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WO2025190968A1 - Nanoparticule lipidique d'apolipoprotéine - Google Patents

Nanoparticule lipidique d'apolipoprotéine

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Publication number
WO2025190968A1
WO2025190968A1 PCT/EP2025/056656 EP2025056656W WO2025190968A1 WO 2025190968 A1 WO2025190968 A1 WO 2025190968A1 EP 2025056656 W EP2025056656 W EP 2025056656W WO 2025190968 A1 WO2025190968 A1 WO 2025190968A1
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WO
WIPO (PCT)
Prior art keywords
apolipoprotein
antigen
lipid nanoparticle
anps
antigens
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/056656
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English (en)
Inventor
Willem J.M. MULDER
Roy VAN DER MEEL
Anne DE DREU
David Pepijn SCHRIJVER
Tom Franciscus Antonius DE GREEF
Anna-Maria MAKRI PISTIKOU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Trip BV
Original Assignee
Bio Trip BV
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Filing date
Publication date
Application filed by Bio Trip BV filed Critical Bio Trip BV
Publication of WO2025190968A1 publication Critical patent/WO2025190968A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of fusion proteins and more particularly fusion proteins that find use in vaccine therapy.
  • the invention further relates to lipid nanoparticles comprising the fusion proteins and methods of making such.
  • the invention relates to methods of treatment using the fusion proteins or lipid nanoparticles.
  • Vaccine development constitutes a paramount accomplishment in modem medicine, offering a preemptive approach to safeguarding public health.
  • vaccines have played a pivotal role in controlling and eradicating a number of infectious diseases.
  • scientists have expanded the focus of vaccine research beyond infectious agents to encompass the realm of inter alia cancer.
  • cancer vaccines are considered an innovative frontier in medical research and are poised to revolutionize the landscape of cancer prevention and treatment, there remain several challenges with respect to their development.
  • inefficient delivery of antigens and adjuvants to draining lymph nodes has shown to be a major hurdle in the development of efficacious cancer vaccines.
  • suboptimal clinical outcomes have been observed for several free soluble cancer vaccines; likely attributable to rapid antigen renal clearance and inadequate codelivery of antigens and adjuvants to antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • Nanoparticles have emerged as a promising technology in the field of cancer vaccine research, as various nano-vaccines have been used for the targeted co-delivery of adjuvants and antigens to lymphoid organs. Nonetheless, the utilization of nanoparticles for the development of cancer vaccines can raise safety concerns, while the ability to scale up production is a noteworthy challenge, as the ideal vaccine platform should be able to readily and easily incorporate novel antigens.
  • next-generation vaccine platforms that can be readily and easily manufactured, while concurrently conferring disease protection, safety, and biocompatibility.
  • Present inventors developed a novel, modular, apolipoprotein-based nanoparticle vaccine platform for the delivery of one or more antigens, optionally in combination with one or more adjuvants, such as squalene, telratolimod or monophosphoryl-Lipid A (MPLA), or trained immunity inducers, such as muramyl tripeptide phosphatidylethanolamine (MTP-PE).
  • adjuvants such as squalene, telratolimod or monophosphoryl-Lipid A (MPLA), or trained immunity inducers, such as muramyl tripeptide phosphatidylethanolamine (MTP-PE).
  • MTP-PE muramyl tripeptide phosphatidylethanolamine
  • apolipoprotein lipid nanoparticles can be used as an antigen carrier, by fusing the antigen to the apolipoprotein component and preparing apolipoprotein lipid nanoparticles (aNPs), such as preferably spherical aNPs, therefrom.
  • aNPs apolipoprotein lipid nanoparticles
  • the present inventors further realized that the aNPs comprising a fusion protein of an apolipoprotein component and an antigen can be used as a scalable and modular vaccine platform, such as for the development of vaccines against infectious diseases or cancer vaccines.
  • an aspect relates to an apolipoprotein lipid nanoparticle (aNP) comprising a fusion protein comprising an apolipoprotein component and one or more antigens; the aNP further comprising phospholipids.
  • aNP apolipoprotein lipid nanoparticle
  • a further aspect provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aNP and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a further aspect provides a method of manufacturing the aNP comprising combining the fusion protein comprising the apolipoprotein component and the one or more antigens with phospholipids, and optionally sterols, lipids and/or one or more adjuvants, to obtain the aNP.
  • aNP or the pharmaceutical composition for use as a medicament; or use in eliciting an immune response to the one or more antigens in a subject.
  • Related aspects provide a method of treating a subject in need thereof comprising administering a therapeutically or prophylactically effective amount of the aNP or the pharmaceutical composition to the subject; or a method for eliciting an immune response to the one or more antigens in a subject in need thereof comprising administering a therapeutically or prophylactically effective amount of the aNP or the pharmaceutical composition to the subject.
  • the aNP or the pharmaceutical composition wherein the one or more antigens are donor-specific antigens, preferably one or more donor Major Histocompatibility Complex (MHC) antigens, for use in treating and/or preventing transplant rejection.
  • MHC Major Histocompatibility Complex
  • the aNP or the pharmaceutical composition wherein the antigen is a disease- associated antigen, for use in treating and/or preventing a disease selected from the list consisting of chronic inflammatory disease, an autoimmune disease, a metabolic disease, an immune -mediated inflammatory disorder (IMID), an autoinflammatory disease, or an allergic disease.
  • a disease selected from the list consisting of chronic inflammatory disease, an autoimmune disease, a metabolic disease, an immune -mediated inflammatory disorder (IMID), an autoinflammatory disease, or an allergic disease.
  • IMID immune -mediated inflammatory disorder
  • aNP or the pharmaceutical composition wherein the one or more antigen is derived from an allergen, for use in the treatment and/or prevention of an allergic disease.
  • the aNP or the pharmaceutical composition wherein the one or more antigen is an autoimmune disease-associated antigen, such as a self-antigen, for use in the treatment and/or prevention of an autoimmune disease.
  • an autoimmune disease-associated antigen such as a self-antigen
  • aNP or the pharmaceutical composition wherein the one or more antigens is a tumour antigen, for use in the treatment and/or prevention of a neoplastic disease.
  • a related aspect provides a method of treating and/or preventing a neoplastic disease in a subject in need thereof comprising administering a therapeutically or prophylactically effective amount of the aNP or the pharmaceutical composition, wherein the one or more antigen is a tumour antigen, to the subject.
  • aNP or the pharmaceutical composition wherein the one or more antigen is from a pathogen, for use in the treatment and/or prevention of an infectious disease caused by or associated with the pathogen.
  • a related aspect provides a method of treating and/or preventing an infectious disease caused by or associated with a pathogen in a subject in need thereof comprising administering a therapeutically or prophylactically effective amount of the aNP or the pharmaceutical composition, wherein the one or more antigen is from the pathogen.
  • Fig. 1 Engineered fusion proteins allow antigen integration in nanoparticles, a, Schematic overview of apolipoprotein Al (apoAl)-based nanoparticle (aNPs) vaccine platform (vaccine-aNPs); composed of a fusion protein of a selected antigen (either ovalbumin (OVA) protein or the OVA- derived immunogenic peptide OT1) to apoAl, distinct adjuvants, cholesterol, phospholipids, and triglycerides, b, ApoAl ’s interaction with receptors expressed on antigen presenting cells (APCs) leads to targeted co-delivery of antigens and adjuvants to pertinent tissues, c, Sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE) gel analyses, showing the presence of apoAl-OVA (left) and apoAl-OTl (right) fusion protein, d, Percentage of proliferation of antigen specific OT1 T cells after
  • apoAl -OVA-mVenus fusion protein e.g. apoAl -OVA-mVenus fusion protein as defined by SEQ ID No:32
  • RPTPo chicken Receptor protein tyrosine phosphatase sigma
  • the fluorescent protein mVenus is fused, through a human rhinovirus (HRV) 3C protease recognition site.
  • HRV human rhinovirus
  • strep-tag II twin strep tag
  • Cysteine-modified apoAl apoAl-S230C or apoAl-S141C
  • maleimide functionalised OT-I i.e. OT1
  • Vaccine-aNP distributes to antigen presenting cells in secondary lymphoid organs, a, Positron emission tomography with computed tomography (PET-CT) render 24 hours following intravenous (I.V.) or intramuscular (I.M.) injection of 89 Zr-OVA-aNPs (left) or 89 Zr-OTl-aNPs (right). Following I.V. administration, aNPs mainly accumulate in lymphoid organs such as the bone marrow and spleen, as well as the liver. I.M.
  • PET-CT Positron emission tomography with computed tomography
  • aNPs primarily accumulate in the lymph nodes
  • Fig. 3 Tailored adjuvant integration improves humoral and cellular immune responses
  • a Chemical structures of adjuvants incorporated in aNPs; squalene, telratolimod, MTP-PE and MPLA.
  • b Schematic of vaccination study design
  • c Adjuvant loaded-aNP composition for 15 distinct formulations with cryo-TEM images of each aNP displayed on the bottom row (scale bars, 50 nm). Colors represent the number of adjuvants incorporated (dark blue, 1; turquoise, 2; light green, 3; dark green, 4).
  • aNP vaccination prevents tumor development
  • a Schematic of vaccination and tumor inoculation study design
  • b Tumor growth curves for mice vaccinated with different vaccine-aNP. No tumor growth is observed in mice vaccinated with formulation 5, 9, 13, and 15, while PBS and apoAl aNP treated mice do develop tumors
  • d Anti-OVA IgG titers measured in the serum of mice at day 35 using ELISA.
  • RBD-aNP engineering a Cryogenic transmission electron microscopy (cryo-TEM) images for RBD-aNPs. Scale bar, 50 nm.
  • b RBD-aNPs size distribution (hydrodynamic radius in nm), as quantified by dynamic light scattering (DLS).
  • Fig. 6 Engineered fusion protein characterization, a, Western blot analysis showing the presence of apoAl (left panel) and OVA (right panel) in the apoAl -OVA fusion protein, b, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cysteine-modified apoAl (apoAl -S230C).
  • P pellet
  • SN supernatant
  • FT flow-through
  • W wash
  • E elution fraction
  • M Marker
  • c LC-MS analysis of the maleimide-OTl peptide.
  • Fig. 7 In vitro fusion protein-specific T cell response, a, Schematic representation of T cell proliferation assay to assess uptake and processing of fusion proteins by antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • bone marrow progenitor cells isolated from murine bone marrow are differentiated to dendritic cells (mBMDCs), by culturing in medium supplemented with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 8 days.
  • mBMDCs are subsequently incubated with the antigen of choice (apoAl -OVA, apoAl -OT1) or related controls for 24 hours.
  • IL-4 interleukin 4
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Fig. 8 OVA-aNP and OTl-aNP engineering and internalization by antigen-presenting cells, a- b, Cryogenic transmission electron microscopy (cryo-TEM) images for OVA-aNPs (a) and OT1- aNPs (b). Scale bar, 50 nm.
  • DLS dynamic light scattering
  • PET-CT Positron emission tomography with computed tomography
  • Fig. 10 In vivo humoral immune response over time after I.M. vaccine-aNP administration.
  • a,b Anti-OVA IgG (a) and total IgG (b) serum concentrations in ng mL-1 over time determined by ELISA. Bars indicate mean activity ⁇ s.d.; individual data points represent independent biological replicates. Significance (one-way ANOVA with Dunnett’s multiple comparisons test) is noted above bars.
  • I.V. intravenously
  • I.M. intramuscularly
  • Fig. 11 Splenic OVA specific CD8 + T cell proliferation 28 days after intramuscular injection with distinct vaccine-aNPs. Percentage OVA specific T cells of CD8 + splenic T cells. Bars indicate mean activity ⁇ s.d.; individual data points represent independent biological replicates. Significance (one-way ANOVA with Dunnett’s multiple comparisons test) is noted above bars.
  • Fig. 13 Stability and morphology of vaccine-aNP particles measured by DLS and cryo-TEM. a. Polydispersity of vaccine-aNP particles over time. aNPs remain monodisperse over two weeks (Pdl ⁇ 0.2). b. Number mean diameter of vaccine-aNP particles over time. aNPs are approximately 50 nm and their size remains stable over 14 days.
  • Fig. 14 Two-adjuvant combination aNP engineering, a aNPs incorporating a total of two adjuvants were engineered; namely MTP-PE-MPLA-aNPs, MTP-PE-tel-aNPs, sq-tel-aNPs, sq- MTP-PE-aNPs, and MPLA-tel-aNPs.
  • Adjuvant amount expressed as weight percentage (w%) of triglycerides for squalene, or POPCs for MTP-PE, MPLA, and telratolimod is shown in Supplementary Table 11.
  • b aNPs stability in phosphate-buffered saline (PBS) for 12 days at 4°C.
  • Graphs show nanoparticle size as indicated by the hydrodynamic radius (in nm; DLS); c aNP homogeneity, as indicated by polydispersity index (PDI; DLS). Individual data points represent independently formulated triplicates.
  • Fig. 15 Three-adjuvant combination aNP engineering, a aNPs incorporating a total of three adjuvants were engineered; namely sq-tel-MTP-PE-aNPs, sq-tel-MPLA-aNPs, sq-MTP-PE-MPLA- aNPs, and tel-MTP-PE-MPLA-aNPs.
  • Adjuvant amount expressed as weight percentage (w%) of triglycerides for squalene, or POPCs for MTP-PE, MPLA, and telratolimod is shown in Supplementary Table 12.
  • b aNPs stability in phosphate-buffered saline (PBS) for 12 days at 4°C.
  • Graphs show nanoparticle size as indicated by the hydrodynamic radius (in nm; DLS); c aNP homogeneity, as indicated by polydispersity index (PDI; DLS). Individual data points represent independently formulated triplicates.
  • Fig. 16 Four-adjuvant combination aNP engineering, a aNPs incorporating a total of four adjuvants were engineered; namely sq-tel-MTP-PE-MPLA-aNPs.
  • Adjuvant amount expressed as weight percentage (w%) of triglycerides for squalene, or POPCs for MTP-PE, MPLA, and telratolimod is shown in Supplementary Table 13.
  • b aNPs stability in phosphate-buffered saline (PBS) for 12 days at 4°C.
  • Graphs show nanoparticle size as indicated by the hydrodynamic radius (in nm; DLS); c aNP homogeneity, as indicated by polydispersity index (PDI; DLS). Individual data points represent independently formulated triplicates.
  • each sub-range between any stated value in a stated range and either the lower endpoint or the upper endpoint of the stated range is also specifically disclosed.
