WO2024110757A1 - Control of nanocage self-assembly - Google Patents

Control of nanocage self-assembly Download PDF

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Publication number
WO2024110757A1
WO2024110757A1 PCT/GB2023/053057 GB2023053057W WO2024110757A1 WO 2024110757 A1 WO2024110757 A1 WO 2024110757A1 GB 2023053057 W GB2023053057 W GB 2023053057W WO 2024110757 A1 WO2024110757 A1 WO 2024110757A1
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Prior art keywords
ferritin
nanocage
construct
subunits
linker
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PCT/GB2023/053057
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French (fr)
Inventor
Kourosh HONARMAND EBRAHIMI
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King's College London
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Priority claimed from GBGB2217590.5A external-priority patent/GB202217590D0/en
Priority claimed from GBGB2312763.2A external-priority patent/GB202312763D0/en
Application filed by King's College London filed Critical King's College London
Publication of WO2024110757A1 publication Critical patent/WO2024110757A1/en

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to constructs that can be used to produce ferritin nanocages and are specifically designed to control nanocage self-assembly. Also described herein are nucleic acid molecules, expression vectors, host cells, and pharmaceutical compositions. Also described herein are methods of preparing a ferritin nanocage, methods of treating or preventing a disease in a subject, and methods of raising an immune response against an antigen. Background to the Invention Natural protein nanocages are made of multiple subunits, which spontaneously self- assemble to form various structures with an internal cavity. Many protein nanocages have been described (1).
  • ferritin nanocage A spherical-shape nanocage that is naturally produced by all kingdoms of life is the ferritin nanocage (2). Most ferritins consist of 24 subunits, but 12-mer ferritins are also known (e.g., Listeria innocua ferritin; Dps mini-ferritin). Ferritin is a universal intracellular protein that stores iron and releases it in a controlled fashion. Ferritin protein is produced by almost all living organisms, including archaea, bacteria, algae, higher plants, and animals. Ferritin is a globular protein complex consisting of 24 identical or similar protein subunits that form a hollow nanocage with multiple metal-protein interactions.
  • the nanocage has an outer diameter of 12-13 nm, an interior cavity diameter of 7-8 nm, and a thickness of 2-2.5 nm. Every two subunits of ferritin form a group in antiparallel, and then these twelve pairs of subunits form an approximately regular dodecahedron, with 4-3-2 axial symmetry.
  • the application of ferritin nanocages in medicine has found significant interest (3,4) because of several advantages of the ferritin nanocage over other systems.
  • the ferritin nanocage is very stable, e.g., at 100°C for minutes to hours. Ferritin is naturally produced in humans and application of bacterial ferritin in animal models has been shown to be non-toxic (9,10).
  • the ferritin nanocage has a spherical-shape structure with an internal cavity of 8 nm (see Figure 1A). Therefore, small molecules, peptides, or small RNAs can be encapsulated inside the ferritin nanocage.
  • Human ferritin can target cancer cells and thus, can be used for targeted delivery of an anticancer drug (5,6).
  • the N-terminal end of the ferritin subunit can be modified with a ligand which results in the ligand being expressed on the surface of the ferritin nanocage; this system can therefore be used to target specific cells, e.g., cancer cells (see Figure 1B).
  • each ferritin subunit is present on the surface of the ferritin nanocage at three-fold symmetry axis; thus viral surface glycoproteins can be added to the N-terminus of the ferritin subunits such that the assembled nanocages can be used to mimic viruses e.g., influenza (7), HIV-1 (8) or SARS-CoV2 (9,10), and be used as novel vaccines. These vaccines generate robust responses in preclinical studies (11). Ferritin protein can be lyophilized and thus, it offers a platform to make room- temperature stable therapeutics like vaccines.
  • ferritin nanocages As a drug delivery system or for making vaccines, a major bottleneck for commercial application is the nanocage itself. Like all other protein nanocages, ferritin nanocages spontaneously form when the protein subunit is overexpressed by E. coli or other hosts. Hence, to encapsulate a drug inside, or add multiple ligands to the N-terminus and create novel mosaic vaccines, first the nanocage must be disassembled. Current approaches to disassemble ferritin include: 1. Decreasing the pH to 2-3 (12,13). 2.
  • a construct comprising: two ferritin subunits connected by a linker, wherein the linker comprises a cleavage site, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage, and wherein cleavage of the linker at the cleavage site does not prevent the ferritin subunits from self-assembling into a ferritin nanocage.
  • the inventors have developed a construct where two ferritin subunits are connected by a cleavable linker.
  • the two ferritin subunits are constrained and cannot self-assemble with other subunits into a ferritin nanocage.
  • This provides the advantage that the ferritin subunits can be retained in a controlled form where they will not spontaneously self-assemble into a nanocage.
  • Self-assembly of a ferritin nanocage can be induced by cleaving the linker, thus separating the ferritin subunits and allowing them to participate in the necessary interactions to form a ferritin nanocage.
  • This provides the advantage that the timing of the assembly of a ferritin nanocage can be precisely controlled by the provision of a cleaving agent designed to cleave the linker.
  • this technology can be used to control the assembly of ferritin nanocages to encapsulate a cargo molecule, such as a drug, and/or to control the assembly of ferritin nanocages with attached surface ligands, such as antigens, without exposing the ferritin to conditions that may result in degradation of the protein or ligands, or generate toxic molecules. Accordingly, this technology can be used in vitro and in vivo to generate ferritin nanocages.
  • the term “construct” is used herein to refer to the molecule comprising the two ferritin subunits connected by a linker. In some embodiments, the construct is a polypeptide.
  • polypeptide As described above, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa.
  • the above term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are amino acids that are not naturally encoded.
  • naturally occurring amino acid molecule generally refers to an amino acid molecule that occurs in nature.
  • a naturally occurring amino acid molecule can be a proteinogenic or non-proteinogenic amino acid.
  • proteinogenic amino acid refers to one of the twenty amino acids used for protein biosynthesis as well as other amino acids that can be incorporated into proteins during translation (including pyrrolysine and selenocysteine).
  • the twenty proteinogenic amino acids include glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, serine, threonine, glutamine, asparagine, arginine, lysine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine and histidine.
  • non-proteinogenic amino acid refers to an amino acid that is not encoded by the standard genetic code, or incorporated into proteins during translation. In some embodiments, the non- proteinogenic amino acid can result from posttranslational modification of proteins. Exemplary naturally occurring non-proteinogenic amino acids include, but are not limited to, hydroxyproline and selenomethionine.
  • non-naturally occurring amino acid molecule refers to an amino acid that is not a naturally occurring amino acid molecule as defined herein.
  • non-naturally occurring amino acid molecule can be used synonymously with the term “amino acid analog.”
  • the non-naturally occurring amino acid molecule is an amino acid formed by synthetic modification or manipulation of a naturally occurring amino acid.
  • a non-naturally occurring amino acid molecule can be a molecule which departs from the structure of the naturally occurring amino acids, but which have substantially the structure of an amino acid, such that they can be substituted within a polypeptide which retains its activity, e.g., ligand- binding activity.
  • amino acids can also include amino acids having side chain modifications or substitutions, and also include related organic acids, amides or the like.
  • non-naturally occurring amino acid molecule examples include, but are not limited to, homocysteine; phosphoserine; phosphothreonine; phosphotyrosine; ⁇ -carboxyglutamate; hippuric acid; octahydroindole-2-carboxylic acid; statine; 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid; penicillamine (3-mercapto-D-valine); ornithine (Orn); citruline; alpha-methyl- alanine; para-benzoylphenylalanine; para-aminophenylalanine; p-fluorophenylalanine; phenylglycine; propargylglycine; N-methylglycins (sarcosine, Sar); and tert- butylglycine; diaminobutyric acid; 7-hydroxy-tetrahydroisoquinoline carboxylic acid; naphthylalan
  • a non-naturally occurring amino acid molecule can be a chemically modified amino acid.
  • chemically modified amino acid refers to an amino acid that has been treated with one or more reagents.
  • a non-naturally occurring amino acid molecule can be a beta- amino acid.
  • beta-amino acids include, but are not limited to, L- ⁇ - Homoproline hydrochloride; ( ⁇ )-3-(Boc-amino)-4-(4-biphenylyl)butyric acid; ( ⁇ )-3- (Fmoc-amino)-2-phenylpropionic acid; (1S,3R)-(+)-3-(Boc- amino)cyclopentanecarboxylic acid; (2R,3R)-3-(Boc-amino)-2-hydroxy-4- phenylbutyric acid; (2S,3R)-3-(Boc-amino)-2-hydroxy-4-phenylbutyric acid; (R)-2- [(Boc-amino)methyl]-3-phenylpropionic acid; (R)-3-(Boc-amino)-2-methylpropionic acid; (R)-3-(Boc-amino)-2-phenylpropionic acid; (R)-3-(
  • the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 70% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 75% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 80% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 85% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 90% sequence identity thereto.
  • the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 95% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 98% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 99% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • the construct will have the functional requirements of (1) the linker being arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage when the linker is not cleaved, and (2) the ferritin subunits being arranged to self-assemble into a ferritin nanocage when the linker is cleaved.
  • a subunit is a well understood term in molecular biology.
  • the ferritin globular protein is typically formed of 24 subunits.
