provided herein are combined therapies with any of the lipid-based nanoparticle or pharmaceutical composition comprising such, as described herein and a suitable second agent, for the treatment of a disease or disorder.

[0524] In some aspects, the lipid-based nanoparticle and the second agent can be present in a unique pharmaceutical composition. Alternatively, the terms “combination therapy” or “combined therapy”, as used herein, embrace administration of these two agents (e.g., a lipid-based nanoparticle as described herein and an additional or second suitable therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., a lipid-based nanoparticle) can be administered intramuscularly, and an additional therapeutic agent (e.g., an anti-cancer agent, an anti-infection agent; or an immune modulator) can be administered intravenously. Alternatively, an agent of the combination selected may be administered by intravenous injection while the other agents of the combination may be administered intramuscularly.

[0525] In some aspects, the additional therapeutic agent can be selected in the non-exhaustive list comprising alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic, antiproliferative, antivirals, aurora kinase inhibitors, apoptosis promoters (for example, Bcl-2 family inhibitors), activators of death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (BiSpecific T cell Engager) antibodies, antibody drug conjugates, biologic response modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, leukemia viral oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormonal therapies, inhibitors of inhibitors of apoptosis proteins (IAPS), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutics, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoids, plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoints inhibitors, peptide vaccine and the like, epitopes or neoepitopes from tumor antigens, as well as combinations of one or more of these agents.

[0526] In one aspect, the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is particularly selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular of adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates. Preferably, suitable agents for co-use with any of the lipid-based nanoparticle or with the pharmaceutical composition according to the invention include an antibody binding to a co-stimulatory receptor (e.g., 0X40, CD40, ICOS, CD27 or HVEM), an agent that induces immunogenic cell death (e.g., a chemotherapeutic agent, a radio- therapeutic agent, an anti-angiogenic agent, or an agent for targeted therapies), an agent that inhibits a checkpoint molecule (e.g., LAG-3, TIM-3, BTLA, or TIGIT), a cancer vaccine, an agent that modifies an immunosuppressive enzyme (e.g., IDO1 or iNOS), an agent that targets Treg cells, an agent for adoptive cell therapy, or an agent that modulates myeloid cells.

[0527] In some aspects, the invention relates to a combined therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator of adaptive immune cells (T and B lymphocytes) selected from the group consisting of anti-CD2, anti-CD40, anti-HVEM, anti- BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4, and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonists, anti-ICOS, ICOS-L and B-cell receptor agonists.

[0528] In some embodiments, the lipid-based nanoparticle or the pharmaceutical composition according to the invention is combined with other forms of immunotherapy such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2, IL-7), or any therapy which provides for enhanced presentation of tumor antigens.

[0529] Subject, regimen and administration

[0530] A subject in need of a treatment may be a human having, at risk for, or suspected of having a disease. Such a patient can be identified by routine medical examination.

[0531] As used herein, the term “subject”, “host”, “individual,” or “patient” refers to human, including adult and child. The subject to treat may particularly be a human, particularly a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult, in particular an adult of at least 30 years old, 40 years old, preferably an adult of at least 50 years old, still more preferably an adult of at least 60 years old, even more preferably an adult of at least 70 years old.

[0532] The form of the pharmaceutical compositions, the route of administration and the dose of administration can be adjusted by the man skilled in the art according to the type and severity of the infection, and to the patient, in particular its age, weight, size, sex, and / or general physical condition. The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired.

[0533] In some embodiments, the subject has already received at least one line of treatment, preferably several lines of treatment, prior to the administration of the lipid-based nanoparticle, the pharmaceutical composition or the combined therapy of the invention.

[0534] Kit

[0535] Any of the lipid-based nanoparticle or compositions described herein may be included in a kit provided by the present invention. The present disclosure particularly provides kits for use in treating an autoimmune disease and / or inflammatory disease or for inhibiting immune cells activation.

[0536] In the context of the present invention, the term “kit” means two or more components (one of which corresponding to the lipid-based nanoparticle) packaged in a container, recipient or otherwise. A kit can hence be described as a set of products and / or utensils that are sufficient to achieve a certain goal, which can be marketed as a single unit. The kits of this invention are in suitable packaging.

[0537] The kit may include, in suitable container means, the pharmaceutical composition or lipid-based nanoparticle of the present invention.

[0538] The instructions related to the use of the lipid-based nanoparticle or pharmaceutical composition described herein generally include information as to dosage, dosing schedule, route of administration for the intended treatment, means for reconstituting the lipid-based nanoparticle and / or means for diluting the lipid-based nanoparticle of the invention. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit in the form of a leaflet or instruction manual).

[0539] All the references cited in this description are incorporated by reference in the present application. Others features and advantages of the invention will become clearer in the following figures and examples which are given for purposes of illustration and not by way of limitation.

[0540] Items

[0541] The application concerns a lipid-based nanoparticle comprising an antigen binding domain of an antibody covalently linked to a nonglycosylated Fc domain and optionally one or more nucleic acid molecules.

[0542] Particularly, the Fc domain is an IgGl, IgG2, IgG3 or IgG4 Fc domain, preferably an IgGl or IgG4 Fc domain, even more preferably an IgGl Fc domain.

[0543] Particularly, the nonglycosylated Fc domain is a deglycosylated Fc domain. Alternatively, said nonglycosylated Fc domain is an aglycosylated Fc domain

[0544] Particularly, the Fc domain i) does not have an Asparagine (N) at position 297 according to Eu numbering and / or ii) has an amino acid residue at position 298 and / or 299 according to Eu numbering that reduces or eliminates glycosylation at position 297.

[0545] Particularly, the aglycosylated Fc domain comprises an amino acid substitution in the CH2 domain selected from the group consisting of N297A, N297D, N297Q, N297G, N297F, N297K, N297R, N297H, N297V, N297L, N297I, N297G, N297P, N297E, from the group consisting of N297A, N297Q and N297G, according to Eu numbering.

[0546] In particular, the Fc domain i) is 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.

[0547] In the LNP disclosed herein, the antigen binding domain is preferably selected from the group consisting ofaFab, aFab’, aF(ab')2, aFv, a crossMAb Fab, a crossMAb Fab’, acrossMAb F(ab')2, a single-chain variable fragment (scFV) and a VHH.

[0548] Typically, the antigen binding domain binds to a target selected from the group consisting of PD- 1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, LOX1, SIGLEC 6, TACVTNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, CD160, CD127 and SIRPg.

[0549] Typically, the antigen binding domain is an anti-PD-1 binding domain comprising:

[0550] (i) a VH comprising a heavy chain CDR1 (HCDR1), CDR2 (HCDR2) and CDR3 (HCDR3), and

[0551] (ii) a VL comprising a light chain CDR1 (LCDR1), CDR2 (LCDR2) and CDR3 (LCDR3), wherein:

[0552] - the HCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 1;

[0553] - the HCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 2;

[0554] - the HCDR3 comprises or consists of an amino acid sequence of SEQ ID NO: 3;

[0555] - the LCDR1 comprises or consists of an amino acid sequence of SEQ ID NO: 4;

[0556] - the LCDR2 comprises or consists of an amino acid sequence of SEQ ID NO: 5, and

[0557] - the LCDR3 comprises or consists of an amino acid sequence of SEQ ID NO:6. In particular, the lipid-based composition of the lipid based nanoparticle comprises or consists of i) a cationic or ionizable lipid, ii) a helper lipid, iii) a sterol and iv) a PEG-lipid or a combination of PEG-lipids, said combination preferably comprising a first PEG-lipid that does not comprise a reactive group for conjugation to the non-glycosylated Fc domain and a second PEG-lipid that comprises a reactive group for conjugation to the non-glycosylated Fc domain.

