Apolipoprotein fusion proteins for cell-specific immune modulation

Abstract

The present invention relates to fusion proteins of apolipoproteins and immunomodulatory biomolecules and / or rerouting molecules. The fusion proteins can be used as carriers for the immunomodulatory biomolecules themselves or can be incorporated into lipid nanoparticles. The fusion proteins find use in the treatment of immune-related disorders or to target payloads to specific target sites.

Description

[Technical field]

[0001] The present invention relates to the field of fusion proteins, more particularly to fusion proteins that find use in the treatment of immune-related disorders.The present invention further relates to lipid nanoparticles comprising the fusion proteins and methods for making the same.Finally, the present invention relates to methods of treatment using the fusion proteins or lipid nanoparticles. [Background technology]

[0002] Many promising therapeutics are hindered by a lack of circulation time due to the rapid removal of the therapeutic from the body. For example, it has been found that cytokines may hold very promising uses in many immunological applications. However, due to their very short half-life in the body, they either fail to exert an effect on the intended target or require a toxic amount to achieve an effect. Therefore, improved methods are needed to safely reduce the circulating half-life of therapeutics.

[0003] Furthermore, many promising therapeutic agents suffer from the fact that they do not reach the intended target site, or do so insufficiently, or exhibit undesirable off-target effects. Thus, there is a further need to improve the targeting of therapeutic agents.

[0004] Among other things, these problems are addressed by the products and methods defined in the appended claims. Summary of the Invention

[0005] The present invention is based on the inventors' discovery that apolipoproteins can be used as therapeutic drug carriers, and apolipoproteins can be further modified to target specific cells, tissues or organs.The inventors have found that the fusion protein of an immunomodulatory biomolecule, such as cytokines, and an apolipoprotein or an apolipoprotein mimic shows a significant increase in half-life in blood, thereby opening up the possibility of using an immunomodulatory biomolecule, such as cytokines, in a therapeutic manner without the need for administration of toxic concentrations.Furthermore, it has been found that apolipoproteins or mimics thereof, when fused together, allow the targeting of an immunomodulatory biomolecule, such as cytokines.

[0006] It has further been recognized that fusion protein or apolipoprotein (or mimic thereof) can be directed to intended target by linking it to rerouting molecule.Furthermore, the inventors have unexpectedly found that fusion of immunomodulatory biomolecules and / or rerouting molecules to apolipoprotein or apolipoprotein mimic allows said immunomodulatory biomolecules and / or rerouting molecules to be easily incorporated into lipid nanoparticles and to expose said immunomodulatory biomolecules and / or rerouting molecules to the environment surrounding said lipid nanoparticles.In this way, apolipoprotein (or mimic thereof) or fusion protein can be targeted to cells, tissues or organs that are otherwise not or poorly reached, or can be used to reduce off-target effects.

[0007] The fusion protein can be used as it is, meaning that it is not part of a lipoprotein or lipid nanoparticle. In this way, the fusion protein can serve as a carrier for delivering immunomodulatory biomolecules to target sites. Alternatively, the fusion protein can be used to prepare lipid nanoparticles.

[0008] A first aspect of the present invention is an apolipoprotein lipid nanoparticle, comprising: A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, and phospholipids, Apolipoprotein lipid nanoparticles are provided in which the immunomodulatory biomolecule is a protein that enhances or suppresses the immune response.

[0009] A further aspect of the invention is an apolipoprotein lipid nanoparticle comprising: A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, and phospholipids, A rerouting molecule is a molecule that enables the fusion protein to bind to a target different from the target that the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with higher affinity, as provided by an apolipoprotein lipid nanoparticle.

[0010] A further aspect of the invention is an apolipoprotein lipid nanoparticle comprising: A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule, and phospholipids, the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response; A rerouting molecule is a molecule that enables the fusion protein to bind to a target different from the target that the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with higher affinity, as provided by an apolipoprotein lipid nanoparticle.

[0011] A further aspect of the invention is an apolipoprotein lipid nanoparticle comprising: a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, the immunomodulatory biomolecule being a protein that enhances or suppresses an immune response; A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, the rerouting molecule being a molecule that enables the fusion protein to bind to a target different from the target that the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind its intended target with higher affinity, and Apolipoprotein lipid nanoparticles comprising a phospholipid are provided.

[0012] A further aspect of the present invention is a method of producing the apolipoprotein lipid nanoparticles described herein, comprising the steps of: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, One or more apolipoprotein fusion proteins Apolipoproteins or apolipoprotein mimetics fused to immunomodulatory biomolecules; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; Apolipoproteins or apolipoprotein mimetics fused to immune-modulating biomolecules and rerouting molecules, and Combinations of these 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, The one or more conjugated apolipoproteins Apolipoproteins or apolipoprotein mimetics conjugated to immunomodulatory biomolecules; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; Apolipoproteins or apolipoprotein mimetics conjugated to immunomodulatory biomolecules and rerouting molecules, and Combinations of these and b) combining one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids, and optionally sterols and / or lipids, to obtain apolipoprotein-lipid nanoparticles. The present invention provides a method comprising:

[0013] A further aspect of the present invention provides apolipoprotein lipid nanoparticles obtained or obtainable by the methods taught herein.

[0014] A further aspect of the present invention provides a pharmaceutical composition comprising the apolipoprotein lipid nanoparticles taught herein and a pharma- ceutically acceptable carrier.

[0015] A further aspect of the present invention provides an apolipoprotein lipid nanoparticle as taught herein or a pharmaceutical composition as taught herein for use as a medicament.

[0016] A further aspect of the present invention provides an apolipoprotein lipid nanoparticle as taught herein or a pharmaceutical composition as taught herein for use in treating an immune-related disorder.

[0017] A further aspect of the present invention provides an apolipoprotein lipid nanoparticle as taught herein or a pharmaceutical composition as taught herein for use in targeting said immunomodulatory biomolecule to a target cell, preferably a myeloid cell.

[0018] A further aspect of the present invention provides the use of the apolipoprotein lipid nanoparticles taught herein for targeted delivery of immunomodulatory biomolecules, preferably wherein the target is a cell, tissue and / or organ.

[0019] A further aspect of the invention provides a fusion protein comprising an apolipoprotein or apolipoprotein mimetic and an immunomodulatory biomolecule, the immunomodulatory biomolecule being a protein that enhances or suppresses an immune response, for use in targeting the immunomodulatory biomolecule to myeloid cells.

[0020] A further aspect of the present invention provides a fusion protein comprising an apolipoprotein or apolipoprotein mimetic and a rerouting molecule, the rerouting molecule being a molecule that enables the fusion protein to bind to a target different from the target that the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind its intended target with higher affinity.

[0021] A further aspect of the invention provides a nucleic acid encoding a fusion protein comprising an apolipoprotein or apolipoprotein mimetic taught herein and a rerouting molecule.

[0022] A further aspect of the invention provides a pharmaceutical composition comprising a fusion protein as taught herein, a nucleic acid as taught herein, and a pharma- ceutically acceptable carrier.

[0023] Further aspects of the present invention provide a fusion protein as taught herein, a nucleic acid as taught herein, or a pharmaceutical composition as taught herein for use as a medicament.

[0024] A further aspect of the present invention provides a fusion protein as taught herein, a nucleic acid as taught herein, or a pharmaceutical composition as taught herein for use in treating an immune-related disorder, preferably the immune-related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorder, allergy, organ transplant rejection and graft-versus-host disease (GVH).

[0025] A further aspect provides a pharmaceutical composition as taught herein when comprising a fusion protein as taught herein, a nucleic acid encoding a fusion protein as taught herein, or an immunomodulatory biomolecule that is a protein that enhances or suppresses an immune response, for use in targeting said immunomodulatory biomolecule to a target cell.

[0026] As supported by the Examples section, the inventors found that a fusion protein of an apolipoprotein or apolipoprotein mimetic, preferably ApoA1 and IL-4, allows IL-4 to be targeted to the myeloid compartment. To their surprise, the inventors found that IL-4 can simultaneously reduce inflammation and induce trained immunity, especially when targeted to the myeloid compartment. Thus, the inventors concluded that an apolipoprotein or apolipoprotein mimetic, preferably ApoA1 and IL-4 fusion protein, can be used to prevent immune-related disorders by promoting trained immunity. Furthermore, the inventors found that the pharmacokinetic profile and bioavailability of IL-4 to innate immune cells can be further improved by incorporating said fusion protein into myeloid cell-affinity lipid nanoparticles. The incorporation of such IL-4 fusion protein into lipid nanoparticles does not hinder its ability to target the myeloid compartment.

[0027] Thus, a further aspect of the invention provides a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4).

[0028] A further aspect of the invention provides a nucleic acid encoding a fusion protein comprising an apolipoprotein or apolipoprotein mimetic taught herein and interleukin-4 (IL-4).

[0029] A further aspect of the present invention provides a pharmaceutical composition comprising a fusion protein comprising an apolipoprotein or apolipoprotein mimetic as taught herein and interleukin-4 (IL-4), or a nucleic acid encoding said fusion protein as taught herein, and a pharma- ceutically acceptable carrier.

[0030] Further aspects of the present invention provide fusion proteins comprising an apolipoprotein or apolipoprotein mimetic as taught herein and interleukin-4 (IL-4), nucleic acids encoding such fusion proteins as taught herein, or pharmaceutical compositions comprising such fusion proteins or nucleic acids for use as pharmaceuticals.

[0031] A further aspect of the present invention provides a fusion protein comprising an apolipoprotein or apolipoprotein mimetic as taught herein and interleukin-4 (IL-4), a nucleic acid encoding such a fusion protein as taught herein, or a pharmaceutical composition comprising such a 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 immunoparalysis, preferably wherein the hyperinflammation and / or immunoparalysis is caused by an infectious disease such as COVID-19 due to sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

[0032] Further aspects of the present invention provide fusion proteins comprising an apolipoprotein or apolipoprotein mimetic as taught herein and interleukin-4 (IL-4), nucleic acids encoding such fusion proteins as taught herein, or pharmaceutical compositions comprising such fusion proteins or nucleic acids for use in targeting IL-4 to target cells, preferably myeloid cells.

[0033] A further aspect of the invention provides a fusion protein comprising a bone marrow targeting molecule and IL-4, wherein the bone marrow targeting molecule is capable of targeting the IL-4 to myeloid cells.

[0034] A further aspect of the present invention provides a nucleic acid encoding a fusion protein comprising a bone marrow targeting molecule as taught herein and IL-4.

[0035] A further aspect of the invention is 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 a means for targeted expression in myeloid cells, said means comprising - a promoter for selective or inducible expression in said myeloid cells operably linked to said nucleic acid, or a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cells, or - a nucleic acid selected from one or more apolipoproteins, a phospholipid, a lipid nanoparticle comprising said nucleic acid and optionally a sterol.

[0036] A further aspect of the invention provides a pharmaceutical composition comprising a fusion protein comprising a myeloid targeting molecule as taught herein and IL-4, or a nucleic acid encoding said fusion protein as taught herein, or a nucleic acid comprising a means for targeted expression in myeloid cells as taught herein, and a pharma- ceutically acceptable carrier.

[0037] In a further aspect of the invention, a fusion protein comprising a myeloid targeting molecule as taught herein and IL-4, or a nucleic acid encoding said fusion protein as taught herein, or a nucleic acid comprising a means for targeted expression in myeloid cells as taught herein, or a pharmaceutical composition comprising said fusion protein or nucleic acid as taught herein for use as a medicament.

[0038] A further aspect of the invention provides a fusion protein comprising a myeloid targeting molecule as taught herein and IL-4, or a nucleic acid encoding said fusion protein as taught herein, or a nucleic acid comprising a means for targeted expression in myeloid cells as taught herein, or a pharmaceutical composition comprising said fusion protein or nucleic acid as taught herein for use in the treatment of an immune related disorder.

[0039] A further aspect of the invention provides the in vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or organism. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows a schematic overview of the optional assembly of exemplary apolipoprotein fusions in lipid nanoparticles (spheres or discs) and subsequent binding to target cells.

[0041] [Diagram 2]A schematic overview of different envisioned apolipoproteins and their subsequent assembly in lipid nanoparticles is shown. The following fusion proteins are shown (schematically): top left shows both an apolipoprotein fused to an immunomodulatory biomolecule and an apolipoprotein fused to a rerouting molecule. bottom left shows an apolipoprotein fused to an immunomodulatory biomolecule. top right shows an apolipoprotein fused to a rerouting molecule. bottom right shows an apolipoprotein fused to an immunomodulatory biomolecule and a rerouting molecule.

[0042] [Figure 3-1] Shown is an SDS-PAGE gel demonstrating the expression and purification of different apolipoprotein fusion constructs (ApoA1-IL1B, ApoA1-IL38, ApoA1-IL2). The rectangle indicates the band corresponding to the desired protein. (P: pellet containing cell debris, SN: supernatant containing the soluble protein fraction, FT: flow-through of SN applied to the Ni-NTA column, A: 1st wash with 10 mM imidazole, A50: 2nd wash with 50 mM imidazole, E1: elution fraction 1, E2: elution fraction 2, E3: elution fraction 3, E4: elution fraction 4, FW: final wash with 500 mM imidazole.) [Figure 3-2] Shown is an SDS-PAGE gel demonstrating the expression and purification of different apolipoprotein fusion constructs (ApoA1-IL1B, ApoA1-IL38, ApoA1-IL2). The rectangle indicates the band corresponding to the desired protein. (P: pellet containing cell debris, SN: supernatant containing the soluble protein fraction, FT: flow-through of SN applied to the Ni-NTA column, A: 1st wash with 10 mM imidazole, A50: 2nd wash with 50 mM imidazole, E1: elution fraction 1, E2: elution fraction 2, E3: elution fraction 3, E4: elution fraction 4, FW: final wash with 500 mM imidazole.)

[0043] [Figure 4]Dynamic light scattering measurements are shown for four lipid nanoparticles, three of which contain different apolipoproteins (ApoA1-IL1B, ApoA1-IL38, ApoA1-IL4) and one containing apoA1 as a control nanoparticle. The mean number diameter (dark grey) and polydispersity index (PdI) (light grey) were determined for these nanoparticles over a period of 11 days.

[0044] [Diagram 5] 1 shows an SDS-PAGE gel showing expression and purification of apoA1 with serine to cysteine ​​mutations at either position 147 or 279. The rectangle indicates the band corresponding to the mutated apoA1.

[0045] [Figure 6-1] Quadrupole time-of-flight (Q-ToF) results for apoA1 mutants are shown. In both graphs, the chromatogram is plotted in the top right corner, below which is the m / z value of the main peak from the chromatogram. The deconvoluted mass spectrum shows that the desired mutant apoA1 protein is present. [Figure 6-2] Quadrupole time-of-flight (Q-ToF) results for apoA1 mutants are shown. In both graphs, the chromatogram is plotted in the top right corner, below which is the m / z value of the main peak from the chromatogram. The deconvoluted mass spectrum shows that the desired mutant apoA1 protein is present.

[0046] [Figure 7] HPLC-MS chromatogram of interleukin (IL)-4 modified to contain a single N-terminal azide. Masses corresponding to the proteins represented by the peaks in the chromatogram are indicated.

[0047] [Figure 8]Shown is an SDS-PAGE gel showing the purity of the IL-4 used, IL-4 modified with an N-terminal azide (reactions at 4 °C or 20 °C), apoA1 linked to a PEG-linker-containing DBCO group, and the reaction product in which IL-4 modified to contain an azide was linked to apoA1 via the linker and DBCO group (reactions at 4 °C or 20 °C). The rectangle indicates the band corresponding to the desired conjugation product.

[0048] [Figure 9] 1 shows the results of a HEK293 IL-4 reporter cell assay evaluating the binding of commercially available IL-4 (mammalian), recombinantly expressed IL-4 (bacterial), recombinantly expressed apoA1-IL4 fusion protein, and chemically conjugated apoA1-IL4 fusion protein. Absorbance corresponds to the level of binding of IL-4 to its receptor.

[0049] [Figure 10] SDS-PAGE analysis of chemical (right panel) and recombinant (left panel) apoA1-IL2 fusion constructs (apoA1-IL2 wild type "ApoA1-IL2" or apoA1-IL2 mutant "ApoA1-IL2v4"). Rectangles indicate bands corresponding to apoA1-IL2 or apoA1-IL2v4 fusion constructs, respectively.

[0050] [Figure 11] Successful formulation of discoidal nanoparticles containing apoA1-IL2 fusion protein using cryogenic transmission electron microscopy (cryo-TEM) (right panel) and analysis of nanoparticle size and stability in PBS over 21 days using dynamic light scattering (DLS) (left panel).

[0051] [Figure 12] Ability of apoA1-IL2 fusion protein to stimulate proliferation of CD4+ or CD8+ T cells. Abbreviations: PHA, phytohemagglutinin.

[0052] [Figure 13] SDS-PAGE analysis of chemical (right panel) and recombinant (left panel) apoA1-IL1β fusion constructs is shown. The arrow (left panel) or rectangle (right panel) indicates the band corresponding to the apoA1-IL1β fusion construct.

[0053] [Figure 14] Successful formulation of discoidal nanoparticles containing apoA1-IL1β fusion protein using cryogenic transmission electron microscopy (cryo-TEM) (right panel) and analysis of nanoparticle size and stability in PBS over 21 days using dynamic light scattering (DLS) (left panel).

[0054] [Figure 15] SDS-PAGE analysis of chemical (lower panel) and recombinant (upper panel) apoA1-IL38 fusion constructs is shown. Arrows (upper panel) or rectangles (lower panel) indicate bands corresponding to the apoA1-IL38 fusion constructs.

[0055] [Figure 16] Successful formulation of discoidal nanoparticles containing apoA1-IL38 fusion protein using cryogenic transmission electron microscopy (cryo-TEM) (right panel) and analysis of nanoparticle size and stability in PBS over 21 days using dynamic light scattering (DLS) (left panel).

[0056] [Figure 17-1] IL4 inhibits acute inflammation but induces trained immunity. (A) Schematic of the in vitro direct inflammation experiment. (B) TNF, IL6 and IL1Ra levels after 24 h stimulation of human primary monocytes. (C) Schematic of the in vitro trained immunity experiment. (D) TNF and IL6 levels after restimulation of β-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 shown as mean ± SD. [Figure 17-2] IL4 inhibits acute inflammation but induces trained immunity. (A) Schematic of the in vitro direct inflammation experiment. (B) TNF, IL6 and IL1Ra levels after 24 h stimulation of human primary monocytes. (C) Schematic of the in vitro trained immunity experiment. (D) TNF and IL6 levels after restimulation of β-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 shown as mean ± SD.

[0057] [Figure 18-1] Immune and epigenetic mechanisms mediating IL4-induced trained immunity. (A) Schematic overview of the primary IL4 signaling pathways mentioned above. (B) TNF and IL6 levels after 24 h stimulation of monocytes while blocking key IL4 signaling pathways. (C) TNF and IL6 levels after restimulation of IL4-trained cells while blocking key IL4 signaling pathways. (D) Heatmap of IL4-trained cell transcriptome before and after restimulation. (E) Transcription factor motif enrichment analysis in IL4-trained immunity (heatmap shows z-score). (F) Pathway enrichment analysis of IL4-trained immune transcriptome. (G) TNF and IL6 levels after restimulation of IL4-trained cells in the presence of SET7 methyltransferase inhibitor. (H) ChIP-qPCR AUC analysis of TNF in IL4-trained cells. Data in bar graphs are presented as mean ± SD. [Figure 18-2]Immune and epigenetic mechanisms mediating IL4-induced trained immunity. (A) Schematic overview of the primary IL4 signaling pathways mentioned above. (B) TNF and IL6 levels after 24 h stimulation of monocytes while blocking key IL4 signaling pathways. (C) TNF and IL6 levels after restimulation of IL4-trained cells while blocking key IL4 signaling pathways. (D) Heatmap of IL4-trained cell transcriptome before and after restimulation. (E) Transcription factor motif enrichment analysis in IL4-trained immunity (heatmap shows z-score). (F) Pathway enrichment analysis of IL4-trained immune transcriptome. (G) TNF and IL6 levels after restimulation of IL4-trained cells in the presence of SET7 methyltransferase inhibitor. (H) ChIP-qPCR AUC analysis of TNF in IL4-trained cells. Data in bar graphs are presented as mean ± SD.

[0058] [Figure 19-1] Engineered apoA1-IL4 fusion protein. (A) Schematic overview of the apoA1-based fusion protein platform. (B) Schematic of the apoA1-IL4 fusion protein structure. (C) SDS-PAGE of recombinantly expressed proteins, 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 IL4Rα using SPR. (G) Activation of HEK-Blue cells expressing IL4Rα and IL13Rα1 by apoA1-IL4. Data are shown as mean ± SD. [Figure 19-2]Engineered apoA1-IL4 fusion protein. (A) Schematic overview of the apoA1-based fusion protein platform. (B) Schematic of the apoA1-IL4 fusion protein structure. (C) SDS-PAGE of recombinantly expressed proteins, 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 IL4Rα using SPR. (G) Activation of HEK-Blue cells expressing IL4Rα and IL13Rα1 by apoA1-IL4. Data are shown as mean ± SD.

[0059] [Figure 20-1] Integration of apoA1-IL4 in nanoparticle platforms. (A) Schematic representation of discoidal (upper panel) and spherical IL4-aNPs (lower panel) and (B) cryo-TEM images. (C) IL4-aNP size distribution and (D) IL4-aNP stability over time as determined by dynamic light scattering. IL4-aNP sizes are reported as number averages. (E) Super-resolution fluorescence microscopy (dSTORM) images of human monocytes incubated with fluorescently labeled apoA1(-IL4), or (IL4-)aNPs and stained with anti-IL4Rα antibody. Colocalization between the proteins and IL4Rα can be recognized by the arrows. In subsequent images on the right, the white areas of interest are enlarged. Data are shown as mean ± SD. [Figure 20-2]Integration of apoA1-IL4 in nanoparticle platforms. (A) Schematic representation of discoidal (upper panel) and spherical IL4-aNPs (lower panel) and (B) cryo-TEM images. (C) IL4-aNP size distribution and (D) IL4-aNP stability over time as determined by dynamic light scattering. IL4-aNP sizes are reported as number averages. (E) Super-resolution fluorescence microscopy (dSTORM) images of human monocytes incubated with fluorescently labeled apoA1(-IL4), or (IL4-)aNPs and stained with anti-IL4Rα antibody. Colocalization between the proteins and IL4Rα can be recognized by the arrows. In subsequent images on the right, the white areas of interest are enlarged. Data are shown as mean ± SD.

[0060] [Figure 21-1] Immunological in vitro, in vivo and ex vivo therapeutic evaluation of IL4-aNPs. (A) Schematic overview of in vitro direct inflammation and trained immunity experiments. (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 trained cells with IL4(-aNPs). (D) Schematic overview of a murine in vivo tolerance model involving IL4-nanotherapy. (E) Serum TNF and IL6 levels after LPS re-exposure in mice treated with IL4m-aNPs. Mann-Whitney U test was used for statistical comparison. (F) Schematic overview of a human experimental endotoxemia model involving ex vivo tolerance reversal. (G) TNF and IL6 levels after ex vivo restimulation of human in vivo LPS tolerized cells. (H) TNF and IL6 are fold increased after ex vivo restimulation of human in vivo LPS tolerized cells. Data are shown as mean ± SD. [Figure 21-2]Immunological in vitro, in vivo and ex vivo therapeutic evaluation of IL4-aNPs. (A) Schematic overview of in vitro direct inflammation and trained immunity experiments. (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 trained cells with IL4(-aNPs). (D) Schematic overview of a murine in vivo tolerance model involving IL4-nanotherapy. (E) Serum TNF and IL6 levels after LPS re-exposure in mice treated with IL4m-aNPs. Mann-Whitney U test was used for statistical comparison. (F) Schematic overview of a human experimental endotoxemia model involving ex vivo tolerance reversal. (G) TNF and IL6 levels after ex vivo restimulation of human in vivo LPS tolerized cells. (H) TNF and IL6 are fold increased after ex vivo restimulation of human in vivo LPS tolerized cells. Data are shown as mean ± SD. [Figure 21-3] Immunological in vitro, in vivo and ex vivo therapeutic evaluation of IL4-aNPs. (A) Schematic overview of in vitro direct inflammation and trained immunity experiments. (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 trained cells with IL4(-aNPs). (D) Schematic overview of a murine in vivo tolerance model involving IL4-nanotherapy. (E) Serum TNF and IL6 levels after LPS re-exposure in mice treated with IL4m-aNPs. Mann-Whitney U test was used for statistical comparison. (F) Schematic overview of a human experimental endotoxemia model involving ex vivo tolerance reversal. (G) TNF and IL6 levels after ex vivo restimulation of human in vivo LPS tolerized cells. (H) TNF and IL6 are fold increased after ex vivo restimulation of human in vivo LPS tolerized cells. Data are shown as mean ± SD.

[0061] [Figure 22] 1 shows a schematic overview of the optional assembly of an exemplary apolipoprotein fusion comprising an apolipoprotein and a rerouting protein on a lipid nanoparticle (disc).

