Modified apolipoproteins with targeting bodies for lipid nanoparticles

Abstract

The present invention relates to modified apolipoproteins with a targeting body. The targeting body may be, for example, an antibody or antigen-binding fragment that allows for targeting, for example, a specific cell, tissue or organ. The modified apolipoprotein can be used as a carrier for a payload in place of itself or when incorporated into lipid nanoparticles. The modified apolipoproteins may be used to treat or prevent a disease, or target a payload to a specific target site.

Description

Field of the Invention

[0001] The present invention relates to the following fields: modified apolipoproteins for treating or preventing disorders. More specifically, the present invention relates to apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics or apolipoprotein mimetic derivatives with targeting bodies. The present invention further relates to lipid nanoparticles comprising modified apolipoproteins and methods for preparing the same. Finally, the present invention relates to methods for treatment using modified apolipoproteins or lipid nanoparticles comprising the same.

[0002] introduction

[0003] Many promising therapeutic agents suffer from the fact that they fail or insufficiently reach the intended target site, or exhibit undesirable off-target effects. In addition, many therapies can trigger unwanted immune responses, which lead to degradation of the therapeutic agent or unwanted inflammatory reactions. Therefore, there is a continuing need to improve or provide alternative ways of targeting therapeutic agents, and to evade immune responses when delivering therapeutic agents to the intended target.

[0004] These problems, among others, are solved by the products and methods defined in the appended claims. Summary of the invention

[0005] The present invention is based on the following findings by the inventors: apolipoproteins, apolipoprotein derivatives, apolipoprotein mimetics or apolipoprotein mimetic derivatives can be modified to target specific cells, tissues or organs by attaching them to targeting bodies, wherein the targeting bodies are capable of binding to molecules on the cell surface of target cells, and such modified apolipoproteins can be used as carriers for therapeutic agents, either when used on their own (meaning not as part of a lipid nanoparticle) or when as part of a lipid nanoparticle, preferably a spherical lipid nanoparticle.

[0006] In addition, the inventors unexpectedly found that attaching the targeting body to apolipoprotein, apolipoprotein derivative, apolipoprotein mimics or apolipoprotein mimics derivatives allows the targeting body to be easily incorporated into the lipid nanoparticle (preferably spherical lipid nanoparticle) comprising an outer layer and a core, and the targeting body is exposed to the environment around the lipid nanoparticle. In this way, modified apolipoprotein can target cells, tissues or organs that it would not or would not reach otherwise, or it can be used to reduce off-target effects.

[0007] In a first aspect, the present invention relates to a modified apolipoprotein comprising an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic or an apolipoprotein mimetic derivative attached to a targeting body, wherein the targeting body is a molecule capable of binding to a molecule on the cell surface of a target cell.

[0008] In a second aspect, the present invention relates to a lipid nanoparticle comprising an outer layer and a core, wherein the outer layer comprises:

[0009] - phospholipids;

[0010] - sterols; and

[0011] - a modified apolipoprotein as disclosed herein; and wherein the core comprises at least one core component selected from:

[0012] A lipid, a cationic lipid, or a multivalent molecule.

[0013] In a third aspect, the present invention relates to a lipid nanoparticle comprising an outer layer and a core, wherein the outer layer comprises:

[0014] - phospholipids;

[0015] - sterols; and

[0016] - a modified apolipoprotein as disclosed herein; and

[0017] The core comprises nucleic acid and cationic lipid or ionizable cationic lipid.

[0018] In a fourth aspect, the present invention relates to a method for preparing lipid nanoparticles as disclosed herein, the method comprising the following steps:

[0019] a1) expressing and isolating a modified apolipoprotein as disclosed herein to obtain an isolated modified apolipoprotein; and / or

[0020] a2) chemically conjugating the targeting body to apolipoprotein or an apolipoprotein mimetic to obtain a modified apolipoprotein and isolating the modified apolipoprotein;

[0021] b) combining the isolated modified apolipoprotein obtained in step a1 and / or step a2 with phospholipids, sterols and optionally lipids to obtain lipid nanoparticles.

[0022] In a fifth aspect, the present invention relates to a method for producing lipid nanoparticles comprising the following steps:

[0023] a) rapidly mixing a lipid component in an organic solvent with nucleic acids in an aqueous buffer to produce lipid nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a cationic lipid, or an ionizable cationic lipid, wherein the aqueous buffer has a pH of 5.0 or less; and

[0024] b) rapidly mixing the lipid nanoparticles with one or more modified apolipoproteins as taught herein;

[0025] The lipid nanoparticles are produced at a pH between 5.5 and 8.0, preferably between 6.0 and 8.0.

[0026] In a sixth aspect, the present invention relates to a modified apolipoprotein according to the first aspect or a lipid nanoparticle according to the second or third aspect, or a lipid nanoparticle obtained or obtainable by a method according to the fourth or fifth aspect of the invention, for use as a medicament.

[0027] In a seventh aspect, the present invention relates to a modified apolipoprotein according to the first aspect or a lipid nanoparticle according to the second or third aspect, or a lipid nanoparticle obtained or obtainable by the method of the fourth or fifth aspect of the invention, for use in treating or preventing immune-related disorders, such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, nervous system disorders or tissue trauma.

[0028] In an eighth aspect, the present invention relates to the use of a modified apolipoprotein according to the second or third aspect, or a lipid nanoparticle obtained or obtainable by the method of the fourth or fifth aspect of the invention, for delivering a compound to a target, preferably wherein the target is a cell, a tissue and / or an organ, even more preferably wherein the target is a lymphoid cell, a myeloid cell, a tumor cell, an endothelial cell, a hematopoietic stem and progenitor cell (HSPC), a hematopoietic stem cell (HSC), a multipotent progenitor cell (MPP), a common myeloid progenitor cell (CMP), or wherein the targeting body binds a bacterial protein or antigen, a viral protein or antigen, a fungal protein or antigen or a parasite protein or antigen,

[0029] Preferably, the lymphoid or myeloid cells are selected from: monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, T helper cells such as Th1, Th2, Th17 or Th22, dendritic cells such as plasmacytoid dendritic cells, conventional DC 1 or conventional DC 2, or tumor-associated macrophages.

[0030] In a ninth aspect, the present invention relates to an in vitro or ex vivo method for introducing a nucleic acid into a cell, the method comprising contacting a lipid nanoparticle as disclosed herein or a lipid nanoparticle obtained or obtainable by a method as disclosed herein with the cell.

[0031] In a tenth aspect, the present invention relates to an in vivo method for introducing a nucleic acid into a cell, the method comprising contacting a lipid nanoparticle as disclosed herein or a lipid nanoparticle obtained or obtainable by a method as disclosed herein with the cell.

[0032] In an eleventh aspect, the present invention relates to a method for in vivo delivery of a nucleic acid, the method comprising administering to a subject a lipid nanoparticle as disclosed herein or a lipid nanoparticle obtained or obtainable by a method as disclosed herein.

[0033] In a twelfth aspect, the present invention relates to a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle as disclosed herein or a lipid nanoparticle obtained or obtainable by a method as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Described is an embodiment of the invention, wherein modified apolipoprotein (in this case apolipoprotein-reselectin fusion protein, and more particularly apolipoprotein-nanobody (VHH) fusion protein) is contained in lipid nanoparticles such as spherical lipid nanoparticles. The main components of apolipoprotein nanoparticles (aNPs) are lipids (e.g., phospholipids), sterols (e.g., cholesterol) and (modified) apolipoproteins.

[0035] Figure 2 is a schematic overview of apolipoprotein (e.g., apolipoprotein A1 (apoA1))-rerouting agent fusion protein nanoparticle technology. (A) Targeting proteins or rerouting agents are molecules that are able to bind to molecules of target cells (on the cell surface) and can include antibodies (fragments), protein ligands, peptides, peptide mimetics, or glycopolymers. (B) Apolipoprotein-rerouting agent fusion proteins are modified apolipoproteins that include an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic, or an apolipoprotein mimetic derivative attached to a targeting body, and more specifically to a rerouting peptide or protein. (C) Apolipoprotein-rerouting agent fusion proteins are easily and stably incorporated into lipid nanoparticles, such as spherical lipid nanoparticles, which include lipids (phospholipids), sterols. In another example, apolipoprotein-rerouting agent fusion proteins are readily and stably incorporated into spherical lipid nanoparticles comprising lipids (phospholipids), sterols, ionizable cationic lipids, and therapeutic payloads including nucleic acids such as small interfering RNA or messenger RNA.

[0036] Figure 3 The expression of VHHCD8-apolipoprotein A1 (apoA1) fusion protein in Clearcoli cells is shown. A small amount of protein contamination is present after IMAC purification [lane E1]. The most prominent band corresponds to the fusion protein with a molecular weight of 43.3 kDa (rectangle).

[0037] Figure 4The mean fluorescence intensity (MFI) of fluorescently labeled VHHCD8-apoA1 and apoA1 in mouse splenocytes is shown (upper panel: CD3+ T cells from splenocytes; lower panel: all cells from spleen).

[0038] Figure 5 Shown are mean fluorescence intensities (MFI) in mouse splenocytes of apolipoprotein nanoparticles (aNPs) formulated with VHHCD8-apoA1 and apoA1 and containing a fluorescent dye in the lipid structure of the particles.

[0039] Figure 6 Shown is the successful formulation of spherical apolipoprotein nanoparticles (aNPs) containing VHHCD8-apoA1 fusion protein using cryo-transmission electron microscopy (cryo-TEM) (bottom panel) and 14-day analysis of nanoparticle size and polydispersity index PDI using dynamic light scattering (DLS) (top panel).

[0040] Figure 7 In vivo biodistribution study setup of spherical apolipoprotein nanoparticles (aNPs) containing the VHHCD8-apoA1 fusion protein.

[0041] Figure 8 In vivo cellular association of VHHCD8-apolipoprotein A1 fusion protein nanoparticles. Flow cytometric analysis of the association of fluorescent apolipoprotein A1 protein nanoparticles (apoA1) and VHHCD8-apolipoprotein A1 fusion protein nanoparticles (VHHCD8) myeloid cells (A), CD8 T cells (B), CD4 T cells (C), CD3 T cells (D) in the bone marrow (BM), spleen and blood after intravenous administration. MFI: mean fluorescence intensity.

[0042] Fig. 9 In vivo cellular association of VHHCD8-Apolipoprotein A1 fusion protein nanoparticles. Flow cytometric analysis of CD8 T cells, CD4 T cells, and CD3 T cells association of fluorescent apolipoprotein A1 protein nanoparticles (apoA1) and VHHCD8-Apolipoprotein A1 fusion protein nanoparticles (VHHCD8) in lymph nodes after intravenous administration. MFI: mean fluorescence intensity.

[0043] Fig.10 In vivo cellular association of VHHCD8-apolipoprotein A1 fusion protein nanoparticles. Flow cytometric analysis of cellular association of fluorescent apolipoprotein A1 protein nanoparticles (apoA1) and VHHCD8-apolipoprotein A1 fusion protein nanoparticles (VHHCD8) in bone marrow (BM), blood, spleen and lymph nodes after intravenous administration.

[0044] Fig.11FRET assay of lipid nanoparticles containing VHHCD8-apolipoprotein A1 fusion protein and / or IL-2-apolipoprotein A1 fusion protein.

[0045] Fig.12 Functional reporter gene expression in mouse splenocytes ex vivo. Flow cytometric analysis of mouse splenocytes incubated with apoA1 aNPs, VHHCD8-apoA1 aNPs, and VHHGFP-apoA1 aNPs containing mRNA encoding the fluorescent mCherry protein (left panel: CD3+CD4-mCherry+ T cells, right panel: CD11b+mCherry+ myeloid cells). DETAILED DESCRIPTION OF THE INVENTION

[0047] For purposes of the present invention, the following terms are defined below.

[0048] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, etc.

[0049] As used herein, the term "and / or" refers to situations where one or more of the stated situations may occur alone or in combination with at least one of the stated situations, up to all of the stated situations.

[0050] As used herein, the term "antigen" refers to a substance to which the binding portion of an antibody can bind. A specific immunoreactive site within an antigen is called an "epitope" (or antigenic determinant). The target of an antibody or its antigen binding portion may include an antigen such as defined herein.

[0051] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" should be understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ..., etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" should be understood to be the same as "5 or less", i.e., 5, 4, 3 ... -10, -11, etc.

[0052] As used herein, the word "comprise" or variations such as "comprises" or "comprising" will be understood to include the stated elements, integers or steps, or groups of elements, integers or steps, but not to exclude any other elements, integers or steps, or groups of elements, integers or steps. The verb "comprise" includes the verbs "consist essentially of" and "consist of."

[0053] As used herein, the term "conventional techniques" refers to situations in which methods of performing conventional techniques used in the methods of the invention will be apparent to those skilled in the art. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields is well known to those skilled in the art and is discussed, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0054] As used herein, the term "identity" refers to a measure of the identity of a nucleotide sequence or an amino acid sequence. Typically, the sequences are aligned so that the highest level of matching is obtained. "Identity" itself has a meaning recognized in the art and can be calculated using disclosed techniques. See, for example: (Computational Molecular Biology, Lesk, AM, ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM, and Griffin, HG, EDS., Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J., ed., M Stockton Press, New York, 1991). Although there are many methods to measure the identity between two nucleotide sequences or amino acid sequences, the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48: 1073). In general, methods for determining the identity or similarity between two sequences include, but are not limited to, those disclosed in Guide To Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48: 1073. Methods for determining identity and similarity are encoded in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol. (1990) 215:403).

[0055] As an illustration, a polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to a reference nucleotide sequence encoding a polypeptide of a certain sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference amino acid sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and / or substituted with another nucleotide, and / or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between these terminal positions, interspersed individually between nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence.

[0056] Similarly, by reference to a polypeptide having an amino acid sequence that is, for example, at least 95% "identical" to a reference amino acid sequence of SEQ ID NO: X, it is meant that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the reference amino acid SEQ ID NO: X. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or up to 5% of the amino acids in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, interspersed individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0057] As used herein, the term "in vitro" refers to experiments or measurements performed using components of an organism that have been isolated from its native condition.

[0058] As used herein, the term "ex vivo" refers to experiments or measurements performed in or on tissues from an organism in an external environment with minimal changes from natural conditions.

[0059] As used herein, the terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are intended to include DNA molecules and RNA molecules. Nucleic acid (molecule) can be single-stranded or double-stranded, but is preferably double-stranded DNA or single-stranded RNA or double-stranded RNA, more preferably single-stranded RNA or double-stranded RNA.

[0060] The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are well known in the art. By further guidance, these terms generally refer to polymers (preferably linear polymers) of any length that are substantially composed of nucleoside units. Nucleoside units generally include heterocyclic bases and sugar groups. Heterocyclic bases can especially include purine and pyrimidine bases, such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), which are widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated), non-natural or derived bases. Exemplary modified nucleobases include, but are not limited to, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In particular, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability. Sugar groups may include pentose (pentofuranosyl) groups, such as the preferred ribose and / or 2-deoxyribose commonly found in naturally occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose groups, as well as modified or substituted sugar groups (such as, but not limited to, 2'-O-alkylated, e.g., 2'-O-methylated or 2'-O-ethylated sugars, such as ribose; 2'-O-alkoxyalkylated, e.g., 2'-O-methoxyethylated sugars, such as ribose; or 2'-O,4'-C-alkylene-linked, e.g., 2'-O-methylene-linked or 2'-O,4'-C-methylene-linked or 2'-O,4'-C-ethylene-linked sugars, such as ribose; 2'-fluoro-arabinose, etc.). The nucleoside units may be linked to one another by any of a number of known internucleoside linkages, including inter alia the phosphodiester linkages commonly found in naturally occurring nucleic acids, as well as further modified phosphate- or phosphonate-based linkages such as phosphorothioates, alkylphosphorothioates (such as methylphosphorothioate), phosphorodithioates, alkylphosphonates (such as methylphosphonate), alkylphosphorothioates, phosphotriesters (such as alkylphosphotriesters), phosphoramidates, phosphoropiperazidates, morpholinophosphoroesters, bridged phosphoramidates, bridged methylenephosphonates, bridged phosphorothioates; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate (such as 3'-N-carbamate), morpholino, borane, thioether, 3'-thioacetal, and sulfone internucleoside linkages. Preferably, the internucleoside linkage may be a phosphate-based linkage, including a modified phosphate-based linkage, such as, more preferably, a phosphodiester, phosphorothioate or phosphorodithioate linkage, or a combination thereof.The term "nucleic acid" also encompasses any other nucleobase-containing polymers, such as nucleic acid mimetics, including but not limited to peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholinophosphodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclic-DNA (tcDNA), and nucleic acids with backbone moieties having alkyl or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628–1644)). As used herein, "alkyl" particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 straight or branched chain hydrocarbons, saturated or unsaturated hydrocarbons, such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl, and isopropyl.

[0061] The nucleic acid contemplated herein can include naturally occurring nucleosides, modified nucleosides or mixtures thereof. Modified nucleosides can include modified heterocyclic bases, modified sugar moieties, modified internucleoside bonds or combinations thereof. The term "nucleic acid" further preferably encompasses DNA, RNA and DNA / RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides and synthetic (e.g., chemically synthesized) DNA, RNA or DNA / RNA heterocompounds. Nucleic acid can be naturally occurring, such as present in nature or separated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and / or can be partially or entirely chemically or biochemically synthesized." Nucleic acid" can be double-stranded, partially double-stranded, or single-stranded. At the single-stranded part, the nucleic acid can be a sense strand or an antisense strand. In addition, the nucleic acid can be circular or linear.

[0062] As used herein, the term "sequence" or "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" when referring to nucleotides refers to the order of nucleotides of a nucleic acid and / or polynucleotide, or the order of nucleotides within a nucleic acid and / or polynucleotide. In the context of the present invention, a first nucleic acid sequence may be contained within or overlap with a further nucleic acid sequence.

[0063] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal", which are used interchangeably, refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, livestock, farm animals and zoo animals, sports animals or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, etc. As defined herein, a subject may be alive or dead. Samples may be taken from a subject post mortem, i.e., after death, and / or samples may be taken from a living subject.

[0064] As used herein, the terms "treatment," "treating," "palliating," "alleviating," or "ameliorating," which are used interchangeably, refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefit. By therapeutic benefit, it is meant eradication or amelioration or reduction (or delay) of the progression of the underlying disease being treated. Furthermore, therapeutic benefit is achieved with eradication or amelioration or reduction (or delay) of the progression of one or more physiological symptoms associated with the underlying disease, such that an improvement or slowing or reduction in decline is observed in the patient, although the patient may still be suffering from the underlying disease.

[0065] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which the nucleic acid molecule capable of transport has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be linked. The term "vector" may also refer to a viral particle (i.e., a viral vector) containing a nucleic acid of interest.

[0066] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, photographs of devices, or any other aspect of this disclosure for which copyright protection is available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patented invention as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0067] Various terms related to the method, composition, purposes and other aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, such terms will be given their ordinary meaning in the relevant field of the present invention. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein. Preferred materials and methods are described herein, although any methods and materials similar or equivalent to those described herein can be used to test the practice of the present invention.

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0069] The present invention is based on the discovery of the inventor, i.e. apolipoprotein can be modified to target specific cells, tissues, organs or pathogens. The inventor has found that the modified apolipoprotein in conjunction with the targeting body allows the targeting of the lipid nanoparticle containing this type of modified apolipoprotein. Because lipid nanoparticle (or modified apolipoprotein) can be loaded with a useful load such as a medicine, this allows the drug to be directly targeted to the desired target site. In addition, modified apolipoprotein of the present invention also allows the preparation of lipid nanoparticles, wherein as a result, the targeting body is exposed to the environment (i.e. aqueous environment) around the apolipoprotein lipid nanoparticle.