[0558] Particularly, in the LNP disclosed herein: 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), 1,2-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), 1,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl )-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn-glycero-3 - ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl 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' -di oleylcarbamyl -3- dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen- 19-yl-4-(dimethylamino)butanoate (DLin-MC3 -DMA), N-(2 -Hydroxy ethyl)-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- (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), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12- 200), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide (SS-OP) and any mixtures thereof, preferably is ALC-0315, SM-102, Dlin-MC3- DMA or SS-OP and any mixture thereof, more preferably is ALC-0315 or SS-OP;

[0559] - 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;

[0560] - the helper lipid is selected from DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DEPC, DOPC and DSPE and any mixture thereof, preferably is DOPE or DSPC or a mixture thereof; and / or

[0561] - the PEG-lipid is selected from 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-DSG, PEG-DSPE or ALC-0159 and any mixture thereof.

[0562] Typically, the lipid-based nanoparticle comprises from about 40 mol % to about 60 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 25 mol% to about 50 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid, preferably 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 30 mol% to about 45 mol% of a sterol, and from about 1 mol% to about 2.5 mol% of a PEG-lipid.

[0563] The LNPs disclosed herein typically comprises one or more nucleic acid molecules that is / are mRNA.

[0564] In particular, the LNPs envisioned herein comprises or consists essentially of: a) a combination of lipids comprising or consisting of:

[0565] - an ionizable lipid;

[0566] - a helper lipid;

[0567] -a sterol;

[0568] - a mixture of PEG lipids, said mixture comprising a first PEG-lipid that is not linked to a targeting moiety and a second PEG-lipid that is covalently linked to a targeting moiety, said targeting moiety comprising an antigen binding domain of an antibody, preferably of an anti-PD-1 antibody, covalently linked to the nonglycosylated Fc domain; and b) optionally a nucleic acid molecule, preferably one or more mRNA molecule(s).

[0569] This application also discloses a pharmaceutical composition comprising at least one lipid-based nanoparticle envisioned herein and optionally a pharmaceutically acceptable carrier or excipient. The lipid-based nanoparticle or the pharmaceutical composition are particularly for use as a medicament, typically for use in the treatment of a cancer, an infectious disease, an inflammatory disease or an auto-immune disease.

[0570] BRIEF DESCRIPTION OF THE DRAWINGS

[0571] Figure 1 - Targeted LNP prepared from OSE-279 IgGl N297A format induce higher transfection selectivity in PD-1 positive cells, compared to OSE-279 IgG4 format. OSE-279 targeted LNP were prepared with various formats of anti-PD-1 mAb OSE-279 (IgG4 vs IgGl N297A vs Monovalent IgGl N297A) and their transfection efficiency were assessed in different cell lines expressing the PD-1 receptors: U937 (Figure 1A), Jurkat (Figure IB) and HPB-ALL (Figure 1C). Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control. Figure 2 - The N297A mutation on OSE-279 IgGl allows to produce efficient OSE-279 targeted LNP. OSE-279 targeted LNP were prepared with IgGl formats of anti-PD-1 mAb OSE- 279 (with or without N297A mutation) and their transfection efficiency were compared in different cell lines expressing the PD-1 receptors: U937 (Figure 2A), Jurkat (Figure 2B) and HPB-ALL (Figure 2C). Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0572] Figure 3 - Targeted LNP with an anti-CLEC-1 antagonist mAb can target CLEC-1 expressing cells, preferentially when the IgGl N297A format of mAb is used in the targeted LNP. U937 and THP-1 cells were transduced to express CLEC-1 receptors. Flow cytometry was used to measure the percentage of CLEC-1 positive cells. Dashed line: cells were stained with a purified control isotype human IgGl (OSE Immunotherapeutics) at 10 pg / mL. Dark grey: cells were stained with a purified anti-human CLEC-1 antibody (OSE Immunotherapeutics) at 10 pg / mL to measure CLEC-1 positive cells. A PE-labelled anti-human IgG antibody (clone: QA19A42 #366904 batch: B359783, BioLegend) was used for detection of primary purified antibodies. Dead cells were stained and excluded from analysis with LIVE / DEAD™ Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies) and human Fc Receptor were saturated with Human FcBlock (#564220 batch: 2122225, BD Biosciences). Transfection efficiency of anti-CLEC-1 targeted LNP was assessed in U937 (Figure 3A) and in THP-1 (Figure 3B) cells expressing CLEC-1 receptors. Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0573] Figure 4 - Targeted LNP with an anti-CD127 antagonist mAb are able to target CD-127 expressing cells, preferentially when the IgGl N297A format of the anti-CD127 mAb is used in the targeted LNP. U937 and Jurkat cells were transduced to express CD-127 receptors. Flow cytometry was used to measure the percentage of CD-127+ cells. Dashed line: cells were stained with a PE-Cy7 labelled control isotype mouse IgGl (#557646 batch: 8155598, BD Biosciences). Dark grey: cells were stained with a PE-Cy7 labelled anti-human CD-127 antibody (#351320 batch: B251081, BioLegend) to measure CD-127 positive cells. Dead cells were stained and excluded from analysis with LIVE / DEAD™ Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies). Transfection efficiency of anti-human CD-127 targeted LNP was assessed in U937 (Figure 4A) and in Jurkat (Figure 4B) cells expressing CD-127 receptors. Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0574] Figure 5 - Using an anti-CD127 antagonist mAb IgGl LALAPG in targeted LNP does not allow to reach similar targeting properties than with the same anti-CD127 antagonist mAb in IgGl N297A format. U937 cells were transduced to express CD-127 receptors. Flow cytometry was used to measure the percentage of CD127+ cells. HPB-ALL cells naturally express CD-127. Dashed line: cells were stained with a PE-Cy7 labelled control isotype mouse IgGl (#557646 batch: 8155598, BD Biosciences). Dark grey: cells were stained with a PE-Cy7 labelled antihuman CD-127 antibody (#351320 batch: B251081, BioLegend) to measure CD-127 positive cells. Dead cells were stained and excluded from analysis with LIVE / DEAD™ Fixable Yellow Dead Cell Stain Kit (#L34968A batch: 2438368, Life Technologies). Transfection efficiency of anti-human CD-127 targeted LNP was assessed in U937 (Figure 5A) and HPB-ALL (Figure 5B) cells expressing CD-127 receptors. Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0575] Figure 6 - OSE-279 targeted LNP prepared after deglycosylation of OSE-279 IgGl leads to improved transfection in PD-1 positive cells. OSE-279 IgGl was deglycosylated and subsequently used to prepare targeted LNP (Figures 6A-C). The transfection efficiency of targeted LNP prepared with either OSE-279 IgGl N297A, OSE-279 IgGl or OSE-279 IgGl deglycosylated was assessed in different cell lines expressing the PD-1 receptors: U937 (Figure 6D) and Jurkat (Figure 6E). Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0576] Figure 7 - Deglycosylation of IgG4 isotype leads to targeted LNP with improved targeting abilities in PD-1 positive cells, compared to targeted LNP prepared with IgG4 format. OSE- 279 IgG4 was deglycosylated and subsequently used to prepare targeted LNP (Figure 7A). The transfection efficiency of targeted LNP prepared with either OSE-279 IgG4 or OSE-279 IgG4 deglycosylated were assessed in different cell lines expressing the PD-1 receptors: U937 (Figure 7B) and Jurkat (Figure 7C). Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0577] Figure 8 - Different lipidic composition can be used for the preparation of efficient targeted LNP with OSE-279 IgGl N297A used as targeting agent. OSE-279 targeted LNP were prepared using the same lipid constituents as the 3 FDA and EMA-approved mRNA-based LNP (SpikeVax®, Onpattro® and Cominarty®) and a formulation using Coatsome® SS-OP as ionizable lipid. For each formulation, targeted LNP were prepared with OSE-279 IgGl, IgGl N297A or a control isotype. Comparative transfection efficiency of non-targeted vs targeted LNP were performed in PD-1 positive cells: U937 and Jurkat cells (Figures 8A-H).