[0062] [Figure 23] Figure 1 shows the expression of the VHHCD8-apoA1 fusion protein in Clearcoli cells. There is a slight protein contamination after IMAC purification [lane E1]. The most prominent band corresponds to the fusion protein with a molecular weight of 43.3 kDa (rectangle).

[0063] [Figure 24] Successful formulation of discoidal nanoparticles containing VHHCD8-apoA1 fusion protein using cryogenic transmission electron microscopy (cryo-TEM) (right panel) and analysis of nanoparticle size and polydispersity index (PDI) over 14 days using dynamic light scattering (DLS) (left panel).

[0064] [Diagram 25] The mean fluorescence intensity (MFI) of fluorescently labeled VHHCD8-apoA1 and apoA1 in mouse splenocytes (upper panel: CD3+ T cells derived from splenocytes; lower panel: total cells from the spleen) is shown.

[0065] [Figure 26] Shown is the mean fluorescence intensity (MFI) of discoidal and spherical aNPs formulated with VHHCD8-apoA1 and apoA1 and containing a fluorescent dye in the lipid structure of the particles in mouse splenocytes.

[0066] [Figure 27-1]In vivo pharmacokinetics, biodistribution and safety profile after intravenous injection. (A) PET / CT 2 rendered at 24 hours after injection of 89Zr-labeled constructs. (B) Blood half-life of 89Zr-labeled constructs (n=5, fitted with a biphasic decay function). (C) Ex vivo gamma counting of tissues 24 hours after injection of 89Zr-labeled constructs (n=5), numbers represent ratios of target and clearance organs. (D) Cell type-specific biodistribution of DiO-labeled discoidal IL4-aNPs in spleen and bone marrow measured by flow cytometry. (E) 89Zr-IL4-aNP blood half-life in non-human primates. (F) Organ SUV averages over time for 89Zr-IL4-aNP injected in non-human primates (n=2). (G) Organ-specific SUV averages 48 hours after 89Zr-IL4-aNP injection in non-human primates (n=2). (H) PET / MRI scan of a non-human primate 48 h after 89Zr-IL4-aNP injection. Data are presented as mean ± SD, where appropriate. [Figure 27-2] In vivo pharmacokinetics, biodistribution and safety profile after intravenous injection. (A) PET / CT 2 rendered at 24 hours after injection of 89Zr-labeled constructs. (B) Blood half-life of 89Zr-labeled constructs (n=5, fitted with a biphasic decay function). (C) Ex vivo gamma counting of tissues 24 hours after injection of 89Zr-labeled constructs (n=5), numbers represent ratios of target and clearance organs. (D) Cell type-specific biodistribution of DiO-labeled discoidal IL4-aNPs in spleen and bone marrow measured by flow cytometry. (E) 89Zr-IL4-aNP blood half-life in non-human primates. (F) Organ SUV averages over time for 89Zr-IL4-aNP injected in non-human primates (n=2). (G) Organ-specific SUV averages 48 hours after 89Zr-IL4-aNP injection in non-human primates (n=2). (H) PET / MRI scan of a non-human primate 48 h after 89Zr-IL4-aNP injection. Data are presented as mean ± SD, where appropriate. [Figure 27-3]In vivo pharmacokinetics, biodistribution and safety profile after intravenous injection. (A) PET / CT 2 rendered at 24 hours after injection of 89Zr-labeled constructs. (B) Blood half-life of 89Zr-labeled constructs (n=5, fitted with a biphasic decay function). (C) Ex vivo gamma counting of tissues 24 hours after injection of 89Zr-labeled constructs (n=5), numbers represent ratios of target and clearance organs. (D) Cell type-specific biodistribution of DiO-labeled discoidal IL4-aNPs in spleen and bone marrow measured by flow cytometry. (E) 89Zr-IL4-aNP blood half-life in non-human primates. (F) Organ SUV averages over time for 89Zr-IL4-aNP injected in non-human primates (n=2). (G) Organ-specific SUV averages 48 hours after 89Zr-IL4-aNP injection in non-human primates (n=2). (H) PET / MRI scan of a non-human primate 48 h after 89Zr-IL4-aNP injection. Data are presented as mean ± SD, where appropriate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The recitation of numerical ranges by endpoints includes all integers and, where appropriate, fractions subsumed within each range, as well as the recited endpoints. This applies to numerical ranges whether introduced by the phrase "from" or "between" or another phrase. Any numerical range recited herein is intended to include all subranges subsumed therein.

[0068] The terms "about" or "approximately" as used herein when referring to a measurable value, such as a parameter, amount, duration, etc., are meant to encompass variation of the specified value and variation from the specified value, e.g., variation of no more than + / - 10%, preferably no more than + / - 5%, more preferably no more than + / - 1%, and even more preferably no more than + / - 0.1% of the specified value, insofar as such variation is appropriate for the practice of the disclosed invention. It is to be understood that the values ​​to which the modifier "about" or "approximately" refers are themselves specifically, and preferably disclosed.

[0069] Moreover, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements, unless otherwise specified, and are not necessarily intended to describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, with the understanding that the embodiments of the invention described herein may operate in sequences other than those described or illustrated herein.

[0070] The discussion of the background of the invention herein is included to explain the context of the invention and should not be construed as an admission that any of the material mentioned was published, publicly known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0071] Throughout this disclosure, various publications, patents and published patent specifications are referenced by specific citations. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents that are specifically mentioned herein are incorporated by reference.

[0072] Unless otherwise defined, all terms used in this disclosure, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which this invention belongs. For further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in relation to a particular aspect of the present invention or a particular embodiment of the present invention, such connotation or meaning is meant to apply throughout this specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise defined.

[0073] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined can be combined with any other aspect or embodiment, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0074] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features included in other embodiments but not other features, but it is meant that combinations of features of different embodiments form different embodiments within the scope of the invention and as would be understood by one of ordinary skill in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0075] Similarly, in describing exemplary embodiments of the invention, it will be understood that various features of the invention may be 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.

[0076] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a combination of two or more cells, and the like.

[0077] As used herein, the term "and / or" refers to a situation in which one or more of the stated instances may occur alone or in combination with at least one of the stated instances, up to all of the stated instances. For example, if a list is stated to include groups A, B, and / or C, the list can include A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0078] As used herein, the term "antigen" refers to a substance to which the binding portion of an antibody can bind. A particular immunoreactive site within an antigen is known as an "epitope" (or antigenic determinant). The target of an antibody, or an antigen-binding portion thereof, may include an antigen as defined herein.

[0079] As used herein, the term "at least" a particular value means greater than or equal to that particular value. For example, "at least two" is understood to be the same as "two or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc. As used herein, the term "up to" a particular value means less than or equal to that particular value. For example, "up to 5" is understood to be the same as "five or less", i.e., 5, 4, 3, ....-10, -11, etc. The term "one or more" or "at least one", e.g., one or more members or at least one member of a group of members, is itself clear by further illustration, but the term specifically encompasses reference to any one of the members, or any two or more of the members, e.g., any three or more, four or more, five or more, six or more, or seven or more, etc., up to all of the members. As another example, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0080] As used herein, the word "comprise" or variations thereof such as "comprises" or "comprising" are understood to include the stated elements, integers or steps, or group of elements, integers or steps, but not to exclude any other elements, integers or steps, or group of elements, integers or steps. The verb "comprise" includes the verbs "consisting essentially of" and "consisting of."

[0081] As used herein, the term "conventional technology" refers to the situation where the method of carrying out the conventional technology used in the method of the present invention is clear to those skilled in the art.The implementation of conventional technology in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields is well known to those skilled in the art, for example, the following literature references: Sambrook et al., Molecular Cloning.A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley&Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0082] As used herein, the term "identity" refers to a measure of the identity of nucleotide or amino acid sequences. Generally, sequences are aligned to obtain the highest degree of agreement. "Identity" itself has an art-recognized meaning and can be calculated using published techniques. See, for example: (Computational Molecular Biology, Lesk, AM, ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, DW, ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, AM, And Griffin, HG, EDS., Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). There are several methods for measuring the identity between two nucleotide or amino acid sequences, but the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48: 1073). Commonly used methods for determining identity or similarity between two sequences include, but are not limited to, the various methods 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. Various methods for determining identity and similarity are coded into computer programs.Exemplary computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

[0083] By way of example, a polynucleotide having a nucleotide sequence with at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a particular sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations for each 100 nucleotides of the reference amino acid sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted and / or replaced with another nucleotide, and / or up to 5% of the number of nucleotides of the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between the terminal positions, and may be interspersed between the nucleotides of the reference sequence individually or in one or more consecutive groups within the reference sequence.

[0084] Similarly, a polypeptide having an amino acid sequence with at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO:X is intended to mean that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the amino acid sequence may contain up to 5 amino acid changes per 100 amino acids of the reference amino acid sequence of SEQ ID NO:X. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues of the reference sequence may be deleted or replaced with another amino acid, or a number of amino acids up to 5% of the total amino acid residues of the reference sequence may be inserted into the reference sequence. These changes of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, and may be interspersed individually among the residues of the reference sequence, or in one or more contiguous groups within the reference sequence.

[0085] As used herein, the term "in vitro" can refer to experiments or measurements performed using living components separated from their natural state.

[0086] As used herein, the term "ex vivo" can refer to experiments or measurements that are performed in or on the tissue of an organism in an external environment with minimal alteration of natural conditions.

[0087] As used herein, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are intended to include DNA molecules and RNA molecules. A nucleic acid (molecule) can be single-stranded or double-stranded, but is preferably double-stranded DNA.

[0088] 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 present invention, a first nucleic acid sequence may be contained within or overlap with an additional nucleic acid sequence.

[0089] As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal" are used interchangeably and refer to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, farm animals, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bovines, bears, and the like. As defined herein, subjects may be living or dead. Samples may be taken from subjects post-mortem, i.e., after death, and / or samples may be taken from living subjects.

[0090] As used herein, the terms "treatment", "treat", "alleviate", "mitigate" or "ameliorate", which are used interchangeably, refer to an approach to obtain a beneficial or desired result, including but not limited to a therapeutic benefit. Therapeutic benefit refers to eradication or amelioration or reduction (or delay) in the progression of the underlying disease being treated. Therapeutic benefit is also achieved by eradication or amelioration or reduction (or delay) in the progression of one or more of the physiological symptoms associated with the underlying disease, such that an improvement or slowdown or reduction in decline is observed in the patient, even though the patient may still be suffering from the underlying disease.

[0091] As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. The term "vector" can also refer to a viral particle (i.e., a viral vector) that contains a nucleic acid of interest.

[0092] A portion of this invention contains material that is subject to copyright protection, such as, but not limited to, diagrams, equipment photographs, or any other aspect 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 the patented invention, as it appears in the patent file or records at the Patent and Trademark Office, but otherwise reserves all copyrights.

[0093] Various terms relating to the method, composition, use and other aspects of the present invention are used throughout the specification and claims.Such terms should be given their ordinary meaning in the art to which the present invention pertains unless otherwise indicated.Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein.Preferred materials and methods are described herein, but any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0095] The present invention is based on the inventors' discovery that apolipoproteins or apolipoprotein mimetics can be used as carriers of therapeutic drugs, and apolipoproteins can be further modified to target specific cells, tissues or organs.The inventors have found that fusion proteins of cytokines and apolipoproteins or apolipoprotein mimetics show a significant increase in half-life in blood, thereby opening up the possibility of using cytokines in a therapeutic manner without the need for administration of toxic concentrations.Furthermore, it has been realized that apolipoproteins or apolipoprotein mimetics allow cytokine targeting when fused to target cells, tissues and / or organs, such as myeloid cells.The inventors have realized that this concept is more broadly applicable and can be used for a wide range of immune-modulating biomolecules, such as cytokines, chemokines, hormones, growth factors, etc.

[0096] It has further been recognized that fusion proteins or apolipoproteins or apolipoprotein mimetics can be directed to their intended targets by linking them to rerouting molecules, which can then be used to target the apolipoproteins (or apolipoprotein mimetics) or fusion proteins to cells, tissues or organs that would otherwise not be reached or are poorly reached, or to reduce off-target effects.

[0097] Thus, in a first aspect, the present 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) the immune response.

[0098] In a second aspect, the present invention relates to a fusion protein comprising, consisting essentially of, or consisting of an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, which when fused to the apolipoprotein enables the apolipoprotein to bind to a target different from the target it would bind if it were not fused to the rerouting molecule and / or to bind its intended target with higher affinity.

[0099] In a third aspect, the present 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.

[0100] Fusion proteins may conveniently be described as fusion proteins of an apolipoprotein or an apolipoprotein mimetic with an immunomodulatory biomolecule and / or a rerouting molecule. The fusion protein can be used as it is, meaning that it is not part of lipoprotein, lipid or apolipoprotein lipid nanoparticles.In this way, the fusion protein can serve as a carrier to deliver immunomodulatory biomolecules to target sites.Alternatively, rerouting molecules can be used to target the fusion protein to specific sites such as cells, tissues or organs.

[0101] Apolipoproteins are proteins that bind lipids such as triglycerides and cholesterol to form lipoproteins. They transport lipids (and fat-soluble vitamins) in the blood, cerebrospinal fluid, and lymph. The lipid components of lipoproteins are insoluble in water. However, due to their detergent-like (amphiphilic) properties, apolipoproteins and other amphipathic molecules such as phospholipids can surround lipids and create lipoprotein particles that are themselves water-soluble and therefore can be transported through the aqueous circulation (i.e., blood, lymph). In addition to stabilizing lipoprotein structure and solubilizing lipid components, apolipoproteins interact with lipoprotein receptors and lipid transport proteins, thereby participating in lipoprotein uptake and clearance.

[0102] 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 affect their functions. For example, apolipoprotein AI (apoA1) is the major structural protein component of high-density lipoprotein (HDL), but is present in smaller amounts in other lipoproteins, and HDL contains other apolipoproteins.

[0103] It is envisaged that the present invention is not limited to a particular type of apolipoprotein or apolipoprotein mimetic (e.g., ApoA1, ApoB or ApoE). Thus, in an embodiment, the apolipoprotein of the fusion protein of the present invention, e.g., the apolipoprotein of the fusion protein according to the first, second or third aspect of the present invention, is selected from apoA1, ApoA-1 Milano, apoA2, apoA4, apoA5, ​​apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoL1, apoL2, apoL3, apoL4, apoL5, apoL6, apoLD1, apoM, apoO, apoOL, or combinations thereof, or mimetics thereof. For example, the apolipoprotein may be selected from apoA1, ApoA-1 Milano, apoA2, apoA4, apoA5, ​​apoB48, apoB100, apoC-I, apoC-II, apoC-III, apoC-IV, apoE, apoL1, apoL2, apoL3, apoL4, apoL5, apoL6, or combinations or mimetics thereof. More preferably, the apolipoprotein may be selected from apoA1, apoA4, apoC3, apoD, apoE, apoL1, apoL3, or combinations or mimetics thereof. In other words, in certain embodiments, the apolipoprotein is ApoA1, ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1, ApoL3, or the apolipoprotein mimetic is a mimetic of ApoA1, ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1, ApoL3. Even more preferably, the apolipoprotein is ApoA1.

[0104] In certain embodiments, the apolipoprotein may also be a fragment of an apolipoprotein. Preferably, the fragment of an apolipoprotein retains the biological activity of the apolipoprotein, such as the ability of the apolipoprotein to be incorporated into lipid nanoparticles or to target immunomodulatory biomolecules, such as the ability to target to the myeloid compartment. In certain embodiments, the fragment of an apolipoprotein 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 region of the full-length apolipoprotein, thereby allowing binding to myeloid cells. In certain embodiments, the fragment of an apolipoprotein comprises at least the alpha helices of the full-length apolipoprotein. These helices are hydrophilic on one side (interacting with the aqueous environment) and hydrophobic on the other side (interacting with lipids within the particle).

[0105] The term "fragment" as used throughout this specification with respect to a peptide, polypeptide or protein generally refers to a portion of a peptide, polypeptide or protein, such as a form in which the peptide, polypeptide or protein is typically truncated at the N-terminus and / or C-terminus. Preferably, a fragment can comprise at least about 30%, such as at least about 50% or at least about 70%, preferably at least about 80%, such as at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% or even about 99% of the length of the amino acid sequence of the peptide, polypeptide or protein. For example, so long as it does not exceed the length of the full-length peptide, polypeptide or protein, a fragment can comprise a sequence of 5 or more consecutive amino acids, or 10 or more consecutive amino acids, or 20 or more consecutive amino acids, or 30 or more consecutive amino acids, such as 40 or more consecutive amino acids, such as 50 or more consecutive amino acids, such as 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, or 200 or more consecutive amino acids of the corresponding full-length peptide, polypeptide or protein.

[0106] In certain embodiments, the fragment of an apolipoprotein comprises a bone marrow-binding portion of a full-length apolipoprotein.

[0107] In certain embodiments, the apolipoprotein may also be an apolipoprotein mutant that includes a mutation that allows chemical conjugation of the apolipoprotein to an immunomodulatory biomolecule and / or a rerouting molecule. In certain embodiments, the apolipoprotein may also be an apolipoprotein mutant that includes a serine to cysteine ​​substitution, such as an ApoA1 mutant as defined by SEQ ID NO: 1, 7, 9, or 11, as described elsewhere herein.

[0108] The peptide sequences of the different proteins described herein, or the nucleic acid sequences of the genes encoding the different proteins described herein, are readily available to those skilled in the art, for example, from the UCSC Genome Browser (http: / / genome.ucsc.edu / ), the Ensembl genome browser (https: / / www.ensembl.org) and NCBI (https: / / www.ncbi.nlm.nih.gov / protein). Consensus sequences of various proteins or genes can be readily derived from these sources, but it is understood that certain variations may exist due to, but are not limited to, genetic mutations and multiple splice variants of genes. Thus, when a particular protein is mentioned, this should be interpreted as including sequence variations due to genetic mutations and splice variants. Thus, as used herein when referring to a particular protein, this refers to a protein sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence retrieved from the Ensembl genome browser, or the consensus nucleic acid (gene) sequence retrieved from the Ensembl genome browser, or the corresponding consensus protein sequence retrieved from the Ensembl genome browser. A consensus sequence should be interpreted as a gene sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus gene sequence retrieved from the Ensembl genome browser, or a nucleic acid sequence that encodes a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding consensus protein sequence retrieved from the Ensembl genome browser.

[0109] Apolipoprotein mimetics are synthetic peptides or proteins that mimic the function or structure of apolipoproteins. Several apolipoprotein mimetics are known, for example Wolska et al. (Cells. 2021 Mar;10(3):597, incorporated by reference in its entirety) reviewing different apoA1, apoE and apoC-II mimetics described in the literature. For example, ApoA1 mimetic peptides have been designed in large part based on their ability to efflux cholesterol from cells. Since this process has not been shown to depend on specific protein-protein interactions, most apoA1 mimetic peptides are simply amphipathic helices, and indeed many have no primary amino acid homology with apoA1. Exemplary ApoA1 mimetics are ApoA1 mimetic 18A, ApoA1 mimetic 2F and ApoA1 mimetic 37pA, which are represented by peptide sequences corresponding to SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0110] For example, apoE has several putative atheroprotective functions, and many different types of apoE-based peptides have been reported. One of the main objectives in the design of these peptides is to promote hepatic clearance of apoB-containing lipoproteins. Because apoE can only bind to its receptor when bound to lipids, these peptides usually have not only a receptor-binding motif derived 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.

[0111] For example, apoC-II mimetics have been described based either on a truncated first helix (18A) linked to the LPL activation domain of apoC-II, or in which both the first and second helices are based on the native apoC-II helix with amino acid substitutions to enhance double-helical binding to lipoproteins.

[0112] Therefore, as used herein, apolipoprotein mimics refers to synthetic proteins or peptides that share structural and / or functional characteristics with their respective apolipoproteins.For example, the shared structural characteristics can be the primary, secondary or tertiary peptide structures, such as peptide sequences, the presence of structures such as alpha helices or beta sheets, or the three-dimensional structure of peptides, or the functional characteristics can be the similarity in binding to specific targets, such as receptors.Preferably, apolipoproteins can bind to lipids in a manner similar to that of corresponding apolipoproteins, and more preferably can form lipid particles.

[0113] In certain embodiments, the apolipoprotein mimics can bind to myeloid cells to the same or similar extent as the respective apolipoproteins. For example, an apolipoprotein mimic of ApoA1 can bind to myeloid cells to the same or similar extent as ApoA1.

[0114] In an embodiment, the fusion protein is a fusion protein of ApoA1 (or a variant thereof), preferably human ApoA1, and an immunomodulatory biomolecule and / or a rerouting molecule.

[0115] As 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.

[0116] In certain embodiments, the ApoA1 is wild-type ApoA1 (e.g., derived from the human precursor of ApoA1 as defined by SEQ ID NO:1, the first 18 amino acids of which form a signal peptide) or an ApoA1 variant (e.g., defined by SEQ ID NO:7, 9, or 11). In certain embodiments, the ApoA1 is wild-type human ApoA1 as defined by SEQ ID NO:78.

[0117] For example, a reactive handle is typically required to chemically conjugate apoA1 to immunomodulatory biomolecules and / or rerouting molecules. Thus, apoA1 variants containing cysteine ​​instead of serine at positions 147 (e.g., as defined by SEQ ID NO:9) or 279 (e.g., as defined by SEQ ID NO:11) may be useful for preparing chemically conjugated ApoA1 fusion proteins.

[0118] In embodiments, ApoA1 is a peptide having 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 ApoA1 comprises, consists essentially of, or consists of, or comprises, consists essentially of, or consists of an amino acid sequence encoded by 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 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.

[0119] It should be noted that the ApoA1 sequences defined by SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11 contain the amino acid sequence GLVPRGSIDD (SEQ ID NO:79) at the N-terminus, which is a thrombin cleavage site. For example, the ApoA1 sequence as defined by SEQ ID NO:5 contains a 6His tag at the N-terminus, followed by the amino acid sequence GLVPRGSIDD (SEQ ID NO:79). Here, the thrombin cleavage site can be used to remove the N-terminal His tag from the peptide.

[0120] In embodiments, ApoA1 is a peptide having 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, or ApoA1 comprises, consists essentially of, or consists of, SEQ ID NO:7, which includes a cysteine ​​at position 7 of SEQ ID NO:7. Such ApoA1 variants are sometimes referred to herein as "S14C" variants.

[0121] In embodiments, ApoA1 is a peptide having 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, or ApoA1 comprises, consists essentially of, or consists of, SEQ ID NO: 9, and SEQ ID NO: 7 contains a cysteine ​​at position 150 of SEQ ID NO: 9. Such ApoA1 variants may be referred to herein as "S147C" or "S157C" variants.

[0122] In embodiments, ApoA1 is, or ApoA1 comprises, consists essentially of, or consists of a peptide having 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, and SEQ ID NO: 7 contains a cysteine ​​at position 239 of SEQ ID NO: 11. Such ApoA1 variants may be referred to herein as "S279C" or "S239C" variants.

[0123] In an embodiment, the fusion protein is a fusion protein of an ApoA1 mimetic and an immunomodulatory biomolecule and / or a rerouting molecule. In an embodiment, the ApoA1 mimetic is a peptide having 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, or the ApoA1 mimetic comprises, consists essentially of, or consists of it.

[0124] In an embodiment, the fusion protein is a fusion protein of ApoE and an immunomodulatory biomolecule and / or a rerouting molecule. In an embodiment, ApoE is a peptide having 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 ApoE comprises, consists essentially of, or consists of, or comprises, consists essentially of, or consists of, an amino acid sequence encoded by 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 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.

[0125] As described elsewhere herein, a fusion protein of ApoA1 can be a fusion protein of ApoA1 with a cytokine, such as IL-1β (IL-1B), IL-2, IL-4, or IL-38, preferably IL-4.

[0126] Thus, in an embodiment, the fusion protein is a fusion protein of ApoA1 (including variants thereof) and interleukin (IL)-1B, preferably human IL-1B.

[0127] In embodiments, the ApoA1-IL1B fusion protein comprises, consists essentially of, or consists of a polypeptide having 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 comprises or consists of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:22 or SEQ ID NO:83.

[0128] In an embodiment, the fusion protein is a fusion protein of ApoA1 (including variants thereof) and IL-2, preferably human IL-2.

[0129] In embodiments, the ApoA1-IL2 fusion protein comprises or consists of a polypeptide having 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 comprises or consists of an amino acid sequence encoded by 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 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.

[0130] In an embodiment, the fusion protein is a fusion protein of ApoA1 (including variants thereof) and IL-4, preferably human IL-4.

[0131] In embodiments, the ApoA1-IL4 fusion protein comprises, consists essentially of, or consists of a polypeptide having 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 comprises or consists of an amino acid sequence encoded by 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 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.

[0132] In an embodiment, the fusion protein is a fusion protein between ApoA1 (including variants thereof) and IL-38 (also known as IL1F10), preferably human IL-38.

