Apolipoprotein lipid nanoparticle

Abstract

The invention relates to fusion proteins of apolipoprotein with an immunomodulatory biomolecule and / or a rerouting molecule. The fusion proteins can be used as a carrier for an immunomodulatory biomolecule as such or incorporated in a lipid nanoparticle. The fusion protein finds use in the treatment of immune related disorders, or targeting a payload, such as a nucleic acid, to a specific target site.

Description

[0001] APOLIPOPROTEIN LIPID NANOPARTICLE

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of fusion proteins and more particularly fusion proteins that find use in the treatment of immune related disorders. The invention further relates to lipid nanoparticles comprising the fusion proteins and methods of making such. Lastly the invention relates to methods of treatment using the fusion proteins or lipid nanoparticles.

[0004] BACKGROUND OF THE INVENTION

[0005] Many promising therapeutics are hampered by poor circulation time due to rapid clearance of the therapeutic from the body. For example, it was found that cytokines may hold very promising uses in many immunological applications. However due to the very short half-life in the body, either no effect can be exerted on the intended target, or toxic amounts are needed to achieve an effect. Therefore, there is a need for improved methods to safely reduce the circulation half-life of therapeutics.

[0006] Additionally, many promising therapeutics suffer from the fact that they either do not, or poorly, reach the intended target site, or present undesired off-target effects. Therefore, there is a further need to improve targeting of therapeutics.

[0007] These, among other, problems are addressed by the products and methods as defined in the appended claims.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is based on the inventors’ finding that apolipoproteins can be used as a carrier for therapeutic agents, and that apolipoproteins may further be modified to target specific cells, tissues, or organs. The inventors found that fusion proteins of immunomodulatory biomolecules, such as cytokines, with apolipoproteins or apolipoprotein mimetics demonstrate strongly increased half-life in blood, thereby opening up the possibility to use immunomodulatory biomolecules, such as cytokines, in a therapeutical manner without requiring toxic concentrations to be administered. Further it was realized that apolipoproteins or mimetics thereof allow targeting of immunomodulatory biomolecules, such as cytokines, when fused together.

[0010] It was further realized that it is possible to direct a fusion protein or apolipoprotein (or mimetic thereof) to an intended target by linking it to a rerouting molecule. Moreover, present inventors unexpectedly found that the fusion of an immunomodulatory biomolecule and / or a rerouting molecule to an apolipoprotein or an apolipoprotein mimetic allows to easily incorporate said immunomodulatory biomolecule and / or a rerouting molecule in a lipid nanoparticle and to expose said immunomodulatory biomolecule and / or a rerouting molecule to the environment surrounding said lipid nanoparticle. In this way the apolipoprotein (or mimetic thereof) or fusion protein can be targeted to cells, tissues, or organs it would otherwise not or insufficiently reach, or it could be used to reduce off-target effects.

[0011] The fusion proteins may be used as such, meaning not as part of a lipoprotein or lipid nanoparticle. In such way the fusion protein may serve as a carrier to deliver an immunomodulatory biomolecule to a target site. Alternatively, the fusion protein may be used to prepare a lipid nanoparticle.

[0012] A first aspect of the invention provides an apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule; and phospholipids; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response.

[0013] A further aspect of the invention provides an apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule; and phospholipids; wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

[0014] A further aspect of the invention provides an apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule; and phospholipids; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response; and wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity. A further aspect of the invention provides an apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response; a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule; wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; and phospholipids.

[0015] A further aspect of the invention provides a method of manufacturing an apolipoprotein lipid nanoparticle as described herein, the method comprising the steps of: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; and / or a2) chemically conjugating one or more apolipoproteins or apolipoprotein mimetics and isolating the one or more conjugated apolipoproteins to obtain one or more isolated conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; and b) combining the one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or the one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids, and optionally sterols and / or lipids, to obtain an apolipoprotein lipid nanoparticle.

[0016] A further aspect of the invention provides an apolipoprotein lipid nanoparticle obtained by or obtainable by the method as taught herein. A further aspect of the invention provides a pharmaceutical composition comprising the apolipoprotein lipid nanoparticle as taught herein, and a pharmaceutically acceptable carrier.

[0017] A further aspect of the invention provides the apolipoprotein lipid nanoparticle as taught herein or the pharmaceutical composition as taught herein for use as a medicament.

[0018] A further aspect of the invention provides the apolipoprotein lipid nanoparticle as taught herein or the pharmaceutical composition as taught herein for use in the treatment of an immune related disorder.

[0019] A further aspect of the invention provides the apolipoprotein lipid nanoparticle as taught herein or the pharmaceutical composition as taught herein for use in targeting said immunomodulatory biomolecule to a target cell, preferably a myeloid cell.

[0020] A further aspect of the invention provides the use of the apolipoprotein lipid nanoparticle as taught herein for delivering an immunomodulatory biomolecule to a target, preferably wherein the target is a cell, tissue, and / or organ.

[0021] A further aspect of the invention provides a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, for use in targeting said immunomodulatory biomolecule to a myeloid cell.

[0022] A further aspect of the invention provides a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

[0023] A further aspect of the invention provides a nucleic acid encoding the fusion protein comprising the apolipoprotein or an apolipoprotein mimetic and the rerouting molecule as taught herein.

[0024] A further aspect of the invention provides a pharmaceutical composition comprising the fusion protein as taught herein or the nucleic acid as taught herein, and a pharmaceutically acceptable carrier.

[0025] A further aspect of the invention provides the fusion protein as taught herein, the nucleic acid as taught herein or the pharmaceutical composition as taught herein for use as a medicament.

[0026] A further aspect of the invention provides the fusion protein as taught herein, the nucleic acid as taught herein or the pharmaceutical composition as taught herein for use in the treatment of an immune related disorder, preferably wherein the immune related disorder is an immune related disorder selected from the group consisting of cancer, inflammation, an infectious disease, an autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

[0027] A further aspect provides the fusion protein as taught herein, the nucleic acid encoding the fusion protein as taught herein or the pharmaceutical composition as taught herein when comprising an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, for use in targeting said immunomodulatory biomolecule to a target cell.

[0028] As supported by the example section, present inventors found that a fusion protein of an apolipoprotein or an apolipoprotein mimetic, preferably apolipoprotein A1 (ApoA1), with interleukin-4 (IL-4) allows targeting IL-4 to the myeloid compartment. To their surprise, present inventors found that IL-4 can simultaneously reduce inflammation and induce trained immunity, particularly when targeted to the myeloid compartment. Therefore, the present inventors concluded that fusion proteins of an apolipoprotein or an apolipoprotein mimetic, preferably ApoA1 , with IL-4 can be used to prevent immune related disorders by promoting trained immunity. Furthermore, present inventors found that IL-4’s pharmacokinetic profile and bioavailability to innate immune cells can be further improved by integrating said fusion proteins into myeloid cell-avid lipid nanoparticles. Incorporation of such IL-4 fusion protein into a lipid nanoparticle does not hamper its ability to target to the myeloid compartment.

[0029] Accordingly, a further aspect of the invention provides a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4).

[0030] A further aspect of the invention provides a nucleic acid encoding the fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4) as taught herein.

[0031] A further aspect of the invention provides a pharmaceutical composition comprising the fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin- 4 (IL-4) as taught herein or the nucleic acid encoding said fusion protein as taught herein, and a pharmaceutically acceptable carrier.

[0032] A further aspect of the invention provides the fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4) as taught herein, the nucleic acid encoding such fusion protein as taught herein or the pharmaceutical composition comprising such fusion protein or nucleic acid for use as a medicament.

[0033] A further aspect of the invention provides the fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4) as taught herein, the nucleic acid encoding such fusion protein as taught herein or the pharmaceutical composition comprising such fusion protein or nucleic acid for use in the treatment of an immune related disorder, preferably wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

[0034] A further aspect of the invention provides the fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4) as taught herein, the nucleic acid encoding such fusion protein as taught herein or the pharmaceutical composition comprising such fusion protein or nucleic acid for use in targeting IL-4 to a target cell, preferably a myeloid cell.

[0035] A further aspect of the invention provides a fusion protein comprising a myeloid- targeting molecule and IL-4, wherein the myeloid-targeting molecule is capable of targeting the IL-4 to a myeloid cell.

[0036] A further aspect of the invention provides a nucleic acid encoding the fusion protein comprising a myeloid-targeting molecule and IL-4 as taught herein.

[0037] A further aspect of the invention provides a nucleic acid comprising a nucleic acid sequence 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. 44 or comprising a nucleic acid sequence encoding a polypeptide having a sequence 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. 43 and further comprising means for targeted expression in a myeloid cell, wherein said mean are selected from:

[0038] - a promoter for selective or inducible expression in said myeloid cell operatively linked to said nucleic acid; or

[0039] - a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cell; or

[0040] - a lipid nanoparticle comprising one or more apolipoproteins, phospholipids, said nucleic acid, and optionally sterol.

[0041] A further aspect of the invention provides a pharmaceutical composition comprising the fusion protein comprising a myeloid-targeting molecule and IL-4 as taught herein or the nucleic acid encoding said fusion protein as taught herein or the nucleic acid comprising means for targeted expression in a myeloid cell as taught herein, and a pharmaceutically acceptable carrier.

[0042] A further aspect of the invention the fusion protein comprising a myeloid-targeting molecule and IL-4 as taught herein or the nucleic acid encoding said fusion protein as taught herein or the nucleic acid comprising means for targeted expression in a myeloid cell as taught herein, or the pharmaceutical composition comprising said fusion protein or nucleic acid as taught herein for use as a medicament.

[0043] A further aspect of the invention provides the fusion protein comprising a myeloid- targeting molecule and IL-4 as taught herein or the nucleic acid encoding said fusion protein as taught herein or the nucleic acid comprising means for targeted expression in a myeloid cell as taught herein, or the pharmaceutical composition comprising said fusion protein or nucleic acid as taught herein for use in the treatment of an immune related disorder.

[0044] A further aspect of the invention provides in vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or an organism.

[0045] BRIEF DESCRIPTION OF THE FIGURES

[0046] Fig. 1 depicts a schematic overview of the optional assembly of an exemplary apolipoprotein fusion in a lipid nanoparticle (sphere or disk) and subsequent binding to a target cell.

[0047] Fig. 2 depicts a schematic overview of different envisioned apolipoproteins and subsequent assembly in lipid nanoparticles. Depicted are the following fusion proteins (schematically): top left depicts both an apolipoprotein fused to an immunomodulatory biomolecule, and an apolipoprotein fused to a rerouting molecule; bottom left depicts an apolipoprotein fused to an immunomodulatory biomolecule; top right depicts an apolipoprotein fused to a rerouting molecule; bottom right depicts an apolipoprotein fused to an immunomodulatory biomolecule and a rerouting molecule.

[0048] Fig. 3 shows SDS-PAGE gels demonstrating the expression and purification of different apolipoprotein fusion constructs (ApoA1-IL1 B, ApoA1-IL38, ApoA1-IL2). The rectangles indicate the bands corresponding to the desired proteins. (P: pellet containing cell debris, SN: supernatant containing soluble protein fraction, FT: flow through of SN applied to Ni-NTA column, A: first wash using 10 mM imidazole, A50: second wash using 50 mM imidazole, E1 : elution fraction 1 , E2: elution fraction 2, E3: elution fraction 3, E4: elution fraction 4, FW: final wash using 500 mM imidazole.)

[0049] Fig. 4 shows results of dynamic light scattering measurements for four lipid nanoparticles, three of which each contain a different apolipoprotein (ApoA1-IL1 B, ApoA1- IL38, ApoA1-IL4) and one containing apoA1 as a control nanoparticle. Mean number diameter (darker grey) and polydispersity index (Pdl) (lighter grey) have been determined for these nanoparticles over the course of 11 days. Fig. 5 shows an SDS-PAGE gel demonstrating the expression and purification of apoA1 in which serine to cysteine mutations have been introduced, at either position 147 or 279. The rectangle indicates the band corresponding to the mutated apoA1.

[0050] Fig. 6 shows the quadrupole time-of-flight (Q-ToF) results for the apoA1 mutants. In both graphs, the chromatogram is plotted in the upper right corner, with below this the m / z values of the main peak from the chromatogram. The deconvoluted mass spectra show the presence of the desired mutant apoA1 proteins.

[0051] Fig. 7 shows the HPLC-MS chromatogram of interleukin (IL)-4 which has been modified to contain one N-terminal azide. The mass corresponding to the proteins represented by the peaks in the chromatogram are indicated.

[0052] Fig. 8 shows SDS-PAGE gels demonstrating the purity of the used IL-4, the IL-4 modified with an N-terminal azide (reaction at 4°C or 20°C), apoA1 coupled to a PEG-linker containing DBCO group, and the reaction products of coupling IL-4 modified to contain an azide to apoA1 with linker and DBCO group (reaction at 4°C or 20°C). The rectangle indicates the bands corresponding to the desired conjugation product.

[0053] Fig. 9 shows the results of a HEK293 IL-4 reporter cell assay in which the binding of commercially obtained IL-4 (mammalian), recombinantly expressed IL-4 (bacterial), recombinantly expressed apoA1-IL4 fusion protein, and chemically conjugated apoA1-IL4 fusion protein are evaluated. The absorbance corresponds to the level of binding of IL-4 to its receptor.

[0054] Fig. 10 shows an SDS-PAGE analysis of the chemical (right panel) and recombinant (left panel) apoA1-IL2 fusion constructs (apoA1-IL2 wild-type “ApoA1-IL2” or apoA1-IL2 mutant “ApoA1-IL2v4”). The rectangle indicates the band corresponding to the apoA1-IL2 or apoA1-IL2v4 fusion construct, respectively.

[0055] Fig. 11 shows the successful formulation of discoidal nanoparticles comprising apoA1-IL2 fusion proteins using cryogenic transmission electron microscopy (cryo-TEM) (Right panel) and the analysis of nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Left panel).

[0056] Fig. 12 shows the ability of the apoA1-IL2 fusion proteins to stimulate CD4+ or CD8+ T-cell proliferation. Abbreviations: PHA, phytohemagglutinin.

[0057] Fig. 13 shows an SDS-PAGE analysis of the chemical (right panel) and recombinant (left panel) apoA1-ILip fusion constructs. The arrow (left panel) or rectangle (right panel) indicates the band corresponding to the apoA1-ILip fusion construct.

[0058] Fig. 14 shows the successful formulation of discoidal nanoparticles comprising apoA1-ILip fusion proteins using cryogenic transmission electron microscopy (cryo-TEM) (Right panel) and the analysis of nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Left panel).

[0059] Fig. 15 shows an SDS-PAGE analysis of the chemical (lower panel) and recombinant (upper panel) apoA1-IL38 fusion constructs. The arrow (upper panel) or rectangle (lower panel) indicates the band corresponding to the apoA1-IL38 fusion construct.

[0060] Fig. 16 shows the successful formulation of discoidal nanoparticles comprising apoA1-IL38 fusion proteins using cryogenic transmission electron microscopy (cryo-TEM) (Right panel) and the analysis of nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Left panel).

[0061] Fig.17 IL4 inhibits acute inflammation, yet induces trained immunity. (A) Schematic of in vitro direct inflammation experiments. (B) TNF, IL6, and IL1 Ra levels after 24 h stimulation of human primary monocytes. (C) Schematic of in vitro trained immunity experiments. (D) TNF and IL6 levels after re-stimulation of p-glucan-trained cells. (E) TNF and IL6 levels after restimulation of IL4-trained cells. (F) Seahorse analysis of glycolytic (left) and mitochondrial (right) metabolism in IL4-trained cells. Data are presented as mean ± SD.

[0062] Fig. 18 I mmune and epigenetic mechanisms mediating IL4-induced trained immunity. (A) Schematic overview of previously described premier IL4 signaling pathways. (B) TNF and IL6 levels after 24 h stimulation of monocytes whilst blocking key IL4 signaling routes. (C) TNF and IL6 levels after re-stimulation of cells that were trained with IL4 whilst blocking key IL4 signaling routes. (D) Heatmap of the transcriptome of IL4-trained cells, before and after re-stimulation. (E) Transcription factor motif enrichment analysis in IL4 trained immunity (heatmap indicates z-scores). (F) Pathway enrichment analyses of the IL4 trained immunity transcriptome. (G) TNF and IL6 levels after re-stimulation of cells that were trained with IL4 in the presence of a SET7 methyltransferase inhibitor. (H) ChlP-qPCR AUG analysis of TNF in IL4-trained cells. Data in bar graphs are presented as mean ± SD.

[0063] Fig. 19 Engineering apoA1-IL4 fusion protein. (A) Schematic overview of apoA1- based fusion protein platform. (B) Schematic of apoA1-IL4 fusion protein structure. (C) SDS- PAGE and (D) Western blot of recombinantly expressed proteins. Antibodies specific for endogenous IL4 and apoA1. (E) Chromatogram and Q-TOF-MS spectrum of apoA1-IL4. (F) Kinetics of apoA1-IL4 binding to IL4Ra using SPR. (G) Activation of HEK-Blue cells expressing IL4Ra and IL13Ra1 by apoA1-IL4. Data are presented as mean ± SD.

[0064] Fig. 20 Integrating apoA1-IL4 in nanoparticles platform. (A) Schematic representation of discoidal (upper panel) and spherical IL4-aNPs (lower panel) and (B) cryoTEM images. (C) IL4-aNP size distribution and (D) stability of IL4-aNPs over time as determined by the dynamic light scattering. IL4-aNP size is reported as the number mean. (E) Super-resolution fluorescence microscopy (dSTORM) images of human monocytes incubated with either fluorescently-labeled apoA1 (-IL4) or (IL4-)aNPs and stained with anti- IL4Ra antibody. The co-localization between the proteins and IL4Ra can be appreciated by the arrows. White regions of interest are magnified in subsequent images on the right. Data are presented as mean ± SD.

[0065] Fig. 21 Immunological in vitro, in vivo and ex vivo therapeutic evaluation of IL4-aNPs.

[0066] (A) Schematic overview of the direct inflammation and trained immunity experiments in vitro.

[0067] (B) TNF and IL6 levels after 24 h stimulation of monocytes in the presence of IL4-aNPs. (C) TNF and IL6 levels after restimulation of IL4(-aNP)-trained cells. (D) Schematic overview of murine in vivo tolerance model, including IL4-nanotherapy. (E) Serum TNF and IL6 levels following LPS re-challenge of mice treated IL4m-aNPs. The Mann-Whitney II test was used for statistical comparisons. (F) Schematic overview of human experimental endotoxemia model, including ex vivo tolerance reversal. (G) TNF and IL6 levels after ex vivo restimulation of human in vivo LPS-tolerized cells. (H) TNF and IL6 fold increase after ex vivo re-stimulation of human in vivo LPS-tolerized cells. Data are presented as mean ± SD.

[0068] Fig. 22 depicts a schematic overview of the optional assembly of an exemplary apolipoprotein fusion comprising an apolipoprotein and a rerouting protein in a lipid nanoparticle (disc).

[0069] Fig. 23 shows the expression of VHHCD8-apoA1 fusion protein in Clearcoli cells. Minor protein contaminants are present after IMAC purification [lane E1], The most prominent band corresponds to the fusion protein with a molecular weight of 43.3 kDa (rectangle).

[0070] Fig. 24 shows the successful formulation of discoidal nanoparticles comprising VHHCD8-apoA1 fusion proteins using cryogenic transmission electron microscopy (cryo- TEM) (Right panel) and the analysis of nanoparticle size and poly dispersity index (PDI) for 14 days using dynamic light scattering (DLS) (Left panel).

[0071] Fig. 25 shows the mean fluorescence intensity (MFI) of fluorescently labelled VHHCD8-apoA1 and apoA1 in mouse splenocytes (upper panel: CD3+ T cells from splenocytes; lower panel: all cells from the spleen).

[0072] Fig. 26 shows the mean fluorescence intensity (MFI) of discoidal and spherical aNPs formulated with VHHCD8-apoA1 and apoA1 and comprising a fluorescent dye in the lipid structure of the particle in mouse splenocytes.

[0073] Fig. 27 In vivo pharmacokinetics, biodistribution and safety profile after intravenous injection. (A) PET / CT 2 render at 24 h after injecting89Zr-labeled constructs. (B)89Zr-labeled construct blood half-life (n=5, as fitted with a two-phase decay function). (C) Ex vivo gamma counting of tissues 24 h after89Zr-labeled construct injection (n=5), number represents ratio target to clearance organs. (D) Cell type-specific biodistribution of DiO-labeled discoid I L4- aNPs in spleen and bone marrow, as measured by flow cytometry. (E)89Zr-IL4-aNP blood half-life in non- human primates. (F) Organ SUVmean over time in89Zr-IL4-aNPs injected non-human primates (n=2). (G) Organ specific SUVmean 48 h after89Zr-IL4-aNPs injection in non-human primates (n=2). (H) PET / MRI scan of non-human primate 48 h after89Zr-IL4- aNPs injection. Data are presented as mean ± SD where appropriate.

[0074] Fig. 28 shows the successful formulation of spherical nanoparticles comprising VHHCD8-apoA1 fusion proteins using cryogenic transmission electron microscopy (cryo- TEM) (bottom panel) and the analysis of nanoparticle size and poly dispersity index (PDI) for 14 days using dynamic light scattering (DLS) (top panel).

[0075] Fig. 29 In vivo biodistribution study setup for of spherical nanoparticles comprising VHHCD8-apoA1 fusion proteins.

[0076] Fig. 30 Cell association of VHHCD8-apolipoprotein A1 fusion protein nanoparticles in vivo. Flow cytometry analysis of fluorescent apolipoprotein A1 protein nanoparticle (ApoA1) and VHHCD8-apolipoprotein A1 fusion protein nanoparticle (VHHCD8) myeloid cell (A), CD8 T cell (B), CD4 T cell (C), CD3 T cell (D) association in bone marrow, spleen, and blood following intravenous administration. MFI: mean fluorescence intensity.

[0077] Fig. 31 Cell association of VHHCD8-apolipoprotein A1 fusion protein nanoparticles in vivo. Flow cytometry analysis of fluorescent apolipoprotein A1 protein nanoparticle (ApoA1) and VHHCD8-apolipoprotein A1 fusion protein nanoparticle (VHHCD8) CD8 T cell, CD4 T cell and CD3 T cell association in lymph nodes following intravenous administration. MFI: mean fluorescence intensity.

[0078] Fig. 32 Cell association of VHHCD8-apolipoprotein A1 fusion protein nanoparticles in vivo. Flow cytometry analysis of fluorescent apolipoprotein A1 protein nanoparticle (ApoA1) and VHHCD8-apolipoprotein A1 fusion protein nanoparticle (VHHCD8) cell association in bone marrow (BM), blood, spleen, and lymph nodes following intravenous administration.

[0079] Fig. 33 FRET assay of lipid nanoparticles containing VHHCD8-apoliprotein A1 fusion proteins and / or IL-2- apolipoprotein A1 fusion proteins.

[0080] DETAILED DESCRIPTION OF THE INVENTION

[0081] The recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to...” or the expression “between... and...” or another expression. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1 % or less, and still more preferably + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0082] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0083] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0084] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0085] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

[0086] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0087] Similarly, it should be appreciated that in the description of illustrative embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

[0088] As used herein, the singular form terms “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, and the like.

[0089] As used herein, the term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. For example, if a list is described as comprising group A, B, and / or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0090] As used herein, the term "antigen" refers to a substance to which a binding portion of an antibody may bind. The specific immunoreactive sites within the antigen are known as “epitopes” (or antigenic determinants). A target for an antibody, or antigen-binding portion thereof, may comprise an antigen, such as is defined herein.

[0091] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is 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" is understood to be the same as "5 or less" i.e., 5, 4, 3, ....-10, -11 , etc. Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1 , 2, 3, 4, 5, 6, 7 or more.

[0092] As used herein, the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to include a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or steps, or groups of elements, integers or steps. The verb “comprising” includes the verbs “essentially consisting of” and “consisting of”.

[0093] As used herein, the term ’’conventional techniques” refers to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 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.

[0094] As used herein, the term “identity" refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (Computational Molecular Biology, Lesk, A. M., ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, D. W., ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, A. M., And Griffin, H. G., 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., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two nucleotide sequences or amino acid sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine 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 to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

[0095] As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference amino acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence 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 a number of nucleotides 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 positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0096] Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: X is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: X. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a 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 a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0097] As used herein, the term “in vitro” refers to experimentation or measurements conducted using components of an organism that have been isolated from their natural conditions.

[0098] As used herein, the term “ex vivo” refers to experimentation or measurements done in or on tissue from an organism in an external environment with minimal alteration of natural condition. As used herein, the term "nucleic acid", “nucleic acid molecule” and “polynucleotide” is intended to include DNA molecules and RNA molecules. A nucleic acid (molecule) may be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0099] The terms "nucleic acid", “nucleic acid molecule” and “polynucleotide” are well understood in the art. By means of further guidance, the terms typically refer to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units. A nucleoside unit commonly includes a heterocyclic base and a sugar group. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (II), which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine), as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Exemplary modified nucleobases include, without limitation, 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 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 inter alia pentose (pentofuranose) groups such as preferably ribose and / or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as, without limitation, 2’-O- alkylated, e.g., 2’-0-methylated or 2’-0-ethylated sugars such as ribose; 2’-O- alkyloxyalkylated, e.g., 2’-O-methoxyethylated sugars such as ribose; or 2’-O,4’-C-alkylene- linked, e.g., 2’-O,4’-C-methylene-linked or 2’-O,4’-C-ethylene-linked sugars such as ribose; 2’-fluoro-arabinose, etc.). Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3’-N- carbamate, morpholino, borano, thioether, 3’-thioacetal, and sulfone internucleoside linkages. Preferably, inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof. The term “nucleic acid” also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexene nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)). “Alkyl” as used in this context particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1- propenyl, 2-propenyl, and isopropyl.

[0100] Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof. A modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination 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 synthesised) DNA, RNA or DNA / RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e. , produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0101] As used herein, the terms “sequence” when referring to nucleotides, or “nucleic acid sequence”, “nucleotide sequence” or “polynucleotide sequence” refer to the order of nucleotides of, or within, a nucleic acid and / or polynucleotide. Within the context of the current invention a first nucleic acid sequence may be comprised within or overlap with a further nucleic acid sequence.

[0102] As used herein, the term “subject” or “individual” or “animal” or “patient” or “mammal,” used interchangeably, refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo-, sports-, or pet-animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears, and so on. As defined herein a subject may be alive or dead. Samples can be taken from a subject post-mortem, i.e. after death, and / or samples can be taken from a living subject.

[0103] As used herein, terms "treatment", "treating", "palliating", “alleviating” or "ameliorating", used interchangeably, refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration or reduction (or delay) of progress of the underlying disease being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration or reduction (or delay) of progress of one or more of the physiological symptoms associated with the underlying disease such that an improvement or slowing down or reduction of decline is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disease.

[0104] 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 transporting has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. The term “vector” may also refer to the viral particle (i.e. viral vector) which contains the nucleic acid of interest.

[0105] A portion of this invention contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent invention, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0106] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention relates, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition as provided herein. The preferred materials and methods are described herein, although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

[0108] The present invention is based on the inventors’ finding that apolipoprotein or an apolipoprotein mimetic can be used as a carrier for therapeutic agents, and that apolipoproteins may further be modified to target specific cells, tissues or organs. The inventors found that fusion proteins of cytokines with apolipoproteins or apolipoprotein mimetics demonstrate strongly increased half-life in blood, thereby opening up the possibility to use cytokines in a therapeutical manner without requiring toxic concentrations to be administered. Further it was realized that apolipoproteins or apolipoprotein mimetics allow targeting of cytokines when fused together, such as to a target cell, tissue and / or organ, like a myeloid cell. The inventors realized that this concept is more broadly applicable and can also be used for a broad spectrum of immunomodulatory biomolecules such as cytokines, chemokines, hormones, growth factors etc. It was further realized that it is possible to direct a fusion protein or apolipoprotein or an apolipoprotein mimetic to an intended target by linking it to a rerouting molecule. In this way the apolipoprotein (or the apolipoprotein mimetic) or the fusion protein can be targeted to cells, tissues or organs it would otherwise not or insufficiently reach, or it could be used to reduce off-target effects.

[0109] Therefore, in a first aspect, the invention relates to a fusion protein comprising, consisting essentially of, or consisting of an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that modulates (e.g. enhances or suppresses) an immune response.

[0110] In a second aspect the invention relates to a fusion protein comprising, consisting essentially of, or consisting of an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows, when fused to the apolipoprotein, the apolipoprotein to bind to a different target than it would bind when the apolipoprotein was not fused to the rerouting molecule and / or to bind to its intended target with a higher affinity.

[0111] In a third aspect the invention relates to a fusion protein comprising, consisting essentially of, or consisting of an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule and a rerouting molecule.

[0112] The fusion protein may be conveniently denoted as being a fusion protein of an apolipoprotein or an apolipoprotein mimetic with an immunomodulatory biomolecule and / or a rerouting molecule.

[0113] The fusion proteins may be used as such, meaning not as part of a lipoprotein, lipid or apolipoprotein lipid nanoparticle. In such way the fusion protein may serve as a carrier to deliver an immunomodulatory biomolecule to a target site. Alternatively, the rerouting molecule can be used to target the fusion protein to a specific site, such as a cell, tissue, or organ.

[0114] Apolipoproteins are proteins that bind 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 (amphipathic) properties, apolipoproteins and other amphipathic molecules (such as phospholipids) can surround the lipids, creating a lipoprotein particle that is itself water-soluble, and can thus be carried through water-based circulation (i.e. , blood, lymph). In addition to stabilizing lipoprotein structure and solubilizing the lipid component, apolipoproteins interact with lipoprotein receptors and lipid transport proteins, thereby participating in lipoprotein uptake and clearance. In lipid transport, apolipoproteins function as structural components of lipoprotein particles, ligands for cell-surface receptors and lipid transport proteins, and cofactors for enzymes. Different lipoprotein particles contain different classes of apolipoproteins, which influence their function. For example, apolipoprotein A1 (apoA1) is the major structural protein component of high-density lipoproteins (HDL), although it is present in other lipoproteins in smaller amounts, and HDL comprises other apolipoproteins.

[0115] It is envisioned that the invention is not limited to a particular type of apolipoprotein or apolipoprotein mimetic (e.g. ApoA1 , ApoB or ApoE). Therefore in an embodiment, the apolipoprotein of the fusion protein of the invention, such as the apolipoprotein of the fusion protein according to the first, second or third aspect of the invention is selected from apoA1 , ApoA-1 Milano, apoA2, apoA4, apoA5, apoB48, apoB100, apoC-l, apoC-ll, apoC-lll, apoC- IV, apoD, apoE, apoF, apoH, apoL1 , apoL2, apoL3, apoL4, apoL5, apoL6, apoLDI , apoM, apoO, apoOL, or combinations thereof, or a mimetic thereof. For example, the apolipoprotein may be selected from apoA1 , ApoA-1 Milano, apoA2, apoA4, apoA5, apoB48, apoB100, apoC-l, apoC-ll, apoC-lll, apoC-IV, apoE, apoL1 , apoL2, apoL3, apoL4, apoL5, apoL6, or combinations thereof or mimetics thereof. More preferably the apolipoprotein may be selected from apoA1 , apoA4, apoC3, apoD, apoE, apoL1 , apoL3, or combinations thereof or mimetics thereof. In other words, in particular embodiments, the apolipoprotein is an ApoA1 , ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3 or the apolipoprotein mimetic is a mimetic of an ApoA1 , ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3. Even more preferably, the apolipoprotein is ApoA1.