[0578] Figure 9 - Targeted LNP produced by Thiol-Michael addition improve transfection in PD-1 positive cells, preferentially with OSE-279 IgGl N297A variant. OSE-279 targeted LNP were prepared using the classical method described in the literature for grafting mAb on LNP surface via Thiol-Michael addition. IgGl N297 and IgG4 formats of OSE-279 were used to prepare targeted LNP and their transfection potency of PD-1 positive cells U937 (Figure 9A), Jurkat (Figure 9B) and HPB-ALL (Figure 9C) was compared.

[0579] Figure 10 - OSE-279 targeted LNP prepared by Thiol-Michael addition with deglycosylated OSE-279 IgGl lead to improved transfection in PD-1 positive cells, compared to OSE-279 IgGl. OSE-279 IgGl was deglycosylated and subsequently used to prepare targeted LNP. The transfection efficiency of targeted LNP prepared by Thiol-Michael addition with either OSE-279 IgGl N297A, OSE-279 IgGl or OSE-279 IgGl deglycosylated was assessed in different cell lines expressing the PD-1 receptors: U937 (Figure 10A), Jurkat (Figure 10B), HPB-ALL (Figure 10C) and CHO (Figure 10D). Non-targeted LNP and targeted LNP prepared with a control isotype mAb were used as negative control.

[0580] EXAMPLES

[0581] Constructs:

[0582] Throughout all the Example section and figure are described constructions comprising IgGl or IgG4 domain wherein:

[0583] IgGl, IgGl wild type or G1 corresponds to a Fc domain of an IgGl immunoglobulin, in particular having a sequence such as described in SEQ ID NO: 58.

[0584] IgGl N297A corresponds to a Fc domain of an IgGl immunoglobulin with the amino acid substitution N297A, in particular having a sequence such as described in SEQ ID NO: 59

[0585] IgGl N297A Mai eimide corresponds to a Fc domain of an IgGl immunoglobulin with the amino acid substitution N297A, in particular having a sequence such as described in SEQ ID NO: 59, with a Thiol-Michael addition to allow the Fc domain to be grafted on a PEG-lipid.

[0586] IgGl LALAPG corresponds to a Fc domain of an IgGl immunoglobulin with mutations L234A, L235A, and P329G, in particular having a sequence such as described in SEQ ID NO: 56.

[0587] IgGl deglyco corresponds to a Fc domain of an IgGl immunoglobulin, in particular having a sequence such as described in SEQ ID NO: 58, submitted to an enzymatic deglycosylation, resulting in the cleavage of the glycosylation motif attached to the N297 residue..

[0588] IgGl deglyco Mai eimide corresponds to a Fc domain of an IgGl immunoglobulin, in particular having a sequence such as described in SEQ ID NO: 58, submitted to an enzymatic deglycosylation, resulting in the cleavage of the glycosylation motif attached to the N297 residue with a Thiol-Michael addition to allow the Fc domain to be grafted on a PEG-lipid. IgG4 or IgG4 mAb wild-type corresponds to a Fc domain of an IgG4 immunoglobulin with mutation S228P, in particular having a sequence such as described in SEQ ID NO: 55.

[0589] IgG4 deglyco corresponds to a Fc domain of an IgG4 immunoglobulin with mutation S228P, in particular having a sequence such as described in SEQ ID NO: 55, submitted to an enzymatic deglycosylation, resulting in the cleavage of the glycosylation motif attached to the N297 residue. OSE-279 (anti-PD-1) comprises a heavy chain having an amino-acid sequence as disclosed in WO2020 / 127366. OSE-279 particularly comprises a VH sequence as set forth in SEQ ID NO: 15 and a VL sequence as set forth in SEQ ID NO: 16. OSE-279 particularly comprises a heavy chain sequence as set forth in SEQ ID NO: 19 and a light chain sequence as set forth in SEQ ID NO: 20. These chains are linked to a Fc domain such as disclosed herein. When no precision is mentioned, OSE-279 is bivalent, e.g., is in the form of a F(ab’)2

[0590] Monovalent OSE-279 corresponds to OSE-279 Fab connected to a knob-into-hole heterodimeric IgGl Fc domain comprising i) a first Fc chain comprising the substitutions T366W / S354C and ii) a second Fc chain comprising the substitutions T366S / L368A / Y407V / Y349C, in particular having a sequence such as described in SEQ ID NO: 66 and 67, respectively.

[0591] Anti-hCLECl was produced by OSE Immunotherapeutics.

[0592] The anti-CD127 antagonist mAb - (anti-IL7R) was produced by OSE Immunotherapeutics. Control isotype IgG corresponds to the targeting moiety of an irrelevant antibody, Motavizumab, linked to a IgGl or IgG4 Fc domain such as disclosed herein.

[0593] Example 1: OSE-279 targeted LNP prepared with either full IgGl or Monovalent IgGl format with N297A mutation are both efficient in preferentially transfecting PD-1 positive cells.

[0594] The influence of monoclonal antibodies (mAb) format on targeting properties of LNP was assessed by preparing OSE-279 targeted LNP with mAb in full-length IgG4, full-length IgGl N297A or Monovalent IgGl N297A formats.

[0595] Results of Figures 1A-C indicate that all OSE-279 mAb formats (full IgG4, full IgGl N297A and Monovalent IgGl N297A) are suitable for producing targeted LNP able to preferentially transfect PD-1 positive cell lines. Among the 3 formats assessed, the bivalent IgGl N297A (i.e., OSE279- IgGl N297A) format leads to the highest increase in targeting compared to non-targeted LNP.

[0596] For each targeted LNP, the improvements in transfection potency were suppressed when a preincubation step with a saturating concentration of an anti-PD-1 mAbs was performed before the transfection protocols, indicating the improvements are mediated by the targeting of PD-1. Interestingly, and unexpectedly, the improvements in transfection are not linked to the loss of FcR binding capacity caused by the N297A, as U937 cells express FcR while Jurkat and HPB-ALL do not.