[0133] In embodiments the ApoA1-IL38 fusion protein comprises, consists essentially of, or consists of a polypeptide having 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 comprises or consists of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:81 or SEQ ID NO:85.

[0134] As described elsewhere herein, 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 VHHCD8 as described in Woodham A Wet al., Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses, Cancer Immunology Research, 2018, Vol. 6, issue 7, and includes the amino acid sequence shown in Supplementary Table 1 of said reference.

[0135] In an embodiment, the fusion protein is a fusion protein of ApoA1 (including variants thereof) and VHH8CD8.

[0136] In embodiments, the VHHCD8-apoA1 fusion protein comprises, consists essentially of, or consists of a polypeptide having 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 comprises or consists of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55 or SEQ ID NO:57.

[0137] As used herein, the fusion protein when referring to an apolipoprotein fusion protein should be interpreted as an apolipoprotein or apolipoprotein mimetic, to which the immunomodulatory biomolecule and / or rerouting molecule is covalently attached. The covalent attachment may be by in-frame encoding of a peptide or protein sequence by the nucleotide sequence encoding the fusion protein. Alternatively, the covalent attachment may be by covalent attachment of the immunomodulatory biomolecule and / or rerouting molecule to the apolipoprotein 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 rerouting molecule and / or apolipoprotein (or mimetic thereof) may include site-specific incorporation of a non-natural amino acid, such as para-azidophenylalanine, which can be used for subsequent (strain-promoted) "click" (conjugation) reaction with an alkyne modification reagent.

[0138] In certain 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 can be a glycine-serine linker, such as a (GGS)n-linker, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably a (GGS)4-linker.

[0139] In certain embodiments, the fusion protein may include one or more tags, such as at the N-terminus and / or C-terminus of the fusion protein. The one or more tags, such as a 6His tag or a strep tag, may allow for purification of the fusion protein.

[0140] The 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 for such covalent attachment.

[0141] In certain embodiments, the immunomodulatory biomolecule is located at the N-terminus or C-terminus of the apolipoprotein or apolipoprotein mimetic of the fusion protein.

[0142] In certain embodiments, the rerouting molecule is located at the N-terminus or C-terminus of the apolipoprotein or apolipoprotein mimetic of the fusion protein.

[0143] In certain embodiments in which the fusion protein comprises an immunomodulatory biomolecule and a rerouting molecule, the apolipoprotein or apolipoprotein mimetic may be located at the N-terminus or C-terminus of both the immunomodulatory biomolecule and the rerouting molecule, or may be located between the immunomodulatory biomolecule and the rerouting molecule (e.g., with the immunomodulatory biomolecule at the N-terminus or C-terminus of the apolipoprotein or apolipoprotein mimetic).

[0144] As used herein, immune response refers to the reaction that occurs in the body of an organism by the immune system.Immune response can be innate immune response, adaptive immune response, or complement immune system.As referred to herein, immune response includes, but is not limited to, the secretion of proinflammatory molecules, the secretion of antiinflammatory molecules, phagocytosis, antibody production, presentation or secretion, antigen presentation, the activation, proliferation, suppression or differentiation of immune cells, the binding of immune cells to targets, the initiation of immune-related cell signaling cascades, or combinations thereof.

[0145] As used herein, an immunomodulatory biomolecule refers to a molecule that enhances or suppresses immune response. A biomolecule may interfere with, change, stimulate or suppress innate or adaptive immune response or complement immune system. A biomolecule may be a protein, peptide or organic compound. A compound may be isolated or derived from natural sources, cloned or synthesized. Non-limiting examples of immunomodulatory biomolecules are cytokines, chemokines, hormones, growth factors and hematopoietic growth factors and antibodies (or their antigen-binding fragments), although those skilled in the art may recognize additional immunomodulatory molecules.

[0146] Thus, in embodiments, 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 a combination thereof.

[0147] In an embodiment, the immunomodulatory biomolecule can be a cytokine. Cytokines are known to those skilled in the art to be small proteins of about 5 to 20 kDa, and are important in cell signaling. For example, the cytokine can represent four alpha helix bundle family cytokines, such as interleukin (IL)-2 subfamily, interferon (IFN) subfamily or IL-10 subfamily, IL-1 family, cysteine ​​knot cytokines, such as transforming growth factor (TGF) beta family, IL-17 family. Thus, the cytokine is preferably IL18, interleukin 18 binding protein (IL18BP), interleukin 1 alpha (IL1A), interleukin 1 beta (IL1B), interleukin 1 family member 10 (IL1F10), IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, interleukin 1 receptor-like 2 (IL1RL2), IL1F9, IL33, B cell activating factor (BAFF), 4-1BBL, TNF superfamily member 8 (TNFSF8), cluster of differentiation 40 (CD40) ligand (CD40LG), CD70, CD95L / CD178, ectodysparsin-A1 (EDA-A1), TNFSF14, lymphotoxin alpha (LTA) / TNFB, lymphotoxin beta (LTB), TNFα, TNFSF10, TNFSF11, TNF SF12, 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, IFNω / IFNW1, cardiotrophin-like cytokine factor 1 (CLCF1), ciliary neurotrophic factor (CNTF), IL11, IL31, IL6, leptin, leukemia inhibitory factor (LIF), onocostatin M (OSM), IL10, IL19, IL20, IL22, IL24, IL28A, IL28B, IL29, TGF beta 1 / TGFB1, TGF-beta 2 / TGFB2, TGF-beta 3 / TGFB3.In preferred embodiments, the cytokine is selected from the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 family, the TGFβ family, or the IL-17 family, or a combination thereof, more preferably, the cytokine is selected from IL-1β, IL-2, IL-4, IL-38, or a combination thereof. More preferably, the cytokine is IL-4.

[0148] In certain embodiments, the cytokine is a human cytokine.

[0149] In an embodiment, the fusion protein is a fusion protein of apolipoprotein or its mimic with IL-1B.IL-1B is also known as IL1B, IL-1β, IL1F2, or interleukin 1β, and is a cytokine protein encoded by IL1B gene in humans.As an example, the human IL-1B 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.

[0150] In embodiments, IL-1B is a peptide having, 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 a peptide comprising, consisting essentially of, or consisting of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:46.

[0151] In an embodiment, the fusion protein is a fusion protein of apolipoprotein or its mimic with IL-2.IL-2, also known as IL2 TCGF, ​​lymphokine or interleukin 2, is an interleukin that regulates the activity of white blood cells involved in immunity.As an example, the human IL-2 protein sequence is annotated in NCBI Genbank (http: / / www.ncbi.nlm.nih.gov / ) accession number NP_000577.2 and Uniprot (www.uniprot.org) accession number P60568.1.

[0152] In certain embodiments, the IL-2 can be wild-type IL-2 or an IL-2 mutant.

[0153] In certain embodiments, the IL-2 may contain 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, OncoImmunology, 2017, Volume 6, issue 3, e1277306.

[0154] In certain embodiments, IL-2 may comprise one or more, e.g. one, two, three or all four, of the following amino acid substitutions: F42A, Y45A, L72G and / or C125A (the positions of the substitutions are shown relative to the sequence of the mature human IL-2 protein). It should be noted that the first 20 amino acids of the human IL-2 amino acid sequence (human IL-2 precursor) defined by SEQ ID NO:47 represent a signal peptide, and thus IL-2 may comprise one or more, e.g. one, two, three or all four, of the following amino acid substitutions: F62A, Y65A, L92G and / or C145A, the positions of which are shown relative to SEQ ID NO:47.

[0155] In embodiments, the IL-2 is a peptide having, 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 a peptide comprising, consisting essentially of, or consisting of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:48.

[0156] In an embodiment, the fusion protein is a fusion protein of apolipoprotein or its mimic with IL-4.IL-4 is also known as BSF-1, IL4 or interleukin 4, and is a cytokine that induces the differentiation of naive helper T cells.As an example, the human IL-4 protein sequence is annotated as 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.

[0157] In embodiments, the IL-4 is a peptide having, 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 a peptide comprising, consisting essentially of, or consisting of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:44.

[0158] In a further embodiment, the fusion protein is a fusion protein of ApoA1 or a mimetic thereof and IL-4.

[0159] In an embodiment, the fusion protein is a fusion protein of apolipoprotein or its mimic with IL-38.IL-38 is also known as IL38, IL1F10 interleukin 38, interleukin 1 family member 10 or IL1-θ, and is a protein encoded by IL1F10 gene in humans.As an example, 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 embodiments, IL-38 is a peptide having, 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 a peptide comprising, consisting essentially of, or consisting of an amino acid sequence encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:50.

[0160] In an embodiment, the immunomodulatory biomolecule may be a chemokine. The chemokine is preferably selected from the group consisting of chemokine (CC motif) ligand 1 (CCL1) / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CC motif chemokine ligand 3-like 3 (CCL3L3), CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 In another embodiment, the chemokine is selected from a CC chemokine, a CXC chemokine ligand 1 (CXCL1), a CXC motif chemokine ligand 1 (CXCL1), a CXCL10, a CXCL11, a CXCL12, a CXCL13, a CXCL14, a CXCL15, a CXCL16, a CXCL17, a CXCL2 / MIP-2, a CXCL3, a CXCL4, a CXCL5, a CXCL6, a CXCL7 / Ppbp, a CXCL9, an IL8 / CXCL8, a XC motif chemokine ligand 1 (XCL1), a XCL2, a FAM19A1, a FAM19A2, a FAM19A3, a FAM19A4, and a FAM19A5. In another embodiment, the chemokine is selected from a CC chemokine, a CXC chemokine, a C chemokine, a CX3C chemokine, or a combination thereof.

[0161] In an embodiment, the immunomodulatory biomolecule may be a hormone. It is known to those skilled in the art that hormones are signaling molecules in multicellular organisms that are transported to distant organs to regulate physiology and behavior. In an embodiment, the hormone may be 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 inhibitory factor / hormone), adiponectin, adrenocorticotropic hormone (also known as corticotropin), and the like. angiotensinogen, angiotensin, antidiuretic hormone (also known as vasopressin, arginine vasopressin), atrial natriuretic peptide (also known as atrial leptin), brain natriuretic peptide, calcitonin, cholecokinin, 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 leutetropic hormone), prolactin-releasing hormone, relaxin, renin, secretin, somatostatin (also known as growth hormone inhibiting hormone or growth hormone releasing inhibiting hormone or somatotropin releasing inhibiting factor or somatotropin releasing inhibiting hormone), thrombopoietin, thyroid stimulating hormone (also known as thyrotropin), thyrotropin releasing hormone, vasoactive intestinal peptide, guanylin, or uroguanylin.

[0162] In an embodiment, the immunomodulatory biomolecule may be a growth factor. Growth factors are known to those skilled in the art as naturally occurring substances that can stimulate cell proliferation, wound healing, and in some cases cell differentiation. In an embodiment, the growth factor is selected from the group consisting of adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A, and the like. 5, 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 (FGF12), fibroblast growth factor 12 (FGF13), fibroblast growth factor 13 (FGF14), fibroblast growth factor 14 (FGF15), fibroblast growth factor 15 (FGF16), fibroblast growth factor 17 (FGF18), fibroblast growth factor 19 (FGF19), fibroblast growth factor 20 (FGF21), fibroblast growth factor 22 (FGF22), fibroblast growth factor 23 (FGF23), fibroblast growth factor 24 (FGF24), fibroblast growth factor 25 (FGF25), fibroblast growth factor 26 (FGF26), fibroblast growth factor 27 (FGF27), fibroblast growth factor 28 (FGF28), fibroblast growth factor 29 (FGF29), fibroblast growth factor 30 (FGF30), fibroblast growth factor 31 (FGF31), fibroblast growth factor 32 (FGF32), fibroblast growth factor 33 (FGF33), fibroblast growth factor 34 (FGF34), fibroblast growth factor 35 (FGF35), fibroblast growth factor 36 (FGF36), fibroblast growth factor 37 (FGF37), fibroblast growth factor 38 (FGF3 FGF11, Fibroblast Growth Factor 12 (FGF12), Fibroblast Growth Factor 13 (FGF13), Fibroblast Growth Factor 14 (FGF14), Fibroblast Growth Factor 15 (FGF15), Fibroblast Growth Factor 16 (FGF16), Fibroblast Growth Factor 17 (FGF17), Fibroblast Growth Factor 18 (FGF18), Fibroblast Growth Factor 19 (FGF19), Fibroblast Growth Factor 20 (FGF20), Fibroblast Growth Factor 21 (FGF21), Fibroblast Growth Factor 22 (FGF22), Fibroblast growth factor 23 (FGF23), fetal bovine growth factor (FBS), glial cell line-derived neurotrophic factor (GDNF), neuron, persephin, artemin, growth differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), hepatocyte-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF),Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like protein (HGFLP), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS)-anti-apoptotic survival factor, T-cell growth factor (TCR)-anti-apoptotic growth factor (ATG)-anti-inflammatory ... The present invention is directed to a method for treating or preventing the progression of a tumor, comprising administering to the patient a therapeutically effective amount of at least one of the following: tumor necrosis factor (TNF-α), cellular growth factor (TCGF), thrombopoietin (TPO), transforming growth factor, transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16. In a preferred embodiment, the growth factor is selected from VEGF, EGF, CNTF, LIF, ephrin, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, neurotrophins, PGF, PDGF, RNLS, TCGF, ​​TGF, TNF, WNT, or a combination thereof.

[0163] In embodiments, the immunomodulatory biomolecule is a hematopoietic growth factor, which in embodiments 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 a combination thereof.

[0164] As used herein, the term target refers to an object to which an apolipoprotein (or apolipoprotein mimetic) or fusion protein preferentially binds. Targets can refer to receptors or cell surface molecules, such as proteins or proteoglycans, cells, cell types, tissues or tissue types or organs.

[0165] In certain embodiments, the fusion protein or lipid nanoparticles comprising said fusion protein can bind to myeloid cells. This may be the result of the inherent property of apolipoproteins such as ApoA1, which target myeloid cells. As used herein, the term myeloid cells refers to blood cells derived from precursor cells of granulocytes, monocytes, erythrocytes or platelets. Myeloid cells are the main cell compartments of the immune system, including monocytes, dendritic cells, tissue macrophages and granulocytes. As used herein, the term myeloid compartment refers to the totality of myeloid cells in an organism.

[0166] As used herein, a rerouting molecule refers to a molecule that, when fused to a protein such as an apolipoprotein or an apolipoprotein-immunomodulatory biomolecule fusion protein, allows the protein to bind to a different target (in other words, a target different from the target to which it would naturally bind) than it would bind if it were not fused to the rerouting molecule, and / or bind to its intended target with higher affinity. Binding to a different target can also include binding to a specific subset of target cells, including binding to a specific subset of the set of cells that would normally be bound by the apolipoprotein or apolipoprotein-immunomodulatory biomolecule fusion protein. Non-limiting examples of rerouting molecules are antibodies or antigen-binding fragments thereof or antibody fragments, rerouting peptides or rerouting proteins, and preferably, the rerouting peptides or rerouting proteins are ligands for receptors present on the target.

[0167] It is understood that apolipoproteins bind to specific ligands. For example, the different apolipoproteins found in different lipoproteins (e.g., HDL, LDL, VLDL, etc.) are believed to be responsible for the differences in targeting and binding, and therefore in the function of lipoproteins. Without wishing to be bound by theory, it is believed that a part of the apolipoprotein has an amphipathic nature and, together with phospholipids and / or sterols, plays a role in binding to lipids in an aqueous environment, while different parts of the molecule are involved in interactions with other molecules, for example binding to protein receptors. It is further hypothesized that apolipoproteins can circulate not only as lipoproteins, but also as proteins. Thus, the possibility of modifying the binding affinity of apolipoproteins (by fusion to rerouting molecules) offers interesting opportunities, as it allows the targeting or binding of apolipoproteins to be fine-tuned. Several uses of such fusion proteins are envisioned.

[0168] First, rerouting molecules can simply be used to reroute apolipoproteins or lipoproteins, for example to change lipid homeostasis. For example, it can be envisioned that by using apolipoprotein fusion proteins with rerouting molecules, the LDL or HDL levels in the blood of a subject can be changed. This could be utilized in the treatment of lipid disorders, such as high blood cholesterol levels.

[0169] Secondly, it can be used to reroute lipoproteins (lipid nanoparticles) with a payload to a given target. Lipoproteins or lipid nanoparticles pose an interesting way to carry a payload, such as pharmaceutical compounds. This allows the delivery of lipophilic compounds via the blood, since the compounds can be dissolved in the lipid core of the lipoprotein / lipid nanoparticle. An additional advantage is that lipoproteins consist of naturally occurring compounds and are therefore considered natural by the immune system, avoiding the elicitation of an immune response by pharmaceutical compounds.

[0170] Third, it may be combined with an immunomodulatory biomolecule (either as a single fusion protein (apolipoprotein fused to both the rerouting molecule and the immunomodulatory biomolecule) or as a combination of two different fusion proteins), thus allowing the rerouting of the immunomodulatory biomolecule. As mentioned above, the apolipoprotein can essentially function as a carrier for the immunomodulatory biomolecule. The advantage is that it greatly reduces the clearance of the immunomodulatory biomolecule, making it feasible to use immunomodulatory biomolecules (e.g., cytokines) that are easily cleared from the blood in therapeutic situations. One problem that may arise is that the immunomodulatory biomolecule / apolipoprotein fusion protein does not reach the intended target of the immunomodulatory biomolecule (i.e., the site, cell, tissue or organ where it is intended to exert its effect). This can be solved by further including a rerouting molecule.

[0171] Fourth, a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule makes it possible to prepare lipid nanoparticles, where the rerouting molecule, as a result of its fusion to the apolipoprotein, is exposed to the environment surrounding said apolipoprotein-lipid nanoparticles (i.e., an aqueous environment).

[0172] Data generated by the inventors suggests that rerouting molecules can be used to successfully reroute apolipoprotein fusion proteins to different targets. Thus, in embodiments, the rerouting molecule is selected from an antibody or antigen-binding fragment thereof, a rerouting peptide or a rerouting protein, preferably the rerouting peptide or protein is a ligand of a receptor present at the target.

[0173] In embodiments, the rerouting molecule may be an antibody or an antigen-binding fragment thereof. It is envisaged that any type of antigen-binding molecule may in principle be used as a rerouting molecule in a fusion protein according to the invention.

[0174] An antibody can be any immunological binding agent, such as, but not limited to, a whole antibody, including chimeric, humanized, human, recombinant, transgenic, grafted, and single-chain antibodies, or any fusion protein, conjugate, fragment, or derivative thereof that contains one or more domains that selectively bind to an antigen of interest. An antibody can be a whole immunoglobulin molecule, a monoclonal antibody, a chimeric, humanized, human antibody, or an immunologically effective fragment of any of these. Thus, an antibody can include intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., bivalent, trivalent, or higher) and / or multispecific antibodies (e.g., bispecific or higher specific antibodies) formed from at least two intact antibodies, as well as antibody fragments so long as they exhibit the desired biological activity (in particular the ability to specifically bind to an antigen of interest), as well as multivalent and / or multispecific complexes of such fragments.

[0175] The terms "specifically bind" or "specifically interact" as used throughout this specification mean that the agent binds to or affects one or more desired molecules or analytes to the substantial exclusion of other random or unrelated molecules, and optionally to the substantial exclusion of other structurally related molecules. These terms do not necessarily require that the agent binds exclusively to its intended target. For example, an agent may be one whose affinity for such intended target under binding conditions is at least about 2-fold greater than its affinity for non-target molecules, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, even more preferably at least about 25-fold greater, even more preferably at least about 50-fold greater, even more preferably at least about 100-fold greater or more, e.g., at least about 1000-fold greater or more, e.g., at least about 1×10 4 More than twice as large, or at least about 1×10 5 If the binding affinity is 2-fold or greater, it can be said to specifically bind the target of interest.

[0176] Thus, in embodiments, an antibody or antigen-binding fragment thereof may be a fragment antigen-binding region (Fab), Fab2, single chain variable fragment (scFv), scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab-2-, Fab3, trispecific Fab3 diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, immunoglobulin novel antigen receptor (IgNAR), monovalent IgG, V h H or variable domains of novel antigen receptors (VNARs). The antibody or antigen-binding fragment may also be a designed antigen-binding protein, such as, but not limited to, an affibody, an FN3 domain, a DARPin, or a de novo designed protein receptor. It is recognized that antibodies or antigen-binding fragments thereof with lower molecular weights are preferred due to their small size, and therefore, in preferred embodiments, the antibody or antigen-binding fragment thereof is selected from Fab, scFv, single domain antibodies, V h H or V.

[0177] In embodiments, the rerouting molecule can be a rerouting peptide. Non-limiting examples of rerouting peptides are receptor binding peptides, ligand mimetic peptides.

[0178] Thus, in embodiments, the rerouting peptide is selected from programmed cell death protein 1 (PD1) or signal regulatory protein alpha (SIRPa). However, it is understood that any peptide that has binding specificity for a cell surface receptor can be used as a rerouting molecule.

[0179] In an embodiment, the rerouting molecule can be a rerouting protein, such as a receptor ligand, a receptor, or an interacting protein. Thus, 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 directly bind to the CD4 T cell coreceptor. However, it is understood that any protein that has binding specificity for a cell surface receptor can be used as a rerouting molecule.

[0180] As used herein, the term lipoprotein refers to particles, generally nanoparticles, of at least one apolipoprotein and lipid molecules dispersed or dissolved in an aqueous environment.

[0181] As used herein, the term rerouting refers to targeting a fusion protein to a different target that it normally binds to, or preventing the binding of the normal target of an apolipoprotein, or preventing off-target binding. For example, apoA1 is known to bind to a receptor on myeloid cells, and thus rerouting molecules can be used to bind to different cells or prevent the binding of myeloid cells. In other words, rerouting molecules can bind, preferably specifically bind, to non-myeloid cells.

[0182] Thus, in certain embodiments, the rerouting molecule is capable of binding, and preferably specifically binding, to a cell that is not a myeloid cell, but can differentiate into a myeloid cell, e.g., a hematopoietic stem cell (HSC), a multipotent progenitor (MPP) or a hematopoietic stem and progenitor cell (HSPC), such as a common myeloid progenitor cell (CMP).

[0183] In certain embodiments, the rerouting molecule can bind, preferably specifically bind, to non-myeloid cells, such as non-myeloid immune cells or endothelial cells. Endothelial cells can be targeted by using rerouting molecules that can bind to surface markers of endothelial cells. For example, endothelial cells can be targeted by using rerouting molecules that can bind to factor VIII-related antigens, such as factor VIII, rerouting molecules that can bind to CD31 / PECAM-1, such as CD31, rerouting molecules that can bind to angiotensin-converting enzyme (ACE / CD143), such as angiotensin, rerouting molecules that can bind to CD34, such as L-selectin, or rerouting molecules that can bind to endoglin (CD105).

[0184] In certain embodiments, the non-myeloid cells are lymphocytes, such as T cells, B cells, or natural killer (NK) cells. Preferably, the lymphocytes are T cells, even more preferably CD8+ T cells.

[0185] In certain embodiments, when the target cell is a T cell, the rerouting molecule can be an antibody or antigen-binding fragment thereof that binds, preferably specifically binds, to CD8. For example, the rerouting molecule can be VHHCD8 as described in Woodham A Wet al., Nanobody-antigen conjugates elicit HPV-specific antitumor immune responses, Cancer Immunology Research, 2018, Vol. 6, issue 7, and comprises the amino acid sequence as shown in Supplementary Table 1 of said reference.

[0186] In certain embodiments, when the target cells are T cells and / or B cells, the rerouting molecule or peptide can be PD1, CD40L, or GP120.

[0187] The term rerouting can also encompass increasing the specificity of a fusion protein for a particular target, such as a particular target cell. For example, apoA1 is known to bind to a receptor on myeloid cells, and thus, a rerouting molecule can be used to bind to a particular subtype of myeloid cells.

[0188] Thus, in certain embodiments, the rerouting molecule can bind, preferably specifically bind, to myeloid cells selected from the group consisting of megakaryocytes, eosinophils, basophils, erythrocytes, monocytes such as dendritic cells or macrophages, and neutrophils. For example, SIRPalpha as a rerouting molecule may allow targeting immunosuppressive macrophages.

[0189] It is envisioned that the apolipoprotein fusion proteins described herein may be used as protein or lipid nanoparticles. As mentioned above, the apolipoproteins may circulate intact (meaning not incorporated into lipoproteins or lipid nanoparticles). This application may be suitable for delivering immunomodulatory biomolecules, such as cytokines, such as IL-4, to target sites. It is known that apolipoproteins can circulate as proteins, but can also form lipoproteins in situ. However, it may also be advantageous to include the fusion protein in a lipid nanoparticle. Thus, in an embodiment, the present invention relates to lipid nanoparticles comprising one or more fusion proteins herein, the lipid nanoparticles further comprising a phospholipid, and optionally a sterol. The lipid nanoparticles are conveniently referred to herein as "apolipoprotein lipid nanoparticles". The fusion protein may be a fusion protein of an apolipoprotein with an immunomodulatory biomolecule, or an apolipoprotein with a rerouting molecule, or a fusion protein of an apolipoprotein with an immunomodulatory biomolecule and a rerouting molecule, or a combination thereof. The apolipoprotein may be an apolipoprotein mimic. Without wishing to be bound by any hypothesis, it is believed that the apolipoprotein or apolipoprotein mimetic may function as a scaffold to aid in the formation of nanoparticles together with phospholipids and optionally sterols.