[0116] In particular embodiments, the apolipoprotein may also be an apolipoprotein fragment. Preferably, the apolipoprotein fragment retains the biological activity of the apolipoprotein, such as the ability of the apolipoprotein to integrate into a lipid nanoparticle or to target an immunomodulatory biomolecule to a target, such as to the myeloid compartment. In particular embodiments, the apolipoprotein fragment comprises at least the ATP Binding Cassette Subfamily A Member 1 (ABCA1), ATP Binding Cassette Subfamily G Member 1 (ABCG1) and / or Scavenger receptor class B type 1 (SR-BI) binding regions of the full-length apolipoprotein, thereby allowing binding to a myeloid cell. In particular embodiments, the apolipoprotein fragment comprises at least the alpha helices of the full- length apolipoprotein. These helices are hydrophilic on one side (interact with aqueous environment) and hydrophobic (interacts with lipids in the particle) on the other side.

[0117] The term “fragment” as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and / or C-terminally truncated form of the peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., > 40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, or > 200, consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein.

[0118] In particular embodiments, the apolipoprotein fragment comprises the myeloid- binding portion of full-length apolipoprotein.

[0119] In particular embodiments, the apolipoprotein may also be an apolipoprotein mutant comprising a mutation that allows chemical conjugation of the apolipoprotein to an immunomodulatory biomolecule and / or a rerouting molecule. In particular embodiments, the apolipoprotein may also be an apolipoprotein mutant comprising a serine to cysteine substitution, such as the ApoA1 mutant as defined by SEQ ID NO: 1 , 7, 9 or 11 as described elsewhere herein.

[0120] Peptide sequences for the different proteins described herein, or nucleic acid sequences for the genes encoding the different proteins described herein, are readily available to the skilled person, for example from the LICSC Genome Browser (http: / / genome.ucsc.edu / ), Ensembl genome browser (https: / / www.ensembl.org) and NCBI (https: / / www.ncbi.nlm.nih.gov / protein). Consensus sequences for different proteins or genes are readily derived from these sources, although it is understood a certain variation may be present due to but not limited to genetic variation and multiple splice variants of the gene. Therefore, when referring to a specific protein, this should be interpreted to encompass sequence variations due to genetic variation and splice variants. Therefore, when used herein when referring to a certain protein, this should be interpreted as the corresponding consensus protein sequence as retrieved from the Ensembl genome browser, or the consensus nucleic acid (gene) sequence as retrieved from the Ensembl genome browser, or a protein sequence 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence as retrieved from the Ensembl genome browser, or a gene sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus gene sequence as retrieved from the Ensembl genome browser, or a nucleic acid sequence encoding a protein 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence as retrieved from the Ensembl genome browser.

[0121] Apolipoprotein mimetics are synthetic peptides or proteins that mimic the function or structure of apolipoproteins. Several apolipoprotein mimetics are known and for example Wolska et al. (Cells. 2021 Mar; 10(3): 597., incorporated by reference in its entirety) review different apoA1 , apoE and apoC-ll mimetics described in the literature. For example, ApoA1 mimetic peptides have largely been designed based on their ability to efflux cholesterol from cells. As this process has not been shown to depend upon a specific protein-protein interaction, most apoA1 mimetic peptides are simply just amphipathic helices and, in fact, many have no primary amino acid homology to apoA1. Exemplary ApoA1 mimetics are ApoA1 mimetic 18A, ApoA1 mimetic 2F and ApoA1 mimetic 37pA, which are represented by the peptides sequences corresponding to SEQ ID Nos 51 , 52 and 53.

[0122] For example, apoE has several putative atheroprotective functions, many different types of apoE-based peptides have been reported. One of the main goals in the design of these peptides is to facilitate the hepatic clearance of apoB-containing lipoproteins. As apoE can only bind to its receptor when bound to lipids, these peptides usually have not only the receptor-binding motif from the N-terminal domain of apoE, but also a lipid-binding region based on the C-terminal domain of apoE or some other sequence.

[0123] For example apoC-ll mimetics have been described either based on a shortened first helix (18A) linked to the LPL-activation domain of apoC-ll, or mimetics where both the first and second helix are based on the native apoC-ll helices with amino acid substitutions to enhance bihelical binding to lipoproteins.

[0124] Therefore, when used herein, an apolipoprotein mimetic refers to a synthetic protein or peptide which shares a structural and / or functional feature with the respective apolipoprotein. For example, the shared structural feature may be a primary, secondary or tertiary peptide structure such as the peptide sequence, presence of structures such as an alpha helix or beta sheet or three-dimensional structure of the peptide, or the functional feature may be a similarity in binding to a certain target such as a receptor. Preferably the apolipoprotein is capable of binding lipids, more preferably forming lipid particles, in a similar manner as the corresponding apolipoprotein.

[0125] In particular embodiments, the apolipoprotein mimetic is able to bind to a myeloid cell to the same or a similar extent as the respective apolipoprotein. For example, the apolipoprotein mimetic of ApoA1 is able to bind to a myeloid cell to the same or a similar extent as ApoA1 . In an embodiment the fusion protein is a fusion protein of ApoA1 (or a mutant thereof), preferably human ApoA1 , with an immunomodulatory biomolecule and / or a rerouting molecule.

[0126] By means of an example, the human ApoA1 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.

[0127] In particular embodiments, the ApoA1 is wild-type ApoA1 (e.g. as derived from the human precursor of ApoA1 as defined by SEQ ID NO. 1 , of which the first 18 amino acids form the signal peptide) or an ApoA1 mutant (e.g. as defined by SEQ ID NO. 7, 9 or 11). In particular embodiments, the ApoA1 is wild-type human ApoA1 as defined by SEQ ID NO. 78.

[0128] For example, in order to chemically conjugate apoA1 to an immunomodulatory biomolecule and / or a rerouting molecule, a reactive handle is typically needed. Therefore, apoA1 mutants comprising a cysteine in the place of a serine at position 147 (e.g. as defined by SEQ ID NO. 9) or 279 (e.g. as defined by SEQ ID NO: 11) could be useful to prepare chemically conjugated ApoA1 fusion proteins.

[0129] In an embodiment, the ApoA1 is a peptide with, or the ApoA1 comprises, consists essentially of or consists of, an amino acid sequence 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. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11 , or comprising, consisting essentially of or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12.

[0130] It is noted that the ApoA1 sequence as defined by SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO. 11 comprise N-terminally the amino acid sequence GLVPRGSIDD (SEQ ID NO. 79), which is a thrombin cleavage site. For example, the ApoA1 sequence as defined by SEQ ID NO. 5 comprises N-terminally a 6His tag followed by the amino acid sequence GLVPRGSIDD (SEQ ID NO. 79). Here, the thrombin cleavage site as could be used to remove the N-terminal His tag from the peptide.

[0131] In an embodiment, the ApoA1 is a peptide with, or the ApoA1 comprises, consists essentially of or consists of, an amino acid sequence 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, wherein SEQ ID NO. 7 comprises a cysteine at position 7 of SEQ ID NO. 7. Such ApoA1 mutant may be referred to herein as “S14C” mutant.

[0132] In an embodiment, the ApoA1 is a peptide with, or the ApoA1 comprises, consists essentially of or consists of, an amino acid sequence 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, wherein SEQ ID NO. 7 comprises a cysteine at position 150 of SEQ ID NO. 9. Such ApoA1 mutant may be referred to herein as “S147C” or “S157C” mutant.

[0133] In an embodiment, the ApoA1 is a peptide with, or the ApoA1 comprises, consists essentially of or consists of, an amino acid sequence 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. 7 comprises a cysteine at position 239 of SEQ ID NO. 11. Such ApoA1 mutant may be referred to herein as “S279C” or “S239C” mutant.

[0134] In an embodiment the fusion protein is a fusion protein of an ApoA1 mimetic with an immunomodulatory biomolecule and / or a rerouting molecule. In an embodiment, the ApoA1 mimetic is a peptide with or the ApoA1 mimetic comprises, consists essentially of or consists of, an amino acid sequence 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. 51 , SEQ ID NO. 52, or SEQ ID NO. 53.

[0135] In an embodiment the fusion protein is a fusion protein of ApoE with an immunomodulatory biomolecule and / or a rerouting molecule. In an embodiment, the ApoE is a peptide with or the ApoE comprises, consists essentially of or consists of, an amino acid sequence 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. 13, SEQ ID NO. 15, SEQ ID NO. 17 or SEQ ID NO. 19, or comprising, consisting essentially of or consisting of, an amino acid sequence encoded by a nucleic acid with a sequence 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. 14, SEQ ID NO. 16, SEQ ID NO. 18 or SEQ ID NO. 20.

[0136] As described elsewhere herein, the fusion protein of ApoA1 may be a fusion protein of ApoA1 and a cytokine, such as IL-1 p (IL-1 B), IL-2, IL-4 or IL-38, preferably IL-4.

[0137] Accordingly, in an embodiment the fusion protein is a fusion protein of ApoA1 (including mutants thereof) with interleukin (IL)-1 B, preferably human IL-1 B. In an embodiment, the ApoA1-IL1 B fusion protein comprises or consists of a polypeptide with an amino acid sequence 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. 21 or 82, or comprising or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 22 or 83.

[0138] In an embodiment the fusion protein is a fusion protein of ApoA1 (including mutants thereof) with IL-2, preferably human IL-2.

[0139] In an embodiment, the ApoA1-IL2 fusion protein comprises or consists of a polypeptide with an amino acid sequence 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. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 58 or SEQ ID NO. 60, or comprising or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 59 or SEQ ID NO. 61.

[0140] In an embodiment the fusion protein is a fusion protein of ApoA1 (including mutants thereof) with IL-4, preferably human IL-4.

[0141] In an embodiment, the ApoA1-IL4 fusion protein comprises or consists of a polypeptide with an amino acid sequence 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. 35, SEQ ID NO. 37 or SEQ ID NO. 39, or comprising or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 36, SEQ ID NO. 38 or SEQ ID NO. 40.

[0142] In an embodiment the fusion protein is a fusion protein of ApoA1 (including mutants thereof) with IL-38 (also known as IL1 F10), preferably human IL-38.

[0143] In an embodiment, the ApoA1-IL38 fusion protein comprises or consists of a polypeptide with an amino acid sequence 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. 80 or 84, or comprising or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 81 or 85.

[0144] As described elsewhere herein, the ApoA1 may also be fused to a rerouting molecule, such as a rerouting molecule capable of binding to lymphocytes, preferably T cells, more preferably CD8+ T cells. For example, the rerouting molecule may be a VHHCD8 as described in Woodham A.W. et al., Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses, Cancer Immunology Research, 2018, Vol. 6, issue 7, and comprising an amino acid sequence as shown in Supplemental Table 1 of said reference.

[0145] In an embodiment the fusion protein is a fusion protein of ApoA1 (including mutants thereof) with VHH8CD8.

[0146] In an embodiment, the VHHCD8-apoA1 fusion protein comprises or consists of a polypeptide with an amino acid sequence 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. 54 or 56, or comprising or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 55 or 57.

[0147] When used herein, fusion protein when referring to an apolipoprotein fusion protein should be interpreted as an apolipoprotein or apolipoprotein mimetic and covalently attached thereto an immunomodulatory biomolecule and / or a rerouting molecule. The covalent attachment may be due to the in frame coding of a peptide or protein sequence by the nucleotide sequence that encodes the fusion protein. Alternatively, the covalent attachment may be due to covalent linkage of the immunomodulatory biomolecule and / or a rerouting molecule to the apolipoprotein, for example via a sulfur bond such as a thioether bond, formed at a cysteine residue of the apolipoprotein. It is understood that the immunomodulatory biomolecule and / or the rerouting molecule and / or the apolipoprotein (or mimetic thereof) may include the site-specific incorporation of non-natural amino acids such as para-azidophenylalanine, which can be used in subsequent (strain-promoted) “click” (conjugation) reactions with alkyne modified reagents.

[0148] In particular embodiments, the fusion protein comprises a linker, such as a flexible linker, between the apolipoprotein or apolipoprotein mimetic and said immunomodulatory biomolecule and / or rerouting molecule. The linker may be a glycine-serine linker, such as a (GGS)n-linker, wherein n is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably a (GGS)4-linker. In particular embodiments, the fusion protein may comprise one or more tags, such as at the N- and / or C-terminal end of the fusion protein. The one or more tags, such as a 6His-tag or strep-tag may allow purification of the fusion protein.

[0149] Said immunomodulatory biomolecule and / or rerouting molecule may be covalently attached to any portion of the apolipoprotein or apolipoprotein mimetic. A linker, such as a flexible linker, may be used to allow such covalent attachment.

[0150] In particular embodiments, said immunomodulatory biomolecule is located N- or C- terminally of said apolipoprotein or apolipoprotein mimetic in said fusion protein.

[0151] In particular embodiments, said rerouting molecule is located N- or C-terminally of said apolipoprotein or apolipoprotein mimetic in said fusion protein.

[0152] In particular embodiments, wherein said fusion protein comprises an immunomodulatory biomolecule and a rerouting molecule, said apolipoprotein or apolipoprotein mimetic may be located N- or C-terminally of both said immunomodulatory biomolecule and said rerouting molecule or may be located in between said immunomodulatory biomolecule and said rerouting molecule (e.g. with the immunomodulatory biomolecule N- or C-terminally of the apolipoprotein or apolipoprotein mimetic).

[0153] When used herein an immune response refers to a reaction which occurs within an organism by the immune system. The immune response may be the innate immune response or the adaptive immune response or the complement immune system. Immune responses when referred herein include but are not limited to: secretion of a pro- inflammatory molecule; secretion of an anti-inflammatory molecule; phagocytosis; antibody production, presentation or secretion; antigen presentation; activation, proliferation, suppression or differentiation of an immune cell; binding of an immune cell to a target, initiation of an immune related cellular signaling cascade, or combinations thereof.

[0154] When used herein an immunomodulatory biomolecule refers to a molecule that enhances or suppresses an immune response. The biomolecule may interfere with, change, stimulate or suppress the innate immune response or the adaptive immune response or the complement immune system. The biomolecule may be a protein, peptide or organic compound. The compound may be isolated or derived from a natural source, cloned or synthesized. Non limiting examples of immunomodulatory biomolecules are cytokines, chemokines, hormones, growth factors and hematopoietic growth factors and antibodies (or antigen binding fragments thereof), although the skilled person may be aware of additional immunomodulatory molecules. Therefore, in an embodiment the immunomodulatory biomolecule is selected from a cytokine, a chemokine, a hormone, a growth factor, a hematopoietic growth factor, an antibody or antigen binding fragment thereof, or combinations thereof.

[0155] In an embodiment the immunomodulatory biomolecule may be a cytokine. Cytokines are known to the skilled person to be small proteins of approximately 5 to 20 kDa and are important in cell signaling. For example, a cytokine may refer to: a four-alpha-helix bundle family cytokine, such as the interleukin (IL)-2 subfamily, the interferon (IFN) subfamily or the IL-10 subfamily; the IL-1 family; the cysteine knot cytokines such as the transforming growth factor (TGF) beta family; the IL-17 family. Therefore, the cytokine is preferably selected from IL18, interleukin 18 binding protein (IL18BP), interleukin 1 alpha (IL1A), interleukin 1 beta (IL1 B), interleukin-1 family member 10 (IL1 F10), IL1 F3 / IL1 RA, IL1 F5, IL1 F6, IL1 F7, IL1 F8, interleukin 1 receptor like 2 (IL1 RL2), IL1 F9, IL33, B-cell activating factor (BAFF), 4-1 BBL, TNF super family member 8 (TNFSF8), cluster of differentiation 40 (CD40) ligand (CD40LG), CD70, CD95L / CD178, ectodysplasin-A1 (EDA-A1), TNFSF14, lymphotoxin alpha (LTA) / TNFB, lymphotoxin beta (LTB), TNFalpha, TNFSF10, TNFSF11 , TNFSF12, TNFSF13, TNFSF15, TNFSF4, interferon alpha (IFNA)1 , IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, interferon beta (IFNB) 1 , interferon epsilon (IFNE), interferon gamma (IFNG), interferon zeta (IFNZ), IFNA8, IFNA5 / IFNaG, IFNw / IFNW1 , cardiotrophin like cytokine factor 1 (CLCF1), ciliary neurotrophic factor (CNTF), IL11 , IL31 , IL6, Leptin, leukemia inhibitory factor (LIF), oncostatin M (OSM), IL10, IL19, IL20, IL22, IL24, IL28B, IL28A, IL29, TGF-beta 1 / TGFB1 , TGF-beta 2 / TGFB2, TGF-beta 3 / 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 TGFbeta family, or the IL-17 family, or combinations thereof, more preferably wherein the cytokine is selected from I L-1 p , IL-2, IL- 4, IL-38, or combinations thereof. More preferably, wherein the cytokine is IL-4.

[0156] In particular embodiments, the cytokine is a human cytokine.

[0157] In an embodiment the fusion protein is a fusion protein of an apolipoprotein or a mimetic thereof with IL-1 B. IL-1 B is also known as IL1 B, IL-1 p, IL1 F2 or interleukin 1 beta, and is a cytokine protein that in humans is encoded by the IL1B gene. By means of an example, the human IL-1 B protein sequence is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_000567.1 (proprotein), and Uniprot (www.uniprot.org) accession number P01584.2.

[0158] In an embodiment, the I L-1 B is a peptide with, or comprising, consisting essentially of or consisting of, an amino acid sequence 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. 45, or comprising, consisting essentially of or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 46.

[0159] In an embodiment the fusion protein is a fusion protein of an apolipoprotein or a mimetic thereof with IL-2. IL-2 is also known as IL2 TCGF, lymphokine or interleukin 2, and is an interleukin that regulates the activities of leukocytes that are responsible for immunity. By means of an example, the human IL-2 protein sequence is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_000577.2, and Uniprot (www.uniprot.org) accession number P60568.1.

[0160] In particular embodiments, IL-2 may be wild-type IL-2 or an IL-2 mutant.

[0161] In particular embodiments, IL-2 may comprise one or more amino acid substitutions as described in Wang A. et al., Science, Site-specific mutagenesis of the human interleukin- 2 gene: structure-function analysis of the cysteine residues., 1984, Vol. 224, No. 4656 or Klein C. et al., Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines, Oncolmmunology, 2017, Volume 6, issue 3, e1277306.

[0162] In particular embodiments, IL-2 may comprise one or more, such as one, two, three or all four of the following amino acid substitutions: F42A, Y45A, L72G and / or C125A (the positions of the substitutions are indicated vis-a-vis the sequence of the mature human IL- 2 protein). It is noted that the first 20 amino acids of the human IL-2 amino acid sequence as defined by SEQ ID NO. 47 (human IL-2 precursor) represent the signal peptide, accordingly, IL-2 may comprise one or more, such as one, two, three or all four of the following amino acid substitutions, F62A, Y65A, L92G and / or C145A, wherein the positions are indicated vis-a-vis SEQ ID NO. 47.

[0163] In an embodiment, the IL-2 is a peptide with, or comprising, consisting essentially of or consisting of, an amino acid sequence 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. 47, or comprising, consisting essentially of or consisting of, an amino acid sequence encoded by a nucleic acid with a sequence 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. 48.

[0164] In an embodiment the fusion protein is a fusion protein of an apolipoprotein or a mimetic thereof with IL-4. IL-4 is also known as BSF-1 , IL4 or interleukin 4, is a cytokine that induces differentiation of naive helper T cells. By means of an example, the human IL- 4 protein sequence is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_000580.1 (isoform 1 precursor), and Uniprot (www.uniprot.org) accession number P05112.1.

[0165] In an embodiment, the IL-4 is a peptide with, or comprising, consisting essentially of or consisting of, an amino acid sequence 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. 43, or comprising, consisting essentially of or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 44.

[0166] In a further embodiment, the fusion protein is a fusion protein of ApoA1 or a mimetic thereof with IL-4.

[0167] In an embodiment the fusion protein is a fusion protein of an apolipoprotein or a mimetic thereof with IL-38. IL-38 is also known as IL38, IL1 F10 interleukin 38, interleukin 1 family member 10 or IL1-theta, and is a protein that in humans is encoded by the IL1 F10 gene. By means of an example, the human IL-38 protein sequence is annotated under NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_775184.1 , and Uniprot (www.uniprot.org) accession number Q8WWZ1 .1 . In an embodiment, the IL-38 is a peptide with, or comprising, consisting essentially of or consisting of, an amino acid sequence 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. 49, or comprising, consisting essentially of or consisting of an amino acid sequence encoded by a nucleic acid with a sequence 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. 50.

[0168] In an embodiment the immunomodulatory biomolecule may be a chemokine. The chemokine is preferably selected from chemokine (C-C motif) ligand 1 (CCL1) / TCA3, CCL11 , CCL12 / MCP-5, CCL 13 / M CP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1 , CCL20, CCL21 , CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, C-C motif chemokine ligand 3 like 3 (CCL3L3), CCL4, CCL4L1 / LAG- 1 , CCL5, CCL6, CCL7, CCL8, CCL9, C-X3-C motif chemokine ligand 1 (CX3CL1), C-X-C motif chemokine ligand 1 (CXCL1), CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, X-C motif chemokine ligand 1 (XCL1), XCL2, FAM19A1 , FAM19A2, FAM19A3, FAM19A4 and FAM19A5. In an alternative embodiment the chemokine is selected from a CC chemokine, a CXC chemokine, a C chemokine, a CX3C chemokine or combinations thereof.

[0169] In an embodiment the immunomodulatory biomolecule may be a hormone. Hormones are known to the skilled person to be signaling molecules in multicellular organisms, that are transported to distant organs to regulate physiology and behavior. In an embodiment the hormone is selected from Adrenaline (also known as epinephrine), Melatonin, Noradrenaline (also known as norepinephrine), Triiodothyronine, Thyroxine, Dopamine, Prostaglandins, Leukotrienes, Prostacyclin, Thromboxane, Amylin (also known as Islet Amyloid Polypeptide), Anti-Mullerian hormone (also known as Mullerian-inhibiting factor / hormone), Adiponectin, Adrenocorticotropic hormone (also known as corticotropin), Angiotensinogen, Angiotensin, Antidiuretic hormone (also known as vasopressin, arginine vasopressin), Atrial natriuretic peptide (also known as atriopeptin), Brain natriuretic peptide, Calcitonin, Cholecystokinin, Corticotropin-releasing hormone, Cortistatin, Enkephalin, Endothelin, Erythropoietin, Follicle-stimulating hormone, Galanin, Gastric inhibitory polypeptide, Gastrin, Ghrelin, 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 known as somatomedin), Leptin, Lipotropin, Luteinizing hormone, Melanocyte stimulating hormone, Motilin, Orexin, Osteocalcin, Oxytocin (also known as pitocin), Pancreatic polypeptide, Parathyroid hormone, Pituitary adenylate cyclase-activating peptide, Prolactin (also known as leuteotropic hormone), Prolactin-releasing hormone, Relaxin, Renin, Secretin, Somatostatin (also known as growth hormone-inhibiting hormone or growth hormone release-inhibiting hormone or somatotropin release-inhibiting factor or somatotropin release-inhibiting hormone), Thrombopoietin, Thyroid-stimulating hormone (also known as thyrotropin), Thyrotropin-releasing hormone, Vasoactive intestinal peptide, Guanylin or Uroguanylin.

[0170] In an embodiment the immunomodulatory biomolecule may be a growth factor. Growth factors are known to the skilled person as naturally occurring substances capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. In an embodiment the growth factor is selected from Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Ciliary neurotrophic factor (CNTF), Leukemia inhibitory factor (LIF), lnterleukin-6 (IL-6), Macrophage colonystimulating 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), Foetal Bovine Somatotrophin (FBS), 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), Migrationstimulating 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 factors, Transforming growth factor alpha (TGF-a), Transforming growth factor beta (TGF-P), Tumor necrosis factor-alpha (TNF-a), 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, Ephrins, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, Neurotrophin, PGF, PDGF, RNLS, TCGF, TGF, TNF, WNT or combinations thereof.

[0171] In an embodiment the immunomodulatory biomolecule is a hematopoietic growth factor. In an embodiment, the hematopoietic growth factor is selected from IL-3, colony stimulating factor 1 (CSF-1 (M-CSF)), granulocyte-macrophage (GM)-CSF, granulocyte (G)- CSF, a member of the IL-12 family of interleukins or erythropoietin or combinations thereof.

[0172] When used herein the term target refers to the object the apolipoprotein (or apolipoprotein mimetic) or fusion protein preferentially binds to. A target may refer to a receptor or cell surface molecule such as a protein or proteoglycan, a cell, a cell type, a tissue or tissue type or an organ. In particular embodiments, the fusion protein or lipid nanoparticle comprising said fusion protein is capable of binding to a myeloid cell. This may for example be a result of the inherent nature of apolipoproteins, such as ApoA1 , to target myeloid cells. When used herein, the terms myeloid cell refers to blood cells that are derived from a progenitor cell for granulocytes, monocytes, erythrocytes, or platelets. Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. The term myeloid compartment, when used herein, refers to the totality of myeloid cells in an organism.

[0173] When used herein a rerouting molecule refers to a molecule that allows, when fused to a protein such as an apolipoprotein or apolipoprotein- immunomodulatory biomolecule fusion protein, the protein to bind to a different target than it would bind when the protein was not fused to the rerouting molecule (or in other words, a different target than that to which it would have innately bound), and / or to bind to its intended target with a higher affinity. Binding to a different target may also encompass binding to a particular subset of target cells, including binding to a particular subset of a set of cells which would normally be bound by the apolipoprotein or apolipoprotein- immunomodulatory biomolecule fusion protein. Non limiting examples of rerouting molecules are an antibody or antigen binding fragment thereof or an antibody fragment, a rerouting peptide or a rerouting protein, preferably wherein the rerouting peptide or rerouting protein is a ligand of a receptor present on the target.

[0174] It is appreciated that apolipoproteins bind to specific ligands. For example, it is thought that the different apolipoproteins found in different lipoproteins (e.g. HDL, LDL, VLDL, etc.) are responsible for differences in targeting and binding and thus function of the lipoproteins. Without wishing to be bound by theory, it is thought that part of the apolipoprotein has an amphipathic nature and is responsible, together with phospholipids and / or sterols to bind lipids in an aqueous environment, while different parts of the molecule are responsible for interacting with other molecules, e.g. binding to protein receptors. It is further assumed that apolipoproteins may circulate also as proteins, meaning not as lipoproteins. Therefore, the possibility to modify binding affinity of the apolipoprotein (by fusion to a rerouting molecule) provides interesting opportunities, as it allows to fine-tune targeting or binding of the apolipoprotein. Several applications are envisioned for such a fusion protein:

[0175] First the rerouting molecule may simply be used to reroute the apolipoprotein or lipoprotein, for example to make changes in the lipid homeostasis. For example, one could envision that LDL or HDL values in the blood of a subject could be altered by using apolipoprotein fusion proteins with a rerouting molecule. This could potentially be exploited for treatment of lipid disorders such as high blood cholesterol levels.

[0176] Second it may be used to reroute a lipoprotein (lipid nanoparticle) with a payload to a predetermined target. Lipoproteins or lipid nanoparticles pose interesting methods of carrying a payload such as a pharmaceutical compound. It allows delivery of lipophilic compounds through blood, as the compound may be dissolved in the lipid core of a lipoprotein I lipid nanoparticle. An additional advantage is that the lipoprotein consists of naturally occurring compounds and thus is seen as native by the immune system, avoiding triggering an immune response by the pharmaceutical compound.

[0177] Third, it may be combined with an immunomodulatory biomolecule (either as a single fusion protein (apolipoprotein fused to both a rerouting molecule and immunomodulatory biomolecule) or as a combination of two distinct fusion proteins), and as such allow the rerouting of the immunomodulatory biomolecule. As described above, apolipoprotein can essentially serve as a carrier for an immunomodulatory biomolecule. Advantages are that it greatly reduces clearing of the immunomodulatory biomolecule, making it feasible to use immunomodulatory biomolecules that are easily cleared from the blood in a therapeutic setting (e.g. cytokines). One issue that might arise is that an immunomodulatory biomolecule / apolipoprotein fusion protein does not arrive at the intended target for the immunomodulatory biomolecule (i.e. the site, cell, tissue or organ where it is intended to exert its effect). This may be solved by further including a rerouting molecule.

[0178] Fourth, the fusion protein comprising the apolipoprotein or apolipoprotein mimetic and rerouting molecule allows to prepare a lipid nanoparticle, wherein the rerouting molecule is exposed to the environment (i.e. aqueous environment) surrounding said apolipoprotein lipid nanoparticle, as a result of its fusion to the apolipoprotein.

[0179] Data generated by the inventors suggests that using a rerouting molecule the apolipoprotein fusion protein can successfully be rerouted to a different target. Therefore, in an embodiment, the rerouting molecule is selected from an antibody or an antigen binding fragment thereof, a rerouting peptide or a rerouting protein, preferably wherein the rerouting peptide or rerouting protein is a ligand of a receptor present on the target.

[0180] In an embodiment the rerouting molecule may be an antibody or an antigen binding fragment thereof. It is envisioned that any type of antigen binding molecule can in principle be used as a rerouting molecule in the fusion protein according to the invention.

[0181] The antibody may be any immunologic binding agent, such as a whole antibody, including without limitation a chimeric, humanized, human, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion proteins, conjugates, fragments, or derivatives thereof that contain one or more domains that selectively bind to an antigen of interest. The antibody may be a whole immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an immunologically effective fragment of any of these. Thus, the antibody may encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., 2-, 3- or more-valent) and / or multispecific antibodies (e.g., bi- or more-specific antibodies) formed from at least two intact antibodies, and antibody fragments insofar they exhibit the desired biological activity (particularly, ability to specifically bind an antigen of interest), as well as multivalent and / or multi-specific composites of such fragments.

[0182] The terms “specifically bind” or “specifically interact” as used throughout this specification mean that an agent binds to or influences one or more desired molecules or analytes substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The terms do not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g., at least about 1000-fold or more greater, at least about 1x104-fold or more greater, or at least about 1x105-fold or more greater, than its affinity for a non-target molecule.