  • ferritin subunit is understood to mean any subunit that can self-assemble into a multi-meric ferritin globular protein nanocage, including universal 24-meric ferritins, haem-containing 24-meric bacterioferritins and prokaryotic 12-meric Dps proteins.
  • the ferritin subunit is a subunit that can self-assemble into a 24-meric ferritin globular protein.
  • the ferritin subunits i.e., ferritin monomers
  • the ferritin subunits may be selected from animal ferritin, plant ferritin, fungal ferritin, bacterial ferritin, archaeal ferritin, or combinations thereof.
  • the ferritin subunits may be selected from vertebrate ferritin, amphibian ferritin, plant ferritin, bacterial ferritin, archaea ferritin, gastropod ferritin, or combinations thereof. In some embodiments, the ferritin subunits are selected from human ferritin, bacterial ferritin, or combinations thereof. In some embodiments, the ferritin subunits are human ferritin subunits.
  • the ferritin subunits are selected from heavy chain ferritin subunits (‘H’ chain, also referred to as ‘higher’), medium chain ferritin subunits (‘M’ chain), light chain (‘L’ chain, also referred to as ‘lower’) ferritin subunits, or a combination thereof.
  • the ferritin subunits are selected from heavy chain ferritin subunits or light chain ferritin subunits, or a combination thereof.
  • the ferritin subunits are a combination of heavy chain ferritin subunits and light chain ferritin subunits.
  • the two ferritin subunits are heavy chain ferritin subunits or the two ferritin subunits are light chain ferritin subunits.
  • the ferritin subunits are identical.
  • the ferritin subunits are selected from heavy chain human ferritin subunits or light chain human ferritin subunits, or a combination thereof.
  • self-assembly is intended to mean that the ferritin subunits, when the linker is cleaved, spontaneously form a ferritin nanocage (e.g., a 24-meric ferritin nanocage), when exposed to nanocage-forming conditions.
  • the construct comprises more than two ferritin subunits connected by linkers, each linker comprising a cleavage site.
  • the construct may comprise three ferritin subunits connected by linkers in a linear chain, as schematically shown below (“N” and “C” refer to the N-terminus and the C-terminus of the molecule).
  • the ferritin subunits are connected by linkers in a closed loop.
  • the construct may comprise four ferritin subunits connected by linkers in a linear chain, as schematically shown below.
  • linker is intended to mean that the two ferritin subunits are attached together via the linker when the cleavage site is uncleaved (i.e., the linker is intact).
  • the linker has a first end and a second end, and the first end is connected to the first ferritin subunit and the second end is connected to the second ferritin subunit.
  • the two ferritin subunits are connected in series (i.e., the C- terminus of the first subunit is connected to a first end of the linker, and the second end of the linker is connected to the N-terminus of the second subunit), as schematically shown below.
  • the linker may be any suitable molecule that: (i) links the two ferritin subunits together and constrains their ability to form the necessary protein-protein interactions with other ferritin subunits such that the subunits cannot self-assemble into a nanocage; and (ii) can be cleaved at a cleavage site to remove the constraints on the ferritin subunits, thereby allowing the subunits to self-assemble into a nanocage (i.e., cleavage of the linker detaches the two ferritin subunits from each other, allowing them to interact with other ferritin subunits to form a ferritin nanocage).
  • a ferritin nanocage cannot form because the linker prevents the interaction of the ferritin subunits necessary for self-assembly (the subunits cannot form the necessary antiparallel side-by-side interactions).
  • the ferritin subunits are detached and are then able to interact with other ferritin subunits to self-assemble into a ferritin nanocage (the subunits are able to form the natural antiparallel side-by-side interactions with other subunits to induce formation of the nanocage).
  • the linker may be a peptide linker or a non-peptide linker (e.g., a PEG linker).
  • the linker is a peptide linker comprising a cleavage site.
  • the linker is a soluble peptide linker.
  • the linker may comprise or consist of 5-1500 amino acid residues. In some embodiments, the linker comprises or consists of 10-1000 amino acid residues.
  • the linker comprises or consists of 15-500 amino acid residues. In some embodiments, the linker comprises or consists of 20-250 amino acid residues. In some embodiments, the linker comprises or consists of 25-150 amino acid residues. Typically, the linker comprises or consists of 5-100 amino acid residues. In some embodiments, the linker is a rigid linker. In other embodiments, the linker is a flexible linker. In some embodiments, the linker comprises an amino acid sequence to increase protein expression. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 70% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 75% sequence identity thereto.
  • the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 80% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 85% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 90% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 95% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 98% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 99% sequence identity thereto.
  • the linker will have the functional requirements of (1) the linker being arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage when the linker is not cleaved at the cleavage site, and (2) the linker being cleavable at the cleavage site to allow the ferritin subunits to self-assemble into a ferritin nanocage.
  • identity is used to refer to the similarity of two sequences.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the nucleotide/amino acid residues at each position are then compared.
  • a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • a sequence comparison is typically carried out over the entire length of the two sequences being compared.
  • the skilled person will be aware of the fact that several different computer programs are available to determine the identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleic acid sequences is determined using the sequence alignment software Clone Manager 9 (Sci-Ed software - www.scied.com) using global DNA alignment; parameters: both strands; scoring matrix: linear (mismatch 2, OpenGap 4, ExtGap 1).
  • the percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a further method to assess the percent identity between two amino acid or nucleic acid sequences can be to use the BLAST sequence comparison tool available on the National Center for Biotechnology Information (NCBI) website (www.blast.ncbi.nlm.nih.gov), for example using BLASTn for nucleotide sequences or BLASTp for amino acid sequences using the default parameters.
  • the cleavage site may be any suitable cleavage site that can be specifically cleaved by a cleaving agent to allow the self-assembly of the ferritin subunits into a ferritin nanocage.
  • the cleavage site and the cleaving agent should be selected such that the construct is not cleaved at any other site that would result in the nanocage not assembling.
  • the cleaving agent should not cleave a site within the ferritin subunits.
  • the cleavage site and the cleaving agent should be selected such that the effect of the cleaving agent is non-toxic.
  • cleavable linkers include pH- sensitive linkers (e.g., hydrazone), photocleavable linkers, glutathione-sensitive (e.g., SPDB) and protease-sensitive linkers (e.g., valine-citrulline dipeptide).
  • the cleavage site is a protease cleavage site (i.e., the linker is a protease-sensitive linker).
  • the cleavage site is an enterokinase or thrombin cleavage site.
  • the cleavage site is an enterokinase cleavage site.
  • Exemplary recognition sequences for cleavage proteases are provided in the table below.
  • the linker comprises a recognition sequence selected from the sequences in the table below.
  • the cleavage site is an extracellular matrix protease cleavage site, such as ACE2 or Thrombin.
  • the cleavage site is a viral protease cleavage site, such as an HIV-1 protease cleavage site, an influenza virus protease cleavage site, or a SARS-CoV-2 protease cleavage site.
  • a viral protease cleavage site such as an HIV-1 protease cleavage site, an influenza virus protease cleavage site, or a SARS-CoV-2 protease cleavage site.
  • Nanocage self-assembly may be achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble (i.e., nanocage-forming conditions).
  • nanocage self-assembly is induced by exposing the cleaved construct to a buffer comprising 50 mM-10 M of a salt and a pH of 6-8.5.
  • the buffer may be any suitable buffer that induces nanocage self-assembly.
  • the buffer is selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate-Bicarbonate buffer, and any combination thereof.
  • the buffer is Tris.
  • the buffer may be present at any suitable concentration that facilitates the self-assembly of the ferritin subunits into a ferritin nanocage.
  • the buffer is present at a concentration of 25-200 mM.
  • the buffer may be present at a concentration of 50-150 mM.
  • the buffer may be present at a concentration of 75-125 mM.
  • the buffer is present at a concentration of 25-75 mM.
  • the buffer is present at a concentration of about 50 mM.
  • the buffer is Tris at a concentration of about 50 mM.
  • the salt may be any suitable salt that induces nanocage self-assembly.
  • the salt is selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl 2 , KCl, CaI 2 , NaI, KI, MgI 2 , and any combination thereof.
  • the salt is NaCl.
  • the salt is present at a concentration of 75 mM – 5 M.
  • the salt is present at a concentration of 100 mM-2 M.
  • the salt is present at a concentration of 200 mM-1 M.
  • the salt is present at a concentration of 500-900 mM.
  • the salt is present at a concentration of 600-800 mM.
  • the pH is between 6.1 and 8.4. In some embodiments, the pH is between 6.2 and 8.3. In some embodiments, the pH is between 6.3 and 8.2. In some embodiments, the pH is between 6.4 and 8.1. In some embodiments, the pH is between 6.5 and 8.0. In some embodiments, the pH is between 6.6 and 7.9. In some embodiments, the pH is between 6.7 and 7.8. In some embodiments, the pH is between 6.8 and 7.7. In some embodiments, the pH is between 6.9 and 7.6. In some embodiments, the pH is between 7.0 and 7.5. In some embodiments, the pH is between about 7.5-8.2. In some embodiments, the buffer further comprises a solvent for solubilisation and encapsulation of a therapeutic agent.
  • the solvent is selected from the group consisting of DMSO, ethanol, acetonitrile, or any combination thereof.
  • the solvent is DMSO.
  • the solvent to construct ratio is from 0:1 to 0.5:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.1:1 to 0.4:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.2:1 to 0.3:1 (v/v).