[0190] In an embodiment of the invention, the lipid nanoparticle comprises a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, and a phospholipid.

[0191] In one aspect of the invention, the lipid nanoparticle comprises a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, and a phospholipid.

[0192] Apolipoprotein lipid nanoparticles may contain both an immunomodulatory biomolecule and a rerouting molecule, which may be accomplished by incorporating one fusion protein containing both an immunomodulatory biomolecule and a rerouting molecule, or by incorporating two fusion proteins, one containing an immunomodulatory biomolecule and one containing a rerouting molecule, into the apolipoprotein lipid nanoparticle.

[0193] Thus, in one aspect of the invention, a lipid nanoparticle comprises a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule, and a phospholipid.

[0194] In one aspect of the invention, the lipid nanoparticles comprise (i) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, and a phospholipid, and (ii) a fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, and a phospholipid.

[0195] As used herein, lipid nanoparticle (LNP) refers to an aggregate of phospholipid and one or more apolipoproteins that are soluble in aqueous solution.Particle can further comprise sterol and / or lipid (e.g. triglyceride).When LNP comprises both sterol and lipid, lipid is encapsulated by phospholipid and sterol.Thus, lipid nanoparticle is structurally different from liposome.

[0196] In certain embodiments, the phospholipid is selected from phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylglycerol, or a combination thereof. In more particular embodiments, the phospholipid is selected from 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 ...lauroylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroylphosphatidylglycerol (DLPG), dilauroylphosphatidylglycerol (DMPG), dilauroylphosphatidylglycerol (DPPG), dilauroylphosphatidylglycerol (DSPG), dilauroylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dilauroylphosphatidylserine (DLPS), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof.

[0197] In certain embodiments, the ratio of apolipoprotein (or apolipoprotein mimetic) to phospholipid based on molar weight percentage is 1:25 to 1:400, more preferably 1:50 to 1:200, and even more preferably 1:75 to 1:150. In certain embodiments, the ratio of a fusion protein taught herein to phospholipid based on molar weight percentage is 1:25 to 1:400, more preferably 1:50 to 1:200, and even more preferably 1:75 to 1:150.

[0198] In certain embodiments, the ratio of apolipoprotein (or apolipoprotein mimetic) to phospholipid on a weight basis is 3:1 to 1:100. In certain embodiments, the ratio of fusion protein taught herein to phospholipid on a weight basis is 3:1 to 1:100.

[0199] In certain embodiments, the sterol is selected from cholesterol, desmosterol, stigmasterol, β-sitosterol, ergosterol, hopanoids, hydroxysteroids, plant sterols, steroids, hydrogenated cholesterol, campesterol, zoosterol, or combinations thereof.

[0200] The nanoparticles may comprise additional components, such as additional proteins or payloads. Thus, in embodiments, the lipid nanoparticles defined herein further comprise a lipid. In further embodiments, the lipid nanoparticles defined herein further comprise a payload.

[0201] As used herein, the term payload refers to a compound contained in lipid nanoparticles that is not an apolipoprotein, a phospholipid, or a sterol. The payload may be, for example, a pharmaceutical compound. Lipid nanoparticles are particularly suitable for lipophilic payloads, but can also be used for amphiphilic molecules. Pharmaceutical compounds are often organic compounds, peptides, proteins, nucleic acids or nucleic acid analogs, biologics, or lipids. Thus, in embodiments, the lipid nanoparticle further comprises a payload, and preferably the payload is selected from nucleic acids or nucleic acid analogs, therapeutic agents, biologics, or combinations thereof.

[0202] For example, the payload can be a nucleic acid or a nucleic acid analog. Examples can be, but are not limited to, mRNA, siRNA, miRNA, piRNA, snRNA, snoRNA, srRNA or tsRNA. The nucleic acid analog can 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 acid (HNA), or mixtures or combinations thereof. Alternatively, the payload can be a small organic compound, such as a small molecule drug. Generally, small organic compounds are synthesized. The therapeutic agent can be, for example, an anti-cancer therapy, such as chemotherapy. Alternatively, the payload can be a biologic. As used herein, the term biologic is used to indicate a biologic, also known as a biological medical product, and can be any pharmaceutical drug product manufactured, extracted, or semi-synthesized from a biological source. The biologic can be composed of sugars, proteins, nucleic acids, or complex combinations of these substances, or can be living cells or tissues.

[0203] In certain embodiments, the lipid nanoparticles comprise a native (e.g., unfused) apolipoprotein, an apolipoprotein mimetic, or a combination thereof, in addition to the apolipoprotein or apolipoprotein mimetic that forms part of a fusion protein described herein.

[0204] In certain embodiments, the lipid nanoparticles have an average size of 10 to 100 nM, for example 30 to 100 nM.

[0205] In certain embodiments, the lipid nanoparticles are spheres, ribbons or discs, preferably spheres or discs, more preferably spheres.

[0206] The apolipoprotein or apolipoprotein mimetic forms part of the lipid nanoparticle structure. In certain embodiments, at least a portion of the fusion protein is exposed to the environment surrounding the apolipoprotein lipid nanoparticle (i.e., aqueous environment). Typically, a portion of the apolipoprotein or apolipoprotein mimetic is exposed to the environment surrounding the apolipoprotein lipid nanoparticle (see, e.g., Figures 1, 2, and 22). Furthermore, fusion of the immunomodulatory biomolecule and / or rerouting molecule to the apolipoprotein or apolipoprotein mimetic typically allows the immunomodulatory biomolecule and / or rerouting molecule to be fully exposed to the environment surrounding the apolipoprotein lipid nanoparticle (see, e.g., Figures 1, 2, and 22). In other words, the immunomodulatory biomolecule and / or rerouting molecule is not embedded within the lipid nanoparticle. As a result, the immunomodulatory biomolecule and / or the rerouting molecule can move freely and exert its natural function, such as its cell targeting function.

[0207] In certain embodiments, the lipid nanoparticle comprises a payload, the lipid nanoparticle comprises a core surrounded by a surface layer, the core comprises the payload, and the surface layer comprises an apolipoprotein or an apolipoprotein mimetic, a phospholipid, an immunomodulatory biomolecule and / or a rerouting molecule, and optionally a sterol.

[0208] In certain embodiments, the lipid nanoparticle is not a phospholipid bilayer. Also provided herein are methods for producing such lipid nanoparticles. Thus, in a further aspect, the present invention provides a method for producing lipid nanoparticles, a method for producing the apolipoprotein lipid nanoparticles taught herein, comprising: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, apolipoproteins or apolipoprotein mimetics, in which one or more apolipoprotein fusion proteins are fused to an immune modulating biomolecule, such as IL-4; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; and / or apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule, such as IL-4, and a rerouting molecule, and combinations thereof; 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, apolipoproteins or apolipoprotein mimetics, wherein one or more of the conjugated apolipoproteins are 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 one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids and optionally sterols and / or lipids to obtain apolipoprotein-lipid nanoparticles.

[0209] In a further aspect, the present invention provides a method for producing a lipid nanoparticle as defined herein, comprising the steps of: a1) expressing and isolating an apolipoprotein fusion protein to obtain an isolated apolipoprotein fusion protein, the apolipoprotein fusion protein being an apolipoprotein fused to a cytokine and a targeting moiety, and / or the apolipoprotein fusion protein being 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, wherein the conjugated apolipoprotein is an apolipoprotein conjugated to a cytokine and a targeting moiety and / or 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 lipid nanoparticles.

[0210] In a further aspect, the present invention relates to apolipoprotein lipid nanoparticles obtained or obtainable by the method of producing apolipoprotein lipid nanoparticles taught herein.

[0211] It is understood that the fusion protein can be expressed as a chimeric fusion protein between an apolipoprotein and an immunomodulatory biomolecule and / or a rerouting molecule, or can be chemically conjugated to an immunomodulatory biomolecule and / or a rerouting molecule, or can be produced in a combination of these. Chimeric protein expression is known to those skilled in the art and can be used when the immunomodulatory biomolecule and / or a rerouting molecule is a peptide or a protein. For example, it is well within the knowledge of those skilled in the art to use molecular techniques to create nucleic acids encoding such proteins by cloning an immunomodulatory biomolecule and / or a rerouting molecule coding sequence in frame with the sequence encoding the apolipoprotein (or mimic), e.g., with nucleotides encoding the C- or N-terminal sequence. The advantage of using chimeric protein expression is that all proteins expressed are fusion proteins.

[0212] Alternatively, chemical conjugation may be used. Methods suitable for chemical conjugation of immunomodulatory biomolecules and / or rerouting molecules to apolipoproteins (or their mimetics) are known to those skilled in the art. Non-limiting examples are strain-promoted cycloaddition, aminolysis and Michael-type addition. For example, existing or introduced cysteine ​​residues can be used on either apolipoproteins or immunomodulatory biomolecules and / or rerouting molecules. For example, as described elsewhere herein, the ApoA1 protein may contain a cysteine ​​instead of a serine at positions 147 or 279. Introduction of a cysteine ​​residue may be accomplished by point mutation of a nucleotide in the encoding nucleotide sequence or by introduction of a codon that codes for a cysteine. 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.

[0213] It is understood that the fusion protein, phospholipids, and any components such as sterols and lipids can be rapidly mixed to obtain lipid nanoparticles. Optionally, lipids and / or payloads as defined herein can be added.

[0214] A further aspect of the present invention provides a pharmaceutical composition comprising a fusion protein as taught herein, a nucleic acid as taught herein, or a lipid nanoparticle as taught herein, and a pharma- ceutically acceptable carrier.

[0215] In a further aspect, the present invention relates to a fusion protein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtained or obtainable by the method described herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for use as a medicament.It is envisaged that either the fusion protein (e.g. by the action of an immunomodulatory biomolecule) or the payload contained in the nanoparticle can be used to treat, ameliorate or alleviate symptoms in a subject.

[0216] In a further aspect, the present invention relates to a fusion protein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtained or obtainable by a method as defined herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for use in the treatment of an immune-related disorder.In other words, the present invention relates to a method of treating an immune-related disorder in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a fusion protein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtained or obtainable by a method as defined herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein.Also provided herein is the use of a fusion protein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle obtained or obtainable by a method as defined herein, or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, for the manufacture of a medicament for the treatment of an immune-related disorder in a subject.

[0217] Immune-related disorder as used herein includes any disorder in which immune system plays a role in disease development. Immune-related disorder can refer to disorders in which immune system is suppressed or (over)activated. Examples of immune-related disorder are cancer, infection, sepsis, autoimmune disease and cardiovascular disease. Examples of autoimmune disease are type 1 diabetes, rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, multiple sclerosis (MS), systemic lupus (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.

[0218] Thus, in embodiments, 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 (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.

[0219] In further particular embodiments, the immune-related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorders such as multiple sclerosis, allergies, organ transplant rejection, and graft-versus-host disease (GVH).

[0220] Unexpectedly, the inventors found that IL-4 can paradoxically reduce inflammation and simultaneously induce trained immunity, especially when targeted to the bone marrow compartment. In many inflammatory problems (such as sepsis, stroke and myocardial infarction), hyperinflammation and immunosuppression occur simultaneously. Therefore, the inventors concluded that apolipoproteins or apolipoprotein mimetics, preferably fusion proteins of ApoA1 and IL-4, can be used to prevent immune-related disorders by simultaneously reducing inflammation and promoting trained immunity.

[0221] Thus, in certain embodiments, where said immunomodulatory biomolecule is IL-4, the immune-related disorder is a disease that would benefit from reducing inflammation and / or promoting trained immunity.

[0222] Thus, in certain embodiments, wherein said immunomodulatory biomolecule is IL-4, the immune-related disorder is a state of hyperinflammation and subsequent immunoparalysis, preferably, the hyperinflammation and / or immunoparalysis is caused by an infectious disease such as COVID-19 due to sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis.

[0223] In certain embodiments, where the immunomodulatory biomolecule is IL-4, the apolipoprotein nanoparticle or fusion protein simultaneously reduces inflammation and induces trained immunity.

[0224] In certain embodiments, where the immunomodulatory biomolecule is IL-2, the apolipoprotein nanoparticles or fusion proteins may be used to stimulate T cell proliferation.

[0225] In certain embodiments, where the immunomodulatory biomolecule is IL-1β, apolipoprotein nanoparticles or fusion proteins can be used to induce trained immunity.

[0226] In certain embodiments, where the immune-modulating biomolecule is IL-38, apolipoprotein nanoparticles or fusion proteins can be used to reduce trained immunity.

[0227] In a further aspect, the present invention relates to the use of a fusion protein as defined herein or a lipid nanoparticle as defined herein, or a lipid nanoparticle or a nucleic acid as defined herein, or a pharmaceutical composition as defined herein, obtained or obtainable by a method as defined herein, in delivering a compound or an immunomodulatory molecule to a target, preferably the target being 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 the rerouting molecule binds to a cell surface protein, such as a receptor. Thus, 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 the fusion protein can be targeted to different proteins by selecting or adapting the rerouting molecule. For example, a receptor binding domain of a ligand can be used to target a specific receptor. Alternatively, a known binding partner of a cell surface protein can be used to reroute the fusion protein.

[0228] In a further aspect, the present invention relates to a nucleic acid encoding a fusion protein as defined herein. The nucleic acid may be comprised in a vector, such as a viral vector for stable integration into a cell, or an expression vector allowing transient expression.

[0229] In certain embodiments, the vector comprises a myeloid-specific or enhanced promoter or promoter element, which can be used in the vector to drive myeloid-specific expression of IL-4. Suitable promoters are known to those skilled in the art, and non-limiting examples are: lysM, Csf1r, CD11c, CX3CR1, Langerin / CD207, MMLV LTR, Visna virus LTR, DC-STAMP, human MSR, MSR-A, huCD68, CD4, CD2 and Iba-AIF-1 (for review, see, for example, Hume., Journal of leukocyte biology, Volume 89, Issue 4, April 2011, Pages 525-538). Such promoters or promoter elements can be incorporated into vectors, such as lentiviral vectors, to stably transfect cells and allow specific expression of transgenes in myeloid cells.

[0230] In our quest to simultaneously resolve hyperinflammation and immune paralysis, we have extensively studied the role of interleukin (IL)-4 in trained immunity and tolerance. Examining 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, WH, Morton, DL & Economou, JS IL-4 down-regulates IL-1 and TNF gene expression in human monocytes. J. Immunol. 142, 3857 (1989); Woodward, EA, Prele, CM, Nicholson, SE, Kolesnik, TB & Hart, PH The anti-inflammatory effects of interleukin-4 are not mediated by suppressor of cytokine signalling-1 (SOCS1). Immunology 131, 118-127 (2010)], we found that IL-4 simultaneously downregulates inflammatory programs and induces trained immunity. We found that the unique properties of IL-4 allow it to overcome lipopolysaccharide-induced immunodeficiency in monocytes.

[0231] However, its non-specific nature and unfavorable pharmacokinetic properties make IL-4 unsuitable as a myeloid cell-regulating therapeutic. To overcome these limitations, we have now found that delivering IL-4 to the myeloid compartment is an attractive therapeutic avenue. In support of this concept, we herein described and developed a fusion protein that binds IL-4 to apolipoprotein A-1 (apoA1), the major protein component of high density lipoprotein (HDL), as described elsewhere herein. The resulting apoA1-IL-4 fusion protein is easily incorporated into myeloid cell-affinity lipid nanoparticles (apoA1-IL4 nanoparticles), significantly improving the pharmacokinetic profile and bioavailability of IL-4 to innate immune cells. We used quantitative nuclear imaging techniques and blood chemistry measurements to evaluate the in vivo behavior and safety profile of apoA1-IL-4-nanoparticles in mice and non-human primates. Finally, we studied the therapeutic potential of apoA1-IL-4-nanoparticles in a multi-translational inflammation and sepsis model, institutionalizing a new paradigm for the management of hyper-inflammation-induced immune paralysis.

[0232] Thus, the inventors concluded that IL-4 is a promising novel therapeutic agent that can be used to prevent immune-related disorders by promoting trained immunity if it can be targeted to the myeloid compartment. As described elsewhere herein, the inventors have demonstrated that this can be accomplished by covalent attachment of IL-4 to apolipoproteins, but it is envisioned that at least the following method can be used to achieve targeting of the myeloid compartment. - a fusion of IL-4 to an apolipoprotein as described elsewhere herein, -fusion of IL-4 to a bone marrow targeting molecule, preferably an antibody or antigen-binding fragment thereof that binds to a bone marrow marker, or a ligand or peptide that allows targeting of the bone marrow compartment; - Targeted expression of IL-4 in or near the myeloid compartment.

[0233] Thus, in a further aspect, the present invention relates to a fusion protein of a bone marrow targeting molecule and IL-4, preferably in which the bone marrow targeting molecule is capable of targeting IL-4 to myeloid cells.

[0234] In certain embodiments, the bone marrow targeting molecule is a chemical, such as a small organic molecule, or a biological molecule, such as a biological polymer, such as a protein, polypeptide or peptide, a nucleic acid, a saccharide, a polysaccharide. Preferably, the molecule is a protein, polypeptide or peptide.

[0235] As detailed above, the present invention is further supported by the experimental examples provided below. -IL-4 is surprisingly able to induce trained immunity, making it an interesting therapeutic agent for immune-related disorders, especially immune paralysis, and -based on the finding that the unfavorable pharmacological properties of injected IL-4 can be circumvented by targeting IL-4 to the bone marrow compartment.

[0236] In certain 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 43, or a circular permutation thereof. In further embodiments, the IL-4 polypeptide is encoded by 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 44.

[0237] It is understood that fusions with the same function can also be constructed using circular permutations. A circular permutation is a relationship between proteins, where the order of amino acids in a peptide sequence is changed. This results in a protein structure with different connectivity but with an overall similar three-dimensional shape. For example, a first protein has the sequence abc, and after permutation, a second protein has the sequence cab while maintaining the same three-dimensional shape. Thus, in a further embodiment, the IL-4 polypeptide comprises two sequences that are both 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.

[0238] As used herein, the term "targeting" when referring to targeting myeloid cells or targeting myeloid compartment should be understood to mean bringing myeloid cells or myeloid compartment close to each other or concentrating the vicinity of myeloid cells or myeloid compartment.This means that on average, when targeted (by myeloid targeting molecule), more IL-4 molecules are close to myeloid cells or myeloid compartment.As used herein, closeness means that IL-4 is located so that it can interact with myeloid cells, for example by binding one of its receptors.

[0239] As used herein, a bone marrow targeting molecule is intended to denote any molecule, but preferably a peptide, protein, or a portion of a protein, such as a protein domain, that when fused to IL-4 allows targeting of IL-4 to myeloid cells. As explained in more detail below, it is demonstrated or assumed that the following options function as bone marrow targeting molecules: apolipoproteins, antibodies or antigen-binding fragments thereof, myeloid cell-specific ligands of receptors or membrane molecules.

[0240] As used herein, the term myeloid cells refers to blood cells derived from precursor cells of granulocytes, monocytes, erythrocytes or platelets. Myeloid cells are the major cell compartments of the immune system, including monocytes, dendritic cells, tissue macrophages and granulocytes. As used herein, the term myeloid compartment refers to the totality of myeloid cells in an organism.

[0241] In certain embodiments, the bone marrow targeting molecule is selected from an antibody or antigen-binding fragment thereof, a bone marrow targeting peptide or a bone marrow targeting protein, preferably the bone marrow targeting peptide or protein is a ligand for a receptor present on the target.

[0242] For example, an antibody or antigen-binding fragment thereof can be used that specifically binds to an antigen that is highly expressed or exclusively present on myeloid cells.Suitable targets are known to those skilled in the art, and non-limiting examples are costimulatory molecules such as CD11b, CD11c, CD14, or CD80, CD83, CD86, CD40 or HLA-DR.Thus, in an embodiment, the myeloid targeting peptide or protein is selected from an antibody or antigen-binding fragment thereof that selectively binds to CD11b, CD11c, CD14, CD80, CD83, CD86, CD40 or HLA-DR.

[0243] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from a Fab, Fab2, scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab-2-, Fab3, trispecific Fab-3-diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, IgNAR, monovalent IgG, VhH or VNAR.

[0244] Alternatively, a ligand or cofactor may be used that specifically or primarily binds to a receptor or factor expressed on myeloid cells, a non-limiting example being CD40L (CD154) and the Fc domain, although the skilled artisan will know of other suitable ligands or cofactors. Thus, in an embodiment, the bone marrow targeting molecule is a bone marrow targeting peptide or protein, and the bone marrow targeting protein or peptide is selected from CD40L (CD154) and the Fc domain.

[0245] In a further aspect, the present invention relates to a nucleic acid encoding a fusion protein of a bone marrow targeting molecule and IL-4 as taught herein. In an embodiment, the present invention relates to a nucleic acid comprising 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 a nucleic acid 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.

[0246] The nucleic acid sequence can be used to express the fusion protein according to the invention, and thus in a preferred embodiment the nucleic acid is included in a vector, such as a protein expression vector or a viral vector. The fusion protein can be expressed ex vivo and, for example, subsequently administered to a subject. Alternatively, the vector can be used to transiently or stably transform cells in a subject. For example, it may be particularly beneficial to transform hepatocytes, fibroblasts or muscle cells to express a fusion protein that can then be released into the blood circulation to allow targeting of the bone marrow compartment. As used herein, the term vector refers to a plasmid or virus designed for gene expression in cells. Vectors are used to introduce specific genes into target cells and can manipulate the cellular machinery 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 result in efficient transcription of the gene carried on the expression vector. Those skilled in the art are aware of how to adapt enhancer and promoter regions, for example, for cell type specific or inducible expression of genes (and subsequent translation into proteins).

[0247] It is further envisaged that instead of expressing an IL-4 fusion protein using a myeloid targeting molecule that targets myeloid cells, IL-4 may be expressed in or near myeloid cells to ensure targeting of IL-4 to myeloid cells. Thus, a further embodiment is 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, further comprising a means for targeted expression in myeloid cells, said means comprising - a promoter for selective or inducible expression in said myeloid cells operably linked to said nucleic acid, or a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cells, or - a nucleic acid selected from one or more apolipoproteins, a phospholipid, a lipid nanoparticle comprising said nucleic acid and optionally a sterol.

[0248] For example, myeloid-specific or enhanced promoters or promoter elements can be used in vectors to drive myeloid-specific expression of IL-4. Suitable promoters are known to those skilled in the art, and non-limiting examples are: lysM, Csf1r, CD11c, CX3CR1, Langerin / CD207, MMLV LTR, Visna virus LTR, DC-STAMP, human MSR, MSR-A, huCD68, CD4, CD2 and Iba-AIF-1 (for review, see, for example, Hume., Journal of leukocyte biology, Volume 89, Issue 4, April 2011, Pages 525-538). Such promoters or promoter elements can be incorporated into vectors, such as lentiviral vectors, to stably transfect cells and allow specific expression of transgenes in myeloid cells.

[0249] For example, viruses such as modified retroviruses can be used to specifically infect and drive expression of IL-4 in myeloid cells.

[0250] For example, lipid nanoparticles comprising apolipoproteins, cholesterol, and phospholipids containing mRNA encoding IL-4 can be used to specifically target myeloid cells where the mRNA is translated into protein.

[0251] The inventors have demonstrated that IL-4 can reduce inflammation and induce trained immunity, especially when targeted to the bone marrow compartment.Accordingly, a further aspect provides a fusion protein of a bone marrow targeting molecule and IL-4, or a nucleic acid encoding said fusion protein, for use as a medicament.A further aspect provides a fusion protein of a bone marrow targeting peptide or protein and IL-4, or a nucleic acid encoding said fusion protein, for use in treating an immune-related disorder, preferably the immune-related disorder is a state of hyperinflammation followed by immunoparalysis, preferably the hyperinflammation and / or immunoparalysis is caused by an infectious disease such as COVID-19 due to sepsis, myocardial infarction or stroke.

[0252] In other words, a further aspect provides a method of treating a subject in need thereof comprising administering a fusion protein of a bone marrow targeting peptide or protein and IL-4 or a nucleic acid encoding said fusion protein, preferably the method of treatment is a method of treating an immune-related disorder.

[0253] The inventors have shown for the first time that IL-4 can induce trained immunity. Furthermore, the inventors have demonstrated that by targeting myeloid cells in the organism, the unfavorable pharmacological properties of IL-4 (e.g., extremely short half-life) can be avoided. The experimental data detailed below demonstrate the use of IL-4 as a targeted therapeutic agent in cases of hyperinflammation and subsequent immune paralysis, such as in cases of infectious diseases such as COVID-19 due to sepsis, myocardial infarction or stroke.