  • the stated value may be an isolated value or an endpoint of a range subsumed by or overlapping with the stated range.
  • a stated range with lower endpoint LI and upper endpoint U1 i.e., stated range Ll-Ul
  • a stated subrange nested within the stated range with lower endpoint L2 and upper endpoint U2 i.e., stated subrange L2-U2
  • one or more or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.
  • “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
  • the term “and/or” when used in a list of two or more items means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and/or C, the list can comprise A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
  • the present inventors provide fusion proteins comprising an apolipoprotein component and one or more antigens and demonstrate that such fusion proteins can effectively deliver antigens to antigen- presenting cells (APCs) and that the APCs subsequently internalize and process the fusion protein of present invention to induce T cell proliferation of clonal T cells capable of recognising processed peptides, presented on the major histocompatibility complex (MHC).
  • APCs antigen- presenting cells
  • MHC major histocompatibility complex
  • present inventors have shown that both apoAl-OVA and apoAl-OTl fusion proteins induce T cell proliferation in vitro, following processing by dendritic cells (DCs), while formulated OVA and OT1 aNPs are successfully taken up by DCs.
  • DCs dendritic cells
  • Present inventors have successfully engineered aNPs carrying the receptor binding domain (RBD) of the SARS-CoV2 (severe acute respiratory syndrome coronavirus 2) spike protein.
  • RBD receptor binding domain
  • aNP apolipoprotein-based nanoparticle
  • aNP apolipoprotein-based nanoparticle
  • aNPs are nanostructures derived from HDL, engineered from naturally occurring molecules found in the human body, including phospholipids, cholesterol, triglycerides, and the protein apoAl, which has an intrinsic avidity for APCs.
  • Free adjuvant administration in vaccines can lead to their dissociation from the antigen, preventing effective entry into the lymphatic system. Additionally, free adjuvants may undergo rapid degradation, further diminishing the quantity that eventually reaches target organs, while the presence of free adjuvant circulating in the body has the potential to trigger autoimmunity. Accordingly, targeted co-delivery of antigens and adjuvants by the aNPs of the present invention would overcome these disadvantages.
  • the apolipoprotein component is an apolipoprotein or an apolipoprotein mimetic.
  • Apolipoproteins are proteins that bind lipids such as triglycerides and cholesterol to form lipoproteins. They transport lipids (and fat-soluble vitamins) in blood, cerebrospinal fluid and lymph. The lipid components of lipoproteins are insoluble in water. However, because of their detergentlike (amphipathic) properties, apolipoproteins and other amphipathic molecules (such as phospholipids) can surround the lipids, creating a lipoprotein particle that is itself water-soluble, and can thus be carried through water-based circulation (i.e., blood, lymph). In addition to stabilizing lipoprotein structure and solubilizing the lipid component, apolipoproteins interact with lipoprotein receptors and lipid transport proteins, thereby participating in lipoprotein uptake and clearance.
  • apolipoproteins function as structural components of lipoprotein particles, ligands for cell-surface receptors and lipid transport proteins, and cofactors for enzymes.
  • Different lipoprotein particles contain different classes of apolipoproteins, which influence their function.
  • apolipoprotein Al (apoAl) is the major structural protein component of high-density lipoproteins (HDL), although it is present in other lipoproteins in smaller amounts, and HDL comprises other apolipoproteins.
  • the invention is not limited to a particular type of apolipoprotein or apolipoprotein mimetic (e.g. apoAl, apoB or apoE). Therefore in certain embodiments, the apolipoprotein of the fusion protein is selected from apoAl, apoA-1 Milano, apoA2, apoA4, apoA5, apoB48, apoBlOO, apoC-I, apoC-II, apoC-III, apoC-IV, apoD, apoE, apoF, apoH, apoLl, apoL2, apoL3, apoL4, apoL5, apoL6, apoLDl, apoM, apoO, apoOL, or combinations thereof, or a mimetic thereof.
  • apolipoprotein of the fusion protein is selected from apoAl, apoA-1 Milano, apoA2, apoA4, apoA5, apoB48, apoBlOO, apoC-I,
  • apoAl may be designated as apoA-I.
  • the apolipoprotein may be selected from apoAl, apoA-1 Milano, apoA2, apoA4, apoA5, apoB48, apoBlOO, apoC-I, apoC-II, apoC-III, apoC-IV, apoE, apoLl, apoL2, apoL3, apoL4, apoL5, apoL6, or combinations thereof or mimetics thereof.
  • the apolipoprotein may be selected from apoA 1 , apoA4, apoC3, apoD, apoE, apoLl, apoL3, or combinations thereof or mimetics thereof.
  • the apolipoprotein is an apoAl, apoA-1 Milano, apoA4, apoC3, apoD, apoE, apoLl, apoL3 or the apolipoprotein mimetic is a mimetic of an apoAl, apoA-1 Milano, apoA4, apoC3, apoD, apoE, apoLl, apoL3.
  • the apolipoprotein is apoAl.
  • the human apoAl protein sequence is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NP_001304947.1 (isoform 1 preproprotein), and Uniprot (www.uniprot.org) accession number P02647.1.
  • apoAl isoform 1 preproprotein An exemplary sequence of human apoAl isoform 1 preproprotein is shown below as SEQ ID NO: 1, of which the first 18 amino acids form the signal peptide. It shall be understood that the term apolipoprotein as used here may suitably and depending on the context denote a preprotein form or a mature form of the protein.
  • the apolipoprotein may also be an apolipoprotein fragment.
  • the apolipoprotein fragment retains the biological activity of the apolipoprotein, such as the ability of the apolipoprotein to integrate into a lipid nanoparticle or to present the antigen to an APC.
  • the apolipoprotein fragment comprises at least the ATP Binding Cassette Subfamily A Member 1 (ABCA1), ATP Binding Cassette Subfamily G Member 1 (ABCG1) and/or Scavenger receptor class B type 1 (SR-BI) binding regions of the full-length apolipoprotein, thereby allowing binding to a myeloid cell such as an APC.
  • fragment as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and/or C-terminally truncated form of the peptide, polypeptide, or protein.
  • a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein.
  • a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., > 40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, or > 250, consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein.
  • the apolipoprotein fragment comprises the myeloid-binding portion, such as the APC-binding portion, of full-length apolipoprotein.
  • the apolipoprotein may by as shown in SEQ ID NO: 2 below.
  • the apolipoprotein may also be an apolipoprotein mutant comprising a mutation that allows chemical conjugation of the apolipoprotein to an antigen.
  • the apolipoprotein may also be an apolipoprotein mutant comprising a serine to cysteine substitution, such as the apoAl mutant as defined by SEQ ID NO: 3, 4, 5, 25 or 30.
  • Peptide sequences for the different proteins described herein, or nucleic acid sequences for the genes encoding the different proteins described herein, are readily available to the skilled person, for example from the UCSC Genome Browser (http://genome.ucsc.edu/), Ensembl genome browser (https://www.ensembl.org) andNCBI (https://www.ncbi.nlm.nih.gov/protein). Consensus sequences for different proteins or genes are readily derived from these sources, although it is understood a certain variation may be present due to but not limited to genetic variation and multiple splice variants of the gene. Therefore, when referring to a specific protein, this should be interpreted to encompass sequence variations due to genetic variation and splice variants.
  • Apolipoprotein mimetics are synthetic peptides or proteins that mimic the function or structure of apolipoproteins.
  • Several apolipoprotein mimetics are known and for example Wolska et al. (Cells. 2021 Mar; 10(3): 597., incorporated by reference in its entirety) review different apoAl, apoE and apoC-II mimetics described in the literature.
  • apoAl mimetic peptides have largely been designed based on their ability to efflux cholesterol from cells.
  • apoAl mimetic peptides are simply just amphipathic helices and, in fact, many have no primary amino acid homology to apoAl.
  • exemplary apoAl mimetics are 3F, 4F, 5F, 6F, and 7F, ELK peptides, FAMP, i-FAMP, apoAl mimetic 18A, R18A, apoAl mimetic 2F and apoAl mimetic 37pA, and combinations thereof.
  • ApoAl mimetic 18A, 2F and 37 pA are represented by the peptides sequences corresponding to SEQ ID Nos 6, 7 and 8.
  • apoE has several putative atheroprotective functions, many different types of apoE- based peptides have been reported.
  • One of the main goals in the design of these peptides is to facilitate the hepatic clearance of apoB-containing lipoproteins.
  • these peptides usually have not only the receptor-binding motif from the N-terminal domain of apoE, but also a lipid-binding region based on the C-terminal domain of apoE or some other sequence.
  • apoC-II mimetics have been described either based on a shortened first helix (18A) linked to the LPL-activation domain of apoC-II, or mimetics where both the first and second helix are based on the native apoC-II helices with amino acid substitutions to enhance bihelical binding to lipoproteins.
  • an apolipoprotein mimetic refers to a synthetic protein or peptide which shares a structural and/or functional feature with the respective apolipoprotein.
  • the shared structural feature may be a primary, secondary or tertiary peptide structure such as the peptide sequence, presence of structures such as an alpha helix or beta sheet or three-dimensional structure of the peptide, or the functional feature may be a similarity in binding to a certain target such as a receptor.
  • the apolipoprotein is capable of binding lipids, more preferably forming lipid particles, in a similar manner as the corresponding apolipoprotein.
  • the apolipoprotein mimetic is able to bind to a myeloid cell to the same or a similar extent as the respective apolipoprotein.
  • the apolipoprotein mimetic of apoAl is able to bind to a myeloid cell, such as an APC, to the same or a similar extent as apoAl .
  • the fusion protein is a fusion protein of apoAl (or a mutant thereof), preferably human apoAl, with one or more antigens.
  • the apoAl is wild-type apoAl (e.g. as derived from the human precursor of apoAl as defined by SEQ ID NO. 1, of which the first 18 amino acids form the signal peptide) or an apoAl mutant (e.g. as defined by SEQ ID NO. 3, 4 or 5).
  • the apoAl is wild-type human apoAl without signal peptide as defined by SEQ ID NO. 9.
  • apoAl mutants comprising a cysteine in the place of a serine at position 147 (e.g. as defined by SEQ ID NO: 4) or 279 (e.g. as defined by SEQ ID NO: 5) could be useful to prepare chemically conjugated apoAl fusion proteins.
  • apoAl proteins as described herein in the Examples section may be used e.g. apoAl-S230C as defined by SEQ ID NO: 25 or apoAl-S141C as defined by SEQ ID NO: 30.
  • the apoAl is a peptide with, or the apoAl comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 10, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO: 25 or SEQ ID NO: 30.
  • SEQ ID NO: 10 is His-tagged apoAl.
  • apoAl of SEQ ID NO: 1 may be encoded by the nucleic acid of SEQ ID NO: 11 and the skilled person would understand how to modify this nucleic acid in keeping with the principles of genetic code and codon optimisation.
  • the apoAl sequence as defined by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 comprise N-terminally the amino acid sequence GLVPRGSIDD (SEQ ID NO: 12) which is a thrombin cleavage site.
  • the apoAl sequence as defined by SEQ ID NO: 10 comprises N-terminally a 6His tag followed by the amino acid sequence GLVPRGSIDD (SEQ ID NO: 12).
  • the thrombin cleavage site could be used to remove the N-terminal His tag from the peptide.
  • the apoAl sequence as defined by SEQ ID NO: 25 comprises N- terminally of SEQ ID NO: 5, a 6His tag (SEQ ID NO: 34).
  • the apoAl sequence as defined by SEQ ID NO: 30 comprises N-terminally of SEQ ID NO: 4, a 6His tag (SEQ ID NO: 34).
  • the apoAl is a peptide with, or the apoAl comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 3, wherein SEQ ID NO. 3 comprises a cysteine at position 7 of SEQ ID NO. 3.
  • S 14C Such apoAl mutant may be referred to herein as “S 14C” mutant.
  • the apoAl is a peptide with, or the apoAl comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 4, wherein SEQ ID NO. 4 comprises a cysteine at position 150 of SEQ ID NO. 4.
  • SEQ ID NO. 4 comprises a cysteine at position 150 of SEQ ID NO. 4.
  • Such apoAl mutant may be referred to herein as “S147C” or “S157C” mutant.
  • the apoAl-S141C mutant as referred to in the examples comprises SEQ ID NO. 5.
  • the apoAl is a peptide with, or the apoAl comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 5, wherein SEQ ID NO. 5 comprises a cysteine at position 239 of SEQ ID NO. 5.
  • S279C or “S239C” mutant.
  • the apoAl -S230C mutant as referred to in the examples comprises SEQ ID NO. 5.
  • the fusion protein is a fusion protein of an apoAl mimetic with one or more antigens.
  • the apoAl mimetic is a peptide with or the apoAl mimetic comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8.
  • the apoAl is a peptide with, or the apoAl comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of the apoAl proteins as described herein in the Examples section, such as the apoAl protein as defined by SEQ ID NO: 25 or 30.
  • the fusion protein is a fusion protein of apoE with one or more antigens.
  • the apoE is a peptide with or the apoE comprises, consists essentially of or consists of, an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15 or SEQ ID NO. 16.
  • apoE of SEQ ID NO: 13 may be encoded by the nucleic acid of SEQ ID NO: 17 and the skilled person would understand how to modify this nucleic acid in keeping with the principles of genetic code and codon optimisation.
  • the lipid nanoparticle comprises a native (e.g. unfused) apolipoprotein, an apolipoprotein mimetic or a combination thereof, in addition to the apolipoprotein or an apolipoprotein mimetic which forms part of the fusion protein as described herein.
  • the lipid nanoparticle may additionally a fusion protein of an apolipoprotein component, such as apolipoprotein or apolipoprotein mimetic, and an immunomodulatory biomolecule, or a rerouting molecule, or both, as disclosed in WO2023/046931.
  • the lipid nanoparticle may additionally a fusion protein of an apolipoprotein component, such as apolipoprotein or apolipoprotein mimetic, and a targeting body, wherein the targeting body is a molecule capable of binding a molecule on the cell surface of a target cell, as disclosed in WO2023/227682.
  • fusion protein when referring to an apolipoprotein fusion protein should be interpreted as an apolipoprotein or apolipoprotein mimetic and covalently attached thereto one or more antigens.
  • the covalent attachment may be due to the in frame coding of a peptide or protein sequence by the nucleotide sequence that encodes the fusion protein, i.e., genetic fusion.