[0070] Thus, in a first aspect, the present invention relates to a modified apolipoprotein comprising an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic or an apolipoprotein mimetic derivative attached to a targeting body, wherein the targeting body is a molecule capable of binding to a molecule on the cell surface of a target cell.

[0071] Modified apolipoprotein can be used by itself, which means not as a part of lipid nanoparticles. In this way, modified apolipoprotein can serve as a carrier to deliver a payload (e.g., a therapeutic agent) to a target site. Payload can be combined with modified apolipoprotein, for example, by a covalent bond. Targeting bodies can be used to target modified apolipoprotein to a specific site, such as a cell, tissue, organ, or pathogen.

[0072] As used herein, when referring to a modified apolipoprotein, the term attached may refer to a stable or semi-stable association, such as, but not limited to, covalent bonds, hydrogen bonds, or van der Waals forces, or combinations thereof, such as, but not limited to, peptide bonds, sulfide bonds, protein-protein interactions, or protein-ligand interactions.

[0073] Targeting body

[0074] As used herein, a targeting body refers to a molecule that, when attached to a protein such as apolipoprotein, allows the protein to bind to a target that is different from the target to which it would bind when the protein was not fused to the targeting body (or in other words, a target that is different from the target to which it would inherently bind), and / or to bind to its intended target with higher affinity.

[0075] In certain embodiments, the targeting body can allow binding to different targets, such as specific subpopulations of target cells, including binding to specific subpopulations of a group of cells that would normally be bound by a modified apolipoprotein. For example, apoA1 is known to bind to receptors on myeloid cells, so the targeting body can be used to bind to a specific subtype of myeloid cells. For example, as described elsewhere herein, SIRPα as a targeting body can allow targeting of immunosuppressive macrophages. Non-limiting examples of targeting bodies are antibodies or antigen-binding fragments thereof or antibody fragments, rerouting peptides or rerouting proteins, preferably wherein the rerouting peptide or rerouting protein is a ligand for a receptor present on the target.

[0076] Thus, in embodiments, the targeting agent is selected from:

[0077] - an antibody or an antigen-binding fragment of an antibody,

[0078] - a protein ligand, a protein binding domain or a protein binding fragment thereof,

[0079] -peptides,

[0080] - a peptide mimetic, or

[0081] -Sugar polymers.

[0082] As used herein, the term target refers to an object to which a modified apolipoprotein or targeting body preferentially binds. A target may refer to a receptor or cell surface molecule, such as a protein or proteoglycan, a lipid, a phospholipid, a sugar, a glycopolymer, a cell, a cell type, a tissue or tissue type, or an organ.

[0083] It should be understood that apolipoprotein is combined with a specific ligand. For example, it is believed that the different apolipoproteins found in different lipoproteins (such as HDL, LDL, VLDL, etc.) are responsible for the targeting and combination of lipoprotein and the differences in function. Without wishing to be bound by theory, it is believed that a part of apolipoprotein has an amphipathic nature, and is responsible for binding the lipid in the aqueous environment together with phospholipids and / or sterols, while the different parts of the molecule are responsible for interacting with other molecules, such as binding protein receptors. It is further assumed that apolipoprotein can also circulate as protein, which means not as lipoprotein. Therefore, the possibility of modifying the binding affinity of apolipoprotein (by adding a targeting body) provides interesting opportunities, because it allows fine-tuning the targeting or combination of apolipoprotein. Several applications of such modified apolipoproteins are envisioned:

[0084] First, targeting bodies can simply be used to reroute apolipoproteins or lipoproteins, such as to effect changes in lipid homeostasis. For example, one can envision that by using apolipoproteins in combination with targeting bodies, LDL or HDL values ​​in a subject's blood can be altered. This can potentially be used to treat lipid disorders such as high blood cholesterol levels.

[0085] Secondly, it can be used to reroute lipoproteins (lipid nanoparticles) with a payload to a predetermined target. Lipoproteins or lipid nanoparticles present an interesting method for carrying payloads such as drug compounds. It allows the delivery of lipophilic compounds through the blood because the compounds can be dissolved in the lipid core of the lipoprotein / lipid nanoparticle. An additional advantage is that lipoproteins are composed of naturally occurring compounds and are therefore considered natural by the immune system, which avoids the drug compound from triggering an immune response.

[0086] The third, it can be further combined with a payload, and therefore allows the targeting of a payload. As mentioned above, apolipoprotein can serve as the carrier of a payload substantially. The advantage is that it greatly reduces the removal of a payload, which makes it feasible to use a payload (such as a cytokine) that is easily removed from blood in a treatment context. A problem that may occur is that the payload / apolipoprotein combination does not reach the expected target (that is, the position, cell, tissue or organ that it is expected to exert its effect) of a payload. This is solved by further including a targeting body.

[0087] The data generated by the inventors indicate that modified apolipoproteins can be successfully rerouted to different targets by using targeting bodies. Thus, in one embodiment, the targeting body is selected from an antibody or antigen binding fragment thereof, a rerouting peptide or a rerouting protein, preferably, wherein the rerouting peptide or rerouting protein is a ligand for a receptor present on the target.

[0088] In an embodiment, the targeting entity may be an antibody or an antigen binding fragment thereof.It is envisaged that any type of antigen binding molecule may in principle be used as a targeting entity in the modified apolipoprotein according to the invention.

[0089] In a specific embodiment, the targeting body binds to an antigen that is highly expressed on myeloid cells or is present only on myeloid cells. Suitable targets are known to the skilled person, and non-limiting examples are CD11b, CD11c, CD14, or co-stimulatory molecules such as CD80, CD83, CD86, CD40, or HLA-DR. Therefore, in an embodiment, the targeting body is a peptide or protein targeting the myeloid system selected from an antibody or antigen-binding fragment thereof that binds to CD11b, CD11c, CD14, CD80, CD83, CD86, CD40, or HLA-DR.

[0090] In other specific embodiments, the targeting body can be a protein ligand that binds to a receptor or factor expressed on myeloid cells, a non-limiting example being CD40L (CD154) and the FC domain, but the skilled person will know other suitable ligands or cofactors. Thus, in one embodiment, the targeting body is a myeloid-targeting peptide or a myeloid-targeting protein, wherein the myeloid-targeting protein or the myeloid-targeting peptide is selected from CD40L (CD154) and the FC domain.

[0091] In an embodiment, the targeting agent is an antibody or an antigen-binding fragment thereof, wherein the antibody or its antigen-binding fragment is selected from: fragment antigen-binding region (Fab), Fab2, single-chain variable fragment (scFv), scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab3, trispecific Fab3, diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, immunoglobulin new antigen receptor (IgNAR), monovalent IgG, VhH, nanobody or variable domain of new antigen receptor (VNAR) or its antigen-binding fragment, preferably camel VhH or shark VhH or its derivative or antigen-binding fragment thereof.

[0092] The antibody or antigen-binding fragment may also be a designed antigen-binding protein, such as, but not limited to, an affibody, an FN3 domain, a DARPin, or a de novo designed protein receptor. It will be appreciated that antibodies or antigen-binding fragments thereof having a lower molecular weight are preferred due to their reduced size, and thus in a preferred embodiment, the antibody or antigen-binding fragment thereof is a Fab, scFv, single domain antibody, V h H or VNAR.

[0093] In an embodiment, the targeting body can be a re-selection peptide. Non-limiting examples of re-selection peptides are receptor binding peptides and / or ligand mimetic peptides. Therefore, in one embodiment, the re-selection peptide is selected from programmed cell death protein 1 (PD1) or signal regulatory protein alpha (SIRPa). SIRPα as a targeting body can allow targeting of immunosuppressive macrophages. However, it should be understood that any peptide with binding specificity to a cell surface receptor can be used as a targeting body.

[0094] In embodiments, the targeting body can be a protein, such as a receptor ligand, a receptor or an interacting protein. Alternatively, the targeting body can be a part of a protein, such as a protein or a ligand binding domain. Therefore, in embodiments, the reselection protein is selected from CD40L or GP120, for example. CD40L can be used to target cells expressing the CD40 receptor. GP120 can be used to directly bind to CD4 T cell co-receptors. However, it should be understood that any protein with binding specificity to a cell surface receptor can be used as a targeting body.

[0095] In embodiments, the targeting body can be a glycopolymer. The glycopolymer can be used to direct the apolipoprotein modified with the glycopolymer to a specific site or receptor. For example, the apolipoprotein can be modified with mannose to direct the modified apolipoprotein to the mannose receptor, which is usually expressed in macrophages, dendritic cells and endothelial cells. The technician will be aware of other glycopolymers that can be used for similar purposes, and which cells can be targeted with these glycopolymers. When used herein, the term glycopolymer refers to any polysaccharide, and therefore refers to any molecule comprising at least two sugars.

[0096] As used herein, the term lipoprotein refers to a particle, typically a nanoparticle, of at least one apolipoprotein and lipid molecules dispersed or dissolved in an aqueous environment.

[0097] As used herein, the term rerouting refers to targeting a modified apolipoprotein to a different target to which it normally binds, or reducing binding to the conventional target of the modified apolipoprotein or preventing off-target binding. For example, apolipoprotein a1 (apoA1) is known to bind to receptors on myeloid cells, so a targeting agent can be used to bind to a different cell and reduce binding to a myeloid cell (e.g., a myeloid cell can be a monocyte, a dendritic cell, a tissue macrophage, or a granulocyte).

[0098] In embodiments, the targeting body binds to a protein on the surface of a lymphoid cell, a myeloid cell, a tumor cell, an endothelial cell, a hematopoietic stem and progenitor cell (HSPC), a hematopoietic stem cell (HSC), a multipotent progenitor cell (MPP), a common myeloid progenitor cell (CMP), or wherein the targeting body binds to a bacterial protein or antigen, a viral protein or antigen, a fungal protein or antigen, or a parasitic protein or antigen,

[0099] Preferably, wherein the targeting body binds to a protein on the surface of a lymphoid cell, a myeloid cell, a tumor cell, or wherein the targeting body binds to a bacterial protein or antigen, a viral protein or antigen, a fungal protein or antigen, or a parasitic protein or antigen,

[0100] Preferably, the lymphoid or myeloid cell is selected from the group consisting of monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, granulocytes, T helper cells such as Th1, Th2, Th17 or Th22, dendritic cells such as plasmacytoid dendritic cells, conventional DC 1 or conventional DC 2, or tumor-associated macrophages,

[0101] More preferably, the lymphoid or myeloid cells are selected from monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, T helper cells such as Th1, Th2, Th17 or Th22, dendritic cells such as plasmacytoid dendritic cells, conventional DC 1 or conventional DC 2, or tumor-associated macrophages.

[0102] In certain embodiments, the myeloid cell can be a blood cell derived from a progenitor cell of a granulocyte, a monocyte, an erythrocyte, or a platelet.

[0103] In an embodiment, the targeting body binds to a protein selected from the following: CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD15u, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD133, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147,CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD157、CD158、CD158a、CD158b、CD159a、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD183、CD184、CD195、CDw197、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207(CLEC4K)、CD208、CD209(CLEC4L)、CDw210、CD212、CD213a1、CD213a2、CDw217、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CCR1、CCR2、CCR3、CCR4、CCR5、CCR7、CCR8、CCR9、CLEC1A、CLEC1B、CLEC2A、CLEC2B、CLEC3A、CLEC3B、CLEC4A、CLEC4C(CD303)、CLEC4D、CLEC4J、CLEC4E、CLEC4F、CLEC4G、ASGR1(CLEC4H1)、ASGR2(CLEC4H2)、FCER2()、CLEC4M、CLEC5A、CLEC6A、CLEC7A、OLR1(CLEC8A)、CLEC9A、CLEC10A、CLEC11A、CLEC12A、CLEC12B、CD302(CLEC13A)、LY75(CLEC13B)、PLA2R1(CLEC13C)、MRC1(CLEC13D)、MRC2(CLEC13E)、CLEC14A、CLEC16A、CLEC17A、KLRA1、KLRB1(CLEC5B)、KLRC1、KLRC2、KLRC3、KLRC4, KLRD1, KLRF1(CLEC5C), KLRG1(CLEC15A), KLRG2(CLEC15B), KLRK1, AGC1, ATRNL1, BCAN, CD248, CHODL, CL-K1-Ia, C L-K1-Ib, CL-K1-Ic, CLECSF5, COLEC10, COLEC11, COLEC12, CSPG3, FCER2, FREM1, HBXBP, LAYN, LOC348174, LOC728276, MAFA 、MBL2、MGC34761、MICL、MRC1L1、PAP、PKD1、PKD1L2、PRG2、PRG3、REG1A、REG1B、REG3A、REG3G、REG4、SELE、SELL、SELP、SFTPA1、SFTPA2、SFTPA2B、SFTPD、SRCL、THBD、VCAN、alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelioma antigen (ETA), tyrosinase and melanoma-associated antigen (MAGE). 、

[0104] Preferred proteins are selected from the following: CD14, CD11b, CD357 (GITR), CD193, CD123, CD117, CD56, CD19, CD38, CD25, CD133, CXCR3, CCR3, CD196, CCR10, CD64, CD206, CLEC4C (CD303), CLEC9A, CD1c, CD163, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelioma antigen (ETA), tyrosinase and melanoma-associated antigen (MAGE).

[0105] These targets can be used to target specific cell types or cell subpopulations. The skilled person knows which markers are suitable for which cell types, for example, the following proteins can be used to target specific cells:

[0106] Cells of the Innate Immune System:

[0107] -Monocytes: CD14

[0108] -Macrophages: CD11b

[0109] - Innate lymphoid cells: CD357 (GITR)

[0110] -Eosinophils: CD193

[0111] - Basophils: CD123

[0112] -Mast cells: CD117

[0113] Cells of the Adaptive Immune System:

[0114] -NK cells: CD56

[0115] -B cells: CD19

[0116] -Plasma cells: CD38

[0117] - Regulatory T cells: CD25

[0118] Immune cell subtypes:

[0119] -Myeloid Progenitor Cells:

[0120] -Hematopoietic stem cells: CD133

[0121] -T helper cell subsets: Th1: CXCR3, Th2: CCR3, Th17: CD196, Th22: CCR10

[0122] - Macrophage polarization state (M1-like or M2-like): M1: CD64, M2: CD206 (mannose receptor) (e.g., targeted by apolipoprotein conjugated with a nanobody specific for this receptor or apolipoprotein conjugated with mannose)

[0123] - Subtypes of dendritic cells: plasmacytoid dendritic cells: CD303, conventional DC 1: CLEC9A, conventional DC 2: CD1c

[0124] -Tumor-associated macrophages: CD163 (or targeted with nanobodies against tumor antigens).

[0125] In certain embodiments, the targeting body is capable of binding to non-myeloid cells, such as non-myeloid immune cells, preferably non-myeloid leukocytes, more preferably lymphocytes, more preferably T cells, even more preferably CD8+ T cells.

[0126] In certain embodiments, the targeting body is capable of binding to cells that are not myeloid cells but can differentiate into myeloid cells, such as hematopoietic stem and progenitor cells (HSPCs), e.g., hematopoietic stem cells (HSCs), multipotent progenitor cells (MPPs), or common myeloid progenitor cells (CMPs).

[0127] In certain embodiments, the targeting body can be bound to non-myeloid cells, such as non-myeloid immune cells or endothelial cells. Endothelial cells can be targeted by using a targeting body that can be bound to endothelial cell surface markers. For example, endothelial cells can be targeted by using a targeting body that can be bound to factor VIII related antigens such as factor VIII, a targeting body that can be bound to CD31 / PECAM-1 such as CD31, a targeting body that can be bound to angiotensin converting enzyme (ACE / CD143) such as angiotensin, a targeting body that can be bound to CD34 such as L-selectin or a targeting body that can be bound to endoglin (CD105).

[0128] In a particular embodiment, the non-myeloid cell is a lymphocyte, such as a T cell, a B cell or a natural killer (NK) cell. Preferably, the lymphocyte is a T cell, even more preferably a CD8+ T cell.

[0129] In certain embodiments, the targeting entity is an antibody or antigen-binding fragment thereof that binds, such as specifically binds, to CD8.

[0130] In a particular embodiment, if the target cell is a T cell, the targeting body can be an antibody or antigen binding fragment thereof that binds (preferably specifically binds) to CD8. For example, the targeting body can be VHHCD8 as described in Woodham AW et al., Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses, Cancer Immunology Research, 2018, Vol.6, issue 7, and comprises an amino acid sequence as shown in Supplementary Table 1 of the reference.

[0131] For example, apolipoprotein nanoparticles targeting CD8 as described herein can also be used to generate chimeric antigen receptor (CAR) T cells in vivo. In particular, apolipoprotein nanoparticles targeting CD8 can be used to deliver coding instructions to reprogram T cells in vivo plasmid DNA (pDNA), linear or circular RNA (e.g., linear or circular mRNA) and / or gene editing components. For example, targeted nucleases can be used to introduce nucleic acids into cells. For example, targeted nucleases include but are not limited to transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZNFs), regular clustered short palindromic repeats (CRISPR), CRISPR / Cas9, CRISPR / CPFL and combinations thereof, such as using single guide RNA or other gene editing components, such as single guide RNA with CRISPR / Cas. In a particular example, pDNA or RNA encoding a chimeric antigen receptor (CAR) can be delivered to T cells so that they can recognize and kill tumor cells with cognate ligands. In another specific example, pDNA or RNA encoding a chimeric antigen receptor (CAR) can be delivered to T cells, enabling them to recognize and eliminate pathological cells, such as activated fibroblasts in fibrotic disorders.

[0132] As described herein, the apolipoprotein nanoparticles targeting CD8 can also be used to modify CD8+T cell functions.For example, CD8+T cell functions can be modified by using the apolipoprotein nanoparticles targeting CD8 to modify cytokine-cytokine receptor interactions, and the apolipoprotein nanoparticles targeting CD8 deliver the plasmid DNA (pDNA), linear or circular RNA (e.g., linear or circular mRNA) and / or gene editing components of the instructions encoding the production of cytokines in T cells as described above. In a particular example, pDNA or mRNA encoding cytokines such as IFN-γ, IL-2, IL-10, IL-12, IL-15 can be delivered to T cells so that they can secrete cytokines. This can result in immunosuppressive tumor microenvironment being reprogrammed to an anti-tumor phenotype. In another particular example, pDNA or mRNA encoding cytokine receptors such as IFN-γR, IL-2R, IL-12R or IL-15R can be delivered to T cells. CD8+T cell function can also be modified by regulating immune checkpoints, for example, CD8+T cell function can be modified by downregulating immune checkpoints using apolipoprotein nanoparticles targeting CD8, which deliver antisense oligonucleotides, small interfering RNA (siRNA), mRNA and / or gene editing components encoding instructions as described above to downregulate immune checkpoints in T cells. In a specific example, siRNA or gene editing components targeting PD1, CTLA4, SHP-2, LAG3 or TIM-3 as described above can be delivered to T cells for downregulating checkpoint molecules that inhibit cytotoxic T cell function in cancer.