  • the cleaved construct is exposed to suitable conditions which allow the nanocage to self-assemble for 0-4 h. In some embodiments, the cleaved construct is exposed to suitable conditions which allow the nanocage to self-assemble for 0.5-3 h.
  • the cleaved construct is exposed to suitable conditions which allow the nanocage to self-assemble for 1-2 h. In some embodiments, the cleaved construct is incubated at 20-30°C. In some embodiments, the cleaved construct is incubated at 22-28°C. In some embodiments, the cleaved construct is incubated at 24-26°C. In some embodiments, the cleaved construct is incubated at 25°C.
  • nanocage self-assembly is induced by exposing the cleaved construct to a buffer selected from the group consisting Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate- Bicarbonate buffer, and any combination thereof, wherein the buffer comprises 50 mM- 10 M of a salt selected from the group consisting of NaCl, CaCl 2 , MgCl 2 , MnCl 2 , KCl, CaI2, NaI, KI, MgI2, and any combination thereof, and a pH of 6-8.5.
  • a buffer selected from the group consisting Tris, MO
  • nanocage self-assembly is induced by exposing the cleaved construct to a buffer selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate- Bicarbonate buffer, and any combination thereof, for 0-4 h at a temperature of 20-30°C, wherein the buffer comprises 50 mM-10 M of a salt selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, and a pH of 6-8.5.
  • a buffer selected from the group consist
  • nanocage self-assembly is induced by exposing the cleaved construct to a buffer selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate- Bicarbonate buffer, and any combination thereof, for 0-4 h at a temperature of 20-30°C, wherein the buffer comprises 50 mM-10 M of a salt selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, a solvent selected from the group consisting of DMSO,
  • nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble after the cleavage step. In some embodiments, nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self- assemble directly after the cleavage step. In some embodiments, nanocage self- assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble within 0-4 h of the cleavage step.
  • nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble within 0-2 h of the cleavage step. In some embodiments, nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self- assemble within 0-1 h of the cleavage step. In some embodiments, nanocage self- assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble simultaneously with the cleavage step.
  • the construct may comprise one or more further elements to enhance the function of the construct and/or aid the purification of the construct and/or enhance, modify or target the expression of the construct.
  • the construct further comprises a purification tag.
  • the purification tag may be any suitable tag that aids or facilitates the purification of the construct.
  • the purification tag may be a polyhistidine tag, a Glutathione-S transferase (GST) tag, a calmodulin binding protein tag, a maltose binding protein (MBP) tag, a myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag, a Spot- tag, a C-tag, a Strep-tag.
  • the purification tag may be at the N-terminal end or the C- terminal end of the construct.
  • the purification tag is a His tag, for example a 6-His tag or a 10-His tag.
  • the purification tag is an N-terminal His tag, for example, an N-terminal 6-His tag (i.e., the His tag is present at the N-terminal end of the construct).
  • the purification tag is cleavable from the construct (e.g., the purification tag is positioned next to a protease cleavage motif, such that cleavage of the motif cleaves the tag from the construct).
  • the cleavage motif may be the same as the cleavage site of the linker, or may differ therefrom. Accordingly, when the cleavage motif is the same as the cleavage site of the linker a single cleavage agent can be used to cleave both the purification tag and the linker.
  • the construct further comprises one or more therapeutic elements and/or one or more non-therapeutic elements. In particular embodiments, the construct comprises one or more therapeutic elements.
  • therapeutic element is intended to have its broadest possible interpretation and refers to any element that when delivered to a bodily conduit of a living being produces a desired, usually beneficial, effect.
  • a therapeutic element relates to any element that can confer a therapeutic benefit on a subject and includes, without limitation, conventional drugs, gene therapy constructs, chemotherapeutic agents, antibiotics, macromolecules, protein bound drugs and antigens.
  • the construct comprises one or more non-therapeutic elements.
  • non-therapeutic element is intended to cover any other element that is not a therapeutic element, i.e., an element that does not confer a therapeutic benefit on a subject. Any combination of therapeutic elements and/or non-therapeutic elements is envisaged.
  • the construct may comprise one therapeutic element, or one therapeutic element and one non-therapeutic element, or one non-therapeutic element, or two therapeutic elements and one non-therapeutic element, etc.
  • the therapeutic element(s) and/or non-therapeutic element(s) may be contained within the linker.
  • the linker may comprise a therapeutic peptide.
  • the therapeutic element(s) and/or non-therapeutic element(s) are contained within the linker and positioned adjacent to the N-terminus of the second ferritin subunit (i.e., the ferritin subunit attached to the linker at the N-terminal end of the subunit).
  • adjacent to is intended to cover directly adjacent to (i.e., directly bonded to, with no intervening amino acid) and in proximity to (i.e., at least one intervening amino acid, and optionally between 1 and 100 intervening amino acids).
  • the cleavage site is not located within the therapeutic element(s)/non- therapeutic element(s).
  • the linker may comprise a therapeutic peptide positioned either N- or C-terminal to the cleavage site.
  • the therapeutic element(s) and/or non-therapeutic element(s) may be located at the N-terminal end of the construct or in the N-terminal portion of the construct.
  • the element(s) will be located on the surface of the ferritin nanocage once assembled.
  • N-terminal end is intended to mean the element is located directly at the N-terminus (i.e., the N-terminal residue of the element forms the N-terminal end of the construct).
  • N-terminal portion is intended to mean the region of the construct from (and including) the N-terminus of the construct up to but not including the N-terminal residue of the first ferritin subunit.
  • an antigen or therapeutic peptide may be located at the N-terminal end of the construct.
  • an antigen or therapeutic peptide may be located in the N- terminal portion of the construct, adjacent to a further element at the N-terminal end (e.g., a purification tag or a secretory signal).
  • a first therapeutic element or non-therapeutic element is positioned in the N-terminal portion of the construct and a second therapeutic element or non-therapeutic element is positioned adjacent to the N-terminus of the second ferritin subunit.
  • the therapeutic element(s) and/or non-therapeutic element(s) may be located at the C-terminal end of the construct or in the C-terminal portion of the construct.
  • C-terminal end is intended to mean the element is located directly at the C-terminus (i.e., the C-terminal residue of the element forms the C- terminal end of the construct).
  • C-terminal portion is intended to mean the region of the construct from (and including) the C-terminus of the construct up to but not including the C-terminal residue of the ferritin subunit positioned closest to the C- terminus of the construct.
  • the construct further comprises one or more therapeutic elements selected from the group consisting of drugs, antibodies, proteins (including enzymes), peptides, genes, oligonucleotides, RNA therapeutics, antigens, other pharmaceutically active ingredients, or a combination thereof.
  • the therapeutic element is a therapeutic peptide.
  • the therapeutic peptide may be Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide, Teduglutide, Linaclotide, Pramlintide, Abarelix, Degarelix, Carfilzomib, Mifamurtide, Aviptadil, Atosiban, Carbetocin, Taltirelin, Bremelanotide, Teriparatide, Abaloparatide, Plecanatide, Nesiritide, Angiotensin II, Icatibant, Enfuvirtide, Tesamorelin, Ziconotide, Romiplostim, Peginesatide, Lucinactant, Etelcalcetide, Afamelanotide, Pasireotide, Lutetium Lu 177inate, Edotreotide gallium Ga-68, Setmelanotide, Taspoglutide, Somapacitan, Selepressin, Lenomoreli
  • the therapeutic peptide is 20 to 200 amino acids in length.
  • the therapeutic element may be an antigen.
  • the antigen may be selected such that when a nanocage is produced and administered, an immune response is raised against the antigen.
  • the antigen is a cancer antigen (e.g., a peptide expressed or overexpressed in a cancer), a self-antigen, or a microbial antigen (e.g., a bacterial or viral antigen).
  • the antigen may be a tumour-associated antigen or a tumour-specific antigen.
  • the antigen may be a microbial antigen, such as a microbial peptide, where it is desired to raise an immune response against the microbe for vaccination.
  • the microbial antigen may be a viral or bacterial protein or peptide. Typically, the antigen is a peptide antigen. The peptide antigen may be 20 to 1500 amino acids in length.
  • the therapeutic element is a plurality of antigens. In various embodiments, the construct comprises a plurality of antigens. The plurality of antigens may be the same antigen or different antigens. In some embodiments, the construct further comprises one or more non-therapeutic elements selected from the group consisting of secretory signals, binding molecules, targeting molecules, detectable moieties, or a combination thereof. The non-therapeutic element may be a secretory signal, such as a secretory signal peptide.
  • the secretory signal may be selected based on the expression system, and various secretory signals are known in the art. Typically, the secretory signal is located at the C-terminal end of the construct. In some embodiments, the secretory signal is cleavable from the construct.
  • the non-therapeutic element may be a binding molecule.
  • binding molecule is intended to mean any molecule that can specifically bind and retain a separate molecule or molecules to the surface of the nanocage. This provides the advantage that the ferritin nanocage can be loaded with a variety of molecules on its surface, such as lipids, drugs etc, once the nanocage has been formed. In some embodiments, the binding molecule is biotin.
  • the binding molecule is streptavidin.
  • a biotin-tagged molecule such as an antigen or drug
  • the binding molecule is a click chemistry moiety.
  • the binding molecule is one or more non-naturally occurring amino acid molecules.
  • the non-therapeutic element may be a targeting molecule.