[0183] Therefore, in an embodiment, the antibody or antigen binding fragment thereof is selected from a Fragment antigen-binding region (Fab), a Fab2, a single-chain variable fragment (scFv), a scFv-Fc, a dAb-Fc, a free light chain antibody, a half antibody, a bispecific Fab2, a Faba, a trispecific Fab3 a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an immunoglobulin new antigen receptor (IgNAR), a monovalent IgG, a VhH or a variable domain of new antigen receptor (VNAR). The antibody or antigen binding fragment may also be a designed antigen binding protein such as but not limited to affibodies, FN3 domains, DARPins or de novo designed protein receptors. It is appreciated that antibodies or antigen binding fragments thereof with a lower molecular weight are preferred due to their reduced size, therefore in a preferred embodiment the antibody or antigen binding fragment thereof is a Fab, scFv, single domain antibody, VhH or VNAR.

[0184] In an embodiment the rerouting molecule may be a rerouting peptide. Non limiting examples of rerouting peptides are receptor binding peptides, ligand mimicking peptides.

[0185] Therefore, in an embodiment, the rerouting peptide is selected from programmed cell death protein 1 (PD1) or signal-regulatory protein alpha (SIRPa). It is however understood that any peptide with binding specificity to a cell surface receptor could be used as a rerouting molecule.

[0186] In an embodiment the rerouting molecule may be a rerouting protein such as a receptor ligand, a receptor, or interacting protein. Therefore, in an embodiment, the rerouting protein is selected from CD40L or GP120. CD40L can be used to target cells expressing the CD40 receptor. GP120 can be used to bind directly to the CD4 T-Cell coreceptor. It is however understood that any protein with binding specificity to a cell surface receptor could be used as a rerouting molecule.

[0187] When used herein, the term lipoprotein refers to a particle, generally a nanoparticle, of at least one apolipoprotein and lipid molecules, dispersed or dissolved in an aqueous environment.

[0188] When used herein, the term rerouting refers to targeting the fusion protein to a different target it would normally bind to, or preventing the binding of the regular target of the apolipoprotein or preventing off-target binding. For example, apoA1 is known to bind receptors on myeloid cells, thus the rerouting molecule may be used to bind different cells or prevent binding of myeloid cells. In other words, the rerouting molecule may bind, preferably specifically bind, to non-myeloid cells.

[0189] Accordingly, in particular embodiments, the rerouting molecule is capable of binding, preferably specifically binding, to cells that are not myeloid cells, but that may differentiate into myeloid cells, such as a hematopoietic stem and progenitor cell (HSPC), like a hematopoietic stem cell (HSC), a multipotent progenitor (MPP), or a common myeloid progenitor cell (CMP).

[0190] In particular embodiments, the rerouting molecule is capable of binding, preferably specifically binding, to a non-myeloid cell, such as a non-myeloid immune cell or an endothelial cell. Endothelial cells may be targeted by use of a rerouting molecule capable of binding to a surface marker of endothelial cells. For example, endothelial cells may be targeted by use of a rerouting molecule capable of binding to Factor VI I l-related antigen such as Factor VIII, a rerouting molecule capable of binding to CD31 / PECAM-1 such as CD31 , a rerouting molecule capable of binding to Angiotensin-converting enzyme (ACE / CD143) such as angiotensin, a rerouting molecule capable of binding to CD34 such as L-selectin or a rerouting molecule capable of binding to endoglin (CD105).

[0191] In particular embodiments, 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.

[0192] In particular embodiments, if the target cell is a T cell, the rerouting molecule may be an antibody or antigen binding fragment thereof binding, preferably specifically binding, to CD8. For example, the rerouting molecule may be a VHHCD8 as described in Woodham A.W. et al., Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses, Cancer Immunology Research, 2018, Vol. 6, issue 7, and comprising an amino acid sequence as shown in Supplemental Table 1 of said reference.

[0193] For example, CD8-targeted apolipoprotein nanoparticles as described herein can also be used to generate chimeric antigen receptor (CAR) T cells in vivo. In particular, the CD8-targeted apolipoprotein nanoparticles can be used to deliver plasmid DNA (pDNA), linear or circular mRNA, and / or gene editing components encoding instructions to reprogram T cells in vivo. For example, targeted nucleases can be used to introduce a nucleic acid in a cell. For example, the targeted nucleases include, but are not limited to, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZNF), clustered regulatory interspaced short palindromic repeats (CRISPR), CRISPR / Cas9, CRISPR / CPFL and combinations thereof, such as for example 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 chimeric antigen receptors (CAR) can be delivered into T cells that enables them to recognize and kill tumor cells with a cognate ligand. In another particular example, pDNA or RNA encoding chimeric antigen receptors (CAR) can be delivered into T cells that enables them to recognize and eliminate pathologic cells such as activated fibroblasts in fibrotic disorders.

[0194] CD8-targeted apolipoprotein nanoparticles as described herein can also be used to modify CD8+ T cell function. For example, CD8+ T cell function may be modified through modifying cytokine-cytokine receptor interactions using CD8-targeted apolipoprotein nanoparticles as described to deliver plasmid DNA (pDNA), linear or circular mRNA, and / or gene editing components as mentioned above encoding instructions for cytokine production in T cells. In a particular example, pDNA or mRNA encoding cytokines such as IFN-y, IL-2, IL-10, IL-12, IL-15 may be delivered into T cells to enable them to secrete cytokines. This may result in reprogramming an immunosuppressive tumor microenvironment into an antitumor phenotype. In another particular example, pDNA or mRNA encoding cytokines receptors such as IFN-yR, IL-2R, IL-12R, or IL-15R may be delivered into T cells. CD8+ T cell function may also be modified through regulating immune checkpoints, e.g. CD8+ T cell function may be modified through downregulating immune checkpoints using CD8-targeted apolipoprotein nanoparticles as described to deliver antisense oligonucleotides, small interfering RNA (siRNA), mRNA, and / or gene editing components as mentioned above encoding instructions to downregulate immune checkpoints in T cells. In a particular example, siRNA or gene editing components as mentioned above targeting PD1 , CTLA4, SHP-2, LAG3, or TIM-3 may b delivered to T cells for downregulation of checkpoint molecules that inhibit cytotoxic T cell function in cancer.

[0195] In yet another example, CD8-targeted apolipoprotein nanoparticles as described herein can be used for re-polarization of pathogenic CD8+ T cells in autoimmunity towards a tolerogenic phenotype. In particular, the CD8-targeted apolipoprotein nanoparticles can be used to deliver antisense oligonucleotides, small interfering RNA (siRNA), mRNA or gene editing components as mentioned above encoding instructions to re-polarize pathogenic T cells. In a particular example, mRNA encoding FOXP3 may be delivered into T cells to alter their phenotype into regulatory T cells (T regs). In another particular example, CD8+ IL17+ T cells may be re-polarized via delivery of siRNA targeting IL-17 or the RORyt transcription factor.

[0196] In particular embodiments, if the target cell is a T and / or B cell, the rerouting molecule or rerouting peptide may be PD1 , CD40L or GP120.

[0197] The term rerouting may also encompass increasing the specificity of a fusion protein to a particular target, such as a particular target cell. For example, apoA1 is known to bind receptors on myeloid cells, thus the rerouting molecule may be used to bind a particular subtype of myeloid cells.

[0198] Accordingly, in particular embodiments, the rerouting molecule is capable of binding, preferably specifically binding, to a myeloid cell selected from the group consisting of megakaryocyte, eosinophil, basophil, erythrocyte, monocyte such as dendritic cell or macrophage, and a neutrophil. For example, SIRPalpha as rerouting molecule may allow to target immunosuppressive macrophages.

[0199] It is envisioned the apolipoproteins fusion proteins as described herein may be used as proteins or as lipid nanoparticles. As described above apolipoprotein may circulate as such (meaning not incorporated in a lipoprotein or lipid nanoparticle). This application may be suitable to deliver an immunomodulatory biomolecule, for example a cytokine like IL-4, to a target site. It is known that apolipoproteins can circulate as proteins but may also form lipoproteins in situ. It may however also be advantageous to include the fusion protein in a lipid nanoparticle. Therefore, in an aspect the invention relates to a lipid nanoparticle comprising one or more fusion proteins as herein, the lipid nanoparticle further comprising phospholipids, and optionally sterols. Lipid nanoparticles are conveniently denoted herein as “apolipoprotein lipid nanoparticles”. The fusion protein may be a fusion protein of an apolipoprotein with an immunomodulatory biomolecule, or a fusion protein of an apolipoprotein with a rerouting molecule, or an apolipoprotein with an immunomodulatory biomolecule and a rerouting molecule or combinations thereof. The apolipoprotein may also be an apolipoprotein mimetic. Without being bound to any hypothesis, it is believed that the apolipoprotein or apolipoprotein mimetic may function as a scaffold to help the formation of the nanoparticle together with the phospholipids, and optionally sterols.

[0200] In an aspect of the invention, the lipid nanoparticle comprises a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule; and phospholipids.

[0201] In an aspect of the invention, the lipid nanoparticle comprises a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule; and phospholipids.

[0202] The apolipoprotein lipid nanoparticle may comprise both an immunomodulatory biomolecule and a rerouting molecule. This may be achieved by incorporating either one fusion protein comprising both the immunomodulatory biomolecule and the rerouting molecule or by incorporating two fusion proteins, one comprising the immunomodulatory biomolecule and one comprising the rerouting molecule, into the apolipoprotein lipid nanoparticle.

[0203] Accordingly, in an aspect of the invention, the lipid nanoparticle comprises a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule; and phospholipids.

[0204] In an aspect of the invention, the lipid nanoparticle comprises (i) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule; and phospholipids and (ii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule; and phospholipids.

[0205] When used herein a lipid nanoparticle (LNP) refers to an assembly of phospholipids and one or more apolipoproteins that is soluble in an aqueous solution. The particles may further comprise sterol and / or lipids (e.g. triglycerides). If the LNP comprises both sterol and lipids, the lipids are encapsulated by the phospholipids and sterols. Accordingly, the lipid nanoparticles thus differ in their structure from liposomes.

[0206] In particular embodiments, the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine and a phosphatidylglycerol or combinations thereof. In further particular embodiments, 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), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dilauroyl phosphatidylserine (DLPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), 1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), or combinations thereof.

[0207] In particular embodiments, the ratio of apolipoprotein (or apolipoprotein mimetic) (in case of two or more fusion proteins, cumulatively) to phospholipid based on percentage molar weight is between 1 :25 and 1 :400, more preferably between 1 :50 and 1 :200, even more preferably between 1 :75 and 1 :150. In particular embodiments, the ratio of fusion protein (in case of two or more fusion proteins, cumulatively) as taught herein to phospholipid based on percentage molar weight is between 1 :25 and 1 :400, more preferably between 1 :50 and 1 :200, even more preferably between 1 :75 and 1 :150.

[0208] In particular embodiments, the ratio of apolipoprotein (or apolipoprotein mimetic) (in case of two or more fusion proteins, cumulatively) to phospholipid based on weight is from 3:1 to 1 :100. In particular embodiments, the ratio of fusion protein (in case of two or more fusion proteins, cumulatively) as taught herein to phospholipid based on weight is from 3:1 to 1 :100.

[0209] Where a nanoparticle includes two or more distinct apolipoprotein components, such as an apolipoprotein and a apolipoprotein fusion protein, or two or more apolipoprotein fusion proteins and optionally one or more apolipoprotein, etc., the cumulative amount of these apolipoprotein components is taken into account when quantitative relationships to other constituents of the nanoparticle are discussed.

[0210] In particular embodiments, the sterol is selected from cholesterol, desmosterol, stigmasterol, p-sitosterol, ergosterol, hopanoids, hydroxysteroid, phytosterol, steroids, hydrogenated cholesterol, campesterol, zoosterol, or combinations thereof.

[0211] In certain embodiments, in apolipoprotein lipid nanoparticles as taught herein, such as in particular but without limitation nanoparticles not containing a nucleic acid: the amount of the fusion protein (in case of two or more fusion proteins, cumulatively) ranges from 0.05 to 2.0 mol%, such as from 0.1 to 1.0 mol%, for example from 0.2 to 0.7 mol%, or from 0.2 to 0.5 mol%, such as preferably about 0.3 mol%; and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 10 to 50 mol%, for example from 10 to 40 mol%, from 10 to 30 mol%, or from 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 the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 60 mol%, from 2.5 to 50 mol%, from 2.5 to 40 mol%, or from 2.5 to 30 mol%, or from 2.5 to 20 mol%, or from 2.5 to 10 mol%, such as from 2.5 to 5 mol%, such as about 3.0 or about 4.0, or about 5.0 mol%; and / or the amount of lipids, such as preferably triglycerides, ranges from 1 to 95 mol%, such as from 10 to 95 mol%, or from 20 to 95 mol%, or from 30 to 95 mol%, or from 40 to 95 mol%, or from 50 to 95 mol%, or from 60 to 95 mol%, such as from 1 to 90 mol%, such as from 10 to 90 mol%, or from 20 to 90 mol%, or from 30 to 90 mol%, or from 40 to 90 mol%, or from 50 to 90 mol%, or from 60 to 90 mol%, such as from 60 to 80 mol%, or from 70 to 80 mol%, such as about 65, about 70, or about 75 mol%, wherein the molar percentage is based solely on the combined amounts of the fusion protein, phospholipids, sterols, and lipids, such as preferably triglycerides in the apolipoprotein lipid nanoparticle.

[0212] The nanoparticles may comprise further components such as additional proteins or a payload. Therefore, in an embodiment the lipid nanoparticle as defined herein further comprises lipids. In a further embodiment, the lipid nanoparticle as defined herein further comprises a payload.

[0213] When used herein the term payload refers to a compound included in a lipid nanoparticle, not being an apolipoprotein, phospholipid or sterol. The payload may for example be a pharmaceutical compound. The lipid nanoparticle is particularly suitable for lipophilic payloads but may also be used for amphipathic molecules. The pharmaceutical compound many be an organic compound, peptide, protein, nucleic acid or nucleic acid analog, biologic or lipid. Therefore, in an embodiment the lipid nanoparticle further comprises a payload, preferably wherein the payload is selected from a nucleic acid or a nucleic acid analog, a therapeutic, a biologic or combinations thereof.

[0214] For example, the payload may be a nucleic acid or a nucleic acid analog. Examples may be but are not limited to mRNA, siRNA, sgRNA, miRNA, piRNA, snRNA, snoRNA, srRNA or tsRNA. The nucleic acid analogue may be peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA), or mixtures or combinations thereof.

[0215] In certain embodiments, the apolipoprotein lipid nanoparticle comprises a nucleic acid and a cationic or ionizable cationic lipid.

[0216] In certain embodiments, the apolipoprotein lipid nanoparticle comprises a core surrounded by a surface layer, the nucleic acid and the cationic or ionizable cationic lipid are comprised by the core, and the fusion protein or fusion proteins as taught herein and the phospholipids, and optionally sterols, are comprised by the surface layer. The present invention thus also encompasses an apolipoprotein lipid nanoparticle comprising a core surrounded by a surface layer, wherein: the core comprises a nucleic acid and a cationic or ionizable cationic lipid; and the surface layer comprises: a phospholipid, a sterol, and

[0217] (i) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response;

[0218] (ii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity;

[0219] (iii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule as defined in (i), and a rerouting molecule as defined in (ii); or

[0220] (iv) the fusion protein as defined in (i) and the fusion protein as defined in (ii).

[0221] Nucleic acid containing apolipoprotein particles of this kind are described in WO / 2022 / 268913, the entire contents of which are incorporate by reference herein. The nanoparticles are engineered to complex nucleic acids, which are hydrophilic in nature, using helper molecules to draw the nucleic acids into the hydrophobic nanoparticle core. To this end, cationic hydrophobic molecules are employed. The cationic group can complex with the anionic phosphate groups in the sugar phosphate backbone via ionic interactions. The hydrophobic part of the helper molecule forms a shell around the hydrophilic nucleic acid molecule. The cationic helper molecules can be either permanently charged or ionizable. They comprise a wide variety of molecules, commercially available or synthesized in house, but they need to adhere to two general criteria: 1) A positively charged group to enable complexation with the negatively charged sugar phosphate backbone. 2) A hydrophobic part to form a hydrophobic shell and enable integration in the nanoparticle core. The content of cationic material in nanoparticle formulations may range from a cationic-to- anionic ratio of 1 :1 to 25:1. This ratio, often referred to as the N / P (nitrogen / phosphate) ratio, is based on the number of positive charges in the (ionizable) cationic lipid (often nitrogen-based) versus the number of negative charges in the nucleic acid payload (usually phosphate). Accordingly, the N / P ratio is the ratio between the cumulative molar amount of cationic and / or ionizable groups in the cationic or ionizable lipid component(s) (N) and the cumulative molar amount of phosphate groups in the nucleic acid component(s) (P). In particular embodiments, the N / P ratio of the nanoparticles as taught herein is from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 12, from 1 to 9, from 1 to 6, or from 1 to 3. For example, the N / P ratio of the nanoparticles as taught herein may be 3, 6, 9 or 12.

[0222] Without being bound to theory, present inventors believe that the nucleic acidcontaining nanoparticles described herein have an outer layer comprising mainly the apolipoprotein fusion protein, phospholipid and optional sterol, and a core comprising cationic or ionizable cationic lipid and the nucleic acid. More particularly, the core of the nanoparticle comprises an assembly of nucleic acid interacting with the (ionizable) cationic lipid, wherein this core of the nanoparticle is surrounded by a lipid shell comprising, consisting essentially of or consisting of the apolipoprotein fusion protein, phospholipid and optional sterol. According to this understanding, the nucleic acid is located within (i.e. on the inside) of the nanoparticle, and is not located at the outer surface of the nanoparticle and / or is not exposed to the surroundings of the nanoparticle. In particular embodiments, the payload (i.e. nucleic acid) of the nanoparticles of this invention is not bound by ionic interactions at the outside (surface) of the particle. Binding of nucleic acid to the outside surface of the particle is undesired as the nucleic is left exposed to the immediate surroundings, presumably making the particles more toxic as well as leading to fast (bio)- degradation of the nucleic acid payload. According to this understanding, the apolipoprotein fusion protein is located at the outer surface of the nanoparticle and / or is exposed to the surroundings of the nanoparticle.

[0223] The core of the nanoparticle may be solid and not have or bear a significant aqueous void or reservoir in the core. In particular embodiments, the core of the nanoparticle is nonaqueous.

[0224] In certain embodiments, the core of the nanoparticle is not surrounded by a lipid bilayer, such as present in vesicle-like or liposomal particles with lipid bi-layers surrounding an aqueous core.

[0225] In particular embodiments, the nanoparticles do not comprise synthetic (non-natural) hydrophilic polymers or (lipid) conjugates of such polymers, such as most notably polyethylene-glycol (PEG). As a result thereof, such nanoparticles do not elicit unwanted immune responses, especially upon repeated administration.

[0226] In certain embodiments, the nanoparticle core further comprises a filler, preferably a filler selected from a triacylglyceride and a cholesterol acyl ester, or combinations thereof, such as wherein the triacylglyceride is tricaprylin and / or wherein the cholesterol acyl ester is cholesteryl caprylate and / or cholesteryl oleate. Cholesteryl acetate may also be employed as filler material. Yet other filler materials that can be applied are di-glycerides or triglycerides or other esters derived from C1-C18 carboxylic acids, preferably C6-C18 fatty acids, where these carboxylic acids and fatty acids may be saturated or unsaturated. Preferably, the filler is a tri-glyceride derived from C6-C18 fatty acids are preferred. Besides nucleic acids and cationic helper molecules, additional hydrophobic filler molecules can be included in the core of nanoparticle formulations. Their main application is to alter nanoparticle physicochemical properties and / or improve stability.

[0227] In certain embodiments, the nucleic acid is RNA, DNA or a nucleic acid analogue.

[0228] In certain embodiments, the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), long noncoding RNA (IncRNA), or guide RNA (gRNA), or combinations thereof and / or modifications thereof.

[0229] In certain embodiments, the DNA is single stranded or double stranded DNA.

[0230] In certain embodiments, the nucleic acid is an antisense oligonucleotide and the antisense oligonucleotide is single strand DNA or RNA consisting of nucleotide or nucleoside analogues containing modifications of the phosphodiester backbone or the 2’ ribose.

[0231] In certain embodiments, the nucleotide or nucleoside analogues are 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 acids (HNA), or mixtures or combinations thereof.

[0232] In certain embodiments, the nucleic acid is conjugated, and the nucleic acid conjugate is incorporated into the nanoparticle 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 are of a hydrophobic nature.

[0233] In certain embodiments, the nucleic acid is incorporated as such or 'as is' within the nanoparticle, meaning that the nucleic acid is not being conjugated.

[0234] When used herein, the term ionizable cationic lipid refers to a lipid which 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 which is protonated or positively charged at a lower pH (e.g. at pH 1 to 5, preferably at pH 1 to 4, such as at pH 4). It is understood that ionizable cationic lipids are particularly useful, as they may be protonated at low pH thus facilitating binding to the hydrophilic nucleic acid. By subsequently raising the pH the lipids may become (partly) neutral further facilitating inclusion in a hydrophobic environment, e.g. the hydrophobic core of a nanoparticle. Alternatively, and without being bound to theory, the ionizable lipids may remain positively charged within the nanoparticles, even though the pH of the surrounding aqueous solution has been raised to physiological pH, such as about 7.4, due to the action of the surface layer of the nanoparticle that comprises phospholipid, optionally sterol, and apolipoprotein fusion protein, and / or due to the non-aqueous environment within the nanoparticle. Furthermore, ionizable cationic lipids are theorized to facilitate the endosomal escape of the nucleic acid in the target cells, where due to the low pH the ionizable cationic lipid will be protonated.

[0235] Non-limiting examples of ionizable cationic lipids are DLin-DMA (2-[2,2-bis(octadeca- 9, 12-dienyl)-1 ,3-dioxolan-4-yl]-N,N-dimethylethanamine), DLin-KC2-DMA (2-[2,2- bis[(9Z,12Z)-octadeca-9,12-dienyl]-1 ,3-dioxolan-4-yl]-N,N-dimethylethanamine) and DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-

[0236] (dimethylamino)butanoate) as represented by formula 1 below:

[0237] Formula 1

[0238] Indeed, a broad range of ionizable cationic lipids (including lipidoids) can be employed for preparing the nanoparticles of this invention, as various series of ionizable cationic lipids have been developed and reported on in 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).

[0239] The ionizable lipid may further be an ionizable triglyceride. A non-limiting example is the compound represented by formula 2:

[0240] Formula 2 The ionizable lipid may further be a cholesterol ester (also named a cholesteryl ester). A non-limiting example is represented by formula 3:

[0241] Formula 3

[0242] When used herein, the term cationic lipid refers to a positively charged lipid at physiological pH (e.g. 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(heptadecyl)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 molecules wherein the 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 resultant quaternary ammonium molecule has a permanent positive (cationic) charge, and accordingly also bears a counter anion, for example a chloride anion.

[0243] In an embodiment, only ionizable cationic lipids are used to prepare the nucleic acidcontaining nanoparticles as taught herein. Accordingly, in an embodiment, the nanoparticles as taught herein do not comprise cationic lipids.

[0244] In an embodiment, only cationic lipids are used to prepare the nucleic acid-containing nanoparticles as taught herein. Accordingly, in an embodiment, the nanoparticles as taught herein do not comprise ionizable cationic lipids.

[0245] In an embodiment, a combination of ionizable cationic lipids and cationic lipids are used to prepare the nucleic acid-containing nanoparticles as taught herein.

[0246] When referring to the nucleic acid-containing nanoparticles as taught herein, the term “payload” in particular refers to the nucleic acid, preferably in combination with the cationic and / or ionizable cationic lipids.

[0247] The term “lipid” is well known in the art, and as used herein may in particular be considered to encompass both lipids, i.e. naturally occurring hydrophobic biomolecules such as for example fatty acids, mono-, di- or tri-glycerides of fatty acids, sterol (derivatives) or phospholipids, and lipid-like biomolecules. It is noted that the cationic lipids or ionizable cationic lipids (or lipidoids) described herein are typically not lipids within the most narrow interpretation of the term, i.e. naturally occurring hydrophobic biomolecules such as for example fatty acids, mono-, di- or tri-glycerides of fatty acids, sterol (derivatives) or phospholipids, but are lipid-like biomolecules that resemble lipid biomolecules, i.e. they preferably contain groups that are biocompatible (such as e.g. esters or amides), and / or are constructed using naturally occurring building blocks (e.g. fatty acids, glycerol, cholesterol).

[0248] In certain embodiments, the cationic 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 combinations thereof.

[0249] The ionizable cationic ester of a long chain alcohol may for example be an ester of a tertiary amine with a carboxy group such as a compound with the formula (CH3)2N(CH2)nCOOH, wherein n is an integer of 1 or more, for instance n is 1 to 12; for example 3-dimethylamino-propionic acid or 4-dimethylamino-butyric acid or 5 dimethylamino-pentanoic acid. The ester is formed with a long chain alcohol. The long chain alcohol is preferably a primary or secondary alcohol with a straight 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.

[0250] The ionizable cationic ester of a diglyceride is preferably a diacyl glycerol (i.e. a diglyceride) coupled at the 1 or 2 position with a tertiary amine with a carboxy group such as a compound with the formula (CH3)2N(CH2)nCOOH, wherein n is an integer of 1 or more, for instance n is 1 to 12; for example 3-dimethylamino-propionic acid or 4-dimethylamino- butyric acid or 5-dimethylamino-pentanoic acid. The diacyl glycerol may comprise medium chain or long chain saturated or unsaturated fatty acids or derivatives or modifications thereof.

[0251] The ionizable cationic ester of a sterol is preferably an ester of sterol coupled at the hydroxyl group to a tertiary amine with a carboxy group such as a compound with the formula (CH3)2N(CH2)nCOOH, wherein n is an integer of 1 or more, , for instance 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 p-sitosterol.

[0252] In the above, a carboxy compound is presented with the formula (CH3)2N(CH2)nCOOH, wherein n is an integer of 1 or more. Instead of this compound, an alternative compound can be employed with the formula NH2-(C=NH)-NH-(CH2)nCOOH, wherein n is an integer of 1 or more, for instance n is 1 to 12. This carboxy compound comprises a guanidine group instead of a tertiary amine group. The ionizable cationic lipid can for example be selected from the molecules as according to Formulas (I) to (V).

[0253] Formula (I) represents a tri-glyceride, wherein the ionizable cationic group (ICG) is comprised in the 1-position.

[0254] Formula (II) represents the same type of tri-glyceride as represented in Formula (I), albeit that the molecule is stereo-specifically defined in the naturally occurring configuration, i.e. as it would in a phospholipid: the ICG group is in the same position as the phosphate group is in a phospholipid.

[0255] Formula (III) represents a tri-glyceride, wherein the ionizable cationic group (ICG) is comprised in the 2-position.

[0256] Formula (IV) represents a di-ester (or a tri-ester), wherein the ionizable cationic group (ICG) is connected via the amide functionality.

[0257] Formula (V) represents a cholesteryl ester, wherein the ionizable cationic group (ICG) is connected via the ester functionality.

[0258] The ionizable cationic group (ICG) is connected via the wavy bond to the rest of the molecule for any of the Formulas (I) to (V), where the ICG can either represent a tertiary amine (ICG type A, or ICG-A) or it can represent a guanidine (ICG type B, or ICG-B).

[0259] In Formulas (I) to (IV), Ri can be independently selected for every position, and it represents a linear or branched C1-C19 alkyl, a linear or branched C1-C19 alkenyl, aryl, arylene-alkyl or alkylene-aryl group, wherein said alkyl or alkenyl group, optionally containing 5 heteroatoms, independently selected from O and N. Preferably, every Ri-group within a specific molecule as according to any of the Formulas (I) to (IV) is the same Ri group. Preferably, the Ri group is a linear or branched C5-C19 alkyl group, or a linear or branched C5-C19 alkenyl group. When Ri is an alkenyl group, this group preferably has one single double bond only. More preferably, the Ri group is a linear or branched C9-C17 alkyl group or a linear or branched C5-C17 alkenyl group. Preferably R1 is a linear C5-C15 alkyl group or a linear C17-C19 alkenyl. Carboxylic acids derived from Ri, i.e. Ri-COOH, are preferably naturally occurring fatty acid molecules such as capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, palmiteic acid, oleic acid or linoleic acid. Preferred are the C10-C16 saturated fatty acids as well as oleic acid (C18, unsaturated).

[0260] The integer p is a discrete number and not an average value; p can be 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.

[0261] The R2 group in Formula (IV) can be selected from a hydrogen, a methyl, an ethyl and a -CH2-O-C(O)-Riagroup (wherein Riahas the same meaning as Ri defined above). Preferably, R2 is a hydrogen, a methyl or a -CH2-O-C(O)-RI group. More preferably, R2 is a methyl.

[0262] The R3 group in Formula (IV) can be selected from a hydrogen, aryl, arylene-alkyl, alkylene-aryl or a linear C1-C6 alkyl group. Preferably, R3 is a hydrogen or a methyl. More preferably, R3 is a hydrogen.

[0263] The Rxgroup in ICG-A can be independently selected for every position, and is selected from a methyl, an ethyl, a propyl and an ethylene-hydroxy (-CH2-CH2-OH) group, preferably it is a methyl group. Preferably, both Rxgroups in ICG-A are the same groups, and they preferably are methyl groups.

[0264] The Rygroup in ICG-B can be independently selected for every position from a hydrogen, a linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein said alkyl group optionally contains up to 5 heteroatoms, independently selected from O and N. Preferably, the Rygroup is selected from a hydrogen and a linear C1-C6 alkyl group. Even more preferably, the Rygroup is a hydrogen. Preferably, all four Ry-groups in ICG-B are the same groups, and they preferably are hydrogens.

[0265] From Formulas (I) to (V), Formulas (I), (II) and (IV) are preferred. More preferred are Formulas (I) and (II).

[0266] From the ICGs ICG-A and ICG-B, ICG-A is preferred, i.e. tertiary amine ionizable cationic lipids are preferred.

[0267] The ionizable cationic lipid molecule as according to any one of the Formulas (I) to (V) has a molecular weight that is higher than 250 Dalton, preferably higher than 350 Dalton, more preferably higher than 450 Dalton. It has a molecular weight that is lower than 3000 Dalton, preferably lower than 1800 Dalton, more preferably lower than 1200 Dalton. The molecules that are represented by Formulas (I) to (V) may exist in various isomeric forms such as rotamers, tautomers, stereoisomers or regiomers, and all of these are included in the scope of the present invention.

[0268] The ionizable cationic lipid as according to any one of the Formulas (I) to (V) preferably is a single compound, i.e. not a mixture of compounds. Accordingly, the purity of the ionizable cationic lipid of Formula (I) to (V) preferably is 50% or higher, preferably 80% or higher, more preferably 90% or higher, most preferably 95% or higher. In case the ionizable cationic lipid is a mixture of compounds, then this is preferably due only to the presence of undefined stereo-centers in the molecule. An example is the use of branched alkyl chains in the ionizable cationic lipid that are of racemic origin. Other examples are triglycerides wherein the substitution pattern over the three hydroxy-groups in the glycerol entity is not stereo-specifically defined.