[0133] In another example, the apolipoprotein nanoparticles targeting CD8 as described herein can be used to repolarize pathogenic CD8+T cells in autoimmunity to a tolerogenic phenotype. In particular, the apolipoprotein nanoparticles targeting CD8 can be used to deliver antisense oligonucleotides, small interfering RNA (siRNA), mRNA or gene editing components of the coding instructions as described above to repolarize pathogenic T cells. In a specific example, the mRNA encoding FOXP3 can be delivered to T cells to change its phenotype into regulatory T cells (T reg). In another specific example, CD8+IL17+T cells can be repolarized via delivery of siRNA targeting IL-17 or RORyt transcription factors.

[0134] In one embodiment, the modified apolipoprotein comprises aapoA1 having VHH8CD8.

[0135] In an embodiment, the apolipoprotein modified by VHHCD8-apoA1 comprises or consists of a polypeptide having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.7 or 1, or comprises or consists of an amino acid sequence that is encoded by a nucleic acid having a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.8 or 2.

[0136] In certain embodiments, if the target cells are T and / or B cells, the targeting agent may be PD1, CD40L or GP120.

[0137] Apolipoprotein

[0138] Apolipoprotein is a protein that combines lipids such as triglycerides and cholesterol to form lipoproteins. They transport lipids (and fat-soluble vitamins) in blood, cerebrospinal fluid and lymph. The lipid components of lipoproteins are insoluble in water. However, because of their detergent-like (amphiphilic) properties, apolipoproteins and other amphiphilic molecules (such as phospholipids) can surround lipids, which produce lipoprotein particles that are themselves water-soluble, and therefore can be carried by circulation (i.e. blood, lymph) based on water. In addition to stabilizing the lipoprotein structure and dissolving the lipid components, apolipoproteins also interact with lipoprotein receptors and lipid transport proteins, thereby participating in the uptake and removal of lipoproteins.

[0139] In lipid transport, apolipoproteins serve as structural components of lipoprotein particles, ligands of cell surface receptors and lipid transport proteins, and cofactors of enzymes. Different lipoprotein particles contain different classes of apolipoproteins, which affect their functions. For example, apolipoprotein A1 (apoA1) is the main structural protein component of high-density lipoprotein (HDL), although it is present in other lipoproteins in smaller amounts, and HDL contains other apolipoproteins.

[0140] It is contemplated that the present invention is not limited to a particular type of apolipoprotein, and thus in embodiments, the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative is selected from apoA1, apoA-1 Milano, apoA2, apoA4, apoA5, ​​apoB, apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL, aapoL1, aapoL2, aapoL3, aapoL4, aapoL5, aapoL6, aapoLD1, apoO, apoOL and apoM, or a combination thereof, or a mimetic or derivative thereof,

[0141] Preferably selected from apoA1, apoA2, apoA4, apoA5, ​​apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL and apoM, or a mimetic or derivative thereof,

[0142] More preferably, it is selected from apoA1, apoA2, apoA4, apoA5, ​​apoB100, apoC-I, apoC-II, apoC-III, apoC-IV and apoE, or a mimetic or derivative thereof,

[0143] Even more preferably selected from apoA1, apoA4, apoA5, ​​apoB100, apoC-III and apoE, or a mimetic or derivative thereof,

[0144] Most preferably, it is selected from apoA1, apoB100 and apoE, or a mimetic or derivative thereof.

[0145] In certain embodiments, the apolipoprotein component of the modified apolipoprotein is apoA1 or an apoA1 mutant.

[0146] In a specific embodiment, the apolipoprotein component can also be an apolipoprotein fragment. Preferably, the apolipoprotein fragment retains the biological activity of the full-length apolipoprotein, such as the ability of apolipoprotein to be integrated into lipid nanoparticles or to target myeloid compartments. In a specific embodiment, the apolipoprotein fragment at least comprises the ATP binding cassette subfamily A member 1 (ABCA1), ATP binding cassette subfamily G member 1 (ABCG1) and / or scavenger receptor B class type 1 (SR-BI) binding region of the full-length apolipoprotein, thereby allowing binding to myeloid cells. In a specific embodiment, the apolipoprotein fragment at least comprises the alpha helix of the full-length apolipoprotein. These helices are hydrophilic on one side (interacting with an aqueous environment) and hydrophobic on the other side (interacting with lipids in the particle).

[0147] In certain embodiments, a fragment may be an N-terminally truncated form and / or a C-terminally truncated form of the full-length peptide, polypeptide or protein from which it is derived.

[0148] In a particular embodiment, a fragment may comprise at least about 30%, such as at least about 50% or at least about 70%, preferably at least about 80%, such as at least about 85%, more preferably at least about 90%, and still more preferably at least about 95% or even about 99% of the length of the amino acid sequence of the full-length peptide, polypeptide or protein from which it is derived. For example, within the range of not exceeding the length of the full-length peptide, polypeptide or protein, a fragment may comprise a sequence of ≥5 consecutive amino acids or ≥10 consecutive amino acids or ≥20 consecutive amino acids or ≥30 consecutive amino acids, such as ≥40 consecutive amino acids, such as, for example, ≥50 consecutive amino acids, for example ≥60, ≥70, ≥80, ≥90, ≥100 or ≥200 consecutive amino acids of the corresponding full-length peptide, polypeptide or protein.

[0149] In certain embodiments, the apolipoprotein component, such as an apolipoprotein fragment, comprises a myeloid-binding portion of the full-length apolipoprotein.

[0150] In a specific embodiment, the apolipoprotein component is an apolipoprotein mutant comprising a mutation that allows chemical conjugation of the apolipoprotein to a targeting entity, wherein the targeting entity is a molecule that is capable of binding to a molecule on the cell surface of a target cell. In a specific embodiment, the apolipoprotein component can also be an apolipoprotein mutant comprising a serine to cysteine ​​substitution, such as a mutant defined by SEQ ID NO: 9, 10, 11 or 12 as described elsewhere herein.

[0151] The peptide sequences of the different proteins described herein or the nucleic acid sequences of the genes encoding the different proteins described herein are readily available to the skilled person, for example from the UCSC Genome Browser (http: / / genome.ucsc.edu / ), the Ensembl genome browser (https: / / www.ensembl.org) and NCBI (https: / / www.ncbi.nlm.nih.gov / protein). The consensus sequences of the different proteins or genes are easily derived from these sources, although it should be understood that certain variations may exist due to, but not limited to, genetic variation and various splicing variations of the genes. Therefore, when referring to a specific protein, this should be interpreted as covering sequence variations due to genetic variation and splicing variation. Therefore, when used herein, when referring to a certain protein, this should be interpreted as the corresponding consensus protein sequence retrieved from the Ensembl genome browser, or the consensus nucleic acid (gene) sequence retrieved from the Ensembl genome browser, or a protein sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence retrieved from the Ensembl genome browser, or the consensus nucleic acid (gene) sequence retrieved from the Ensembl genome browser. The invention further comprises a gene sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus gene sequence retrieved from the Ensembl genome browser, or a nucleic acid sequence that encodes a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence retrieved from the Ensembl genome browser.

[0152] Apolipoprotein mimics are synthetic peptides or proteins that simulate the function or structure of apolipoprotein. Several apolipoprotein mimics are known, and for example Wolska et al. (Cells. 2021 Mar; 10 (3): 597., incorporated by reference in its entirety) reviewed the different apoA1, apoE and apoC-II mimics described in the literature. For example, aapoA1 mimetic peptides are designed to a large extent based on their ability to efflux cholesterol from cells. Since the process has not yet been shown to rely on specific protein-protein interactions, most apoA1 mimetic peptides are merely amphipathic helices, and in fact, many have no primary amino acid homology with apoA1. Exemplary aapoA1 mimics are aapoA1 mimic 18A, aapoA1 mimic 2F and aapoA1 mimic 37pA.

[0153] For example, apoE has several putative anti-atherosclerotic (atheroprotective), and many different types of apoE-based peptides have been reported. One of the main goals of designing these peptides is to promote the hepatic clearance of apoB-containing lipoproteins. Since apoE can bind to its receptor only when it is bound to lipids, these peptides usually have not only a receptor binding motif from the N-terminal domain of apoE, but also a lipid binding region or some other sequence based on the C-terminal domain of apoE.

[0154] For example, apoC-II mimetics have been described that are based on a shortened first helix (18A) linked to the LPL activation domain of apoC-II, or in which both the first and second helices are based on mimetics of the native apoC-II helix with amino acid substitutions to enhance binding to the double helix of lipoproteins.

[0155] Therefore, when used herein, apolipoprotein mimetics refers to synthetic proteins or peptides that share structural features and / or functional features with respective apolipoproteins. For example, the shared structural features may be primary, secondary or tertiary peptide structures, such as peptide sequences, structures such as the presence of alpha helices or beta folds or three-dimensional structures of peptides, or functional features may be similarities in binding to certain targets such as receptors. Preferably, apolipoproteins are capable of binding lipids in a manner similar to the corresponding apolipoproteins, more preferably forming lipid particles.

[0156] In certain embodiments, an apolipoprotein mimetic may be capable of binding to myeloid cells to the same or similar extent as the respective apolipoprotein. For example, an apolipoprotein mimetic of apoA1 preferably is capable of binding to myeloid cells to the same or similar extent as apoA1.

[0157] In an embodiment, the modified apolipoprotein is a combination of apoAl and a targeting body. In an embodiment, the modified apolipoprotein is a combination of an apoAl mimetic and a targeting body. In an embodiment, the modified apolipoprotein is a combination of apoE and a targeting body.

[0158] In an embodiment, the modified apolipoprotein is a combination of an apolipoprotein component (e.g., an apoA1 mutant, preferably a human apoA1 mutant) and a targeting agent. In an embodiment, the modified apolipoprotein is a combination of an apoA1 mimetic and a targeting agent. In an embodiment, the modified apolipoprotein is a combination of apoE and a targeting agent.

[0159] By way of example, the human apoAl protein sequence is annotated under NCBI Genbank http: / / www.ncbi.nlm.nih.gov / ) accession number NP_001304947.1 (isoform 1 preproprotein) and Uniprot www.uniprot.org) accession number P02647.1.

[0160] In certain embodiments, the apolipoprotein component is apoA1 (e.g., derived from a human precursor of apoA1 as defined by SEQ ID NO.9, the first 18 amino acids of which form a signal peptide) or an apolipoprotein component derived from apoA1 (e.g., as defined by SEQ ID NO.10, 11 or 12). In certain embodiments, the apolipoprotein component is human apoA1 as defined by SEQ ID NO.13.

[0161] For example, in order to chemically conjugate the apolipoprotein component to the targeting body, a reactive handle can be used. Thus, an apolipoprotein component derived from apoA1 can be used to prepare a modified apolipoprotein, the apolipoprotein component derived from apoA1 comprising a cysteine ​​in place of the serine at position 147 (e.g., as defined by SEQ ID NO. 11) or 279 (e.g., as defined by SEQ ID NO: 12). In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.9, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12, or an amino acid sequence encoded by SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22 has a nucleic acid with a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

[0162] It should be noted that the sequence defined as SEQ ID NO. 14, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12 contains the amino acid sequence GLVPRGSIDD (SEQ ID NO. 16) at the N-terminus, which is a thrombin cleavage site. For example, the sequence defined as SEQ ID NO. 15 contains a 6His tag at the N-terminus, followed by the amino acid sequence GLVPRGSIDD (SEQ ID NO. 16). Here, the thrombin cleavage site can be used to remove the N-terminal His tag from the peptide.

[0163] In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.10, wherein SEQ ID NO.10 comprises cysteine ​​at position 7 of SEQ ID NO.10.

[0164] In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.11, wherein SEQ ID NO.11 comprises cysteine ​​at position 150 of SEQ ID NO.11.

[0165] In one embodiment, the apolipoprotein component comprises, consists essentially of, or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.12, wherein SEQ ID NO.12 comprises cysteine ​​at position 239 of SEQ ID NO.12.

[0166] When used in this article, when mentioning modified apolipoprotein, the term fusion protein should be interpreted as apolipoprotein and the targeting body covalently attached thereto. Covalent attachment can be attributed to the in-frame coding of the nucleotide sequence encoding the fusion protein to a peptide or protein sequence. Or, covalent attachment can be due to the covalent bonding of the targeting body and apolipoprotein, for example, via the sulfide bond such as thioether bond formed at the cysteine ​​residues of apolipoprotein. In addition, if the effective load is a peptide or protein, it can optionally be a fusion protein with modified apolipoprotein. It should be understood that the targeting body and / or effective load and / or apolipoprotein (or its analogs or derivatives) can include the site-specific incorporation of non-natural amino acids such as p-azidophenylalanine, which can be used for subsequent (strain promoted) "click" (conjugation) reaction with alkyne-modified reagents.

[0167] The modified apolipoprotein may comprise a linker between the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative and the targeting entity, such as a flexible linker. The linker may be a glycine-serine linker, such as (GGS) n -linker, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably (GGS)4 (SEQID 23)-linker.

[0168] In certain embodiments, the modified apolipoprotein may comprise one or more tags, such as at the N-terminus and / or C-terminus of the modified apolipoprotein. One or more tags, such as a 6His-tag or a strep-tag may allow purification of the modified apolipoprotein.

[0169] The targeting body can be covalently attached to any part of the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic, or apolipoprotein mimetic derivative. Linkers, such as flexible linkers, can be used to allow such covalent attachment.

[0170] In certain embodiments, the targeting body is located at the N-terminus or C-terminus of the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative in the modified apolipoprotein.

[0171] As used herein, an immune response refers to a reaction that occurs within an organism by the immune system. An immune response can be an innate immune response or an adaptive immune response or a complement immune system. When referred to herein, an immune response includes, but is not limited to: secretion of pro-inflammatory molecules; secretion of anti-inflammatory molecules; phagocytosis; antibody production, presentation or secretion; antigen presentation; activation, proliferation, inhibition or differentiation of immune cells; binding of immune cells to targets, or initiation of immune-related cell signaling cascades.

[0172] Payload

[0173] In embodiments, modified apolipoprotein further comprises a payload. Payload can be, for example, covalently bound to modified apolipoprotein. When used herein, payload refers to a molecule to be delivered to a target site. Although the technician may know additional types of payloads, non-limiting examples are nucleic acids or nucleic acid analogs, therapeutic agents, biologics, cytokines, chemokines, hormones, growth factors, or combinations thereof. Payload can be an immunomodulatory molecule, and interferes, changes, stimulates, or inhibits innate immune response or adaptive immune response or complement immune system. Payload can be a protein, peptide, or organic compound. Payload can be separated or derived, cloned, or synthesized from a natural source.

[0174] Thus, in an embodiment, the payload is selected from a nucleic acid or nucleic acid analog, a therapeutic agent, a biologic, a cytokine, a chemokine, a hormone, a growth factor, or a combination thereof.

[0175] In an embodiment, the payload can be a cytokine. It is known to technicians that cytokines are small proteins of about 5-20 kDa and are important in cell signaling. For example, cytokines can refer to: four α-helical bundle family cytokines, such as the IL-2 subfamily, the interferon (IFN) subfamily, or the IL-10 subfamily; the IL-1 family; cysteine ​​knot cytokines such as the TGFβ family; the IL-17 family. Therefore, the cytokine is preferably selected from IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF, 4-1BBL, TNFSF8, CD40LG, CD70, CD95L / CD178, EDA-A1, TNFSF14, LTA / TNFB, LTB, TNFα, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TNFS F4, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNω / IFNW1, CLCF1, CNTF, IL11, IL31, IL6, leptin, LIF, OSM, IL10, IL19, IL20, IL22, IL24, IL28B, IL28A, IL29, TGF-β1 / TGFB1, TGF-β2 / TGFB2, TGF-β3 / TGFB3. In a preferred embodiment, the cytokine is selected from the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 family, the TGFβ family or the IL-17 family or a combination thereof, more preferably, wherein the cytokine is selected from IL-1β, IL-2, IL-4, IL-38 and or a combination group.

[0176] In an embodiment, the modified apolipoprotein is a fusion protein of a modified apolipoprotein and IL-1B. IL-1B is also referred to as IL1B, IL-1βIL1F2 or interleukin 1β, and is a cytokine protein encoded by the IL1B gene in humans. In an embodiment, the modified apolipoprotein is a fusion protein of a modified apolipoprotein and IL-2. IL-2 is also referred to as IL2 TCGF, ​​lymphokine or interleukin 2, and is an interleukin that regulates the activity of leukocytes responsible for immunity. In an embodiment, the modified apolipoprotein is a fusion protein of a modified apolipoprotein and IL-4. IL-4 is also referred to as BSF-1, IL4 or interleukin 4, and is a cytokine that induces initial (naive) helper T cell differentiation. In an embodiment, the modified apolipoprotein is a fusion protein of a modified apolipoprotein and IL-38. IL-38 is also known as IL38, IL1F10 interleukin 38, interleukin 1 family member 10, or IL1-theta, and is a protein encoded by the IL1F10 gene in humans.

[0177] In an embodiment, the payload can be a chemokine. The chemokine is preferably selected from CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6 , CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4, and FAM19A5. In an alternative embodiment, the chemokine is selected from CC chemokines, CXC chemokines, C chemokines, CX3C chemokines, or a combination thereof.

[0178] In an embodiment, the payload is a hormone. Hormones are known to the skilled artisan as signaling molecules in multicellular organisms that are transported to distant organs to regulate physiology and behavior. In an embodiment, the hormone is selected from the group consisting of adrenaline (also known as epinephrine), melatonin, noradrenaline (also known as norepinephrine), triiodothyronine, thyroxine, dopamine, prostaglandins, leukotrienes, prostacyclins, thromboxanes, amylin (also known as islet amylin), anti-mullerian hormone (also known as mullerian inhibitory factor / hormone), adiponectin, adrenocorticotropic hormone (also known as corticotropin), angiotensinogen, angiotensin, vasopressin (also known as vasopressin, arginine vasopressin), atrial natriuretic peptide (also known as atrial peptide), brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, cortistatin, enkephalin, endothelin , erythropoietin, follicle-stimulating hormone, galanin, gastrin, gastrin, somatotropin, glucagon, glucagon-like peptide-1, gonadotropin-releasing hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor (also called somatomedin), leptin, lipolysis, luteinizing hormone, melanocyte-stimulating hormone, motilin, orexin, osteocalcin, oxytocin (also called pitocin), pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, prolactin (also called leuteotropic hormone), prolactin-releasing hormone, relaxin, renin, secretin, somatostatin (also called somatostatin or growth hormone-releasing hormone or somatostatin or growth hormone-releasing inhibitory hormone), thrombopoietin, thyrotropin (also called thyroid-stimulating hormone) hormone) (also called thyrotropin), thyrotropin-releasing hormone, vasoactive intestinal polypeptide, guanylin, or uroguanylin.