[0597] Preparation of LNP

[0598] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 9.1 mM.

[0599] FLuc-mRNA (50 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.9xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0600] The selected experimental conditions allow for a N / P ratio of 6.

[0601] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0602] In vitro cellular uptake

[0603] LNP were diluted to 3.9 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL of LNP / well corresponding to 96 ng RNA / well). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 24 h post-treatment in a 96-wells white cell culture plate and TEC AN Spark plate reader were used to quantify the luminescence. In mAb pre-incubation experiments, 5 pL of mAb (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP. Example 2: OSE-279 targeted LNP prepared with OSE-279 IgGl N297A shows superior transfection in PD-1 positive cells, compared to targeted LNP prepared with OSE-279 IgGl wild-type (without N297A mutation).

[0604] The N297A mutation is known to suppress glycosylation and to markedly decrease mAb capacity to bind Fc y-receptors. Mutation of N297 to Ala (N297A) eliminates IgG glycosylation, and the aglycosylated IgG consequently undergoes a conformational change in the CH2 domain that significantly decreases binding to Fc y-receptors.

[0605] The influence of this mutation (and the conformational change induced) on the ability of targeted LNP to transfect PD-1 positive cells was assessed by comparing the transfection efficacy of targeted LNP prepared with either OSE-279 IgGl N297A or OSE-279 IgGl.

[0606] The transfection experiments (Figures 2A-C) show that targeted LNP prepared with OSE-279 IgGl N297A induce a clear improvement in transfection of PD-1 positive cells, compared to targeted LNP prepared with OSE-279 IgGl . The N297A mutation appears to play a crucial role in the targeting of PD-1 positive cells with targeted LNP. Of note, OSE-279 IgGl targeted LNP also display targeting properties compared to non-targeted LNP, albeit with lower improvement in comparison to targeted LNP obtained with OSE-279 IgGl N297A.

[0607] Preparation of LNP

[0608] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 4.55 mM.

[0609] FLuc-mRNA (25 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.041 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.3xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0610] The selected experimental conditions allow for a N / P ratio of 6.

[0611] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0612] Table 2. Characteristics of the LNPs produced

[0613] In vitro cellular uptake

[0614] LNP were diluted to 3.6 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 90 ng RNA / well for U937 cells and 2.5 pL LNP / well corresponding to 9 ng RNA / well for Jurkat and HPB-ALL cells). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 24 h post-treatment in a 96-wells white cell culture plate and TEC AN Spark plate reader were used to quantify the luminescence. In mAh pre-incubation experiments, 5 pL of mAh (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP.

[0615] Example 3: Anti-CLEC-1 targeted LNP prepared with IgGl N297A mAh format show superior transfection in CLEC-1 positive cells, compared to targeted LNP prepared with IgGl or IgG4 mAh wild-type (without N297A mutation) formats.

[0616] As for PD-1 targeting described in the previous examples, the influence of both the mAb isotype (IgG4 vs IgGl) and the N297A mutation was evaluated on CLEC-1 positive cells by comparing the transfection efficacy of targeted LNP prepared with anti -CLEC-1 mAb in either in IgG4, IgGl or IgGl N297A formats.

[0617] Transfection results (Figure 3) indicate that CLEC-1 targeting is best observed with targeted LNP prepared with anti-CLEC-1 IgGl N297A. Targeted LNP with anti-CLEC-1 IgG4 also induce improved transfection compared to non-targeted LNP and to control-isotype targeted LNP.

[0618] Preparation of LNP

[0619] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 9.1 mM.

[0620] FLuc-mRNA (50 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.3xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0621] The selected experimental conditions allow for a N / P ratio of 6. The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0622] Table 3. Characteristics of the LNPs produced

[0623] In vitro cellular uptake

[0624] LNP were diluted to 5 ng / pL with PBS IX and added (5 pL of LNP / well corresponding to 25 ng RNA) on cells previously prepared in a final volume of 60 pL per well in their culture medium and pre-treated with 5 pL of pure serum albumin bovine (SAB pre-treatment 15 min before LNP addition). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 48 h post-treatment in a 96-wells white cell culture plate and TEC AN Spark plate reader were used to quantify the luminescence.

[0625] Example 4: Anti-CD127 targeted LNP prepared with an anti-CD127 antagonist mAh IgGl N297A show superior transfection in CD-127 positive cells, compared to targeted LNP prepared with anti-CD127 mAh IgGl or anti-CD127 mAh IgG4 wild-type (without N297A mutation) format.

[0626] As for PD-1 targeting described in the previous examples, the influence of both the mAb isotype (IgG4 vs IgGl) and the N297A mutation were evaluated on CD-127 positive cells by comparing the transfection efficacy of targeted LNP prepared with either anti-CD127 IgG4, anti-CD127 IgGl N297A or OSE-279 IgGl.

[0627] Transfection results (Figure 4) indicate that CD127 targeting is best observed with targeted LNP prepared with anti-CD127 IgGl N297A. Targeted LNP with anti-CD127 IgGl and to a lesser extent with anti-CD127 IgG4 also induce improved transfection compared to non-targeted LNP and to control-isotype targeted LNP.

[0628] Preparation of LNP LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 4.55 mM.

[0629] FLuc-mRNA (25 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.041 mg / mL. For the preparation of targeted LNP, the mAh (0.95 pg / pL in PBS, 1.3xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0630] The selected experimental conditions allow for a N / P ratio of 6.

[0631] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0632] Table 4. Characteristics of the LNPs produced

[0633] In vitro cellular uptake

[0634] LNP were diluted to 3.9 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 97 ng RNA / well for U937 cells and 2.5 pL LNP / well corresponding to 9.7 ng RNA / well for Jurkat cells). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 48 h post-treatment in a 96-wells white cell culture plate and TECAN Spark plate reader were used to quantify the luminescence. In mAb pre-incubation experiments, 5 pL of each mAb (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP.

[0635] Example 5: Anti-CD127 targeted LNP prepared with an anti-CD127 antagonist mAb -IgGl N297A shows superior transfection in CD-127 positive cells, compared to targeted LNP prepared with the anti-CD127 mAB IgGl LALAPG. LALAPG mutations in the Fc region (L234A, L235A, and P329G) are known to inhibit binding to Fc y-receptors. Therefore, N297A and LALAPG represent two distinct mutations both leading to Fc-silencing. The impact of these mutations on targeted LNP properties was compared to assess whether the glycosylation or the Fc-silencing is responsible for the enhanced transfection effect. Figure 5 shows that transfection of CD-127 positive cells show that N297A mutation on 127the anti-CD127 mAB IgGl isotype induce higher targeting properties than the LALAPG mutation on the same isotype. The high targeting efficiency observed with targeted LNP prepared with the anti- CD127 mAB IgGl N297A therefore seems to arise from aglycosylation and / or from the conformational change due to the removal of glycans rather than its Fc-silencing properties. It is therefore possible to enhance the targeting properties of targeted LNP comprising a targeting moiety with a Fc domain, by blocking or suppressing glycosylation of the Fc domain (e.g., with a N297A mutation).

[0636] Preparation of LNP

[0637] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 9.1 mM.

[0638] FLuc-mRNA (50 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.9xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing. The selected experimental conditions allow for a N / P ratio of 6.