[0254] Thus, in certain embodiments, the fusion proteins or nucleic acids for use as taught herein include reducing inflammation and / or stimulating or promoting trained immunity.

[0255] A further aspect provides for the in vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or organism.

[0256] It will be appreciated by those skilled in the art that numerous variations and / or modifications can 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 merely illustrative and not restrictive. The present invention includes the following non-limiting examples.

[0257] The present application also provides aspects and embodiments described in the description* below. Description 1*. A fusion protein of an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, the immunomodulatory biomolecule being a protein that enhances or suppresses an immune response. Statement 2*. A fusion protein of an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, wherein the rerouting molecule is a molecule that enables the fusion protein to bind to a target different from the target to which the apolipoprotein or apolipoprotein mimetic would have bound (in other words, the rerouting molecule is a molecule that, when fused to an apolipoprotein, enables the apolipoprotein to bind to a target different from the target to which it would have bound if it had not been fused to the rerouting molecule). Description 3*. Fusion proteins of apolipoproteins or apolipoprotein mimetics with immunomodulatory biomolecules and rerouting molecules. Statement 4*. The fusion protein according to any one of statements 1* to 3*, wherein the immunomodulatory biomolecule is selected from a cytokine, a chemokine, a hormone, a growth factor, a hematopoietic growth factor or a combination thereof. Statement 5*. The cytokine is selected from the IL-2 subfamily, the interferon subfamily, the IL-10 subfamily, the IL-1 family, the TGFβ family or the IL-17 family or a combination thereof, more preferably the cytokine is selected from IL-1β, IL-2, IL-4, IL-38 or a combination thereof, and / or and / or the chemokine is selected from a CC chemokine, a CXC chemokine, a C chemokine, a CX3C chemokine or a combination thereof; The growth factor is selected from VEGF, EGF, CNTF, LIF, ephrin, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, neurotrophins, PGF, PDGF, RNLS, TCGF, ​​TGF, TNF and WNT or combinations thereof; and / or The fusion protein of statement 4*, 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 a combination thereof. Statement 6*. A fusion protein described in any one of statements 1* to 5*, wherein the rerouting molecule is selected from an antibody or an antigen-binding fragment thereof, a rerouting peptide or a rerouting protein, preferably the rerouting peptide or rerouting protein is a ligand of a receptor present in the target. Description 7*. The antibody or antigen-binding fragment thereof is a Fab, Fab2, scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab3, trispecific Fab3 diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, IgNAR, monovalent IgG, V h H or V; and / or the rerouting peptide is selected from PD1 or SIRPα, and / or The fusion protein according to statement 6*, wherein the rerouting protein is selected from CD40L or GP120. Statement 8*. The fusion protein according to any one of statements *, wherein the apolipoprotein or apolipoprotein mimetic is ApoA1, ApoA4, ApoC3, ApoD, ApoE, ApoL1, ApoL3 or a mimetic thereof. Description 9*. Lipid nanoparticles comprising one or more fusion proteins as defined in description 1* or 4* or 5* or 8* and / or one or more fusion proteins as defined in description 2* or 6* or 7* or 8* and / or one or more fusion proteins as defined in descriptions 3* to 8*, further comprising a phospholipid and a sterol. Statement 10*. A lipid nanoparticle as defined in statement 9*, wherein the lipid nanoparticle further comprises a lipid. Statement 11*. The lipid nanoparticle according to statement 9* or 10*, wherein the lipid nanoparticle further comprises a payload, preferably the payload is selected from a nucleic acid or a nucleic acid analog, a therapeutic agent, a biologic, or a combination thereof. Description 12*. A method for producing lipid nanoparticles as defined in any one of descriptions 9* to 11*, comprising: a1) expressing and isolating an apolipoprotein fusion protein to obtain an isolated apolipoprotein fusion protein, the apolipoprotein fusion protein being an apolipoprotein fused to a cytokine and a targeting moiety, and / or the apolipoprotein fusion protein being 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, wherein the conjugated apolipoprotein is an apolipoprotein conjugated to a cytokine and a targeting moiety and / or 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, a sterol, and optionally lipids to obtain lipid nanoparticles. Description 13*. A fusion protein according to any one of descriptions 1* to 8* or a lipid nanoparticle according to any one of descriptions 9* to 11* or obtained or obtainable by the method of description 12* for use as a medicament. Statement 14*. 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* for use in the treatment of an immune-related disorder. 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 the target being a cell, tissue and / or organ. Description 16*. A nucleic acid encoding a fusion protein as defined in any one of descriptions 1* to 8*.

[0258] The present application also provides aspects and embodiments that are described in the following description. Description 1. Apolipoprotein lipid nanoparticles, A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and an immunomodulatory biomolecule, and phospholipids, Apolipoprotein lipid nanoparticles, where the immunomodulatory biomolecule is a protein that enhances or suppresses the immune response. Statement 2. Apolipoprotein lipid nanoparticles, A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, and phospholipids, A rerouting molecule is a molecule that enables the fusion protein to bind to a target different from the target that the apolipoprotein or apolipoprotein mimetic would have bound, and / or to bind to its intended target with higher affinity, an apolipoprotein-lipid nanoparticle. Statement 3. Apolipoprotein lipid nanoparticles, A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic, an immunomodulatory biomolecule, and a rerouting molecule, and phospholipids, the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response; A rerouting molecule is a molecule that enables the fusion protein to bind to a target different from the target that the apolipoprotein or apolipoprotein mimetic would have bound, and / or to bind to its intended target with higher affinity, an apolipoprotein-lipid nanoparticle. Description 4*. A fusion protein as defined in description 1, A fusion protein as defined in statement 2, and Apolipoprotein lipid nanoparticles comprising phospholipids. Statement 5. The apolipoprotein lipid nanoparticle of any one of statements 1 to 4, wherein the apolipoprotein lipid nanoparticle further comprises a sterol. Statement 6. The apolipoprotein lipid nanoparticle of any one of statements 1 to 5, wherein the apolipoprotein lipid nanoparticle further comprises a lipid, preferably a triglyceride. Statement 7. The apolipoprotein lipid nanoparticle of any one of statements 1 to 6, wherein the apolipoprotein lipid nanoparticle is a sphere, ribbon, or disc. Statement 8. The apolipoprotein lipid nanoparticle of any one of statements 1 to 7, wherein at least a portion of the fusion protein is exposed to the environment surrounding the apolipoprotein lipid nanoparticle, and preferably the immunomodulatory biomolecule and / or the rerouting molecule is exposed to the environment surrounding the apolipoprotein lipid nanoparticle. Statement 9. The apolipoprotein lipid nanoparticle of any one of statements 1 or 3-8, wherein the immunomodulatory biomolecule is selected from the group consisting of cytokines, chemokines, hormones, growth factors, hematopoietic growth factors, and combinations thereof. Statement 10. 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 TGFβ family or the IL-17 family and combinations thereof, more preferably the cytokine is selected from the group consisting of IL-1β, IL-2, IL-4, IL-38 and combinations thereof, and / or and / or the chemokine is selected from the group consisting of CC chemokines, CXC chemokines, C chemokines, CX3C chemokines, and combinations thereof; The growth factor is selected from the group consisting of VEGF, EGF, CNTF, LIF, ephrin, FGF, GDNF, HDF, HDGF, IGF, KGF, MSF, NRG, BDNF, NGF, neurotrophins, PGF, PDGF, RNLS, TCGF, ​​TGF, TNF, and WNT, and combinations thereof; and / or 10. The apolipoprotein lipid nanoparticle of statement 9, 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. Statement 11. The apolipoprotein lipid nanoparticle of statement 9 or 10, wherein the cytokine is IL-4. Statement 12. An 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 the rerouting peptide or rerouting protein is a ligand for a receptor present at the target. Statement 13. The antibody or antigen-binding fragment thereof is a Fab, Fab2, scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab3, trispecific Fab3 diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, IgNAR, monovalent IgG, V h 13. The apolipoprotein lipid nanoparticle of statement 12, wherein the apolipoprotein lipid nanoparticle is selected from the group consisting of: H, and variable novel antigen receptor (VNAR). Statement 14. The apolipoprotein-lipid nanoparticle of any one of 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). Statement 15. The apolipoprotein lipid nanoparticle of 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 megakaryocytes, eosinophils, basophils, erythrocytes, monocytes, such as dendritic cells or macrophages, and neutrophils. Statement 16. The apolipoprotein lipid nanoparticle of statement 15, wherein the rerouting peptide is SIRPα. Statement 17. The apolipoprotein lipid nanoparticle of 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. Statement 19. The apolipoprotein-lipid nanoparticle of statement 17, wherein the rerouting molecule is an antibody or antigen-binding fragment thereof that specifically binds to CD8, or the rerouting peptide is PD1, CD40L, or GP120. Statement 20. The apolipoprotein lipid nanoparticle of any one of statements 1 to 19, wherein the apolipoprotein is ApoA1, ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1, ApoL3 or the apolipoprotein mimetic is a mimetic of ApoA1, ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1, ApoL3. Statement 21. The apolipoprotein lipid nanoparticle of 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 agent, a biologic, or a combination thereof. Statement 22. A method for producing an apolipoprotein lipid nanoparticle as defined in any one of statements 1 to 21, comprising: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins, one or more apolipoprotein fusion proteins comprising an apolipoprotein or apolipoprotein mimetic fused to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; apolipoproteins or apolipoprotein mimetics fused to immune-modulating biomolecules and rerouting molecules, 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, one or more conjugated apolipoproteins are apolipoproteins or apolipoprotein mimetics conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule and a rerouting molecule, and combinations thereof; and b) combining one or more isolated apolipoprotein fusion proteins obtained in step a1 and / or one or more isolated conjugated apolipoproteins obtained in step a2 with phospholipids, and optionally a sterol and / or lipids, to obtain apolipoprotein-lipid nanoparticles. Statement 23. An apolipoprotein lipid nanoparticle obtained or obtainable by the method according to statement 22. Statement 24. A pharmaceutical composition comprising an apolipoprotein lipid nanoparticle according to any one of statements 1 to 21 or 23, and a pharma- ceutically acceptable carrier. Statement 25. An apolipoprotein lipid nanoparticle according to any one of statements 1 to 21 or 23, or a pharmaceutical composition according to statement 24, for use as a medicament. Statement 26. An apolipoprotein lipid nanoparticle according to any one of statements 1 to 21 or 23, or a pharmaceutical composition according to statement 24, for use in treating an immune-related disorder. Statement 27. The apolipoprotein lipid nanoparticle for use according to statement 26, or the pharmaceutical composition for use according to statement 26, wherein the immune-related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorders, allergies, organ transplant rejection, and graft-versus-host disease (GVH). Statement 28. Apolipoprotein-lipid nanoparticles for use according to statement 26 or a pharmaceutical composition for use according to statement 26, wherein the immunomodulatory biomolecule is IL-4 and the immune-related disorder is a state of hyperinflammation and subsequent immunoparalysis, preferably where the hyperinflammation and / or immunoparalysis is caused by sepsis, myocardial infarction, stroke, cancer, or an infectious disease such as COVID-19 due to multiple sclerosis. Statement 29. An apolipoprotein lipid nanoparticle according to any one of statements 1, 3 to 21 or 23 or a pharmaceutical composition according to statement 24 for use in targeting said immunomodulatory biomolecule to a target cell. Statement 30. An apolipoprotein lipid nanoparticle according to any one of statements 1, 3 to 13, 15, 16, 20, 21 or 23, or a pharmaceutical composition according to statement 24, for use in targeting said immunomodulatory biomolecule to a myeloid cell. Statement 31. The use of an apolipoprotein lipid nanoparticle according to any one of statements 1, 3 to 21 or 23 for targeted delivery of an immunomodulatory biomolecule, preferably wherein the target is a cell, tissue and / or organ. Statement 32. A fusion protein comprising an apolipoprotein or apolipoprotein mimetic and an immunomodulatory biomolecule, the immunomodulatory biomolecule being a protein that enhances or suppresses an immune response for use in targeting the immunomodulatory biomolecule to myeloid cells. Statement 33. A fusion protein for use according to statement 32, wherein the fusion protein further comprises a rerouting molecule, the rerouting molecule being a molecule that enables the fusion protein to bind to a target different from the target to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with higher affinity, preferably the rerouting molecule being a rerouting molecule as defined in statement 15. Statement 34. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and a rerouting molecule, the rerouting molecule being a molecule that enables the fusion protein to bind to a target different from the target to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with higher affinity. Statement 35. The fusion protein according to statement 34, wherein the rerouting molecule is a rerouting molecule as defined in any one of statements 12 to 19. Statement 36. The fusion protein according to statement 34 or 35, wherein the fusion protein further comprises an immunomodulatory biomolecule, the immunomodulatory biomolecule being a protein that enhances or suppresses an immune response, preferably the immunomodulatory biomolecule is an immunomodulatory biomolecule as defined in statement 9 or 10. Statement 37. A nucleic acid encoding a fusion protein according to any one of statements 34 to 36. Statement 38. A pharmaceutical composition comprising a fusion protein according to any one of statements 34 to 36 or a nucleic acid according to statement 37 and a pharma- ceutically acceptable carrier. Statement 39. A fusion protein according to any one of statements 34 to 36, a nucleic acid according to statement 37, or a pharmaceutical composition according to statement 38 for use as a medicament. Statement 40. The fusion protein of any one of statements 34 to 36, the nucleic acid of statement 37, or the pharmaceutical composition of statement 38 for use in treating an immune related disorder, preferably wherein the immune related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorders, allergy, organ transplant rejection, and graft versus host disease (GVH). Statement 41. A fusion protein according to statement 36, a nucleic acid encoding a fusion protein according to statement 36, or a pharmaceutical composition according to statement 38, when dependent on statement 36, for use in targeting said immunomodulatory biomolecule to a target cell. Statement 42. A fusion protein comprising an apolipoprotein or an apolipoprotein mimetic and interleukin-4 (IL-4). Statement 43. The fusion protein according to statement 42, wherein the fusion protein further comprises a rerouting molecule, the rerouting molecule being a molecule that enables the fusion protein to bind to a target different from the target to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with higher affinity, preferably the rerouting molecule being a rerouting molecule as defined in any one of statements 12 to 19. Statement 44. The fusion protein according to statement 42 or 43, wherein the apolipoprotein or apolipoprotein mimetic is as defined in statement 20. Statement 45. A nucleic acid encoding a fusion protein according to any one of statements 42 to 44. Statement 46. A pharmaceutical composition comprising a fusion protein according to any one of statements 42 to 44 or a nucleic acid according to statement 45 and a pharma- ceutically acceptable carrier. Statement 47. A fusion protein according to any one of statements 42 to 44, a nucleic acid according to statement 45, or a pharmaceutical composition according to statement 46 for use as a medicament. Statement 48. A fusion protein according to any one of statements 42 to 44, a nucleic acid according to statement 45, or a pharmaceutical composition according to statement 46 for use in treating an immune related disorder. Statement 49. The fusion protein for use according to statement 48, the nucleic acid for use according to statement 48, or the pharmaceutical composition for use according to statement 48, wherein the immune-related disorder is a state of hyperinflammation and subsequent immunoparalysis, preferably where the hyperinflammation and / or immunoparalysis is caused by an infectious disease such as COVID-19 due to sepsis, myocardial infarction, stroke, cancer, or multiple sclerosis. Statement 50. A fusion protein according to any one of statements 42 to 44, a nucleic acid according to statement 45, or a pharmaceutical composition according to statement 46 for use in targeting IL-4 to a target cell. Statement 51. A fusion protein according to any one of statements 42 to 44, a nucleic acid according to statement 45, or a pharmaceutical composition according to statement 46 for use in targeting IL-4 to a myeloid cell. Statement 52. A fusion protein comprising a bone marrow targeting molecule and IL-4, wherein the bone marrow targeting molecule is capable of targeting the IL-4 to a myeloid cell. Statement 53. The fusion protein of statement 52, wherein 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 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:43, or a circular permutation thereof. Statement 54. The fusion protein of statement 52 or 53, wherein the bone marrow targeting molecule is selected from an antibody or antigen-binding fragment thereof, a bone marrow targeting peptide or a bone marrow targeting protein, preferably the bone marrow targeting peptide or the bone marrow targeting protein is a ligand for a receptor present on the target. Statement 55. The fusion protein of statement 54, wherein the antibody or antigen-binding fragment thereof is selected from a Fab, Fab2, scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab-2-, Fab3, trispecific Fab-3-diabody, bispecific diabody, triabody, trispecific triabody, minibody, IgG, IgNAR, monovalent IgG, VhH, or VNAR. Statement 56. A nucleic acid encoding a fusion protein according to any one of statements 52 to 55. Statement 57. 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 a means for targeted expression in myeloid cells, said means comprising - a promoter for selective or inducible expression in said myeloid cells operably linked to said nucleic acid, or a viral expression vector comprising said nucleic acid capable of stably expressing said nucleic acid in said myeloid cells, or - a nucleic acid selected from one or more apolipoproteins, phospholipids, lipid nanoparticles comprising said nucleic acid and optionally a sterol. Statement 58. A pharmaceutical composition comprising a fusion protein according to any one of statements 52 to 55 or a nucleic acid according to statement 56 or 57, and a pharma- ceutically acceptable carrier. Statement 59. A fusion protein according to any one of statements 52 to 55, a nucleic acid according to statement 56 or 57, or a pharmaceutical composition according to statement 58 for use as a medicament. Statement 60. A fusion protein according to any one of statements 52 to 55, a nucleic acid according to statement 56 or 57, or a pharmaceutical composition according to statement 58 for use in treating an immune related disorder. Statement 61. A fusion protein for use according to statement 60, a nucleic acid for use according to statement 60, or a pharmaceutical composition for use according to statement 60, wherein the immune-related disorder is a state of hyperinflammation and subsequent immunoparalysis, preferably wherein the hyperinflammation and / or immunoparalysis is caused by an infectious disease such as COVID-19 due to sepsis, myocardial infarction or stroke. Statement 62. The in vivo, in vitro or ex vivo use of IL-4 in stimulating or promoting trained immunity in a cell, organ, tissue or organism.

[0259] While the present 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 within the spirit and broad scope of the appended claims.

[0260] The presently disclosed aspects and embodiments of the present invention are further supported by the following non-limiting examples. EXAMPLES

[0261] Example 1. Preparation of fusion proteins of apolipoproteins fused to immunomodulatory biomolecules and incorporation into lipid nanoparticles material and method Protein expression, purification and characterization of ApoA1-S147C (e.g., SEQ ID NO: 9) and ApoA1-S279C (e.g., SEQ ID NO: 11) variants, apoA1-IL4 fusion protein (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-IL1β fusion protein (e.g., SEQ ID NO: 21) and apoA1-IL38 fusion protein (e.g., SEQ ID NO: 80) (Figures 1 to 9)

[0262] Bacterial cells containing the pET-vectors encoding the desired proteins were grown in 40 mL of 2YT medium supplemented with additional NaCl (10 g / L) and 100 μg / mL ampicillin, and the transformed bacteria were inoculated and grown overnight. The following day, the overnight culture was inoculated into 2YT medium containing 10 g / L NaCl and 100 μg / mL ampicillin and incubated at 37 °C at 150 rpm until the OD600 reached 0.6-0.8. Isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The culture was further incubated overnight at 20 °C, 150 rpm. Bacteria were pelleted using centrifugation and lysed using Bugbuster® Protein Extraction Reagent (Novagen) per gram of cell pellet supplied with Benzonase® Nuclease (Merck) according to the manufacturer's protocol. The lysate was centrifuged and the resulting supernatant containing the protein of interest was then applied to an IMAC column containing Ni-NTA HisBind® Resin (Merck Millipore).

[0263] The resulting fractions were analyzed using SDS-PAGE (samples were combined with sample buffer (1:1) and run on Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad). The resulting gels were stained using Coomassie G-250 stain and destained using dH2O. Figure 3, Figure 5).

[0264] The obtained purified proteins of interest were further characterized using Q-ToF. Samples were diluted to concentrations of 0.01 to 0.1 mg / mL with 0.1% formic acid in dH2O. After filtration using a PD Spintrap™ G-25 column (0.5 mL, Cytiva), samples were measured using a Waters ACQUITY UPLC I-Class system equipped with a Xevo G2 quadrupole time-of-flight mass spectrometer. Proteins were separated by a C8A reversed-phase column. A gradient of 15% to 75% acetonitrile in 0.1% formic acid in dH2O was used. The obtained data was analyzed using MassLynx (Waters) using the MaxEnt algorithm (Figure 6). Protein expression, purification and characterization of ApoA1-S147C (e.g., SEQ ID NO: 9) and ApoA1-S279C (e.g., SEQ ID NO: 11) variants, apoA1-IL4 fusion protein (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-IL1β 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 to 16)

[0265] Bacterial expression and protein purification): This was carried out as described in Example 2, "Bacterial expression and protein purification."

[0266] Bacterial lysis and protein purification: This was performed as described in Example 2 under the heading "Bacterial lysis and protein purification."

[0267] Discoidal lipid nanoparticle (LNP) formulation To formulate HDL-based LNPs, phospholipids, apoA1 fusion protein 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 molar ratio of 1:10:100 using a staggered herringbone microfluidic mixer. Dynamic light scattering measurements were performed on a Zetasizer Nano ZSP containing the nanobiologics to assess size and heterogeneity. A sample volume of 100 μL was pipetted into a transparent cuvette (Sarstedt), which was inserted into a Malvern Zetasizer Nano ZSP. Samples were measured in triplicate at a temperature of 25 °C, for 10 cycles in one run.

[0268] Azide introduction into immunomodulatory biomolecules and conjugation to apoA1 or its cysteine ​​mutants ApoA1-IL4 Fusions (for data shown in Figures 1-9):IL-4 was buffer exchanged into DEA buffer (50 mM diethanolamine, pH 7.5) and concentrated to 0.75 mM. Imidazole-1-sulfonyl azide hydrochloride (Fluorochem) was dissolved in dH2O to give a stock solution of 20 mg / mL. The pH was adjusted to 7. This was then added to IL-4 at a molar ratio of 227.5:1 (17.5 molar equivalents per amine in IL-4). The reaction mixture was incubated overnight at 4 °C. 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 overnight at 4 °C. The resulting functionalized IL-4-Az and apoA1-DBCO were then buffer-exchanged into PBS (pH 7.9) to remove excess azide transfer reagent and DBCO-maleimide linker. The solutions were added together at a 1:2 apoA1:IL-4 molar ratio and incubated overnight at 4 °C. The next day, the mixture was purified on an 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 above) and HEK293 IL-4 reporter assay.

[0269] ApoA1-cytokine fusions (for data presented in Figures 10-16):In an alternative, a stock solution of imidazole-1-sulfonyl azide (2 mg / mL) in MQ was prepared. The pH of this stock was set to 7.5. To 100 μg of 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, 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 5-fold molar excess of maleimide-PEG4-DBCO linker was added to ApoA1 or its cysteine ​​mutants and allowed to react overnight to form the apoA1-PEG4-DBCO conjugate. Excess linker was removed using Amicon Ultra-0.5 centrifugal filter units with MWCO 10 kDa (Merck). ApoA1-PEG4-DBCO was then combined with azide-containing cytokines and incubated for 4 h at room temperature or overnight at 4 °C. The resulting products were analyzed using SDS-page.

[0270] HEK293 IL-4 reporter assay HEK-Blue™ IL-4 cells (Invivogen) were seeded in T25 culture flasks and grown at 37°C until 80% cell confluency was reached. Cell viability was confirmed using a microscope. Cells were washed with sterile PBS and harvested using trypsinization at 37°C for 5 min. Cells were then seeded in 96-well plates such that each well contained 50,000 cells (180 μL DMEM, 10% FBS, 1% Pen-Strep). For each condition, a 2-fold dilution series was prepared. The dilution series was added in triplicate to the plate containing the cells. The cells were then incubated at 37°C for 24 hours. QUANTI-Blue™ (Invivogen) solution was prepared according to the manufacturer's instructions. In another 96-well plate, 20 μL of conditioned cell medium was added along with 180 μL of QUANTI-Blue™ solution. This was then incubated at 37°C for 3 hours. Absorbance was analyzed at 635 nM using a Spark® multimode microplate reader (Tekan).