[0179] In an embodiment, the payload is a growth factor. Growth factors are naturally occurring substances known to the skilled artisan that are capable of stimulating cell proliferation, wound healing, and occasionally cell differentiation. In an embodiment,Growth factors are selected from adrenomedullin (AM), angiogenin (Ang), autotaxin, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), Ephrin A1, Ephrin A2, Ephrin A3, Ephrin A4, Ephrin A5, Ephrin B1, Ephrin B2, Ephrin B3, erythropoietin (EPO), fibroblast growth factor (FGF), fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 3 (FGF3), fibroblast growth factor 4 (FGF4), fibroblast growth factor 5 (FGF5), fibroblast growth factor 6 (FGF6), fibroblast growth factor 7 (FGF7), fibroblast growth factor 8 (FGF8), fibroblast growth factor 9 (FGF9), fibroblast growth factor 10 (FGF10), fibroblast growth factor 11 (FGF11), fibroblast growth factor 12 (FGF12), Fibroblast growth factor 13 (FGF13), fibroblast growth factor 14 (FGF14), fibroblast growth factor 15 (FGF15), fibroblast growth factor 16 (FGF16), fibroblast growth factor 17 (FGF17), fibroblast growth factor 18 (FGF18), fibroblast growth factor 19 (FGF19), fibroblast growth factor 20 (FGF20), fibroblast growth factor 21 (FGF21), fibroblast growth factor 22 (FGF22), fibroblast growth factor 23 (FGF23), fetal bovine growth hormone (FBS), glial cell line-derived neurotrophic factor (Glial cell line-derived neurotrophic factor)(GDNF), Neurturin, Persephin, Artemin, growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP),Also known as hepatocyte growth factor-like protein (HGFLP), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) - anti-apoptotic survival factor , T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor, transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11 and WNT16. In a preferred embodiment, the growth factor is selected from: VEGF, EGF, CNTF, LIF, ephrin, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, neurotrophic factor, PGF, PDGF, RNLS, TCGF, ​​TGF, TNF, WNT, or a combination thereof. ,

[0180] In embodiments, the payload is a hematopoietic growth factor. In embodiments, the hematopoietic growth factor is selected from: IL-3, CSF-1 (M-CSF), GM-CSF, G-CSF, a member of the IL-12 family of interleukins or erythropoietin, or a combination thereof.

[0181] Lipid Nanoparticles

[0182] It is envisioned that modified apolipoprotein as described herein can be used as protein or lipid nanoparticles. As mentioned above, apolipoprotein can be circulated by itself (meaning not incorporated into lipoprotein or lipid nanoparticles). This application can be suitable for delivering payload (such as cytokine) to target site. Known apolipoprotein can be used as protein circulation, but lipoprotein can also be formed in situ. However, it can also be advantageous to include modified apolipoprotein in lipid nanoparticles. Therefore, in one aspect, the present invention relates to lipid nanoparticles comprising one or more modified apolipoproteins as described herein. Without being bound by any hypothesis, it is believed that apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimics derivatives can be used as scaffolds to help form nanoparticles with phospholipids and sterols.

[0183] Apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative forms part of the lipid nanoparticle structure. In certain embodiments, at least a portion of the modified apolipoprotein is exposed to the environment (i.e., aqueous environment) surrounding the lipid nanoparticle. Typically, apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or a portion of the apolipoprotein mimetic derivative is exposed to the environment (e.g., aqueous environment) surrounding the lipid nanoparticle. Figure 2 ). In addition, the fusion of the targeting body to the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative generally allows the targeting body to be fully exposed to the environment surrounding the apolipoprotein lipid nanoparticle (see, for example Figure 2 ). In other words, in certain embodiments, the targeting body is not embedded in the lipid nanoparticle. Therefore, the targeting body can move freely and perform its natural function, such as its cell targeting function.

[0184] In certain embodiments, the lipid nanoparticle is not a phospholipid bilayer.

[0185] In a specific embodiment, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylglycerol or a combination thereof. In a further specific embodiment, the phospholipid is selected from the group consisting of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (PHPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), acylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dilauroylphosphatidylserine (DLPS), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.

[0186] In certain embodiments, the sterol is selected from cholesterol, desmosterol, stigmasterol, β-sitosterol, ergosterol, hopanoids, hydroxysteroids, phytosterols, steroids, hydrogenated cholesterol, campesterol, zoosterols, or combinations thereof.

[0187] Modified apolipoprotein can be further bound to the effective load. Apolipoprotein can also be an apolipoprotein mimetic or derivative. Two types of nanoparticles can be generally distinguished: discoid nanoparticles and spherical nanoparticles. Both contain the outer layer of phospholipids, sterols and modified apolipoproteins as described herein. The difference between the two is that spherical nanoparticles further include a substantially hydrophobic core, while discoid nanoparticles lack a core and thus form a discoid shape. The core can generally include lipids, but not necessarily so. Preferably, lipid nanoparticles are spherical. Compared with, for example, discoid-shaped nanoparticles, spherical nanoparticles enable the connection of (many) more fusion proteins, depending on size. In addition, compared with discoid nanoparticles, spherical nanoparticles facilitate encapsulation of larger effective loads, such as mRNA or gene editing components, or larger amounts of effective loads (e.g., in the case of small molecule drugs or small lipophilic effective loads).

[0188] In certain embodiments, wherein the lipid nanoparticle comprises a payload, the lipid nanoparticle comprises a core surrounded by a surface layer, wherein the core comprises the payload and the surface layer comprises an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic or an apolipoprotein mimetic derivative, a phospholipid, a targeting body, and a sterol.

[0189] In one embodiment, the present invention relates to a lipid nanoparticle comprising an outer layer and optionally a core, wherein the outer layer comprises:

[0190] - phospholipids;

[0191] - sterols; and

[0192] - A modified apolipoprotein as defined in any one of the preceding claims; and wherein the core, if present, comprises at least one core component selected from:

[0193] Lipids, cationic lipids or multivalent molecules. When referring to the outer layer of a lipid nanoparticle, the outer layer is essentially a phospholipid monolayer comprising additional components, at least cholesterol and modified apolipoproteins.

[0194] When used herein, lipid nanoparticles refer to phospholipids and sterols soluble in aqueous solutions and an assembly of one or more apolipoproteins. The particles can include lipids, in which case the lipids are encapsulated by phospholipids and sterols. The nanoparticles can include further components, such as additional proteins or payloads. Therefore, in embodiments, the lipid nanoparticles as defined herein further include lipids.

[0195] In a particular embodiment, in addition to forming a part of modified apolipoprotein as described herein, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimics derivatives, lipid nanoparticles also include natural (e.g., unfused) apolipoprotein, apolipoprotein derivatives, apolipoprotein mimics or apolipoprotein mimics derivatives. In a further embodiment, the lipid nanoparticles as defined herein further include a payload as defined above. Payload can be, for example, a drug compound. The lipid nanoparticle core is particularly suitable for lipophilic payloads, but can also be used for amphiphilic molecules. The drug compound can be an organic compound, a peptide, a protein, a nucleic acid or a nucleic acid analog, a biological product or a lipid. Therefore, in an embodiment, the lipid nanoparticle further includes a payload, preferably wherein the payload is selected from nucleic acid or a nucleic acid analog, a therapeutic agent, a biological product or a combination thereof. Alternatively, the payload can be combined with the outer layer of the lipid nanoparticle, for example, combined with (modified) apolipoprotein, or the payload can be included in the phospholipid layer.

[0196] For example, the effective load can be a nucleic acid or a nucleic acid analog. Examples can be but are not limited to mRNA, siRNA, sgRNA, miRNA, piRNA, snRNA, snoRNA, srRNA or tsRNA. Nucleic acid analogs can be peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA), threose nucleic acids (TNA) and hexitol nucleic acids (HNA), or mixtures or combinations thereof. It should be understood that if the nucleic acid effective load is included in the lipophilic core of the nanoparticle, the charged backbone of the nucleic acid needs to be neutralized, for example, by neutralization of a cationic lipid.

[0197] In certain embodiments, the lipid nanoparticle comprises a nucleic acid and a cationic lipid or an ionizable cationic lipid.

[0198] In certain embodiments, the nucleic acid and cationic lipid or ionizable cationic lipid are comprised by the core, and the modified apolipoprotein and phospholipid are comprised by the outer layer.

[0199] Thus, the present invention also encompasses lipid nanoparticles comprising an outer layer and a core, wherein the outer layer comprises:

[0200] - phospholipids;

[0201] - sterols; and

[0202] - a modified apolipoprotein as taught herein; and

[0203] The core comprises nucleic acid and cationic lipid or ionizable cationic lipid.

[0204] Nucleic acids containing such apolipoprotein particles are described in WO / 2022 / 268913, the entire contents of which are incorporated herein by reference. Auxiliary molecules are used to attract nucleic acids into the hydrophobic nanoparticle core to engineer nanoparticles to compound hydrophilic nucleic acids in nature. To this end, cationic hydrophobic molecules are used. Cationic groups can be complexed with anionic phosphate groups in the sugar phosphate backbone via ionic interactions. The hydrophobic portion of the auxiliary molecule forms a shell around the hydrophilic nucleic acid molecule. Cationic auxiliary molecules can be permanently charged or ionizable. They include a variety of commercially available or internally synthesized molecules, but they need to comply with two general criteria: 1) a positively charged group that enables complexation with a negatively charged sugar phosphate backbone. 2) A hydrophobic portion to form a hydrophobic shell and enable integration into the nanoparticle core. The content of cationic material in the nanoparticle formulation can range from a cation to anion ratio of 1:1 to 25:1. This ratio (commonly referred to as the N / P (nitrogen / phosphate) ratio) is based on the number of positive charges in the (ionizable) cationic lipid (commonly based on nitrogen) and the number of negative charges in the nucleic acid payload (commonly phosphate). Therefore, the N / P ratio is the ratio between the cumulative molar amount of the cations and / or ionizable groups in the cation or ionizable lipid component (N) and the cumulative molar amount of the phosphate groups in the nucleic acid component (P). In a specific embodiment, the N / P ratio of the nanoparticles as taught herein is 1 to 25, 1 to 20, 1 to 15, 1 to 12, 1 to 9, 1 to 6 or 1 to 3. For example, the N / P ratio of the nanoparticles taught herein can be 3, 6, 9 or 12.

[0205] Without being bound by theory, the inventors believe that the nucleic acid-containing nanoparticles described herein have an outer layer mainly comprising modified apolipoproteins, phospholipids and optional sterols, and a core comprising cationic lipids or ionizable cationic lipids and nucleic acids. More specifically, the core of the nanoparticle comprises an assembly of nucleic acids interacting with (ionizable) cationic lipids, wherein the core of the nanoparticle is surrounded by a lipid shell, which comprises, is essentially composed of, or is composed of: modified apolipoproteins, phospholipids and optional sterols. According to this understanding, the nucleic acid is located within the nanoparticle (i.e., inside), and is not located on the outer surface of the nanoparticle and / or is not exposed to the surrounding environment of the nanoparticle. In a specific embodiment, the effective load of the nanoparticle of the present invention (i.e., nucleic acid) is not bound by ionic interactions outside the particle (surface). The combination of nucleic acids with the outer surface of the particle is undesirable because the nucleic acid is exposed to the immediate surrounding environment, which presumably makes the particle more toxic and causes rapid (bio) degradation of the nucleic acid effective load. According to this understanding, the modified apolipoprotein is located on the outer surface of the nanoparticle and / or is exposed to the surrounding environment of the nanoparticle.

[0206] The core of the nanoparticle may be solid and not have or carry a significant aqueous void or reservoir in the core. In certain embodiments, the core of the nanoparticle is non-aqueous.

[0207] In certain embodiments, the core of the nanoparticle is not surrounded by a lipid bilayer, such as found in vesicle-like or liposomal particles, where a lipid bilayer surrounds an aqueous core.

[0208] In certain embodiments, the nanoparticles do not contain synthetic (non-natural) hydrophilic polymers or (lipid) conjugates of such polymers, such as, most notably, polyethylene glycol (PEG). Thus, such nanoparticles do not elicit an unwanted immune response, especially after repeated administration.

[0209] In certain embodiments, the nanoparticle core also comprises a filler, preferably a filler selected from triacylglycerol and cholesterol acyl ester or a combination thereof, such as wherein the triacylglycerol is tricaprylin and / or wherein the cholesterol acyl ester is cholesterol caprylate and / or cholesterol oleate. Cholesterol acetate may also be used as a filler material. Applicable other filler materials are diglycerides or triglycerides or other esters derived from C1-C18 carboxylic acids, preferably C6-C18 fatty acids, wherein these carboxylic acids and fatty acids may be saturated or unsaturated. Preferably, preferably, the filler is a triglyceride derived from C6-C18 fatty acids. In addition to nucleic acids and cationic auxiliary molecules, additional hydrophobic filler molecules may be included in the core of the nanoparticle formulation. Their main application is to change the physicochemical properties of the nanoparticle and / or to improve stability.

[0210] In certain embodiments, the nucleic acid is RNA, DNA, or a nucleic acid analog.

[0211] In certain embodiments, the RNA is a microRNA (miRNA), a small interfering RNA (siRNA), a piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a transfer RNA (tRNA), a tRNA-derived small RNA (tsRNA), a small regulatory RNA (srRNA), a messenger RNA (mRNA), a modified mRNA, a ribosomal RNA (rRNA), a long noncoding RNA (lncRNA), or a guide RNA (gRNA), or a combination thereof, and / or modifications thereof.

[0212] In certain embodiments, the DNA is single-stranded DNA or double-stranded DNA.

[0213] In certain embodiments, the nucleic acid is an antisense oligonucleotide, and the antisense oligonucleotide is a single-stranded DNA or RNA composed of nucleotide or nucleoside analogs containing a phosphodiester backbone or a modified 2' ribose sugar.

[0214] In certain embodiments, the nucleotide or nucleoside analog is selected from: locked nucleic acid (LNA), bridged nucleic acid (BNA), morpholino or peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), or a mixture or combination thereof.

[0215] In certain embodiments, the nucleic acid is conjugated and the nucleic acid conjugate is incorporated into the nanoparticles of the invention. Nucleic acid conjugates include lipid conjugates with, for example, phospholipids or with sterols such as cholesterol or with hydrophobic alkyl chains. Nucleic acid conjugates also include conjugates with oligomers or polymers. Preferably, these oligomers or polymers have hydrophobic properties.

[0216] In certain embodiments, the nucleic acid is incorporated into the nanoparticle as is or "as is," meaning that the nucleic acid is not conjugated.

[0217] When used herein, the term ionizable cationic lipid refers to a lipid that has a neutral charge at physiological pH (e.g., at pH 7 to 7.5, preferably at pH 7.3 to 7.5, such as at -pH 7.4) and is protonated or positively charged at a relatively low pH (e.g., at pH 1 to 5, preferably at pH 1 to 4, such as at pH 4). It should be understood that ionizable cationic lipids are particularly useful because they can be protonated at low pH to promote the combination with hydrophilic nucleic acids. By subsequently increasing pH, lipids can become (partially) neutral, further promoting the inclusion of, for example, a hydrophobic core of nanoparticles in a hydrophobic environment. Alternatively, and without being bound by theory, even if the pH value of the surrounding aqueous solution has been raised to physiological pH (e.g., about 7.4), ionizable lipids can still remain positively charged in nanoparticles due to the effect of the nanoparticle surface layer comprising phospholipids, optional sterols, and modified apolipoproteins, and / or due to the non-aqueous environment in the nanoparticles. Furthermore, ionizable cationic lipids theoretically facilitate endosomal escape of nucleic acids in target cells, where they would be protonated due to the low pH.

[0218] Non-limiting examples of ionizable cationic lipids are DLin-DMA (2-[2,2-bis(octadec-9,12-dienyl)-1,3-dioxolan-4-yl]-N,N-dimethylethylamine), DLin-KC2-DMA (2-[2,2-bis[(9Z,12Z)-octadec-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethylamine)

[0219] and DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptadeca-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butyrate), as shown in Formula 1 below:

[0220] Formula 1

[0221]

[0222] In fact, a wide range of ionizable cationic lipids (including lipids) can be used to prepare the nanoparticles of the present invention, because various series of ionizable cationic lipids have been developed and reported in the literature. Further non-limiting examples include molecules cKK-E12, C12-200, L319, Acuitas-A9, Moderna-L5, TT3 and ssPalmE (such as described in, for example, Witzigmann et al., Advanced Drug Delivery Reviews 159 (2020) 344–363; doi.org / 10.1016 / j.addr.2020.06.026).

[0223] The ionizable lipid may further be an ionizable triglyceride. A non-limiting example is a compound represented by Formula 2:

[0224] Formula 2

[0225]

[0226] The ionizable lipid may further be a cholesterol ester (also known as cholesterol ester). A non-limiting example is represented by Formula 3:

[0227] Formula 3

[0228]

[0229] As used herein, the term cationic lipid refers to a lipid that is positively charged at physiological pH (eg, pH 7.4). Non-limiting examples of cationic lipids are DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DOGS (2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecanyl)amino]-2-oxoethyl]pentanamide), DOSPA (2-[3-[4-(3-aminopropylamino)butylamino]propylcarbamoylamino]ethyl-[2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl]-dimethylazanium)), and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). Other examples include any ionizable cationic lipid molecule in which a tertiary amine moiety has been converted to a quaternary ammonium moiety, for example by alkylation, such as by methylation (-Me), ethylation (-Et), benzylation (-Bn) or ethoxylation (-CH2CH2-OH). The resulting quaternary ammonium molecule has a permanent positive (cationic) charge and therefore also carries a counter anion, such as a chloride anion.

[0230] In an embodiment, only ionizable cationic lipids are used to prepare nucleic acid-containing nanoparticles as taught herein. Thus, in an embodiment, the nanoparticles as taught herein do not comprise cationic lipids.

[0231] In an embodiment, only cationic lipids are used to prepare nucleic acid-containing nanoparticles as taught herein. Thus, in an embodiment, the nanoparticles as taught herein do not comprise ionizable cationic lipids.

[0232] In embodiments, ionizable cationic lipids and combinations of cationic lipids are used to prepare nucleic acid-containing nanoparticles as taught herein.

[0233] When referring to nucleic acid-containing nanoparticles as taught herein, the term "payload" refers in particular to nucleic acids, preferably in combination with cationic lipids and / or ionizable cationic lipids.

[0234] The term "lipid" is well known in the art and, as used herein, may be considered to encompass both lipids, i.e. naturally occurring hydrophobic biomolecules, such as fatty acids, mono-, di- or tri-glycerides of fatty acids, sterols (derivatives) or phospholipids, and lipid-like biomolecules. It should be noted that the cationic lipids or ionizable cationic lipids (or lipids) described herein are generally not lipids in the narrowest interpretation of the term, i.e. naturally occurring hydrophobic biomolecules, such as, for example, fatty acids, mono-, di- or tri-glycerides of fatty acids, sterols (derivatives) or phospholipids, but rather lipid-like biomolecules similar to lipid biomolecules, i.e. they preferably contain biocompatible groups (such as esters or amides), and / or are constructed using naturally occurring building blocks (e.g., fatty acids, glycerol, cholesterol).

[0235] In certain embodiments, the cationic lipid or ionizable cationic lipid is selected from: an ionizable cationic ester of a long chain alcohol, an ionizable cationic ester of a diglyceride, or an ionizable cationic ester of a sterol, or a combination thereof.

[0236] Ionizable cationic esters of long-chain alcohols may be, for example, esters of tertiary amines having a carboxyl group, such as those having the formula (CH3)2N(CH2) n COOH compounds, wherein n is an integer of 1 or greater, for example, n is 1 to 12; for example, 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. Esters are formed with long chain alcohols. Long chain alcohols are preferably primary or secondary alcohols having a linear or branched chain length of 8 or more carbon atoms, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms.

[0237] The ionizable cationic ester of the diglyceride is preferably a diacylglycerol (i.e., diacylglycerol) coupled at position 1 or 2 to a tertiary amine having a carboxyl group, such as a tertiary amine having the formula (CH3)2N(CH2) n COOH compounds, wherein n is an integer of 1 or greater, for example, n is 1 to 12; for example, 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. Diacylglycerols may contain medium-chain or long-chain saturated or unsaturated fatty acids or derivatives or modifications thereof.

[0238] The ionizable cationic ester of a sterol is preferably a sterol ester coupled at the hydroxyl group to a tertiary amine having a carboxyl group, such as a tertiary amine having the formula (CH3)2N(CH2) n COOH compounds, wherein n is an integer of 1 or greater, for example, n is 1 to 12; for example, 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5-dimethylamino-pentanoic acid. The sterol may be cholesterol, stigmasterol or β-sitosterol.