  • the covalent attachment may be due to covalent linkage of the one or more antigens to the apolipoprotein, for example via a sulphur bond such as a thioether bond, formed at a cysteine residue of the apolipoprotein.
  • a cysteine in the apolipoprotein component may allow linking the same with a maleimide-functionalised antigen or with maleimide-functionalised ligandantigen conjugate via classical maleimide chemistry.
  • the antigen(s) and/or the apolipoprotein (or mimetic thereof) may include the site-specific incorporation of non-natural amino acids such as para-azidophenylalanine, which can be used in subsequent (strain-promoted) “click” (conjugation) reactions with alkyne modified reagents.
  • the apolipoprotein component and the one or more antigens are genetically fused or chemically conjugated.
  • the one or more antigens may be directly or indirectly (such as through a suitable linker) linked to the apolipoprotein component.
  • the fusion protein comprises a linker, such as a flexible, semi-flexible or rigid linker, particularly flexible linker, between the apolipoprotein component, such as an apolipoprotein or apolipoprotein mimetic, and the one or more antigens.
  • the nature of the linker is not central to the operation of the invention.
  • the linker may comprise between 1 and 50 amino acids, between 1 and 40, between 1 and 30, between 1 and 25, or between 1 and 20 amino acids, such as consecutive amino acids.
  • the linker may be a glycine-serine linker, such as a (GGS)n-linker, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably a (GGS)4 (SEQ ID NO: 18)-linker or a (GGS)7 (SEQ ID NO: 19)-linker.
  • a (GGS)n-linker wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably a (GGS)4 (SEQ ID NO: 18)-linker or a (GGS)7 (SEQ ID NO: 19)-linker.
  • the fusion protein may comprise one or more tags, such as at the N- and/or C-terminal end of the fusion protein.
  • the one or more tags such as a 6His-tag or strep-tag may allow purification of the fusion protein.
  • the fusion protein may comprise a fluorescent label, such as a fluorescent protein, like mVenus or GFP.
  • a fluorescent label such as mVenus or GFP.
  • the presence of mVenus may help to solubilize the fusion protein and/or increase stability, detection and/or expression.
  • Said one or more antigens may be covalently attached to any portion of the apolipoprotein or apolipoprotein mimetic.
  • a linker such as a flexible linker, may be used to allow such covalent attachment.
  • one or more antigen(s) are located N- and/or C-terminally, such as C- terminally, of said apolipoprotein or apolipoprotein mimetic in said fusion protein.
  • the fusion protein as such i.e., a fusion protein as described herein comprising the apolipoprotein component and the one or more antigens. Also disclosed is the fusion protein as such for use in the therapeutic indication as described herein for the aNPs, as well as the fusion protein as such for use in targeting the one or more antigens to a myeloid cell, preferably to an antigen-presenting cell (APC), such as a dendritic cell (DC).
  • APC antigen-presenting cell
  • DC dendritic cell
  • nucleic acid encoding the fusion protein as taught herein, preferably wherein the fusion protein is a genetic fusion.
  • the aNPs described herein have an outer layer comprising mainly the apolipoprotein fusion protein, phospholipid and optionally sterol, and a core.
  • the apolipoprotein fusion protein, and hence also the one or more antigens or at least part thereof (i.e. part of an individual antigen) are located at the outer surface of the nanoparticle and/or is exposed to the surroundings of the nanoparticle.
  • the adjuvants may be present in the core and/or the outer layer of the aNPs, depending on the type of adjuvant(s) used.
  • strong hydrophobic adjuvants such as squalene
  • amphiphilic adjuvants such as MPLA
  • the apolipoprotein lipid nanoparticle further comprises sterols.
  • the apolipoprotein lipid nanoparticle further comprises sphingolipids.
  • the apolipoprotein lipid nanoparticle further comprises lipids, preferably triglycerides.
  • the apolipoprotein lipid nanoparticle further comprises sterols and sphingolipids.
  • the apolipoprotein lipid nanoparticle further comprises sterols and lipids, preferably triglycerides. In certain embodiments, the apolipoprotein lipid nanoparticle further comprises sphingolipids and lipids, preferably triglycerides. In certain embodiments, the apolipoprotein lipid nanoparticle further comprises sterols, sphingolipids, and lipids, preferably triglycerides. If the aNP comprises both sterol and lipids, the inventors’ understanding is that the lipids are encapsulated by the phospholipids and sterols. Accordingly, the lipid nanoparticles thus differ in their structure from liposomes.
  • the phospholipid is selected from a phosphatidylcholine (PC), a phosphatidylethanolamine (PE), a phosphatidylserine and a phosphatidylglycerol or combinations thereof.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PEG phosphatidylserine
  • phosphatidylglycerol phosphatidylglycerol
  • the phospholipid is selected from the group consisting of l,2-diphytanoyl-sn-glycero-3 -phosphocholine (PHPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dilauroyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DLPE), dim
  • the ratio of the fusion protein (in case of two or more fusion proteins, cumulatively) to phospholipid based on weight is from 3: 1 to 1: 100, from 1: 1.5 to 2: 1 or from 1: 100 to 2: 1.
  • the ratio of the fusion protein to phospholipid is dependent on the size of the fusion protein.
  • a nanoparticle includes two or more distinct apolipoprotein-containing components, such as an apolipoprotein and an apolipoprotein fusion protein, or two or more apolipoprotein fusion proteins and optionally one or more apolipoprotein, etc.
  • the cumulative amount of these apolipoprotein components is taken into account when quantitative relationships to other constituents of the nanoparticle are discussed.
  • the sterol is selected from cholesterol, desmosterol, stigmasterol, P- sitosterol, ergosterol, hopanoids, hydroxysteroid, phytosterol, steroids, hydrogenated cholesterol, campesterol, zoosterol, or combinations thereof.
  • the apolipoprotein lipid nanoparticle is a sphere, a ribbon or a disc.
  • the aNPs may typically form nano-discs or nanospheres, while other shapes are included.
  • the shape of the nanoparticle may depend on the absence or presence of core material.
  • a core material may for example be lipids such as triglycerides which become enclosed in the core of the particle together with optional payload. It is understood that including more core material will presumably render the nanoparticles larger, up to a certain extent, where the particle become instable.
  • inclusion of a core material may contribute to stabilize the nanoparticles or it may stabilize the inclusion of the payload, or it may modulate or enhance the delivery of the payload.
  • the apolipoprotein lipid nanoparticle is spherical.
  • the aNP comprises a fusion protein comprising the apolipoprotein component and two or more different antigens, such as for example 2, 3, 4, 5 or more than 5 different antigens.
  • the aNP comprises two or more different fusion proteins, such as for example 2, 3, 4, 5 or more than 5 different fusion proteins, wherein each fusion protein may independently comprise an apolipoprotein component (which may be the same or different in the different fusion proteins) and one or more antigens.
  • each fusion protein may independently comprise an apolipoprotein component (which may be the same or different in the different fusion proteins) and one or more antigens.
  • the aNP comprises two or more different fusion proteins, such as for example 2, 3, 4, 5 or more than 5 different fusion proteins, wherein each fusion protein may independently comprise an apolipoprotein component (which may be the same or different in the different fusion proteins) and two or more different antigens, such as for example 2, 3, 4, 5 or more than 5 different antigens.
  • each fusion protein may independently comprise an apolipoprotein component (which may be the same or different in the different fusion proteins) and two or more different antigens, such as for example 2, 3, 4, 5 or more than 5 different antigens.
  • the platform allows for highly modular design with inter alia multitude of antigens which can thereby by co-delivered. Such designs may be particularly interesting for example for multivalent vaccines.
  • the term “antigen” generally refers to a substance that evokes an immune response, including humoral immunity and/or cellular immunity response, and that is capable of binding with a product, e.g., an antibody or a T cell, of the immune response.
  • An antigen as intended herein may in an alternative be such as to induce immuno-tolerance, e.g., may be an auto-antigen (including auto- and allo-antigens) or may be allergen.
  • an antigen requires a functioning immune system of a subject to which it is administered to elicit a physiological response from such a subject.
  • the “antigen” as intended herein also encompasses “self-antigens” which do not provoke an immune response in a healthy individual but would do so in a person suffering from auto-immune disease, i.e., the failure of an organism to recognise its own constituent parts (down to the sub-molecular levels) as “self’, which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease.
  • Non-limiting examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), myositis, Sjogren’s syndrome, scleroderma (e.g.
  • the “antigen” as intended herein also encompasses a (physiologically active) protein which would not provoke an immune response in a healthy individual but would do so in a person genetically deficient in said protein.
  • the “antigen” as intended herein also encompasses an allergen which would not provoke an immune response in a healthy individual but would do so in a person suffering from an allergic disease.
  • An antigen as intended herein may be derived from any polypeptide to which an immune response in a human or animal subject would be therapeutically useful, e.g., from a pathogen, e.g., from a viral, prokaryotic (e.g., bacterial) or eukaryotic pathogen, from a non-physiological protein (e.g., a protein derived from cancer tissue), from allergen (e.g., for eliciting immune tolerance), etc.
  • An antigen could also be a metabolite of a protein.
  • the antigen could be a polysaccharide, a lipid or other.
  • protein generally encompasses macromolecules comprising one or more polypeptide chains.
  • polypeptide generally encompasses linear polymeric chains of amino acid residues linked by peptide bonds.
  • a “peptide bond”, “peptide link” or “amide bond” is a covalent bond formed between two amino acids when the carboxyl group of one amino acid reacts with the amino group of the other amino acid, thereby releasing a molecule of water.
  • protein and polypeptide may be used interchangeably to denote such a protein. The terms are not limited to any minimum length of the polypeptide chain.
  • Polypeptide chains consisting essentially of or consisting of 50 or less ( ⁇ 50) amino acids, such as ⁇ 45, ⁇ 40, ⁇ 35, ⁇ 30, ⁇ 25, ⁇ 20, ⁇ 15, ⁇ 10 or ⁇ 5 amino acids may be commonly denoted as a “peptide”.
  • a “sequence” is the order of amino acids in the chain in an amino to carboxyl terminal direction in which residues that neighbour each other in the sequence are contiguous in the primary structure of the protein, polypeptide or peptide.
  • the terms may encompass naturally, recombinantly, semi -synthetically or synthetically produced proteins, polypeptides or peptides.
  • a protein, polypeptide or peptide can be present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or a protein, polypeptide or peptide can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised.
  • a protein, polypeptide or peptide can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.
  • a suitable host or host cell expression system e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system
  • the terms also encompasses proteins, polypeptides or peptides that carry one or more co- or post-expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, lipidation, acetylation, amidation, phosphorylation, sulphonation, methylation, pegylation (covalent attachment of polyethylene glycol typically to the N-terminus or to the side-chain of one or more Lys residues), ubiquitination, sumoylation, cysteinylation, glutathionylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc.
  • modifications of the polypeptide chain(s) such as, without limitation, glycosylation, lipidation, acetylation, amidation, phosphorylation, sulphonation, methylation, pegylation (co
  • co- or post-expression-type modifications may be introduced in vivo by a host cell expressing the proteins, polypeptides or peptides (co- or post-translational protein modification machinery may be native to the host cell and/or the host cell may be genetically engineered to comprise one or more (additional) co- or post-translational protein modification functionalities), or may be introduced in vitro by chemical (e.g., pegylation) and/or biochemical (e.g., enzymatic) modification of the isolated proteins, polypeptides or peptides.
  • chemical e.g., pegylation
  • biochemical e.g., enzymatic
  • amino acid encompasses naturally occurring amino acids, naturally encoded amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all in their D- and L-stereoisomers, provided their structure allows such stereoisomeric forms.
  • Amino acids are referred to herein by either their name, their commonly known three letter symbols or by the one- letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • a “naturally encoded amino acid” refers to an amino acid that is one of the 20 common amino acids or pyrrolysine, pyrroline -carboxy-lysine or selenocysteine.
  • the 20 common amino acids are: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).
  • non-naturally encoded amino acid refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine, pyrroline -carboxy-lysine or selenocysteine.
  • the term includes without limitation amino acids that occur by a modification (such as a post- translational modification) of a naturally encoded amino acid, but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex, as exemplified without limitation by N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O- phosphotyrosine.
  • non-naturally encoded, un-natural or modified amino acids include 2-Aminoadipic acid, 3 -Aminoadipic acid, beta- Alanine, beta- Aminopropionic acid, 2- Aminobutyric acid, 4-Aminobutyric acid, piperidinic acid, 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3 -Aminoisobutyric acid, 2-Aminopimelic acid, 2,4 Diaminobutyric acid, Desmosine, 2,2’-Diaminopimelic acid, 2,3 -Diaminopropionic acid, N-Ethylglycine, N- Ethylasparagine, homoserine, homocysteine, Hydroxylysine, allo-Hydroxylysine, 3- Hydroxyproline, 4-Hydroxyproline, Isodesmosine, allo-Isoleucine, N-Methylglycine
  • amino acid analogues in which one or more individual atoms have been replaced either with a different atom, an isotope of the same atom, or with a different functional group.
  • un-natural amino acids and amino acid analogues described in Ellman et al. Methods Enzymol. 1991, vol. 202, 301-36.
  • the one or more antigen is a tumor antigen.
  • the tumor antigen may be a tumor-associated antigen (TAA), a tumor specific antigen (TSA), or a neoantigen.
  • tumor antigen refers to an antigenic substance produced in tumor cells that is able to trigger an immune response in a host.
  • tumor specific antigen refers to antigens that are present on tumor cells and not on any other cells.
  • tumor-associated antigen refers to antigens that are present on some tumor cells and also on some normal cells.
  • TSA and/or TAA include CD19, CD319/CS1, ROR1, CD20, CD5, CD7, CD22, CD70, CD30, BCMA, CD25, NKG2D ligands, MICA/MICB, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CA-125, MUC-1, CO17-1A, melanoma-associated antigen (MAGE), mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, GD2, CD 123, CD33, CD37, CD30, CD56, c-Met, mesothelin (MSLN), GD3, HERV-K, IL-l lRa, CSPG4,WT-1, EGFRvIII, TRAIL/DR4, VEGFR2, glycoprotein 100 (gplOO/Pmel 17), NY-BR-1, NY-CO-58, NY-ESO-1, MARTI, 5T4, a
  • Protein descriptions for the protein abbreviations used in the specification can be consulted via the protein database of U.S. government’s National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) or via the homepage ofUniProt (https://www.uniprot.org/).