[0639] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0640] Table 5. Characteristics of the LNPs produced In vitro cellular uptake

[0641] LNP were diluted to 3.7 ng / pL with PBS IX and added (25 pL of LNP / well corresponding to 92 ng RNA) on cells previously prepared in a final volume of 60 pL per well in their culture medium. The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 48 h posttreatment in a 96-wells white cell culture plate and TECAN Spark plate reader were used to quantify the luminescence.

[0642] Example 6: OSE-279 targeted LNP prepared with deglycosylated OSE-279 IgGl shows superior transfection in PD-1 positive cells, compared to targeted LNP prepared with OSE- 279 IgGl.

[0643] The N297A mutation eliminates an N-glycosylation site that is critical for Fc receptors binding.

[0644] To address the importance of this glycosylation on the targeting abilities of OSE-279 targeted LNP, a deglycosylated version of OSE-279 IgGl was prepared by enzymatic treatment. Enzymatic deglycosylation results in the cleavage of the glycosylation motif attached to the N297 residue. Targeted LNP prepared with OSE-279 IgGl N297A, OSE-279 IgGl and OSE-279 IgGl deglycosylated were compared. “OSE-279 IgGl deglycosylated” corresponds to OSE279 IgGl deglycosylated in non-denaturating reaction conditions with Deglycosylation Mix II.

[0645] The results of Figure 6 show that the use of deglycosylated OSE-279 IgGl in targeted LNP improved the transfection in U937 and Jurkat PD-1 positive cells, compared to targeted LNP produced with OSE-279 IgGl. This proves that the presence of the glycans in the mAb structure greatly impairs the targeting properties of targeted LNP, in particular comprising targeting moiety with a Fc domain.

[0646] Targeted LNP prepared with OSE-279 N297A still display improved targeting properties compared to IgGl and IgGl deglycosylated versions of targeted LNP, and the binding studies demonstrates that the difference in targeting does not originate from difference in affinity to the PD-1 receptors (as the 3 mAbs tested display similar properties in terms of binding to PD-1). Therefore, any method or modification which suppresses or blocks glycosylation or strip glycans from the Fc may be used to enhance the targeting properties of targeted LNP.

[0647] Deglycosylation of OSE-279 IgGl

[0648] OSE-279 IgGl was deglycosylated in non-denaturating reaction conditions with Deglycosylation Mix II (NEB # P6044), according to manufacturer’s protocol. Briefly, 400 pg of OSE-279 IgGl was mixed (at a concentration of Img / mL) with 40 pL of Deglycosylation mix Buffer 1 (10X) and 40 pL of Deglycosylation mix II, and volume was adjusted with water to 400 pL. Reactions were mixed gently and incubated for 30 minutes at room temperature. Then, reactions were transferred at 37°C and incubated for 16 hours at 37°C. Deglycosylated antibodies were purified on protein A with Nab protein A plus (Thermofisher #89952) for glycosidase removal. Deglycosylation of antibodies was verified by SDS-PAGE analysis (Figure 6A).

[0649] ELISA assay - mAb Binding PD-1

[0650] For activity ELISA assay, recombinant hPD-1 (Sino Biologicals, Beijing, China; reference 10377- H08H) was immobilized on plastic at 0.5 pg / mL in carbonate buffer (pH 9.2). OSE-279 IgGl and OSE-279 IgGl deglycosylated were then added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035- 149) was added and revealed by conventional methods. Both samples showed similar binding to PD-1 (Figure 6B).

[0651] Preparation of LNP

[0652] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 4.55 mM.

[0653] FLuc-mRNA (25 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.041 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.3xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0654] The selected experimental conditions allow for a N / P ratio of 6.

[0655] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0656] Table 6. Characteristics of the LNPs produced

[0657] ELISA assay - t-LNP Binding PD-1

[0658] For activity ELISA assay, recombinant hPD-1 (Sino Biologicals, Beijing, China; reference 10377- H08H) was immobilized on plastic at 0.5 pg / mL in carbonate buffer (pH 9.2). Targeted LNP (diluted to an initial concentration of 1.03 pg / mL of RNA) were then added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference 709-035-149) was added and revealed by conventional methods. Standard curves consist of i) OSE-279 alone at 5 pg / mL and ii) OSE-279 at different concentrations (5 to 0.04 pg / mL) spiked with non-targeted LNP. The 3 targeted LNP prepared from OSE-279 variants showed similar binding to PD-1 (Figure 6C).

[0659] In vitro cellular uptake

[0660] LNP were diluted to 2.6 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 64 ng RNA / well for U937 cells and 2.5 pL LNP / well corresponding to 6.4 ng RNA / well for Jurkat cells). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 24 h post-treatment in a 96-wells white cell culture plate and TECAN Spark plate reader were used to quantify the luminescence. In mAb pre-incubation experiments, 5 pL of mAb (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP.

[0661] Example 7: OSE-279 targeted LNP prepared with deglycosylated OSE-279 IgG4 shows superior transfection in PD-1 positive cells, compared to targeted LNP prepared with OSE- 279 IgG4.

[0662] To decipher if the deglycosylation step (and the subsequent conformational change) could lead to an increase of targeting with IgG4 isotype (as observed with IgGl isotype), enzymatic deglycosylation was performed on OSE-279 IgG4. Comparison of transfection efficacy on PD-1 positive cells was assessed with targeted LNP prepared with OSE-279 in either IgG4 or IgG4 deglycosylated format.

[0663] Results of Figure 7 show that both in U937 and Jurkat PD-1 positive cells, targeted LNP prepared with the deglycolsylated variant of OSE-279 IgG4 lead to better targeting compared to targeted LNP prepared with OSE-279 IgG4. These results confirmed that the removal of glycans both in IgGl and IgG4 isotypes allow for the preparation of targeted LNP with improved targeting properties.

[0664] Deglycosylation of OSE-279 IgG4

[0665] OSE-279 IgG4 was deglycosylated in non-denaturationg reaction conditions with Deglycosylation Mix II (NEB # P6044), according to manufacturer’s protocol. Briefly, 400 pg of OSE-279 IgG4 was mixed (at concentration of Img / ml) with 40 pL of Deglycosylation mix Buffer 1 (10X) and 40 pL of Deglycosylation mix II, and volume was adjusted with water to 400 pL. Reactions were mixed gently and incubated for 30 minutes at room temperature. Then, reactions were transferred Ill at 37°C and incubated for 16 hours at 37°C. Deglycosylated antibodies were purified on protein A with Nab protein A plus (Thermofisher #89952) for glycosidase removal. Deglycosylation of antibodies was verified by SDS-PAGE analysis (Figure 7A).

[0666] Preparation of LNP

[0667] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.5 %, at a total lipid concentration of 4.55 mM.

[0668] FLuc-mRNA (25 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.041 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.3xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0669] The selected experimental conditions allow for a N / P ratio of 6.

[0670] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C.

[0671] Table 7. Characteristics of the LNPs produced

[0672] In vitro cellular uptake

[0673] LNP were diluted to 4 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 100 ng RNA / well for U937 cells and 2.5 pL LNP / well corresponding to 10 ng RNA / well for Jurkat cells). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 24 h posttreatment in a 96-wells white cell culture plate and TECAN Spark plate reader were used to quantify the luminescence.