  • targeting molecule any molecule that targets the assembled nanocage to a particular location, environment or cell type.
  • the targeting molecule may be a small molecule targeting ligand, an antibody/antibody-binding domain, a fragment antigen-binding (Fab) domain, a lectin, a protein receptor binding domain, or a targeting peptide.
  • Folic acid is an example of a small molecule targeting ligand which is a high affinity ligand of endogenous folate receptor which is frequently upregulated in many types of cancers.
  • small molecule targeting ligands are monosaccharides such as glucose, mannose and galactose, urea derivatives such as glutamate urea and 2-[3-(1,3-dicarboxypropyl)- ureido] pentanedioic acid (DUPA), glycyrrhetinic acid (GA) derivatives, sulfonamide derivatives, benzamides, and phenyl boronic acid.
  • An antibody or antibody-binding domain can be used to target a specific antigen, such as CD33, HER2, EGFR, or PSMA.
  • targeting peptides include the RGD-4C peptide which targets tumours through RGD integrin ⁇ v ⁇ 3 interaction, octreotide, rabies virus glyocoprotein-29, angiopep-1, and iRGD.
  • the non-therapeutic element may be a detectable moiety, for example a fluorescent tag or a non-fluorescent tag.
  • Various fluorescent tags are known in the art.
  • the fluorescent tag may be a fluorescent protein, such as GFP, EGFP, mNeonGreen, TurboGFP, RFP, mCherry, etc.
  • the fluorescent tag may be an extrinsically fluorescent tag, such as a SNAP tag, CLIP tag, Halo tag, LOV, iLOV, etc.
  • non-fluorescent tags include enzymatic labelling tags (e.g., Q-tag)
  • the construct further comprises one or more therapeutic elements selected from the group consisting of a drug, an antibody, a protein, a peptide, a gene, an oligonucleotide, an RNA therapeutic, an antigen, another pharmaceutically active ingredient, and a combination thereof, and/or one or more non-therapeutic elements selected from the group consisting of a secretory signal, a binding molecule, a targeting molecule, a detectable moiety, and a combination thereof.
  • the construct further comprises one or more of a therapeutic element selected from the group consisting of a therapeutic peptide, a peptide antigen, and a combination thereof, and/or one or more of a non-therapeutic element selected from the group consisting of a secretory signal peptide, a binding peptide, a targeting peptide, a fluorescent protein tag, and a combination thereof.
  • the construct consists of or consists essentially of: two ferritin subunits connected by a linker, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage, and wherein cleavage of the linker at the cleavage site does not prevent the ferritin subunits from self-assembling into a ferritin nanocage.
  • the term “consists essentially of” is intended to mean that other elements may be present in the polypeptide that do not materially affect the function of the polypeptide.
  • the construct consists of amino acids.
  • the construct consists of naturally-occurring amino acids.
  • a construct comprising: two ferritin subunits connected by a linker, wherein the linker comprises a cleavage site, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage when the linker is not cleaved at the cleavage site, and wherein the ferritin subunits are arranged to self-assemble into a ferritin nanocage when the linker is cleaved at the cleavage site.
  • the description above in relation to the first aspect is equally applicable to this aspect.
  • Nucleic acid molecule In a third aspect, there is provided a nucleic acid molecule encoding the construct described herein.
  • nucleic acid molecule refers to either single- or double-stranded deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof.
  • DNA molecules e.g., cDNA or genomic DNA
  • RNA molecules e.g., RNA, mRNA
  • expression vector comprising the nucleic acid molecule described herein.
  • the expression vector may be any suitable expression vector.
  • the expression vector may be a plasmid or a vector.
  • the vector may be an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector (such as a lentiviral vector), an alphaviral vector, a flaviviral vector, a herpes simplex viral vector, a rhabdoviral vector, a measles viral vector, a pox viral vector, a newcastle disease viral vector, a coxsackieviral vector, or a non-viral vector, such as a polyvalent cation, lipid nanoparticle, chitosan nanoparticle, PLGA dendrimer or other conjugate allowing cellular uptake.
  • the vector is a viral vector.
  • the vector is an AAV vector or a lentiviral vector.
  • the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 70% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 75% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 80% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 85% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO.
  • the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 90% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 95% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 98% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 99% sequence identity thereto. As will be appreciated by one skilled in the art, the expression vector will encode a functional construct (e.g., the construct does not self-assemble into a ferritin nanocage when uncleaved, but can be cleaved to result in ferritin nanocage self assembly).
  • a functional construct e.g., the construct does not self-assemble into a ferritin nanocage when uncleaved, but can be cleaved to result in ferritin nanocage self
  • a host cell comprising the expression vector described herein.
  • the host cell may be any suitable host cell.
  • the term "host cell” refers to cells which harbour an expression vector of the invention, as well as cells that are suitable for use in expressing a recombinant gene or protein. It is not intended that the present invention be limited to any particular type of cell. Indeed, it is contemplated that any suitable cell will find use in the present invention as a host cell.
  • a host cell according to the invention may permit the expression of a nucleic acid molecule of the invention.
  • the host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell.
  • the host cell may be any suitable eukaryotic cell into which the vector may be introduced.
  • the host cell may be a mammalian cell or a plant cell.
  • the host cell may be a human cell.
  • the host cell may be in vivo, in vitro or ex vivo.
  • Typical prokaryotic host cells include various bacterial cells such as Escherichia coli.
  • Typical eukaryotic host cells include mammalian host cells, insect host cells, plant host cells, fungal host cells, eukaryotic algae host cells, nematode host cells, protozoan host cells, fish host cells, and the like.
  • Mammalian host cells include Chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK293) cells, baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells.
  • Typical fungal host cells include Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaculomyces.
  • compositions comprising the construct described herein or the nucleic acid molecule described herein or the expression vector described herein or the host cell described herein and one or more pharmaceutically acceptable excipients.
  • the one or more excipients include carriers, diluents and/or other medicinal agents, pharmaceutical agents or adjuvants, etc.
  • Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to, the excipient, any suitable binder, lubricant, suspending agent, coating agent or solubilising agent.
  • Preservatives, stabilizers and dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
  • the pharmaceutical composition may be administered using any suitable administration method.
  • the pharmaceutical composition may be administered parenterally in which the composition is formulated in an injectable form, for delivery, by, for example, an intravenous, intradermal, intramuscular, subcutaneous or intraperitoneal route.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • Intradermal administration routes include any dermal-access means, for example, using microneedle-based injection and infusion systems (or other means to accurately target the intradermal space), needleless or needle-free ballistic injection of fluids or powders into the intradermal space, Mantoux-type intradermal injection, enhanced iontophoresis through microdevices, and direct deposition of fluid, solids, or other dosing forms into the skin, including the use of patches to deposit the composition onto the skin.
  • composition may also be formulated to be administered by oral or topical routes, including nasally, orally or epicutaneously.
  • the composition is formulated to be delivered by an oral or nasal route.
  • the method further comprises exposing the cleaved construct to nanocage-forming conditions (i.e., conditions which induce the self-assembly of the ferritin subunits into a ferritin nanocage).
  • the method further comprises incubating the cleaved constructs with a buffer comprising 50 mM-10 M of a salt and a pH of 6-8.5 to induce the self-assembly of the ferritin subunits into a ferritin nanocage.
  • the method further comprises adjusting the salt concentration and/or the pH to induce self-assembly of the ferritin subunits into a ferritin nanocage.
  • the description above in relation to the conditions, for example the buffer (including all of the optional components of the buffer), that allow self-assembly of the nanocages is equally applicable to the method of preparing a ferritin nanocage.
  • the plurality of constructs should be a sufficient number of constructs that, when cleaved, will self-assemble into a ferritin nanocage (either by themselves or with other ferritin subunits). In some embodiments, the plurality of constructs is at least 12 constructs.
  • the method may be performed in vivo, in vitro or ex vivo.
  • the method is performed in vitro or ex vivo.
  • the term “contacting” as used herein refers to bringing the plurality of constructs and the cleaving agent together in such a manner that the cleaving agent can cleave the cleavage site of the linker.
  • the plurality of constructs may be contacted with a cleaving agent by any suitable method.
  • the plurality of constructs is mixed with a cleaving agent in solution.
  • the plurality of constructs is introduced into an environment where the cleaving agent is present and functional.
  • the plurality of constructs may be added to a cell culture where a cleaving agent is inherently present (e.g., an extracellular matrix protease) or where a cleaving agent has been added.
  • the contacting step may be any suitable length such that the cleaving agent cleaves the linkers at the cleavage site.
  • the contacting step is 2-18 h.
  • the contacting step is 4-16 h.
  • the contacting step is 6-14 h.
  • the contacting step is 8-12 h.
  • the contacting step may be carried out in any suitable conditions that allow the cleaving agent to cleave the linkers at the cleavage site.
  • the contacting step is carried out in a reaction buffer.
  • the reaction buffer is selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate-Bicarbonate buffer, and any combination thereof.
  • the reaction buffer comprises 1-500 mM salt.
  • the salt is selected from the group consisting of CaCl 2 , MgCl 2 , MnCl 2 , KCl, CaI 2 , NaI, KI, MgI 2 , and combinations thereof.
  • the contacting step is carried out at a pH between 6.0-8.5. In some embodiments, the contacting step is carried out at a pH between 6.1 and 8.4. In some embodiments, the contacting step is carried out at a pH between 6.2 and 8.3. In some embodiments, the contacting step is carried out at a pH between 6.3 and 8.2. In some embodiments, the contacting step is carried out at a pH between 6.4 and 8.1.