[0269] Hence, in certain embodiments, the ionizable cationic lipid is a molecule according to any one of Formulae (I), (II), (III), (IV) or (V) wherein ICG is or , wherein the wavy line indicates the point of attachment to the compound of formulae (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 Ri is independently selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl group, wherein said alkyl or alkenyl group optionally contains up to 5 heteroatoms, independently selected from O and N;

[0270] R2 is selected from the group consisting of hydrogen, methyl, ethyl and a -CH2-O-C(O)-Ria;

[0271] R3 is selected from the group consisting of hydrogen, aryl, arylene-alkyl, alkylene-aryl and linear C1-C6 alkyl group;

[0272] Ria is selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl group, wherein said alkyl or alkenyl group optionally contains up to 5 heteroatoms, independently selected from O and N; each Rxis independently selected from the group consisting of methyl, ethyl, propyl and - CH2-CH2-OH; each Rygroup is independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein said alkyl group optionally contains up to 5 heteroatoms, independently selected from O and N; or rotamers, tautomers stereoisomers or regioisomers thereof.

[0273] The term "alkyl" by itself or as part of another substituent refers to a hydrocarbyl group of formula CnH2n+i wherein n is a number greater than or equal to 1 . Alkyl groups may be linear or branched and may be substituted as indicated herein. Generally, alkyl groups of this invention comprise from 1 to 18 carbon atoms, preferably from 1 to 17 carbon atoms, preferably from 1 to 15 carbon atoms, preferably from 1 to 6 carbon atoms, preferably from 1 to 5 carbon atoms, preferably from 1 to 4 carbon atoms, more preferably from 1 to 3 carbon atoms, still more preferably 1 to 2 carbon atoms. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term "Ci-ealkyl", as a group or part of a group, refers to a hydrocarbyl group of formula -CnH2n+i wherein n is a number ranging from 1 to 6. Thus, for example, “Ci-ealkyl” includes all linear or branched alkyl groups with between 1 and 6 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, hexyl and its isomers. For example, “Ci-salkyl” includes all includes all linear or branched alkyl groups with between 1 and 5 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers. For example, “Ci-4alkyl” includes all linear or branched alkyl groups with between 1 and 4 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl). For example “Ci-salkyl” includes all linear or branched alkyl groups with between 1 and 3 carbon atoms, and thus includes methyl, ethyl, n-propyl, i-propyl.

[0274] When the suffix "ene" is used in conjunction with an alkyl group, i.e. “alkylene”, this is intended to mean the alkyl group as defined herein having two single bonds as points of attachment to other groups. As used herein, the term “Ci-ealkylene”, by itself or as part of another substituent, refers to Ci-ealkyl groups that are divalent, i.e., with two single bonds for attachment to two other groups. Alkylene groups may be linear or branched and may be substituted as indicated herein. Non-limiting examples of alkylene groups include methylene (-CH2-), ethylene (-CH2-CH2-), methylmethylene (-CH(CH3)-), 1-methyl-ethylene (-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 its chain isomers, hexylene and its chain isomers.

[0275] The term “alkenyl” as a group or part of a group, refers to an unsaturated hydrocarbyl group, which may be linear, or branched, comprising one or more carbon-carbon double bonds. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term “C2- ealkenyl” refers to an unsaturated hydrocarbyl group, which may be linear, or branched comprising one or more carbon-carbon double bonds and comprising from 2 to 6 carbon atoms. For example, C2-4alkenyl includes all linear, or branched alkenyl groups having 2 to 4 carbon atoms. Examples of C2-ealkenyl 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.

[0276] The term “aryl”, as a group or part of a group, refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthyl), or linked covalently, 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 aryl include Ce- aryl, more preferably Ce-saryl. Non-limiting examples of C6-i2aryl comprise phenyl; biphenylyl; biphenylenyl; or 1-or 2-naphthanelyl; 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, this is intended to mean the aryl group as defined herein having two single bonds as points of attachment to other groups. Suitable “C6-i2arylene” groups include 1 ,4-phenylene, 1 ,2-phenylene, 1 ,3-phenylene, biphenylylene, naphthylene, indenylene, 1-, 2-, 5- or 6-tetralinylene, and the like. Where at least one carbon atom in an aryl group is replaced with a heteroatom, the resultant ring is referred to herein as a heteroaryl ring. The heteroatom may be selected from the group consisting of O, N, P and S; preferably O or N.

[0277] The term "alkylene-aryl", as a group or part of a group, means a alkylene as defined herein, wherein at least one hydrogen atom is replaced by at least one aryl as defined herein. Alkylene-aryl groups typically contain 7 to 25 carbon atoms. Non-limiting examples of alkylene-aryl group include benzyl, phenethyl, dibenzylmethyl, methylphenylmethyl, 3-(2- naphthyl)-butyl, and the like. The term "arylene-alkyl", as a group or part of a group, means a arylene as defined herein, wherein at least one hydrogen atom is replaced by at least one alkyl group as defined herein. Arylene-alkyl groups typically contain 7 to 25 carbon atoms.

[0278] Ester, amide, carboxylic acid and alcohol groups are defined hereunder, where Rp represents a hydrogen atom or a cyclic, linear or branched alkyl or alkylene groups. In groups that contain more than one Rp element, then these elements can be independently selected. An ester (functional) group or moiety as indicated in this document is to be understood as a group according to the formula: -C(O)-O-. An amide (functional) group or moiety as indicated in this document is to be understood as a group according to the formula: -NRp-C(O)-. A carboxylic acid (functional) group or moiety as indicated in this document is to be understood as a moiety or group according to the formula: -C(O)OH. An alcohol (or hydroxy) functional group or moiety as indicated in this document is to be understood as a group according to the formula: -OH.

[0279] The ionizable cationic lipids as according to any one of the Formulas (I) to (V) can be prepared by synthetic methods that are known in the art, such as without limitation as described in WO / 2022 / 268913, such as in particular in the Examples section and especially Example 9 of WO / 2022 / 268913.

[0280] The (ionizable) cationic lipid preferably can be processed from solutions. Accordingly, the (ionizable) cationic lipid is preferably soluble in solvents ranging in polarity. Therefore, the (ionizable) cationic lipid is preferably soluble in tricaprylin, in ethanol or in iso-propanol, more preferably in all three of these solvents. The solubility can be checked by stirring about 20 mg of the (ionizable) cationic lipid in about 1 gram of tricaprylin, ethanol or iso-propanol, and assessing whether all material spontaneously dissolves to create a clear / transparent solution with a concentration of about 2 w / w%. The test can be done at about 20 °C (room temperature) or at about 37 °C. Preferably, the (ionizable) cationic lipid is soluble at room temperature. The (ionizable) cationic lipid is preferably non-toxic, or it may have a limited and low toxicity, either on its own, or when bound to or tested together with nucleic acids, or assayed in the nanoparticle as taught herein. Toxicity cell tests can be executed by methods that are known in the art, such as for example by cell viability MTT assays, or by similar or comparable tests.

[0281] In certain embodiments, the amount of the fusion protein (in case of two or more fusion proteins, cumulatively), in particular the cumulative amount in case of two or more fusion proteins, ranges from 0.08 to 2.0 mol%, such as from 0.10 to 2.0 mol%; and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 15 to 90 mol%; and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol%; and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 8.0 to 80 mol%, wherein the molar percentage is based solely on the combined amounts of the fusion protein, phospholipids, sterols and cationic or ionizable cationic lipids in the apolipoprotein lipid nanoparticle.

[0282] In certain embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of fusion protein (in case of two or more fusion proteins, cumulatively) ranges from 0.05 to 2.0 mol%, such as from 0.10 to 2.0 mol% or from 0.08 to 0.5 mol%; and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 15 to 90 mol% or from 8.0 to 50 mol%; and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol% or from 4 to 65 mol%; and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 8.0 to 80 mol% or from 5 to 65 mol% wherein the molar percentage is based solely on the combined amounts of the fusion protein, phospholipids, sterols and cationic and / or ionizable cationic lipids in the nanoparticle. These ranges contribute positively to the stability of the nanoparticles and their ability to incorporate nucleic acids.

[0283] In certain embodiments of nucleic acid-containing nanoparticles as taught herein, the amount of fusion protein (in case of two or more fusion proteins, cumulatively) ranges from 0.01 to 2.0 mol%, such as from 0.05 to 1.0 mol%, or from 0.05 to 0.5 mol%, or from 0.05 to 0.4 mol%, or from 0.05 to 0.3 mol%, or from 0.05 to 0.2 mol%, or from 0.05 to 0.1 mol%, such as about 0.06, about 0.07, about 0.08, or about 0.09 mol%; and / or the amount of phospholipid ranges from 2 to 90 mol%, such as from 2 to 80 mol%, or from 2 to 70 mol%, or from 2 to 60 mol%, or from 2 to 50 mol%, or from 2 to 40 mol%, or from 2 to 30 mol%, or from 2 to 20 mol%, such as from 5 to 10 mol%, such as about 6, about

[0284] 7, about 8, or about 9 mol%; and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol%, or from 5 to 50 mol%, or from 5 to 40 mol%, or from 5 to 30 mol%, or from 10 to 30 mol%, such as from 15 to 25 mol%, such as about 20, or about 21 , or about 22 mol%; and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 10 to 80 mol%, or from 10 to 70 mol%, or from 10 to 60 mol%, or from 10 to 50 mol%, or from 10 to 40 mol%, or from 10 to 30 mol%, or from 15 to 25 mol%, such as about 18, or about 19 or about 21 mol%; and / or the amount of lipids, such as preferably triglycerides, ranges from 0 to 95 mol%, or from 0 to 90 mol%, such as from 1 to 95 mol%, or from 1 to 90 mol%, such as from 10 to 90 mol%, or from 20 to 80 mol%, or from 30 to 70 mol%, or from 40 to 60 mol%, or from 45 to 55 mol%, such as about 50, or about 51 , or 52 mol%; and / or the amount of nucleic acid, such as preferably RNA, e.g., mRNA, ranges from 0.01 to 1 .0 mol%, such as from 0.01 to 0.1 mol%, or from 0.01 to 0.05 mol%, such as about 0.02, about 0.03, or about 0.04 mol%; wherein the molar percentage is based solely on the combined amounts of the fusion protein, phospholipids, sterols, cationic and / or ionizable cationic lipids, lipids, such as preferably triglycerides, and nucleic acid, such as preferably RNA, in the nanoparticle. These ranges contribute positively to the stability of the nanoparticles and their ability to incorporate nucleic acids.

[0285] In certain embodiments of nucleic-acid containing nanoparticles as taught herein, the amount of the fusion protein (in case of two or more fusion proteins, cumulatively) ranges from 0.1 to 90 weight%; the amount of nucleic acid ranges from 0.01 to 90 weight%; the amount of phospholipid ranges from 0.1 to 95 weight%; the amount of sterol ranges from 0.1 to 95 weight%; and / or the amount of cationic and / or ionizable cationic lipid ranges from 0.1 to 95 weight%, wherein these weight percentages are based on the combined amounts of the fusion protein, the nucleic acid, the phospholipid, the sterol and the cationic and / or ionizable cationic lipid.

[0286] In an embodiment of nucleic-acid containing nanoparticles as taught herein, the amount of the fusion protein (in case of two or more fusion proteins, cumulatively) ranges from 0.2 to 50 weight%, such as from 3 to 20 weight% or from 4 to 20 weight%, more preferably from 0.5 to 30 weight%, more preferably from 1 to 20 weight%. In an embodiment of nucleic-acid containing nanoparticles as taught herein, the amount of nucleic acid ranges from 0.02 to 30 weight%, more preferably from 0.05 to 20 weight%, more preferably from 0.1 to 15 weight%, such as from 0.5 to 5 weight%.

[0287] In an embodiment of nucleic-acid containing nanoparticles as taught herein, the amount of phospholipid ranges from 0.2 to 60 weight%, more preferably from 1 to 50 weight%, such as from 10 to 50 weight%, more preferably from 3 to 40 weight%, such as from 10 to 40 weight%.

[0288] In an embodiment of nucleic-acid containing nanoparticles as taught herein, the amount of sterol ranges from 0.2 to 90 weight%, more preferably from 0.5 to 70 weight%, such as from 2 to 65 weight%, more preferably from 1 to 50 weight%, such as from 2 to 45 weight%, from 10 to 45 weight% or from 10 to 20 weight%.

[0289] In an embodiment of nucleic-acid containing nanoparticles as taught herein, the amount of cationic and / or ionizable cationic lipid ranges from 0.2 to 90 weight%, more preferably from 0.5 to 80 weight%, more preferably from 1 to 70 weight%, such as from 5 to 60 weight%, from 8 to 60 weight%, from 9 to 60 weight%, from 10 to 60 weight%, from 15 to 25 weight%, or from 20 to 60 weight%.

[0290] In an embodiment of nucleic-acid containing nanoparticles as taught herein, the amount of optional filler or filler molecule ranges from 0 to 90 weight%, more preferably from 0 to 80 weight%, more preferably from 0 to 70 weight%, such as from 0 to 65 weight%.

[0291] In particular embodiments of nucleic-acid containing nanoparticles as taught herein, the amount of optional filler or filler molecule ranges from 20 to 80 weight%, more preferably from 30 to 70 weight%, even more preferably from 30 to 65 weight%, such as from 40 to 65 weight%, from 45 to 55 weight% or from 30 to 60 weight%.

[0292] These weight percentages as indicated above are based on the combined amounts of the apolipoprotein and / or apolipoprotein mimetic, the nucleic acid, the phospholipid, the sterol and the cationic and / or ionizable cationic lipid, and optionally the filler material, i.e. these five or six components add up to 100% of the weight of the nanoparticle in the context of these statements. These weight percentage ranges contribute positively to the stability of the nanoparticles and their ability to incorporate nucleic acids.

[0293] In particular embodiments, the nanoparticle as taught herein does not comprise a filler or filler molecule.

[0294] In certain embodiments, the ratio of the fusion protein (in case of two or more fusion proteins, cumulatively) to phospholipid based on percentage molar weight is between 1 :25 and 1 :400, more preferably between 1 :50 and 1 :200, even more preferably between 1 :75 and 1 :150.

[0295] In certain embodiments, the ratio of the fusion protein (in case of two or more fusion proteins, cumulatively) to phospholipid based on weight is from 2:1 to 1 :10, more preferably from 1 :1 to 1 :5, even more preferably from 1 :1.5 to 1 :4. These ranges contribute positively to the stability of the nanoparticles.

[0296] Alternatively, the payload may be a small organic compound such as a small molecule drug. Generally, the small organic compound is synthesized. The therapeutic may for example be an anticancer therapy such as a chemotherapy. Alternatively, the payload may be a biologic. When used herein, the term biologic is used to indicate a biopharmaceutical, also known as a biologic(al) medical product, and can be any pharmaceutical drug product manufactured in, extracted from, or semi-synthesized from biological sources. Biologies can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or may be living cells or tissues.

[0297] In particular embodiments, the lipid nanoparticle comprises a native (e.g. unfused) apolipoprotein, an apolipoprotein mimetic or a combination thereof, in addition to the apolipoprotein or an apolipoprotein mimetic which forms part of the fusion protein as described herein.

[0298] In particular embodiments, the lipid nanoparticle has an average size of 10 to 100 nm, such as from 30 to 100 nm.

[0299] In particular embodiments, the lipid nanoparticle is a sphere, a ribbon or a disc, preferably a sphere or a disc, more preferably a sphere.

[0300] The apolipoprotein or apolipoprotein mimetic forms part of the lipid nanoparticle structure. In particular embodiments, at least a part of said fusion protein is exposed to the environment (i.e. aqueous environment) surrounding said apolipoprotein lipid nanoparticle. Typically, part of the apolipoprotein or apolipoprotein mimetic is exposed to the environment surrounding said apolipoprotein lipid nanoparticle (e.g. see Figure 1 , Figure 2 and Figure 22). Furthermore, fusion of an immunomodulatory biomolecule and / or a rerouting molecule to the apolipoprotein or apolipoprotein mimetic typically allows said immunomodulatory biomolecule and / or rerouting molecule to be wholly exposed to the environment surrounding said apolipoprotein lipid nanoparticle (e.g. see Figure 1 , Figure 2 and Figure 22). In other words, the immunomodulatory biomolecule and / or a rerouting molecule are not embedded within the lipid nanoparticle. As a result thereof, said immunomodulatory biomolecule and / or said rerouting molecule will be able to move freely and exert its natural function(s), such as its cell targeting function. In particular 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 the apolipoprotein or apolipoprotein mimetic, the phospholipids, the immunomodulatory biomolecule and / or rerouting molecule, and optionally the sterols.

[0301] In particular embodiments, the lipid nanoparticle is not a phospholipid bilayer.

[0302] Also provided herein are methods of manufacturing such lipid nanoparticles. Accordingly, in a further aspect, the invention relates to a method of manufacturing a lipid nanoparticle a method of manufacturing an apolipoprotein lipid nanoparticle as taught herein, the method comprising the steps of: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule, such as IL-4; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule, such as IL-4, and a rerouting molecule; and combinations thereof; and / or a2) chemically conjugating one or more apolipoproteins or apolipoprotein mimetics and isolating the one or more conjugated apolipoproteins to obtain one or more isolated conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule, such as IL-4; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule, such as IL-4, and a rerouting molecule; and combinations thereof; and b) combining the one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or the one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids and optionally sterols and / or lipids to obtain an apolipoprotein lipid nanoparticle.

[0303] In a further aspect, the invention relates to a method of manufacturing a lipid nanoparticle as defined herein, the method comprising the steps of: a1) expressing and isolating an apolipoprotein fusion protein to obtain an isolated apolipoprotein fusion protein, where the apolipoprotein fusion protein is an apolipoprotein fused to a cytokine and a targeting moiety and / or wherein the apolipoprotein fusion protein is an apolipoprotein fused to a cytokine and / or an apolipoprotein fused to a targeting moiety; and / or a2) chemically conjugating an apolipoprotein and isolating the conjugated apolipoprotein to obtain an isolated conjugated apolipoprotein, where the conjugated apolipoprotein is an apolipoprotein conjugated to a cytokine and a targeting moiety and / or wherein the conjugated apolipoprotein is an apolipoprotein conjugated to a cytokine and / or an apolipoprotein conjugated to a targeting moiety; b) combining the isolated apolipoprotein fusion protein obtained in step a1 and / or the isolated conjugated apolipoprotein obtained in step a2 with phospholipids, and optionally sterols and / or lipids, to obtain a lipid nanoparticle.

[0304] In a further aspect, the invention relates to an apolipoprotein lipid nanoparticle obtained by or obtainable by the method of manufacturing an apolipoprotein lipid nanoparticle as taught herein.

[0305] It is understood that the fusion protein can be expressed as a chimeric fusion protein of the apolipoprotein with the immunomodulatory biomolecule and / or the rerouting molecule or can be chemically conjugated to the immunomodulatory biomolecule and / or the rerouting molecule or can be produced be a combination of these. Expression of chimeric proteins is known to the skilled person and can be used when the immunomodulatory biomolecule and / or the rerouting molecule is a peptide or protein. It is well within the knowledge of the skilled person to use molecular techniques to produce a nucleic acid encoding such protein, for example by cloning an immunomodulatory biomolecule and / or the rerouting molecule encoding sequence in frame with an apolipoprotein (or mimetic) encoding sequence, for example at C or N terminal sequence encoding nucleotides. An advantage of using chimeric protein expression is that all expressed protein will be fusion protein.

[0306] Alternatively chemical conjugation may be used. Suitable methods for chemical conjugation of the immunomodulatory biomolecule and / or the rerouting molecule to the apolipoprotein (or mimetic thereof) are known to the skilled person. Non limiting examples are strain promoted cycloaddition, aminolysis and Michael type addition. For example, an existing or introduced cysteine residue may be used on either the apolipoprotein or the immunomodulatory biomolecule and / or the rerouting molecule.

[0307] For example, as described elsewhere herein, the ApoA1 protein may comprise a cysteine in the place of a serine at position 147 or 279. Introduction of a cysteine residue may be achieved by point mutation of a nucleotide in the encoding nucleotide sequence, or by introduction of a cysteine encoding codon. An advantage of chemical conjugation is that it is not limited to the use of peptide or protein sequences but can be applied to any type of organic molecule. It is understood that the fusion protein, phospholipids, and optional components such as sterols and lipids may be rapidly mixed to obtain a lipid nanoparticle. Optionally added may be lipids and / or a payload as defined herein.

[0308] The present invention also encompasses a method for producing an apolipoprotein lipid nanoparticle, comprising the step of: a) rapid mixing of lipid components in organic solvent with a nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid components comprise a phospholipid, a sterol, a cationic lipid or ionizable cationic lipid, wherein the aqueous buffer has a pH of 5.0 or lower; and b) rapid mixing of the lipid nanoparticles with: one or more apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, and the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; and / or one or more conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, and the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; to produce the apolipoprotein lipid nanoparticle at a pH between 5.5 and 8.0, such as between 6.0 and 8.0.

[0309] According to the above method, a two-step reaction is performed, where in the first step, a nucleic acid containing nanoparticle is formed, and in the next second step, apolipoprotein fusion protein is included in the nanoparticle. Preferably, the first step is performed at low pH and the second step is performed at physiological pH. The organic solvent may be an alcohol such as ethanol, iso-propanol, methanol, acetonitrile, dimethyl sulfoxide (DMSO), chloroform or combinations thereof. Preferred organic solvents are water mixable and non-toxic, for example ethanol and DMSO, or combinations thereof.

[0310] For example, the organic solvent may be from 96% to 100% of ethanol, preferably 100% ethanol.

[0311] Rapid mixing is known in the field and has for example been described in Hirota et al. BIOTECHNIQUES VOL. 27, NO. 2, p286-289; Jeffs et al., Pharm Res 22, 362-372 (2005); Kulkarni et al., ACS Nano 2018, 12, 5, 4787-4795

[0312] The aqueous buffer in step a) has a low pH to ensure that the ionizable cationic lipid is positively charged, allowing binding within and inclusion of the nucleic acid I cationic lipid complex in the particle. For example, the buffer may 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 may be any buffer that does not damage the nucleic acid. An exemplary buffer is sodium acetate at pH 4.0. The nanoparticle is then taken in an aqueous buffer with a pH of around 6 to 8, preferably 7 to 8 more preferably around 7.4. This may for example be achieved by dialysis with an aqueous buffer in the indicated pH range. A non-limiting example of an aqueous buffer suitable for this step is 155 mM PBS at pH 7.4, but it is understood that any buffer may be used that does not damage the nucleic acid.

[0313] In step b) the nanoparticle in an aqueous buffer at a pH between 5.5 and 8, such as between 6 to 8, preferably between pH 7 to 8, is rapidly mixed with the fusion protein in an aqueous buffer at pH between 5.5 and 8, such as between 6 to 8, preferably between pH 7 to 8, to obtain the nanoparticles.

[0314] The above described two-step formulation process as taught herein results in nucleic acid-containing nanoparticles with a broad set of desired and beneficial characteristics (stability, low toxicity or non-toxicity, high nucleic acid retention, nucleic acid activity, etc.). However, the described formulation method is non-limiting as other processes may also lead to nanoparticles with beneficial features.

[0315] A further aspect of the invention provides a pharmaceutical composition comprising the fusion protein as taught herein, the nucleic acid as taught herein or the lipid nanoparticle as taught herein, and a pharmaceutically acceptable carrier.

[0316] In a further aspect the invention relates to fusion protein as defined herein or the lipid nanoparticle as defined herein, or the lipid nanoparticle obtained or obtainable by the method as described herein, or the nucleic acid as defined herein, or the pharmaceutical composition as defined herein for use as a medicament. It is envisioned that either the fusion protein (e.g. by the action of the immunomodulatory biomolecule) or the payload comprised in the nanoparticle may be used to treat, ameliorate or alleviate a symptom in a subject.

[0317] In a further aspect, the invention relates to the fusion protein as defined herein or the lipid nanoparticle as defined herein, or the lipid nanoparticle obtained or obtainable by the method as defined herein, or the nucleic acid as defined herein, or the pharmaceutical composition as defined herein for use in the treatment of an immune related disorder. In other words, the invention relates to a method of treating an immune related disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the fusion protein as defined herein or the lipid nanoparticle as defined herein, or the lipid nanoparticle obtained or obtainable by the method as defined herein, or the nucleic acid as defined herein, or the pharmaceutical composition as defined herein to the subject in need thereof. Also provided herein is the use of the fusion protein as defined herein or the lipid nanoparticle as defined herein, or the lipid nanoparticle obtained or obtainable by the method as defined herein, or the nucleic acid as defined herein, or the pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of an immune related disorder in a subject.

[0318] A further aspect of the invention provides an in vitro or ex vivo method for introducing a nucleic acid in a cell, the method comprising contacting the nucleic acid-containing apolipoprotein lipid nanoparticle as taught herein, or a pharmaceutical composition comprising the nanoparticle and a pharmaceutically acceptable carrier, with a cell.

[0319] A further aspect of the invention provides an in vivo method for introducing a nucleic acid in a cell, the method comprising contacting the nucleic acid-containing apolipoprotein lipid nanoparticle as taught herein, or a pharmaceutical composition comprising the nanoparticle and a pharmaceutically acceptable carrier, with a cell.

[0320] A further aspect of the invention provides the nucleic acid-containing apolipoprotein lipid nanoparticle as taught herein, or a pharmaceutical composition comprising the nanoparticle and a pharmaceutically acceptable carrier, for use in the in vivo delivery of a nucleic acid to a subject.

[0321] A further aspect of the invention provides a method for the in vivo delivery of a nucleic acid, the method comprising administering the nucleic acid-containing apolipoprotein lipid nanoparticle as taught herein, or a pharmaceutical composition comprising the nanoparticle and a pharmaceutically acceptable carrier, to a subject.

[0322] A further aspect of the invention provides a method for treating a disease or disorder, such as a disease or disorder as described herein, for example by stimulating or inhibiting an innate immune response, the method comprising administering a therapeutically effective amount of the nucleic acid-containing apolipoprotein lipid nanoparticle as taught herein, or a pharmaceutical composition comprising the nanoparticle and a pharmaceutically acceptable carrier, to the subject. In certain embodiments, the disease is selected from cancer, cardiovascular disease, autoimmune disorder or xenograft rejection.

[0323] A purpose of the nucleic acid-containing nanoparticles described herein is to deliver a nucleic acid to a cell or to deliver a nucleic acid therapy to a subject. The nucleic acid may be for example an mRNA encoding a peptide or protein of interest which is to be expressed in the cell, or may comprise a short nucleic acid such as an siRNA, shRNA intended to interfere in gene expression (e.g. gene silencing), or it may comprise a component of the CRISPR-Cas or a related gene editing system (e.g. gRNA) to induce a mutation in the genome of the cell. Therefore in general the mode of action of the nucleic acid (the payload of the nanoparticle) is in the cytoplasm or the nucleus. Therefore the nanoparticle preferably has at least the following properties: 1) it allows targeting of the intended target cell, and 2) it allows delivery of the payload where it can assert its action (thus in most cases in the cytoplasm or nucleus of the target cell).

[0324] It is understood that the nucleic acid therapy comprising nanoparticles may be administered to a subject in need thereof. Depending on the target cells or tissue, the administration may be parenteral, e.g. intravenous, intramuscular or subcutaneous. The administration may further be oral, sublingual, topical, rectal, nasal (inhaled) or vaginal. Further the targeting of the target tissue or cells is determined by the proper choice of the fusion protein. In an embodiment, the use of the nanoparticle or composition comprises delivering a nucleic acid to the myeloid compartment or the spleen. This may for example be achieved by intravenous parenteral administration.

[0325] The nanoparticles of certain embodiments of the invention, after systemic injection, can target tissues (spleen, bone marrow) that are associated with the presence of immune cells.

[0326] In certain embodiments, the nucleic acid-containing nanoparticle as taught herein, or the composition as taught herein for use in immunotherapy.

[0327] In an aspect the invention relates to the nanoparticle, including the nucleic acid containing nanoparticles, as taught herein, or the composition comprising them for use in the treatment of a disease, such as a disease or disorder as described herein, for example by stimulating or inhibiting an innate immune response, preferably wherein said disease is a disease that would benefit from stimulating or inhibiting the innate immune response in a subject, such as a disease characterized by a defective innate immune response, more preferably wherein said disease to be treated is a cancer, a cardiovascular disease, an autoimmune disorder or xenograft rejection. Therefore the nanoparticles may be used in the treatment of any disease relating the immune system such as any immune disorder, or for the treatment of any disease or disorder where modulating the immune response is deemed a viable treatment option.

[0328] An immune related disorder as used herein comprises any disorder wherein the immune system plays a role in the disease development. An immune related disorder may refer to a disorder where the immune system is suppressed or where it is (over)activated. Examples of immune related disorders are cancer, infection, sepsis, autoimmune diseases, and cardiovascular diseases. 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, Sjogren’s syndrome, Hashimoto’s thyroiditis, Myasthenia gravis, Autoimmune vasculitis, Pernicious anemia, and Celiac disease.

[0329] Therefore, 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, Sjogren’s syndrome, Hashimoto’s thyroiditis, Myasthenia gravis, Autoimmune vasculitis, Pernicious anemia, and Celiac disease.

[0330] In further particular embodiments, the immune related disorder is selected from the group consisting of cancer, inflammation, an infectious disease, an autoimmune disorder such as multiple sclerosis, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

[0331] Unexpectedly, present inventors found that IL-4 can paradoxically reduce inflammation and induce trained immunity simultaneously, particularly when targeted to the myeloid compartment. In many inflammatory problems (such as sepsis, stroke and myocardial infarction), hyperinflammation and immunosuppression concurrently happen. Therefore, the present inventors concluded that fusion proteins of an apolipoprotein or an apolipoprotein mimetic, preferably ApoA1 , with IL-4 can be used to prevent immune related disorders by simultaneously reducing inflammation and promoting trained immunity.

[0332] Accordingly, in particular embodiments, wherein said immunomodulatory biomolecule is IL-4, the immune related disorder is a disease that benefits from the reduction of inflammation and / or the promotion of trained immunity.

[0333] Accordingly, in particular embodiments, wherein said immunomodulatory biomolecule is IL-4, the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

[0334] In particular embodiments, wherein said immunomodulatory biomolecule is IL-4, the apolipoprotein nanoparticle or fusion protein simultaneously reduces inflammation and induces trained immunity.

[0335] In particular embodiments, wherein said immunomodulatory biomolecule is IL-2, the apolipoprotein nanoparticle or fusion protein may be used to stimulate T-cell proliferation.

[0336] In particular embodiments, wherein said immunomodulatory biomolecule is I L-1 p, the apolipoprotein nanoparticle or fusion protein may be used to induce trained immunity.