[0271] Effect of IL2 constructs on T cell proliferation Human CD3 positive T cells were stained with CFSE (Thermofisher) according to the 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, stained for CD3, CD4 and CD8 and measured on a Cytoflex (Beckman Coulter Inc.). Flow cytometry data were analyzed using FlowJo software (BD). Commercially available IL2 was purchased from Sinobiological. Recombinant IL2 was prepared as follows:

[0272] Bacterial expression of recombinant IL-2 The SUMO-IL2 construct was transformed into Shuffle T7 competent E. coli. Bacteria were inoculated into 40 mL of 2YT medium containing 50 μg / mL kanamycin and grown overnight at 250 rpm and 37 °C to form small cultures. The next day, the small culture was inoculated into 2YT medium containing 50 μg / mL kanamycin to form large cultures. The culture was incubated at 150 rpm and 37 °C until an OD600 of 0.6-0.8 was reached. IPTG was then added to a final concentration of 0.1 mM to induce protein expression. The culture was further incubated overnight at 20 °C, 150 rpm. The bacterial pellet was then obtained by centrifugation at 10,000 x g for 10 min at 4 °C. The resulting supernatant was discarded. The resulting pellet was resuspended in 10 mL of lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.9) per gram of cell pellet. Then, 25 U Benzonase® Nuclease (Merck) was added. One cOmplete™ Protease Inhibitor Cocktail tablet was added per 50 mL of extraction buffer. The resulting solution was stirred for 30 min at 4 °C until no aggregates remained. The solution was then homogenized three times using an Avestin Emulsiflex C3 at 15,000-20,000 psi while kept on ice. The cell lysate was then centrifuged at 20,000xg for 30 min at 4 °C. The resulting supernatant was applied to an IMAC column at 4 °C, which was preloaded with 0.1 M NiSO4 solution. All flow-through fractions were collected. The column was washed with 8 column volumes of buffer A (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9) and then with 8 column volumes of buffer A50 (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 7.9). To elute SUMO-IL2, 8 column volumes of buffer A500 (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.9) were added to the column. The resulting 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 at a ratio of 1 mg hydrolase / 500 mg protein.The solution was then dialyzed into storage buffer (20 mM Tris, 500 mM NaCl, pH 7.9) using a Snakeskin™ 10 kDa cut-off dialysis bag (Thermo Scientific) with gentle stirring overnight at 4 °C. The resulting protein solution was then centrifuged at 4000 x g for 20 min and the supernatant was filtered using a 0.2 μM syringe filter to remove aggregated proteins. The IMAC column protocol was repeated and fractions were analyzed again using SDS-PAGE. If the fractions containing IL2 were contaminated with other proteins, the samples were purified using size-exclusion chromatography (SEC). Otherwise, the fractions containing IL2 were buffer-exchanged into PBS (pH 7.9). SEC was performed on an NGC 10 Medium-Pressure Chromatography System (Bio-Rad) equipped with a GE Hiload 16 / 60 Superdex 75 pg column. The column was first equilibrated with filtered PBS (pH 7.9) before the sample was applied. The collected fractions were analyzed using SDS-PAGE. The fractions containing IL2 were pooled and the final concentration was determined using a Nanodrop™ 1000 spectrophotometer. The protein was then flash frozen in liquid nitrogen and stored at -80°C. Analysis was performed using Q-tof and SPR.

[0273] Cryo-transmission electron microscopy (cryo-TEM): It was carried out as described in Example 2.

[0274] Determination of aNP size and dispersity by DLS: This was carried out as described in Example 2.

[0275] SDS page: Performed as described in Example 2

[0276] result Our fusion proteins can be produced by recombinant expression or by chemical conjugation. So far, we have successfully recombinantly expressed four different fusion proteins. These are apoA1-IL4, apoA1-IL1B, apoA1-IL38 and apoA1-IL2. As can be observed in Figure 3, all were successfully expressed and yielded pure proteins. The black rectangles indicate the elution fractions containing the fusion proteins of interest.

[0277] These recombinantly expressed proteins were then used to formulate nanobiological discs (Figure 4). It can be seen that apoA1-IL4 and apoA1-IL38 remain stable over 11 days and exhibit similar diameters and PdIs as apoA1. ApoA1-IL1b also exhibits this, however after 11 days these nanobiological formulations appear to be aggregated.

[0278] To chemically conjugate apoA1 to cytokines, nanobodies, or other biomolecules, reactive handles are required. Thus, apoA1 mutants containing a cysteine ​​instead of serine at position 147 (herein referred to as "S147C" or "S157C" mutants) or 279 (herein referred to as "S279C" or "S239C" mutants) were generated. For example, apoA1 mutants can be defined by the peptide sequences shown in SEQ ID NO: 9 or 11. As can be seen in Figure 5, expression and purification of these proteins was successful, resulting in yields and purities similar to wild-type apoA1. The resulting proteins were further characterized using quadrupole time-of-flight (Q-ToF), which showed that the proteins were pure and the correct masses were found (Figure 6).

[0279] Conjugation of cytokines to this apoA1 variant can be performed using a maleimide-PEG-DBCO linker, where the maleimide binds to apoA1 and the DBCO binds to the azide that we introduce to the cytokine. We optimized the introduction of a single azide to the N-terminus of IL-4 until ±90% of the product consisted of IL-4-Az (IL-4 with a 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 with IL4, a band appears at approximately 40 kDa, corresponding to the expected molecular weight of the fusion protein. This confirms that we have chemically conjugated IL4 to apoA1 using a PEG-linker (Figure 8). Although this conjugation reaction is still being optimized, we have already obtained a yield of ±20%. The HEK293 IL-4 reporter assay was then used to analyze the biological activity of the recombinantly produced fusion protein as well as the chemical apoA1-IL4 conjugate. This cell line has a fully active STAT6 pathway and carries a STAT6-inducible secreted alkaline phosphatase (SEAP) reporter gene, where the production of SEAP is related to the binding of IL4 to its receptor. HEK-Blue™ IL-4 / IL-13 cells produce SEAP in response to IL-4 and IL-13. IL-4 binding can be quantified by examining the enzymatic activity of SEAP using the QUANTI-Blue colorimetric assay.

[0280] As can be seen from Figure 9, both fusion proteins exert a dose-dependent effect, and therefore it can be concluded that the IL4 in both fusion proteins is still functional. Chemically conjugated apoA1-IL4 performs slightly less preformation than commercial recombinant IL-4, which may be due to the fusion of apoA1 with IL-4. This may slightly hinder the binding of IL-4 to the receptor. However, chemically conjugated apoA1-IL4 performs better than recombinant apoA1-IL4 protein, indicating that the affinity of chemically conjugated apoA1-IL4 is likely to be higher than that of recombinantly expressed apoA1-IL4.

[0281] ApoA1-IL2 fusion protein In addition to producing apoA1-IL4 chemical and recombinant fusion proteins, we also produced apoA1-IL2 fusion proteins. Here, we recombinantly fused wild-type IL2 or IL2 mutants to apoA1. SDS-page analysis of the expressed and IMAC-purified proteins showed that the correct protein was present in our elution fractions (Figure 10, left panel). Furthermore, we chemically conjugated wild-type IL2 to apoA1. This was also verified by SDS-page (Figure 10, right panel).

[0282] Next, we incorporated apoA1-IL2 fusion protein into discoid lipid nanoparticles to obtain IL2-aNP and IL2v4-aNP (Figure 11). The successful formulation of discoid nanoparticles was confirmed by cryo-transmission electron microscopy (cryo-TEM) (Figure 11, right panel). We further analyzed nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Figure 11, left panel).

[0283] We evaluated the ability of apoA1-IL2 fusion proteins to stimulate T cell proliferation. We find that our fusion proteins can induce T cell proliferation. In particular, our apoA1-IL2 chemical conjugate (FIG. 12, center bottom) can induce T cell proliferation to a similar extent as commercial IL2 (FIG. 12, top left).

[0284] ApoA1-IL1β fusion protein We further expanded our library by creating a chemically conjugated, recombinantly expressed apoA1-IL1β fusion protein. Here, we recombinantly fused IL1β to apoA1. SDS-page analysis of the expressed and IMAC purified protein showed that the correct protein was present in our elution fractions, as indicated by a band at approximately 40 kDa (Figure 13, upper panel). Furthermore, we also chemically conjugated IL1β to apoA1. This was also verified by SDS-page (Figure 13, lower panel).

[0285] Next, we incorporated apoA1-IL1β fusion protein into discoid lipid nanoparticles to obtain IL1β-aNP. The successful formulation of discoid nanoparticles was confirmed by cryo-transmission electron microscopy (cryo-TEM) (Figure 13, lower panel). We further analyzed nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Figure 14, upper panel).

[0286] ApoA1-IL38 fusion protein In addition, we have also chemically conjugated and recombinantly expressed apoA1-IL38 fusion protein. Here, we recombinantly fused IL38 to apoA1. SDS-page analysis of the expressed and IMAC purified protein showed that the correct protein was present in our elution fractions, as indicated by a band at approximately 40 kDa (Figure 15, upper panel). In addition, we have also chemically conjugated IL38 to apoA1. This was also verified by SDS-page (Figure 15, lower panel).

[0287] Next, we incorporated apoA1-IL38 fusion protein into discoid lipid nanoparticles to obtain IL38-aNP. The successful formulation of discoid nanoparticles was confirmed by cryo-transmission electron microscopy (cryo-TEM) (Figure 16, lower panel). We further analyzed nanoparticle size and stability in PBS for 21 days using dynamic light scattering (DLS) (Figure 16, upper panel).

[0288] Example 2. ApoA1-IL-4 fusion protein and its incorporation into lipid nanoparticles material and method Isolation of PBMCs and monocytes: Buffy coats (Sanquin) or EDTA whole blood from healthy volunteers were obtained after obtaining written informed consent. This material was diluted at least 1:1 with calcium / magnesium-free PBS (Lonza) and layered on top of Ficoll-Paque (GE Healthcare). Peripheral blood mononuclear cell (PBMC) interface was isolated using density gradient centrifugation at 615 × g for 30 min. After 3–5 washes with cold PBS, PBMC yield and composition were assessed using a Sysmex hematology analyzer (XN-450; Sysmex).

[0289] 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 were assessed on a Sysmex hematology analyzer.

[0290] In some experiments (shown in the main text), monocytes were alternatively enriched from PBMCs by hyperosmotic density gradient centrifugation on Percoll (Sigma-Aldrich). 6 PBMCs were layered on top of a hypertonic Percoll solution (48.5% v / v Percoll in sterile water, 0.16 M NaCl) and centrifuged at 580×g for 15 min at room temperature. The interface was collected, washed once with cold PBS, and resuspended in RPMI.

[0291] Primary human monocyte culture: All primary human monocytes / macrophages were cultured in RPMI-1640 with Dutch modification (Invitrogen) further supplemented with GlutaMAX (2 mM; GIBCO), sodium pyruvate (1 mM; GIBCO) and gentamicin (50 μg / 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").

[0292] 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, GP & 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 allowed to adhere to flat-bottom cell culture plates for 1 h and washed with warm PBS to remove any non-adherent cells and cell debris. They were then stimulated for 24 hours ("training") with any of the stimuli detailed in Table 1 or with medium alone ("untrained" control). Table 1. Stimulation of primary human monocytes and in vivo experiments. [Table 1]

[0293] For pharmacological inhibition experiments, monocytes were preincubated with one of the inhibitors listed in Table 2 for 1 h before the addition of the training stimulus. Table 2. Inhibitors for primary human monocytes. [Table 2]

[0294] After the first 24 h of stimulation, the cells were washed with warm PBS and warm cell culture medium was added. The monocytes were then allowed to settle and differentiate into macrophages for 5 days. On day 6, the induction of trained immunity was evaluated. To this end, the cells were typically restimulated with LPS for another 24 h to induce cytokine production. The supernatants were collected and stored at -20 °C until further analysis, e.g., using ELISA for IL6 and TNF.

[0295] In most other trained immune readout methods, cells were harvested as follows: first, cells were incubated in Versene cell dissociation reagent (Life technologies) for 30 min in a cell culture incubator. Then, a cell scraper was used to remove the cells from the culture plate. To maximize the yield, the culture plate was scraped a second time after adding ice-cold PBS. Macrophages were centrifuged at 300 × g for 10 min at 4 °C and counted before continuing with downstream applications.

[0296] In Vitro Inflammation Inhibition: Monocytes were allowed to adhere to flat-bottom cell culture plates for 1 hour and washed with warm PBS to remove any non-adherent cells and cell debris.

[0297] They were then incubated with IL4 and LPS for 24 h. After the first 24 h of stimulation, the supernatants were collected and stored at −20°C until further analysis by ELISA for IL6 and TNF.

[0298] Generation of primary monocyte-derived dendritic cells (moDCs): For the experiments where moDCs were compared to macrophages (untrained control or IL4-trained), moDCs were differentiated as follows. First, negatively selected monocytes were obtained as described above. After 1 h of adhesion and PBS washing, they were cultured in RPMI+++ with 10% HPS additionally supplemented with IL4 (25 ng / ml) and GM-CSF (1000 IU / ml; premium grade, Miltenyi Biotec). Cells were differentiated until day 6, with one moderate refresh on day 3. On day 6, non-adherent cells were harvested in addition to adherent cells (as described above). moDCs and macrophages were then subjected to analysis by flow cytometry, as described below.

[0299] In vivo experimental human endotoxemia model and ex vivo analysis: Eight healthy (as confirmed by medical history, physical examination, and routine testing) male volunteers provided written informed consent to participate in an experimental endotoxemia experiment performed in the Research Unit of the Intensive Care Unit of the Radboud University Medical Center. All study procedures were approved by the local ethical committee (CMO Arnhem-Nijmegen, registration numbers NL71293.091.19 and 2019-5730) and were carried out in accordance with the latest version of the Declaration of Helsinki.

[0300] A continuous endotoxin infusion regimen was used, as described in detail elsewhere (van Lier, D., Geven, C., Leijte, GP & 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, subjects were admitted to the research unit and the antecubital vein and radial artery were cannulated to allow administration of fluids and endotoxin, as well as blood sampling and hemodynamic monitoring, respectively. A 3-lead ECG was recorded continuously throughout the experiment. After isotonic water pretreatment (1.5 L NaCl 0.45% / glucose 2.5%, administered intravenously for the hour before the start of endotoxin infusion), volunteers were exposed intravenously to a loading dose of 1 ng / kg body weight endotoxin (Escherichia coli lipopolysaccharide [LPS] type O113, lot no. 94332B1; List Biological Laboratories), followed by a direct continuous infusion of 0.5 ng / kg / h for 3 h. Participants were monitored for 8 h after the endotoxin loading dose, after which participants were discharged from the study unit.

[0301] In this project, blood samples were obtained at two time points, 1 h before and 4 h after administration of the loading dose. Negatively selected monocytes were obtained as described above. Cells were allowed to adhere and stimulated for 24 h with recombinant human IL4, discoidal IL4-aNP, LPS (to assess early immune tolerance), or medium alone (as control). After PBS washing, cells were left in culture medium for 48 h and restimulated with LPS for an additional 24 h. Supernatants were collected and stored at -20 °C.

[0302] Cytokine and lactate measurements:TNF, IL6 and IL1Ra were measured in cell culture supernatants using DuoSet ELISA kits (R&D systems) according to the manufacturer's instructions. For lactate measurements, a fluorescent assay was used. 30 μl of sample, medium control or known standard was added to a black 96-well plate. Then, 30 μl of reaction mixture (PBS, pH 7.4, horseradish peroxidase (0.2 U / ml), lactate oxidase (2 U / ml), Amplex red (100 μM; Fisher scientific)) was added and the reaction was incubated for 20 minutes at room temperature in the dark. Immediately after, fluorescence was measured at 530 / 25 nm and 590 / 35 nm. Cytokine and lactate concentrations in the original samples were calculated using Gen5 software (v3.03, BioTek) in conjunction with Microsoft Excel.

[0303] Macrophage Surface Markers Flow Cytometry: Macrophages were harvested as above and transferred to v-bottom 96-well plates for staining. Cells were centrifuged at 1500 rpm for 5 min at 4° C. The supernatant was removed and cells were washed once with 200 μl PBA (PBS, pH 7.4, 1% w / v BSA (Sigma)).

[0304] Fc receptors were blocked by incubation in PBS supplemented with 10% pooled human serum for 15 min at 4° C. After another wash, surface markers and viability were stained using the antibodies and viability dyes listed in Table 3 for 30 min at 4° C. in a volume of 50 μl. Table 3. Flow cytometry antibodies for experiments with primary human monocytes / macrophages (and for MLR experiments). [Table 3]

[0305] After two washes, cells were resuspended in 150 μl PBA and measured on a Cytoflex flow cytometer (Beckman Coulter) or a BD FACSVerse system (BD Biosciences). Compensation was performed using VersaComp compensation beads (Beckman Coulter) for single antibody staining. Mixtures of live and heat-killed cells were used for single staining of viability dyes (following the manufacturer's recommendations). Data analysis was performed with Flowjo (v10.7.1, BD Biosciences). Our gating strategy was as follows: first, a time gate was used when necessary. Single cell events were then 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 intensity was calculated as a measure of surface marker expression.

[0306] T cell polarization readout: For MLR experiments, the harvested macrophages were used for the subsequent T cell polarization assay. Allogeneic naive T cells were seeded with macrophages at a ratio of 10 T cells per macrophage. Cells were cultured in standard cell culture medium in flat-bottom 96-well plates for 7 days. In this model, HLA mismatch causes non-specific activation of the T cell receptor. On the last day, cells were stimulated with PMA (25 ng / mL) + ionomycin (0.5 μg / mL) for 4 hours in the presence of 100 ng / mL brefeldin A (golgi-plug). Cells were harvested and separated into two flow cytometry antibody panels (one for CD4 T cells and one for CD8; see also Table 3). Cells were stained in a similar manner as above, with an additional step for permeabilization of T cells to allow 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 that described above, with the addition of selection for CD3-positive events. The percentage of cells positive for hallmark cytokines of T cell polarization was calculated to estimate the percentage of T cell subsets.

[0307] Phospho-STAT6 measurement by flow cytometry: Monocytes were stimulated with RPMI, IL4, or different concentrations of IL4-aNP (as indicated in the figure) for 20 min at 37 °C. Cells were transferred to v-bottom 96-well plates and kept on ice during the staining procedure. After staining for viability and CD14 (as described above), cells were fixed and permeabilized using fix / perm buffer set (eBioscience) for 45 min at 4 °C in the dark. Cells were washed twice with perm buffer and incubated in freezer-cooled absolute methanol overnight at -20 °C. After two more washes with perm buffer, cells were stained for phospho-STAT6 using the antibodies listed in Table 3 for 45 min at 4 °C in the dark. Cells were washed two more times with perm buffer and finally resuspended in PBA for acquisition on a Cytoflex cytometer. The gating strategy was largely similar to that for macrophage surface markers with the addition of selection for CD14 positive events.

[0308] Phagocytosis assay: Macrophages were harvested as above and incubated with FITC-labeled Candida albicans (kindly provided by Dr. Martin Jaeger, Radboudumc) at an MOI of 1:5 for 1 h at 37 °C. Cells were washed twice with ice-cold PBA and kept on ice to stop phagocytosis. Cells were stained for CD45 (Table 3) for 30 min in the dark at 4 °C. After two washes, trypan blue was added to a final concentration of 0.01% to quench extracellular FITC-Candida. Cells were then acquired on a Cytoflex flow cytometer.

[0309] During data analysis, CD45+ events were first selected to remove Candida-only events, then single cells were gated as above and the percentage of Candida-FITC positive macrophages in each sample was calculated.

[0310] Seahorse metabolic analysis: Macrophages were harvested as described above. Cells were resuspended in RPMI+++ and plated at 10 cells per well in overnight calibrated cartridges. 5Cells were seeded with 100 μg / ml of 100% glycerol. After 1 h of adhesion, the medium was replaced with assay medium (Agilent; see below) and the cells were incubated at 37 °C with ambient CO2 levels for 1 h. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured as proxies for glycolysis and mitochondrial metabolism using the Seahorse XF Glycolysis Stress Test kit or the Seahorse XF Cell Mito Stress Test kit (both Agilent; measurements performed according to the manufacturer's instructions).

[0311] RNA isolation, sequencing and analysis: Monocytes or macrophages were lysed in RLT buffer (Qiagen) and stored at -80°C. RNA extraction was performed using RNeasy mini columns (Qiagen) with on-column DNAse I treatment (RNase-free; Qiagen). Preliminary quality control and concentration measurements were performed using a Nanodrop instrument. Samples were sent to the Beijing Genome Institute (BGI Denmark) for RNA sequencing using the DNBseq platform.

[0312] To infer gene expression levels, RNA-seq reads were aligned to the hg 19 human transcriptome using Bowtie. Quantification of gene expression levels as RPKM was performed using MMSEQ. Reads / transcripts were normalized using DEseq2 and pairwise comparisons were performed. Differentially expressed genes were identified using DEseq2 with fold changes >2 and p-values ​​<0.05, with average RPKM >1. To identify genes 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 / RPMI-d6+LPS. Gene Ontology and TF motif analysis were performed on gene promoters using the HOMER findMotifs tool.

[0313] Chromatin immunoprecipitation: Macrophages were harvested as above and resuspended in RPMI+++. Cells were fixed in 1% methanol-free formaldehyde for 10 min. The reaction was then quenched by adding 125 mM glycine for 3 min. Fixed cells were washed three times with ice-cold PBS and lysed at approximately 15×10 in lysis buffer (20 mM HEPES, pH 7.6, 1% SDS, 1× protease inhibitor cocktail (PIC; Roche)). 6 Cells / ml were lysed, sonicated (Bioruptor Pico, Diagenode), and centrifuged (10 min, 13000 rpm, room temperature).

[0314] An aliquot of chromatin was reverse cross-linked in 0.5× TBE buffer (supplemented with 0.5 mg / ml proteinase K (Qiagen)) at 65°C for 1 h and run on a 1% agarose gel to confirm target fragment sizes of 200–800 bp.

[0315] The remaining chromatin was split into ChIP and input samples. ChIP samples were diluted 10-fold in dilution buffer (16.7 mM Tris, pH 8.0, 1.0% Triton, 1.2 mM EDTA, 167 mM NaCl, 1x PIC in MiliQ) and 1 μg of ChIP grade antibody (Diagenode) was added. Samples were rotated overnight at 4 °C.

[0316] Magnetic protein A / G beads (Dynabeads) were washed twice with dilution buffer supplemented with 0.15% SDS and 0.1% BSA. The washed beads were added to the ChIP samples and rotated for 1 h at 4 °C. The bead-bound chromatin was subsequently washed as follows (rotation for 5 min, 4 °C): once with low-salt wash buffer (20 mM Tris, pH 8.0, 1.0% Triton, 0.1% SDS, 2 mM EDTA, 150 mM NaCl in MilliQ); twice with high-salt wash buffer (same as low-salt wash buffer but containing 500 mM NaCl); twice with no-salt wash 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 min at room temperature. Input samples were diluted 12-fold with elution buffer. After addition of NaCl (0.2 M) and proteinase K (0.1 mg / ml), all samples were reverse cross-linked in a shaking heat block (65 °C, 1000 rpm) for at least 4 h. DNA fragments were purified using Minelute PCR purification columns (Qiagen). DNA fragments were stored at 4 °C until downstream analysis by qPCR.

[0317] qPCR and analysis: qPCR analysis for ChIP samples and input was performed as follows. qPCR was performed with the primers detailed in Table 4 using the SYBR green method. The comparative Ct method was used to compare ChIP to input samples and calculate relative abundance to the negative control region. Untranslated regions of GAPDH and ZNF were used as negative and positive controls for H3K9me3, respectively. TNF was examined using six primer pairs for AUC analysis as previously described (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)). Table 4. Primers for ChIP-qPCR analysis [Table 4]

[0318] Bacterial expression and protein purification: ClearColi BL21(DE3) (Lucigen) was transformed with the pET20b(+)ApoA1-IL4 expression vector. The transformed bacteria were inoculated into 40 mL of lysogeny broth (LB) (Sigma-Aldrich) supplemented with 100 μg / L ampicillin and grown overnight at 37 °C. The overnight culture was then inoculated into 2YT medium (16 g / L peptone, 10 g / L yeast extract and 10 g / L NaCl) supplemented with 100 μg / L ampicillin and grown at 37 °C. When the absorbance at 600 nm reached >1.5, pET20b(+)ApoA1-IL4 expression was induced by adding 1.0 mM isopropyl β-d-thiogalactopyranoside (IPTG) and the cells were incubated overnight at 20 °C. Cells were harvested by centrifugation prior to lysate preparation and purification.

[0319] Bacterial lysis and protein purification:ApoA1-IL4 fusion protein expressing ClearColi cells were harvested by centrifugation at 8000 rpm and 4 ° C for 10 min. The harvested cells were resuspended in PBS and centrifuged at 4000 rpm and 4 ° C for 15 min. The cells were lysed with 20 mL BugBuster® Protein Extraction Reagent (Merck) and 20 μL Benzonase® Nuclease (Merck) per liter of culture on a shaker for 30 min at room temperature. The cell lysate was centrifuged at 18000 rpm and 4 ° C for 30 min. The insoluble pellet was washed with 10 mL BugBuster per liter and centrifuged at 18000 rpm and 4 ° C for 20 min. The pellet containing the 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 room temperature. The suspension was centrifuged at 18000 rpm and 4 °C for 30 min to remove the insoluble fraction. The filtered soluble fraction was loaded onto a nickel column and washed with 15 column volumes of IMAC wash buffer. ApoA1-IL4 was refolded on the nickel column at 2.5 mL / min using a 60 mL linear gradient unfolding (7 mM urea, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 50 mM potassium phosphate, and 100 mM NaCl, pH 6.8) to 60 mL of refolding (1 mM reduced glutathione, 0.1 mM oxidized glutathione, 50 mM potassium phosphate, and 100 mM NaCl, pH 6.8). The refolded apoA1-IL4 was eluted from the column with 0.5 M imidazole, 20 mM Tris, 0.5 M NaCl, pH 7.9. The eluate was collected, concentrated, further purified, and buffer exchanged by size-exclusion chromatography (HiLoad 16 / 600 Superdex 75 Increase; GE Healthcare) equilibrated with PBS storage buffer. Fractions were analyzed by SDS-PAGE, pooled, concentrated, flash frozen in liquid nitrogen, and then stored at -80°C.The mass of ApoA1-IL4 was confirmed by Q-ToF LC-MS (WatersMassLynx v4.1) using MagTran V1.03 for MS.