[0674] In mAb pre-incubation experiments, 5 pL of mAb (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP. Example 8: OSE-279 IgGl N297A targeted LNP prepared using various lipid formulations are efficient in preferentially transfecting PD-1 positive cells.

[0675] To further evidence the potency of OSE-279 IgGl N297A targeted LNP to efficiently transfect PD-1 positive cells, different formulations were used to produce LNP. The gold standard lipidic compositions of SpikeVax®, Onpattro® and Cominarty® were tested, as well as a formulation using Coatsome® SS-OP as ionizable lipid. For each formulation, targeted LNP were prepared using either OSE-279 IgGl, IgGl N297A or a control isotype.

[0676] Results of Figure 8 show that the nature of the lipids used to formulate mRNA does not influence the capacity of OSE-279 IgGl N297A targeted LNP to induce improved transfection in PD-1 expressing cells. Indeed, improved transfection was observed with targeted LNP, compared to non-targeted LNP and control isotype targeted LNP, for the 4 formulations tested (A1-A4) in the 2 cell types tested (U937 and Jurkat). Moreover, as observed in the previous experiments, the improvements in transfection potency were suppressed when a pre-incubation step with a saturating concentration of an anti-PD-1 mAbs was performed before the transfection protocols. In all tested conditions, for a given formulation, targeted LNP prepared with IgGl N297A performed better compared to targeted LNP prepared with IgGl WT.

[0677] Preparation of LNP

[0678] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios, at a total lipid concentration of 9.1 mM. :

[0679] • Formulation Al : SM-102 : 50% / DSPC : 10% / Cholesterol : 38.5 % / DMG-PEG-2000 : 1.5%,

[0680] • Formulation A2 : MC3-DLin-DMA: 50% / DSPC : 10% / Cholesterol: 38.5 % / DMG- PEG-2000 1.5%,

[0681] • Formulation A3 : ALC-0315 : 46.3% / DSPC : 9.4% / Cholesterol : 42.7 % / ALC-0159 : 1.6%,

[0682] • Formulation A4 : SS-OP: 50% / DSPC : 10% / Cholesterol: 38.5 % / DSPE-PEG-2000 : 1.5%.

[0683] Fluc-mRNA (25 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. For the preparation of targeted LNP, the mAb (0.95 pg / pL in PBS, 1.3xl0'6pmol mAb / pg RNA) was added to the mRNA solution prior to microfluidic mixing.

[0684] The selected experimental conditions allow for a N / P ratio of 6.

[0685] The lipids (200 pL) and the mRNA solutions (600 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the LNP were stored in a final volume of 1 to 1.5 mL in PBS at 4°C for characterization.

[0686] Table 8. Formulation and characteristics of the LNPs produced In vitro cellular uptake (Figures 8A-H).

[0687] LNP were diluted to 4 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 100 ng RNA / well forU937 and Jurkat cells). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 48 h post-treatment in a 96-wells white cell culture plate and TECAN Spark plate reader were used to quantify the luminescence. In mAh pre-incubation experiments, 5 pL of each mAh (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP. Example 9: OSE-279 targeted LNP prepared by Thiol-Michael addition show improvements in transfecting PD-1 positive cells, compared to non-targeted LNP. Better targeting is observed when the IgGl N297 variant of OSE-279 is used in the targeted LNP, compared to IgG4 wild-type (without N297A mutation) isotype.

[0688] The objective of this set of experiments was to determine if the superiority in targeting of IgGl N297 variant was confirmed in a different class of targeted LNP.

[0689] The preparation of targeted LNP using the Thiol-Michael addition is a classical approach for grafting mAh on LNP surface and consists of i) preparing a non-targeted LNP including a PEG- lipid decorated with a maleimide moiety, ii) reducing the mAh to expose thiol residue and iii) coupling both moieties via Thiol-Michael addition to allow the mAb to be grafted on the extremity of the PEG-lipid.

[0690] OSE-279 IgG4, OSE-279 IgGl N297A and control isotype IgG were reduced and coupled to a non-targeted LNP carrying the maleimide moiety to provide the corresponding targeted LNP.

[0691] Results of Figure 9 show the transfection of PD-1 positive cells was improved when targeted LNPs with either OSE-279 IgG4 or IgGl N297A were used, compared to non-targeted LNPs and / or control isotype targeted LNPs. As in the previous experiments, a better targeting was observed with targeted LNPs with OSE-279 IgGl N297A compared to IgG4. These results confirmed the superiority in targeting of the IgGl N297A variant, irrespective of the method used to produce targeted LNPs.

[0692] Preparation of LNP with DSPE-PEG-Maleimide

[0693] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.4 % / DSPE-PEG-Maleimide : 0.1 %, at a total lipid concentration of 9.1 mM.

[0694] FLuc-mRNA (150 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. The selected experimental conditions allow for a N / P ratio of 6. The lipids (600 pL) and the mRNA solutions (1800 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the Maleimide-LNP solution was used for coupling with the reduced mAbs.

[0695] Reduction of mAbs and coupling with Maleimide-LNP by thiol-Michael addition

[0696] OSE-279 mAbs (0.5 molar equivalent vs DSPE-PEG-Maleimide) were diluted with PBS to obtain a 5 mg / mL solution, before addition of EDTA (5 mM) and TCEP (50 mM, 3 molar equivalent vs OSE-279). The mixture was heated for 30 min at 37°C and stirred for 30 min at room temperature. Excess TCEP was removed by using 7K desalting columns (ThermoFisher) according to manufacturer protocol. The reduced antibody was then immediately conjugated with Maleimide- LNP by incubation for 1 h at room temperature and overnight at 4°C. The conjugated LNP was then purified by gel filtration chromatography (HiLoad 16 / 600 Superdex 200 column, Cytiva) using PBS as a mobile phase (flow rate : 1 mL / min). LNP fractions were collected and concentrated by 100K Amicon tubes (Millipore).

[0697] Table 9. Characteristics of the LNPs produced

[0698] In vitro cellular uptake

[0699] LNP were diluted to 2.6 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 64 ng RNA / well for U937 cells and 2.5 pL LNP / well corresponding to 6.4 ng RNA / well for Jurkat and HPB-ALL cells). The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 24 h post-treatment in a 96-wells white cell culture plate and TEC AN Spark plate reader were used to quantify the luminescence. In mAb pre-incubation experiments, 5 pL of mAb (9 mg / mL) were added on the cells for a final concentration of 500 pg / mL (saturating conditions) and incubated at room temperature for 30 minutes before treatment with LNP.

[0700] Example 10: OSE-279 targeted LNP prepared by Thiol-Michael addition with deglycosylated OSE-279 IgGl shows superior transfection in PD-1 positive cells, compared to targeted LNP prepared with OSE-279 IgGl wild-type (without N297A mutation).

[0701] Previous experiments have demonstrated that deglycosylation of IgGl variant of OSE-279 induced higher targeting properties of targeted LNP. Here, another class of targeted LNP was prepared and tested to verify the hypothesis that the deglycosylation of OSE-279 IgGl (and the subsequent conformational change) of mAb is beneficial for targeting PD-1 positive cells.