[0337] In particular embodiments, wherein said immunomodulatory biomolecule is IL-38, the apolipoprotein nanoparticle or fusion protein may be used to reduce trained immunity.

[0338] In a further aspect, the invention relates to the use of a fusion protein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtained or obtainable by the method as defined herein or the nucleic acid as defined herein, or the pharmaceutical composition as defined herein in delivering a compound or an immunomodulatory molecule to a target, preferably wherein the target is a cell, tissue, and / or organ. In an embodiment the method is an ex vivo or in vitro method. In an alternative embodiment the method is an in vivo method. Typically, the apolipoprotein or rerouting molecule will bind to a cell surface protein, such as a receptor. Therefore, the target may be a protein such as a receptor, a cell or cell type (expressing said protein), a tissue or tissue type (expressing said protein) or an organ (expressing said protein). It is understood that by choosing or adapting the rerouting molecule the fusion protein can be targeted to different proteins. For example, the receptor binding domain of a ligand can be used to target a specific receptor. Alternatively known binding partners of cell surface proteins can be used to reroute the fusion protein.

[0339] In a further aspect, the invention relates to a nucleic acid encoding the fusion protein as defined herein. The nucleic acid may be comprised in a vector such as a viral vector for stable integration in a cell, or an expression vector to enable transient expression.

[0340] In particular embodiments, the vector comprises a myeloid specific or enhanced promoter or promoter element may be used in a vector to drive myeloid specific expression of IL-4. Suitable promoters are known to the skilled person, non limiting examples are: lysM, Csfl r, CD11c, CX3CR1 , Langerin / CD207, MMLV LTR, Visna virus LTR, DC-STAMP, Human MSR, MSR-A, huCD68, CD4, CD2 and lba-AIF-1 (see e.g. Hume., Journal of leukocyte biology, Volume89, Issue4, April 2011 , Pages 525-538 for a review). Such promoter or promoter elements can be incorporated in a vector such as for example a lentiviral vector to stable transfect cells and allowing specific expression of the transgene in myeloid cells. In the inventors’ quest to simultaneously resolve hyperinflammation and immune paralysis, they extensively studied the role of interleukin (IL)-4 in trained immunity and tolerance. When investigating its effects on monocytes in vitro [Czimmerer, Z. et al. The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages. Immunity 48, 75-90. e6 (2018); Essner, R., Rhoades, K., McBride, W. H., Morton, D. L. & Economou, J. S. IL-4 downreg ulates IL-1 and TNF gene expression in human monocytes. J. Immunol. 142, 3857 (1989); Woodward, E. A., Prele, C. M., Nicholson, S. E., Kolesnik, T. B. & Hart, P. H. The anti-inflammatory effects of interleukin-4 are not mediated by suppressor of cytokine signalling-1 (SOCS1). Immunology 131, 118-127 (2010)], the inventors discovered that IL- 4 simultaneously downregulates inflammatory programs and induces trained immunity. They found that IL-4’s unique properties allow overcoming lipopolysaccharide-induced immunoparalysis in monocytes.

[0341] However, owing to its unspecific nature and unfavorable pharmacokinetic properties, IL-4 is unsuitable as a myeloid cell-regulating therapeutic. To overcome these limitations, the inventors now found that routing IL-4 to the myeloid compartment is an attractive therapeutic avenue. In support of this concept, the inventors herein describe and developed a fusion protein combining IL-4 with apolipoprotein A-1 (apoA1), the main protein constituent of high-density lipoprotein (HDL), as described elsewhere herein. The resulting apoA1-IL-4 fusion protein readily integrates into myeloid cell-avid lipid nanoparticles (apoA1-IL4- nanoparticles), significantly improving IL-4’s pharmacokinetic profile and bioavailability to innate immune cells. The inventors evaluated apoA1-IL-4- nanoparticles’ in vivo behavior and safety profile in mice and non-human primates using quantitative nuclear imaging techniques and blood chemistry measurements. Finally, the inventors studied apoA1-IL-4- nanoparticles’ therapeutic potential in multiple translational inflammation and sepsis models, institutionalizing a new paradigm for the management of hyperinflammation- induced immunoparalysis.

[0342] Therefore the inventors concluded that IL-4 is a promising new therapeutic which can be used to prevent immune related disorders by promoting trained immunity, provided that the IL-4 can be targeted to the myeloid compartment. As described elsewhere herein, the inventors demonstrate this can be achieved though covalent attachment of IL-4 to an apolipoprotein, however it is envisioned that at least the following methods may be used to achieve targeting of the myeloid compartment:

[0343] - fusion of IL-4 to an apolipoprotein as described elsewhere herein; - fusion of IL-4 to a myeloid-targeting molecule, preferably wherein the myeloid- targeting molecule is an antibody or antigen binding fragment thereof which binds a myeloid marker, or a ligand or peptide that allows targeting of the myeloid compartment;

[0344] - targeted expression of IL-4 in or near the myeloid compartment.

[0345] Accordingly, in a further aspect, the invention relates to a fusion protein of a myeloid- targeting molecule and IL-4, preferably wherein the myeloid-targeting molecule is capable of targeting IL-4 to the myeloid cell.

[0346] In particular embodiments, the myeloid-targeting molecule is a chemical substance, such as a small organic molecule, or is a biological molecule, such as a biological polymer, such as for example a protein, polypeptide or peptide, nucleic acid, saccharide, polysaccharide. Preferably, said molecule is a protein, polypeptide or peptide.

[0347] As detailed above, the invention is based on the following findings further supported by the experimental examples provided below:

[0348] - IL-4 surprisingly is able to induce trained immunity, making IL-4 an interesting therapeutic for immune related disorders, particularly for immune paralysis; and

[0349] - unfavorable pharmacological properties of injected IL-4 can be avoided by targeting IL-4 to the myeloid compartment.

[0350] In particular embodiments, the IL-4 is a polypeptide comprising an amino acid sequence at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0351] 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 43, or a circular permutation thereof. In a further embodiment the IL-4 polypeptide is encoded be a nucleic acid having a sequence 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

[0352] 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 44.

[0353] It is understood a fusion with identical function may also be constructed using circular permutation. A circular permutation is a relationship between proteins whereby the proteins have a changed order of amino acids in their peptide sequence. The result is a protein structure with different connectivity, but overall similar three-dimensional shape. For example a first protein has a sequence a-b-c, after the permutation a second protein has a sequence c-a-b while maintaining the same three-dimensional shape. Therefore in a further embodiment the IL-4 polypeptide comprises two sequence which together are 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. 43.

[0354] When used herein, the term “targeting”, when referring to targeting a myeloid cell or targeting the myeloid compartment should be understood to mean bring in proximity of the myeloid cell or the myeloid compartment, or to enrich in the proximity of the myeloid cell or the myeloid compartment. This implies that when targeting (by the myeloid-targeting molecule) on average more molecules of IL-4 are in proximity of a myeloid cell or the myeloid compartment. In proximity herein means being located such that the IL-4 can interact with the myeloid cell, e.g. by binding of one of its receptors.

[0355] When used herein, a a myeloid-targeting molecule is intended to indicate any molecule, but preferably a peptide, protein or part of a protein such as a protein domain, that, when fused to IL-4 allows IL-4 to be targeted to a myeloid cell. As explained in more detail below, the following options are demonstrated or envisioned to serve as myeloid- targeting molecules: apolipoproteins, antibodies or antigen binding fragments thereof, myeloid cell specific ligands of receptors or membrane molecules.

[0356] When used herein, the terms myeloid cell refers to blood cells that are derived from a progenitor cell for granulocytes, monocytes, erythrocytes, or platelets. Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. The term myeloid compartment, when used herein, refers to the totality of myeloid cells in an organism.

[0357] In particular embodiments, the myeloid-targeting moleculeis selected from an antibody or an antigen binding fragment thereof, a myeloid-targeting peptide or a myeloid- targeting protein, preferably wherein the myeloid-targeting peptide or myeloid-targeting protein is a ligand of a receptor present on the target.

[0358] For example, an antibody or antigen binding fragment thereof can be used that specifically binds to an antigen which is highly expressed or exclusively present on myeloid cells. Suitable targets are known to the skilled person, non-limiting examples are CD11 b, CD11c, CD14 or co-stimulatory molecules such as CD80, CD83, CD86, CD40 or HLA-DR. Therefore in an embodiment the myeloid-targeting peptide or protein is selected from an antibody or an antigen binding fragment thereof which selectively binds to CD11 b, CD11c, CD14, CD80, CD83, CD86, CD40 or HLA-DR.

[0359] In particular embodiments, the antibody or antigen binding fragment thereof is selected from a Fab, a Fab2, a scFv, a scFv-Fc, a dAb-Fc, a free light chain antibody, a half antibody, a bispecific Fab-,2"', a Fab3, a trispecific Fab-,3"' a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an IgNAR, a monovalent IgG, a VhH or a VNAR.

[0360] Alternatively a ligand or cofactor may be used which specifically or predominantly binds to a receptor or factor expressed on a myeloid cell, non-limiting examples being CD40L (CD154) and FC domains, but the skilled person is aware of other suitable ligands or cofactors. Therefore, in an embodiment the a myeloid-targeting molecule is a myeloid- targeting peptide or a myeloid-targeting protein wherein the myeloid-targeting protein or myeloid-targeting peptide is selected from CD40L (CD154) and FC domains.

[0361] In a further aspect, the invention relates to a nucleic acid encoding the fusion protein of a myeloid-targeting molecule and IL-4 as taught herein. In an embodiment the invention relates to a nucleic acid having a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 44 or comprising a nucleic acid sequence encoding a polypeptide having a sequence 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. 43.

[0362] The nucleic acid sequence can be used to express the fusion protein according to the invention, therefore in a preferred embodiment the nucleic acid is or is comprised in a vector, such as a protein expression vector or viral vector. The fusion protein can be expressed ex vivo, e.g. to be administrated to a subject later. Alternatively, the vector may be used to transiently or stably transform a cell in the subject. For example it may be particularly beneficial to transform hepatocytes, fibroblasts or myocytes, allowing them to express the fusion protein which can subsequently be released in the blood circulation to allow targeting of the myeloid compartment. When used herein, the term vector refers to a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The skilled person is aware how to adapt the enhancer and promoter regions for example for cell type specific or inducible expression of the gene (and subsequent translation into a protein).

[0363] It is further envisioned that instead of expressing an IL-4 fusion protein with a myeloid-targeting molecule which targets a myeloid cell, IL-4 can be expressed in or near a myeloid cell to ensure targeting of the IL-4 to the myeloid cell. Therefore, a further aspect provides a nucleic acid comprising a nucleic acid sequence 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. 44 or comprising a nucleic acid sequence encoding a polypeptide having a sequence 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. 43 and further comprising means for targeted expression in a myeloid cell, wherein said mean are selected from: - a promoter for selective or inducible expression in said myeloid cell operatively linked to said nucleic acid; or

[0364] - a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cell; or

[0365] - a lipid nanoparticle comprising one or more apolipoproteins, phospholipids, said nucleic acid and optionally sterol.

[0366] For example, a myeloid specific or enhanced promoter or promoter element may be used in a vector to drive myeloid specific expression of IL-4. Suitable promoters are known to the skilled person, non limiting exampkes are: lysM, Csfl r, CD11c, CX3CR1 , Langerin / CD207, MMLV LTR, Visna virus LTR, DC-STAMP, Human MSR, MSR-A, huCD68, CD4, CD2 and lba-AIF-1 (see e.g. Hume., Journal of leukocyte biology, Volume89, Issue4, April 2011 , Pages 525-538 for a review). Such promoter or promoter elements can be incorporated in a vector such as for example a lentiviral vector to stable transfect cells and allowing specific expression of the transgene in myeloid cells.

[0367] For example, viruses such as modified retroviruses may be used to specifically infect and drive expression of IL-4 in myeloid cells.

[0368] For example, lipid nanoparticles comprising apolipoproteins, cholesterol and phospholipids comprising mRNA encoding IL-4 may be used to specifically target myeloid cells and translate the mRNA into protein in the myeloid cells.

[0369] The inventors have demonstrated that IL-4 can reduce inflammation and induce trained immunity, particularly when targeted to the myeloid compartment. Therefore, a further aspect provides the fusion protein of a myeloid-targeting molecule and IL-4 or a nucleic acid encoding said fusion protein for use as a medicament. A further aspect provides the fusion protein of a myeloid-targeting peptide or protein and IL-4 or a nucleic acid encoding said fusion protein for use in the treatment of an immune related disorder, preferably wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction or stroke.

[0370] In other words, a further aspect provides a method of treating a subject in need thereof comprising administering the fusion protein of a myeloid-targeting peptide or protein and IL-4 or a nucleic acid encoding said fusion protein, preferably wherein the method of treatment is a method of treating an immune-related disorder.

[0371] The present inventors have shown for the first time that IL-4 can induce trained immunity. Further the inventors demonstrate that the unfavorable pharmacological properties of IL-4 (e.g. the extreme short half-life) can be avoided by targeting myeloid cells in an organism. Experimental data detailed below demonstrates the use of IL-4 as a targeted therapeutic in cases of hyperinflammation followed by immune paralysis, such as is the case in infectious disease such as COVID-19, by sepsis, myocardial infarction or stroke.

[0372] Accordingly, in particular embodiments, the fusion protein or nucleic acid for use as taught herein comprises reducing inflammation and / or stimulating or promoting trained immunity.

[0373] A further aspect provides in vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or an organism.

[0374] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described aspects and / or embodiments, without departing from the broad general scope of the present invention. The present aspects and / or embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The present invention includes the following non-limiting examples.

[0375] The present application also provides aspects and embodiments as set forth in the following Statements*:

[0376] Statement 1*. A fusion protein of an apolipoprotein or an apolipoprotein mimetic with an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response.

[0377] Statement 2*. A fusion protein of an apolipoprotein or an apolipoprotein mimetic with a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound (or in other words, wherein the rerouting molecule is a molecule that allows, when fused to the apolipoprotein, the apolipoprotein to bind to a different target than it would bind when the apolipoprotein was not fused to the rerouting molecule).

[0378] Statement 3*. A fusion protein of an apolipoprotein or an apolipoprotein mimetic with an immunomodulatory biomolecule and a rerouting molecule.

[0379] Statement 4*. The fusion protein according to any one of the previous statements*, wherein the immunomodulatory biomolecule is selected from a cytokine, a chemokine, a hormone, a growth factor, a hematopoietic growth factor or combinations thereof.

[0380] Statement 5*. The fusion protein according to statement 4*, wherein the cytokine is selected from the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 family, the TGFbeta family, or the IL-17 family, or combinations thereof, more preferably wherein the cytokine is selected from I L-1 p, IL-2, IL-4, IL-38, or combinations thereof; and / or wherein the chemokine is selected from a CC chemokine, a CXC chemokine, a C chemokine, a CX3C chemokine or combinations thereof; and / or wherein the growth factor is selected from VEGF, EGF, CNTF, LIF, Ephrins, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, Neurotrophin, PGF, PDGF, RNLS, TCGF, TGF, TNF and WNT or combinations thereof; and / or wherein 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 combinations thereof.

[0381] Statement 6*. The fusion protein according to any one of the previous statements*, wherein the rerouting molecule is selected from an antibody or an antigen binding fragment thereof, a rerouting peptide or a rerouting protein, preferably wherein the rerouting peptide or rerouting protein is a ligand of a receptor present on the target.

[0382] Statement 7*. The fusion protein according to statement 6*, wherein the antibody or antigen binding fragment thereof is selected from a Fab, a Fab2, a scFv, a scFv-Fc, a dAb-Fc, a free light chain antibody, a half antibody, a bispecific Fab2, a Faba, a trispecific Fab3 a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an IgNAR, a monovalent IgG, a VhH or a VNAR; and / or wherein the rerouting peptide is selected from PD1 or SIRPa; and / or wherein the rerouting protein is selected from CD40L or GP120.

[0383] Statement 8*. The fusion protein according to any one of the previous statements*, wherein the apolipoprotein or apolipoprotein mimetic is an ApoA1 , ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3 or a mimetic thereof.

[0384] Statement 9*. A lipid nanoparticle comprising one or more fusion proteins as defined in statement 1* or 4* or 5* or 8* and / or one or more fusion protein as defined in statement 2* or 6* or 7* or 8* and / or one or more fusion protein as defined in statements 3* to 8*, the lipid nanoparticle further comprising phospholipids, and sterols.

[0385] Statement 10*. Lipid nanoparticle as defined in statement 9*, wherein the lipid nanoparticle further comprises lipids.

[0386] Statement 11*. Lipid nanoparticle according to statement 9* or 10*, wherein the lipid nanoparticle further comprises a payload, preferably wherein the payload is selected from a nucleic acid or a nucleic acid analog, a therapeutic, a biologic or combinations thereof.

[0387] Statement 12*. Method of manufacturing a lipid nanoparticle as defined in any one of statements 9* to 11*, the method comprising the steps of: a1) expressing and isolating an apolipoprotein fusion protein to obtain an isolated apolipoprotein fusion protein, where the apolipoprotein fusion protein is an apolipoprotein fused to a cytokine and a targeting moiety and / or wherein the apolipoprotein fusion protein is an apolipoprotein fused to a cytokine and / or an apolipoprotein fused to a targeting moiety; and / or a2) chemically conjugating an apolipoprotein and isolating the conjugated apolipoprotein to obtain an isolated conjugated apolipoprotein, where the conjugated apolipoprotein is an apolipoprotein conjugated to a cytokine and a targeting moiety and / or wherein the conjugated apolipoprotein is an apolipoprotein conjugated to a cytokine and / or an apolipoprotein conjugated to a targeting moiety; b) combining the isolated apolipoprotein fusion protein obtained in step a1 and / or the isolated conjugated apolipoprotein obtained in step a2 with phospholipids, sterols and optionally lipids to obtain a lipid nanoparticle.

[0388] Statement 13*. The fusion protein according to any one of statements 1* to 8* or the lipid nanoparticle according to any one of statements 9* to 11*, or the lipid nanoparticle obtained or obtainable by the method of statement 12* for use as a medicament.

[0389] Statement 14*. The fusion protein according to any one of statements 1* to 8* or the lipid nanoparticle according to any one of statements 9* to 11*, or the lipid nanoparticle obtained or obtainable by the method of statement 12* for use in the treatment of an immune related disorder.

[0390] Statement 15*. Use of a fusion protein according to any one of statements 1* to 8* or a lipid nanoparticle according to any one of statements 9* to 11*, or a lipid nanoparticle obtained or obtainable by the method of statement 12* in delivering a compound to a target, preferably wherein the target is a cell, tissue, and / or organ.

[0391] Statement 16*. A nucleic acid encoding the fusion protein as defined in any one of statements 1* to 8*.

[0392] The present application also provides aspects and embodiments as set forth in the following Statements:

[0393] Statement 1. An apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule; and phospholipids; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response.

[0394] Statement 2. An apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule; and phospholipids; wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

[0395] Statement 3. An apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule; and phospholipids; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response; and wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

[0396] Statement 4. An apolipoprotein lipid nanoparticle comprising the fusion protein as defined in Statement 1 ; the fusion protein as defined in Statement 2; and phospholipids.

[0397] Statement 5. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to

[0398] 4, wherein the apolipoprotein lipid nanoparticle further comprises sterols.

[0399] Statement 6. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to

[0400] 5, wherein the apolipoprotein lipid nanoparticle further comprises lipids, preferably triglycerides.

[0401] Statement 7. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to

[0402] 6, wherein said apolipoprotein lipid nanoparticle is a sphere, a ribbon or a disc.

[0403] Statement 8. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to

[0404] 7, wherein at least a part of said fusion protein is exposed to the environment surrounding said apolipoprotein lipid nanoparticle, preferably wherein said immunomodulatory biomolecule and / or said rerouting molecule is exposed to the environment surrounding said apolipoprotein lipid nanoparticle.

[0405] Statement 9. The apolipoprotein lipid nanoparticle according to any one of Statements 1 or 3 to 8, wherein the immunomodulatory biomolecule is selected from the group consisting of a cytokine, a chemokine, a hormone, a growth factor, a hematopoietic growth factor, and combinations thereof.

[0406] Statement 10. The apolipoprotein lipid nanoparticle according to Statement 9, wherein the cytokine is selected from the group consisting of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 family, the TGFbeta family, or the IL-17 family, and combinations thereof, more preferably wherein the cytokine is selected from the group consisting of IL-1 p, IL-2, IL-4, IL-38, and combinations thereof; and / or wherein the chemokine is selected from the group consisting of a CC chemokine, a CXC chemokine, a C chemokine, a CX3C chemokine, and combinations thereof; and / or wherein the growth factor is selected from the group consisting of VEGF, EGF, CNTF, LIF, Ephrins, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, Neurotrophin, PGF, PDGF, RNLS, TCGF, TGF, TNF and WNT, and combinations thereof; and / or wherein the hematopoietic growth factor is selected from the group consisting of IL-3, CSF- 1 (M-CSF), GM-CSF, G-CSF, a member of the IL-12 family of interleukins or erythropoietin, and combinations thereof.

[0407] Statement 11. The apolipoprotein lipid nanoparticle according to Statement 9 or 10, wherein the cytokine is IL-4.

[0408] Statement 12. The apolipoprotein lipid nanoparticle according to any one of Statements 2 to 11 , wherein the rerouting molecule is selected from an antibody or an antigen binding fragment thereof, a rerouting peptide or a rerouting protein, preferably wherein the rerouting peptide or rerouting protein is a ligand of a receptor present on the target.

[0409] Statement 13. The apolipoprotein lipid nanoparticle according to Statement 12, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of a Fab, a Fab2, a scFv, a scFv-Fc, a dAb-Fc, a free light chain antibody, a half antibody, a bispecific Fab2, a Faba, a trispecific Fab3 a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an IgNAR, a monovalent IgG, a VhH, and a variable new antigen receptor (VNAR).

[0410] Statement 14. The apolipoprotein lipid nanoparticle according to any one of

[0411] Statements 2 to 13, wherein the rerouting molecule is capable of binding to a hematopoietic stem and progenitor cell (HSPC), such as a hematopoietic stem cell (HSC), a multipotent progenitor (MPP), or a common myeloid progenitor cell (CMP).

[0412] Statement 15. The apolipoprotein lipid nanoparticle according to any one of Statements 2 to 13, wherein the rerouting molecule is capable of binding to a myeloid cell selected from the group consisting of megakaryocyte, eosinophil, basophil, erythrocyte, monocyte such as dendritic cell or macrophage, and a neutrophil.

[0413] Statement 16. The apolipoprotein lipid nanoparticle according to Statement 15, wherein the rerouting peptide is SIRPa.

[0414] Statement 17. The apolipoprotein lipid nanoparticle according to any one of Statements 2 to 13, wherein the rerouting molecule is capable of binding to a non-myeloid cell, such as a non-myeloid immune cell or an endothelial cell. Statement 18. The apolipoprotein lipid nanoparticle according to Statement 17, wherein the rerouting molecule is capable of binding to lymphocytes, preferably T cells, more preferably CD8+ T cells.

[0415] Statement 19. The apolipoprotein lipid nanoparticle according to Statement 17, wherein the rerouting molecule is an antibody or antigen binding fragment thereof specifically binding to CD8 or wherein the rerouting peptide is PD1 , CD40L or GP120.

[0416] Statement 20. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to 19, wherein the apolipoprotein is an ApoA1 , ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3 or the apolipoprotein mimetic is a mimetic of an ApoA1 , ApoA- 1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3.

[0417] Statement 21. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to 20, wherein the apolipoprotein lipid nanoparticle comprises a payload, preferably wherein the payload is selected from a nucleic acid or a nucleic acid analog, a therapeutic, a biologic or combinations thereof.

[0418] Statement 22. The apolipoprotein lipid nanoparticle according to Statement 21 , wherein the apolipoprotein lipid nanoparticle comprises a nucleic acid and a cationic or ionizable cationic lipid.

[0419] Statement 23. The apolipoprotein lipid nanoparticle according to Statement 22, wherein the apolipoprotein lipid nanoparticle comprises a core surrounded by a surface layer, the nucleic acid and the cationic or ionizable cationic lipid are comprised by the core, and the fusion protein or fusion proteins as defined in any one of Statements 1 to 3 and the phospholipids are comprised by the surface layer.

[0420] Statement 24. An apolipoprotein lipid nanoparticle comprising a core surrounded by a surface layer, wherein: the core comprises a nucleic acid and a cationic or ionizable cationic lipid; and the surface layer comprises: a phospholipid, a sterol, and

[0421] (i) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response;

[0422] (ii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; (iii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule as defined in (i), and a rerouting molecule as defined in (ii); or

[0423] (iv) the fusion protein as defined in (i) and the fusion protein as defined in (ii).

[0424] Statement 25. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 24, wherein the nanoparticle core further comprises a filler, preferably a filler selected from a triacylglyceride and a cholesterol acyl ester, or combinations thereof, such as wherein the triacylglyceride is tricaprylin and / or wherein the cholesterol acyl ester is cholesteryl caprylate and / or cholesteryl oleate.

[0425] Statement 26. The apolipoprotein lipid nanoparticle according to any one of Statements 21 to 25, wherein the nucleic acid is RNA, DNA or a nucleic acid analogue.

[0426] Statement 27. The apolipoprotein lipid nanoparticle according to Statement 26, wherein the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), long non-coding RNA (IncRNA), or guide RNA (gRNA), or combinations thereof and / or modifications thereof.

[0427] Statement 28. The apolipoprotein lipid nanoparticle according to Statement 26, wherein the DNA is single stranded or double stranded DNA.

[0428] Statement 29. The apolipoprotein lipid nanoparticle according to any one of Statements 21 to 25, wherein the nucleic acid is an antisense oligonucleotide and the antisense oligonucleotide is single strand DNA or RNA consisting of nucleotide or nucleoside analogues containing modifications of the phosphodiester backbone or the 2’ ribose.

[0429] Statement 30. The apolipoprotein lipid nanoparticle according to Statement 29, wherein the nucleotide or nucleoside analogues are 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 acids (HNA), or mixtures or combinations thereof.

[0430] Statement 31. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 30, wherein the cationic 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 combinations thereof. Statement 32. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 31 , wherein the ionizable cationic lipid is a molecule according to any one of Formulae (I), (II), (III), (IV) or (V) wherein ICG is or , wherein the wavy line indicates the point of attachment to the compound of formulae (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 Ri is independently selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl group, wherein said alkyl or alkenyl group optionally contains up to 5 heteroatoms, independently selected from O and N;

[0431] R2 is selected from the group consisting of hydrogen, methyl, ethyl and a -CH2-O-C(O)-Ria;

[0432] R3 is selected from the group consisting of hydrogen, aryl, arylene-alkyl, alkylene-aryl and linear C1-C6 alkyl group;

[0433] Ria is selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl group, wherein said alkyl or alkenyl group optionally contains up to 5 heteroatoms, independently selected from O and N; each Rxis independently selected from the group consisting of methyl, ethyl, propyl and - CH2-CH2-OH; each Rygroup is independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein said alkyl group optionally contains up to 5 heteroatoms, independently selected from O and N; or rotamers, tautomers stereoisomers or regioisomers thereof.

[0434] Statement 33. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 32, wherein: the amount of the fusion protein ranges from 0.08 to 2.0 mol%, such as from 0.10 to 2.0 mol%; and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 15 to 90 mol%; and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol%; and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 8.0 to 80 mol%, wherein the molar percentage is based solely on the combined amounts of the fusion protein, phospholipids, sterols and cationic or ionizable cationic lipids in the apolipoprotein lipid nanoparticle.

[0435] Statement 34. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 33, wherein: the amount of the fusion protein ranges from 0.1 to 90 weight%; the amount of nucleic acid ranges from 0.01 to 90 weight%; the amount of phospholipid ranges from 0.1 to 95 weight%; the amount of sterol ranges from 0.1 to 95 weight%; and / or the amount of cationic and / or ionizable cationic lipid ranges from 0.1 to 95 weight%, wherein these weight percentages are based on the combined amounts of the fusion protein, the nucleic acid, the phospholipid, the sterol and the cationic and / or ionizable cationic lipid. Statement 35. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 34, wherein the ratio of the fusion protein (in case of two or more fusion proteins, cumulatively) to phospholipid based on percentage molar weight is between 1 :25 and 1 :400, more preferably between 1 :50 and 1 :200, even more preferably between 1 :75 and 1 :150. Statement 36. The apolipoprotein lipid nanoparticle according to any one of Statements 22 to 35, wherein the ratio of the fusion protein (in case of two or more fusion proteins, cumulatively) to phospholipid based on weight is from 2:1 to 1 :10, more preferably from 1 :1 to 1 :5, even more preferably from 1 :1.5 to 1 :4.

[0436] Statement 37. Method of manufacturing an apolipoprotein lipid nanoparticle as defined in any one of Statements 1 to 36, the method comprising the steps of: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; and / or a2) chemically conjugating one or more apolipoproteins or apolipoprotein mimetics and isolating the one or more conjugated apolipoproteins to obtain one or more isolated conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; and b) combining the one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or the one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids, and optionally sterols and / or lipids, to obtain an apolipoprotein lipid nanoparticle.

[0437] Statement 38. Method for producing an apolipoprotein lipid nanoparticle, comprising the step of: a) rapid mixing of lipid components in organic solvent with a nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid components comprise a phospholipid, a sterol, a cationic lipid or ionizable cationic lipid, wherein the aqueous buffer has a pH of 5.0 or lower; and b) rapid mixing of the lipid nanoparticles with: one or more apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, and the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; and / or one or more conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, and the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; to produce the apolipoprotein lipid nanoparticle at a pH between 5.5 and 8.0, such as between 6.0 and 8.0.

[0438] Statement 39. An apolipoprotein lipid nanoparticle obtained by or obtainable by the method of Statement 37 or 38.

[0439] Statement 40. A pharmaceutical composition comprising the apolipoprotein lipid nanoparticle according to any one of Statements 1 to 36 or 39, and a pharmaceutically acceptable carrier.

[0440] Statement 41. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to 36 or 39 or the pharmaceutical composition according to Statement 40 for use as a medicament.

[0441] Statement 42. The apolipoprotein lipid nanoparticle according to any one of Statements 1 to 36 or 39 or the pharmaceutical composition according to Statement 40 for use in the treatment of an immune related disorder.

[0442] Statement 43. The apolipoprotein lipid nanoparticle for use according to Statement 42 or the pharmaceutical composition for use according to Statement 42, wherein the immune related disorder is selected from the group consisting of cancer, inflammation, an infectious disease, an autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH). Statement 44. The apolipoprotein lipid nanoparticle for use according to Statement 42 or the pharmaceutical composition for use according to Statement 42, wherein the immunomodulatory biomolecule is IL-4 and wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

[0443] Statement 45. The apolipoprotein lipid nanoparticle according to any one of Statements 1 , 3 to 36 or 39 or the pharmaceutical composition according to Statement 40 for use in targeting said immunomodulatory biomolecule to a target cell.