[0239] In the above, the carboxyl compound has the formula (CH3)2N(CH2) n COOH, wherein n is an integer of 1 or more. Instead of this compound, a compound having the formula NH2-(C=NH)-NH-(CH2) may be used. n A replacement compound for COOH, wherein n is an integer of 1 or greater, for example, n is 1 to 12. The carboxyl compound contains a guanidine group instead of a tertiary amine group.

[0240] The ionizable cationic lipids may for example be selected from molecules according to formula (I) to (V).

[0241]

[0242] Formula (I) represents a triglyceride in which an ionizable cationic group (ICG) is contained at the 1-position.

[0243] Formula (II) represents the same type of triglyceride as represented in formula (I), although the molecule is stereospecifically defined in the naturally occurring configuration, ie as it is in phospholipids: the ICG group is in the same position as the phosphate group in the phospholipids.

[0244] Formula (III) represents a triglyceride in which an ionizable cationic group (ICG) is contained at the 2-position.

[0245] Formula (IV) represents a diester (or triester) in which the ionizable cationic group (ICG) is linked via an amide function.

[0246] Formula (V) represents a cholesterol ester in which the ionizable cationic group (ICG) is attached via the ester function.

[0247] For any of formulae (I) to (V), the ionizable cationic group (ICG) is linked to the rest of the molecule via a wavy bond, wherein ICG may represent a tertiary amine (ICG type A, or ICG-A) or it may represent a guanidine (ICG type B, or ICG-B).

[0248] In formulas (I) to (IV), for each position, R1 can be independently selected and represents a straight or branched C1-C19 alkyl, a straight or branched C1-C19 alkenyl, an aromatic group, an arylene-alkyl or an alkylene-aromatic group, wherein the alkyl or alkenyl group optionally contains 5 heteroatoms independently selected from O and N. Preferably, each R1 group in a specific molecule according to any one of formulas (I) to (IV) is the same R1 group. Preferably, the R1 group is a straight or branched C5-C19 alkyl group or a straight or branched C5-C19 alkenyl group. When R1 is an alkenyl group, the group preferably has only one single double bond. More preferably, the R1 group is a straight or branched C9-C17 alkyl group or a straight or branched C5-C17 alkenyl group. Preferably, R1 is a straight C5-C15 alkyl or a straight C17-C19 alkenyl group. The carboxylic acid derived from R1 (i.e. R1-COOH) is preferably a naturally occurring fatty acid molecule such as capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, palmitic acid, oleic acid or linoleic acid. Preferred are C10-C16 saturated fatty acids and oleic acid (C18, unsaturated).

[0249] The integer p is a discrete number rather than an average value; p can be from 0 to 11. Preferably, p is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9. More preferably, p is 1, 2, 3 or 4.

[0250] The R2 group in formula (IV) can be selected from hydrogen, methyl, ethyl and -CH2-OC(O)-R 1a Group (where R 1a has the same meaning as R1 defined above). Preferably, R2 is hydrogen, methyl or a -CH2-OC(O)-R1 group. More preferably, R2 is methyl.

[0251] The R3 group in formula (IV) can be selected from hydrogen, aromatic, aromatic-alkyl, alkylene-aromatic or straight-chain C1-C6 alkyl. Preferably, R3 is hydrogen or methyl. More preferably, R3 is hydrogen.

[0252] For each position, R in ICG-A x The group can be independently selected and is selected from methyl, ethyl, propyl and ethylene-hydroxy (-CH2-CH2-OH) groups, preferably it is a methyl group. Preferably, the two Rx groups in ICG-A are the same group, and they are preferably methyl groups.

[0253] For each position, R in ICG-B yThe groups may be independently selected from hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl groups, wherein the alkyl group optionally contains up to 5 heteroatoms independently selected from O and N. Preferably, R y The group is selected from hydrogen and a straight-chain C1-C6 alkyl group. Even more preferably, R y Preferably, all four R y The groups are identical groups and preferably they are hydrogen.

[0254] Among formulae (I) to (V), formulae (I), (II) and (IV) are preferred. More preferred are formulae (I) and (II).

[0255] Among ICG ICG-A and ICG-B, ICG-A is preferred, that is, a tertiary amine ionizable cationic lipid is preferred.

[0256] The ionizable cationic lipid molecule according to any one of formulae (I) to (V) has a molecular weight greater than 250 Daltons, preferably greater than 350 Daltons, more preferably greater than 450 Daltons. It has a molecular weight less than 3000 Daltons, preferably less than 1800 Daltons, more preferably less than 1200 Daltons.

[0257] The molecules represented by formulae (I) to (V) may exist in various isomeric forms, such as rotamers, tautomers, stereoisomers or regioisomers, and all of these are included within the scope of the present invention.

[0258] The ionizable cationic lipid according to any one of formula (I) to (V) is preferably a single compound, i.e., not a mixture of compounds. Therefore, the purity of the ionizable cationic lipid of formula (I) to (V) is preferably 50% or higher, preferably 80% or higher, more preferably 90% or higher, most preferably 95% or higher. If the ionizable cationic lipid is a mixture of compounds, this is preferably only due to the presence of undefined stereocenters in the molecule. An example is the use of branched alkyl chains of racemic origin in ionizable cationic lipids. Other examples are triglycerides, in which the substitution pattern on the three hydroxyl groups in the glycerol entity is not stereospecifically defined.

[0259] Thus, in certain embodiments, the ionizable cationic lipid is a molecule according to any of formula (I), (II), (III), (IV), or (V).

[0260]

[0261] ICG is wherein the wavy line indicates the point of attachment to the compound of formula (I), (II), (III), (IV) or (V);

[0262] p is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11;

[0263] Each R1 is independently selected from the group consisting of a linear or branched C1-C19 alkyl, a linear or branched C1-C19 alkenyl, an aryl, an arylene-alkyl and an alkylene-aryl group, wherein the alkyl or alkenyl group optionally contains up to 5 heteroatoms independently selected from O and N;

[0264] R2 is selected from the group consisting of hydrogen, methyl, ethyl and -CH2-OC(O)-R 1a ;

[0265] R3 is selected from the group consisting of hydrogen, aromatic groups, arylene-alkyl groups, alkylene-aromatic groups, and linear C1-C6 alkyl groups;

[0266] R 1a Selected from the group consisting of: straight chain or branched C1-C19 alkyl, straight chain or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl groups, wherein the alkyl or alkenyl group optionally contains up to 5 heteroatoms independently selected from O and N;

[0267] Each R x independently selected from the group consisting of: methyl, ethyl, propyl, and -CH2-CH2-OH;

[0268] Each R y The groups are independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl groups, wherein the alkyl group optionally contains up to 5 heteroatoms independently selected from O and N;

[0269] or a rotamer, tautomer, stereoisomer or regioisomer thereof.

[0270] The term "alkyl" by itself or as part of another substituent refers to a group of the formula C n H 2n+1wherein n is a number greater than or equal to 1. The alkyl group may be straight or branched and may be substituted as indicated herein. Typically, the alkyl groups of the present invention contain 1 to 18 carbon atoms, preferably 1 to 17 carbon atoms, preferably 1 to 15 carbon atoms, preferably 1 to 6 carbon atoms, preferably 1 to 5 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, and even more preferably 1 to 2 carbon atoms. When a subscript is used after a carbon atom herein, the subscript refers to the number of carbon atoms that the group may contain. For example, as a group or part of a group, the term "C 1-6 "Alkyl" refers to a group of the formula -C n H 2n+1 wherein n is a number ranging from 1 to 6. Thus, for example, "C 1-6 "Alkyl" includes all straight or branched chain alkyl groups having between 1 and 6 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl); pentyl and isomers thereof, hexyl and isomers thereof. For example, "C 1-5 "Alkyl" includes all straight or branched chain alkyl groups having between 1 and 5 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl); pentyl and isomers thereof. For example, "C 1-4 "Alkyl" includes all straight or branched chain alkyl groups having between 1 and 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, isopropyl, butyl and isomers thereof (e.g., n-butyl, isobutyl and tert-butyl). For example, "C 1-3 "Alkyl" includes all straight or branched chain alkyl groups having between 1 and 3 carbon atoms, thus including methyl, ethyl, n-propyl, isopropyl.

[0271] When the suffix "ene" is used in conjunction with an alkyl group (i.e., "alkylene"), it is intended to refer to an alkyl group as defined herein having two single bonds as points of attachment to other groups. As used herein, the term "C 1-6 "Alkylene" by itself or as part of another substituent refers to a divalent C 1-6Alkyl group, i.e., has two single bonds for being attached to two other groups.Alkylene can be straight or branched, and can be substituted as shown herein.Non-limiting examples of alkylene include methylene (-CH2-), ethylidene (-CH2-CH2-), methylmethylene (-CH(CH3)-), 1-methyl-ethylidene (-CH(CH3)-CH2-), n-propylene (-CH2-CH2-CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), 3-methylpropylene (-CH2-CH2-CH(CH3)-), n-butylene (-CH2-CH2-CH2-CH2-), 2-methylbutylene (-CH2-CH(CH3)-CH2-CH2-), 4-methylbutylene (-CH2-CH2-CH2-CH(CH3)-), pentylene and chain isomers thereof, hexylene and chain isomers thereof.

[0272] The term "alkenyl" as a group or part of a group refers to an unsaturated hydrocarbon group, which may be straight or branched, containing one or more carbon-carbon double bonds. When a subscript is used after a carbon atom herein, the subscript refers to the number of carbon atoms that the group may contain. For example, the term "C 2-6 "Alkenyl" refers to an unsaturated hydrocarbon group which may be straight or branched, contains one or more carbon-carbon double bonds and contains from 2 to 6 carbon atoms. For example, C 2-4 Alkenyl includes all straight-chain or branched alkenyl groups having 2 to 4 carbon atoms. 2-6 Examples of alkenyl groups are ethenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl and its isomers, 2-hexenyl and its isomers, 2,4-pentadienyl and the like.

[0273] The term "aromatic" as a group or part of a group refers to a polyunsaturated aromatic hydrocarbon group having a single ring (i.e., phenyl) or multiple aromatic rings fused together (e.g., naphthyl) or covalently linked aromatic rings, typically containing 6 to 24 carbon atoms, preferably 6 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic. Examples of suitable aromatic groups include C 6-10 aromatic group, more preferably C 6-8 Aromatic group. C 6-12Non-limiting examples of aromatic groups include phenyl; biphenylyl; biphenylenyl; or 1- or 2-naphthyl; 1-, 2-, 3-, 4-, 5-, or 6-tetralinyl (also known as "1,2,3,4-tetrahydronaphthalene"); 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-azulenyl, 4-, 5-, 6, or 7-indenyl, 4- or 5-indanyl, 5-, 6-, 7-, or 8-tetrahydronaphthyl; 1,2,3,4-tetrahydronaphthyl; and 1,4-dihydronaphthyl; 1-, 2-, 3-, 4-, or 5-pyrenyl. When the suffix "ene" is used in conjunction with an aryl group (i.e., arylene); it is intended to mean an aryl group as defined herein having two single bonds as points of attachment to other groups. Suitable "C 6-12 "Arylene" groups include 1,4-phenylene, 1,2-phenylene, 1,3-phenylene, biphenylene, naphthylene, indenylene, 1-, 2-, 5- or 6-tetranaphthylene, and the like. When at least one carbon atom in an aryl group is replaced with a heteroatom, the resulting ring is referred to herein as a heteroaryl ring. The heteroatom may be selected from O, N, P and S; preferably O or N.

[0274] As the term "alkylene-aryl" as a group or a part of a group, it means alkylene as defined herein, wherein at least one hydrogen atom is replaced by at least one aromatic group as defined herein. Alkylene-aromatic groups usually contain 7 to 25 carbon atoms. Non-limiting examples of alkylene-aromatic groups include benzyl, phenethyl, dibenzylmethyl, methylphenylmethyl, 3-(2-naphthyl)-butyl, etc. As the term "arylene-alkyl" as a group or a part of a group, it means arylene as defined herein, wherein at least one hydrogen atom is replaced by at least one alkyl group as defined herein. Arylene-alkyl groups usually contain 7 to 25 carbon atoms.

[0275] Ester, amide, carboxylic acid and alcohol groups are defined below, where Rp represents a hydrogen atom or a cyclic, linear or branched alkyl or alkylene group. In groups containing more than one Rp element, these elements can be selected independently. Ester (functional group) groups or moieties as shown herein are understood to be groups according to the formula: -C(O)-O-. Amide (functional group) groups or moieties as shown herein are understood to be groups according to the formula: -NRp-C(O)-. Carboxylic acid (functional group) groups or moieties as shown herein are understood to be moieties or groups according to the formula: -C(O)OH. Alcohol (or hydroxyl) functional groups or moieties as shown herein are understood to be groups according to the formula: -OH.

[0276] Ionizable cationic lipids according to any one of formula (I) to (V) can be prepared by synthetic methods known in the art, such as but not limited to as described in WO / 2022 / 268913, such as in particular the Examples section of WO / 2022 / 268913 and in particular Example 9.

[0277] (Ionizable) cationic lipids preferably can be processed from solution.Therefore, (ionizable) cationic lipids are preferably soluble in solvents in the polar range.Therefore, (ionizable) cationic lipids are preferably soluble in tricaprylin, soluble in ethanol or soluble in isopropanol, more preferably soluble in all three solvents.Can be by about 20mg (ionizable) cationic lipids being stirred in about 1 gram of tricaprylin, ethanol or isopropanol, and assess whether all materials spontaneously dissolve to produce a clear / transparent solution that concentration is about 2w / w% to check solubility.Test can be carried out at about 20 ℃ (room temperature) or at about 37 ℃.Preferably, (ionizable) cationic lipids are soluble at room temperature.

[0278] Preferably, (ionizable) cationic lipid is nontoxic, or itself, or when combined with nucleic acid or tested together, or when measured in nanoparticles as taught herein, can have limited and low toxicity.Toxicity cell test can be carried out by methods known in the art, such as measured by cell viability MTT, or by similar or comparable test.

[0279] In certain embodiments,

[0280] The amount of modified apolipoprotein, in particular the cumulative amount in case of two or more modified apolipoproteins, ranges from 0.08 to 2.0 mol %, such as 0.10 to 2.0 mol %; and / or

[0281] The amount of phospholipids ranges from 5-90 mol%, such as 15-90 mol%; and / or

[0282] The amount of sterol ranges from 2.5 to 65 mol%, such as 2.5 to 50 mol%; and / or

[0283] The amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as 8.0 to 80 mol%.

[0284] The mole percentages are based only on the combined amounts of the modified apolipoprotein, phospholipid, sterol and cationic lipid or ionizable cationic lipid in the lipid nanoparticle.

[0285] When the nanoparticles include two or more different apolipoprotein components, such as apolipoprotein and a modified apolipoprotein, or two or more modified apolipoproteins and optionally one or more apolipoproteins, etc., the cumulative amounts of these apolipoprotein components are considered when discussing the quantitative relationship with other components of the nanoparticles.

[0286] In some embodiments of the nanoparticle containing nucleic acid as taught herein, the amount of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) ranges from 0.05 to 2.0mol%, such as 0.10 to 2.0mol% or 0.08 to 0.5mol%; and / or the amount of phospholipid ranges from 5 to 90mol%, such as 15 to 90mol% or 8.0 to 50mol%; and / or the amount of sterol ranges from 2.5 to 65mol%, such as 2.5 to 50mol% or 4 to 65mol%; and / or the amount of cationic lipid or ionizable cationic lipid ranges from 5.0 to 80mol%, such as 8.0 to 80mol% or 5 to 65mol%, wherein the molar percentage is based only on the combined amount of modified apolipoprotein, phospholipid, sterol and cationic lipid and / or ionizable cationic lipid in the nanoparticle. These ranges make positive contributions to the stability of nanoparticles and their ability to incorporate nucleic acids.

[0287] In certain embodiments of the nucleic acid-containing nanoparticles as taught herein,

[0288] The amount of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) ranges from 0.01 to 2.0 mol%, such as 0.05 to 1.0 mol%, or 0.05 to 0.5 mol%, or 0.05 to 0.4 mol%, or 0.05 to 0.3 mol%, or 0.05 to 0.2 mol%, or 0.05 to 0.1 mol%, such as about 0.06, about 0.07, about 0.08 or about 0.09 mol%; and / or

[0289] The amount of phospholipids ranges from 2 to 90 mol%, such as 2 to 80 mol%, or 2 to 70 mol%, or 2 to 60 mol%, or 2 to 50 mol%, or 2 to 40 mol%, or 2 to 30 mol%, or 2 to 20 mol%, such as 5 to 10 mol%, such as about 6, about 7, about 8 or about 9 mol%; and / or

[0290] The amount of sterol ranges from 2.5 to 65 mol%, such as 2.5 to 50 mol%, or 5 to 50 mol%, or 5 to 40 mol%, or 5 to 30 mol%, or 10 to 30 mol%, such as 15 to 25 mol%, such as about 20 or about 21 or about 22 mol%; and / or

[0291] The amount of the cationic lipid or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as 10 to 80 mol%, or 10 to 70 mol%, or 10 to 60 mol%, or 10 to 50 mol%, or 10 to 40 mol%, or 10 to 30 mol%, or 15 to 25 mol%, such as about 18 or about 19 or about 21 mol%; and / or

[0292] The amount of lipids (such as preferably triglycerides) ranges from 0 to 95 mol%, or 0 to 90 mol%, for example 1 to 95 mol%, or 1 to 90 mol%, for example 10 to 90 mol%, or 20 to 80 mol%, or 30 to 70 mol%, or 40 to 60 mol%, or 45 to 55 mol%, for example about 50 or about 51 or 52 mol%; and / or

[0293] The amount of nucleic acid (such as preferably RNA, e.g. mRNA) ranges from 0.01 to 1.0 mol%, e.g. 0.01 to 0.1 mol%, or 0.01 to 0.05 mol%, e.g. about 0.02, about 0.03 or about 0.04 mol%;

[0294] Wherein the molar percentage is based only on the combined amount of modified apolipoprotein, phospholipid, sterol, cationic lipid and / or ionizable cationic lipid, lipid (such as preferably triglyceride) and nucleic acid (such as preferably RNA) in the nanoparticle.These ranges make positive contributions to the stability of the nanoparticle and its ability to incorporate nucleic acid.

[0295] In certain embodiments of the nucleic acid-containing nanoparticles as taught herein,

[0296] The amount of modified apolipoprotein (cumulatively in the case of two or more modified apolipoproteins) ranges from 0.1 to 90% by weight;

[0297] The amount of nucleic acid ranges from 0.01 to 90% by weight;

[0298] The amount of phospholipids ranges from 0.1 to 95% by weight;

[0299] The amount of sterol ranges from 0.1 to 95% by weight; and / or

[0300] The amount of cationic lipids and / or ionizable cationic lipids ranges from 0.1 to 95% by weight.

[0301] These weight percentages are based on the combined amounts of modified apolipoprotein, nucleic acid, phospholipid, sterol and cationic lipid and / or ionizable cationic lipid.

[0302] In embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of apolipoprotein (cumulatively in the case of two or more modified apolipoproteins) ranges from 0.2 to 50 wt%, such as 3 to 20 wt% or 4 to 20 wt%, more preferably 0.5 to 30 wt%, more preferably 1 to 20 wt%.