[0702] In this context, targeted LNP prepared by Thiol-Michael addition with OSE-279 IgGl N297A, OSE-279 IgGl were compared in terms of transfection. Targeted LNP prepared by Thiol-Michael addition with OSE-279 IgGl deglycosylated were also compared in CHO PD1+ cell lines.

[0703] In all PD-1 positive cell lines tested Figure 10, and as observed in previous experiments, the most efficient transfection was observed when OSE-279 IgGl N297A was used as targeting agent of the LNP. The deglycosylation step of IgGl variant had a positive impact of targeting and transfection properties in CHO PD1+ cell lines. The conformational change induced by the removal of glycans therefore improve the targeting potency of targeted LNP, irrespective of the method used for producing such LNP.

[0704] Deglycosylation of OSE-279 IgGl

[0705] OSE-279 IgGl was deglycosylated in non-denaturationg reaction conditions with Deglycosylation Mix II (NEB # P6044), according to manufacturer’s protocol. Briefly, 400 pg of OSE-279 IgGl was mixed (at a minimal concentration of Img / ml) with 40 pL of Deglycosylation mix Buffer 1 (10X) and 40 pL of Deglycosylation mix II, and volume was adjusted with water to 400 pL. Reactions were mixed gently and incubated for 30 minutes at room temperature. Then, reactions were transferred at 37°C and incubated for 16 hours at 37°C. Deglycosylated antibodies were purified on protein A with Nab protein A plus (Thermofisher #89952) for glycosidase removal. Deglycosylation of antibodies was verified by SDS-PAGE analysis.

[0706] Preparation of lipid-based nanoparticles with PEG-Maleimide

[0707] LNP were prepared using microfluidic mixing. Briefly, lipids were dissolved in ethanol with the following molar ratios ALC-0315: 50% / DOPE: 10% / Cholesterol: 38.5 % / DMG-PEG-2000: 1.4 % / DSPE-PEG-Maleimide : 0.1 %, at a total lipid concentration of 9.1 mM.

[0708] FLuc-mRNA (150 pL, 1 mg / mL) was diluted with 25 mM acetate buffer (pH 4.3) to a final concentration of 0.083 mg / mL. The selected experimental conditions allow for a N / P ratio of 6. The lipids (600 pL) and the mRNA solutions (1800 pL) were then injected into a microfluidic mixer (LNP Pack, Inside Therapeutics, France) at a flow rate ratio of 1 :3 and a combined flow rate of 4 mL / min. The resultant formulation was then immediately dialyzed against PBS IM (pH 7.4) for at least 3.5 h using 3.5 MWCO dialysis cassettes (Pur-A-Lyzer, Sigma Aldrich). After dialysis, the Maleimide-LNP solution was used for coupling with the reduced mAbs.

[0709] Reduction of mAbs and coupling with Maleimide-LNP by thiol-Michael addition

[0710] OSE-279 mAbs (0.5 molar equivalent vs DSPE-PEG-Maleimide) were diluted with PBS to obtain a 5 mg / mL solution, before addition of EDTA (5 mM) and TCEP (50 mM, 3 molar equivalent vs OSE-279). The mixture was heated for 30 min at 37°C and stirred for 30 min at room temperature. Excess TCEP was removed by using 7K desalting columns (ThermoFisher) according to manufacturer protocol. The reduced antibody was then immediately conjugated with Maleimide- LNP by incubation for 1 h at room temperature and overnight at 4°C. The conjugated LNP was then purified by gel filtration chromatography (HiLoad 16 / 600 Superdex 200 column, Cytiva) using PBS as a mobile phase (flow rate : 1 mL / min). LNP fractions were collected and concentrated by 100K Amicon tubes (Millipore).

[0711]

[0712] In vitro cellular uptake

[0713] LNP were diluted to 2.2 ng / pL with PBS IX and added on cells previously prepared in a final volume of 60 pL per well in their culture medium (addition of 25 pL LNP / well corresponding to 56 ng RNA / well for U937 cells and 2.5 pL LNP / well corresponding to 5.6 ng RNA / well for Jurkat,

[0714] HPB-ALL and CHO cells).

[0715] The luciferase assay (ONE-Glo™ Luciferase Assay System, Promega) was performed 24 h posttreatment in a 96-wells white cell culture plate and TECAN Spark plate reader were used to quantify the luminescence.

Claims

1. Claims1. A lipid-based nanoparticle comprising an antigen binding domain of an antibody covalently linked to a N-nonglycosylated Fc domain and optionally one or more nucleic acid molecules.

2. The lipid-based nanoparticle of claim 1, wherein the Fc domain is an IgGl or IgG4 Fc domain.

3. The lipid-based nanoparticle of claim 2, wherein the Fc domain is an IgGl domain.

4. The lipid-based nanoparticle of any one of claims 1-3, wherein said N-nonglycosylated Fc domain is a N-deglycosylated Fc domain.

5. The lipid-based nanoparticle of claim 4, wherein the N-deglycosylated Fc domain is also O-deglycosylated.

6. The lipid-based nanoparticle of any one of claims 1-3, wherein said N-nonglycosylated Fc domain is an N-aglycosylated Fc domain.

7. The lipid-based nanoparticle of claim 6, wherein said Fc domain does not have an Asparagine (N) at position 297 in its amino acid sequence, according to Eu numbering.

8. The lipid-based nanoparticle of claim 6 or 7, wherein the N-aglycosylated Fc domain comprises an amino acid substitution in the CH2 domain selected from the group consisting of N297A, N297D, N297Q, N297G, N297F, N297K, N297R, N297H, N297V, N297L, N297I, N297G, N297P, N297E, according to Eu numbering.

9. The lipid-based nanoparticle of claim 6 or 7, wherein the N-aglycosylated Fc domain comprises an amino acid substitution in the CH2 domain selected from the group consisting of N297A, N297Q and N297G, according to Eu numbering.

10. The lipid-based nanoparticle of claim 6 or 7, wherein the N-aglycosylated Fc domain comprises an amino acid substitution N297A in the CH2 domain, according to Eu numbering.

11. The lipid-based nanoparticle of claim 6 or 7, wherein the N-aglycosylated Fc domain comprises an amino acid sequence comprising or consisting of SEQ ID NO: 59 or 63.

12. The lipid-based nanoparticle of claim 6, wherein the N-aglycosylated Fc domain has in its an amino acid sequence an amino acid residue at position 298 and / or 299 that reduces or eliminates glycosylation at position 297, according to Eu numbering.

13. The lipid-based nanoparticle of any one of the preceding claims, wherein the Fc domain i) is not covalently bound to any of the lipids of the lipid-based nanoparticle, and / or ii) does not comprise any modification for coupling or grafting the antigen binding domain to a lipid.

14. The lipid-based nanoparticle of any one of the preceding claims, wherein the Fc domain further comprises a knob-into-hole modification, preferably wherein the Fc domain comprise a first domain comprising or consisting of SEQ ID NO: 66 and a second domain comprising or consisting of SEQ ID NO: 67.

15. The lipid-based nanoparticle of any one of the preceding claims, wherein the antigen binding domain is selected from the group consisting of a Fab, a Fab’, a F(ab')2, a Fv, a crossMAb Fab, a crossMAb Fab’, a crossMAb F(ab')2, a single-chain variable fragment (scFV) and a VHH.