  • the contacting step is carried out at a pH between 6.5 and 8.0. In some embodiments, the contacting step is carried out at a pH between 6.6 and 7.9. In some embodiments, the contacting step is carried out at a pH between 6.7 and 7.8. In some embodiments, the contacting step is carried out at a pH between 6.8 and 7.7. In some embodiments, the contacting step is carried out at a pH between 6.9 and 7.6. In some embodiments, the contacting step is carried out at a pH between 7.0 and 7.5. In some embodiments, the contacting step is carried out at a pH between about 7.5-8.2. In some embodiments, the reaction buffer comprises a solvent for solubilisation and encapsulation of a therapeutic agent.
  • the solvent is selected from the group consisting of DMSO, ethanol, acetonitrile, and combinations thereof. In some embodiments, the solvent is DMSO. In some embodiments, the solvent to construct ratio is from 0:1 to 0.5:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.1:1 to 0.4:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.2:1 to 0.3:1 (v/v). In some embodiments, the reaction buffer further comprises one or more metal ions. The one or more metal ions may be selected from the group consisting of iron, nickel, cobalt, copper, gold, manganese, palladium, platinum, molybdenum, tungsten and combinations thereof.
  • the contacting step is carried out in 25-75 mM reaction buffer comprising 50-150 mM salt at pH 6-8.5 for 2-16 h.
  • the plurality of constructs are provided by expressing the nucleic acid molecule described herein or the expression vector described herein.
  • the cleaving agent may be any suitable cleaving agent that cleaves the cleavage site of the linker. Typically, the cleaving agent specifically cleaves the cleavage site and does not cleave any other site in the constructs. As an example, if the cleavage site is a protease cleavage site, the cleaving agent will be a protease that cleaves that cleavage site.
  • the cleaving agent will be enterokinase or thrombin, respectively.
  • the term “facilitating self-assembly” is intended to mean that the constraints on the ferritin subunits provided by the uncleaved linker are removed by the cleavage of the linker, thereby enabling the separated ferritin subunits to spontaneously form a ferritin nanocage (e.g., a 24-meric ferritin nanocage) when exposed to nanocage-forming conditions.
  • the cleaved subunits are not subsequently purified following the cleavage step.
  • the method is intended to form ferritin nanocages, and not to separately purify the ferritin subunits.
  • the ferritin nanocages may subsequently be purified following their self-assembly using any suitable purification technique.
  • the method further comprises contacting the plurality of constructs with a cargo molecule, such that the cargo molecule is encapsulated inside the ferritin nanocage during self-assembly of the ferritin subunits into the ferritin nanocage.
  • the cargo molecule may be any molecule that is intended to be encapsulated inside the ferritin nanocage.
  • the cargo molecule is extrinsic to the construct described herein.
  • the term “encapsulated” is intended to mean that the cargo molecule is trapped inside the hollow cavity of the nanocage and is generally not covalently bound to the nanocage.
  • the cargo molecule can be any biological molecule, chemical molecule, synthetic molecule, or any other molecule that can be encapsulated by the ferritin nanocage described herein.
  • the cargo molecule is a therapeutic element.
  • the therapeutic element may be a drug, an antibody, a protein, a peptide, a gene, an oligonucleotide, an RNA therapeutic, an antigen, another pharmaceutically active ingredient, or a combination thereof.
  • the therapeutic element may be any other ingredient intended to have a therapeutic effect (e.g., metal nanoparticles).
  • the cargo molecule may be DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumour proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti- inflammatories, anti-histamines, anti-infectives, and/or chemotherapeutics.
  • Other suitable cargo molecules include sensitizers (e.g., radiosensitizers) that can make a cell or subject more responsive (or sensitive) to a treatment or prevention and imaging or other diagnostic agents.
  • the ferritin nanocages can be used as a monotherapy or in combination with other active agents for treatment or prevention of a disease.
  • Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g., melatonin and thyroxine), small peptide hormones and protein hormones (e.g., thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eiconsanoids (e.g., arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydro testosteron cortisol).
  • amino-acid derived hormones e.g., melatonin and thyroxine
  • small peptide hormones and protein hormones e.g., thyrotropin-releasing hormone, vaso
  • Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6- MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26 and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers).
  • interleukins e.g., IL-2, IL-7, and IL-12
  • cytokines e.g., interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , I
  • Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammants (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylae, and sodium salicaylate), paracetamol/acetaminophen, metamizole, nabumetone, phenazone, and quinine.
  • non-steroidal anti-inflammants e.g., ibuprofen, naproxen, ketoprofen, and nimesulide
  • aspirin and related salicylates e.g., choline salicylate, magnesium salicylae, and sodium salicaylate
  • paracetamol/acetaminophen metamizole
  • nabumetone nabumetone
  • phenazone phenazone
  • quinine quinine
  • Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotenergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepresents, and monoamine oxidase inhibitors), mebicar, afobazole, selank, bromantane, emoxypine, azapirones, barbituates, hyxdroxyzine, pregabalin, validol, and beta blockers.
  • benzodiazepines e.g., alprazolam, bromazepam, chlordiazepoxide, clo
  • Suitable antipsychotics include, but are not limited to, benperidol, bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, car
  • Suitable analgesics include, but are not limited to, paracetamol/acetaminophen, non- steroidal anti-inflammants (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupiretine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g., choline salicylate, magnesium salicylae, and sodium salicaylate).
  • Suitable antispasmodics include, but are not limited to, mebeverine, papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene.
  • Suitable anti-inflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammants (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immune selective anti- inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives).
  • Suitable anti-histamines include, but are not limited to, H1-receptor antagonists (e.g.
  • H2- receptor antagonists e.g., acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebasine, embramine, fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine),
  • cimetidine famotidine, lafutidine, nizatidine, rafitidine, and roxatidine
  • tritoqualine catechin, cromoglicate, nedocromil, and ⁇ 2-adrenergic agonists.
  • Suitable anti-infectives include, but are not limited to, amebicides (e.g., nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, abendazole, miltefosine, thiabendazole, oxamniquine), antifungals (e.g., azole antifungals (e.g., itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g.,
  • cephalosporins e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g., tigecycline), leprostatics (e.g., clofazimine and thalidomide),
  • cephalosporins e.g. cefadroxil, cephradine, cef
  • chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytar
  • Suitable sensitizing agents can include, but are not limited to, radiosensitizers, insulin sensitizers (e.g., metformin, thiazolidinediones,) and photosensitizers for photodynamic therapy (e.g., aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m- tetrahydroxyphenylchlorin (mTHPC) and mono-L-aspartyl chlorin e6 (NPe6)).
  • radiosensitizers e.g., insulin sensitizers (e.g., metformin, thiazolidinediones,) and photosensitizers for photodynamic therapy (e.g., aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m- tetrahydroxyphenylchlorin (mTHPC) and mono-L-aspartyl chlorin e6 (NPe6)).
  • ALA aminolevulinic acid
  • mTHPC Silicon Ph
  • Suitable imaging agents include but are not limited to, fluorescent molecules (e.g., Cy3, Cy5, and other commercially available fluorflores), paramagnetic ions, nanoparticles that can contain a paramagnetic ion, super-paramagnetic iron oxide molecules and nanoparticles thereof, 18F-fluorodeoxyglucose and other PET imaging agents, gadolinium containing contrast agents, radionuclides and compositions thereof.
  • the method comprises binding a cargo molecule to the surface of the ferritin nanocage.
  • the cargo molecule may be any suitable cargo molecule as discussed above; however, in this instance, the cargo molecule is intended to be bound to the surface of the nanocage rather than being encapsulated inside the nanocage.
  • the cargo molecule may be a therapeutic element as defined above, such as a drug, peptide, protein, antigen, gene, oligonucleotide, or other pharmaceutically active ingredient.
  • the cargo molecule may be any other ingredient intended to have a therapeutic effect (e.g., metal nanoparticles).
  • the step of binding the cargo molecule (e.g., an antigen) to the binding molecule may comprise contacting the nanocage with the cargo molecule.
  • the cargo molecule may be cross-linked to the nanocage via transamidation of a glutamine tag in the construct using transglutaminase.
  • the cargo molecule is bound to the binding molecule via click chemistry.
  • the cargo molecule is bound to a non-naturally occurring amino acid molecule in the construct using chemical biology.
  • the construct may include an azidoalanine residue at its N-terminus; a linker (e.g., a DBCO-NHS-ester linker) can be added to a lysine residue of a cargo molecule (e.g., an antibody) which can be linked to the azidoalanine residue via click chemistry.
  • a linker e.g., a DBCO-NHS-ester linker
  • a linker e.g., a DBCO-NHS-ester linker
  • the assembled ferritin nanocage will differ from the ferritin nanocages of the prior art in that the ferritin subunits will remain connected to portions of the cleaved linker. Accordingly, the ferritin nanocage will comprise portions of the cleaved linker. In some embodiments, at least one of the ferritin subunits is connected to a portion of the cleaved linker. In various embodiments, each of the ferritin subunits is connected to a portion of the cleaved linker.
  • a method of treating or preventing a disease in a subject comprising administering a ferritin nanocage described herein to a subject, wherein the ferritin nanocage comprises a therapeutic element.