[0320] Mammalian expression and purification of apoA1-IL4m:HEK293T cells were co-transfected with fuGENE (Promega) containing transfer vector pHR-apoA1-IL4m, packaging pCMVR8.74, and enveloped pMD2.G in Opti-MEM (GIBCO) for 24 h at 37 °C. Cells were washed with DMEM + 2% heat-inactivated FBS and incubated for 48 h. To obtain lentivirus containing pHR-apoA1-IL4m, the supernatant was centrifuged at 1000 rpm to remove cell debris filtered through a 0.45 μm PES syringe filter and centrifuged at 50,000 g for 2 h at 4 °C. The pellet containing lentivirus of pHR-apoA1-IL4m was resuspended in medium, flash 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 h. The cells were then cultured in expression medium (50% EX-CELL® 293 Serum-Free Medium (Merck) and 50% FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific) for HEK293 cells supplemented with Glutamax, 1% Pen-Strep and 1 μg / mL doxycycline (Merck) for 3 days at 37°C on a shaker at 150 rpm. The culture supernatant containing apoA1-IL4m was centrifuged at 4000 rpm at 4°C for 15 min and filtered through a 0.22 μm PES syringe filter to remove cell debris. The filtered soluble fraction was loaded onto a StrepTactin XT 4flow 5 mL column (Cytiva) and eluted with 5 column volumes of W-buffer (150 mM NaCl, 100 mM Tris, 1 mM The column was washed with 100 mM EDTA (pH 8) at a flow rate of 1–2 mL / min. ApoA1-IL4m was eluted from the column with W buffer supplemented with 50 mM biotin. The eluate was collected, concentrated, flash frozen in liquid nitrogen, and then stored at -80 °C. The mass of ApoA1-IL4m was confirmed by Q-ToF LC-MS (Waters MassLynx v4.1) using MagTran V1.03 for MS.

[0321] SDS-PAGE and Western Blot: To confirm the fusion of apoA1 with IL4, 100 ng of IL4 (BioLegend), apoA1 and apoA1-IL4 were loaded onto a 4–20% polyacrylamide gel (Bio-Rad). After gel electrophoresis, samples were transferred to a nitrocellulose membrane containing blotting buffer (10× TG buffer, 20% methanol). The membrane was then incubated overnight at 4 °C with blocking buffer (5% milk in PBS, 0.1% Tween (PBST)). The blot was 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 h 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 an Image Quant gel imager (GE Healthcare).

[0322] 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). A log2 dilution concentration series of apoA1-IL4 ranging from 200 nM to 6.25 nM and human IL4 ranging from 20 nM to 0.65 nM. All samples were prepared in HPS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v / v) P20, pH 7.4). Association was monitored for 180 s and dissociation for 180 s at a flow rate of 30 μL / min. The sensor chip was regenerated with glycine 1.5 (10 mM glycine-HCl, pH 1.5, GE Healthcare). Kinetics were determined by fitting the interaction SPR data for 1:1 binding.

[0323] Human Embryonic Kidney 293 IL4 Reporter Cell Assay: HEK-Blue™ IL4 / IL13 cells were purchased from InvivoGen. This cell line has a fully active STAT6 pathway and carries a STAT6-inducible SEAP reporter gene. HEK-Blue™ IL4 / IL13 cells produce SEAP in response to IL4 and IL13. The level of secreted SEAP can be determined using QUANTI-Blue™ (Invivogen). 5×10 4 180 μL of DMEM with 10% FBS and 1% Pen-Strep containing 10 cells was added per well to a 96-well plate. Then, 20 μL of stimuli or vehicle were added and cells were incubated at 37 °C for 20-24 h. Subsequently, 180 μL of QUANTI-Blue per well was added to another 96-well plate (flat bottom) followed by 20 μL of cell supernatant. Plates were incubated at 37 °C for 1-3 h and SEAP levels were determined by measuring absorbance at 640 nM on a Tecan Spark plate reader.

[0324] Compounded nanoparticles:All phospholipids were purchased from Avanti Polar Lipids Inc. Four different apoA1-based nanoparticles (aNPs) were formulated. For discoidal aNPs, DMPC (133.5 μL), cholesterol (Sigma-Aldrich) (7.5 μL) from a stock solution in chloroform (10 mg / mL) and for spherical aNPs, POPC (66.5 μL), PHPC (17.5 μL), cholesterol (4.5 μL) and tricaprylin (Sigma-Aldrich) (2.79 μL from a 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%, total volume 800 μL). For the apoA1-based formulation, cholesterol (15 μL) was used. Separately, solutions 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) were prepared. Both solutions were simultaneously pumped into a Zeonor Herringbone Mixer (Microfluidic Chipshop, product code: 10000076) using a Microfluidic Pump Fusion 100 (Chemyx Inc) at a flow rate of 0.75 mL / min for the lipid solution and 6 mL / min for the apoA1 solution. The resulting solution was concentrated by centrifugal filtration at 4000 rpm using either a MWCO of 10 kDa for the disk-shaped and a MWCO of 100 kDa for the Vivaspin spherical aNPs 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 μm PES syringe filter to obtain the finished aNPs. Protein concentrations in the aNP samples were quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). For 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 membrane.

[0325] Determination of aNP size and dispersity by DLS: The resulting aNP formulations in PBS were filtered through a 0.22 μm PES syringe filter and analyzed by dynamic light scattering on a Malvern Zetasizer Nano ZS analyzer. Values ​​are reported as mean number average size distribution.

[0326] Radiolabeled aNP: IL4, apoA1-IL4 and IL4-aNP were incubated with a 2 molar excess of DFO-p-NCS (5 mg / mL in DMSO) for 2 hours and washed three times with 10 kDa MWCO Vivaspin tubes to remove any unreacted DFO-p-NCS. For radiolabeling, the DFO-bound proteins and aNP were incubated for 1 hour at 600 rpm using a thermomixer. 89 Incubate at 37 °C with Zr and wash three times using 10 kDa MWCO Vivaspin tubes to remove any unreacted 89 Zr was removed.

[0327] Cryo-transmission electron microscopy (cryo-TEM) of IL4-aNPso: First, the surface of a 200 mesh lacey carbon supported copper grid (Electron Microscopy Sciences) was plasma treated for 40 seconds using a Cressington 208 carbon coater. Subsequently, 3 mL of IL4-aNP sample (approximately 1 mg protein / ml) was applied onto the grid and vitrified into a thin film by plunge vitrification in liquid ethane using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was performed on a cryoTITAN (Thermo Fisher Scientific) equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002) and a post-GIF 2k × 2k Gatan CCD camera (model 794). Images were captured at 6,500x magnification (1.64 electrons / A). 2 s dose rate) or 24,000 times (11.8 electrons / A 2 The images were acquired in bright-field TEM mode with zero-loss energy filtering (dose rate of 100 s), an accelerating voltage of 300 kV, and an acquisition time of 1 s.

[0328] Super-resolution fluorescence microscopy of the interaction of IL4-aNP with the IL4 receptor in human monocytes: Human monocytes were isolated from peripheral blood of healthy donors as described above. 100,000 monocytes were seeded per well on cell culture-treated chambered coverslips (µ-slide 8 well, IBID). After 2 h of incubation at 37 °C (cell attachment), cells were incubated with Cy5-labeled variants of either naked apoA1 or apoA1-IL4, discoidal aNPs or IL4-aNPs, or spherical aNPs or IL4-aNPs for 2 h at 37 °C. Cells were then washed with PBS and fixed with 4% PFA for 20 min. IL4 receptor was stained with polyclonal rabbit IgG1 anti-human IL4R (Thermo Fisher Scientific; 1:100 dilution) primary antibody for 24 h at 4 °C, followed by goat anti-rabbit Alexa Fluor 488-conjugated secondary antibody (Thermo Fischer Scientific; 1:500 dilution) for 1 h at room temperature. Stained cells were stored in PBS at 4 °C. For direct stochastic optical reconstruction microscopy (dSTORM), cells were immersed in GLOXY imaging buffer (40 μg / 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 imaging. Acquisition was performed in total internal reflection fluorescence (TIRF) mode using an ONI Nanoimager (ONI, Oxford, UK), equipped with a 100× / 1.4NA oil immersion objective, an sCMOS camera, and in this study, 488 nm (200 mW) and 640 nm (1000 mW) lasers were used. 10,000 frames were acquired with a 10 ms exposure time in a 50 × 80 μm field of view. Raw data were processed using the ThunderSTORM software to obtain images with a spatial resolution of 10 nM.

[0329] Animal models:Female C57BL / 6 mice were purchased from The Jackson Laboratory. For non-human primate studies, two male cynomolgus monkeys (Macaca fascicularis) were used. All animals were co-housed in climate-controlled conditions with a 12-h light / dark cycle and provided with water ad libitum. Mice were fed a standard chow diet and non-human primates were fed a Teklad Global 20% Protein Primate Diet. Animal care and experimental procedures were based on an approved institutional protocol from the Icahn School of Medicine at Mount Sinai. All mice were randomly assigned to experimental groups.

[0330] Pharmacokinetics and biodistribution in mice and non-human primates: C57BL / 6 mice, respectively. 89 Zr-labeled IL4 variants, IL4 (53.6 ± 6.6 μCi), apoA1-IL4 (30.1 ± 0.9 μCi), discoidal IL4-aNPs (146.1 ± 46.5 μCi) and spherical IL4-aNPs (108.6 ± 16.9 μCi) were intravenously injected into two non-human primates. 89 Zr-labeled IL4-aNPs (1079 μCi and 682 μCi) were injected. At the designated time points, 1, 2, 5, 10, 30 min, and 1, 2, 4, 8, 24 h after injection in mice, and 5, 30, 90 min, and 48 h after injection in non-human primates, blood was collected, weighed, and analyzed by Wizard. 2 Radioactivity was measured using a 2480 automated gamma counter (Perkin Elmer, Waltham, MA). Data were corrected for radioactive decay and the percentage of injected dose per gram of blood (%ID / g) was calculated. Data were fitted using nonlinear biphasic decay regression in GraphPad Prism, using the formula (%rate x t 1 / 2 Fast+%Slow 1 / 2 Weighted blood half-life was calculated via the formula: 1 / 100. Biodistribution was measured in mice 24 hours after injection. After PBS perfusion, tissues of interest were harvested, weighed, and analyzed using the Wizard. 2Radioactivity was measured using a 2480 automated gamma counter (Perkin Elmer, Waltham, Mass.). Data were corrected for radioactive decay and the percentage of injected dose per gram of tissue (%ID / g) was calculated.

[0331] PET / CT imaging of aNP biodistribution in mice: C57BL / 6 mice, respectively. 89 Zr-labeled IL4 variants, IL4 (53.6 ± 6.6 μCi), apoA1-IL4 (30.1 ± 0.9 μCi), discoidal IL4-aNPs (146.1 ± 46.5 μCi) and spherical IL4-aNPs (108.6 ± 16.9 μCi) were injected intravenously. After 24 h, mice were anesthetized using 1.0% isoflurane in O2 at a flow rate of approximately 1.0 liters / min. PET / CT scans were acquired using a Mediso nanoScan PET / CT (Mediso, Budapest, Hungary). A whole-body CT scan was performed (energy, 50 kVp; current, 180 μAs; isotropic voxel size, 0.25 mm), followed by a 20-min PET scan. Reconstructions were performed with attenuation correction using the TeraTomo 3D reconstruction algorithm from Mediso Nucline software. This match was excluded by an energy window between 400 and 600 keV. The voxel size was 0.4 mm wide and isotropic, and the reconstruction was applied to four complete iterations, six subsets per iteration.

[0332] Autoradiography: To determine the radioactivity distribution, tissues were placed in film cassettes against phosphorimaging plates (BASMS-2325, Fujifilm) at −20° C. Plates were read using a Typhoon 7000IP plate reader (GE Healthcare) at a pixel resolution of 25 mm.

[0333] Cell-specific flow cytometry:For cell specificity, mice were intravenously injected with DiO-labeled IL4-aNPs, which were allowed to circulate for 24 hours. Afterwards, mice were sacrificed and single cell suspensions were made 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 a viability stain. Cells were then washed and resuspended in FACS buffer. All data were acquired on an Aurora 5L flow cytometer (Cytek Biosciences). Diol-IL4-aNPs were detected in the FITC channel.

[0334] PET / MRINon-human primate biodistribution: After an overnight fast, non-human primates were anesthetized using ketamine (5 mg / kg) and dexmedetomidine (0.0075–0.015 mg / kg). Non-human primates were administered 1.114 mCi and 0.682 mCi discoids at a dose of approximately 0.1 mg / kg. 89 Zr-labeled IL4-aNPs were injected. Dynamic PET imaging was performed for 60 min after injection, and additional static PET / MRI scans were performed at 1 h and 48 h after injection. In addition, blood was drawn during imaging at 5, 30, and 120 min after injection. PET and MRI images were acquired using a 3T PE / MRI system (Biograph mMR, Siemens Healthineer). Dynamic PET imaging was performed using one couch position covering the chest and abdomen, starting simultaneously with the injection of aNPs. 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; 0.5 × 0.5 × 1.0 mm 3 and acquisition duration, 42 min and 42 s. After the dynamic PET image acquisition, static whole-body PET images were acquired from skull to pelvis using four consecutive couch positions of 15 min each. Simultaneous signal averaging per couch, number of slices 160, and spatial resolution 0.6 × 0.6 × 1.0 mm 3MR images were acquired as above, except using only 1000 x 1000 mm (acquisition duration, 14 min 56 s per couch). Whole-body PET and MR imaging were also performed using the four PET couch positions for 30 min each, with the following MR parameters: acquisition plane, coronal; repetition time, 1,000 ms; echo time, 79 ms; number of slices, 224; number of averages, 2; spatial resolution, 0.6 × 0.6 × 1.0 mm. 3 acquisition durations of 29 min and 56 s were used, performed 48 h after injection. Whole-body MR images from each couch were automatically matched together by the scanner. After acquisition, the raw PET data from each couch were reconstructed and matched together offline using Siemens' proprietary e7 tool with an ordered subset expectation maximization (OSEM) algorithm with point spread function (PSF) correction for three iterations and 24 subsets. A 4 mm Gaussian filter was also applied to the images. Three-compartment (soft tissue, lung, and air) attenuation maps were used for attenuation.

[0335] Imaging-based analysis of 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 the coronal plane. Regions of interest (ROIs) were drawn over tissues of interest, including spleen, liver, kidneys, lungs, heart, cerebellum, and cerebrum, and traced throughout them to determine bone marrow uptake using three vertebrae of the lumbar spine. For each ROI, the mean standardized uptake value (SUV) was calculated. Discoids per organ 89 Zr-labeled IL4-aNP uptake was expressed as the average of all mean SUV values ​​per organ.

[0336] In vivo tolerance model:For the in vivo tolerance model, 11-week-old female C57BL / 6 mice were intraperitoneally tolerized with 0.1 mg / kg body weight LPS. At 24 and 48 h, mice were treated intravenously with either 200 μg IL4m-aNP or PBS. Mice were then re-exposed at 72 h with intraperitoneal 0.1 mg / kg LPS injection. After 90 min, mice were sacrificed, blood was collected for ELISA, and single cell suspensions were made from blood, spleen, and bone marrow. Staining protocol. Blood samples for ELISA were allowed to clot for 30 min at room temperature. Serum was collected after centrifugation at 1000 × g for 10 min at 4 °C. Mouse TNF and IL6 ELISA (Biolegend) were performed according to the manufacturer's protocol. Animal care and experimental procedures were based on the institutional protocols approved by the Nijmegen Animal Experiments Committee.

[0337] Statistical analysis: Data are presented as mean + / - SD unless otherwise stated. Individual data points in the graphs are biological replicates, not technical replicates. When the number of data points cannot be clearly discerned from the figures, n is indicated in the figure legend. Statistical analyses were performed with Graphpad Prism (V9, Graphpad Software) unless otherwise stated. For trained immunity and acute stimulation experiments with primary human monocytes, the (paired, non-parametric) Wilcoxon signed-rank test was used. Statistical methods for RNA sequencing analysis are described above. Two-tailed P values ​​less than 0.05 were considered statistically significant. Statistical significance in the figures is indicated as follows: *=P<0.05, **=P<0.01, ***=P<0.001, NS=P≥0.05.

[0338] Data and Code Availability: Data are available upon request to the Lead Contact. Raw RNA sequencing data have been deposited in the NCBI Gene Expression Omnibus under the accession number GSE185433.

[0339] result IL4 inhibits acute inflammation but induces trained immunity In the context of myeloid cell immunology, IL4 is primarily known for its anti-inflammatory properties. Therefore, we first verified some of the known inhibitory effects of IL4 on inflammation in primary human monocytes (Figure 17A). We 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-1Ra compared to controls (Figure 17B). Since glycolysis is upregulated in activated myeloid cells, we measured lactate production in otherwise unstimulated monocytes treated with IL4 or medium control. We found that IL4 slightly but significantly lowered baseline lactate production (data not shown), confirming its acute anti-inflammatory properties.

[0340] Based on these anti-inflammatory properties, we hypothesized that IL4 might also inhibit the induction of trained immunity (Figure 17C). To test this hypothesis, monocytes were trained with β-glucan, a prototypic trained immune stimulus, for 24 hours, followed by washing out the stimulus and a 5-day resting period in culture medium. On day 6, we restimulated the cells with LPS for another 24 hours and measured TNF and IL6 (Figure 17D). β-glucan induced trained immunity as expected, but the addition of IL4 for the first 24 hours did not inhibit the training effect (Figure 17D). Contrary to our initial hypothesis, exposure of monocytes to IL4 alone for 24 hours induced a trained immune phenotype on day 6 (Figure 17E). In addition to 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 C. albicans compared to untrained controls (data not shown). Collectively, our data demonstrate that IL4 inhibits inflammation and induces trained immunity at both metabolic and functional immunological levels.

[0341] Encouraged by these observations, we comprehensively studied the metabolic changes following IL4-induced trained immunization. To this end, we used Seahorse metabolic flux analysis to examine the glycolytic and oxidative metabolism of IL4-trained cells and non-stimulated controls. IL4 training on day 0 had a significant effect on metabolic parameters measured on day 6 (Figure 17F), with a trend toward higher basal glycolysis and a significant increase in oligomycin-induced maximum glycolytic capacity (Figure 17F left panel). Moreover, both baseline and FCCP-induced maximum respiratory rates were significantly increased by IL4 training (Figure 17F right panel).

[0342] We 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 following IL4 activation on day 0. CD80 was slightly increased by IL4 training, but overall expression remained low in these otherwise naive macrophages. Monocyte-derived dendritic cells (moDCs differentiated with IL4+GM-CSF) are also known to downregulate CD14 while strongly upregulating CD1c. IL4-trained cells expressed slightly more CD1c than untrained cells, but much less than moDCs (data not shown). These results indicate that IL4 induces a program of trained immunity that incorporates features known from classical IL4 immunological functions.

[0343] Immune and epigenetic mechanisms mediating IL4-induced trained immunity The signaling mechanism of IL4: IRS-2 / PI3K / mTOR axis and STAT6 signaling pathway are well described (Figure 18A). We performed pharmacological inhibition experiments to examine the role of these pathways for both the inhibition of acute inflammation and trained immune induction by IL4. Inhibition of PI3K or mTOR (using wortmannin or torin-1, respectively) did not abolish the effect of IL4 on acute inflammation, but reduced trained immune responses (Figure 18B, Figure 18C, data not shown). IL4 training, as measured by increased TNF and IL6 production, was significantly blunted in the presence of torin-1 (Figure 18C, data not shown). In contrast, the STAT6 inhibitor AS1517499 partially restored cytokine production in acute inflammatory responses but did not affect trained immune induction by IL4 (Figure 18B and Figure 18C). Thus, each signaling pathway induced downstream of the binding of IL4 to its receptor has a distinct function: IL4 exerts its known acute anti-inflammatory function via STAT6, but at the same time induces trained immunity via PI3K / mTOR (a previously unknown pro-inflammatory effect).

[0344] To gain insight into the molecular program induced by IL4 training, we performed transcriptomics analysis on naive and IL4-trained macrophages, both before and after LPS restimulation on day 6. Overall, 140 genes were more strongly induced ("upregulated") in IL4-trained macrophages, whereas 249 genes were attenuated (Figure 18D). Notably, upregulated genes were proinflammatory cytokines such as IL6 and IL12B, known to be involved in trained immunity. Among the notably attenuated genes were CCL19 and SOCS2, which are important for lymphocyte trafficking and suppression of cytokine signaling, respectively.

[0345] We next performed transcription factor (TF) motif enrichment analysis (Figure 18E) and gene ontology / pathway enrichment analysis (Figure 18F) to gain further insight into the transcriptome profile. Promoters of genes upregulated in IL4-trained macrophages were highly enriched for motifs recognized by TFs such as ATF2 / ATF7, PPARα and STAT5, whereas interferon regulatory factor (IRF) motifs were particularly depleted. This pattern was almost the reverse for non-affected and attenuated genes, with the exception of TATA-box, NFκB-p65, NFκB-p65-Rel and FRA2: these motifs were highly enriched in attenuated gene promoters but reduced in both non-affected and upregulated genes (Figure 18E). Gene ontology (Biological Process; BP and Molecular Function; MF) and KEGG pathway enrichment showed that immunological activity was 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). We performed similar transcriptome analysis on monocytes stimulated immediately after isolation with IL4, LPS, or a combination of IL4 and LPS and confirmed the acute anti-inflammatory transcriptome response to IL4 (data not shown). Taken together, these data reveal specific transcriptional programs in both the acute anti-inflammatory effect and the long-term trained immune response triggered by IL4.

[0346] We then investigated the importance and presence of epigenetic reprogramming, especially histone 1 modifications. Addition of the antiallergic drug cyproheptadine, a histone 2 methyltransferase inhibitor SET7 (also known as SET9), abolished the induction of trained immunity by IL4 (Figure 18G). SET7 has been previously described as a key epigenetic mediator of trained immunity. Furthermore, we evaluated H3K9me3-mediated suppression of TNF in IL4-induced trained immunity using chromatin immunoprecipitation (ChIP)-qPCR analysis. Using area under the curve (AUC) analysis of primer pairs, we showed a 6-fold decrease in H3K9me3 in IL4-induced trained immunity, but this did not reach statistical significance (Figure 18H, data not shown). Taken together, these data indicate that epigenetic reprogramming is important for and a hallmark of IL4-induced trained immunity.

[0347] Development of apoA1-IL4 fusion protein incorporated into lipid nanoparticles Despite its unique ability to inhibit acute inflammation while simultaneously inducing trained immunity, the clinical translation of recombinant IL4 is hindered by its unfavorable pharmacokinetic properties. To overcome this limitation, we developed an apoA1-based fusion protein that is easily incorporated into lipid nanoparticles to yield IL4-containing nanoparticles (IL4-aNPs). ApoA1-based nanoparticles (aNPs) essentially accumulate in hematopoietic organs and efficiently target myeloid cells and their progenitors (Schrijver, DP et al. in Advanced Therapeutics Vol. 4 2100083-2100083 (John Wiley & Sons, Ltd, 2021; van Leent, MMT 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, we 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). We used molecular characterization techniques to confirm the nature and purity of apoA1-IL4. Each purified protein sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA) and ELISA. By performing GE, we confirmed the presence of proteins with molecular weights of 25 kDa (apoA1), 18 kDa (IL4) and 37 kDa (apoA1-IL4) (Figure 19C), while Western blot showed the presence of apoA1 and IL4 (Figure 19D). Due to its amphipathic properties, the protein consisting of apoA1 migrates lower on the gel than expected. These observations were supported by quadrupole time-of-flight (Q-TOF) mass spectrometry, which showed a single mass peak of 47576.03 Da, corresponding to the expected molecular weight of 47582.57 Da for apoA1 (Figure 19E).