[0303] In embodiments of nucleic acid-containing nanoparticles taught herein, the amount of nucleic acid ranges from 0.02 to 30 wt%, more preferably 0.05 to 20 wt%, more preferably 0.1 to 15 wt%, such as 0.5 to 5 wt%.

[0304] In embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of phospholipid ranges from 0.2 to 60 wt%, more preferably 1 to 50 wt%, such as 10 to 50 wt%, more preferably 3 to 40 wt%, such as 10 to 40 wt%.

[0305] In embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of sterol ranges from 0.2 to 90 wt%, more preferably from 0.5 to 70 wt%, such as 2 to 65 wt%, more preferably from 1 to 50 wt%, such as 2 to 45 wt%, 10 to 45 wt% or 10 to 20 wt%.

[0306] In embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of cationic lipid and / or ionizable cationic lipid ranges from 0.2 to 90 wt%, more preferably from 0.5 to 80 wt%, more preferably from 1 to 70 wt%, for example 5 to 60 wt%, 8 to 60 wt%, 9 to 60 wt%, 10 to 60 wt%, 15 to 25 wt% or 20 to 60 wt%.

[0307] In embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of optional filler or filler molecules ranges from 0 to 90 wt%, more preferably 0 to 80 wt%, more preferably 0 to 70 wt%, such as 0 to 65 wt%.

[0308] In specific embodiments of the nucleic acid-containing nanoparticles taught herein, the amount of optional filler or filler molecules ranges from 20 to 80 wt %, more preferably from 30 to 70 wt %, even more preferably from 30 to 65 wt %, for example 40 to 65 wt %, 45 to 55 wt % or 30 to 60 wt %.

[0309] These weight percentages as described above are based on the combined amounts of modified apolipoprotein, nucleic acid, phospholipid, sterol and cationic lipid and / or ionizable cationic lipid and optional filler material, i.e., in the context of these statements, these five or six components add up to 100% of the nanoparticle weight. These weight percentage ranges contribute positively to the stability of the nanoparticle and its ability to incorporate nucleic acid.

[0310] In certain embodiments, nanoparticles as taught herein do not comprise fillers or filler molecules.

[0311] In certain embodiments, the ratio of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipid is between 1:25 and 1:400, more preferably between 1:50 and 1:200, even more preferably between 1:75 and 1:150, based on molar weight percentage.

[0312] In certain embodiments, the ratio of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipid is 2: 1 to 1: 10, more preferably 1: 1 to 1: 5, even more preferably 1: 1.5 to 1: 4, based on weight. These ranges contribute positively to the stability of the nanoparticles.

[0313] Alternatively, the effective load can be a small organic compound, such as a small molecule drug. Typically, the small organic compound is synthetic. The therapeutic agent can be, for example, an anticancer therapy, such as chemotherapy. Alternatively, the effective load can be a biologic. When used herein, the term biologic is used to represent a biopharmaceutical, also referred to as a biologic medical product (biomedical product), and can be any pharmaceutical drug product prepared in a biological source, extracted from a biological source, or semi-synthesized from a biological source. Biologics can be composed of a complex combination of sugars, proteins, nucleic acids, or these substances, or can be living cells or tissues.

[0314] In certain embodiments, the lipid nanoparticles have an average size of 10 to 100 nm, such as 30 to 100 nm.

[0315] In a particular embodiment, the ratio of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipids is between 1:25 and 1:400, more preferably between 1:50 and 1:200, even more preferably between 1:75 and 1:150, based on molar weight percentages. In a particular embodiment, the ratio of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipids as taught herein is between 1:25 and 1:400, more preferably between 1:50 and 1:200, even more preferably between 1:75 and 1:150, based on molar weight percentages.

[0316] In a specific embodiment, the ratio of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipids is from 3: 1 to 1: 100 based on weight. In a specific embodiment, the ratio of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipids as taught herein is from 3: 1 to 1: 100 based on weight.

[0317] In certain embodiments, in a lipid nanoparticle as taught herein (such as, in particular but not limited to, a nanoparticle that does not contain nucleic acids):

[0318] The amount of modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) ranges from 0.05 to 2.0 mol%, such as 0.1 to 1.0 mol%, for example 0.2 to 0.7 mol%, or 0.2 to 0.5 mol%, such as preferably about 0.3 mol%; and / or

[0319] The amount of phospholipids ranges from 5 to 90 mol%, such as 10 to 50 mol%, for example 10 to 40 mol%, 10 to 30 mol%, or 10 to 20 mol%, such as about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18 or about 19 mol%; and / or

[0320] The amount of sterol ranges from 2.5 to 65 mol%, such as 2.5 to 60 mol%, 2.5 to 50 mol%, 2.5 to 40 mol%, or 2.5 to 30 mol%, or 2.5 to 20 mol%, or 2.5 to 10 mol%, such as 2.5 to 5 mol%, such as about 3.0 or about 4.0 or about 5.0 mol%; and / or

[0321] The amount of lipid (such as preferably triglyceride) ranges from 1 to 95 mol%, such as 10 to 95 mol%, or 20 to 95 mol%, or 30 to 95 mol%, or 40 to 95 mol%, or 50 to 95 mol%, or 60 to 95 mol%, such as 1 to 90 mol%, such as 10 to 90 mol%, or 20 to 90 mol%, or 30 to 90 mol%, or 40 to 90 mol%, or 50 to 90 mol%, or 60 to 90 mol%, such as 60 to 80 mol%, or 70 to 80 mol%, such as about 65, about 70 or about 75 mol%,

[0322] The mole percentages are based solely on the combined amounts of the modified apolipoprotein, phospholipids, sterols, and lipids (such as preferably triglycerides) in the lipid nanoparticles.

[0323] In a further aspect, the present invention relates to a method for preparing a lipid nanoparticle as described herein, the method comprising the following steps:

[0324] a1) expressing and isolating a modified apolipoprotein as disclosed herein to obtain an isolated modified apolipoprotein; and / or

[0325] a2) chemically conjugating the targeting body to apolipoprotein or an apolipoprotein mimetic to obtain a modified apolipoprotein and isolating the modified apolipoprotein;

[0326] b) combining the isolated modified apolipoprotein obtained in step a1 and / or step a2 with phospholipids, sterols and optionally lipids to obtain lipid nanoparticles.

[0327] In an embodiment, step b) comprises:

[0328] b1) mixing a lipid component in an organic solvent with nucleic acids in an aqueous buffer, preferably rapidly, to produce nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a core component and optionally a lipid; and wherein the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; and

[0329] b2) mixing the lipid nanoparticles with the modified apolipoprotein, preferably rapidly, to produce nanoparticles at a pH between 5.5 and 9.0, preferably between 6.0 and 8.0, more preferably at a pH of 6.5 to 8.0.

[0330] It should be understood that modified apolipoprotein can be expressed as a chimeric fusion protein of apolipoprotein and a targeting body, or can be chemically conjugated to the targeting body, or can be produced as a combination of these. The expression of chimeric proteins is known to the technician, and can be used when the targeting body is a peptide or protein. It is well known to the technician that molecular techniques are used to produce nucleic acids encoding such proteins, such as by cloning the targeting body coding sequence with the apolipoprotein (or analogies or derivative) coding sequence frame, such as at the C-terminal or N-terminal sequence of the encoding nucleotide. The advantage of using chimeric protein expression is that all expressed proteins will be fusion proteins.

[0331] Alternatively, chemical conjugation can be used. Suitable methods for chemically conjugating the targeting body to apolipoprotein (or its mimetics) are known to the skilled person. Non-limiting examples are cycloadditions, aminolysis, and Michael-type additions promoted by strains. For example, existing or introduced cysteine ​​residues can be used on apolipoprotein or a targeting body. The introduction of cysteine ​​residues can be achieved by point mutations of nucleotides in the encoding nucleotide sequence or by introducing cysteine ​​encoding codons. The advantage of chemical conjugation is that it is not limited to the use of peptides or protein sequences, but can be applied to any type of organic molecules.

[0332] It will be appreciated that the modified apolipoprotein, phospholipids, sterols and optional components may be mixed rapidly to obtain lipid nanoparticles.Optionally added may be lipids and / or payloads as defined herein.

[0333] The present invention also encompasses a method for producing lipid nanoparticles comprising the following steps:

[0334] a) rapidly mixing a lipid component in an organic solvent with nucleic acids in an aqueous buffer to produce lipid nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a cationic lipid, or an ionizable cationic lipid, wherein the aqueous buffer has a pH of 5.0 or less; and

[0335] b) rapidly mixing the lipid nanoparticles with one or more modified apolipoproteins as taught herein;

[0336] Lipid nanoparticles are produced at a pH between 5.5 and 8.0, preferably between 6.0 and 8.0.

[0337] According to the above method, a two-step reaction is performed, wherein in the first step, nanoparticles containing nucleic acids are formed, and in the following second step, modified apolipoproteins are included in the nanoparticles. Preferably, the first step is performed at low pH and the second step is performed at physiological pH.

[0338] The organic solvent may be an alcohol such as ethanol, isopropanol, methanol, acetonitrile, dimethyl sulfoxide (DMSO), chloroform or a combination thereof. Preferred organic solvents are those that are miscible with water and non-toxic, such as ethanol and DMSO or a combination thereof.

[0339] For example, the organic solvent may be 96% to 100% ethanol, preferably 100% ethanol.

[0340] Rapid mixing is known in the art and has been described, for example, in Hirota et al. BIOTECHNIQUESVOL. 27, NO. 2, p286-289; Jeffs et al., Pharm Res 22, 362–372 (2005); Kulkarni et al., ACS Nano 2018, 12, 5, 4787–4795.

[0341] The aqueous buffer in step a) has a low pH to ensure that the ionizable cationic lipid is positively charged, which allows the binding within the nucleic acid / cationic lipid complex in the particle and contains the nucleic acid / cationic lipid complex in the particle. For example, the buffer can have a pH of 5.0 or lower, such as 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5 or lower. The aqueous buffer can be any buffer that does not damage the nucleic acid. An exemplary buffer is sodium acetate at pH 4.0. The nanoparticles are then placed in an aqueous buffer having a pH of about 6 to 8, preferably about 7 to 8, and more preferably about 7.4. This can be achieved, for example, by dialysis with an aqueous buffer in a specified pH range. A non-limiting example of an aqueous buffer suitable for this step is 155mM PBS at pH 7.4, but it should be understood that any buffer that does not damage the nucleic acid can be used.

[0342] In step b), the nanoparticles in an aqueous buffer at a pH between 6 and 8, preferably between pH 7 and 8, are rapidly mixed with the modified apolipoprotein in an aqueous buffer at a pH between 5.5 and 8, preferably between 6 and 8, more preferably between pH 7 and 8, to obtain nanoparticles.

[0343] The above two-step formulation process as taught herein produces nucleic acid-containing nanoparticles having a wide range of desired and beneficial characteristics (stability, low or no toxicity, high nucleic acid retention, nucleic acid activity, etc.). However, the formulation method described is non-limiting, as other processes may also produce nanoparticles having beneficial characteristics.

[0344] A further aspect of the invention provides a pharmaceutical composition comprising a modified apolipoprotein as taught herein, a nucleic acid as taught herein or a lipid nanoparticle as taught herein, and a pharmaceutically acceptable carrier.

[0345] In a further aspect, the present invention relates to a modified apolipoprotein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtained or obtainable by the method as described herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein for use as a medicament. It is envisioned that the modified apolipoprotein (e.g., by the effect of an attached payload) or a payload contained in a nanoparticle can be used to treat, improve or alleviate the symptoms of a subject. Therefore, in an embodiment, the modified apolipoprotein according to the first aspect or the lipid nanoparticle according to the second aspect, or the lipid nanoparticle obtained or obtainable by the method of the third aspect of the present invention or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, is used to treat or prevent immune-related disorders, such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, nervous system disorders or tissue trauma, preferably immune-related disorders, such as transplant rejection, atherosclerosis, infection, autoimmunity, cancer, genetic disorders, metabolic disorders, nervous system disorders or tissue trauma. A further embodiment relates to a method for treating an immune-related disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a modified apolipoprotein according to the first aspect or a lipid nanoparticle according to the second aspect, or a lipid nanoparticle obtained or obtainable by the method of the third aspect of the invention, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein. Also provided herein is the use of a modified apolipoprotein according to the first aspect or a lipid nanoparticle according to the second aspect, or a lipid nanoparticle obtained or obtainable by the method of the third aspect of the invention, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein in the preparation of a medicament for treating an immune-related disorder in a subject.

[0346] Immune-related disorders as used herein include any disorder in which the immune system plays a role in the development of the disease. Immune-related disorders may refer to disorders in which the immune system is suppressed or in which the immune system is (over)activated. Examples of immune-related disorders are cancer, infection, sepsis, autoimmune diseases, and cardiovascular disease. Examples of autoimmune diseases are type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Addison's disease, Graves' disease, Syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia and celiac disease. In one embodiment, the immune-related disorder is selected from the group consisting of transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, nervous system disorders and tissue trauma, preferably transplant rejection, atherosclerosis, infection, autoimmunity, cancer, genetic disorders, metabolic disorders, nervous system disorders or tissue trauma.

[0347] In an embodiment, the immune-related disorder is selected from the group consisting of: cancer, infection, sepsis, type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Addison's disease, Graves' disease, syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, and celiac disease.

[0348] In a further aspect, the present invention relates to the use of a modified apolipoprotein as described herein or a lipid nanoparticle as described herein, or a lipid nanoparticle obtained or obtainable by a method as described herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for delivering a payload (e.g. a compound) to a target, preferably wherein the target is a cell, a tissue and / or an organ, even more preferably wherein the target is a lymphoid cell, a myeloid cell, a tumor cell, or wherein the targeting body binds a bacterial protein or antigen, a viral protein or antigen, a fungal protein or antigen or a parasitic protein or antigen, preferably wherein the lymphoid or myeloid cell is selected from: a monocyte, a macrophage, an M1-like macrophage, an M2-like macrophage, an eosinophil, a basophil, a mast cell, a NK cell, a B cell, a plasma cell, a regulatory T cell, a hematopoietic stem cell, a T helper cell such as Th1, Th2, Th17 or Th22, a dendritic cell such as a plasmacytoid dendritic cell, a conventional DC 1 or a conventional DC 2, or a tumor-associated macrophage. In an embodiment, the use is in vitro or in vitro use. In an alternative embodiment, the use is in vivo use. Typically, apolipoprotein or targeting body will bind to a cell surface protein such as a receptor. Therefore, the target can be a protein, such as a receptor, a cell or a cell type (which expresses the protein), a tissue or a tissue type (which expresses the protein) or an organ (which expresses the protein). It should be understood that by selecting or adjusting the targeting body, the modified apolipoprotein can target different proteins. For example, the receptor binding domain of a ligand can be used to target a specific receptor. Alternatively, a known binding partner of a cell surface protein can be used to reselect a fusion protein (i.e., a modified apolipoprotein).

[0349] A further aspect of the invention provides an in vitro or ex vivo method for introducing a nucleic acid into a cell, the method comprising contacting a nucleic acid-containing lipid nanoparticle as taught herein or a nucleic acid-containing lipid nanoparticle obtained or obtainable by a method as taught herein with a cell.

[0350] A further aspect of the invention provides an in vivo method for introducing a nucleic acid into a cell, the method comprising contacting a nucleic acid-containing lipid nanoparticle as taught herein or a nucleic acid-containing lipid nanoparticle obtained or obtainable by a method as taught herein with the cell.

[0351] A further aspect of the invention provides a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtained or obtainable by a method as taught herein, for use in delivering a nucleic acid to a subject in vivo.

[0352] A further aspect of the invention provides a method of delivering a nucleic acid in vivo, the method comprising administering to a subject a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtained or obtainable by a method as taught herein.

[0353] A further aspect of the invention provides a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid-containing lipid nanoparticle as taught herein, or a nucleic acid-containing lipid nanoparticle obtained or obtainable by a method as taught herein.

[0354] The disease can be any of the diseases discussed herein, such as, for example, an immune-related disorder such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, a genetic disorder, a metabolic disorder, a nervous system disorder, or tissue trauma, preferably transplant rejection, atherosclerosis, infection, autoimmunity, cancer, a genetic disorder, a metabolic disorder, a nervous system disorder, or tissue trauma.

[0355] The purpose of the nucleic acid nanoparticles described herein is to deliver nucleic acid to cells or to deliver nucleic acid therapy to subjects. Nucleic acid can be, for example, mRNA encoding a peptide or protein of interest to be expressed in a cell, or can include short nucleic acids such as siRNA, shRNA intended to interfere with gene expression (e.g., gene silencing), or it can include components (e.g., gRNA) of CRISPR-Cas or related gene editing systems to induce mutations in cell genomes. Therefore, the mode of action of nucleic acid (payload of nanoparticles) is usually in cytoplasm or nucleus. Therefore, nanoparticles preferably have at least the following characteristics: 1) it allows targeting of expected target cells, and 2) it allows the delivery of payloads where it can play its role (therefore in most cases in the cytoplasm or nucleus of target cells).

[0356] It should be understood that the nucleic acid therapy comprising nanoparticles can be administered to a subject in need thereof. Depending on the target cell or tissue, administration can be parenteral, such as intravenous, intramuscular or subcutaneous. Administration can also be oral, sublingual, topical, rectal, nasal (inhaled) or vaginal. In addition, the targeting of the target tissue or cell is determined by appropriately selecting a modified apolipoprotein. In an embodiment, the use of nanoparticles or compositions includes delivering nucleic acid to a myeloid compartment or spleen. This can be achieved, for example, by intravenous parenteral administration.

[0357] Nanoparticles of certain embodiments of the invention can target tissues associated with the presence of immune cells (spleen, bone marrow) following systemic injection.

[0358] In certain embodiments, the nucleic acid-containing nanoparticles as taught herein or the compositions as taught herein are used in immunotherapy.

[0359] In one aspect, the present invention relates to nanoparticles as taught herein, including nanoparticles containing nucleic acids, or compositions comprising them, for treating a disease by stimulating or inhibiting an innate immune response, preferably wherein the disease is a disease that will benefit from stimulating or inhibiting an innate immune response of a subject, such as a disease characterized by a defective innate immune response, more preferably wherein the disease to be treated is cancer, cardiovascular disease, autoimmune disorder or xenograft rejection. Therefore, nanoparticles can be used to treat any disease associated with the immune system, such as any immune disorder, or for treating any disease or disorder in which modulating an immune response is considered a viable treatment option.

[0360] In a further aspect, the invention relates to a nucleic acid encoding a fusion protein as defined herein (ie a modified apolipoprotein).The nucleic acid may be comprised in a vector, such as a viral vector for stable integration in a cell, or an expression vector enabling transient expression.

[0361] Those skilled in the art will appreciate that, without departing from the wide general scope of the present invention, many changes and / or modifications may be made to the above aspects and / or embodiments. Therefore, aspects and / or embodiments of the present invention are considered to be illustrative rather than restrictive in all respects. The present invention includes the following non-limiting examples. Example

[0362] Example 1. Generation of cell marker-specific apolipoprotein-nanobody fusion proteins

[0363] ANP is a nano-sized formulation based on lipids (about 30-200nm in diameter) with a hydrophobic core and an apolipoprotein covering the outer surface. Apolipoprotein is a spiral protein that has an intrinsic affinity for lipid layers due to its amphipathic characteristics. There are several types of apolipoproteins, and all can be used as structural components of aNP formulations. The presence of apolipoproteins regulates the biological behavior of aNP. For example, apolipoprotein A1 interacts with cells via scavenger receptor B type 1 (SRB1) and ATP-binding cassette transporter ABCA1. This increases the interaction of aNP with myeloid cells in lymphoid organs. However, functionalized aNPs in treatment have been shown to have almost no effect on immune cell types without these mentioned receptors. In addition, increasing the targeting specificity within the myeloid compartment can also increase the efficiency of therapeutic agents. Therefore, rerouting aNP to specific cell types will increase the power of the modular platform.