  • a method of treating or preventing a disease in a subject comprising administering a construct described herein to a subject, wherein the construct comprises a therapeutic element.
  • a method of treating or preventing a disease in a subject comprising administering a nucleic acid molecule described herein or an expression vector described herein or a host cell described herein or a pharmaceutical composition described herein to a subject, wherein the nucleic acid molecule or the expression vector or the host cell or the pharmaceutical composition comprises a therapeutic element.
  • treating means the treatment of a disease in a subject, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development or progression; (b) relieving the disease, i.e., causing regression of the disease state; (c) relieving one or more symptoms of the disease; and (d) curing the disease.
  • preventing means the prevention of a disease in a subject, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; and (c) preventing or delaying the onset of at least one symptom of the disease.
  • the disease may be any disease.
  • Diseases that may be treated or prevented include cancers, neurological disorders, genetic disorders (e.g., protein deficiency), heart disease, stroke, arthritis, viral and bacterial infections, as well as immune system disorders, but are not limited to these.
  • the disease is selected from cancers or microbial infections.
  • the disease is cancer and the therapeutic element is an anti-cancer therapeutic (e.g., chemotherapeutic drug, cancer antigen, antibody, etc).
  • the disease is a microbial infection and the therapeutic element is an antigen specific to the microbe.
  • the term “administering” refers to any method of providing the therapy (e.g., the ferritin nanocage, or the construct) to a subject.
  • Such methods include, but are not limited to, intracardiac administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • the ferritin nanocage may be administered to the subject via any suitable administration route.
  • the nanocage may be administered to the subject orally, intravenously, occularly, intraoccularly, intramuscularly, intravaginally, intraperitoneally, rectally, parenterally, topically, intranasally, or subcutaneously.
  • the term “subject” refers to the target of administration, e.g., an animal.
  • the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.
  • 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 subject is a patient.
  • the therapeutic element may be any therapeutic element that is suitable for treating or preventing the disease in the subject. Examples of therapeutic elements are discussed above.
  • ferritin nanocage described herein for use in therapy wherein the ferritin nanocage comprises a therapeutic element.
  • a construct described herein or a nucleic acid molecule described herein or an expression vector described herein or a host cell described herein or a pharmaceutical composition described herein described herein for use in therapy wherein the construct or the nucleic acid molecule or the expression vector or the host cell or the pharmaceutical composition comprises a therapeutic element.
  • Methods of raising an immune response against an antigen comprising: administering a ferritin nanocage described herein to a subject, wherein the ferritin nanocage comprises an antigen.
  • the ferritin nanocage comprising an antigen may be prepared by the methods as described above.
  • the nanocage may be prepared in a manner which results in antigen being present on the surface of the nanocage.
  • the nanocage may be prepared by contacting a plurality of constructs described herein with a cleaving agent, wherein at least one of the plurality of constructs comprises an antigen, and wherein the cleaving agent is configured to cleave the cleavage site of each linker, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage.
  • the nanocage may be prepared in a manner which results in antigen being bound to binding molecules on the surface of the nanocage.
  • the nanocage may be prepared by contacting a plurality of constructs described herein with a cleaving agent, wherein at least one of the plurality of constructs comprises a binding molecule, and wherein the cleaving agent is configured to cleave the cleavage site of each linker, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage, and binding an antigen to the binding molecule.
  • the nanocage may be prepared in a manner which results in antigen being encapsulated inside the nanocage.
  • the nanocage may be prepared by contacting a plurality of constructs described herein with a cleaving agent and a cargo molecule, wherein the cargo molecule is an antigen, and wherein the cleaving agent is configured to cleave the cleavage site of each linker, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage and the encapsulation of the cargo molecule inside the ferritin nanocage.
  • the description above in relation to the construct, the methods of preparing a ferritin nanocage and the methods of treatment is equally applicable to these aspects.
  • the contacting step may be achieved in any suitable manner as described above
  • the cleaving agent may be any suitable cleaving agent as described above
  • the subject may be any subject as described above
  • the administration step may be performed in any manner as discussed above.
  • the antigen may be any antigen where it is intended to raise an immune response against that antigen.
  • the antigen is a cancer antigen (e.g., a peptide expressed or overexpressed in a cancer), a self-antigen, or a microbial antigen (e.g., a bacterial or viral antigen).
  • the antigen may be a microbial antigen, such as a microbial peptide, where it is desired to raise an immune response against the microbe for vaccination.
  • the antigen is a peptide antigen.
  • the peptide antigen may be 20 to 1500 amino acids in length.
  • the plurality of constructs each comprise an antigen.
  • the antigen may be the same antigen (i.e., all of the constructs contain the same single antigen) or different antigens (i.e., a first construct may contain a first antigen and a second construct may contain a second antigen), the latter resulting in nanocages presenting multiple antigens.
  • the antigen may be positioned at a suitable position within the construct as discussed above in relation to the polypeptide.
  • the binding molecule may be any suitable binding molecule as discussed above in relation to the construct.
  • the step of binding the antigen to the binding molecule may comprise contacting the nanocage with the antigen.
  • the antigen may be cross-linked to the nanocage via transamidation of a glutamine tag in the construct using transglutaminase.
  • the antigen is bound to the binding molecule via click chemistry.
  • the antigen is bound to a non-naturally occurring amino acid molecule in the construct using chemical biology.
  • the present disclosure provides an ACE2-peptide mimetic comprising the sequence shown in SEQ ID NO:3.
  • the ACE2-peptide mimetic was designed by combining the sequences of two polypeptides of the human ACE2 protein that interact with the SARS-CoV-2 receptor binding domain.
  • Figure 1 shows various methods known in the prior art used for controlling ferritin self- assembly in biomedical applications.
  • A Structure of 24-meric nanocage showing the internal cavity of 8 nm.
  • B The N-terminus of each subunit is on the surface at 3-fold symmetry axis and can be used to add a ligand, such as an antigen.
  • C Disassembly and assembly mediated by pH, temperature, and pressure.
  • FIG. 1 shows supporting data for the disclosure.
  • A A schematic presentation of an exemplary embodiment of the invention combining two subunits of ferritin to generate PINCs.
  • B SDS-Page analysis of purified protein. Lane (1) and (2): Nickel column was washed and a sample was run on the gel. Lane (3): Dimeric protein was eluted using 150 mM imidazole and a sample was run on the gel.
  • FIG. 3 shows various applications of the invention.
  • a drug delivery vehicle for various medicines A drug delivery vehicle for various medicines.
  • B Creating mosaic vaccines.
  • C Creating multifunctional medicines.
  • D Developing novel AAV-based vaccines mimicking a virus replication process to create efficacious and long-lasting immune response. In this process the AAV construct of ferritin-dimer carrying a viral antigen and a secretory signal is delivered to the cell.
  • the dimer has a peptide sequence for cleavage by an extracellular protease.
  • FIG. 4 shows the designed Ferritin-ACE2-D construct.
  • a 6xHis-tag polypeptide was added to the N-terminus of first subunit and the N-terminus of the second subunit (with an ACE2 peptide mimetic between the 6xHis-tag and the subunit in each case).
  • the novel ACE2-peptide mimetic was designed from combining the sequences of two polypeptides of the human ACE2 protein interacting with the SARS-CoV-2 receptor binding domain.
  • FIG. 5 shows the exemplary construct in the original plasmid before subcloning into pBAD/His C.
  • Figure 6 shows PINC technology can be used to decorate the surface of nanocages with multiple proteins.
  • PfEDU-2 with an N-terminus Sumo was cleaved using enterokinase to generate two subunits, i.e., Subunit-1 and Subunit-2.
  • Figure 9 shows analysis PfEDU-2 and PINCs using (a) size exclusion chromatography and (b) Native-PAGE. Analysis suggested PINCs generated from PfEDU-2 have a wide range of MW between 600-720 nm. NativePAGETM 4-16% Bis-Tris Protein Gel was used.
  • Figure 10 shows LC-MS/MS analysis of PINCs generated from PfEDUs, confirming decoration with Sumo protein. The list of peptides detected using LC-MS/MS. The Native-PAGE band associated with PINCs generated from PfEDUs were cut and subject to trypsin digestion and analysis.
  • Figure 11 shows LC-MS/MS spectrum of the peptides assigned to Sumo protein.
  • FIG. 12 shows generation of PINCs using HpEDUs.
  • the HpEDUs with an N- terminus Sumo and insecticide protein were cleaved by enterokinase to generate two subunits.
  • Figure 13 shows comparison of the initial rate of Fe(III) formation for PfEDU-2, PINCs, WT-PfFtn, and background oxidation of Fe(II) by molecular oxygen.
  • Figure 14 shows kinetics of Fe(III) mineral core formation.
  • Figure 15 shows Fe(III) oxidation and mineralization by HpEDUs and PINCs.
  • Figure 19 shows Native-PAGE analysis of PINCs encapsulating CPT.
  • the inventors compared PINCs after formation without dialysis with PINCs+CPT after extensive dialysis and with control. The control was PINCs incubated with the drug and extensively dialyzed to remove free CPT. NativePAGETM 4-16% Bis-Tris Protein Gel was used.
  • Figure 20 shows standard curve for CPT. Standard curve was measured by addition of CPT dissolved in DMSO to protein buffer to reach a DMSO concentration of 10% and desired concentration of CPT.