  • NCBI National Center for Biotechnology Information
  • the one or more antigen is an antigen from a pathogen, i.e., an antigen of a pathogen.
  • the one or more antigen is an antigen from a viral, prokaryotic, or eukaryotic pathogen, or a combination thereof.
  • the prokaryotic pathogen is a bacterial pathogen.
  • the pathogen may be a virus, bacteria, fungus or parasite.
  • the antigen is a viral antigen or virus-associated antigen, such as a viral antigen or virus-associated antigen selected from the group consisting of Human Cytomegalovirus (HCMV) antigens, gpl60, Hepatitis C virus (HCV) antigens, Human papilloma virus (HPV) antigens, lysozymes (LYZ), pp50, Tat, VSV8, Epstein-Barr Virus (EBV) antigens, gp33, Hepatitis Delta Virus (HDV), Influenza virus, nef, pp65, tuberculosis antigens, gag, Hepatitis B virus (HBV) antigens, Human immunodeficiency virus (HIV) antigens, LMP2, p21 protein, severe acute respiratory syndrome (SARS)-CoV antigens, and vpr.
  • HCMV Human Cytomegalovirus
  • HCV Hepatitis C virus
  • HPV Human papilloma virus
  • NAbs neutralizing antibodies
  • S viral spike
  • NAbs specific for the N-terminal SI domain which contains the angiotensin-converting enzyme 2 (ACE2) receptorbinding domain — have previously been shown to prevent viral infection in several animal models.
  • ACE2 angiotensin-converting enzyme 2
  • the antigen of the pathogen comprises, consists essentially of or consists of the Spike protein of a coronavirus or part thereof.
  • the antigen of the pathogen comprises, consists essentially of or consists of a receptor-binding domain (RBD) of an SI subunit of a Spike (S) protein of a coronavirus.
  • RBD receptor-binding domain
  • the RBD of an SI subunit of a Spike protein of a coronavirus may comprise, consist essentially of or consist of the sequence defined by SEQ ID NO: 28, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence defined by SEQ ID NO: 28.
  • the coronavirus is COVID-19 (or SARS-CoV-2).
  • the Spike protein may be the Spike protein of any variant of the SARS-CoV-2 virus.
  • the Spike protein is the Spike protein from the SARS-CoV-2 isolate Wuhan-Hu-1 as annotated under NCBI Genbank accession number MN908947.3, the Spike protein from the Alpha variant (also known as the UK variant) of the SARS-CoV-2 virus (e.g. VOC 202012/01, B. l.1.7) , the Spike protein from the Gamma variant (also known as the Brazilian-Japanese variant) of the SARS-CoV- 2 virus (e.g. B. 1.1.28 or Pl), the Spike protein of the Beta variant (also known as the South African variant) of the SARS-CoV-2 virus (e.g.
  • the Spike protein of the Epsilon variant also known as the Californian variant of the SARS-CoV-2 virus (e.g. B.1.427 or B. 1.429)
  • the Spike protein of the Iota variant also known as the New York variant of the SARS-CoV-2 virus (e.g. B. 1.526 or B.1.526.1)
  • the Spike protein of the Eta variant also known as the UK/Nigeria variant of the SARS-CoV-2 virus (e.g. B.1.525)
  • the Spike protein of the Kappa variant also known as the Indian variant of the SARS-CoV-2 virus (e.g.
  • B.1.617, B.1.617.1, B.1.617.2 or B. 1617.3 the Spike protein of the Zeta variant (also known as the Brazilian variant) of the SARS-CoV-2 virus (e.g. P.2), the Theta variant of the SARS-CoV-2 virus (e.g. P3), the Lambda variant of the SARS-CoV-2 virus (e.g. C. 37), the Mu variant of the SARS-CoV-2 virus (e.g. B. 1.621), the Delta variant of the SARS-CoV-2 virus (e.g. B.1.617.2), or the Omicron variant of the SARS-CoV-2 virus (e.g. B.1.1.529).
  • the Spike protein of the Zeta variant also known as the Brazilian variant
  • SARS-CoV-2 virus e.g. P.2
  • Theta variant of the SARS-CoV-2 virus e.g. P3
  • the Lambda variant of the SARS-CoV-2 virus e.g
  • SARS-CoV-2 Spike protein An exemplary amino acid sequence of SARS-CoV-2 Spike protein is annotated under Uniprot (www.uniprot.org) accession number P0DTC2.1 and is depicted below:
  • 1.351 variant typically comprises the following modifications: D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V.
  • the Spike protein from the Pl variant typically comprises the following modifications: L18F, T20N, P26S, D138Y, R190s, K417T, E484K, N501Y, D614G, H655Y, T1027E
  • the Spike protein from the B.l.1.7 variant typically comprises the following modifications: 69del, 70del, 144del (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T716I, S982A, DI 118H, (KI 19N*).
  • 70del refers to the deletion of the amino acid at position 70 of the Spike protein
  • N501Y designates a replacement of the N residue at position 501 of the Spike protein by a Y residue.
  • the mutations are mutations compared to the sequence of the Spike protein of the ancestor SARS-CoV-2 isolate Wuhan-Hu-1. Examples of pathogenic bacteria against which vaccination has been adopted in clinical practice and which are thus particularly interesting candidates for adoption in the present platform include without limitation:
  • DTaP diphtheria, tetanus, and pertussis
  • Haemophilus influenzae type h can cause meningitis, pneumonia, and epiglottitis. Vaccination against Hib is available and recommended for children.
  • Vaccines (MenACWY and MenB) are available against certain serogroups of this bacterium.
  • PCV13 pneumococcal conjugate vaccine
  • PPSV23 pneumococcal polysaccharide vaccine
  • Mycobacterium tuberculosis - causes tuberculosis (TB).
  • the BCG vaccine is available and is used in many countries to protect against TB, especially in children.
  • Salmonella Typhi - causes typhoid fever.
  • Vaccination against typhoid fever is available and recommended for travelers to certain areas and in outbreak situations.
  • bacterial pathogens include:
  • Staphylococcus aureus - can cause a wide range of infections, from skin and wound infections to more serious conditions such as pneumonia, bloodstream infections, and sepsis.
  • Methicillin- resistant Staphylococcus aureus (MRSA) is a notable antibiotic-resistant strain.
  • Escherichia coli E. coli
  • certain strains such as E. coli O157:H7
  • HUS hemolytic uremic syndrome
  • Streptococcus pyogenes (Group A Streptococcus) - causes strep throat, scarlet fever, impetigo, and invasive diseases such as necrotizing fasciitis (flesh-eating disease) and rheumatic fever.
  • Chlamydia trachomatis - causes chlamydia, a sexually transmitted infection (STI) that can lead to serious reproductive and other health problems with both short- and long-term consequences.
  • STI sexually transmitted infection
  • Vibrio cholerae - causes cholera, a severe diarrheal illness that can lead to dehydration and death if untreated. It is often spread through contaminated water.
  • Lyme disease causes Lyme disease, transmitted to humans through the bite of infected black-legged ticks. Lyme disease can lead to symptoms ranging from fever, fatigue, and skin rash, to more severe symptoms affecting the joints, heart, and nervous system if left untreated.
  • Legionella pneumophila causes Legionnaires' disease, a serious type of pneumonia, and Pontiac fever, a milder illness resembling the flu. It is usually spread through inhalation of aerosolized water containing the bacteria, not by person-to-person contact.
  • Mycobacterium leprae - causes leprosy (Hansen's disease), a chronic disease leading to skin lesions and nerve damage. It is transmitted via droplets, from the nose and mouth, during close and frequent contacts with untreated cases.
  • Treponema pallidum - causes syphilis, an STI that can present in stages (primary, secondary, latent, and tertiary) and can affect various organ systems over time if not treated.
  • MALT mucosa-associated lymphoid tissue
  • Non-limiting examples of fungal pathogens include:
  • Candida spp. - This genus includes several species, with Candida albicans being the most well- known. They can cause infections known as candidiasis, which may affect various parts of the body including the mouth (thrush), vagina (yeast infection), skin, and can also lead to invasive candidiasis when the bloodstream or internal organs are involved.
  • Dermatophytes (Trichophyton, Microsporum, and Epidermophyton spp.) - These fungi cause dermatophytoses, also known as ringworm, affecting the skin, hair, and nails. Common conditions include athlete's foot (tinea pedis), jock itch (tinea cruris), and scalp ringworm (tinea capitis).
  • Histoplasma capsulatum This fungus causes histoplasmosis, a disease that primarily affects the lungs and can vary in severity from mild to life-threatening. It is commonly found in bird and bat droppings and in certain geographical areas, such as the Ohio and Mississippi River valleys.
  • PCP pneumocystis pneumonia
  • Blastomyces dermatitidis This fungus causes blastomycosis, which can affect the lungs, skin, and other body areas. It is endemic to parts of North America, especially in areas around the Ohio and Mississippi River valleys and the Great Lakes.
  • Paracoccidioides hrasiliensis This fungus causes paracoccidioidomycosis, primarily affecting the lungs, mucous membranes, and skin. It is endemic to certain regions of Latin America.
  • Sporothrix schenckii This fungus causes sporotrichosis, also known as "rose gardener's disease,” affecting the skin and lymph nodes. It is commonly contracted through the skin from the environment, particularly when handling contaminated plant material or soil.
  • Non-limiting examples of parasite pathogens include:
  • Plasmodium spp. - Protozoan parasites that cause malaria a serious disease transmitted by Anopheles mosquitoes. The most severe form is caused by Plasmodium falciparum, but other species such as P. vivax, P. ovale, and P. malariae also cause malaria.
  • Giardia lamblia also known as Giardia intestinalis or Giardia duodenalis
  • Giardia lamblia also known as Giardia intestinalis or Giardia duodenalis
  • Entamoeba histolytica A protozoan that causes amebiasis, which can range from asymptomatic infection to dysentery and invasive extraintestinal diseases such as liver abscess. Transmission occurs via ingestion of contaminated food or water.
  • Toxoplasma gondii - A protozoan that causes toxoplasmosis, which can be asymptomatic in healthy individuals but serious in pregnant women and immunocompromised patients. Transmission can occur through ingestion of undercooked contaminated meat, contaminated water, or exposure to infected cat feces.
  • Trichuris trichiura whipworm
  • a nematode that causes trichuriasis, leading to gastrointestinal symptoms such as diarrhea and abdominal pain. It is transmitted through ingestion of eggs from contaminated soil.
  • Taenia spp. - Tapeworms including Taenia saginata (beef tapeworm) and Taenia solium (pork tapeworm), which cause taeniasis.
  • T. solium can also cause cysticercosis, a more serious condition when humans ingest eggs leading to the formation of cysts in tissues.
  • the one or more antigen is a donor-specific antigen, such as a donor MHC antigen.
  • the one or more antigen is an antigen associated with a disease selected from the list consisting of chronic inflammatory disease, an autoimmune disease, a metabolic disease, an immune-mediated inflammatory disorder (IMID), an autoinflammatory disease, or an allergic disease.
  • the one or more antigens is an allergen.
  • the one or more antigens is an autoimmune disease-associated antigen, such as a self-antigen.
  • ovalbumin (OVA) (Mw: 45 kDa) and the OVA-derived immunogenic OT-I peptide (the OT-I peptide may also be referred to herein as “OT1” or “OT-I”), composed of a total of eight amino acids, that the fusion protein as taught herein may comprise various sizes of antigen(s).
  • the antigen is a peptide, protein or polypeptide comprising, consisting essentially of or consisting of at most about 2000 amino acids, preferably at most about 1500 amino acids, more preferably at most about 1000 amin acids, such as ⁇ 800, ⁇ 700, ⁇ 600, ⁇ 500, ⁇ 400, ⁇ 300, ⁇ 200, ⁇ 100, or ⁇ 50.
  • the antigen is a peptide, protein or polypeptide comprising, consisting essentially of or consisting of in increasing order of preference at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, or at least 8 amino acids.
  • the antigen is a peptide, protein or polypeptide comprising, consisting essentially of or consisting of from 4 to about 1000 amino acids, from 5 to about 800 amino acids, from 6 to about 600 amino acids, from 7 to about 500 amino acids, or from 8 to about 400 amin acids, such as from 4 to 385 amino acids.
  • these values may apply to each antigen individually or to all antigens combined. Free adjuvant administration in cancer vaccines can lead to their dissociation from the antigen, preventing effective entry into the lymphatic system.
  • free adjuvants may undergo rapid degradation, further diminishing the quantity that eventually reaches target organs, while the presence of free adjuvant circulating in the body has the potential to trigger autoimmunity.
  • Targeted co-delivery of antigens and adjuvants using the aNPs as taught herein addresses these challenges.
  • the aNP further comprises one or more adjuvant.
  • the aNP as taught herein comprises at least one, at least two, at least three, or at least four, adjuvants, such as precisely 2, 3, 4, or 5 adjuvants, preferably 3 or 4 adjuvants.
  • the one or more adjuvant is not part of the fusion protein as taught herein.
  • adjuvant refers to any material that increases the humoral and/or cellular immune response to an antigen. Adjuvants are typically used to accomplish two objectives: slowing down the release of antigens from the injection site, and/or stimulating the immune system. Conventional adjuvants, well-known in the art, are diverse in nature. Non-limiting examples of adjuvants include squalene or squalene-based adjuvants, such as MF59, AS03 or AF03, telratolimod (i.e.
  • 3M-052 a known TLR7/8 agonist
  • imiquimod resiquimod
  • alum or alum-based adjuvants tyrosine crystals
  • LP40 poly I:C
  • Flagellin microcrystalline tyrosine (MCT)
  • MCT microcrystalline tyrosine
  • MCT microcrystalline tyrosine
  • MPL lipopolysaccharide
  • MPLA and LPS are examples of endotoxins.
  • the adjuvant is one or more endotoxins.
  • the one or more adjuvants may be hydrophobic, hydrophilic, amphiphilic or a combination thereof.
  • the adjuvant is a lipid-based adjuvant (e.g.
  • oil-in-water emulsions such as squalene-based oil-in-water emulsions like MF59, or a water-in-oil emulsion
  • a mineral-based adjuvant e.g. a minieral-salt based adjuvant, like aluminum salts such as aluminum hydroxide or aluminum phosphate, or calcium phosphate
  • a saponin-based adjuvant e.g. Quil-A, QS-21
  • nucleic acid-based adjuvant e.g.