[0444] Statement 46. The apolipoprotein lipid nanoparticle according to any one of Statements 1 , 3 to 13, 15, 16, 20, 21 to 36 or 39 or the pharmaceutical composition according to Statement 40 for use in targeting said immunomodulatory biomolecule to a myeloid cell.

[0445] Statement 47. Use of the apolipoprotein lipid nanoparticle according to any one of Statements 1 , 3 to 36 or 39 for delivering an immunomodulatory biomolecule to a target, preferably wherein the target is a cell, tissue, and / or organ.

[0446] Statement 48. An in vitro or ex vivo method for introducing a nucleic acid in a cell, the method comprising contacting the apolipoprotein lipid nanoparticle according to any one of Statements 21 to 36 or 39 or the composition according to Statement 40 with a cell.

[0447] Statement 49. An in vivo method for introducing a nucleic acid in a cell, the method comprising contacting the apolipoprotein lipid nanoparticle according to any one of Statements 21 to 36 or 39 or the composition according to Statement 40 with a cell.

[0448] Statement 50. The apolipoprotein lipid nanoparticle according to any one of Statements 21 to 36 or 39 or the composition according to Statement 40 for use in the in vivo delivery of a nucleic acid to a subject.

[0449] Statement 51. A method for the in vivo delivery of a nucleic acid, the method comprising administering the apolipoprotein lipid nanoparticle according to any one of Statements 21 to 36 or 39 or the composition according to Statement 40 to a subject.

[0450] Statement 52. A method for treating a disease or disorder, such as a disease or disorder as described herein, in a subject in need thereof, for example by stimulating or inhibiting an innate immune response, the method comprising administering a therapeutically effective amount of the apolipoprotein lipid nanoparticle according to Statements 21 to 36 or 39 or the composition according to Statement 40 to the subject.

[0451] Statement 53. The method according to Statement 52, wherein the disease is selected from cancer, cardiovascular disease, autoimmune disorder or xenograft rejection. Statement 54. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, for use in targeting said immunomodulatory biomolecule to a myeloid cell.

[0452] Statement 55. The fusion protein for use according to Statement 54, wherein the fusion protein further comprises a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity, preferably wherein the rerouting molecule is a rerouting molecule as defined in Statement 15.

[0453] Statement 56. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

[0454] Statement 57. The fusion protein according to Statement 56, wherein the rerouting molecule is a rerouting molecule as defined in any one of Statements 12 to 19.

[0455] Statement 58. The fusion protein according to Statement 56 or 57, wherein the fusion protein further comprises an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, preferably wherein the immunomodulatory biomolecule is an immunomodulatory biomolecule as defined in Statement 9 or 10.

[0456] Statement 59. A nucleic acid encoding the fusion protein according to any one of Statements 56 to 58.

[0457] Statement 60. A pharmaceutical composition comprising the fusion protein according to any one of Statements 56 to 58 or the nucleic acid according to Statement 59, and a pharmaceutically acceptable carrier.

[0458] Statement 61. The fusion protein according to any one of Statements 56 to 58, the nucleic acid according to Statement 59 or the pharmaceutical composition according to Statement 60 for use as a medicament.

[0459] Statement 62. The fusion protein according to any one of Statements 56 to 58, the nucleic acid according to Statement 59 or the pharmaceutical composition according to Statement 60 for use in the treatment of an immune related disorder, preferably wherein the immune related disorder is an immune related disorder selected from the group consisting of cancer, inflammation, an infectious disease, an autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

[0460] Statement 63. The fusion protein according to Statement 58, the nucleic acid encoding the fusion protein according to Statement 58 or the pharmaceutical composition according to Statement 60 when being dependent from Statement 58 for use in targeting said immunomodulatory biomolecule to a target cell.

[0461] Statement 64. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4).

[0462] Statement 65. The fusion protein according to Statement 64, wherein the fusion protein further comprises a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity, preferably wherein the rerouting molecule is a rerouting molecule as defined in any one of Statements 12 to 19.

[0463] Statement 66. The fusion protein according to Statement 64 or 65, wherein the apolipoprotein or apolipoprotein mimetic is as defined in Statement 20.

[0464] Statement 67. A nucleic acid encoding the fusion protein according to any one of Statements 64 to 66.

[0465] Statement 68. A pharmaceutical composition comprising the fusion protein according to any one of Statements 64 to 66 or the nucleic acid according to Statement 67, and a pharmaceutically acceptable carrier.

[0466] Statement 69. The fusion protein according to any one of Statements 64 to 66, the nucleic acid according to Statement 67, or the pharmaceutical composition according to Statement 68 for use as a medicament.

[0467] Statement 70. The fusion protein according to any one of Statements 64 to 66, the nucleic acid according to Statement 67, or the pharmaceutical composition according to Statement 68 for use in the treatment of an immune related disorder.

[0468] Statement 71. The fusion protein for use according to Statement 70, the nucleic acid for use according to Statement 70, or the pharmaceutical composition for use according to Statement 70, wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

[0469] Statement 72. The fusion protein according to any one of Statements 64 to 66, the nucleic acid according to Statement 67, or the pharmaceutical composition according to Statement 48 for use in targeting IL-4 to a target cell.

[0470] Statement 73. The fusion protein according to any one of Statements 64 to 66, the nucleic acid according to Statement 67, or the pharmaceutical composition according to Statement 68 for use in targeting IL-4 to a myeloid cell. Statement 74. A fusion protein comprising a myeloid-targeting molecule and IL-4, wherein the myeloid-targeting molecule is capable of targeting the IL-4 to a myeloid cell. Statement 75. The fusion protein according to Statement 74, wherein the IL-4 is a polypeptide comprising an amino acid sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 43, or a circular permutation thereof. Statement 76. The fusion protein according to Statement 74 or 75, wherein the myeloid-targeting molecule is selected from an antibody or an antigen binding fragment thereof, a myeloid-targeting peptide or a myeloid-targeting protein, preferably wherein the myeloid-targeting peptide or myeloid-targeting protein is a ligand of a receptor present on the target.

[0471] Statement 77. The fusion protein according to Statement 76, wherein the antibody or antigen binding fragment thereof is selected from a Fab, a Fab2, a scFv, a scFv-Fc, a dAb- Fc, a free light chain antibody, a half antibody, a bispecific Fab2, a Faba, a trispecific Fab3 a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an IgNAR, a monovalent IgG, a VhH or a VNAR.

[0472] Statement 78. A nucleic acid encoding the fusion protein according to any one of Statements 74 to 77.

[0473] Statement 79. A nucleic acid comprising a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 44 or comprising a nucleic acid sequence encoding a polypeptide having a sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 43 and further comprising means for targeted expression in a myeloid cell, wherein said mean are selected from:

[0474] - a promoter for selective or inducible expression in said myeloid cell operatively linked to said nucleic acid; or

[0475] - a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cell; or

[0476] - a lipid nanoparticle comprising one or more apolipoproteins, phospholipids, said nucleic acid, and optionally sterol.

[0477] Statement 80. A pharmaceutical composition comprising the fusion protein according to any one of Statements 74 to 77 or the nucleic acid according to Statement 78 or 79, and a pharmaceutically acceptable carrier.

[0478] Statement 81. The fusion protein according to any one of Statements 74 to 77, the nucleic acid according to Statement 78 or 79, or the pharmaceutical composition according to Statement 80 for use as a medicament. Statement 82. The fusion protein according to any one of Statements 74 to 77, the nucleic acid according to Statement 78 or 79, or the pharmaceutical composition according to Statement 80 for use in the treatment of an immune related disorder.

[0479] Statement 83. The fusion protein for use according to Statement 82, the nucleic acid for use according to Statement 82, or the pharmaceutical composition for use according to Statement 82, wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction or stroke.

[0480] Statement 84. In vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or an organism.

[0481] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

[0482] The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples.

[0483] EXAMPLES

[0484] Example 1. Preparation of fusion proteins of an apolipoprotein fused to an immunomodulatory biomolecule and incorporation thereof into lipid nanoparticles

[0485] Materials & methods

[0486] Protein expression, purification, and characterization of ApoA1-S147C (e.g. SEQ ID NO. 9) and ApoA1-S279C (e.g. SEQ ID NO. 11) mutants, apoA1-IL4 fusion proteins (e.g. SEQ ID NO. 35), apoA1-IL2 (e.g. SEQ ID NO. 23) or apoA1-IL2v4 (e.g. SEQ ID NO. 31) fusion protein, apoA1-ILi p fusion protein (e.g. SEQ ID NO. 21), and apoA1-IL38 fusion protein (e.g. SEQ ID NO. 80) (Figures 1-9)

[0487] Bacterial cells containing the pET-vector coding for the desired protein were grown in 40 mL of 2YT medium supplemented with extra NaCI (10 g / L) and 100 pg / mL ampicillin was inoculated with transformed bacteria and grown overnight. The following day, 2YT medium containing 10 g / L NaCI and 100 pg / mL ampicillin was inoculated with the overnight culture and incubated at 37 °C at 150 rpm until an OD600 of 0.6-0.8 was reached. Isopropyl p-d- thiogalacopyranoside (IPTG) was added to a final concentration of 0.1 mM. The culture was further incubated at 20 °C at 150 rpm overnight. Bacteria were pelleted using centrifugation and lysed using Bugbuster® protein extraction reagent (Novagen) per gram cell pellet supplied with Benzonase® Nuclease (Merck), following the manufacturer’s protocols. Lysates were centrifuged and resulting supernatant containing protein of interest was then processed on a IMAC column with Ni-NTA HisBind® Resin (Merck Millipore).

[0488] Obtained fractions were analyzed using SDS-PAGE (Samples were combined with sample buffer (1 :1) and run on a Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad). Resulting gels were stained using Coomassie G-250 Stain and de-stained using dH2O Figures 3, 5).

[0489] Obtained purified proteins of interest were further characterized using Q-ToF. Samples were diluted in 0.1 % formic acid in dH2O to a concentration of 0.01 to 0.1 mg / mL. After filtration using a PD Spintrap™ G-25 column (0.5 mL, Cytiva), the samples were measured using a Waters ACQUITY LIPLC l-Class system with a Xevo G2 Quadrupole Time of Flight mass spectrometer. The proteins were separated by a C8A reverse-phase column. A gradient of 15% to 75% acetonitrile in 0.1 % formic acid in dH2O was used. Resulting data was analyzed using MassLynx (Waters) with the MaxEnt algorithm (Figure 6).

[0490] Protein expression, purification, and characterization of ApoA1-S147C (e.g. SEQ ID NO. 9) and ApoA1-S279C (e.g. SEQ ID NO. 11) mutants, apoA1-IL4 fusion proteins (e.g. SEQ ID NO. 35), apoA1-IL2 (e.g. SEQ ID NO. 58) or apoA1-IL2v4 (e.g. SEQ ID NO. 60) fusion protein, apoA1-ILi p fusion protein (e.g. SEQ ID NO. 21 or SEQ ID NO. 82), and apoA1-IL38 fusion protein (e.g. SEQ ID NO. 80 or SEQ ID NO. 84) (Figures 10-16)

[0491] Bacterial expression and protein purification): performed as described in the section “bacterial expression and protein purification” of Example 2.

[0492] Bacterial lysis and protein purification: performed as described in the section “bacterial lysis and protein purification” as described in Example 2.

[0493] Discoidal lipid nanoparticle (LNP) formulation

[0494] To formulate HDL-based LNPs, phospholipids, apoA1 fusion proteins and cholesterol are used. For discoidal LNPs, the fusion protein (in PBS) is mixed with a mixture of cholesterol and 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (in 95% acetonitrile / 5% methanol) in a 1 :10:100 molar ratio, using a staggered herringbone microfluidic mixer. Dynamic Light Scattering measurements were performed on a Zetasizer Nano ZSP with the nanobiologics to assess size and heterogeneity. 100 pL of sample volume was pipetted into a transparent cuvette (Sarstedt) which was inserted into a Malvern Zetasizer Nano ZSP. The sample was measured in triplicate with 10 cycles per run at a temperature of 25 °C.

[0495] Azide introduction in immunomodulatory biomolecule and conjugation to apoA1 or a cysteine mutant thereof

[0496] ApoA 1-IL4 fusion (for data represented in Figures 1-9): IL-4 was buffer exchanged to DEA buffer (50 mM diethanolamine, pH 7.5) and concentrated to 0.75 mM. lmidazole-1-sulfonyl azide hydrochloride (Fluorochem) was dissolved in dH2O to obtain a 20 mg / mL stock solution. The pH was adjusted to 7. This was then added to the IL-4 in a 227.5:1 molar ratio (17.5 molar equivalents per amine in IL-4). The reaction mixture was incubated at 4 °C overnight. The DBCO-PEG12-maleimide linker (Sigma-Aldrich) was dissolved in DMSO. The dissolved linker was then added to apoA1 S279C (0.5 mM) to a final concentration of 5 mM. This reaction mixture was also incubated at 4 °C overnight. The resulting functionalized IL-4-Az and apoA1-DBCO were then buffer exchanged to PBS (pH 7.9) to remove excess azide transfer reagent and DBCO-maleimide linker. The solutions were added together in a 1 :2 apoA1 : IL-4 molar ratio, and incubated at 4 °C overnight. The following day the mixture was purified on a IMAC column with Ni-NTA HisBind® Resin (Merck Millipore). Fractions were collected and analyzed using SDS-PAGE. Analysis was performed using Q-ToF (same method as described above) and a HEK293 IL-4 reporter assay.

[0497] ApoA 1 -cytokine fusion (for data represented in Figures 10-16): Alternatively, stock solutions of imidazole-1-sulfonyl azide (2 mg / mL) in MQ were prepared. The pH of this stock was set to 7.5. To 100 pg cytokine in PBS (pH=7.5), 17,5 molar equivalents of imidazole-1-sulfonyl azide per primary amine were added. The reaction was incubated overnight at 4°C. After overnight incubation the excess imidazole-1-sulfonyl azide was removed by using a PD minitrap G-25 desalting column (Cytiva) and the resulting product was analyzed by Q-ToF LC-MS (WatersMassLynx v4.1), using MagTran V1.03 for MS. A 5x molar excess of a maleimide-PEG4-DBCO linker was added to ApoA1 or a cysteine mutant thereof and reacted overnight to form the apoA1-PEG4-DBCO complex. The excess linker was removed using an Amicon Ultra-0.5 Centrifugal Filter Unit (Merck) with MWCO 10 kDa. ApoA1-PEG4- DBCO was then combined with the azide-containing cytokine and incubated for 4 hours at RT or overnight at 4 °C. Resulting product was analyzed using SDS-page.

[0498] HEK293 IL-4 reporter assay HEK-Blue™ IL-4 cells (Invivogen) were seeded in T25 culture flasks and grown at 37 °C until a confluency of 80% was reached. Cell viability was checked using a microscope. The cells were washed with sterile PBS and harvested using trypsinization for 5 minutes at 37 °C. The cells were then seeded into a 96 wells plate so that each well contained 50.000 cells (180 pL DMEM, 10% FBS, 1 % Pen-Strep). For each condition a 2-fold dilution series was prepared. The dilution series were added to the plate containing cells in triplo. The cells were then incubated for 24 hours at 37 °C. A QUANTI-Blue™ (Invivogen) solution was prepared according to manufacturer’s instructions. In another 96 wells plate, 20 pL of conditioned cell medium was added together with 180 pL of QUANTI-Blue™ solution. This was then incubated for 3 hours at 37 °C. The absorbance was analyzed using a Spark® multimode microplate reader (Tekan) at 635 nm.

[0499] Effect of IL2 constructs on T-cell proliferation

[0500] Human CD3 positive T cells were stained with CFSE (Thermofisher) according to manufacturer’s protocol. After incubation 100.000 T cells were seeded in 96 well round bottom plates and stimulated with IL2 or IL2 constructs for 6 days. T cells were harvested, washed, and stained for CD3, CD4 and CD8 and measured on Cytoflex (Beckman Coulter Inc.). Flow cytometry data was analyzed using FlowJo software (BD). Commercial IL2 was purchased from Sinobiological. Recombinant IL2 was prepared as described below.

[0501] Bacterial expression of recombinant IL-2

[0502] The SUMO-IL2 construct was transformed into Shuffle T7 competent E. Coli. 40 mL of 2YT medium containing 50 pg / mL kanamycin, was inoculated with bacteria and grown overnight at 250 rpm and 37 °C to form a small culture. The following day, 2YT medium containing 50 pg / mL kanamycin was inoculated with the small culture to form a large culture. The culture was incubated at 150 rpm and 37 °C until an QD600 of 0.6-0.8 was reached. Then to induce protein expression, IPTG was added to a final concentration of 0.1 mM. The culture was further incubated at 20 °C at 150 rpm overnight. Bacterial pellets were then obtained by centrifugation at 10.000xg for 10 minutes at 4°C. The resulting supernatant was discarded. The obtained pellet obtained was resuspended in 10 mL lysis buffer (20 mM TRIS, 500 mM NaCI, pH 7.9) per gram cell pellet. 25 U Benzonase® Nuclease (Merck) was then added. One complete™ Protease Inhibitor Cocktail Tablet was added per 50 mL of extraction buffer. The resulting solution was stirred at 4 °C for 30 minutes until no clumps remained. The solution was then homogenized three times using the Avestin Emulsiflex C3 at 15.000- 20.000 psi while being kept on ice. The cell lysate was then centrifuged at 20.000xg for 30 minutes at 4 °C. The resulting supernatant was applied to an IMAC column, at 4 °C, which was previously charged with an 0.1 M NiSO4 solution. All flowthrough fractions were collected. The column was washed with 8 column volumes of buffer A (20 mM Tris, 500 mM NaCI, 10 mM imidazole, pH 7.9), then 8 column volumes of buffer A50 (20 mM Tris, 500 mM NaCI, 50 mM imidazole, pH 7.9). To elute SUMO-IL2, 8 column volumes of buffer A500 (20 mM Tris, 500 mM NaCI, 500 mM imidazole, pH 7.9) was applied to the column. Obtained fractions were analyzed using SDS-PAGE. The eluted fractions containing SUMO-IL2 were pooled. SUMO hydrolase was added to the pooled fractions of SUMO-IL2 in a ratio of 1 mg hydrolase I 500 mg protein. The solution was then dialyzed to storage buffer (20 mM TRIS, 500 mM NaCI, pH 7.9) using a Snakeskin™ 10 kDa cutoff dialysis bag (Thermo Scientific) while gently stirring at 4 °C overnight. Then the resulting protein solution was centrifuged at 4000xg for 20 minutes and the supernatant was filtered using a 0.2 pM syringe filter to remove aggregated protein. The IMAC column protocol was repeated and the fractions were again analyzed using SDS-PAGE. If the fraction containing IL2 was contaminated with other protein, the sample was purified using Size Exclusion Chromatography (SEC). Otherwise the fraction containing IL2 was buffer exchanged to PBS (pH 7.9). SEC was performed a NGC 10 Medium-Pressure Chromatography System (Bio-Rad) with a GE Hiload 16 / 60 Superdex 75 pg column. The column was first equilibrated using filtered PBS (pH 7.9) after which the sample was applied. The collected fractions were analyzed using SDS-PAGE. Fractions containing IL2 were pooled and the final concentration was determined using a Nanodrop™ 1000 spectrophotometer. The protein was then snap-frozen in liquid nitrogen, and stored at -80 °C. Analysis was performed using Q-tof, and SPR.

[0503] Cryo-transmission electron microscopy (cryo-TEM): performed as described in Example 2.

[0504] Determining aNP size and dispersity by DLS: performed as described in Example 2.

[0505] SDS page: performed as described in Example 2

[0506] Results

[0507] Our fusion proteins can be made by recombinant expression, or by chemical conjugation. Thus far, we have managed to express four different fusion proteins recombinantly. These are apoA1-IL4, apoA1-IL1 B, apoA1-IL38 and apoA1-IL2. All expressions were successful and resulted in pure protein, as can be observed in figure 3. The black rectangle indicates the elution fractions containing our fusion protein of interest. These recombinantly expressed proteins were then used to formulate nanobiologic discs (figure 4). It can be appreciated that apoA1-IL4 and apoA1-IL38 remain stable over 11 days and show similar diameters and Pdl as apoA1. ApoA1-IL1b shows this as well, although after 11 days these nanobiologics appear to aggregate.

[0508] In order to chemically conjugate apoA1 to cytokines, nanobodies, or other biomolecules, a reactive handle is needed. Therefore, apoA1 mutants were created that contain a cysteine in the place of a serine at position 147 (referred to herein as the “S147C” or “S157C” mutant) or 279 (referred to herein as the “S279C” or “S239C” mutant). For example, an apoA1 mutant may be defined by a peptide sequence as set forth in SEQ ID NO. 9 or 11 . Expression and purification of these proteins was successful, as can be seen in figure 5, and resulted in yields and purities similar to wild type apoA1. The obtained proteins were further characterized using quadrupole time-of-fligh (Q-ToF), which showed that the protein was pure, and the correct mass was found (figure 6).

[0509] Conjugating cytokines to this apoA1 mutant can be done using a Maleimide-PEG-DBCO linker, where the maleimide couples to the apoA1 and the DBCO couples to an azide that we introduce in the cytokines. Introduction of a single azide at the N-terminus of IL-4 was optimized until ±90% of the product consisted of IL-4-Az (IL-4 with single azide incorporated). This was analyzed using Q-ToF (figure 7) and SDS-PAGE (figure 8). From the SDS-page it can be seen that upon conjugation of ApoA1S279C and IL4, a band appears around 40 kDa, corresponding to the expected molecular mass of the fusion protein. This confirms that we have chemically conjugated IL4 to apoA1 using a PEG-linker (figure 8). While this conjugation reaction is still being optimized, we have already got a yield of ±20%. The bioactivity of the recombinantly produced fusion proteins as well as the chemical apoA1- IL4 conjugate was then analyzed using a HEK293 IL-4 reporter assay. This cell line has a fully active STAT6 pathway and carries a STAT6-inducible secreted alkaline phosphatase (SEAP) reporter gene. Here, SEAP production is linked to binding of IL4 to its receptor. The HEK-BlueTM IL-4 / IL-13 cells produce SEAP in response to IL-4 and IL-13. The IL-4 binding can be quantified by checking the enzymatic activity of SEAP using QUANTI-Blue colorimetric assay.

[0510] As can be seen from figure 9, both fusion proteins exert a dose-dependent effect and thus we can conclude that the IL4 in both fusion proteins is still functional. The chemically conjugated apoA1-IL4 preforms slightly less than the commercial and recombinant IL-4, which can be attributed to the fusion of apoA1 to IL-4. This likely hinders the binding of IL- 4 to the receptor slightly. However, the chemically conjugated apoA1-IL4 performs better than the recombinant apoA1-IL4 protein, indicating that the affinity of the chemically conjugate apoA1-IL4 is likely higher than that of the recombinantly expressed apoA1-IL4.

[0511] ApoA 1-IL2 fusion proteins

[0512] Besides the production of the apoA1-IL4 chemical and recombinant fusion proteins, we have also produced apoA1-IL2 fusion proteins. Here we have recombinantly fused wild-type IL2 or an IL2 mutant to apoA1 . SDS-page analysis of the expressed and IMAC purified proteins indicated that the correct proteins are present in our elution fractions (Figure 10, left panel). Additionally, we have also chemically conjugated wild-type IL2 to apoA1. This was also verified by SDS-page (Figure 10, right panel).

[0513] We have next integrated the apoA1-IL2 fusion proteins in discoidal lipid nanoparticles to yield IL2-aNPs and IL2v4-aNPs (Figure 11). The successful formulation of discoidal nanoparticles was confirmed by cryogenic transmission electron microscopy (cryo-TEM) (Figure 11 , right panel). We additionally analyzed nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Figure 11 , left panel). -

[0514] We assessed the ability of the apoA1-IL2 fusion proteins to stimulate T-cell proliferation. It can be seen that our fusion proteins are able to induce T-cell proliferation. Especially our apoA1-IL2 chemical conjugate (Figure 12, middle bottom) can induce T-cell proliferation to a similar extend as commercially available IL2 (Figure 12, top left).

[0515] ApoA 1-IL1l3 fusion proteins

[0516] We further expanded our library by creating chemically conjugated and recombinantly expressed apoA1-ILip fusion proteins. Here we have recombinantly fused I L1 p to apoA1. SDS-page analysis of the expressed and IMAC purified protein showed that the correct protein is present in our elution fractions as indicated by the band at ~40 kDa (Figure 13, upper panel). Additionally, we have also chemically conjugated ILip to apoA1. This was also verified by SDS-page (Figure 13, lower panel).

[0517] Next, we have integrated the apoA1-ILi p fusion protein in discoidal lipid nanoparticles to yield ILi p-aNPs. The successful formulation of discoidal nanoparticles was confirmed by cryogenic transmission electron microscopy (cryo-TEM) (Figure 13, lower panel). We additionally analyzed nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Figure 14, upper panel)

[0518] ApoA 1-IL38 fusion proteins

[0519] Additionally, we also chemically conjugated and recombinantly expressed apoA1-IL38 fusion proteins. Here we have recombinantly fused IL38 to apoA1. SDS-page analysis of the expressed and IMAC purified protein showed that the correct protein is present in our elution fractions as indicated by the band at ~40 kDa (Figure 15, upper panel). Additionally, we have also chemically conjugated IL38 to apoA1. This was also verified by SDS-page (Figure 15, lower panel).

[0520] We have next integrated the apoA1-IL38 fusion protein in discoidal lipid nanoparticles to yield IL38-aNPs. The successful formulation of discoidal nanoparticles was confirmed by cryogenic transmission electron microscopy (cryo-TEM) (Figure 16, lower panel)). We additionally analyzed nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Figure 16, upper panel)

[0521] Example 2. ApoA1-IL-4 fusion protein and incorporation thereof into lipid nanoparticles

[0522] Materials and methods

[0523] PBMC and monocyte isolation: Buffy coats (Sanquin) or EDTA whole blood from healthy volunteers was acquired after obtaining written informed consent. The material was diluted at least 1 :1 with calcium / magnesium-free PBS (Lonza) and layered on top of Ficoll-Paque (GE Healthcare). Density-gradient centrifugation for 30 minutes at 615 x g was used to separate the peripheral blood mononuclear cell (PBMC) interphase. Following 3-5 washes with cold PBS, PBMC yield and composition were assessed using a Sysmex hemoanalyzer (XN-450; Sysmex).

[0524] Negatively selected monocytes were obtained using MACS, according to the manufacturer’s instructions (MACS Pan monocyte isolation kit, human; Miltenyi Biotec). Monocyte yield and purity was assessed on a Sysmex hemoanalyzer.

[0525] For some experiments (indicated in the text), monocytes were alternatively enriched from PBMCs by hyperosmotic density-gradient centrifugation over Percoll (Sigma-Aldrich). 150- 200 x 106 PBMCs were layered on top of a hyperosmotic Percoll solution (48.5% v / v Percoll, 0.16 M NaCI, in sterile water) and centrifuged for 15 minutes at 580 x g, RT. The interphase was collected, washed once with cold PBS, and resuspended in RPMI.

[0526] Primary human monocyte culture: All primary human monocytes / macrophages were cultured in RPMI-1640 with Dutch modifications (Invitrogen) which was further supplemented with GlutaMAX (2 mM; GIBCO), sodium pyruvate (1 mM; GIBCO), and gentamicin (50 pg / ml; Centrafarm). This medium is further referred to as RPMI+++. Additionally, 10% (v / v) human pooled serum was added to the medium during cell culture (also referred to as “cell culture medium”). In vitro model of trained immunity in primary human monocytes: To induce trained immunity in primary human monocytes, a previously optimized and published method was used (Dominguez-Andres, J. et al. In vitro induction of trained immunity in adherent human monocytes. STAR Protoc 2, 100365, doi:10.1016 / j.xpro.2021 .100365 (2021); van Lier, D., Geven, C., Leijte, G. P. & Pickkers, P. Experimental human endotoxemia as a model of systemic inflammation. Biochimie 159, 99-106, doi: 10.1016 / j . biochi.2018.06.014 (2019)). Briefly: monocytes were adhered to a flat-bottom cell culture plate for 1 hour and washed with warm PBS to remove any non-adherent cells and cell debris. Then, they were stimulated (“trained”) for 24 hours with one of the stimuli detailed in Table 1 , or medium only (“untrained” control).

[0527] Table 1. Stimuli for primary human monocytes and in vivo experiments.

[0528] For pharmacological inhibition experiments, the monocytes were pre-incubated for 1 hour with one of the inhibitors described in Table 2 before addition of the training stimulus.

[0529] Table 2. Inhibitors for primary human monocytes.

[0530] Following the initial 24-hour stimulation, the cells were washed with warm PBS and warm cell culture medium was added. The monocytes were then allowed to rest and differentiate into macrophages for 5 days. On day 6, the induction of trained immunity was assessed. To this end, the cells were typically re-stimulated with LPS for an additional 24 hours to elicit cytokine production. The supernatants were collected and stored at -20 °C until further analysis, e.g. with ELISA for IL6 and TNF.

[0531] For most other trained immunity readout methods, the cells were harvested as follows: first, the cells were incubated in Versene cell dissociation reagent (Life technologies) for 30 minutes in a cell culture incubator. A cell scraper was then used to remove the cells from the culture plates. To maximize yield, the culture plates were scraped a second time after adding ice-cold PBS. The macrophages were centrifuged for 10 minutes at 300 x g, 4 °C and counted before continuing to downstream applications.

[0532] In vitro inflammation inhibition: Monocytes were adhered to a flat-bottom cell culture plate for 1 hour and washed with warm PBS to remove any non-adherent cells and cell debris. Then, they were incubated with IL4 and LPS for 24 hours. Following the initial 24-hour stimulation, the supernatants were collected and stored at -20 °C until further analysis with ELISA for IL6 and TNF.

[0533] Primary monocyte-derived dendritic cell (moDC) generation: For experiments were moDCs were compared to macrophages (untrained control or IL4-trained), moDCs were differentiated as follows. First, negatively selected monocytes were obtained as described above. Following 1 h adherence and a PBS wash, they were cultured in RPMI+++ with 10% HPS, further supplemented with IL4 (25 ng / ml) and GM-CSF (1000 lll / ml; premium grade, Miltenyi Biotec). The cells were differentiated until day 6, with one medium refresh on day 3. On day 6, the non-adherent cells were harvested in addition to the adherent cells (as described above). The moDCs and macrophages were then subjected to analysis by flow cytometry as described below.

[0534] In vivo experimental human endotoxemia model and ex vivo analyses: Eight healthy (as confirmed by medical history, physical examination, and routine laboratory testing) male volunteers provided written informed consent to participate in experimental endotoxemia experiments conducted at the research unit of the intensive care department of the Radboud university medical center. All study procedures were approved by the local ethics committee (CMC Arnhem-Nijmegen, registration numbers NL71293.091 .19 and 2019-5730), and were conducted in accordance with the latest version of the declaration of Helsinki.