  • Ferritin nanocages are typically made of 24 (maxi-ferritin) or 12 (mini-ferritin) subunits.
  • the subunits spontaneously self-assemble to form a spherical shape structure (Figure 1A) with an internal cavity of 8 nm.
  • the protein has eight three-fold axes where the N- termini of the ferritin subunits are positioned towards the outside ( Figure 1B). Due to narrow and uniform size distribution of nanocages, an internal cavity, and an N- terminus accessible for modification, there is a growing interest in using ferritin nanocages in drug delivery, antibody therapy, and vaccine development. Despite all the process in using the nanocage for developing therapeutics, a major bottleneck in the application of ferritin is the self-assembly of the protein.
  • the first step of spontaneous self- assembly of a ferritin nanocage requires that two subunits interact in an anti-parallel fashion via their interface. Therefore, the inventors hypothesised that if the C-terminus of one subunit is linked to the N-terminus of a second subunit (i.e., if the dimers are linked in series), the resulting protein dimers will not physically be able to come together in an anti-parallel fashion and initiate self-assembly. For the self-assembly to initiate, the linker between the dimers must be cleaved, and hence the inventors introduced the recognition sequence of a specific protease, Enterokinase, in the linker peptide between the two subunits.
  • Example 1 As a proof of principle, the inventors aimed to design a prophylactic against SARS- CoV-2. Their aim was to create a nanocage furnished with ACE2-peptide mimetics for use as a novel inhaled prophylactic and decoy of SARS-CoV-2 to prevent infection of cells. Such an inhaled prophylactic could replace or supplement masks. Therefore, the inventors aimed to furnish the surface with a novel peptide sequence mimicking the key sequences of the human ACE2 receptor interacting with SARS-CoV-2 receptor binding domain (RBD). The ACE2-peptide mimetic was added to the N-terminus of the first subunit and the second subunit.
  • Construct design Figure 2A shows an exemplary construct according to an embodiment of the disclosure.
  • the construct includes two ferritin subunits linked by a linker containing a protease cleavage site.
  • the N-terminus of the construct has a 6-His tag.
  • the connection of the two subunits via the linker prevents the subunits from self-assembling into a ferritin nanocage.
  • Cleavage of the linker at the cleavage site by a protease allows the self- assembly of the subunits (along with other monomeric subunits) into a ferritin nanocage (referred to as a Protease Induced Ferritin Nanocage; PINC).
  • PINC Protease Induced Ferritin Nanocage
  • 24 ferritin subunits are required to form the ferritin nanocage.
  • ACE2-ferritin dimer A construct for expression of a Pyrococcus furiosus ferritin dimer containing an ACE2 peptide-mimetic at each terminus of the subunit was designed (see Figure 4).
  • the construct had an amino acid sequence according to SEQ ID NO: 1.
  • Each ferritin subunit (SEQ ID NO:4) has a poly histidine peptide tag (SEQ ID NO: 5) and an ACE2- peptide mimetic (SEQ ID NO:3) at the N-terminus of each subunit.
  • the two subunits are linked by a peptide sequence (SEQ ID NO:2) cleaved specifically by Enterokinase enzyme.
  • the construct is referred to as ACE2-Fr-D.
  • the codon optimised gene for expression of ACE2-Fr-D in E. coli was obtained from GeneArt (ThermoFisher Scientific; SEQ ID NO: 6) (see Figure 5).
  • GeneArt ThermoFisher Scientific; SEQ ID NO: 6
  • SEQ ID NO: 6 The codon optimised gene for expression of ACE2-Fr-D in E. coli was obtained from GeneArt (ThermoFisher Scientific; SEQ ID NO: 6) (see Figure 5).
  • SEQ ID NO: 6 The sequences for two restriction enzymes were added.
  • the sequence for NcoI restriction enzyme at the 5′ end the sequence for EcoRI restriction enzyme at the 3′ end.
  • This designed gene was then cloned into PBAD/His C expression plasmid using NcoI and EcoRI restriction sites.
  • the vector sequence had a nucleic acid sequence according to SEQ ID NO: 7.
  • the resulting construct was then transformed into E. coli Top10 cells.
  • Top10 cells carrying the ACE2-Fr-D plasmid were inoculated into LB media (typically 10-50 ml) and grown overnight in a shaker, 37 °C with 200 rpm. After 12-16 hours the overnight culture was inoculated into Terrific Broth (TB) media (100-500 ml). Cells were grown for approximately 2 hours until the Optical Density at 600 nm reached 0.5-0.8. Then expression of protein was induced by addition of arabinose (final concentration of 0.04% (W/V)). Cells were grown for another 5-6 hours and then collected using centrifugation (5000 rpm, 4 °C).
  • lysis buffer 50 mM Tris-HCl, 300 mM NaCl, 1 mM PMSF and 0.1mM DNase, pH 8.0.
  • the buffer contained 0.02 w/v TritonX- 100 and 0.5 mM dithiothreitol (DTT) to reach maximum cell lysis efficiency using sonification.
  • Cells were sonicated for 5 minutes (30 seconds each cycle and 10 cycles in total) by using a probe sonicator (Branson Sonifier). The % amplitude of sonication from 30%. Protein concentration was measured by using Implen NanoPhotometer. Sonication was performed on ice.
  • the cell-free extract was used for purification of the protein using his-tag affinity purification using cOmpleteTM His-tag purification resin (Roche).
  • the resin was equilibrated with buffer A containing 50 mM NaH2PO4, pH 7.6, 300 mM NaCl, 0.02 w/v TritonX-100 and 0.5mM DTT and 10 mM Imidazole.
  • the cell-free extract obtain from E. coli was incubated with the cOmplete TM His- tag purification resin and incubated in cold room (4 °C) for 2-4 hours with continuous shaking.
  • ThermoFisher Scientific was washed with wash buffer A (approximately 10 ml) and protein was eluted using buffer A containing 150 mM Imidazole.
  • the concentration of protein was measured using BCA assay or alternatively by measuring the absorbance at 260 nm using a Nano spectrophotometer.
  • the purified protein was flash-frozen in liquid nitrogen and kept in -80 °C freezer for future experiments. Analysis of protein assembly using SDS-PAGE Gel electrophoresis
  • the gel electrophoresis apparatus, pre-casted polyacrylamide gels (Any kDTM) and running buffers were purchased from Bio-Rad Laboratories.
  • the protein ladder Precision Plus Protein TM All Blue Standard 10-250kDa (Bio-Rad Laboratories) was used.
  • the protein loading dye 4xLaemmli sample buffer (Bio-Rad Laboratories) was used for running SDS-PAGE.
  • the native gel protein loading buffer (4x) was made of 2.5x Tris-Borate-EDTA buffer, 50% glycerol, 0.1% bromophenol blue and ddH2O. Gel electrophoresis was run at 120V for 30 minutes.
  • the polyacrylamide gel was immersed in the Coomassie blue dye PageBlue Protein Staining Solution (Thermo Scientific) and incubated for 4 to 12 hours on a shaker.
  • FIG. 2B shows SDS- PAGE gel analysis of the purified construct, clearly showing purification of the 58 kDa dimer in lane (3).
  • Enterokinase cleavage of ACE2-Fr-D Enterokinase were purchased from Sigma (100 U/mg). The lyophilised protein was dissolved in 400 ⁇ l milli-Q water.
  • the ACE2-Fr-D sample was washed multiple times (20 times of its volume) using Amicon Ultracentrifugation filter (10 kDa) to exchange the buffer to 50 mM Tris, pH 8.0 (a buffer which facilitates enterokinase cleavage and nanocage self-assembly).
  • Amicon Ultracentrifugation filter (10 kDa) to exchange the buffer to 50 mM Tris, pH 8.0 (a buffer which facilitates enterokinase cleavage and nanocage self-assembly).
  • an Enterokinase:ACE2-Fr-D ratio of 1:20 or 1:200 (mg/ml) was used.
  • Enterokinase the sample was incubated at 25 °C for 16 hr. Subsequently, the reaction was stopped by addition of SDS denaturing solution and heating the sample at 70 C for 10 min.
  • FIG. 2C shows SDS- PAGE gel analysis of the constructs following enterokinase cleavage, clearly showing purification of PINCs in lane (3)+P at the expected theoretical molecular weight of 696 kDa.
  • SEC-MALS analysis The actual molecular weight (MW) of ACE2-Fr 24-mer nanocage was detected by SEC- MALS (Size-Exclusion Chromatography Multi-Angle Light Scattering). SEC-MALS measured the UV absorbance at the wavelength (280 nm and 230nm) for four protein samples including bovine serum albumin standard (BSA standard, theoretical MW: 66.4 kDa), natural P.
  • BSA standard bovine serum albumin standard
  • the measured molecular weight of the ACE2-Fr 24-mer nanocage (692.00 ⁇ 52.72 kDa) was similar to the theoretical molecular weight (696kDa). It was not possible to accurately measure the molecular weight of ACE2-Fr-D because the protein is not globular and is flexible due to the presence of the linker; accordingly a very broad band with multiple small peaks was observed, reflecting the various shapes of the dimer in solution.
  • Table 1 Measurement of molecular weight (MW) of ACE2-Ferritin Nanocage using SEC-MALS.
  • Example 2 The inventors used PINC technology to decorate the surface of nanocages with a protein.