[0348] Prior to incorporating apoA1-IL4 into lipid nanoparticles, biophysical cell analysis using surface plasmon resonance (SPR) and HEK-Blue™ IL4 / IL13 (HEK-IL4) reporter cells was performed to determine the preservation of biological activity after the purification and refolding process. We determined that the equilibrium dissociation constant Kd of apoA1-IL4 to human IL4 receptor alpha (IL4Rα) using SPR was 4.5 nM ± 1.1 nM (Figure 19F). HEK-IL4 cells carry an IL4Rα / STAT6 inducible reporter gene encoding secreted alkaline phosphatase (SEAP), which was significantly produced upon introduction of apoA1-IL4 into their culture wells. This indicates the biological activity of apoA1-IL4 (Figure 19G). Fusion with apoA1 significantly altered the biophysical properties of IL4, allowing its incorporation into lipid nanoparticles, while binding to its receptor was preserved with a kd of 0.28 ± 0.1 nM (data not shown). In summary, we have developed an apoA1-IL4 fusion protein that preserves the biological activity of IL4 after extraction, purification, and refolding, and contains the desired physicochemical characteristics for apoA1-mediated integration into lipid nanoparticles.

[0349] Incorporation of apoA1-IL4 into lipid nanoparticles To improve the pharmacokinetic properties of IL4 and its bioavailability to myeloid cells, we incorporated apoA1-IL4 fusion protein into lipid nanoparticles to obtain IL4-aNPs. By varying the composition of the nanomaterial, we obtained nanoparticles of different sizes and morphologies (Figure 20A). The successful formulation of discoidal and spherical nanoparticles was confirmed by cryo-transmission electron microscopy (cryo-TEM) (Figure 20B). We further analyzed the nanoparticle size and stability in PBS for 14 days using dynamic light scattering (DLS) (Figures 20C and 20D). IL4-aNPs remained stable for 14 days and have similar size and stability compared to our conventional aNPs (data not shown). Conventional aNPs contain apolipoproteins as described in Schrijver, DP et al. in Advanced Therapeutics Vol.4 2100083-2100083).

[0350] Next, we investigated the interaction of IL4-aNP with primary human monocytes. Direct stochastic optical reconstruction microscopy (dSTORM) analysis revealed the expression of IL4Rα on the membrane. Furthermore, the binding of naked apoA1, naked apoA1-IL4, and IL4-aNP was confirmed by a rim covering the cell surface. Focusing on the enlarged cross-section of the membrane, we found that naked apoA1-IL4 and IL4-aNP associated with IL4Rα and formed co-clusters enriched on the cell surface, which we did not observe for naked apoA1 and conventional aNP (Figure 20E). Taken together, our DLS size stability assay, cryoTEM and dSTORM analysis revealed that the incorporation of apoA1-IL4 fusion protein into lipid nanoparticles yielded biologically functional IL4-aNP.

[0351] Study of IL4, apoA1-IL4 and IL4-aNP formulations in mice To investigate the pharmacokinetics and biodistribution in C57BL / 6 mice, we administered four different IL4 therapeutics: recombinant IL4; naked apoA1-IL4 fusion protein; and zirconium-89 (89 We radioactively labeled the protein components of discoidal and spherical IL4-aNPs with ZnO (ZnS). Note that these experiments were performed with a human variant of IL4 that does not show biological activity in mice. Positron emission tomography / computed tomography (PET / CT) imaging 24 hours after intravenous administration demonstrated that 89 Zr-IL4 and 89 We showed that Zr-apoA1-IL4 accumulated primarily in the kidney and liver. In contrast, in addition to accumulating in the liver and kidney, 89 Zr-IL4-aNP accumulated relatively large amounts in immune cell-rich organs including spleen and bone marrow (Figure 27A). We performed ex vivo gamma counting to determine the blood half-life and uptake in major organs of these nanomaterials (Figure 27B and Figure 27C), which we corroborated with autoradiography (data not shown). Comparison of uptake ratios by target organs (bone marrow + spleen) / clearance organs (kidney + liver) showed a significant increase in uptake ratios of IL4-aNP formulations compared to unformulated fusion protein and naked IL4 (data not shown). We then used flow cytometry to measure cell type-specific biodistribution in target organs. Dio-labeled discoidal IL4-aNP accumulated in myeloid cells, most notably monocytes and neutrophils, in both spleen and bone marrow, but did not interact (or only slightly) with lymphocytes (Figure 27D). Based on its favorable (and bone marrow-specific) uptake in hematopoietic organs, we selected the discoidal IL4-aNP formulation for further studies in non-human primates and translational models of inflammation and sepsis.

[0352] IL4-aNP immunotherapy shows favorable uptake profile in non-human primates To evaluate the clinical translation potential of IL4-aNP immunotherapy, we determined their biodistribution and safety in non-human primates. 89Zr-IL4-aNPs were injected intravenously. Their in vivo behavior was studied in vivo using fully integrated three-dimensional PET combined with magnetic resonance imaging (PET / MRI). After injection, dynamic PET / MRI (data not shown) demonstrated rapid accumulation of IL4-aNPs in the liver, kidney (data not shown), spleen and bone marrow (Figure 27E-H). In accordance with our mouse data, no unwanted uptake of IL4-aNPs was observed in non-target organs, including the brain and heart (data not shown). Taken together, these results demonstrating the favorable biodistribution and safety profile of IL4-aNPs were retained across species, supporting the translational potential of this immunotherapy.

[0353] IL4-aNP therapy reverses immunological disorders in vitro and in vivo After establishing that native IL4 simultaneously attenuates acute inflammatory responses and induces a program of trained immunity, we evaluated the effect of IL4-aNP on monocytes in vitro (Figure 21A). We based the dose for in vitro experiments on the efficiency of phospho-STAT6 induction in primary human monocytes compared with naked IL4 (data not shown). Indeed, IL4-aNP (molar equivalent of 200 ng / mL naked IL4) significantly reduced TNF and IL6 production of LPS-stimulated monocytes (Figure 21B), while enhancing the long-term responsiveness of monocytes on day 6 (Figure 21C). These data indicate that IL4-aNP, like IL4, suppresses acute inflammation and induces trained immunity in vitro. Although our in vivo biodistribution data show that IL4-aNP specifically targets myeloid cells (Figure 27D), IL4-induced trained immunity alters surface marker expression of macrophages, which are antigen-presenting cells (data not shown). This may in time affect polarization signals during T cell activation. To examine these potential indirect effects of IL4-aNP on T cells, allogeneic naive T cells were cultured in the presence of IL4-trained macrophages. In this model, HLA mismatch causes antigen-nonspecific T cell activation and polarization. No significant differences were observed in the abundance of T cell subtypes, Th1 (CD4+IFNγHigh+), Th2 (CD4+IL4+), Treg (CD4+IL10+), Th17 (CD4+IL17+) and cytotoxic T cells (CD8+Granzyme B+Perforin+) between trained macrophages and controls (data not shown). Taken together, these findings demonstrate no ability of IL4 training to indirectly skew T-cell responses, suggesting a primarily myeloid-specific effect.

[0354] Sepsis patients may experience both an exaggerated inflammatory response and immune paralysis, creating a therapeutic paradox. Induction of trained immunity could theoretically be used to reverse immune tolerance, but this has not been translated to an in vivo model. One reason is that human IL4 does not show biological activity in mice. Therefore, we designed and produced a chimeric fusion protein consisting of human apoA1 and mouse IL4 for formulation with lipids to obtain IL4m-aNPs. Here, we investigated whether IL4m-aNPs could reverse LPS-induced tolerance in mice. To that end, we injected C57B / 6 mice intraperitoneally with LPS (0.1 mg / kg) to induce immune paralysis or PBS (as a control). We administered IL4m-aNPs (200 μg / dose) intravenously 24 and 48 hours after LPS treatment. We re-exposed the mice to another intraperitoneal injection of LPS (0.1 mg / kg) 72 hours after the first exposure (Figure 21D). Indeed, treatment with IL4m-aNP improved the innate immune response, as shown by a significant (p=0.0079) increase in serum IL6 concentration after LPS re-exposure in tolerized mice (Figure 21E). Although TNF levels in some mice were clearly elevated, statistical significance was not achieved (p=0.1508) due to the heterogeneity of the treatment response (Figure 21E). Taken together, our in vitro and in vivo data demonstrate that IL4-aNP can reduce tolerance.

[0355] Human endotoxemia model After we observed tolerance reversal in that mouse model, we substantiated these results using a model that more closely mimics human clinical immunodialysis. We obtained blood from healthy individuals undergoing experimental human endotoxemia, a standardized controlled model of systemic inflammation that captures features of both the hyperinflammatory and immunoparalytic phenotypes of sepsis (Figure 21F). In this controlled human model, LPS was administered intravenously to healthy volunteers, resulting in a systemic inflammatory response and subsequent tolerization of circulating monocytes, a phenomenon also observed in sepsis-induced immunoparalysis. Blood was collected before and 4 hours after the start of LPS administration. Monocytes isolated after LPS administration showed insufficient cytokine production upon immediate re-exposure to LPS, indicating tolerization (data not shown). Tolerant monocytes from LPS-exposed volunteers, when exposed ex vivo to either IL4 or IL4-aNP for 24 hours, showed significant improvement in TNF but not IL6 production when restimulated with LPS on day 3 (Figure 21G (concentration), 21H (fold change), data not shown). In contrast, untreated monocytes remained completely resistant. Taken together, these data highlight the ability of IL4 and IL4-aNP to at least partially reverse LPS tolerance ex vivo.

[0356] Consideration Although IL4 is generally considered to be an anti-inflammatory cytokine, its long-term effects on monocyte / macrophage function are unclear. We initially expected that IL4 would inhibit trained immunity, similar to IL37 and IL38. Surprisingly, we observed that IL4 induces trained immunity, as assessed by increased cytokine production responsiveness, in addition to its known inhibitory effects on acute inflammation. Although the induction of trained immunity by IL4 was unexpected, our observations are consistent with IL4's activation of the mTOR5 signaling cascade, a central mechanism in trained immunity. We then investigated the long-term effects of pre-IL4 exposure, as well as the ability of IL4 to reverse immune tolerance induced by experimental endotoxemia. In this context, the results demonstrate that an anti-inflammatory STAT6-dependent cellular program predominates during acute exposure of cells to IL4, but that this transitions over time to an mTOR-driven program of long-term trained immunity. IL4 training fits all the parameters typically used to describe trained immunity, including enhanced cytokine production, epigenetic rewiring, increased metabolic activity, and altered transcriptomic responses upon restimulation. These findings are consistent with growing evidence for dynamic and timing-dependent models of monocyte differentiation, such as the one proposed in 2017 by Sander et al. (Sander, J. et al. Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent 7 Interleukin-4 Signaling and the Transcriptional Regulator NCOR2. Immunity 47, 1051-1066 e1012, 8 doi:10.1016 / j.immuni.2017.11.024(2017))

[0357] The unique ability of IL4 to simultaneously suppress acute inflammation while inducing a trained immune program that has been reported to improve host defense can potentially be used to treat severe infectious diseases. For example, both sepsis and COVID-19 are characterized by dysregulated immune responses, creating a therapeutic paradox that requires both management of an excessive inflammatory response to (opportunistic) secondary infections and improvement of host defense responses. To exploit the unique features of IL4, we developed a nanoparticle protein engineering strategy, thereby overcoming the unfavorable in vivo pharmacokinetic properties of this cytokine. We demonstrated that our aNP strategy favorably altered the blood half-life and biodistribution profile of IL4, leading to elevated neutrophil- and monocyte-specific accumulation in organs rich in myeloid lineage cells, such as bone marrow and spleen, as previously observed in models of trained immunity. While biodistribution was studied using human IL4-aNP, we further developed the murine mutant IL4m-aNP to evaluate in vivo efficacy. Indeed, we demonstrated the immune isolation-reversing effect of IL4 nanoparticles in an LPS-induced cytokine storm mouse model. Although the data demonstrate that the innate immune response was restored with a significant increase in IL6 levels and a clear trend towards increased TNF concentrations in the serum of mice treated with IL4m-aNPs, full-scale dose-ranging studies are needed to expand the potential of IL4 nanoparticle therapy in various immune-mediated diseases characterized by concurrent hyperinflammation and immune paralysis. Encouragingly, using a human fusion protein, we showed that IL4-aNPs can reverse immune tolerance of cells obtained from a human model that mimics clinical immune paralysis.

[0358] We anticipate that their cytokine nanoparticle platform may be used in immune paralysis following sepsis-induced hyperinflammation, as well as other myeloid-directed applications. Since cancer is also characterized by local tumor-promoting inflammation and simultaneous suppression of antitumor responses (often mediated by myeloid cells), immuno-oncological applications may be considered. Myocardial infarction and stroke are also characterized by sterile inflammation followed by immune paralysis, and severe trauma patients suffer from a similar immune paralyzed state. In all these situations, reducing inflammation and overcoming immune paralysis may be beneficial for patient recovery and prevention of secondary infections. Our IL4-aNP platform has the potential to develop into a pivotal therapeutic modality for treating all these conditions.

[0359] Example 3. Fusion protein of an apolipoprotein fused to a rerouting molecule and its incorporation into lipid nanoparticles material and method Expression and purification of VHHCD8-apoA1 fusion protein:Small cultures of ClearColi cells transformed with the pET20b-VHHCD8-apoA1 and pDisconTune plasmids were started in LB medium containing 100 μg / mL ampicillin. The next day, 40 mL of the small culture was diluted into 1 liter of 2YT medium to start a large culture, and rhamnose was added to a final concentration of 50 μM to induce T7 lysozyme on the pDisconTune plasmid. The cultures were grown at 37 °C and 150 rpm to an OD600 of 0.6-0.8, and then isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce expression. The induced cultures were incubated overnight at 18 °C and 150 rpm. The induced bacterial cultures were pelleted and the cells were resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.9). Benzonase Nuclease (Merck Millipore) and one cOmplete™ EDTA-Free Protease Inhibitor Cocktail tablet (Roche) per 50 mL cell suspension were added and the cell suspension was incubated at 4 °C with agitation. The suspension was subsequently homogenized three times at 15000-20000 psi using an Avestin Emulsiflex C3. The cell lysate was kept on ice at all times. After lysis, the cell lysate was centrifuged to pellet insoluble cell debris and the supernatant was run on an Immobilized Metal Chelate Affinity Chromatography (IMAC) column containing immobilized nickel ions. The column was washed with 8 column volumes of Buffer A (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9) and then with 8 column volumes of Buffer A50 (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 7.9). To elute VHHCD8-apoA1, 8 column volumes of buffer A500 (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.9) were added to the column. All fractions from the purification step were collected and analyzed by SDS-PAGE.The buffer of the fractions containing purified VHHCD8-ApoA1 was changed to PBS using Amicon Ultracentrifugal Filters (Amicon). To preserve VHHCD8-apoA1, aliquots were flash frozen in liquid nitrogen and stored at -70°C.

[0360] The VHHCD8-apoA1 fusion protein has the sequence defined by SEQ ID NO:54 or is encoded by the sequence defined by SEQ ID NO:55, which includes a linker between apoA1 and VHHCD8 that contains a cysteine.

[0361] SDS-page: This is carried out as described in Example 2.

[0362] aNP formulations: The nanoparticles were prepared as described in Example 2 under the heading "Nanoparticle Formulation."

[0363] DLS: It was carried out as described in Example 2.

[0364] Cryo-TEM: It was carried out as described in Example 2.

[0365] In vitro binding of VHHCD8-apoA1 in mouse splenocytes: Spleens were obtained from mice, cut into small pieces, and pulled through a 70 μm strainer (Corning) multiple times to obtain a splenocyte suspension. Cells were spun down at 1500 rpm for 10 min, the supernatant was removed, and cells were lysed in 2 mL of 1× red blood cell lysis buffer (Thermofisher). The suspension was incubated at room temperature for 5 min, 10 mL of Roswell Park Memorial Institute (RPMI) medium (Thermofisher) was added, and cells were spun down again at 1500 rpm for 10 min. Cells were then redissolved in RPMI medium and seeded at 150,000 cells / well in 96-well plates.

[0366] Proteins were labeled by adding a 5-fold molar excess of sulfo-cyanine 5-maleimide (Lumiprobe) in dimethyl sulfoxide (DMSO). The mixture was stirred for 2 hours at room temperature. Excess dye was removed using PD minitrap G-25 desalting columns (Cytiva). Fluorescently labeled aNPs were formulated by adding 6.4 μg DiI for discoidal formulations and 21 μg DiI for spherical formulations (see the "Nanoparticle Formulation" section in Example 2). Fluorescently labeled fusion proteins or aNPs and controls were added to the wells and incubated at 4°C (for proteins) or 37°C (for aNPs) for 30 minutes, after which the cells were harvested, washed, stained for CD3 and CD4, and measured on a Cytoflex (Beckman Coulter Inc). FlowJo software (BD) was used to analyze flow cytometry data. result

[0367] The VHHCD8-apoA1 fusion protein was successfully expressed in Clearcoli cells. Minor protein contamination was present after IMAC purification [lane E1] (Figure 23). The most prominent band corresponds to the fusion protein with a molecular weight of 43.3 kDa (Figure 23). The correct mass was later confirmed by mass spectrometry (data not shown). Discoidal apolipoprotein nanoparticles were formulated incorporating VHHCD8-apoA1. Dynamic light scattering (DLS) was used to determine the size and polydispersity index (PDI) of the particles. The size of the particles remained stable for 7 days (Figure 24 (left panel)). At day 14, the size increased slightly (Figure 24 (left panel)). The PDI remained stable for 14 days (Figure 24 (left panel)). Cryo-TEM images of the nanoparticles showed the expected discoid shape (Figure 24 (right panel)).

[0368] VHHCD8-apoA1 and apoA1 were fluorescently labeled and subsequently added to mouse splenocytes. For the VHHCD8-apoA1 fusion protein, a dose-dependent increase in mean fluorescence intensity (MFI) was observed, indicating binding of the fusion protein to the CD8 receptor (Figure 25; lower panel). The apoA1 condition did not show this dose-dependent behavior and had a similar MFI to the control sample.

[0369] Disk-shaped and spherical aNPs were formulated with VHHCD8-apoA1 and apoA1. A fluorescent dye was incorporated into the lipid structure of the particles. Mouse splenocytes were incubated with the nanoparticles. Both VHHCD8-apoA1 particles showed a dose-dependent increase in MFI, indicating nanoparticle binding to the CD8 receptor (Figure 26). Disk-shaped nanoparticles showed a greater increase than spherical nanoparticles. No increase in MFI was observed in the apoA1 nanoparticle condition.

[0370] Sequence Listing This patent application is filed with a corresponding sequence listing, which is incorporated by reference in its entirety. Below is a summary of the sequences and their brief description. [Table 5-1] [Table 5-2] [Table 5-3]

Claims

1. 1. An apolipoprotein lipid nanoparticle, comprising: a fusion protein comprising an apolipoprotein or apolipoprotein mimetic and a rerouting molecule; Phospholipids and Including, The rerouting molecule is a molecule that enables the fusion protein to bind to a target different from that to which the apolipoprotein or apolipoprotein mimetic would have bound and / or to bind to its intended target with higher affinity, apolipoprotein lipid nanoparticles.

2. The fusion protein further comprises an immunomodulatory biomolecule, The apolipoprotein lipid nanoparticle of claim 1 , wherein the immunomodulatory biomolecule is a protein that enhances or suppresses an immune response.

3. The apolipoprotein lipid nanoparticle of claim 1 , wherein the apolipoprotein lipid nanoparticle further comprises a sterol.

4. The apolipoprotein lipid nanoparticle of claim 1 , wherein the apolipoprotein lipid nanoparticle further comprises a lipid, preferably a triglyceride.

5. The apolipoprotein lipid nanoparticle of claim 1 , wherein the apolipoprotein lipid nanoparticle is a sphere, ribbon, or disc.

6. The apolipoprotein lipid nanoparticle of claim 1, wherein at least a portion of the fusion protein is exposed to the environment surrounding the apolipoprotein lipid nanoparticle, and preferably the immunomodulatory biomolecule and / or the rerouting molecule is exposed to the environment surrounding the apolipoprotein lipid nanoparticle.

7. 3. The apolipoprotein lipid nanoparticle of claim 2, wherein the immunomodulatory biomolecule is selected from the group consisting of cytokines, chemokines, hormones, growth factors, hematopoietic growth factors, and combinations thereof.

8. The apolipoprotein lipid nanoparticle of claim 1, wherein the rerouting molecule is selected from an antibody or its antigen-binding fragment, a rerouting peptide or a rerouting protein, and preferably the rerouting peptide or rerouting protein is a ligand for a receptor present in the target.

9. The antibody or antigen-binding fragment thereof may be Fab, Fab ２ , scFv, scFv-Fc, dAb-Fc, free light chain antibody, half antibody, bispecific Fab2, Fab ３ , trispecific Fab3 diabodies, bispecific diabodies, triabodies, trispecific triabodies, minibodies, IgG, IgNAR, monovalent IgG, V ｈ 9. The apolipoprotein lipid nanoparticle of claim 8, wherein the apolipoprotein lipid nanoparticle is selected from the group consisting of H, and variable novel antigen receptor (VNAR).

10. The apolipoprotein lipid nanoparticle of claim 1, wherein the rerouting molecule is capable of binding to hematopoietic stem and progenitor cells (HSPCs), such as hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), or common myeloid progenitor cells (CMPs).

11. The apolipoprotein lipid nanoparticle of claim 1, wherein the rerouting molecule is capable of binding to myeloid cells selected from the group consisting of megakaryocytes, eosinophils, basophils, erythrocytes, monocytes, such as dendritic cells or macrophages, and neutrophils.

12. The apolipoprotein lipid nanoparticle of claim 11, wherein the rerouting peptide is SIRPα.

13. The apolipoprotein lipid nanoparticle of claim 1 , wherein the rerouting molecule is capable of binding to a non-myeloid cell, such as a non-myeloid immune cell or an endothelial cell.

14. The apolipoprotein lipid nanoparticle of claim 13, wherein the rerouting molecule is capable of binding to lymphocytes, preferably T cells.

15. The apolipoprotein lipid nanoparticle of claim 13, wherein the rerouting molecule is an antibody or antigen-binding fragment thereof that specifically binds to CD8, or the rerouting peptide is PD1, CD40L, or GP120.

16. 2. The apolipoprotein lipid nanoparticle of claim 1, wherein the apolipoprotein is ApoA1, ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1, or ApoL3, or the apolipoprotein mimetic is a mimetic of ApoA1, ApoA-1 Milano, ApoA4, ApoC3, ApoD, ApoE, ApoL1, or ApoL3.

17. The apolipoprotein lipid nanoparticle of claim 1, wherein the apolipoprotein lipid nanoparticle comprises a payload, preferably the payload is selected from a nucleic acid or nucleic acid analog, a therapeutic agent, a biologic, or a combination thereof.

18. 18. A method for producing apolipoprotein lipid nanoparticles as defined in any one of claims 1 to 17, comprising: a1) expressing and isolating one or more apolipoprotein fusion proteins to obtain one or more isolated apolipoprotein fusion proteins; the one or more apolipoprotein fusion proteins an apolipoprotein or apolipoprotein mimetic fused to a rerouting molecule; apolipoproteins or apolipoprotein mimetics fused to immunomodulatory biomolecules and rerouting molecules, and combinations of these and / or a2) chemically conjugating one or more apolipoproteins or apolipoprotein mimetics and isolating said one or more conjugated apolipoproteins to obtain one or more isolated conjugated apolipoproteins, the one or more conjugated apolipoproteins an apolipoprotein or apolipoprotein mimetic conjugated to an immunomodulatory biomolecule; an apolipoprotein or apolipoprotein mimetic conjugated to a rerouting molecule; apolipoproteins or apolipoprotein mimetics conjugated to immunomodulatory biomolecules and rerouting molecules, and combinations of these 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 apolipoprotein-lipid nanoparticles. A method comprising:

19. A pharmaceutical composition comprising the apolipoprotein lipid nanoparticles of any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

20. 20. Apolipoprotein lipid nanoparticles according to any one of claims 1 to 17 for use as a medicament.

21. The pharmaceutical composition of claim 19 for use as a medicine.

22. 20. An apolipoprotein lipid nanoparticle according to any one of claims 1 to 17 for use in the treatment of an immune-related disorder.

23. The pharmaceutical composition of claim 19 for use in treating an immune-related disorder.

24. 23. The apolipoprotein lipid nanoparticle of claim 22, wherein the immune-related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorders, allergies, organ transplant rejection, and graft-versus-host disease (GVH).

25. The pharmaceutical composition of claim 23, wherein the immune-related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

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

27. 27. The fusion protein of claim 26, wherein the rerouting molecule is a rerouting molecule as defined in any one of claims 8 to 15.

28. The fusion protein of claim 26, wherein the fusion protein further comprises an immunomodulatory biological molecule, the immunomodulatory biological molecule being a protein that enhances or suppresses an immune response, preferably the immunomodulatory biological molecule being an immunomodulatory biological molecule as defined in claim 7.

29. 27. The fusion protein of claim 26, for use in treating an immune-related disorder, preferably wherein the immune-related disorder is selected from the group consisting of cancer, inflammation, infectious disease, autoimmune disorder, allergy, organ transplant rejection, and graft-versus-host disease (GVH).

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