[0364] Nanobodies are single antigen-specific polypeptides found in Camelidae that consist of only the heavy chain variable domains (VH domains) of heavy chain antibodies, also referred to as VHH domain antibodies or single domain antibodies (sdAbs). Nanobodies have low complexity, and are highly stable and soluble, which gives them greater therapeutic potential compared to human antibody equivalents. They consist of a conserved framework of β-folds and three variable complementary determining regions (CDRs). The specificity of nanobodies stems from changes in these CDR loops. Nanobodies lack hydrophobic patches (hydrophobic patches) that are present in human antibody frameworks to increase the interaction between VH and VL domains. Nanobodies have four landmark mutations in this region, which are key to their stability and solubility. Point mutations can be used to modify the conserved framework to humanize nanobodies to limit immunogenicity.

[0365] Apolipoprotein-nano antibody fusion protein combines the particle formation ability of apolipoprotein and the targeting ability of nano antibody. Fusion protein is generated by molecular cloning of the genes of apolipoprotein and nano antibody, and apolipoprotein and nano antibody are fused with appropriate amino acid linkers to allow enough space between apolipoprotein and nano antibody. Fusion protein will be recombinantly expressed in Escherichia coli subsequently, and purification tags such as histidine tags are used for purification. During aNP production, fusion protein is included and then cell type specific aNP is generated. By changing the nano antibody sequence to a sequence specific to the desired cell marker, the cell type of targeting can be easily determined.

[0366] Nanobodies specific for desired cell markers were engineered via yeast surface display methods and directed evolution. Generative modeling guided by machine learning was used to introduce mutations during directed evolution to create nanobody sequences with higher affinity for their targets. The process was repeated until a sequence with the desired binding affinity was selected.

[0367] Example 2. Evaluation of rerouting aNP

[0368] In vitro evaluation of cell-specific aNPs was performed to analyze aNP-cell interactions. aNPs were incubated with human peripheral blood mononuclear cells (PBMCs) and flow cytometry was used to evaluate the binding of aNPs to immune cells. The therapeutic efficacy of aNPs was evaluated in vitro after incorporation of therapeutic compounds.

[0369] Subsequently, the in vivo efficiency of cell targeting by rerouted aNPs was evaluated in mice with a humanized immune system. The organ-specific and cell-specific biodistribution of aNPs, as well as toxicological and therapeutic efficacy were investigated.

[0370] Example 3. Implementation Method

[0371] Reselected aNPs have the potential to specifically deliver therapeutic compounds to desired immune cell types, thereby modulating immune responses. The main advantage of the Reselected aNP platform technology is that nanobodies and therapeutic payloads can be easily replaced. Therefore, Reselected aNPs can be implemented as immunotherapies that promote immune responses to treat, for example, cancer or infectious diseases, or suppress immune responses to treat, for example, autoimmune diseases or suppress immune responses during organ transplantation.

[0372] Example 4. Fusion proteins of apolipoprotein fused to rerouting proteins or peptides (targeting bodies) and their incorporation into lipids Nanoparticles

[0373] Materials and methods

[0374] Expression and purification of VHHCD8-apoA1 fusion protein:A small culture of ClearColi cells transformed with pET20b-VHHCD8-apoA1 plasmid and pDiscoTune plasmid was started in LB medium containing 100 μg / mL ampicillin. The next day, 40 mL of the small culture was diluted in 1 liter of 2YT medium to start a large culture, and rhamnose was added at a final concentration of 50 μM to induce T7 lysozyme on the pDiscoTune plasmid. The culture was grown at 37 ° C and 150 rpm until an OD600 of 0.6-0.8, and then isopropyl β-d-1-thiogalactoside (IPTG) was added at a final concentration of 0.1 mM to induce expression. The induced culture was incubated overnight at 18 ° C and 150 rpm. The induced bacterial culture was precipitated and the cells were resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.9). Benzonase nuclease (Merck Millipore) and one cOmplete were added to each 50 mL cell suspension. TM The cell suspension was incubated at 4°C with EDTA-free protease inhibitor cocktail tablets (Roche) and stirred. Subsequently, Avestin Emulsiflex C3 was used to homogenize the suspension 3 times at 15000-20000psi. Cell lysates were kept on ice at all times. After lysis, the cell lysates were centrifuged to precipitate insoluble cell debris, and the supernatant was flowed through an immobilized metal chelate affinity chromatography (IMAC) column containing immobilized nickel ions. The column was washed with 8 column volumes of buffer A (20mM Tris, 500mM NaCl, 10mM imidazoles, pH 7.9), followed by 8 column volumes of buffer A50 (20mM Tris, 500mM NaCl, 50mM imidazoles, pH 7.9). To elute VHHCD8-apoA1, 8 column volumes of buffer A500 (20 mMTris, 500 mMNaCl, 500 mM imidazole, pH 7.9) were applied to the column. All fractions of the purification step were collected and analyzed with SDS-PAGE. The buffer of the fraction containing purified VHHCD8-apoA1 was changed to PBS using an Amicon ultracentrifugal filter (Amicon). To store VHHCD8-apoA1, aliquots were quickly frozen in liquid nitrogen and stored at -70 °C.

[0375] The VHHCD8-apoA1 fusion protein has a sequence as defined by SEQ ID NO: 1 or is encoded by a sequence as defined by SEQ ID NO: 2 comprising a linker between apoA1 and VHHCD8 comprising cysteine.

[0376] SEQ ID NO:1

[0377]

[0378] SEQ ID NO:2

[0379]

[0380] aNP formulations: All phospholipids were purchased from Avanti Polar Lipids Inc. For discoid lipid nanoparticles: from a stock solution (10 mg / mL) in chloroform, DMPC (128 μL), cholesterol (Sigma-Aldrich) (7.5 μL), and for spherical lipid nanoparticles: POPC (66.5 μL), PHPC (17.5 μL), cholesterol (4.5 μL) and tricaprylin (Sigma-Aldrich) (2.8 μL, from a 0.956 g / mL stock solution) were combined in a glass vial and dried under vacuum. The resulting film was redissolved in an acetonitrile / methanol mixture (95:5 wt%, total volume 900 μL). Separately, a solution of VHHCD8-apolipoprotein A1 protein in PBS (6.5 mL, 0.14 mg / mL) was prepared twice. The two solutions were infused simultaneously into a Zeonor herringbone mixer (Microfluidic Chipshop) using a microfluidic pump fusion 100 (Chemyx Inc), wherein the flow rate of the lipid solution was 0.8 mL / min, and the flow rate of the apolipoprotein A1 solution was 6 mL / min. A Vivaspin tube of 10 kDa MWCO was used for discoid aNPs or 50 kDa MWCO was used for spherical aNPs, and the obtained solution was concentrated by centrifugal filtration at 4000 rpm to obtain a volume of 1 mL. PBS (5 mL) was added, and the solution was concentrated to 1 ml; this was repeated twice. The washed solution was concentrated to approximately 1.5 ml and filtered through a 0.22 μm PES syringe filter to obtain the final nanoparticles containing VHHCD8-apolipoprotein A1.

[0381] DLS: The obtained PBS solution containing the nanoparticle formulation of VHHCD8-apolipoprotein A1 was filtered through a 0.22 μm PES syringe filter and analyzed by dynamic light scattering on a Malvern Zetasizer Nano ZS analyzer. Values ​​are reported as the mean numerical average size distribution.

[0382] Cryo-TEM:First, the surface of a 200 mesh lace carbon-supported copper grid (Electron Microscopy Sciences) was plasma treated for 40 seconds using a Cressington 208 carbon coater. Subsequently, 3 μL of VHHCD8-apolipoprotein A1-containing nanoparticles (approximately 1 mg protein / ml) were applied to the grid and vitrified into a thin film by plunge vitrification in liquid ethane using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was performed on a CryoTITAN (Thermo Fisher Scientific) equipped with a field emission gun (FEG), a post-column Gatan imaging filter (Model 2002), and a post-GIF 2k×2k Gatan CCD camera (Model 794). Images were acquired at 300 kV accelerating voltage, 6500× magnification (dose rate of 1.64 electrons / A2·s) or 24000× magnification (dose rate of 11.8 electrons / A2·s) and 1 s acquisition time in light-field TEM mode with zero-loss energy filtering.

[0383] In vitro binding of VHHCD8-apoA1 in mouse splenocytes:

[0384] Spleen is obtained from mice, sliced, and filtered repeatedly with 70 μm filters (Corning), to obtain splenocyte suspension. Cells were lysed 10 minutes with the spin settling of 1500rpm, supernatant was removed, and cells were dissolved in 2mL 1X erythrocyte lysis buffer (Thermofisher). Suspension was incubated at room temperature for 5 minutes, 10mL Roswell ParkMemorial Institute (RPMI) culture medium (Thermofisher) was added, and cells were lysed 10 minutes with the spin settling of 1500rpm again. Cells were then redissolved in the RPMI culture medium and plated in 96 orifice plates with 150.000 cells / well.

[0385] Proteins were labeled by adding a 5x molar excess of sulfo-cyanine 5-maleimide (Lumiprobe) in dimethyl sulfoxide (DMSO) solution. The mixture was incubated at room temperature for 2 hours. Excess dye was removed using a PD minitrap G-25 desalting column (Cytiva). Fluorescently labeled aNPs were prepared by adding 6.4 μg DiI for discoid formulations and 21 μg DiI for spherical formulations. Fluorescently labeled fusion proteins or aNPs and controls were added to wells and incubated at 4°C (for proteins) or 37°C (for aNPs) for 30 minutes, then harvested, washed cells, stained for CD3 and CD4 and measured on Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD).

[0386] result

[0387] VHHCD8-apoA1 fusion protein was successfully expressed in Clearcoli cells. A small amount of protein contamination was found after IMAC purification [lane E1]( Figure 3 The most prominent band corresponds to a fusion protein with a molecular weight of 43.3 kDa ( Figure 3 The correct mass was subsequently confirmed by mass spectrometry (data not shown).

[0388] Formulation of discoidal apolipoprotein nanoparticles incorporated VHHCD8-apoA1. The size and polydispersity index (PDI) of the particles were determined using dynamic light scattering (DLS). The size of the particles remained stable for 7 days (data not shown). At day 14, there was a slight increase in size (data not shown). The PDI remained stable for 14 days (data not shown). Cryo-TEM images of the nanoparticles showed the expected discoid shape (data not shown)

[0389] VHHCD8-apoA1 and apoA1 were fluorescently labeled and subsequently added to mouse splenocytes. For the VHHCD8-apoA1 fusion protein, a dose-dependent increase in mean fluorescence intensity (MFI) was observed, indicating binding of the fusion protein to the CD8 receptor ( Figure 4 ; lower panel). The apoA1 condition did not show this dose-dependent behavior and had a similar MFI to the control sample.

[0390] Discoidal aNPs and spherical aNPs were formulated with VHHCD8-apoA1 and apoA1. Fluorescent dyes were integrated into the lipid structure of the particles. Mouse splenocytes were incubated with the nanoparticles. Both VHHCD8-apoA1 particles showed a dose-dependent increase in MFI, indicating binding of the nanoparticles to the CD8 receptor ( Figure 5). Discoidal nanoparticles showed a greater increase than spherical nanoparticles. No increase in MFI was observed for the apoA1 nanoparticle condition.

[0391] Example 5. In vivo biodistribution of lipid nanoparticles comprising Apolipoprotein fusion protein fused to apolipoprotein

[0392] Materials and methods

[0393] Formulation of spherical VHHCD8-aNPs

[0394] All phospholipids were purchased from Avanti Polar Lipids Inc. For spherical aNPs, POPC (66.5 μL), PHPC (17.5 μL), cholesterol (4.5 μL) and tricaprylin (Sigma-Aldrich) (2.8 μL from 0.956 g / mL stock solution) were combined in a glass vial and dried under vacuum. The resulting film was redissolved in an acetonitrile / methanol mixture (95:5%, total volume 900 μL).

[0395] Separately, a solution of VHHCD8-apoA1 protein (SEQ ID NO: 1) in PBS (6.5 mL, 0.14 mg / mL) was prepared twice.

[0396] The two solutions were infused simultaneously into a Zeonor herringbone mixer (Microfluidic Chipshop) using a microfluidic pump fusion 100 (Chemyx Inc), with a lipid solution flow rate of 0.8 mL / min and apoA1 solution flow rate of 6 mL / min. A 50 kDa MWCO Vivaspin tube was used for spherical aNPs, and the obtained solution was concentrated by centrifugal filtration at 4000 rpm to obtain a volume of 1 mL. PBS (5 mL) was added, and the solution was concentrated to 1 ml; this was repeated twice. The washed solution was concentrated to approximately 1.5 ml and filtered through a 0.22 μm PES syringe filter to obtain the final aNPs.

[0397] Table 1. Composition of VHHCD8-apolipoprotein A1 fusion protein nanoparticles in molar percentage. DiR: 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine; Lyso-PC: 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine; POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

[0398] Mol% POPC 12.4％ Lyso-PC 4.9％ Triglycerides 76.1％ cholesterol 4.3％ DiR 2％ protein 0.3％

[0399] In vivo biodistribution of VHHCD8-apoA1 in mice ( Figure 7 )

[0400] Spherical VHHCD8-aNPs with 2 mol% DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindoltricarbonylcyanine iodide) were intravenously injected into C57BL / 6 mice at a dose of 1 mg / kg DiR. After 24 hours, the mice were sacrificed, blood was harvested, and other tissues of interest (bone marrow, spleen, and lymph nodes) were harvested after PBS perfusion. Tissues were homogenized into single-cell suspensions and stained for myeloid (CD115, F4 / 80, Ly6C, CD11b, CD45, Ly6G, CD11c), lymphoid (CD45, CD11b, CD3, CD19, CD4, CD8a), and progenitor (CD117, CD34, Ly-6A / E, CD135, CD16 / 32, CD48, CD41, CD150, CD3, CD11b, CD45R / B220, Ly-76, Ly6G, Ly6C) cell markers and analyzed on a Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD).

[0401] result

[0402] The spherical lipid nanoparticles containing VHHCD8-apolipoprotein A1 were characterized using DLS to determine their hydrodynamic size (mean value) and polydispersity index (Pdi), and cryo-electron microscopy to determine their size and morphology ( Figure 6 ).

[0403] In vivo biodistribution data showed that VHHCD8-apolipoprotein A1 fusion protein nanoparticles were significantly less associated with myeloid cells in the bone marrow, spleen, and blood when compared to control apolipoprotein A1 protein nanoparticles ( Figure 8 A). VHHCD8-apolipoprotein A1 fusion protein nanoparticles were significantly more associated with CD8+ T cells in the bone marrow, spleen, and blood when compared to control apolipoprotein A1 protein nanoparticles ( Figure 8 B). VHHCD8-apolipoprotein A1 fusion protein nanoparticles were less associated with CD4+ T cells in the bone marrow, spleen, and blood when compared to control apolipoprotein A1 protein nanoparticles ( Figure 8 C). In addition, when compared with control apolipoprotein A1 protein nanoparticles, VHHCD8-apolipoprotein A1 fusion protein nanoparticles were more associated with CD3+ T cells in the bone marrow, spleen, and blood ( Figure 8 D). In lymph nodes, VHHCD8-apolipoprotein A1 fusion protein nanoparticles were more associated with CD8+ or CD3+ when compared with control apolipoprotein A1 protein nanoparticles, and VHHCD8-apolipoprotein A1 fusion protein nanoparticles were less associated with CD4+ T cells in lymph nodes when compared with control apolipoprotein A1 protein nanoparticles ( Fig. 9 ).

[0404] When compared to control apolipoprotein A1 protein nanoparticles, VHHCD8-apolipoprotein A1 fusion protein nanoparticles were more associated with CD8+ T cells in the bone marrow, blood, spleen, and lymph nodes ( Fig.10 There were no differences in CD4+ T cell engagement between treatment groups ( Fig.10 ).

[0405] Example 6. Fusion proteins comprising apolipoprotein fused to a rerouting peptide or protein and / or fused to an immunomodulatory biopeptide Lipid Nanoparticles Fusion Proteins with Apolipoprotein

[0406] Materials and methods

[0407] ApoA1-IL-2 was reduced to break disulfide bonds between cysteines in the protein by incubation in 1 mM DTT for 2 hours at room temperature.ApoA1-IL-2 and VHHCD8-apoA1 were fluorescently labeled with cy3 and cy5, respectively, as described in Example 4.

[0408] The apoA1-IL-2 fusion protein has a sequence as defined by SEQ ID NO: 3 or 5, or is encoded by a sequence as defined by SEQ ID NO: 4 or 6.

[0409]

[0410]

[0411]

[0412]

[0413] Disk-shaped aNPs were prepared with 513 μg ApoA1-IL-2 and 467 μg VHHCD8-ApoA1. Spherical aNPs were prepared in two sizes, in which only the triglyceride content was different. Large spheres were prepared as described in Example 5, and small spheres were prepared with half the triglyceride content of large spheres. The spheres with only apoA1-IL-2 (donor) contained 1025 μg ApoA1-IL-2. The spheres with only VHHCD8-apoA1 (acceptor) contained 921 μg VHHCD8-apoA1. Large spheres and small spheres with 1:1 mixed donors and acceptors contained 513 μg ApoA1-IL-2 and 467 μg VHHCD8-ApoA1.

[0414] The PBS solution of aNPs was pipetted into a black 96-well plate (Thermo Fisher Scientific-Nunclon) with 100 μL per well. For 1:1 mixed donor spheres and recipient spheres only, 50 μL of each formulation was placed in the well. Fluorescently labeled apoA1-IL-2 and VHHCD8-apoA1 of 1:1 mixing (molar ratio) were included as controls. The sample was excited at 520nm, and the emission was detected between 560nm and 780nm in a Spark plate reader (Tecan) with a step size of 5nm. The data were normalized by dividing the intensity of a sample at each wavelength by the intensity of the sample at 565 (donor emission maximum).

[0415] result

[0416] FRET results showed that both VHHCD8-apolipoprotein A1 and IL-2-apolipoprotein A1 fusion proteins could be easily and stably incorporated into one lipid nanoparticle formulation ( Fig.11 ).

[0417] Example 6. Production, analysis and evaluation of CD8+ T cell-targeted apolipoprotein nanoparticles containing mRNA

[0418] Materials and methods

[0419] Apolipoprotein nanoparticle production

[0420] CD8-aNP-mRNA is prepared by T-connecting mixer and mixing rapidly.Cationic ionizable lipid ALC-0315, dimyristoylfosfatidylcholine (DMPC), cholesterol and tricaprylin are dissolved in ethanol with appropriate molar ratio (Table 2), to a final concentration of 10mM total lipid.mRNA is dissolved in 25mM sodium acetate buffer at pH 4.0, to obtain a final mixture of nucleic acid and lipid weight / μmol ratio with a determination of 0.0278 (N / P6).In the first mixing step, organic solution and aqueous solution are mixed with a flow ratio of 1:3 (v:v) and a total flow of 28mL / min.Gained mixture is dialyzed overnight for 1000 times of volume of phosphate buffered saline (PBS) pH 7.4. In the second mixing step, lipid nanoparticles containing mRNA and CD8VHH-apolipoprotein A1 fusion protein were mixed at a flow ratio of 1:3 (v:v) and a total flow rate of 28 mL / min. The resulting CD8-aNP-mRNA was sterile filtered (0.2 μM) and concentrated using centrifugal flow filtration.