[0535] A continuous endotoxin infusion regimen was employed, as is described in detail elsewhere (van Lier, D., Geven, C., Leijte, G. P. & Pickkers, P. Experimental human endotoxemia as a model of systemic inflammation. Biochimie 159, 99-106, doi : 10.1016 / j. biochi.2018.06.014 (2019)). In short: subjects were admitted to the research unit and an antecubital vein and radial artery were cannulated to allow administration of fluids and endotoxin, and blood sampling and hemodynamic monitoring, respectively. A 3-lead ECG was recorded continuously throughout the experiment. After iso-osmolar pre-hydration (1.5 L NaCI 0.45 % I glucose 2.5 % administered intravenously in the hour before start of endotoxin infusion), volunteers were intravenously challenged with a loading dose of 1 ng / kg bodyweight endotoxin (E. coli lipopolysaccharide [LPS] type 0113, lot no. 94332B1 ; List Biological laboratories), directly followed by continuous infusion of 0.5 ng / kg / hour for 3 hours. Participants were monitored for 8 hours after the endotoxin loading dose after which they were discharged from the research unit.

[0536] For this project, blood samples were obtained at two timepoints: 1 hour before, and 4 hours after administration of the loading dose. Negatively selected monocytes were acquired as described above. The cells were adhered and stimulated for 24 hours with recombinant human IL4, discoidal IL4-aNPs, LPS (to assess initial immune tolerance), or medium only (as a control). Following a PBS wash, the cells were rested in culture medium for 48 hours and re-stimulated with LPS for an additional 24 hours. Supernatants were collected and stored at -20 °C.

[0537] Cytokine and lactate measurements: TNF, IL6, and IL1 Ra were measured in cell culture supernatants using duoset ELISA kits (R&D systems), according to the manufacturer’s instructions. For lactate measurements, a fluorometric assay was used. 30 pl sample, medium control, or known standard was added to a black 96-well plate. Then, 30 pl reaction mix (PBS pH 7.4, horse radish peroxidase (0.2 U / ml), lactate oxidase (2 U / ml), Amplex red (100 pM; Fisher scientific)) was added and the reaction was incubated for 20 minutes in the dark at RT. Immediately thereafter, fluorescence was measured at 530 / 25 nm and 590 / 35 nm. Gen5 software (v3.03, BioTek) was used in conjunction with Microsoft Excel to calculate cytokine and lactate concentrations in the original samples.

[0538] Macrophage surface marker flow cytometry: Macrophages were harvested as described above and transferred to a v-bottom 96 well plate for staining. The cells were centrifuged at 1500 rpm, 5 minutes, 4 °C. The supernatant was removed, and the cells were washed once with 200 pl PBA (PBS pH 7.4, 1 % w / v BSA (Sigma)).

[0539] Fc-receptors were blocked by incubation in PBS supplemented with 10% human pooled serum for 15 minutes at 4 °C. After washing once more, surface markers and viability were stained for in a volume of 50 pl for 30 minutes at 4 °C, using the antibodies and viability dye described in table 3.

[0540] Table 3. Flow cytometry antibodies for experiments with primary human monocytes / macrophages (and MLR experiments).

[0541] Following two washes, the cells were resuspended in 150 pl PBA and measured on a Cytoflex flow cytometer (Beckman Coulter) or BD FACSVerse system (BD Biosciences). Compensation was performed using VersaComp compensation beads (Beckman Coulter) for single antibody stains; a mixture of live and heat-killed cells was used for single stains of the viability dye (as per the manufacturer’s recommendations). Data analysis was performed in Flowjo (v10.7.1 , BD Biosciences) Our gating strategy was as follows: first, a time gate was used if necessary. Then, single cell events were selected using subsequent FSC-A / SSC-A and FSC-A / FSC-H gates. Dead cells were removed from the analysis by selecting the viability dye-negative population. Geometric mean fluorescence intensities were calculated as a measure of surface marker expression.

[0542] T cell polarization readout: For MLR experiments, harvested macrophages were used for subsequent T cell polarization assays. Allogeneic naive T cells were seeded with macrophages in a ratio of 10 T cells for every macrophage. The cells were cultured in flat- bottom 96 well plates for 7 days in standard cell culture medium. In this model, HLA mismatch causes non-specific activation of the T cell receptor. On the final day, the cells were stimulated with PMA (25 ng / mL) + ionomycin (0.5 pg / mL) for 4 hours in the presence of 100 ng / mL Brefeldin A, a ‘golgi-plug’. The cells were harvested and split over 2 flowcytometry antibody panels (one for CD4 T cells and one for CD8; see also table 3). The cells were stained in a similar manner as described above, with an extra step for permeabilization of the T cells to allow for intracellular cytokine staining. This was performed using the Fix / Perm buffer set (eBioscience), according to the manufacturer’s instructions. The gating strategy was similar to what is described above, with the addition of a selection for CD3- positive events. The percentage of cells positive for hallmark cytokines of T cell polarization were calculated to estimate T cell subset proportions.

[0543] Phospho-STAT6 measurement by flow cytometry: Monocytes were stimulated with RPMI, IL4, or different concentrations of IL4-aNPs (indicated in the figure) for 20 minutes at 37 °C. The cells were transferred to a v-bottom 96 wells plate and kept on ice for the duration of the staining procedure. After staining for viability and CD14 (in the manner described above), the cells were fixed and permeabilized using the fix / perm buffer set (eBioscience) for 45 minutes at 4 °C in the dark. The cells were washed twice with perm buffer and incubated overnight in freezer-chilled absolute methanol at -20 °C overnight. Following two more washes in perm buffer the cells were stained for phospho-STAT6 using the antibody described in table 3, for 45 minutes at 4 °C in the dark. The cells were washed two more times in perm buffer and finally resuspended in PBA for acquisition on the Cytoflex cytometer. The gating strategy was largely similar to the one for macrophage surface marker with the addition of a selection for CD14-positive events.

[0544] Phagocytosis assay: Macrophages were harvested as described above and incubated at 37 °C for 1 hour with FITC-labeled Candida albicans (kindly provided by Dr. Martin Jaeger, Radboudumc) at an MOI of 1 :5. The cells were washed 2 times with ice-cold PBA and kept on ice to halt the phagocytosis. The cells were stained for CD45 (table 3) during 30 minutes in the dark at 4 °C. Following two washes, trypan blue was added to a final concentration of 0.01 % to quench extracellular FITC-Candida. The cells were then acquired on a Cytoflex flow cytometer.

[0545] During data analysis, CD45+ events were first selected to remove Candida-only events. Single cells were then gated on as described above and the percentage of Candida-FITC positive macrophages in each sample was calculated. Seahorse metabolic analyses: Macrophages were harvested as described above. The cells were resuspended in RPMI+++ and seeded into overnight-calibrated cartridges at 105cells per well. After adhering for 1 hour, the medium was changed to assay medium (Agilent; see below) and the cells were incubated for 1 hour at 37 °C in ambient CO2 levels. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured as proxies for glycolytic and mitochondrial metabolism, using a Seahorse XF Glycolysis Stress Test kit or a Seahorse XF Cell Mito Stress Test kit (both Agilent; measurements performed according to manufacturer’s instructions).

[0546] RNA isolation, sequencing, and analysis'. Monocytes or macrophages were lysed in RLT buffer (Qiagen) and stored at -80 °C. RNA extractions were performed using RNeasy mini columns (Qiagen) with on-column DNAse I treatment (RNase-free; Qiagen). Preliminary quality control and measurements of concentration were performed using a Nanodrop apparatus. Samples were sent to the Beijing Genome Institute (BGI Denmark) for RNA sequencing using the DNBseq platform.

[0547] To infer gene expression levels, RNA-seq reads were aligned to hg19 human transcriptome using Bowtie. Quantification of gene expression levels as RPKM was performed using MMSEQ. Reads / transcript were normalized using DEseq2 and pair-wise comparisons were performed. Differentially expressed genes were identified using DEseq2 with fold change > 2 and p-value < 0.05, with a mean RPKM > 1. To identify genes that were upregulated or attenuated by IL4 training, RPMI-d6 and IL4-d6 macrophages were compared with RPMI- d6+LPS and IL4-d6+LPS samples, respectively. Gene lists were merged and ranked based on IL4-d6+LPS I RPMI-d6+LPS. Gene ontology and TF motif analysis was performed on gene promoters using the HOMER findMotifs tool.

[0548] Chromatin immunoprecipitation: Macrophages were harvested as described above and resuspended in RPMI+++. The cells were fixed for 10 minutes in 1 % methanol-free formaldehyde. The reaction was then quenched for 3 minutes by adding 125 mM glycine. The fixed cells were washed three times with ice-cold PBS and lysed at approximately 15*106cells / ml in lysis buffer (20 mM HEPES pH 7.6, 1% SDS, 1x protease inhibitor cocktail (PIC; Roche)), sonicated (Bioruptor Pico, Diagenode), and centrifuged (10 minutes, 13000 rpm, RT).

[0549] Aliquots of chromatin were de-crosslinked in 0.5x TBE buffer (supplemented with 0.5 mg / ml proteinase K (Qiagen)) for 1 hour at 65 °C and run on a 1 % agarose gel to confirm target fragment size of 200-800 bp. The remaining chromatin was divided into ChIP and input samples. ChIP samples were diluted 10x in dilution buffer (16.7 mM Tris pH 8.0, 1.0% Triton, 1.2 mM EDTA, 167 mM NaCI, 1x PIC in MiliQ) and 1 pg of ChlP-grade antibody (Diagenode) was added. The samples were rotated overnight at 4 °C.

[0550] Magnetic protein A / G beads (Dynabeads) were washed 2 times in dilution buffer supplemented with 0.15% SDS and 0.1 % BSA. The washed beads were added to the ChIP samples and rotated at 4 °C for 1 hour. The bead-bound chromatin was subsequently washed (rotation for 5 minutes, 4 °C) as follows: 1x with low-salt washing buffer (20 mM Tris pH 8.0, 1.0% Triton, 0.1 % SDS, 2 mM EDTA, 150 mM NaCI in MilliQ); 2x with high-salt washing buffer (same as low-salt washing buffer but with 500 mM NaCI); 2x with no-salt washing buffer (20 mM Tris pH 8.0, 1 mM EDTA, in MilliQ). Chromatin was eluted from the beads in elution buffer (0.1 M NaHCO3, 1 % SDS, in MilliQ) for 20 minutes, RT. Input samples were diluted 12x in elution buffer. After addition of NaCI (0.2 M) and proteinase K (0.1 mg / ml), all samples were decrosslinked for at least 4 hours on a shaking heatblock (65° C, 1000 rpm). Minelute PCR purification columns (Qiagen) were used to purify DNA fragments. DNA fragments were stored at 4 °C until downstream analysis by qPCR. qPCR and analysis: qPCR analysis for ChIP samples and inputs was performed as follows. The SYBR green method was used to perform qPCR with the primers detailed in table 4. A comparative Ct method was used to compare ChIP against input samples and calculate relative abundance over a negative control region. GAPDH and the untranslated region of ZNF were respectively used as negative and positive controls for H3K9me3. TNF was interrogated using 6 primer pairs for AUC analysis as described previously (Bekkering, S. et al. Treatment with Statins Does Not Revert Trained Immunity in Patients with Familial Hypercholesterolemia. Cell Metabolism 30, 1-2, doi:10.1016 / j.cmet.2019.05.014 (2019)).

[0551] Table 4. Primers for ChlP-qPCR analysis.

[0552] Bacterial expression and protein purification: ClearColi BL21 (DE3) (Lucigen) were transformed with a pET20b(+)ApoA1-IL4 expression vector. Transformed bacteria were inoculated in 40 mL lysogeny broth (LB) (Sigma-Aldrich) supplemented with 100 pg / L ampicillin and grown overnight at 37 °C. Subsequently, the overnight culture was inoculated in 2YT medium (16 g / L Peptone, 10 g / L yeast extract and 10 g / L NaCI) supplemented with 100 pg / L ampicillin and grown at 37 °C. At the point that absorbance at 600 nm reached > 1.5, 1.0 mM isopropyl p-d-thiogalacopyranoside (IPTG) was added to induce pET20b(+)ApoA1-IL4 expression, cells were incubated overnight at 20 °C. Cells were harvested by centrifugation before preparation of lysates and purification.

[0553] Bacterial lysis and protein purification: ApoA1-IL4 fusion protein expressing ClearColi cells were harvested by centrifugation at 8000 rpm and 4 °C for 10 minutes. Harvested cells were resuspended in PBS and centrifuged at 4000 rpm and 4 °C for 15 minutes. Cells were lysed using 20 mL BugBuster® Protein Extraction Reagent (Merck) and 20 pL Benzonase® Nuclease (Merck) per liter culture on a shaker for 30 min at RT. Cell lysates were centrifuged at 18000 rpm and 4 °C for 30 minutes. Insoluble pellets were washed with 10 mL BugBuster per liter and centrifuged at 18000 rpm and 4 °C for 20 minutes. Pellet containing inclusion bodies was resuspended in extraction buffer (6 M guanidine hydrochloride, 50 mM potassium phosphate and 1 mM reduced glutathione) and incubated on a shaker for 15 min at RT. Suspension was centrifuged at 18000 rpm and 4 °C for 30 minutes to remove insoluble fraction. Filtered soluble fraction was loaded on a nickel column and washed with 15 column volumes IMAC wash buffer. ApoA1-IL4 was refolded on the nickel column using a linear gradient unfolding 60 mL (7 M urea, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 50 mM potassium phosphate and 100 mM NaCI pH 6.8) to refolding 60 mL (1 mM reduced glutathione, 0.1 mM oxidized glutathione, 50 mM potassium phosphate and 100 mM NaCI pH 6.8) 2.5 mL / minute. Refolded apoA1-IL4 was eluted from the column with 0.5 M imidazole, 20 mM Tris, 0.5 M NaCI pH 7.9. Eluate was collected, concentrated, and further purified and buffer-exchanged via size exclusion chromatography (HiLoad 16 / 600 Superdex 75 Increase; GE Healthcare) equilibrated with PBS storage buffer. Fractions were analyzed by SDS-PAGE, pooled, concentrated, and snap-frozen in liquid nitrogen before storing at -80 °C. ApoA1-IL4 mass was confirmed by Q-ToF LC-MS (WatersMassLynx v4.1), using MagTran V1.03 for MS.

[0554] Mammalian expression and purification of apoA 1-IL4m: HEK293T cells were co transfected with fuGENE (Promega) including transfer vector pHR-apoA1-IL4m, packaging pCMVR8.74 and envelop pMD2.G in Opti-MEM (GIBCO) at 37 °C for 24 hours. Cells were washed with DMEM + 2 % heat inactivated FBS and incubated for 48 hours. To obtain the lentivirus containing pHR-apoA1-IL4m, supernatant was centrifuged at 1000 rpm to remove cell debris filtered through 0.45 pm PES syringe filter and centrifuged at 50,000g for 2 hours at 4 °C. Pellet containing pHR-apoA1-IL4m lentivirus was resuspend in culture medium, snap frozen in liquid nitrogen and sorted at -80 °C. HEK293F cells were transduced with pHR-apoA1- IL4m containing lentivirus in transfection medium (DMEM, 10 % HI FBS, 1x Polybrene (Sigma-Aldrich) for 24 hours. Subsequently, cells were cultured in expression medium (50 % EX-CELL® 293 Serum-Free Medium for HEK293 Cells (Merck) and 50 % Freestyle™ 293 Expression Medium (Thermo Fisher Scientific), supplemented with Glutamax, 1 % Pen- Strep and 1 pg / mL doxycycline (Merck) on a shaker at 150 rpm for 3 days at 37 °C. Culture supernatant containing apoA1-IL4m was centrifuged at 4000 rpm, 4 °C for 15 minutes and filtered through 0.22 pm PES syringe filter to remove cell debris. Filtered soluble fraction was loaded on a StrepTactin XT 4flow 5 mL column (Cytiva) and washed with 5 column volumes W-buffer (150 mM NaCI, 100 mM Tris, 1 mM EDTA pH 8) with flow rate 1-2 mL / minute. ApoA1-IL4m was eluted from the column with W-buffer supplemented with 50 mM biotin. Eluate was collected, concentrated, and snap-frozen in liquid nitrogen before storing at -80 °C. Apoa1-IL4m mass was confirmed by Q-ToF LC-MS (WatersMassLynx v4.1), using MagTran V1.03 for MS.

[0555] SDS-PAGE and Western blot: To confirm fusion of apoA1 and IL4, 100 ng IL4 (BioLegend), apoA1 and apoA1-IL4 were loaded on a 4-20% polyacrylamide gel (Bio-Rad). After gel electrophoresis, samples were transferred to nitrocellulose membranes with blot buffer (10x TG buffer, 20% methanol). Subsequently, membranes were incubated with blocking buffer (5% milk, 0.1 % tween in PBS (PBST)) overnight at 4 °C. The blots were incubated with primary monoclonal antibodies monoclonal anti-IL4 (HIL41 , 1 :200; sc-12723, Santa Cruz Biotechnology) and anti-apoA1 (B10, 1 :100; sc-376818, Santa Cruz Biotechnology) for 1 hour at 4 °C. After incubation with primary antibodies, membranes were washed and incubated with rabbit anti-mouse IgG (H+L)-HRP conjugate (1 :5000, 31457, Pierce). HRP- conjugated secondary antibodies were detected with TMB (Thermo Fisher Scientific) and visualized using the Image Quant gel imager (GE Healthcare).

[0556] Surface plasmon resonance: SPR measurements were performed using a Biacore X100 SPR system (GE Healthcare). Human IL4 receptor alpha-FC chimera (Biolegend) was immobilized on a protein G sensor chip (GE Healthcare). Log2 dilution concentration series of apoA1-IL4 ranging from 200 nM to 6.25 nM and of human IL4 ranging from 20 nM to 0.65 nM. All samples were prepared in HPS-EP buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.005 % (v / v) P20 pH 7.4). Association was monitored for 180 seconds and dissociation for 180 seconds with a flow rate of 30 pL / minute. Sensor chip was regenerated with glycine 1.5 (10 mM glycine-HCI pH 1.5, GE Healthcare). Kinetics was determined by fitting the interaction SPR data for 1 :1 binding.

[0557] Human embryonic kidney 293 IL4 reporter cell assay: HEK-BlueTM IL4 / IL13 cells were purchased from InvivoGen. This cell line has a fully active STAT6 pathway and carries a STAT6-inducible SEAP reporter gene. The HEK-BlueTM IL4 / IL13 cells produce SEAP in response to IL4 and IL13. The levels of secreted SEAP can be determined with QUANTI- BlueTM (Invivogen). 180 pL DMEM with 10 % FBS and 1 % Pen-Strep containing 5*104 cells was added per well in a 96-well-plate. Subsequently, 20 pL stimulus or vehicle was added and cells were incubated for 20-24 hours at 37 °C. Subsequently, 180 pL QUANTI- Blue was added per well to a separate 96-well-plate (flat-bottom) and 20 pL of the cell supernatant was added. The plate was incubated for 1-3 hours at 37 °C and absorbance at 640 nm was measured on a Tecan Spark plate reader to determine SEAP levels.

[0558] Formulating nanoparticles: All phospholipids were purchased from Avanti Polar Lipids Inc. Four different apoA1 based nanoparticles (aNPs) were formulated. For discoidal aNPs from stock solutions (10 mg / mL) in chloroform, DMPC (133.5 pL), cholesterol (Sigma-Aldrich) (7.5 pL) and for spherical aNPs, POPC (66.5 pL), PHPC (17.5 pL), cholesterol (4.5 pL), and tricaprylin (Sigma-Aldrich) (2.79 pL from 0.956 g / mL stock) were combined in a glass vial and dried under vacuum. The resulting film was redissolved in an acetonitrile / methanol mixture (95:5%, 800 pL total volume). For formulation based on apoA1 , cholesterol (15 pL) was used. Separately, a solution of apoA1 protein in PBS (6 mL, 0.1 mg / mL), apoA1-IL4 protein in PBS (6 mL, 0.17 mg / mL) or apoA1-IL4m in PBS (6 mL, 0.18 mg / mL) was prepared. Both solutions were simultaneously injected using a microfluidic pump fusion 100 (Chemyx Inc) into a Zeonor herringbone mixers (Microfluidic Chipshop, product code: 10000076) with a flow rate of 0.75 mL / minute for the lipid solution and a rate of 6 mL / minute for the apoA1 solution. The obtained solution was concentrated by centrifugal filtration using either a 10 kDa MWCO for discoidal and a 100 kDa MWCO for spherical aNPs Vivaspin tube at 4000 rpm to obtain a volume of 1 mL. PBS (5 mL) was added, and the solution was concentrated to 5 ml; this was repeated twice. The washed solution was concentrated to approximately 1.5 ml and filtered through a 0.22 pm PES syringe filter to obtain the finished aNPs. Protein concentration in aNP samples was quantified with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). To formulated fluorescent aNPs, 0.5 mg of DiOC18(3) dye (DiO) (Thermo Fisher Scientific) was dissolved in the chloroform solution used to prepare the lipid film.

[0559] Determining aNP size and dispersity by DLS: Obtained aNP formulations in PBS were filtered through a 0.22 pm PES syringe filter and analyzed by dynamic light scattering on a Malvern Zetasizer Nano ZS analyzer. Values are reported as the mean number average size distribution. apoA1-IL4 and IL4-aNPs were incubated with 2 molar excesses of

[0560] DFO-p-NCS (5 mg / mL in DMSO) for 2 hours washed three times using a 10 kDa MWCO

[0561] Vivaspin tubes to remove any unreacted DFO-p-NCS. For radiolabeling, DFO coupled proteins and aNPs were incubated with89Zr at 37 °C using a thermomixer at 600 rpm for 1 hour and washed three times using 10 kDa MWCO Vivaspin tubes to remove any unreacted

[0562] 89Zr. First, the surface of 200- mesh lacey carbon supported copper grids (Electron Microscopy Sciences) was plasma treated for 40 seconds using a Cressington 208 carbon coater. Subsequently, 3 ml of I L4- aNPs sample (~1 mg protein / ml) was applied on a grid and vitrified into a thin film by plunge vitrification in liquid ethane by using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was performed on the cryoTITAN (Thermo Fisher Scientific), equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002) and a post-GIF 2k x 2k Gatan CCD camera (model 794). The images were acquired at 300 kV acceleration voltage in bright-field TEM mode with zero-loss energy filtering at either 6,500x (dose rate of 1.64 electrons / A2s) or 24,000x magnification (dose rate of 11.8 electrons / A2s), and 1s acquisition time. Super-resolution fluorescence microscopy of IL4-aNPs’ interactions with IL4 receptor in human monocytes: Human monocytes were isolated from a healthy donor’s peripheral blood as described above. 100,000 monocytes were seeded per well on a cell culture-treated chambered coverslip (p-Slide 8-well, IBID). After 2 hours of incubation at 37 °C (cell attachment), the cells were incubated for 2h at 37 °C with Cy5-labeled variant of either bare apoA1 or apoA1-IL4, discoidal aNPs or IL4-aNPs, spherical aNPs or IL4-aNPs. Subsequently, the cells were washed with PBS and fixed with 4 % PFA for 20 minutes. IL4 receptor was stained with a polyclonal rabbit lgG1 anti-human IL4R (Thermo Fisher Scientific; 1 :100 dilution) primary antibody for 24 hours at 4 °C, followed by a goat antirabbit Alexa Fluor 488-conjugated secondary antibody (Thermo Fischer Scientific; dilution 1 :500) for 1 hour at room temperature. The stained cells were stored in PBS at 4°C. For the direct stochastic optical reconstruction microscopy (dSTORM), the cells were immersed in GLOXY imaging buffer (40 pg / ml catalase, 0.5 mg / ml glucose oxidase, 5 % glucose and 0.01 M cysteamine in PBS, pH 8.0) for a few minutes before and during the imaging. The acquisition was performed in Total Internal Reflection Fluorescence (TIRF) mode, using ONI Nanoimager (ONI, Oxford, UK). It is equipped with a 100x / 1.4NA oil immersion objective, a sCMOS camera, and, in this study, 488 nm (200 mW) and 640 nm (1000 mW) lasers were used. 10000 frames were acquired with a 10 ms exposure time with the field of view of 50 x 80 pm. Raw data were processed using the ThunderSTORM software, yielding images with a spatial resolution of 10 nm.

[0563] Animal models-. Female C57BL / 6 mice were purchased from The Jackson Laboratory. For nonhuman primate studies, two male cynomolgus monkeys (Macaca fascicularis) were used. All animals were cohoused in climate-controlled conditions with 12-hour light-dark cycles and provided water ad libitum. Mice were fed a standard chow diet and nonhuman primates were fed Teklad Global 20 % Protein Primate Diet. Animal care and experimental procedures were based on approved institutional protocols from the Icahn School of Medicine at Mount Sinai. All mice were randomly assigned to experimental groups.

[0564] Pharmacokinetics and biodistribution in mice and non-human primates: C57BL / 6 mice were intravenously injected with89Zr-labeled-IL4 variants, respectively, IL4 (53.6 ± 6.6 pCi), apoA1-IL4 (30.1 ± 0.9 pCi), discoidal IL4-aNPs (146.1 ± 46.5 pCi), and spherical IL4-aNPs (108.6 ± 16.9 pCi). Two non-human primates were injected with discoidal89Zr-labeled-IL4- aNPs (1079 pCi and 682 pCi). At predetermined time points, 1 , 2, 5, 10, and 30 minutes, and 1 , 2, 4, 8, and 24 hours for mice and 5, 30 and 90 minutes and 48 hours for non-human primates, after injection blood was drawn, weighed, and radioactivity was measured using a Wizard22480 automatic gamma counter (Perkin Elmer, Waltham, MA). Data was corrected for radioactive decay, percentage of injected dose per gram of blood (%l D / g) was calculated. Data was fitted using a non-linear two-phase decay regression in GraphPad Prism and weighted blood half-life was calculated via the equation (% fast x ti / 2 fast + % slow x ti / 2) / 100. Biodistribution in mice was determined 24 hours post injection. After PBS perfusion, tissues of interest were harvested, weighed, and radioactivity was measured using a Wizard22480 automatic gamma counter (Perkin Elmer, Waltham, MA). Data was corrected for radioactive decay, percentage of injected dose per gram of tissue (%l D / g) was calculated.

[0565] PET / CT imaging of aNP biodistribution in mice: C57BL / 6 mice were injected intravenously with89Zr-labeled-IL4 variants, respectively, IL4 (53.6 ± 6.6 pCi), apoA1-IL4 (30.1 ± 0.9 pCi), discoidal IL4-aNPs (146.1 ± 46.5 pCi), and spherical IL4-aNPs (108.6 ± 16.9 pCi). After 24 hours, mice were anesthetized using 1 .0 % isoflurane in O2 at a flow rate of ~1 .0 liter / minute. PET / CT scans were acquired using a Mediso nanoScan PET / CT (Mediso, Budapest, Hungary). A whole-body CT scan was executed (energy, 50 kVp; current, 180 pAs; isotropic voxel size, 0.25 mm) followed by a 20-minute PET scan. Reconstruction was performed with attenuation correction using the TeraTomo 3D reconstruction algorithm from the Mediso Nucline software. The coincidences were excluded by an energy window between 400 and 600 keV. The voxel size was isotropic with 0.4-mm width, and the reconstruction was applied for four full iterations, six subsets per iteration.

[0566] Autoradiography: Tissues were placed in a film cassette against a phosphorimaging plate (BASMS-2325, Fujifilm) at -20 °C to determine the radioactivity distribution. The plates were read at a pixel resolution of 25 mm with a Typhoon 7000IP plate reader (GE Healthcare).

[0567] Cellular specificity flow cytometry: For cellular specificity, mice were intravenously injected with DiO labeled IL4-aNPs that was allowed to circulate for 24 hours. Subsequently, mice were sacrificed, and single cell suspensions were created from blood, spleen and bone marrow as previously described. Cell suspensions were incubated with anti-CD115, anti- CD11b, anti-Ly6C, anti-Ly6G, anti-CD19, anti-CD45, anti-CD11c, anti-CD3, anti-F4 / 80. Live / Dead Aqua was used as viability stain. Cells were subsequently washed and resuspended in FACS-buffer. All data were acquired on an Aurora 5L flow cytometer (Cytek Biosciences). DiO-IL4-aNPs were detected in the FITC channel. PET / MRI non-human primate biodistribution: After overnight fasting, non-human primates were anaesthetized using ketamine (5 mg / kg) and dexmedetomidine (0.0075-0.015 mg / kg). Non-human primates were injected with 1.114 mCi and 0.682 mCi discoidal89Zr-labeled- IL4-aNPs, at a dose of approximately 0.1 mg / kg. Dynamic PET imaging as performed for 60 minutes following infusion, and additional static PET / MRI scans were performed at 1 hour and 48 hours after injection. Furthermore, blood was drawn during imaging at 5, 30, and 120 minutes after injection. PET and MRI images were acquired using a 3T PE / MRI system (Biograph mMR, Siemens Healthineers). Beginning concurrently with the injection of aNPs, dynamic PET imaging was performed using one bed position covering the chest and abdomen. MR imaging parameters were as follows: acquisition plane, coronal; repetition time, 1 ,000 ms; echo time, 79 ms; number of slices, 144; number of averages, 4; spatial resolution of 0.5 x 0.5 x 1.0 mm3and acquisition duration, 42 minutes and 42 seconds. After dynamic PET image acquisition, static whole-body PET images were acquired from the cranium to the pelvis, using 4 consecutive bed positions of 15 min each. Simultaneously with each bed, MR images were acquired as described above, except using only 1.4 signal averages, number of slices 160, and spatial resolution 0.6 x 0.6 x 1.0 mm3(acquisition duration, 14 minutes 56 seconds per bed). Whole-body PET and MR imaging was also performed at 48 hours after injection, using 4 PET bed positions of 30 minutes each, with MR parameters as follows: acquisition plane, coronal; repetition time, 1 ,000 ms; echo time, 79 ms; number of slices, 224; number of averages, 2; spatial resolution of 0.6 x 0.6 x 1.0 mm3; acquisition duration, 29 minutes and 56 seconds. Whole-body MR images from each bed were automatically collated together by the scanner. After acquisition, PET raw data from each bed were reconstructed and collated together offline using the Siemens proprietary e7tools with an Ordered Subset Expectation Maximization (OSEM) algorithm with Point Spread Function (PSF) correction for 3 iterations and 24 subsets. Also, Gaussian filter of 4 mm was applied to the images. A three-compartment (soft tissue, lung and air) attenuation map was used for attenuation.