  • CpG oligodeoxynucleotides ODN
  • polinosinic:polycytidylic acid poly(I:C)
  • an endotoxin or derivative thereof e.g. monophosphoryl lipid A (MPLA), LPS, glucopyranosyl lipid adjuvant (GLA), baceterial toxoids
  • a protein-based adjuvant e.g. TLR5 agonists such as flagellin, a cytokine or chemokine
  • a glycan-based adjuvant e.g. beta-glucans or Advax delta inulin
  • the adjuvant is a TLR agonist (e.g. a TLR4 agonist or a TLR7/8 agonist).
  • the adjuvant is selected from the group consisting of squalene, telratolimod, MPLA, and combinations thereof.
  • Vaccines which are typically viewed as biological preparations that provide active acquired immunity to a particular disease-causing entity such as a pathogen or a cancerous cell, principally focus on raising the adaptive immune response against specific antigens, yet it has been proposed that certain vaccines can have beneficial effects for heterologous infections as they provide an enduring, non-specific immunological asset, coined trained immunity.
  • Trained immunity-inducing nanoparticles thus consist of valuable targets for the development of next-generation nano-vaccines that can not only engage the innate as well as the adaptive immune response, but integrate multiple epitopes for antigen presentation, along with the targeted delivery of additional adjuvants, to enhance immune response.
  • vaccines are conceived more broadly here as encompassing compositions which induce adaptive immune response and/or trained immunity.
  • the apolipoprotein lipid nanoparticle further comprises one or more trained immunity inducer.
  • the trained immunity inducer is muramyl tripeptide phosphatidylethanolamine (MTP-PE).
  • the aNPs may contain various combinations of adjuvants and/or trained immunity inducers.
  • they may comprise or contain precisely (i) MTP-PE and MPLA; (ii) MTP-PE and telratolimod; (iii) squalene and telratolimod; (iv) squalene and MTP-PE; (v) squalene and MPLA; (vi) MPLA and telratolimod; (vii) squalene, telratolimod, MPLA and MTP-PE; (viii) squalene, telratolimod, and MTP-PE, (ix) squalene, MPLA and MTP-PE, (x) squalene, telratolimod, and MPLA, or (xi) telratolimod, MPLA and MTP-PE.
  • the adjuvants are a combination of squalene, telratolimod and MPLA. In certain embodiments, the adjuvants are a combination of squalene, telratolimod, MPLA and MTP- PE .
  • the aNP comprises one or more adjuvants in an amount of from 0 to 30%, from 0 to 20%, from 0 to 10%, such as from 1 to 10%, from 2 to 10%, from 3 to 10%, of the total amount of adjuvants, phospholipids, lipids and sterols in the aNP. In certain embodiments, the ratio of the one or more adjuvants to phospholipids and/or lipids (e.g. triglycerides) based on weight is from 3: 1 to 1:500, such as from 3:25 to 1: 1 or from 3:95 to 4:45.
  • the ratio of the one or more adjuvants to phospholipids based on weight is from 3: 1 to 1:500, such as from 3:25 to 1: 1.
  • the aNP comprises one or more adjuvants, e.g. amphiphilic and/or hydrophilic adjuvants, in an amount of from 0 to 30 wt.% or from 1 to 30 wt.% of phospholipids , such as from 2 to 20 wt.% of phospholipids, from 3 to 15 wt.% of phospholipids or from 10 to 20 wt.% of phospholipids.
  • adjuvants e.g. amphiphilic and/or hydrophilic adjuvants
  • the total amount of adjuvant, such as the total amount of amphiphilic and/or hydrophilic adjuvant, in the aNP is from 0 to 20 wt.% or from 1 to 30 wt.% of phospholipids , such as from 2 to 20 wt.% of phospholipids, from 3 to 15 wt.% of phospholipids or from 10 to 20 wt.% of phospholipids.
  • the total amount of adjvuvants such as the total amount of hydrophobic adjuvants, in the aNP as taught herein is from 0 to 20 wt.% or from 1 to 20 wt.% of lipids (e.g. triglycerides), such as from 2 to 15 wt.% of lipids (e.g. triglycerides), or from 2 to 10 wt.% of lipids (e.g. triglycerides).
  • the aNP is capable of eliciting an immune response to the one or more antigens, when administered to a subject, optionally wherein the immune response is adaptive immune response or innate immune response such as trained immunity, optionally wherein the subject is human.
  • the aNP may further comprise immune suppressive molecules, such as in addition to or instead of the one or more adjuvants.
  • immune suppressive molecules may allow to use the aNP as an antigen specific tolerogenic vaccine.
  • the aNP may comprise a payload, preferably wherein the payload is selected from the group consisting of a nucleic acid or a nucleic acid analogue, a therapeutic, a biologic, and combinations thereof.
  • the term payload refers to a compound included in a lipid nanoparticle, not being an apolipoprotein, phospholipid, adjuvant, antigen or sterol.
  • the payload may for example be a pharmaceutical compound.
  • the lipid nanoparticle is particularly suitable for lipophilic payloads but may also be used for amphipathic molecules.
  • the pharmaceutical compound many be an organic compound, peptide, protein, nucleic acid or nucleic acid analogue, biologic or lipid.
  • the payload may be a nucleic acid or a nucleic acid analogue.
  • examples may be but are not limited to mRNA, siRNA, sgRNA, miRNA, piRNA, snRNA, snoRNA, srRNA or tsRNA.
  • the nucleic acid analogue may be peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA), or mixtures or combinations thereof.
  • Hofstraat et al. 49 have demonstrated the successful integration of small interfering RNAs (siRNAs) into aNPs.
  • the aNP comprises a nucleic acid and a cationic or ionizable cationic lipid.
  • the aNP comprises a core surrounded by a surface layer, the nucleic acid and the cationic or ionizable cationic lipid are comprised by the core, and the fusion protein or fusion proteins or combinations thereof as taught herein and the phospholipids, and optionally sterols, are comprised by the surface layer.
  • the aNPs of present invention have a similar size and stability in PBS as aNPs formulated with an apolipoprotein or an apolipoprotein mimetic that is not fused to an antigen.
  • the lipid nanoparticle has an average size of 10 to 100 nm, such as from 30 to 100 nm.
  • the lipid nanoparticle is a sphere.
  • the lipid nanoparticles are spherical with average size of 10 to 100 nm, such as from 30 to 100 nm, like about 50 nm.
  • nanoparticle size can be determined by dynamic light scattering as common in the art.
  • the fusion protein or the aNP as taught herein can be stored at fridge temperature, such as at a temperature of about 4°C, for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days or at least 12 days.
  • the method may also contain the steps: al) expressing and isolating the fusion protein which is a genetic fusion comprising the apolipoprotein component and the one or more antigens, and/or a2) chemically conjugating the apolipoprotein component and the one or more antigens to obtain the fusion protein and isolating the fusion protein.
  • the method may further comprise the steps of: bl’) mixing, preferably rapid mixing, of lipid components (e.g. phospholipid and optionally sterols, lipids and/or one or more adjuvants) in an organic solvent to produce nanoparticles; b2’) mixing, preferably rapid mixing, of lipid nanoparticles with the apolipoprotein fusion to produce the nanoparticle at a pH between 5.5 and 9.0, preferably at a pH between 6.0 and 8.0, more preferably at a pH between 6.5 and 8.0.
  • step b 1 ’ comprises forming a lipid fdm of the lipid components and dissolving the lipid fdm in an organic solvent, prior to step b2’ .
  • the combining step may comprise: bl”) mixing, preferably rapid mixing, of lipid components in organic solvent with a nucleic acid in an aqueous buffer to produce nanoparticles, wherein the aqueous buffer has a pH of 5.5 or lower, preferably 5.0 or lower; and b2”) mixing, preferably rapid mixing, of lipid nanoparticles with the apolipoprotein fusion to produce the nanoparticle at a pH between 5.5 and 9.0, preferably at a pH between 6.0 and 8.0, more preferably at a pH between 6.5 and 8.0.
  • the lipid components may typically comprise a phospholipid, a sterol, a cationic or ionizable cationic lipid, and optionally a lipid.
  • a further aspect provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein or aNP as taught herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical composition is a vaccine.
  • pharmaceutically acceptable as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
  • “Acceptable carrier, diluent or excipient” refers to an additional substance that is acceptable for use in human and/or veterinary medicine, with particular regard to vaccines.
  • an acceptable carrier, diluent or excipient may be a solid or liquid fdler, diluent or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers, well known in the art may be used.
  • These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate and carbonates, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulphates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
  • Any safe route of administration may be employed for providing a patient with the vaccine.
  • oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intranasal, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigens are donor-specific antigens (i.e. alloantigens), such as one or more donor MHC antigens, for use in treating and/or preventing transplant rejection, such as by causing transplant or immune tolerance.
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigens are donor-specific antigens is being administered to the subject receiving the transplant using sequential doses prior to and/or after the transplantation.
  • provided herein is a method of treating and/or preventing a transplant rejection in a subject, comprising administering a therapeutically and/or prophylactically effective amount of the fusion protein or the aNP or the pharmaceutical composition as taught herein to the subject.
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein wherein the one or more antigens are donor-specific antigens for use in the manufacture of a medicament for treating and/or preventing a transplant rejection in a subject.
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the antigen is a disease-associated antigen, for use in treating and/or preventing a disease caused by an abnormal immune response, such as a chronic inflammatory disease (e.g. inflammatory bowel disease, atherosclerosis), an autoimmune disease, an immune -mediated inflammatory disorder (IMID), an autoinflammatory disease, or an allergic disease).
  • a chronic inflammatory disease e.g. inflammatory bowel disease, atherosclerosis
  • IMID immune -mediated inflammatory disorder
  • the one or more antigens in the fusion protein or aNP may comprise nucleotide-binding oligomerization domain containing 2 (N0D2).
  • the one or more antigens may comprise Oxidized low-density lipoprotein (oxLDL).
  • a disease caused by an abnormal immune response such as a chronic inflammatory disease (e.g. inflammatory bowel disease, atherosclerosis), an autoimmune disease, an immune-mediated inflammatory disorder (IMID), an autoinflammatory disease, or an allergic disease)
  • IMID immune-mediated inflammatory disorder
  • an allergic disease comprising administering a therapeutically and/or prophylactically effective amount of the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the antigen is a disease-associated antigen, to the subject.
  • the antigen is a disease-associated antigen, for use in the manufacture of a medicament for treating and/or preventing a disease caused by an abnormal immune response, such as a chronic inflammatory disease (e.g. inflammatory bowel disease, atherosclerosis), an autoimmune disease, an immune-mediated inflammatory disorder (IMID), an autoinflammatory disease, or an allergic disease.
  • a chronic inflammatory disease e.g. inflammatory bowel disease, atherosclerosis
  • IMID immune-mediated inflammatory disorder
  • an autoinflammatory disease e.g. asthma, asthma, asthma, asthma, or an allergic disease.
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein may also be used for allergy de sensitization, such as by delivering allergen-derived epitopes in a controlled manner.
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is an allergen or is derived from an allergen, for use in the treatment and/or prevention of an allergic disease or reaction, such as an immediate hypersensitivity reaction (IHR).
  • IHR immediate hypersensitivity reaction
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein is being administered to the subject suffering from the allergy using sequential, incremental doses.
  • the allergic disease may be allergic rhinitis, asthma, or a food allergy.
  • the antigen may be Ara h 2 for a subject suffering from a peanut allergy
  • the antigen may be Bet v 1 for a subject suffering from a Birch pollen allergy or the antigen may be Fel d 1 for a subject suffering from a cat allergy.
  • a method of treating and/or preventing an allergic disease or reaction in a subject comprising administering a therapeutically and/or prophylactically effective amount of the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is an allergen or is derived from an allergen, to the subject.
  • fusion protein or the aNP or the pharmaceutical composition as taught herein for use in the manufacture of a medicament for treating and/or preventing an allergic disease or reaction in a subject, wherein the one or more antigen is an allergen or is derived from an allergen.
  • IHR immediate hypersensitivity reaction
  • a substance such as a small compound or a drug
  • IgE immunoglobulin E
  • IHR may manifest as clinical urticaria, vomiting, wheezing, angioedema, bronchospasm and/or anaphylaxis.
  • anaphylaxis is the most severe IHR and typically occurs within minutes of exposure to the small compound or drug and can cause itchy rash, throat or tongue swelling, shortness of breath, vomiting, lightheadedness and low blood pressure and eventually death.
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein for use in treating and/or preventing a metabolic disease, such as type 1 diabetes (T1D), wherein if the metabolic disease is T1D, the antigen may be proinsulin, glutamic acid decarboxylase 65 or IA-2.
  • a metabolic disease such as type 1 diabetes (T1D)
  • the antigen may be proinsulin, glutamic acid decarboxylase 65 or IA-2.
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein for use in the manufacture of a medicament for treating and/or preventing a metabolic disease, such as type 1 diabetes (T1D), wherein if the metabolic disease is T1D, the antigen may be proinsulin, glutamic acid decarboxylase 65 or IA-2.
  • T1D type 1 diabetes
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein may also be used for autoimmune disease modulation, such as by reprogramming or dampening the overactive immune response in patients suffering from an autoimmune disease.
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is an autoimmune disease-associated antigen, such as a self-antigen (autoantigen), for use in the treatment and/or prevention of an autoimmune disease.
  • an autoimmune disease-associated antigen such as a self-antigen (autoantigen)
  • the autoimmune disease may be rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), myositis, Sjogren’s syndrome, scleroderma (e.g.
  • a method of treating and/or preventing an autoimmune disease in a subject comprising administering a therapeutically and/or prophylactically effective amount of the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is an autoimmune disease-associated antigen, such as a self-antigen, to the subject.
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein wherein the one or more antigen is an autoimmune disease-associated antigen, such as a self-antigen, for use in the manufacture of a medicament for treating and/or preventing an autoimmune disease.
  • the antigens may be citrullinated proteins (e.g., citrullinated fibrinogen, vimentin).
  • the antigens may be myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG).
  • MBP myelin basic protein
  • PBP proteolipid protein
  • MOG myelin oligodendrocyte glycoprotein
  • the antigens may be Ro (SS-A) or La (SS-B) .
  • the antigens may be signal recognition particle (SRP) or aminoacyl-tRNA synthetase (e.g., Jo-1). If the autoimmune disease is Sjogren’s syndrome, the antigens may be Ro (SS-A) or La (SS-B). If the autoimmune disease is scleroderma (systemic sclerosis), the antigens may be topoisomerase I or centromere proteins (CENP-B). If the autoimmune disease is Hashimoto’s thyroiditis, the antigens may be thyroid peroxidase (TPO) or thyroglobulin. If the autoimmune disease is Graves’ disease, the antigen may be the thyroid-stimulating hormone receptor (TSHR).