16. The lipid-based nanoparticle of any one of the preceding claims, wherein the antigen binding domain binds to a target selected from the group consisting of PD-1, BCMA / TNFRSF17, BTLA, CD101 / IGSF2, CD103, CD119, CD137 / 4-1BB / TNFRSF9, CD150, CD153, CD154, CD223, CD226, CD25, CD254, CD26, CD27, CD275, CD39 / ENTPD1, CD40L, CD44, CD45RO, CD45RC, LGR6, CD69, GPR18, GPR35, FPR2, CD80, CD83, CD86, CD95, CMKLR1, CRTAM, CST7, CTLA4, CXCR3, CXCR4, CXCR5, CXCR6, FasL / TNFSF6, GITR / TNFRSF18, GPR32, TIM3 / HAVCR2, ICOS, IL18Rl / CXCRl / CD218a, ITGAE, LAG3, TRAILR, OX40L, LY108 / SlamF6, NKG2D, OX40 / TNFRSF4, PTPN22, RGS1, LOX1, SIGLEC 6, TACVTNFRSF13B, TIGIT, CD163, CD206, LTBR / CD70, TNFSF14, SLAMF1, SLAMF7, NKG2A, KIR2DL2, CD96, CD112R, CD28H, IL2RB, TRAIL, CD48, CD53, CD164, CD138 (SDC1), CD38, CD39, FCRL4, CD30 / TNFRSF8, CD78, TRAF1, TRAF2, TRAF3 / CD40BP, TRAF3IP1, TRAF4, TRAF7, TRAP1, TNFR1 / TNFRSF1A / CD120A, TRAP100 / MED24, TNFR2 / TNFRSF1811 / CD120B, CDCR3 / TNFRSF6B, TNFRSF12A / FN14 / TWEAKR, BAFFR / TNFRSF13C / CD268, HVEM / TNFRSF14 / CD270, GITR / TNFRSF8 / CD357, RELT / TNFRSF19L, TNFRSF19 / TROY, TNFRSF21 / DR6, TNFRSF25 / DR3 / TNFRSF12, CD301, IL4R, CLEC-1A, CD21, CLEC-9A, CD180, CD59, CD54, CD71, CD35, CD218a, CD74, CD165, 4-1BBL / CD137L, ICOSL, CD160, CD127 and SIRPg.

17. The lipid-based nanoparticle of any one of the preceding claims, wherein the antigen binding domain binds to a target selected from the group consisting of PD-1, CLEC-1A and CD127.

18. The lipid-based nanoparticle of any one of the preceding claims, wherein the antigen binding domain is an anti-PD-1 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:- 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.

19. The lipid-based nanoparticle of any one of the claims 1-14, wherein the lipid-based nanoparticle comprises a targeting moiety comprising the antigen binding domain covalently linked to the N-nonglycosylated Fc domain, said targeting moiety being an anti-PD-1 antibody comprising a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 70 or 71 and a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 20.

20. The lipid-based nanoparticle of any one of the preceding claims, wherein the lipid-based nanoparticle comprises targeting moieties comprising the antigen binding domain covalently linked to the N-nonglycosylated Fc domain, said targeting moieties being antibodies; wherein in said lipid based nanoparticles at least 50% of the targeting moieties comprises a N-nonglycosylated Fc domain.

21. The lipid-based nanoparticle according to any one of the preceding claims, wherein the lipid-based composition of the lipid based nanoparticle comprises or consists of i) a cationic or ionizable lipid, ii) a helper lipid, iii) a sterol and iv) a PEG-lipid or a combination of PEG-lipids, said combination preferably comprising a first PEG-lipid that does not comprise a reactive functional group for conjugation to the non-glycosylated Fc domain and a second PEG-lipid that comprises a reactive functional group for conjugation to the non-glycosylated Fc domain.

22. The lipid-based nanoparticle of claim 21, 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), 1,2-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), 1,2- distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl )-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn-glycero-3 -121 ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl 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),1.2-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' -di oleylcarbamyl -3- dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), l,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2- Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-di oxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen- 19-yl-4-(dimethylamino)butanoate (DLin-MC3 -DMA), N-(2 -Hydroxy ethyl)-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-((4-122(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), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12- 200), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), bis[2-(4-{2-[4-(cis-9-octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide (SS-OP) and any mixtures thereof, preferably is ALC-0315, SM-102, Dlin-MC3- DMA or SS-OP and any mixture thereof, more preferably is ALC-0315 or SS-OP;- 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;- the helper lipid is selected from DOPE, DOPS, DODMA, DOTAP, DODAP, DDAB, POPE, DSPC, DEPC, DOPC and DSPE and any mixture thereof, preferably is DOPE or DSPC or a mixture thereof; and / or- the PEG-lipid is selected from 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-DSG, PEG-DSPE or ALC-0159 and any mixture thereof.

23. The lipid-based nanoparticle of claim 21 or 22, wherein the lipid-based nanoparticle comprises from about 40 mol % to about 60 mol % of a cationic or ionizable lipid, from about 5 mol% to about 20 mol % of a helper lipid, from about 25 mol% to about 50 mol% of a sterol, and from about 0.5 mol% to about 4 mol% of a PEG-lipid, preferably 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 30 mol% to about 45 mol% of a sterol, and from about 1 mol% to about 2.5 mol% of a PEG-lipid.

24. The lipid-based nanoparticle of any one of the preceding claims, wherein the LNP comprises one or more nucleic acid molecules, said one or more nucleic acid molecules being mRNA.

25. The lipid-based nanoparticle of any one of the preceding claims, wherein the LNP comprises or consists essentially of a) a combination of lipids comprising or consisting of123- an ionizable lipid;- a helper lipid;-a sterol;- a mixture of PEG lipids, said mixture comprising a first PEG-lipid that is not linked to a targeting moiety and a second PEG-lipid that is covalently linked to a targeting moiety, said targeting moiety comprising an antigen binding domain of an antibody, preferably of an anti-PD-1 antibody, covalently linked to the N-nonglycosylated Fc domain; and b) optionally a nucleic acid molecule, preferably one or more mRNA molecule(s).

26. The lipid-based nanoparticle of any one of the claims 21-25, wherein:- the ionizable lipid is selected from the group consisting of ALC-0315, SM-102, Dlin- MC3-DMA and SS-OP;- the helper lipid is DOPE or DSPC;-the sterol is cholesterol;- the mixture of PEG lipids is PEG-DSPE and / or PEG-DMG.

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

28. The lipid-based nanoparticle according to any one of claims 1-26 or the pharmaceutical composition according to claim 27, for use as a medicament.

29. The lipid-based nanoparticle or the pharmaceutical composition of claim for use according to claim 28, for use in the treatment of a cancer, an infectious disease, an inflammatory disease or an auto-immune disease.

30. Use of the lipid-based nanoparticle according to any one of claims 1-26 or of the pharmaceutical composition according to claim 27, in the manufacture or a medicament for treating a disease, preferably selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an auto-immune disease.

31. A method for treating a disease, preferably selected from the group consisting of cancer, an infectious disease, an inflammatory disease and an auto-immune disease, in a subject in need thereof, wherein the method comprises administering to said subject a therapeutic amount of the lipid-based nanoparticle according to any one of claims 1-26 or of the pharmaceutical composition according to claim 27.

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