[0421] Table 2. CD8-aNP-mRNA composition (molar ratio)

[0422]

[0423] Physicochemical Analysis of Apolipoprotein Nanoparticles

[0424] CD8-aNP-mRNA was characterized based on size distribution by dynamic light scattering, zeta potential by laser Doppler electrophoresis, mRNA encapsulation efficiency as determined by Ribogreen, and cholesterol / phospholipid content using a dedicated colorimetric assay.

[0425] In vitro evaluation of apolipoprotein nanoparticles

[0426] Spleen was obtained from mice, sliced ​​and filtered multiple times with 70 μm filters (Corning) to obtain splenocyte suspension. Cells were spun down at 1500 rpm for 10 minutes, supernatant was removed, and cells were dissolved in 2 mL 1X red blood cell lysis buffer (Thermofisher). The suspension was incubated at room temperature for 5 minutes, 10 mL RPMI base (Thermofisher) was added, and cells were spun down at 1500 rpm for 10 minutes. The cells were then redissolved in RPMI culture medium and plated in 96-well plates with 150.000 cells / well. CD8-aNP-mRNA encoding mCherry and control formulations were added to the wells at appropriate concentrations, and incubated at 37 ° C for 6 hours, then harvested cells, washed and CD3 and CD4 were stained, and measured on Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD).

[0427] In vivo evaluation of apolipoprotein nanoparticles

[0428] CD8-aNP-mRNA encoding mCherry and control formulations were intravenously injected into C57BL / 6 mice at a dose of 0.5 mg / kg mRNA. Six hours later, mice were sacrificed, blood was harvested, and other tissues of interest (bone marrow, spleen, and lymph nodes) were harvested after PBS perfusion. Tissues were homogenized into single-cell suspensions and stained for myeloid (CD115, F4 / 80, Ly6C, CD11b, CD45, Ly6G, CD11c), lymphoid (CD45, CD11b, CD3, CD19, CD4, CD8a), and progenitor (CD117, CD34, Ly-6A / E, CD135, CD16 / 32, CD48, CD41, CD150, CD3, CD11b, CD45R / B220, Ly-76, Ly6G, Ly6C) cell markers and analyzed on a Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD).

[0429] result

[0430] Fig.12Flow cytometric analysis of mouse splenocytes incubated ex vivo with apoA1 aNP, VHHCD8-apoA1aNP, and VHHGFP-apoA1 aNP containing mRNA encoding the fluorescent mCherry protein is shown in (left panel: CD3+CD4-mCherry+T cells, right panel: CD11b+mCherry+ myeloid cells). The results showed that VHHCD8-apoA1 aNP induced functional mCherry reporter gene expression in significantly more CD3+CD4-T cells when compared with apoA1 aNP, VHHGFP-apoA1 aNP, and lipofectamine controls. ApoA1 aNP induced functional mCherry reporter gene expression in significantly more CD11b+ myeloid cells when compared with VHHCD8-apoA1 aNP, VHHGFP-apoA1 aNP, and lipofectamine controls. These results suggest that apoA1 aNPs have a natural propensity to interact with myeloid cells and that these aNPs can be rerouted to other immune cells (such as CD8+ T cells) by incorporating fusion proteins of apolipoprotein and a targeting moiety.

Claims

1. A modified apolipoprotein comprising an apolipoprotein, an apolipoprotein derivative, an apolipoprotein mimetic or an apolipoprotein mimetic derivative attached to a targeting body, wherein the targeting body is a molecule that is capable of binding to a molecule on the cell surface of a target cell.

2. The modified apolipoprotein according to claim 1, wherein the targeting body is selected from: - an antibody or an antigen-binding fragment of an antibody, - a protein ligand, a protein binding domain or a protein binding fragment thereof, -peptides, - a peptide mimetic, or -Sugar polymers.

3. The modified apolipoprotein according to claim 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of: Fab, Fab2, scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab3, trispecific Fab3, diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, IgNAR, monovalent IgG, VhH, nanobody or VNAR or an antigen-binding fragment thereof, preferably camel VhH or shark VhH or a derivative or an antigen-binding fragment thereof.

4. A modified apolipoprotein according to any of the preceding claims, wherein the targeting body binds to a protein on the surface of a lymphoid cell, a myeloid cell, a tumor cell, an endothelial cell, a hematopoietic stem and progenitor cell (HSPC), a hematopoietic stem cell (HSC), a multipotent progenitor cell (MPP), a common myeloid progenitor cell (CMP), or wherein the targeting body binds to a bacterial protein or antigen, a viral protein or antigen, a fungal protein or antigen, or a parasitic protein or antigen, Preferably, the lymphoid or myeloid cells are selected from: monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, granulocytes, T helper cells such as Th1, Th2, Th17 or Th22, dendritic cells such as plasmacytoid dendritic cells, conventional DC 1 or conventional DC 2, or tumor-associated macrophages.

5. A modified apolipoprotein according to any of the preceding claims, wherein the targeting body binds to a protein selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDwl2, CD13, CD14, CD15, CD15s, CD15u, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD 34. CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, C Dw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD1 07a, CD107b, CD108, CD109, CD110, CD111, CD112, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, C D125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD133, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143,CD144、CD145、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD157、CD158、CD158a、CD158b、CD159a、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD183、CD184、CD195、CDw197、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207(CLEC4K)、CD208、CD209(CLEC4L)、CDw210、CD212、CD213a1、CD213a2、CDw217、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CCR1、CCR2、CCR3、CCR4、CCR5、CCR7、CCR8、CCR9、CLEC1A、CLEC1B、CLEC2A、CLEC2B、CLEC3A、CLEC3B、CLEC4A、CLEC4C(CD303)、CLEC4D、CLEC4J、CLEC4E、CLEC4F、CLEC4G、ASGR1(CLEC4H1)、ASGR2(CLEC4H2)、FCER2()、CLEC4M、CLEC5A、CLEC6A、CLEC7A、OLR1(CLEC8A)、CLEC9A、CLEC10A、CLEC11A、CLEC12A、CLEC12B、CD302(CLEC13A)、LY75(CLEC13B)、PLA2R1(CLEC13C)、MRC1(CLEC13D)、MRC2(CLEC13E)、CLEC14A、CLEC16A、CLEC17A、KLRA1、KLRB1(CLEC5B), KLRC1, KLRC2, KLRC3, KLRC4, KLRD1, KLRF1(CLEC5C), KLRG1(CLEC15A), KLRG2(CLEC15B), KLRK1, AGC1, ATRNL1, BCA N, CD248, CHODL, CL-K1-Ia, CL-K1-Ib, CL-K1-Ic, CLECSF5, COLEC10, COLEC11, COLEC12, CSPG3, FCER2, FREM1, HBXBP, LAYN, LOC34817 4. LOC728276, MAFA, MBL2, MGC34761, MICL, MRC1L1, PAP, PKD1, PKD1L2, PRG2, PRG3, REG1A, REG1B, REG3A, REG3G, REG4, SELE, SELL, SELP, SFTPA1, SFTPA2, SFTPA2B, SFTPD, SRCL, THBD, VCAN, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelioma antigen (ETA), tyrosinase, and melanoma-associated antigen (MAGE); Preferred proteins are selected from the following: CD14, CD11b, CD357 (GITR), CD193, CD123, CD117, CD56, CD19, CD38, CD25, CD133, CXCR3, CCR3, CD196, CCR10, CD64, CD206, CLEC4C (CD303), CLEC9A, CD1c, CD163, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelioma antigen (ETA), tyrosinase and melanoma-associated antigen (MAGE).

6. A modified apolipoprotein according to any one of the preceding claims, wherein the apolipoprotein, apolipoprotein derivative, apolipoprotein mimetic or apolipoprotein mimetic derivative is selected from apoA1, apoA-1 Milano, apoA2, apoA4, apoA5, ​​apoB, apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL, apoL1, apoL2, apoL3, apoL4, apoL5, apoL6, apoLD1, apoO, apoOL and apoM, or a combination thereof, or a mimetic or derivative thereof, Preferably selected from apoA1, apoA2, apoA4, apoA5, ​​apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL and apoM, or a mimetic or derivative thereof, More preferably, it is selected from apoA1, apoA2, apoA4, apoA5, ​​apoB100, apoC-I, apoC-II, apoC-III, apoC-IV and apoE, or a mimetic or derivative thereof, Even more preferably selected from apoA1, apoA4, apoA5, ​​apoB100, apoC-III and apoE, or a mimetic or derivative thereof, Most preferably, it is selected from apoA1, apoB100 and apoE, or a mimetic or derivative thereof.

7. A lipid nanoparticle comprising an outer layer and a core, wherein the outer layer comprises: - phospholipids; - sterols; and - A modified apolipoprotein as defined in any one of the preceding claims; and wherein the core comprises at least one core component selected from: A lipid, a cationic lipid, or a multivalent molecule.

8. The lipid nanoparticle of claim 7, wherein the nanoparticle further comprises a payload, preferably wherein the payload is contained in the core, the phospholipid layer, or wherein the payload is associated with a component of the outer layer.

9. The lipid nanoparticle of claim 8, wherein the effective load is selected from a nucleic acid or nucleic acid analog, a therapeutic agent, a biologic, a cytokine, a chemokine, a hormone, a growth factor, or a combination thereof.

10. The lipid nanoparticle of claim 9, wherein the lipid nanoparticle comprises a nucleic acid and a cationic lipid or an ionizable cationic lipid.

11. The lipid nanoparticle of claim 10, wherein the nucleic acid and the cationic lipid or ionizable cationic lipid are contained by the core, and the modified apolipoprotein and the phospholipid are contained by the outer layer.

12. A lipid nanoparticle comprising an outer layer and a core, wherein the outer layer comprises: - phospholipids; - sterols; and - a modified apolipoprotein as defined in any one of claims 1 to 6; and The core comprises a nucleic acid and a cationic lipid or an ionizable cationic lipid.

13. The lipid nanoparticle according to any one of claims 10 to 12, wherein the nanoparticle core further comprises a filler, preferably a filler selected from triacylglycerol and cholesterol acyl ester or a combination thereof, such as wherein the triacylglycerol is tricaprylin and / or wherein the cholesterol acyl ester is cholesterol caprylate and / or cholesterol oleate.

14. The lipid nanoparticle of any one of claims 9 to 13, wherein the nucleic acid is RNA, DNA or a nucleic acid analog.

15. The lipid nanoparticle of claim 14, wherein the RNA is a microRNA (miRNA), a small interfering RNA (siRNA), a piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a transfer RNA (tRNA), a tRNA-derived small RNA (tsRNA), a small regulatory RNA (srRNA), a messenger RNA (mRNA), a modified mRNA, a ribosomal RNA (rRNA), a long noncoding RNA (lncRNA) or a guide RNA (gRNA) or a combination thereof, and / or modifications thereof.

16. The lipid nanoparticle of claim 14, wherein the DNA is single-stranded or double-stranded DNA.

17. The lipid nanoparticle of any one of claims 9 to 14, wherein the nucleic acid is an antisense oligonucleotide, and the antisense oligonucleotide is a modified single-stranded DNA or RNA consisting of nucleotides or nucleoside analogs containing a phosphodiester backbone or 2' ribose.

18. The lipid nanoparticle of claim 17, wherein the nucleotide or nucleoside analog is selected from the group consisting of locked nucleic acid (LNA), bridged nucleic acid (BNA), morpholino or peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), or a mixture or combination thereof.

19. The lipid nanoparticle according to any one of claims 10 to 18, wherein the cationic lipid or ionizable cationic lipid is selected from: an ionizable cationic ester of a long-chain alcohol, an ionizable cationic ester of a diglyceride, or an ionizable cationic ester of a sterol, or a combination thereof.

20. The lipid nanoparticle according to any one of claims 10 to 19, wherein the ionizable cationic lipid is a molecule according to any one of formula (I), (II), (III), (IV) or (V): ICG is wherein the wavy line indicates the point of attachment to the compound of formula (I), (II), (III), (IV) or (V); p is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11; Each R1 is independently selected from the group consisting of a linear or branched C1-C19 alkyl, a linear or branched C1-C19 alkenyl, an aryl, an arylene-alkyl and an alkylene-aryl group, wherein the alkyl or alkenyl group optionally contains up to 5 heteroatoms independently selected from O and N; R2 is selected from the group consisting of hydrogen, methyl, ethyl and -CH2-OC(O)-R 1a ; R3 is selected from the group consisting of hydrogen, aromatic groups, arylene-alkyl groups, alkylene-aromatic groups, and linear C1-C6 alkyl groups; R 1a Selected from the group consisting of: straight chain or branched C1-C19 alkyl, straight chain or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl groups, wherein the alkyl or alkenyl group optionally contains up to 5 heteroatoms independently selected from O and N; Each R x independently selected from the group consisting of: methyl, ethyl, propyl, and -CH2-CH2-OH; Each R y The groups are independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl groups, wherein the alkyl group optionally contains up to 5 heteroatoms independently selected from O and N; or a rotamer, tautomer, stereoisomer or regioisomer thereof.

21. The lipid nanoparticle according to any one of claims 10 to 19, wherein: The amount of the modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) ranges from 0.08 to 2.0 mol %, such as 0.10 to 2.0 mol %; and / or The amount of phospholipids ranges from 5-90 mol%, such as 15-90 mol%; and / or The amount of sterol ranges from 2.5 to 65 mol%, such as 2.5 to 50 mol%; and / or The amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as 8.0 to 80 mol%. The molar percentages are based only on the combined amounts of the modified apolipoprotein, phospholipid, sterol and cationic lipid or ionizable cationic lipid in the lipid nanoparticle.

22. The apolipoprotein lipid nanoparticle according to any one of claims 10 to 21, wherein: The amount of the modified apolipoprotein (cumulatively in the case of two or more modified apolipoproteins) ranges from 0.1 to 90% by weight; The amount of nucleic acid ranges from 0.01 to 90% by weight; The amount of phospholipids ranges from 0.1 to 95% by weight; The amount of sterol ranges from 0.1 to 95% by weight; and / or The amount of cationic lipids and / or ionizable cationic lipids ranges from 0.1 to 95% by weight. These weight percentages are based on the combined amounts of the modified apolipoprotein, the nucleic acid, the phospholipid, the sterol and the cationic lipid and / or the ionizable cationic lipid.

23. The lipid nanoparticle of any one of claims 10 to 22, wherein the ratio of the modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipid is between 1:25 and 1:400, more preferably between 1:50 and 1:200, even more preferably between 1:75 and 1:150, based on molar weight percentage.

24. The lipid nanoparticle of any one of claims 10 to 23, wherein the ratio of the modified apolipoprotein (in the case of two or more modified apolipoproteins, cumulatively) to phospholipid is 2:1 to 1:10, more preferably 1:1 to 1:5, even more preferably 1:1.5 to 1:4, based on weight.

25. A method for preparing a lipid nanoparticle as defined in any one of claims 7 to 9, comprising the steps of: a1) expressing and isolating a modified apolipoprotein as defined in any one of claims 1 to 5 to obtain an isolated modified apolipoprotein; and / or a2) chemically conjugating the targeting body to apolipoprotein or an apolipoprotein mimetic to obtain a modified apolipoprotein and isolating the modified apolipoprotein; b) combining the isolated modified apolipoprotein obtained in step a1 and / or step a2 with phospholipids, sterols and optionally lipids to obtain lipid nanoparticles.

26. The method according to claim 25, wherein step b) comprises: b1) mixing a lipid component in an organic solvent with nucleic acids in an aqueous buffer, preferably rapidly, to produce nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a core component and optionally a lipid; and wherein the aqueous buffer has a pH of 5.5 or less, preferably 5.0 or less; and b2) mixing lipid nanoparticles with said modified apolipoprotein, preferably rapidly, to produce said nanoparticles at a pH between 5.5 and 9.0, preferably between 6.0 and 8.0, more preferably at a pH of 6.5 to 8.

0.

27. A method for producing lipid nanoparticles, comprising the steps of: a) rapidly mixing a lipid component in an organic solvent with a nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid component comprises a phospholipid, a sterol, a cationic lipid, or an ionizable cationic lipid, wherein the aqueous buffer has a pH of 5.0 or less; and b) rapidly mixing the lipid nanoparticles with one or more modified apolipoproteins as defined in any one of claims 1 to 6; The lipid nanoparticles are produced at a pH between 5.5 and 8.0, preferably between 6.0 and 8.

0.

28. A modified apolipoprotein according to any one of claims 1 to 6 or a lipid nanoparticle according to any one of claims 7 to 24, or the lipid nanoparticle obtained or obtainable by the method according to any one of claims 25 to 27, for use as a medicament.

29. A modified apolipoprotein according to any one of claims 1 to 6 or a lipid nanoparticle according to any one of claims 7 to 24, or a lipid nanoparticle obtained or obtainable by a method according to any one of claims 25 to 27, for use in treating or preventing immune-related disorders, such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, genetic disorders, metabolic disorders, nervous system disorders or tissue trauma.

30. Use of a modified apolipoprotein according to any one of claims 1 to 6 or a lipid nanoparticle according to any one of claims 7 to 24, or the lipid nanoparticle obtained or obtainable by the method of any one of claims 25 to 27, in delivering a compound to a target, preferably wherein the target is a cell, a tissue and / or an organ, even more preferably wherein the target is a lymphoid cell, a myeloid cell, a tumor cell, an endothelial cell, a hematopoietic stem and progenitor cell (HSPC), a hematopoietic stem cell (HSC), a multipotent progenitor cell (MPP), a common myeloid progenitor cell (CMP), or wherein the targeting body binds a bacterial protein or antigen, a viral protein or antigen, a fungal protein or antigen or a parasitic protein or antigen, Preferably, the lymphoid or myeloid cells are selected from: monocytes, macrophages, M1-like macrophages, M2-like macrophages, eosinophils, basophils, mast cells, NK cells, B cells, plasma cells, regulatory T cells, hematopoietic stem cells, T helper cells such as Th1, Th2, Th17 or Th22, dendritic cells such as plasmacytoid dendritic cells, conventional DC 1 or conventional DC 2, or tumor-associated macrophages.

31. An in vitro or ex vivo method for introducing a nucleic acid into a cell, the method comprising contacting a lipid nanoparticle according to any one of claims 9 to 24 or obtained or obtainable by a method according to any one of claims 25 to 27 with a cell.

32. An in vivo method for introducing a nucleic acid into a cell, the method comprising contacting a lipid nanoparticle according to any one of claims 9 to 24 or obtained or obtainable by a method according to any one of claims 25 to 27 with a cell.

33. A lipid nanoparticle according to any one of claims 9 to 24, or obtained or obtainable by a method according to any one of claims 25 to 27, for use in delivering a nucleic acid to a subject in vivo.

34. A method for delivering a nucleic acid in vivo, the method comprising administering to a subject a lipid nanoparticle according to any one of claims 9 to 24 or a lipid nanoparticle obtained or obtainable by a method according to any one of claims 25 to 27.

35. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle according to any one of claims 9 to 24 or a lipid nanoparticle obtained or obtainable by the method of any one of claims 25 to 27.

36. The method of claim 35, wherein the disease is an immune-related disorder, such as transplant rejection, graft-versus-host disease (GVH), atherosclerosis, infection, inflammation, autoimmunity, allergy, cancer, a genetic disorder, a metabolic disorder, a nervous system disorder, or tissue trauma.

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