[0568] Imaging-based analysis of the IL4-aNP biodistribution in non-human primates: Image analysis was performed using Osirix MD, version 11.0. Whole-body MR images were fused with PET images and analyzed in a coronal plane. Regions of interest (ROIs) were drawn on tissues of interest including, spleen, liver, kidneys, lungs, heart, cerebellum, and cerebrum were traced in their entirety, and bone marrow uptake was determined using three vertebrae in the lumbar spine. For each ROI, mean standardized uptake values (SLIVs) were calculated. Discoidal89Zr-labeled-IL4-aNP uptake per organ was expressed as the average of all mean SUV values per organ. In vivo tolerance model: For in vivo tolerance model, 11-weeks-old female C57BL / 6 mice were intraperitoneal tolerized with 0.1 mg / kg body weight LPS. At 24 and 48 hours, mice were treated intravenously with either 200 pg IL4m-aNPs or PBS. Subsequently, mice were rechallenged with intraperitoneal 0.1 mg / kg LPS injection at 72 hours. After 90 minutes, mice were sacrificed, blood collected for ELISA and single cell suspensions were created from blood, spleen and bone marrow. Staining protocol. Blood samples for ELISA were allowed to clot at RT for 30 minutes. Serum was taken after centrifugation at 1000 x g for 10 minutes at 4 °C. .Mouse TNF and IL6 ELISAs (Biolegend) were performed according to manufacturer’s protocols. Animal care and experimental procedures were based on approved institutional protocols from the Nijmegen Animal Experiments Committee.

[0569] Statistical analysis: Data are shown as mean + / - SD, unless otherwise indicated. Individual data points in graphs are biological replicates, not technical repeats. Wherever the number of data points cannot be clearly discerned from the figure, n is indicated in the figure legend. Unless otherwise indicated, statistical analyses were performed in Graphpad Prism (V9, Graphpad Software). For trained immunity- and acute stimulation experiments with primary human monocytes, (paired, non-parametric) Wilcoxon signed-rank tests were used. Statistical methods for RNA-sequencing analysis are described above. Two-sided P values under 0.05 were considered statistically significant. Statistical significance in figures is indicated as follows: * = P < 0.05, ** = P < 0.01 , *** = P < 0.001 , NS = P > 0.05.

[0570] Data and Code availability: Data are available upon request to the Lead Contact. Raw RNA sequencing data are deposited in the NCBI Gene Expression Omnibus under accession number: GSE185433.

[0571] Results

[0572] IL4 inhibits acute inflammation, yet induces trained immunity

[0573] In the context of myeloid cell immunology, IL4 is known primarily for its anti-inflammatory properties. Therefore, present inventors first validated several known inhibitory effects of IL4 on inflammation in primary human monocytes (Figure 17A). Present inventors stimulated Percoll-enriched monocytes with LPS for 24 hours, in the presence or absence of IL4 (25 ng / mL). As expected, IL4 potently inhibited the secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF) and IL6 (Figure 17B). Interestingly, IL4-treated cells secreted significantly more IL-1 Ra compared to controls (Figure 17B). As glycolysis is upregulated in activated myeloid cells, present inventors measured lactate production in otherwise unstimulated monocytes treated with IL4 or a medium control. Present inventors found IL4 to slightly, but significantly, lower baseline lactate production (data not shown), confirming its acute anti-inflammatory properties.

[0574] Based on these anti-inflammatory properties, present inventors hypothesized that IL4 might also inhibit the induction of trained immunity (Figure 17C). To test this hypothesis, monocytes were trained with p-glucan, a prototypical trained immunity stimulus, for 24 hours, followed by washing the stimulus and a 5-day resting period in culture medium. On day 6, present inventors re-stimulated the cells with LPS for another 24 hours and measured TNF and IL6 (Figure 17D). While p-glucan induced trained immunity as expected, addition of IL4 in the first 24 hours did not inhibit the training effect (Figure 17D). Contrary to present inventors’ initial hypothesis, exposing monocytes to IL4 alone for 24 hours induced a trained immunity phenotype on day 6 (Figure 17E). Besides enhanced production of pro- inflammatory cytokines, IL4-trained cells produced more lactate at baseline (data not shown). IL4-trained cells were slightly less effective at phagocytosing heat-killed Candida albicans than untrained controls (data not shown). Collectively, the data of present inventors demonstrate that IL4 inhibits inflammation and induces trained immunity, at both metabolic and functional immunologic level.

[0575] Encouraged by these observations, present inventors comprehensively studied the metabolic alterations following IL4-induced trained immunity. To this aim, present inventors employed Seahorse metabolic flux analyses to probe glycolytic and oxidative metabolism of IL4-trained cells and unstimulated controls. IL4 training on day 0 had a marked effect on metabolic parameters measured on day 6 (Figure 17F), with a trend towards higher basal glycolysis, and a significant increase of oligomycin-triggered maximum glycolytic capacity (Figure 17F left panel). In addition, both baseline- and FCCP-triggered maximum respiration rates were significantly augmented by IL4 training (Figure 17F right panel).

[0576] Present inventors then used flow cytometry to measure several parameters commonly associated with IL4 activation of monocytes / macrophages (data not shown). IL4 training caused a strong downregulation of CD14 expression on day 6. In contrast, CD200R and especially CD206 were significantly enhanced on day 6 subsequent to IL4-activation on day 0. CD80 was marginally increased by IL4 training, but overall expression was still low on these otherwise naive macrophages. It is known that monocyte-derived dendritic cells (moDCs, which are differentiated using IL4+GM-CSF) also downregulate CD14, whilst strongly upregulating CD1c. IL4-trained cells expressed slightly more CD1c than untrained cells, but far less than moDCs (data not shown). These results indicate IL4 induces a program of trained immunity which incorporates features known from classical IL4 immunological functions.

[0577] Immune and epigenetic mechanisms mediating IL4-induced trained immunity

[0578] The signaling mechanisms of IL4 are well-described: the IRS-2 / PI3K / mTOR axis and the STAT6 signaling pathway (Figure 18A). Present inventors performed pharmacological inhibition experiments to investigate the role of these pathways for both inhibition of acute inflammation as well as trained immunity induction by IL4. Inhibition of PI3K or mTOR (using wortmannin or torin-1 , respectively) did not abrogate the effect of IL4 on acute inflammation, but diminished the trained immunity responses (Figure 18B, 18C and data not shown). IL4 training, as measured by an increased TNF and IL6 production, was significantly blunted in the presence of torin-1 (Figure 18C and data not shown). In contrast, the STAT6 inhibitor AS1517499 partly restored cytokine production in acute inflammatory responses, but did not affect trained immunity induction by IL4 (Figure 18B and 18C). Thus, each of the signaling pathways induced downstream of IL4 engagement with its receptors has distinct functions: IL4 exerts its known acute anti-inflammatory function through STAT6, but simultaneously induces trained immunity via PI3K / mTOR, a previously unknown pro-inflammatory effect.

[0579] To gain insight into the molecular program induced by IL4 training, present inventors performed transcriptomics analysis on naive and IL4-trained macrophages, both before and after LPS re-stimulation on day 6. Overall, 140 genes were more strongly induced (‘upregulated’) in IL4-trained macrophages, whereas 249 genes were attenuated (Figure 18D). Amongst the top upregulated genes were pro-inflammatory cytokines such as IL6 and IL12B, that are known to be involved in trained immunity. Among the prominent attenuated genes were CCL19 and SOCS2, which are important for lymphocyte trafficking and suppression of cytokine signaling, respectively.

[0580] Present inventors next performed transcription factor (TF) motif enrichment analysis (Figure 18E) and gene ontology / pathway enrichment analyses (Figure 18F) to gain further insight into the transcriptome profiles. Promoters of genes upregulated in IL4-trained macrophages were highly enriched for motifs recognized by TFs such as ATF2 / ATF7, PPARa, and STAT5, whereas interferon regulatory factor (IRF) motifs were especially depleted. This pattern was mostly reversed for unaffected- and attenuated genes, except for TATA-box, NFKB-P65, NFKB-p65-Rel, and FRA2: these motifs were highly enriched in attenuated gene promoters, but decreased in both unaffected- and upregulated genes (Figure 18E). Gene ontology (biological process; BP, and molecular function; MF) and KEGG pathway enrichment showed that immunological activities were present in both upregulated (e.g. BP “Response to organism”, KEGG “TNF signaling”) and attenuated (e.g. BP “Immune response”, MF “Cytokine activity”) gene sets (Figure 18F). Present inventors performed a similar transcriptome analysis on monocytes stimulated immediately after isolation with IL4, LPS, or IL4 and LPS combined, which confirmed an acute anti-inflammatory transcriptomic response to IL4 (data not shown). Together, these data reveal specific transcriptional programs in both the acute anti-inflammatory effects and the long-term trained immunity responses invoked by IL4.

[0581] Present inventors subsequently investigated the importance and presence of epigenetic reprogramming, specifically histone 1 modifications. Addition of the anti-allergy drug cyproheptadine, a SET7 (also known as SET9) histone 2 methyltransferase inhibitor, abrogated the induction of trained immunity by IL4 (Figure 18G). SET7 has been described earlier as an important epigenetic mediator of trained immunity22. Furthermore, present inventors evaluated H3K9me3-mediated repression of TNF using chromatinimmunoprecipitation (ChlP)-qPCR analyses in IL4 induced trained immunity. Using an area under the curve (AUG) analysis of primer pairs showed a decrease of H3K9me3 6 in I L4- induced trained immunity, although this did not reach statistical significance (Figure 18H and data not shown). Together, these data indicate epigenetic reprogramming is crucial for, and characteristic of, IL4-induced trained immunity.

[0582] Developing an apoA1-IL4 fusion protein that integrates in lipid nanoparticles

[0583] Despite its unique ability to inhibit acute inflammation while simultaneously inducing trained immunity, recombinant IL4’s clinical translation is hampered by its unfavorable pharmacokinetic properties. To overcome this limitation, present inventors developed an apoA1-based fusion protein that readily integrates in lipid nanoparticles to yield IL4- containing nanoparticles (IL4-aNPs). ApoA1-based nanoparticles (aNPs) inherently accumulate in hematopoietic organs and efficiently target myeloid cells and their progenitors (Schrijver, D. P. et al. in Advanced Therapeutics Vol. 4 2100083-2100083 (John Wiley & Sons, Ltd, 2021 ; van Leent, M. M. T. et al. Regulating trained immunity with nanomedicine. Nature Reviews Materials 7, 46 465-481 , doi:10.1038 / s41578-021-00413-w (2022)) (Figure 19A). Specifically, present inventors designed a fusion protein consisting of human apoA1 and human IL4 (apoA1-IL4) connected via a flexible linker and flanked by two purification tags, a 6his-tag located at the N-terminus and a strep-tag at the C-terminus (Figure 19B). Present inventors used molecular characterization techniques to confirm the nature and purity of apoA1-IL4. By performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on each purified protein sample, present inventors confirmed the presence of protein with molecular weights of 25 kDa (apoA1), 18 kDa (IL4) and 37 kDa (apoA1-IL4) (Figure 19C), while Western-blots indicated the presence of apoA1 and IL4 (Figure 19D). Due to its amphophilic properties, proteins consisting of apoA1 run lower on the gel than expected. These observations were corroborated by quadrupole time-of-flight (Q-TOF) mass spectrometry showing a single mass peak at 47576.03 Da corresponding to apoATs expected molecular weight of 47582.57 Da (Figure 19E).

[0584] Before integrating apoA1-IL4 in lipid nanoparticles, biophysical and cellular analysis - using surface plasmon resonance (SPR) and HEK-Blue™ IL4 / IL13 (HEK-IL4) reporter cells - were performed to determine the preservation of biological activity after the purification and refolding ...

Claims

CLAIMS1. An apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule; and phospholipids; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response.

2. An apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule; and phospholipids; wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

3. An apolipoprotein lipid nanoparticle comprising a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule; and phospholipids; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response; and wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

4. An apolipoprotein lipid nanoparticle comprising the fusion protein as defined in claim 1 ; the fusion protein as defined in claim 2; and phospholipids.

5. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 4, wherein the apolipoprotein lipid nanoparticle further comprises sterols.

6. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 5, wherein the apolipoprotein lipid nanoparticle further comprises lipids, preferably triglycerides.

7. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 6, wherein said apolipoprotein lipid nanoparticle is a sphere, a ribbon or a disc.

8. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 7, wherein at least a part of said fusion protein is exposed to the environment surrounding said apolipoprotein lipid nanoparticle, preferably wherein said immunomodulatory biomolecule and / or said rerouting molecule is exposed to the environment surrounding said apolipoprotein lipid nanoparticle.

9. The apolipoprotein lipid nanoparticle according to any one of claims 1 or 3 to 8, wherein the immunomodulatory biomolecule is selected from the group consisting of a cytokine, a chemokine, a hormone, a growth factor, a hematopoietic growth factor, and combinations thereof.

10. The apolipoprotein lipid nanoparticle according to claim 9, wherein the cytokine is selected from the group consisting of the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 family, the TGFbeta family, or the IL-17 family, and combinations thereof, more preferably wherein the cytokine is selected from the group consisting of IL-1 p, IL-2, IL-4, IL-38, and combinations thereof; and / or wherein the chemokine is selected from the group consisting of a CC chemokine, a CXC chemokine, a C chemokine, a CX3C chemokine, and combinations thereof; and / or wherein the growth factor is selected from the group consisting of VEGF, EGF, CNTF, LIF, Ephrins, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, Neurotrophin, PGF, PDGF, RNLS, TCGF, TGF, TNF and WNT, and combinations thereof; and / or wherein the hematopoietic growth factor is selected from the group consisting of IL-3, CSF- 1 (M-CSF), GM-CSF, G-CSF, a member of the IL-12 family of interleukins or erythropoietin, and combinations thereof.11 . The apolipoprotein lipid nanoparticle according to claim 9 or 10, wherein the cytokine is IL-4.

12. The apolipoprotein lipid nanoparticle according to any one of claims 2 to 11 , wherein the rerouting molecule is selected from an antibody or an antigen binding fragment thereof, a rerouting peptide or a rerouting protein, preferably wherein the rerouting peptide or rerouting protein is a ligand of a receptor present on the target.

13. The apolipoprotein lipid nanoparticle according to claim 12, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of a Fab, a Fab2, a scFv, a scFv-Fc, a dAb-Fc, a free light chain antibody, a half antibody, a bispecific Fab2, a Faba, a trispecific Fab3 a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an IgNAR, a monovalent IgG, a VhH, and a variable new antigen receptor (VNAR).

14. The apolipoprotein lipid nanoparticle according to any one of claims 2 to 13, wherein the rerouting molecule is capable of binding to a hematopoietic stem and progenitor cell (HSPC), such as a hematopoietic stem cell (HSC), a multipotent progenitor (MPP), or a common myeloid progenitor cell (CMP).

15. The apolipoprotein lipid nanoparticle according to any one of claims 2 to 13, wherein the rerouting molecule is capable of binding to a myeloid cell selected from the group consisting of megakaryocyte, eosinophil, basophil, erythrocyte, monocyte such as dendritic cell or macrophage, and a neutrophil.

16. The apolipoprotein lipid nanoparticle according to claim 15, wherein the rerouting peptide is SIRPa.

17. The apolipoprotein lipid nanoparticle according to any one of claims 2 to 13, wherein the rerouting molecule is capable of binding to a non-myeloid cell, such as a non-myeloid immune cell or an endothelial cell.

18. The apolipoprotein lipid nanoparticle according to claim 17, wherein the rerouting molecule is capable of binding to lymphocytes, preferably T cells, more preferably CD8+ T cells.

19. The apolipoprotein lipid nanoparticle according to claim 17, wherein the rerouting molecule is an antibody or antigen binding fragment thereof specifically binding to CD8 or wherein the rerouting peptide is PD1 , CD40L or GP120.

20. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 19, wherein the apolipoprotein is an ApoA1 , ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3or the apolipoprotein mimetic is a mimetic of an ApoA1 , ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1 , ApoL3.21 . The apolipoprotein lipid nanoparticle according to any one of claims 1 to 20, wherein the apolipoprotein lipid nanoparticle comprises a payload, preferably wherein the payload is selected from a nucleic acid or a nucleic acid analog, a therapeutic, a biologic or combinations thereof.

22. The apolipoprotein lipid nanoparticle according to claim 21 , wherein the apolipoprotein lipid nanoparticle comprises a nucleic acid and a cationic or ionizable cationic lipid.

23. The apolipoprotein lipid nanoparticle according to claim 22, wherein the apolipoprotein lipid nanoparticle comprises a core surrounded by a surface layer, the nucleic acid and the cationic or ionizable cationic lipid are comprised by the core, and the fusion protein or fusion proteins as defined in any one of claims 1 to 3 and the phospholipids are comprised by the surface layer.

24. An apolipoprotein lipid nanoparticle comprising a core surrounded by a surface layer, wherein: the core comprises a nucleic acid and a cationic or ionizable cationic lipid; and the surface layer comprises: a phospholipid, a sterol, and(i) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response;(ii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity;(iii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule as defined in (i), and a rerouting molecule as defined in (ii); or(iv) the fusion protein as defined in (i) and the fusion protein as defined in (ii).

25. The apolipoprotein lipid nanoparticle according to any one of claims 22 to 24, wherein the nanoparticle core further comprises a filler, preferably a filler selected from a triacylglyceride and a cholesterol acyl ester, or combinations thereof, such as wherein the triacylglyceride is tricaprylin and / or wherein the cholesterol acyl ester is cholesteryl caprylate and / or cholesteryl oleate.

26. The apolipoprotein lipid nanoparticle according to any one of claims 21 to 25, wherein the nucleic acid is RNA, DNA or a nucleic acid analogue.

27. The apolipoprotein lipid nanoparticle according to claim 26, wherein the RNA is microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), transfer RNA (tRNA), tRNA-derived small RNA (tsRNA), small regulatory RNA (srRNA), messenger RNA (mRNA), modified mRNA, ribosomal RNA (rRNA), long non-coding RNA (IncRNA), or guide RNA (gRNA), or combinations thereof and / or modifications thereof.

28. The apolipoprotein lipid nanoparticle according to claim 26, wherein the DNA is single stranded or double stranded DNA.

29. The apolipoprotein lipid nanoparticle according to any one of claims 21 to 25, wherein the nucleic acid is an antisense oligonucleotide and the antisense oligonucleotide is single strand DNA or RNA consisting of nucleotide or nucleoside analogues containing modifications of the phosphodiester backbone or the 2’ ribose.

30. The apolipoprotein lipid nanoparticle according to claim 29, wherein the nucleotide or nucleoside analogues are 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 acids (HNA), or mixtures or combinations thereof.31 . The apolipoprotein lipid nanoparticle according to any one of claims 22 to 30, wherein the cationic 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 combinations thereof.

32. The apolipoprotein lipid nanoparticle according to any one of claims 22 to 31 , wherein the ionizable cationic lipid is a molecule according to any one of Formulae (I), (II), (III), (IV) or (V)wherein ICG is or , wherein the wavy line indicates the point of attachment to the compound of formulae (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 Ri is independently selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl group, wherein said 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 a -CH2-O-C(O)-Ria; R3 is selected from the group consisting of hydrogen, aryl, arylene-alkyl, alkylene-aryl and linear C1-C6 alkyl group;Ria is selected from the group consisting of linear or branched C1-C19 alkyl, linear or branched C1-C19 alkenyl, aryl, arylene-alkyl and alkylene-aryl group, wherein said alkyl oralkenyl group optionally contains up to 5 heteroatoms, independently selected from O and N; each Rxis independently selected from the group consisting of methyl, ethyl, propyl and - CH2-CH2-OH; each Rygroup is independently selected from the group consisting of hydrogen, linear or branched C1-C18 alkyl, aryl, arylene-alkyl or alkylene-aryl group, wherein said alkyl group optionally contains up to 5 heteroatoms, independently selected from O and N; or rotamers, tautomers stereoisomers or regioisomers thereof.

33. The apolipoprotein lipid nanoparticle according to any one of claims 22 to 32, wherein: the amount of the fusion protein ranges from 0.08 to 2.0 mol%, such as from 0.10 to 2.0 mol%; and / or the amount of phospholipid ranges from 5 to 90 mol%, such as from 15 to 90 mol%; and / or the amount of sterol ranges from 2.5 to 65 mol%, such as from 2.5 to 50 mol%; and / or the amount of cationic or ionizable cationic lipid ranges from 5.0 to 80 mol%, such as from 8.0 to 80 mol%, wherein the molar percentage is based solely on the combined amounts of the fusion protein, phospholipids, sterols and cationic or ionizable cationic lipids in the apolipoprotein lipid nanoparticle.

34. The apolipoprotein lipid nanoparticle according to any one of claims 22 to 33, wherein: the amount of the fusion protein ranges from 0.1 to 90 weight%; the amount of nucleic acid ranges from 0.01 to 90 weight%; the amount of phospholipid ranges from 0.1 to 95 weight%; the amount of sterol ranges from 0.1 to 95 weight%; and / or the amount of cationic and / or ionizable cationic lipid ranges from 0.1 to 95 weight%, wherein these weight percentages are based on the combined amounts of the fusion protein, the nucleic acid, the phospholipid, the sterol and the cationic and / or ionizable cationic lipid.

35. The apolipoprotein lipid nanoparticle according to any one of claims 22 to 34, wherein the ratio of the fusion protein to phospholipid based on percentage molar weight is between 1 :25 and 1 :400, more preferably between 1 :50 and 1 :200, even more preferably between 1 :75 and 1 :150.

36. The apolipoprotein lipid nanoparticle according to any one of claims 22 to 35, wherein the ratio of the fusion protein to phospholipid based on weight is from 2:1 to 1 :10, more preferably from 1 :1 to 1 :5, even more preferably from 1 :1.5 to 1 :4.

37. Method of manufacturing an apolipoprotein lipid nanoparticle as defined in any one of claims 1 to 36, the method comprising the steps of: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; and / or a2) chemically conjugating one or more apolipoproteins or apolipoprotein mimetics and isolating the one or more conjugated apolipoproteins to obtain one or more isolated conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; and b) combining the one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or the one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids, and optionally sterols and / or lipids, to obtain an apolipoprotein lipid nanoparticle.

38. Method for producing an apolipoprotein lipid nanoparticle, comprising the step of: a) rapid mixing of lipid components in organic solvent with a nucleic acid in an aqueous buffer to produce lipid nanoparticles, wherein the lipid components comprise a phospholipid, a sterol, a cationic lipid or ionizable cationic lipid, wherein the aqueous buffer has a pH of 5.0 or lower; and b) rapid mixing of the lipid nanoparticles with:one or more apolipoprotein fusion proteins, wherein the one or more apolipoprotein fusion proteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, and the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; and / or one or more conjugated apolipoproteins, wherein the one or more conjugated apolipoproteins are selected from the group consisting of: an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule; and combinations thereof; wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, and the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity; to produce the apolipoprotein lipid nanoparticle at a pH between 5.5 and 8.0, such as between 6.0 and 8.0.

39. An apolipoprotein lipid nanoparticle obtained by or obtainable by the method of claim 37 or 38.

40. A pharmaceutical composition comprising the apolipoprotein lipid nanoparticle according to any one of claims 1 to 36 or 39, and a pharmaceutically acceptable carrier.

41. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 36 or 39 or the pharmaceutical composition according to claim 40 for use as a medicament.

42. The apolipoprotein lipid nanoparticle according to any one of claims 1 to 36 or 39 or the pharmaceutical composition according to claim 40 for use in the treatment of an immune related disorder.

43. The apolipoprotein lipid nanoparticle for use according to claim 42 or the pharmaceutical composition for use according to claim 42, wherein the immune related disorder is selected from the group consisting of cancer, inflammation, an infectious disease, an autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

44. The apolipoprotein lipid nanoparticle for use according to claim 42 or the pharmaceutical composition for use according to claim 42, wherein the immunomodulatory biomolecule is IL-4 and wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

45. The apolipoprotein lipid nanoparticle according to any one of claims 1 , 3 to 36 or 39 or the pharmaceutical composition according to claim 40 for use in targeting said immunomodulatory biomolecule to a target cell.

46. The apolipoprotein lipid nanoparticle according to any one of claims 1 , 3 to 13, 15, 16, 20, 21 to 36 or 39 or the pharmaceutical composition according to claim 40 for use in targeting said immunomodulatory biomolecule to a myeloid cell.

47. Use of the apolipoprotein lipid nanoparticle according to any one of claims 1 , 3 to 36 or 39 for delivering an immunomodulatory biomolecule to a target, preferably wherein the target is a cell, tissue, and / or organ.

48. An in vitro or ex vivo method for introducing a nucleic acid in a cell, the method comprising contacting the apolipoprotein lipid nanoparticle according to any one of claims 21 to 36 or 39 or the composition according to claim 40 with a cell.

49. An in vivo method for introducing a nucleic acid in a cell, the method comprising contacting the apolipoprotein lipid nanoparticle according to any one of claims 21 to 36 or 39 or the composition according to claim 40 with a cell.

50. The apolipoprotein lipid nanoparticle according to any one of claims 21 to 36 or 39 or the composition according to claim 40 for use in the in vivo delivery of a nucleic acid to a subject.

51. A method for the in vivo delivery of a nucleic acid, the method comprising administering the apolipoprotein lipid nanoparticle according to any one of claims 21 to 36 or 39 or the composition according to claim 40 to a subject.

52. A method for treating a disease or disorder, such as a disease or disorder as described herein, in a subject in need thereof, for example by stimulating or inhibiting an innate immune response, the method comprising administering a therapeutically effective amount of the apolipoprotein lipid nanoparticle according to claims 21 to 36 or 39 or the composition according to claim 40 to the subject.

53. The method according to claim 52, wherein the disease is selected from cancer, cardiovascular disease, autoimmune disorder or xenograft rejection.

54. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response, for use in targeting said immunomodulatory biomolecule to a myeloid cell.

55. The fusion protein for use according to claim 54, wherein the fusion protein further comprises a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity, preferably wherein the rerouting molecule is a rerouting molecule as defined in claim 15.

56. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity.

57. The fusion protein according to claim 56, wherein the rerouting molecule is a rerouting molecule as defined in any one of claims 12 to 19.

58. The fusion protein according to claim 56 or 57, wherein the fusion protein further comprises an immunomodulatory biomolecule, wherein the immunomodulatory biomoleculeis a protein that enhances or suppresses an immune response, preferably wherein the immunomodulatory biomolecule is an immunomodulatory biomolecule as defined in claim 9 or 10.

59. A nucleic acid encoding the fusion protein according to any one of claims 56 to 58.

60. A pharmaceutical composition comprising the fusion protein according to any one of claims 56 to 58 or the nucleic acid according to claim 59, and a pharmaceutically acceptable carrier.61 . The fusion protein according to any one of claims 56 to 58, the nucleic acid according to claim 59 or the pharmaceutical composition according to claim 60 for use as a medicament.

62. The fusion protein according to any one of claims 56 to 58, the nucleic acid according to claim 59 or the pharmaceutical composition according to claim 60 for use in the treatment of an immune related disorder, preferably wherein the immune related disorder is an immune related disorder selected from the group consisting of cancer, inflammation, an infectious disease, an autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

63. The fusion protein according to claim 58, the nucleic acid encoding the fusion protein according to claim 58 or the pharmaceutical composition according to claim 60 when being dependent from claim 58 for use in targeting said immunomodulatory biomolecule to a target cell.

64. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4).

65. The fusion protein according to claim 64, wherein the fusion protein further comprises a rerouting molecule, wherein the rerouting molecule is a molecule that allows that fusion protein to bind to a different target than to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with a higher affinity, preferably wherein the rerouting molecule is a rerouting molecule as defined in any one of claims 12 to 19.

66. The fusion protein according to claim 64 or 65, wherein the apolipoprotein or apolipoprotein mimetic is as defined in claim 20.

67. A nucleic acid encoding the fusion protein according to any one of claims 64 to 66.

68. A pharmaceutical composition comprising the fusion protein according to any one of claims 64 to 66 or the nucleic acid according to claim 67, and a pharmaceutically acceptable carrier.

69. The fusion protein according to any one of claims 64 to 66, the nucleic acid according to claim 67, or the pharmaceutical composition according to claim 68 for use as a medicament.

70. The fusion protein according to any one of claims 64 to 66, the nucleic acid according to claim 67, or the pharmaceutical composition according to claim 68 for use in the treatment of an immune related disorder.

71. The fusion protein for use according to claim 70, the nucleic acid for use according to claim 70, or the pharmaceutical composition for use according to claim 70, wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

72. The fusion protein according to any one of claims 64 to 66, the nucleic acid according to claim 67, or the pharmaceutical composition according to claim 48 for use in targeting IL- 4 to a target cell.

73. The fusion protein according to any one of claims 64 to 66, the nucleic acid according to claim 67, or the pharmaceutical composition according to claim 68 for use in targeting IL- 4 to a myeloid cell.

74. A fusion protein comprising a myeloid-targeting molecule and IL-4, wherein the myeloid-targeting molecule is capable of targeting the IL-4 to a myeloid cell.

75. The fusion protein according to claim 74, wherein the IL-4 is a polypeptide comprising an amino acid sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 43, or a circular permutation thereof.

76. The fusion protein according to claim 74 or 75, wherein the myeloid-targeting molecule is selected from an antibody or an antigen binding fragment thereof, a myeloid- targeting peptide or a myeloid-targeting protein, preferably wherein the myeloid-targeting peptide or myeloid-targeting protein is a ligand of a receptor present on the target.

77. The fusion protein according to claim 76, wherein the antibody or antigen binding fragment thereof is selected from a Fab, a Fab2, a scFv, a scFv-Fc, a dAb-Fc, a free light chain antibody, a half antibody, a bispecific Fab2, a Faba, a trispecific Fab3 a diabody, a bispecific diabody, a triabody, a trispecific triabody, a minibody, an IgG, an IgNAR, a monovalent IgG, a VhH or a VNAR.

78. A nucleic acid encoding the fusion protein according to any one of claims 74 to 77.

79. A nucleic acid comprising a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO.44 or comprising a nucleic acid sequence encoding a polypeptide having a sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO. 43 and further comprising means for targeted expression in a myeloid cell, wherein said mean are selected from:- a promoter for selective or inducible expression in said myeloid cell operatively linked to said nucleic acid; or- a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cell; or- a lipid nanoparticle comprising one or more apolipoproteins, phospholipids, said nucleic acid, and optionally sterol.

80. A pharmaceutical composition comprising the fusion protein according to any one of claims 74 to 77 or the nucleic acid according to claim 78 or 79, and a pharmaceutically acceptable carrier.

81. The fusion protein according to any one of claims 74 to 77, the nucleic acid according to claim 78 or 79, or the pharmaceutical composition according to claim 80 for use as a medicament.

82. The fusion protein according to any one of claims 74 to 77, the nucleic acid according to claim 78 or 79, or the pharmaceutical composition according to claim 80 for use in the treatment of an immune related disorder.

83. The fusion protein for use according to claim 82, the nucleic acid for use according to claim 82, or the pharmaceutical composition for use according to claim 82, wherein the immune related disorder is a state of hyperinflammation followed by immune paralysis, preferably wherein the hyperinflammation and / or the immune paralysis is caused by an infectious disease such as COVID-19, by sepsis, myocardial infarction or stroke.

84. In vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or an organism.

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