  • SRP signal recognition particle
  • aminoacyl-tRNA synthetase e.g., Jo-1
  • the antigens may be Ro (SS-A) or La (SS-B). If the autoimmune disease is scleroderma (systemic sclerosis), the antigens may be top
  • the antigens may be insulin, glutamic acid decarboxylase (GAD65), or IA-2 (Insulinoma-associated protein 2). If the autoimmune disease is Addison’s disease, the antigen may be 21 -hydroxylase. If the autoimmune disease is myasthenia gravis (MG), the antigens may be acetylcholine receptor (AChR) or MuSK (Muscle-specific kinase). If the autoimmune disease is Guillain-Barre syndrome, the antigens may be gangliosides (e.g., GM1, GDla, GQlb).
  • the antigen may be HLA-Cw6. If the autoimmune disease is dermatomyositis, the antigens may be signal recognition particle (SRP) or transcription intermediary factor 1 (TIPI). If the autoimmune disease is vasculitis, the antigens may be proteinase 3 (PR3) or myeloperoxidase (MPO). If the autoimmune disease is rheumatoid vasculitis, the antigens may be citrullinated proteins, PR3, or MPO. If the autoimmune disease is urticarial vasculitis, the antigen may be complement components (e.g., Clq).
  • SRP signal recognition particle
  • TIPI transcription intermediary factor 1
  • MPO myeloperoxidase
  • the antigens may be citrullinated proteins, PR3, or MPO. If the autoimmune disease is urticarial vasculitis, the antigen may be complement components (e.g., Clq).
  • the antigens may be melanocyte antigens (e.g., tyrosinase, MART-1). If the autoimmune disease is Crohn’s disease, the antigen may be NOD2 (nucleotide-binding oligomerization domain containing 2). If the autoimmune disease is celiac disease, the antigen may be gliadin (component of gluten). If the autoimmune disease is ulcerative colitis, the antigens may be intestinal epithelial cells and goblet cell antigens. If the autoimmune disease is autoimmune gastritis, the antigen may be parietal cell antigen (H+/K+ ATPase). If the autoimmune disease is chronic inflammatory demyelinating polyneuropathy (CIPD), the antigens may be myelin-associated glycoprotein (MAG), neurofascin, or contactin- 1.
  • CIPD chronic inflammatory demyelinating polyneuropathy
  • the antigens may be myelin-associated glycoprotein (MAG),
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is a tumour antigen, for use in the treatment and/or prevention of a neoplastic disease.
  • a method of treating and/or preventing a neoplastic disease in a subject comprising administering a therapeutically and/or prophylactically effective amount of the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is a tumour antigen, to the subject.
  • fusion protein or the aNP or the pharmaceutical composition as taught herein wherein the one or more antigen is a tumour antigen, for use in the manufacture of a medicament for treating and/or preventing a neoplastic disease in a subject.
  • a further aspect provides the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is from a pathogen, for use in the treatment and/or prevention of an infectious disease caused by or associated with the pathogen.
  • provided herein is a method of treating and/or preventing an infectious disease caused by or associated with a pathogen in a subject, comprising administering a therapeutically and/or prophylactically effective amount of the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is from the pathogen, to the subject.
  • the fusion protein or the aNP or the pharmaceutical composition as taught herein, wherein the one or more antigen is from a pathogen, for use in the manufacture of a medicament for treating and/or preventing an infectious disease caused by or associated with the pathogen.
  • the pathogen may be any as discussed elsewhere in this specification.
  • the infection may be a coronavirus infection, more preferably a SARS-CoV-2 infection.
  • the SARS-CoV-2 infection may be an infection with any variant of the SARS-CoV-2 virus, such as the SARS-CoV-2 variants as described elsewhere herein.
  • the fusion protein as taught herein comprises an S protein SARS-CoV- 2 variant Beta or a fragment thereof
  • the SARS-CoV-2 infection to be treated is preferably a SARS- CoV-2 infection with the WT (Wuhan) and/or Beta SARS-CoV-2 variant.
  • a method for preventing an infection with a pathogen such as for preventing a coronavirus infection (e.g. a method of vaccinating against a coronavirus), like a SARS-CoV-2 infection, in a subject comprising administering a prophylactically effective amount of the fusion protein, the aNP or the pharmaceutical composition as taught herein.
  • a pathogen such as for preventing a coronavirus infection
  • a coronavirus infection e.g. a method of vaccinating against a coronavirus
  • a coronavirus infection e.g. a method of vaccinating against a coronavirus
  • the fusion protein, the aNP or the pharmaceutical composition as taught herein is administered intramuscular.
  • treatment refers to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit.
  • therapeutic benefit is meant eradication or amelioration or reduction (or delay) of progress of the underlying disease being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration or reduction (or delay) of progress of one or more of the physiological symptoms associated with the underlying disease such that an improvement or slowing down or reduction of decline is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disease.
  • Reference to “therapy” or “treatment” broadly encompasses both curative and preventative treatments, and the terms may particularly refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder.
  • the terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies. Measurable lessening includes any statistically significant decline in a measurable marker or symptom.
  • the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease .
  • the terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition.
  • the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment.
  • the term may encompass ex vivo or in vivo treatments as appropriate in the context of the present invention.
  • subject typically and preferably denote humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably non-human mammals. Particularly preferred are human subjects including both genders and all age categories thereof. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The term subject is further intended to include transgenic non-human species.
  • subject in need of treatment refers to subjects diagnosed with or having a disease as recited herein and/or those in whom said disease is to be prevented.
  • terapéuticaally effective amount generally denotes an amount sufficient to elicit the pharmacological effect or medicinal response in a subject that is being sought by a medical practitioner such as a medical doctor, clinician, surgeon, veterinarian, or researcher, which may include inter alia alleviation of the symptoms of the disease being treated, in either a single or multiple doses.
  • a medical practitioner such as a medical doctor, clinician, surgeon, veterinarian, or researcher
  • prophylactically effective amount in particular relates to situations where the pharmacological effect or medicinal response in a subject to be elicit relates to prevention of the disease.
  • Appropriate effective doses of the present molecules may be determined by a qualified physician with due regard to the nature and severity of the disease, and the age and condition of the patient.
  • neoplastic disease generally refers to any disease or disorder characterised by neoplastic cell growth and proliferation, whether benign (not invading surrounding normal tissues, not forming metastases), pre-malignant (pre-cancerous), or malignant (invading adjacent tissues and capable of producing metastases).
  • neoplastic disease generally includes all transformed cells and tissues and all cancerous cells and tissues. Neoplastic diseases or disorders include, but are not limited to abnormal cell growth, benign tumors, premalignant or precancerous lesions, malignant tumors, and cancer.
  • neoplastic diseases or disorders are benign, pre-malignant, or malignant neoplasms located in any tissue or organ, such as in the prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, or urogenital tract.
  • tissue or organ such as in the prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, or urogenital tract.
  • tumor or tumor tissue refer to an abnormal mass of tissue that results from excessive cell division.
  • a tumor or tumor tissue comprises tumor cells which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign, pre-malignant or malignant, or may represent a lesion without any cancerous potential.
  • a tumor or tumor tissue may also comprise tumor-associated non-tumor cells, e.g., vascular cells which form blood vessels to supply the tumor or tumor tissue. Non-tumor cells may be induced to replicate and develop by tumor cells, for example, the induction of angiogenesis in a tumor or tumor tissue.
  • the neoplastic disease is cancer.
  • cancer refers to a malignant neoplasm characterised by deregulated or unregulated cell growth.
  • the term “cancer” includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject’s body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
  • metastasis generally refers to the spread of a cancer from one organ or tissue to another non-adjacent organ or tissue. The occurrence of the neoplastic disease in the other non- adjacent organ or tissue is referred to as metastasis.
  • cancer examples include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include without limitation: squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including smallcell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung and large cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as CNS cancer, mel
  • cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS- Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Urethra, Central Nerv
  • any substance as taught herein may be administered as the sole pharmaceutical agent (active pharmaceutical ingredient) or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects.
  • two or more substances as taught herein may be co-administered.
  • one or more substances as taught herein may be co-administered with a pharmaceutical agent that is not a substance as envisaged herein.
  • the substances as taught herein may be combined with known anti -cancer therapy or therapies, such as for example surgery, radiotherapy, chemotherapy, biological therapy, or combinations thereof.
  • chemotherapy as used herein is conceived broadly and generally encompasses treatments using chemical substances or compositions.
  • Chemotherapeutic agents may typically display cytotoxic or cytostatic effects.
  • a chemotherapeutic agent may be an alkylating agent, a cytotoxic compound, an anti-metabolite, a plant alkaloid, a terpenoid, a topoisomerase inhibitor, or a combination thereof.
  • biological therapy as used herein is conceived broadly and generally encompasses treatments using biological substances or compositions, such as biomolecules, or biological agents, such as viruses or cells.
  • a biomolecule may be a peptide, polypeptide, protein, nucleic acid, or a small molecule (such as primary metabolite, secondary metabolite, or natural product), or a combination thereof.
  • biomolecules include without limitation interleukins, cytokines, anti-cytokines, tumor necrosis factor (TNF), cytokine receptors, vaccines, interferons, enzymes, therapeutic antibodies, antibody fragments, antibody-like protein scaffolds, or combinations thereof.
  • biomolecules include but are not limited to aldesleukine, alemtuzumab, atezolizumab, bevacizumab, blinatumomab, brentuximab vedotine, catumaxomab, cetuximab, daratumumab, denileukin diftitox, denosumab, dinutuximab, elotuzumab, gemtuzumab ozogamicin, 90 Y-ibritumomab tiuxetan, idarucizumab, interferon A, ipilimumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, ramucirumab, rituximab, tasonermin, 131 I-tositumomab
  • Suitable oncolytic viruses include but are not limited to talimogene laherparepvec.
  • Further categories of anti-cancer therapy include inter alia hormone therapy (endocrine therapy), immunotherapy, and stem cell therapy, which are commonly considered as subsumed within biological therapies.
  • suitable hormone therapies include but are not limited to tamoxifen; aromatase inhibitors, such as atanastrozole, exemestane, letrozole, and combinations thereof; luteinizing hormone blockers such as goserelin, leuprorelin, triptorelin, and combinations thereof; anti-androgens, such as bicalutamide, cyproterone acetate, flutamide, and combinations thereof; gonadotrophin releasing hormone blockers, such as degarelix; progesterone treatments, such as medroxyprogesterone acetate, megestrol, and combinations thereof; and combinations thereof.
  • the term “immunotherapy” broadly encompasses any treatment that modulates a subject’s immune system.
  • the term comprises any treatment that modulates an immune response, such as a humoral immune response, a cell-mediated immune response, or both.
  • Immunotherapy comprises cell-based immunotherapy in which immune cells, such as T cells and/or dendritic cells, are transferred into the patient.
  • the term also comprises an administration of substances or compositions, such as chemical compounds and/or biomolecules (e.g., antibodies, antigens, interleukins, cytokines, or combinations thereof), that modulate a subject’s immune system.
  • substances or compositions such as chemical compounds and/or biomolecules (e.g., antibodies, antigens, interleukins, cytokines, or combinations thereof), that modulate a subject’s immune system.
  • cancer immunotherapy include without limitation treatments employing monoclonal antibodies, for example Fc-engineered monoclonal antibodies against proteins expressed by tumor cells, immune checkpoint inhibitors, prophylactic or therapeutic cancer vaccines, adoptive cell therapy, and combinations thereof.
  • Adoptive cell therapy can refer to the transfer of cells, most commonly immune-derived cells, such as in particular cytotoxic T cells (CTLs), back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing tissue rejection and graft vs. host disease issues.
  • TCR T cell receptor
  • P chains selected peptide specificity
  • CARs chimeric antigen receptors
  • T cells specific for selected targets, such as malignant cells
  • CAR constructs include without limitation 1) CARs consisting of a single -chain variable fragment of an antibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD 8 a hinge domain and a CD 8 a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3 ⁇ or FcRy; and 2) CARs further incorporating the intracellular domains of one or more costimulatory molecules, such as CD28, 0X40 (CD 134), or 4- IBB (CD 137) within the endodomain, or even including combinations of such costimulatory endodomains.
  • costimulatory molecules such as CD28, 0X40 (CD 134), or 4- IBB (CD 137
  • Stem cell therapies in cancer commonly aim to replace bone marrow stem cells destroyed by radiation therapy and/or chemotherapy, and include without limitation autologous, syngeneic, or allogeneic stem cell transplantation.
  • the stem cells in particular hematopoietic stem cells, are typically obtained from bone marrow, peripheral blood or umbilical cord blood. Details of administration routes, doses, and treatment regimens of anti-cancer agents are known in the art, for example as described in “Cancer Clinical Pharmacology” (2005) ed. By Jan H. M. Schellens, Howard L. McLeod and David R. Newell, Oxford University Press.
  • a combination therapy with any molecule as taught herein with one or more of a MEK inhibitor e.g.
  • a SHP2 inhibitor e.g., TNO155
  • an mTOR inhibitor e.g., rapamycin or a rapamycin derivative (“rapalog”), including sirolimus, temsirolimus (CCI-779), temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus (AP -23573)
  • rapamycin or a rapamycin derivative rapalog
  • active components of any combination therapy may be admixed or may be physically separated, and may be administered simultaneously or sequentially in any order.
  • pathogen treatments or prevents with the present substances may combined with other antibacterials, antifimgals, or anti-parasite treatments as known in the art.
  • nextgeneration vaccine platforms based on apolipoprotein Al (apoAl) nanoparticles (aNPs) (Fig. la-b).
  • aNP platform consists of (phospho-)lipid nanoaggregates that integrate and are stabilized by apoAl .
  • the resulting vaccine-aNP displays intrinsic tropism for antigen presenting cells (APCs) synergistically facilitates for the targeted delivery of antigens and adjuvants into pertinent tissues.
  • the vaccine-aNP can be administered intramuscularly (I.M.) for effective lymph node accumulation, or intravenously (I V.) for preferential engagement of the hematopoietic organs.
  • I.M. intramuscularly
  • I V. intravenously
  • the developed aNP- vaccine has the potential to serve as a cancer vaccine as well as a vaccine against infectious disease.
  • We evaluated the vaccine-aNP’ s potential for effective vaccination in mice using a combination of protein engineering tools, in vivo biodistribution studies and ex vivo analyses, in vitro immunological assays, as well as preventative vaccination studies in mice.
  • Vaccine development constitutes a paramount accomplishment in modem medicine, offering a preemptive approach to safeguarding public health 1 .
  • vaccines have played a pivotal role in controlling and eradicating a number of infectious diseases 2 .
  • scientists have expanded the focus of vaccine research beyond infectious agents to encompass the realm of cancer 3 - 4 .
  • cancer vaccines are considered an innovative frontier in medical research and are poised to revolutionize the landscape of cancer prevention and treatment, there remain several challenges with respect to their development.
  • inefficient delivery of antigens and adjuvants to draining lymph nodes has shown to be a major hurdle in the development of efficacious cancer vaccines 5 .
  • suboptimal clinical outcomes have been observed for several free soluble cancer vaccines; likely attributable to rapid antigen renal clearance and inadequate codelivery of antigens and adjuvants to antigen presenting cells (APCs) 5 s .
  • APCs antigen presenting cells
  • Nanoparticles have emerged as a promising technology in the field of cancer vaccine research'' l s . as various nanovaccines have been used for the targeted co-delivery of adjuvants and antigens to lymphoid organs 1 ' 21 . Nonetheless, the utilization of nanoparticles for the development of cancer vaccines can raise safety concerns, while the ability to scale up production is a noteworthy challenge, as the ideal vaccine platform should be able to readily and easily incorporate novel antigens.
  • apoAl as an antigen carrier, by fusing the model antigen ovalbumin (OVA) and the antigenic peptide OT1 (ovalbumin residue) to apoAl, and construct 50 nm sized, spherical aNPs.
  • OVA model antigen ovalbumin
  • OT1 antigenic peptide OT1 (ovalbumin residue)
  • squalene a dual TLR7/8 agonist
  • MPLA monophosphoryl-Lipid A
  • aNPs nanostructures based on high-density lipoprotein (HDL), engineered from molecules native to the human body; namely phospholipids, cholesterol, triglycerides and apoAl, which is the principal protein component of HDL 30 .
  • HDL high-density lipoprotein
  • apoAl -OVA fusion protein of apoAl to OVA
  • a genetic construct consisting of human apoAl and OVA, bearing a chicken RPTPo signal sequence for secretion in the 5 ’ and a twin strep-purification tag (strep-tag II) in the 3 ’ end, used for subsequent purification.
  • ApoAl is coupled to OVA through a flexible (GGS)? (SEQ ID NO: 19) linker; seven consecutives glycine-glycine-serine repeats (see Supplementary Table 1, 2).
  • the fusion protein construct was expressed in mammalian cells (human embryonic kidney cells (HEK239S)) and subsequent purification resulted in the recovery of apoAl -OVA, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Fig. 1c, left and Fig. 6a
  • the apoAl-OVA may further also comprise a fluorescent protein or tag linked to the C-terminus, such as mVenus (e.g. Fig. Ih) in order to solubilize and/or enhance expression. Successive mVenus cleavage and strep-tag purification will than yield pure ApoAl-OVA fusion protein (data not shown).
  • apoAl-OTl apoAl-OTl
  • apoAl-OT- I apoAl-OTl
  • Fig. 6b, d and Supplementary Table 1, 2 Fig. 12
  • Fig. 6c and Fig. li a maleimide- modified OT1 peptide
  • mBMDCs Murine Bone Marrow-derived Dendritic Cells
  • mBMDCs murine bone marrow-derived dendritic cells
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • TCR T-cell receptor
  • MHC-I major histocompatibility complex I
  • This variation in T cell proliferation levels is consistent with the expected variations in OT1 availability on the major histocompatibility complex I (MHC-I) for the different experimental conditions, given that mBMDCs are exposed to equal protein quantities in each case resulting in the presentation of not only OT1 peptides but also other processed peptide epitopes on their surface.
  • MHC-I major histocompatibility complex I
  • spherical aNPs 25 composed of phospholipids, cholesterol, triglycerides and apoAl-OVA or apoAl-OTl (”apoAl-OTl”).
  • apoAl-OT1 and apoAl -OVA fusion proteins resulted in homogenous, spherical particles, as confirmed by cryogenic transmission electron microscopy (cryo-TEM; Fig. le and Fig. 8a-bOTl).
  • DLS Dynamic light scattering revealed similar size for both aNP formulations, approximating 50 nm in diameter (Fig. If).
  • OVA-aNPs and OT1 aNPs are internalised by APCs.
  • OT-I-aNP OVA-aNPs and OT1 aNPs
  • mBMDCs mouse bone marrow derived DCs
  • Fig. 1g Prior to imaging, the cell membrane was stained with the lipophilic fluorescent label DiB (Fig. 1g). Confocal fluorescence microscopy confirmed OVA-aNP and OT1 aNP uptake, as verified by the presence of Cy3 within the cell cytoplasm (Fig. 1g).
  • Cy3- aNPs formulated with apoAl that is not fused to an antigen are internalised by both RAW 264.7 macrophages and BMDCs (Fig. 8). These data indicate that antigens incorporated in the vaccine- aNP platform are efficiently internalized by APCs.
  • C57BL/6 mice were injected intravenously (IV) or intramuscularly (IM) with zirconium-89 ( 89 Zr) radiolabelled aNPs ( 89 Zr-aNPs) OVA-aNPs ( 89 Zr-OVA-aNPs), and OTl-aNPs ( 89 Zr-OTl-aNPs).
  • Positron emission tomography with computed tomography (PET-CT) imaging at 24 h post administration showed that I.V. administered 89 Zr-labelled aNPs primarily accumulated in the liver, spleen and bone marrow in contrast to aNPs administered through the I.M. route (Fig. 2a). In particular, I.M.
  • IM administration typically employed for vaccines against infectious diseases showed favourable uptake of aNP vaccine platform by lymph nodes. This is of significance, since lymph nodes serve as central hubs for APCs where the adaptive immune response, vital for effective vaccine development, is primarily initiated 46 .
  • the unconventional IV administration route has shown promise for the development of cancer vaccines by significantly engaging cytotoxic T lymphocytes 47,48 .
  • the aNP vaccine platform developed here exhibits potential for successful use both as a cancer vaccine and as a vaccine against infectious diseases, owing to its ability to accommodate different modes of administration and its favourable interaction with immune cells in lymph nodes and other myeloid rich organs.
  • OVA-aNPs and OTl-aNPs interact with APCs, such as macrophages, dendritic cells, and other myeloid cells including monocytes and neutrophils, in both spleen (Fig. 2d) and lymph nodes (Fig. 2e) regardless of the administration route.
  • APCs such as macrophages, dendritic cells, and other myeloid cells including monocytes and neutrophils
  • lymphocytes including T and B cells.
  • the route of administration does not alter cell-specific biodistribution, it does affect distribution on organ level.
  • I.M. administration of antigen carrying aNPs is the most favorable administration route for vaccination purposes, as it effectively targets the lymph nodes, where adaptive immune responses are orchestrated
  • Free adjuvant administration in cancer vaccines can lead to their dissociation from the antigen, preventing effective entry into the lymphatic system 33 . Additionally, free adjuvants may undergo rapid degradation 34 , further diminishing the quantity that eventually reaches target organs, while the presence of free adjuvant circulating in the body has the potential to trigger autoimmunity 35 . To overcome this challenge, we devised a strategy to create a vaccine platform with tunable adjuvanticity.
  • aNPs that can integrate a diversity of selected adjuvants based on their hydrophobic or amphiphilic character and their interaction with diverse receptors: namely squalene 36 , telratolimod 37 , muramyl tripeptide phosphatidyl ethanolamine (MTP-PE), and monophosphoryl Lipid A (MPLA) 38 (Fig. 3a).
  • Squalene a triterpenoid derived from shark liver oil, is a main component of the licenced vaccine adjuvant MF59 39 and has been shown to successfully stimulate the immune system and increase host response.
  • squalene will successfully incorporate in the core of our formulated aNPs, more particularly OVA-aNPs.
  • sq-aNPs squalene adjuvanted aNPs
  • MPLA a known TLR4 agonist
  • MPLA-aNPs MPLA adjuvanted aNPs
  • MPLA-aNPs were successfully engineered, yielding homogenous, spherical nanoparticles, approximately 50 nm in diameter, that are stable at 4°C for a total of 14 days in PBS (data not shown).
  • Vaccines principally focus on raising the adaptive immune response against specific antigens 40 , yet it has been proposed that certain vaccines can have beneficial effects for heterologous infections as they provide an enduring, non-specific immunological asset 41 , coined trained immunity 42 .
  • Trained immunity-inducing nanoparticles thus consist of valuable targets for the development of nextgeneration vaccines that can not only engage the innate as well as the adaptive immune response, but integrate multiple epitopes for antigen presentation, along with the targeted delivery of additional adjuvants, to enhance immune response.
  • MTP-PE is known to bind nucleotide -binding oligomerization domain-containing protein 2 (N0D2) and has demonstrated its capacity as an inducer of trained immunity 27 2 '.
  • Adjuvant amount expressed as weight percentage (w%) of triglycerides for squalene, or POPCs for MTP-PE, MPLA, and telratolimod.
  • Supplementary Table 12 Amount of adjuvant incorporated in aNPs for three-adjuvant combinations.
  • Adjuvant amount expressed as weight percentage (w%) of triglycerides for squalene, or POPCs for MTP-PE, MPLA, and telratolimod.
  • aNPs containing all four specific adjuvants (Fig. 16a and Supplementary Table 13).
  • the resulting aNPs exhibited uniform spherical morphology, and an average diameter of approximately 50 nm. Additionally, they demonstrated notable stability, maintaining their structural integrity for a duration of 12 days when stored at 4°C in PBS (Fig. 16b, c).
  • Supplementary Table 13 Amount of adjuvant incorporated in aNPs for four-adjuvant combinations.
  • Adjuvant amount expressed as weight percentage (w%) of triglycerides for squalene, or POPCs for MTP-PE, MPLA, and telratolimod.
  • aNPs have similar size and stability in PBS as aNPs formulated with apoAl that is not fused to an antigen (data not shown) as well OVA-aNPs and OTl-aNPs (data not shown), confirming the versatility of our platform, enabling the incorporation of antigenic epitopes for infectious disease.
  • mice that received vaccine-aNPs tumor development was prevented, whereas mice that received PBS and apoAl-aNP controls lacking adjuvant and antigen did develop tumors (Fig. 4b-c).
  • Mice vaccinated with a mix of free OVA antigen and alum adjuvant showed intermediate tumor growth.
  • the weight of the mice did not change following vaccination with different vaccine-aNPs, indicating that vaccine-aNPs are well tolerated and non-toxic (data not shown).
  • We determined anti-OVA specific IgG titers on day 35 for all vaccinated mice (Fig. 4d). As anticipated, titers corresponded to values determined from vaccinated healthy mice (Fig. 3d) with vaccine-aNP formulations 13 and 15 resulting in the highest anti-OVA IgG levels.
  • antigen which initiates signal 1 by facilitating antigen presentation 1
  • adjuvant which is important for the engagement of signals 2 (co-stimulatory signals) and 3 (release of immunomodulatory cytokines) 45 .
  • mRNA-LNPs mRNA encapsulated in lipid nanoparticles
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the vaccine-aNPs high potency
  • our platform can also be employed in a therapeutic setting.
  • the approach might therefore be effective against already established B16F10- OVA tumors.
  • the differences in potency between aNPs with varying adjuvanticity may result in differences in therapeutic efficacy.
  • the vaccine-aNP platform technology can be deployed in a broad range of indications, spanning from preventing infectious diseases to immune-oncology and autoimmune diseases. Owing to its high degree of modularity, the aNPs can efficiently be adapted to diverse scenarios.
  • the ability to couple peptides to apoAl using click chemistry but also the incorporation of larger antigens, e.g.
  • a library of (neo-)epitopes opens an interesting new avenue for the vaccine-aNP platform in personalized treatment of cancers.
  • antigens and immune suppressive molecules instead of adjuvants, might be incorporated in the vaccine-aNP platform creating an antigen specific tolerogenic vaccine. Consequently, a tolerogenic phenotype is induced, characterized by reduced co-stimulatory marker expression and increased anti-inflammatory cytokine secretion by APCs decreased T helper cell activation and increased regulatory T cell activation 31 .
  • All vaccine-aNPs integrate a recombinant protein that is formulated into a nanoparticle with lipids and adjuvants.
  • lipids and adjuvants For applications where easy, cost-effective, and widescale manufacturing are key, such as for seasonal flu vaccines, creating a vaccine-aNP that contains no, or only one, adjuvant is preferred.
  • incorporating multiple adjuvants into the vaccine-aNP might be warranted. This ability to finetune the vaccine-aNP platform depending on the application, development speed and costs, demonstrates the large bandwidth of our approach.
  • mice and OT1 C57BL/6-Tg(TcraTcrb)1100Mjb/Crl were purchased from Charles River. All animals were co-housed in climate-controlled conditions, respectively, at 20-24 °C, 45-65 % humidity with 12 h light-dark cycles and provided water ad libitum and fed a standard chow diet.
  • Maleimide-functionalised OT1 peptides (Maleimide-SIINFEKL (SEQ ID NO: 21)) were synthesized (25 pmole scale) on a rink amide MBHA resin using automated Fmoc-based Solid-Phase peptide synthesis (SPPS) on a MultiPep RSi automated peptide synthesizer (Intavis). 6-Maleimidohexanoic acid was used to introduce a maleimide at the N-terminus of the peptide.
  • the peptides were cleaved using a mixture of 96.5% trifluoric acid, 2.5% dH2O, and 1% triisopropylsilane. This was incubated at room temperature for two hours. The peptides were then twice precipitated in ice cold diethyl ether and centrifuged. The ether was then decanted, and peptide was dried by evaporation. Peptides were dissolved in 50% acetonitrile in dH 2 O. The solution was lyophilized overnight to form the final dry peptides. The peptides were analyzed using LC-MS (Fig. 6c).
  • cysteine-fimctionalised apoAl (apoAl-S230C or apoAl- S141C) was incubated with five molar excesses of mal-OTl peptide in PBS (pH 7.9) and incubated at 4 °C overnight.
  • PBS PBS
  • the mixture was loaded on a PD-10 column (Cytiva) and purified apoAl-OTl fusion protein was collected. Purity and successful conjugation were confirmed by SDS-PAGE and Q-ToF LC-MS(WatersMassLynx v4.1), using MagTran VI.03 for MS.. Protein was snap-frozen in liquid nitrogen before storing at -80 °C.
  • Lentivirus was produced by cotransfecting human embryonic kidney 293T cells (HEK293T, ATCC® CRL3216TM) that have reached -80-90% confluency with the second generation pHR plasmid carrying the desired transgene and the vectors encoding for the packaging proteins (pCMVR8.74; gift from Didier Trono (Addgene plasmid #22036; http://n2t.nct/addgene:22036; RRID:Addgene_22036) and VSV-G envelope (pMD2.G, gift from Didier Trono (Addgene plasmid #12259; http://n2t.nct/addgene: 12259; RRID:Addgene_12259)) at a ratio of 1:2:
  • the produced lentivirus was pelleted by centrifugation at 50,000 x g, 4°C for two hours, resuspended in DMEM (41966; Thermo Fisher Scientific) supplemented with 2% (v/v) FBS (S-FBS-SA-015; Serana) and either used directly for cell line transduction or snap-frozen and stored at -80°C.
  • HEF293S GnTI- TetR Human embryonic kidney 293 S
  • DMEM transfection medium
  • the HEK293S could also be transduced with lentivirus harbouring apoAl-OVA-mVenus fusion protein, under the control of the major immediate-early (MIE) human cytomegalovirus (CMV) enhancer and promoter and two TetO operator sequences. .
  • MIE major immediate-early
  • CMV human cytomegalovirus
  • Cells were bulk-sorted for GFP expression (or alternatively mVenus) to enrich fusion protein expression by fluorescence-activated cell sorting (FACS) in growth media (DMEM + 10 % FBS + lx Pen/Strep) using a BD FACSAria III Cell sorter (BD Biosciences) equipped with operating at low-middle pressure and equipped with a 70 pm nozzle, 488 nm laser and a 530/30 nm detector for GFP. Cells were cultured under standard incubation conditions (37°C, in a humidified atmosphere of 5% CO2) and passaged upon reaching confluency (approximately every 2 days).
  • FACS fluorescence-activated cell sorting
  • cells were cultured in expression medium (50 % EX -CELL 293 Serum -Free Medium for HEK293 Cells (Merck) and 50 % FreeStyle 293 Expression Medium (Thermo Fisher Scientific)), supplemented with 2 mM Glutamax, 1 % Pen-Strep and 1 pg mL 1 doxycycline (Merck) on a shaker at 150 r.p.m. for 3 days at 37 °C. Culture supernatant containing fusion proteins was collected, centrifuged at 3,500 g, 4 °C for 15 minutes and filtered through 0.2 pm PES syringe filter to remove cell debris.
  • expression medium 50 % EX -CELL 293 Serum -Free Medium for HEK293 Cells (Merck) and 50 % FreeStyle 293 Expression Medium (Thermo Fisher Scientific)
  • Filtered soluble fraction was loaded on a StrepTactin XT 4flow 5 mL column (Cytiva) and washed with 5 column volumes W-buffer (150 mM NaCl, 100 mM Tris, 1 mM EDTA pH 8.0) with flow rate 1-2 m E min ' . Fusion proteins were eluted from the column with W-buffer supplemented with 50 mM biotin. Eluate was collected, concentrated and snap-frozen in liquid nitrogen before storing at -80 °C. Purity of the protein was assessed SDS-PAGE.
  • samples were mixed with SDS loading buffer (100 mM Tris-Cl, 4 % sodium dodecyl sulphate, 0.2 % bromophenol blue, 20 % glycerol, 200 mM DTT, pH 6.8) in 1: 1 ratio and heated for 5 minutes at 95°C.
  • SDS loading buffer 100 mM Tris-Cl, 4 % sodium dodecyl sulphate, 0.2 % bromophenol blue, 20 % glycerol, 200 mM DTT, pH 6.8
  • a solution of apoAl or apoAl -antigen, fusion protein in PBS (6.5 mb, 3.67 pM) was prepared.
  • the dissolved lipid film and the diluted protein were simultaneously injected using a microfluidic pump fusion 100 (Chemyx) into a Zeonor herringbone mixer (Microfluidic ChipShop, product code 10000076), with a flow rate of 0.8 mb min -1 for the lipid solution and a flow rate of 6 mb min -1 for the protein solution.
  • Biodistribution in mice was determined 24 hours post injection. After PBS perfusion, tissues of interest (blood, spleen, liver, lung, bone, muscle, bone marrow, kidney, heart, lymph nodes, intestines, side of injection) were collected and weighed, and radioactivity was measured using a WIZARD2 Automatic Gamma Counter (PerkinElmer). Data were corrected for radioactive decay, and percentage of injected dose per gram of tissue (%ID/g) was calculated.
  • aNPs were incubated with two molar excesses of DFO-p-NCS (5 mg mF 1 in DMSO) for 2 hours in PBS (pH 8.8) and separated from unreacted DFO-p-NCS via PD10 desalting column (GE Healthcare).
  • DFO coupled aNPs were incubated with 89 Zr at 37°C using a thermomixer at 600 r.p.m. for 1 hour. Radiolabelled aNPs were separated from unbound 89 Zr using Zeba spin desalting column filtration. Radioactivity was measured after desalting to determine the radiolabeling efficiency.
  • mBMDCs murine bone marrow cells were differentiated to dendritic cells.
  • Bone marrow progenitor cells of a C57BL/6 mice were flushed with PBS from the femurs, , filtered through a 70 mm cell strainer (BD Falcon). Cell suspension was incubated with Red Blood Cell (RBC) Lysis buffer (BioLegend) for 30 seconds, and washed and dissolved in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10 % FBS and 1 % Pen/Strep (culture medium). This medium is further referred to as “culture medium”.
  • GM-CSF granulocyte -macrophage colony-stimulating factor
  • IL-4 interleukin 4
  • mBMDCs were collected and cultured in a 96- well flat bottom plate, with 40,000 cells per well in culture medium. After 4 hours of incubation, cells were stimulated with LPS (Sigma Aldrich) and one of the proteins detailed in Supplementary Table 7. The next day, spleens were obtained from OT1 C57BL/6-Tg(TcraTcrb)1100Mjb/Crl mice and mashed, filtered through a 70 mm cell strainer, incubated with RBC lysis buffer for 4 minutes and washed with PBS. Next, T cells were isolated using the mouse Pan T Cell Isolation Kit II (Miltenyi Biotec).
  • Negative selected T cells were stained with carboxyl fluorescein succinimidyl ester (CFSE) (Invitrogen) according to the manufacturer’s protocol and dissolved in RPMI 1640, complemented with 5 % FCS, 1 % GlutaMax (Thermo Fischer Scientific) and 1 % gentamicin (Thermo Fischer Scientific).
  • CFSE carboxyl fluorescein succinimidyl ester
  • mBMDC stimulation medium was removed, and 200,000 OT1 T cells were added and co-cultured for 72 hours.
  • non-adherend cells were collected and transferred to a 96-well V-bottom plate for staining. The cells were centrifuged at 400 g for 5 minutes at 4°C.
  • the supernatant was removed, and the cells were washed once with 200 pL PBS-BSA (PBS 1 % w/v BSA (Sigma Aldrich), pH 7.4,). Next, surface markers and cell viability were determined by staining in 50 pL for 30 minutes at 4°C, using the antibodies and viability dye described in Supplementary Table 8. After two washes, the cells were re-suspended in 100 pL PBS-BSA and analyzed on a CytoFlex flow cytometer (Beckman Coulter).
  • mBMDCs were seeded in 96-well glass bottom plates (Cellvis), 22,000 cells per well and incubated with Cy3 labelled variants of either aNPs, OTl-aNPs or OVA-aNPs for 48 hours. Prior to imaging, cells were stained with 5 pg/ml DiB membrane stain (60036, Biotium) according to manufacturer’s instructions. The acquisition of images was performed using of a Leica SP8 confocal microscope, using a Leica HC PL APO 20x/0.75 dry objective and 405 nm, 488 nm, 552 nm, and 638 nm continuous wave diode lasers. For image analysis, the Leica LAS X software (version 5.1.0) and Image J were used.
  • Cryogenic Transmission Electron Microscopy (Cryo-TEM) samples were prepared by treating the surface of 200-mesh lacey carbon supported copper grids (Electron Microscopy Sciences) with plasma for 40 seconds using a Cressington 208 carbon coater. Subsequently, 3 pL of aNP samples ( ⁇ 1 mg protein per mL) was applied on a grid and vitrified into a thin film by plunge vitrification in liquid ethane by using an automatic robot (FEI Vitrobot Mark IV).
  • cryo-TEM imaging was performed on the cryoTITAN (Thermo Fisher Scientific) equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002) and a post-GIF 2k x 2k Gatan CCD camera (model 794).
  • the images were acquired at 300 kV acceleration voltage in bright-field EM mode with zeroloss energy filtering at either 6,500x (dose rate of 1.64 electrons A 2 s ') or 24,000x (dose rate of 11.8 electrons A 2 s ') magnification and Is acquisition time.
  • C57BL/6 mice were intravenously or intramuscularly injected with 89 Zr-labelled OTl-aNPs, OVA- aNPs or aNPs without adjuvant.
  • 89 Zr-labelled OTl-aNPs OVA- aNPs or aNPs without adjuvant.
  • blood was drawn and weighed, and radioactivity was measured using a Wizard2 2480 automatic gamma counter (Perkin Elmer).
  • Data were corrected for radioactive decay, and percentage of injected dose per gram of blood (%ID g 1 ) was calculated.
  • Data were fitted using a nonlinear two-phase decay regression in GraphPad Prism for the intravenously injected groups, and weighted blood half-life was calculated via the equation (% fast x 11/2 fast + % slow x 11/2)/ 100.
  • a spline fit was done for the intramuscular injected groups. Biodistribution in mice was determined 24 hours post injection. After PBS perfusion, tissues of interest were collected and weighed, and radioactivity was measured using a Wizard2 2480 automatic gamma counter (Perkin Elmer). Data were corrected for radioactive decay, and percentage of injected dose per gram of tissue (%ID g 1 ) was calculated.
  • mice were intravenously or intramuscularly injected with DiR-labelled OT1- aNPs, OVA-aNPs or control aNPs. After 24 hours mice were euthanized, and single cell suspensions were created from spleen and lymph nodes as previously described. Cells suspensions were incubated with antibodies and viability dye described in Supplementary Table 9 according to the manufacturer’s instructions. Subsequently, cells were washed and re-suspended in PBA-buffer (PBS pH 7.4, 1 % w/v BSA (Sigma)). All data were acquired on a CytoFlex LX flow cytometer (Beckman Coulter).
  • PBA-buffer PBS pH 7.4, 1 % w/v BSA (Sigma)
  • FSC-A x SSC-A was constructed to identify the broader cellular population and a second gate on FSC-A x FSC-H was constructed to discriminate single cells from duplets. Live cells were discriminated using a conventional live-death stain. Clonal populations were identified by staining the cells for representative surface markers. Geometric mean fluorescence intensities were calculated as a measure of surface marker expression.
  • mice C57BL/6 mice were intramuscularly injected on day 0 and day 10 with 25 pg of OVA-aNPs containing one or multiple adjuvants, OVA-aNPs mixed with alum, OVA-aNPs without adjuvants, or PBS only. Composition of the aNPs can be found in Supplementary Tables 4-6.
  • ELISA enzyme-linked immunosorbent assay
  • mice were euthanized, blood was collected for ELISA and spleens were collected for flow cytometry. Blood samples for ELISA were allowed to clot on ice for 60 minutes. Serum was taken after centrifugation at 6000 r.p.m.
  • ELISA was used to measure anti-OVA immunoglobulin G.
  • Anti-OVA IgG from serum was captured in Nunc® MaxiSorpTM 96 well plates using chicken egg albumin (InvivoGen) and detected with a goat antimouse IgG conjugated to HRP (AP503P, Merck).
  • HRP HRP
  • General IgG levels were determined using Mouse IgG (Total) Uncoated ELISA Kit (Invitrogen). 4.21 Dextramer staining after vaccination
  • Spleens were obtained from vaccinated C57BL/6 mice after euthanization at day 28 single cell suspensions were prepared as previously described. Cells were resuspended in 200 pL PBA (PBS pH 7.4, 1 % w/v BSA (Sigma) and surface markers and cell viability were stained according to the manufacturer's instructions, using the antibodies, OVA specific dextramer, and viability dye described in Supplementary Table 10. Data was acquired on a CytoFlex LX flow cytometer (Beckman Coulter).
  • a first gate on FSC-A x SSC-A was constructed to identify the broader cellular population and a second gate on FSC-A x FSC-H was constructed to discriminate single cells from duplets. Live cells were discriminated using a conventional live-death stain. CD8+ 1 cells were identified by staining the cells for representative surface markers. OT-1 specific CD8+ T cells were identified by staining with the OVA specific dextramer. Geometric mean fluorescence intensities were calculated as a measure of surface marker expression.
  • Plasmid backbones used to design and express various proteins and lentivirus development Plasmid backbones used to design and express various proteins and lentivirus development.

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WO2016154544A1 (fr) * 2015-03-25 2016-09-29 The Regents Of The University Of Michigan Compositions et procédés permettant d'administrer des agents de type biomacromolécule
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WO2023046931A1 (fr) 2021-09-23 2023-03-30 Bio-Trip B.V. Protéines de fusion d'apolipoprotéine pour la régulation immunitaire spécifique de cellules
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Publication number Priority date Publication date Assignee Title
WO2016154544A1 (fr) * 2015-03-25 2016-09-29 The Regents Of The University Of Michigan Compositions et procédés permettant d'administrer des agents de type biomacromolécule
WO2019030574A1 (fr) * 2017-08-10 2019-02-14 Cerenis Therapeutics Holding Cargomères
WO2022268913A1 (fr) 2021-06-22 2022-12-29 Bio-Trip B.V. Nanoparticules contenant un acide nucléique
WO2023046931A1 (fr) 2021-09-23 2023-03-30 Bio-Trip B.V. Protéines de fusion d'apolipoprotéine pour la régulation immunitaire spécifique de cellules
CA3231980A1 (fr) * 2021-09-23 2023-03-30 Roy VAN DER MEEL Proteines de fusion d'apolipoproteine pour la regulation immunitaire specifique de cellules
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