  • the resulting construct (PfEDU-2) was cloned into a pBAD vector or pET28a vector, and PfEDU-2 was overexpressed in E. coli.
  • the inventors then characterized the Fe(II) oxidation and Fe(III) mineralisation activity of EDUs and the resulting PINCs. They determined the initial rates of Fe(III) core formation for both PfEDU-2 and PINCs. They compared the results with Fe(II) oxidation by WT-PfFtn and the background oxidation of Fe(II) by molecular oxygen (Figure 13). The results of the initial rate measurement show that PfEDUs have the highest activity ( Figure 13). It is suggested that in natural ferritin nanocages, the Fe(II) ions must transfer through the pores at 3-fold symmetry to reach the ferroxidase centre, where they are oxidized.
  • the inventors used the anticancer drug doxorubicin (DOX).
  • DOX anticancer drug doxorubicin
  • CPT anticancer drug camptothecin
  • the inventors mixed PfEDU-2 with DOX and then, added enterokinase to form PINCs and encapsulate DOX ( Figure 7a).
  • the inventors first generated PINCs and then added DOX to the nanocages. Both samples were subject to extensive dialysis using dialysis tubes with a molecular weight cutoff of 300 kDa. Subsequently samples were analysed using Native-PAGE to confirm the presence of PINCs ( Figure 16).
  • PINC technology unlocks new opportunities for delivery of a wide range of drugs and developing advanced multifunctional therapeutics to tackle diseases such as viral infection and cancer.
  • Chemicals and Reagents All chemicals were reagent grades and were purchased from Merck or Fisher Scientific. Enterokinase from the porcine intestine (40 UN) was from Merck. The lyophilized power of enterokinase was dissolved in 400 ⁇ l 50mM Tris 100mM NaCl pH 8.0, and the solution was divided into small aliquots, flash frozen using liquid nitrogen, and stored at -80 °C freezer for future use. Protein constructs.
  • the inventors designed three constructs ( Figure 6(f)), two engineered dimer units (EDUs) using Pyrococcus furiosus (Pf) ferritin subunits and one construct using H. pylori (Hp) ferritin subunits.
  • the first PfEDU construct (PfEDU-1) had an N-terminus peptide
  • the second construct (PfEDU-2) had an N-terminus Sumo protein.
  • the HpEDU had Sumo protein and an insecticide protein at the N- terminus of subunit-1 and the pesticide protein at the N-terminus of subunit-2.
  • coli codon-optimized gene encoding PfEDU-2 was obtained from GeneArt (Thermo Fisher) in pBAD/His A plasmid. This gene was then subcloned into the Escherichia coli (E. coli) expression vector pET-28a using the SacI and HindIII restriction sites. The E. coli codon-optimized gene encoding PfEDU-1, and HpEDU was obtained from GeneArt in pMA vector. These genes were subsequently subcloned into pBAD/His C vector using KpnI and EcoRI restriction sites. The presence of the correct insert was confirmed by double digestion and agarose gel electrophoresis.
  • coli cells were cultured overnight in 50 ml Luria-Bertani (LB) medium supplemented with 100 ⁇ g/mL Kanamycin (PfEDU-2) or 100 ⁇ g/ml ampicillin (PfEDU-1 or HpEDU). The flasks were incubated in a shaker at 200 rpm and 37°C. After the overnight incubation, the cells were inoculated into 500 ml of terrific broth (TB) medium, with a ratio of LB:TB as 1:10. The flasks were incubated in a shaker at 200 rpm and 37°C.
  • TB terrific broth
  • OD@600 nm reached a value of 0.5-0.8
  • 1 mM (final concentration) IPTG was added to induce the expression of PfEDUs or 0.04% (final concentration) of L-arabinose was added to induce the expression of HpEDUs.
  • the cells were incubated at 37°C and 200 rpm for another 8 hours to allow protein overproduction.
  • the cells were collected using centrifugation at 3750 rpm for 20 minutes and suspended in 2 ml lysis buffer (50 mM Tris, 300 mM NaCl, Ph 8.0 containing 2% triton, 0.5 mg PMSF, 0.05 mg DNase, and 1 mM DTT, 10mM Imidazole 0.1mg/ml lysozyme). These cells were stored at -80°C freezer for the subsequent purification step.
  • 2 lysis buffer 50 mM Tris, 300 mM NaCl, Ph 8.0 containing 2% triton, 0.5 mg PMSF, 0.05 mg DNase, and 1 mM DTT, 10mM Imidazole 0.1mg/ml lysozyme.
  • lysis buffer containing (50 ml of 50 mM Tris, 300 mM NaCl, pH 8.0 containing 2% triton, 0.5 mg PMSF, 0.05 mg DNase, and 1 mM DTT, 10mM Imidazole 0.1mg/ml lysozyme).
  • Cells were subject to sonication (10 cycles of 30 seconds with 10-15 seconds off intervals with 50% amplitude). A total of 25 ml of cell lysate was processed each time. Next, the lysed cell mixture was subjected to ultracentrifugation at 4°C and 20,000 rpm for 20 minutes to remove cell debris.
  • Ni 2+ resin 250 ⁇ l of resin per 500 ml of bacterial growth culture
  • HisPurTM Ni-NTA Resin HisPurTM Ni-NTA Resin, ThermoFisher
  • This solution was then incubated at 4 °C for 1 hour.
  • the Ni 2+ resin, now containing the bound EDUs was loaded onto a gravity column.
  • the resins were washed 5 times (each time 2-time of the volume of the Ni2+ resin) using the wash buffer (50 mM Tris, 300 mM NaCl, pH 7.5, 20 mM Imidazole, 0.2% Triton, 1 mM DTT).
  • EDUs were eluted using the elution buffer (50 mM Tris, 300 mM NaCl, pH 7.5, 500 mM Imidazole, 0.2% Triton, 1 mM DTT).
  • PD10 desalting column was used to exchange the buffer to 50mM Tris, 300mM NaCl pH 8.
  • the concentration of EDUs was determined using the BCA assay.
  • the protein solution was subsequently diluted to a final concentration of 1.5 ⁇ M and divided into aliquots, and flash-frozen using liquid nitrogen. The samples were stored at -80 °C freezer for further analysis.
  • Overexpression and Purification of WT-PfFtn were eluted using the elution buffer (50 mM Tris, 300 mM NaCl, pH 7.5, 500 mM Imidazole, 0.2% Triton, 1 mM DTT).
  • PD10 desalting column was used to exchange the buffer to 50mM Tris, 300mM NaC
  • an inducer buffer composed of 100ul of 1M MOPS and 1M NaCl at pH 7 was added to the reaction mixture and incubated for an additional 1 hour at 37°C.
  • the protein solution was then concentrated using an Amicon ultracentrifugal filter with a 10kDa cutoff. The concentration of protein was determined using polyacrylamide gel electrophoresis (PAGE). Polyacrylamide gel electrophoresis (PAGE). Each protein was mixed with 4x Native sample buffer (Invitrogen) (2.5 ⁇ l). Then, 10 ⁇ l of each sample or the standard was loaded into each well. NovexTM 4-12% Tris-Glycine Mini Gels, or NativePAGETM 4- 16% Bis-Tris Protein Gels were used.
  • the NativeMarkTM Unstained Protein Standard (ThermoFisher) (5 ⁇ l) mixed with 2.5 ⁇ l and 2.5 ⁇ l Milli-Q water was used for NativePAGE. 7 ⁇ l of the PageRulerTM Plus Prestained Protein Ladder (Fisher Scientific) was used with NovexTM 4-12% Tris-Glycine Mini Gels. The Mini Gel Tank (Life Technologies) was used for running gel with Anode and Cathode buffer (Invitrogen) at 150V for 100 mins. After running, the gel was washed with MiliQ water 3 times, each 5 minutes. Then the gels were stained with SimplyBlue SafeStain (novex, life technologies) for 1 hr.
  • a stock solution of PfEDU-2 (8.3 ⁇ M), its PINC (0.7 ⁇ M on 24-mer basis), or WT-PfFtn (8.3 ⁇ M) was used for Fe(II) oxidation studies.
  • the Fe(II) stock (24mM) was prepared from Fe(NH4)2(SO4)2 in MOPS buffer (100mM MOPS 100mM NaCl pH 7) under anaerobic conditions.
  • MOPS buffer 100mM MOPS 100mM NaCl pH 7
  • SEC-MALS Size Exclusion Chromatography-Multiangle Light Scattering
  • BSA, WT-PfFtn, PfEDU-2, and PINC were prepared and concentrated to 200 ⁇ l (circa 2 mg/ml). 100 ⁇ l of each sample was injected. The column was WTC- 030S5. Absorbance was recorded for 25 min at 280 nm. Encapsulation of Doxorubicin (DOX).
  • Both sample (a) and control (b) were subject to extensive dialysis (Float-A-LyzerTM G2 Dialysis Devices, Spectrum), to remove free DOX.
  • the dialysis process was repeated three times, each with 3 L of fresh dialysis buffer 5 mM MOPS and 300 mM NaCl at pH 7.
  • the length of dialysis steps was 6 hours, overnight, and 20 hours. All dialysis steps were done at 4°C.
  • UV-visible spectrophotometry was utilized to quantify the amount of DOX encapsulated within the nanocages. For UV-visible spectroscopy, a quartz cuvette was used.
  • the length of dialysis steps was 6 hours, overnight, and 20 hours. All dialysis steps were done at 4°C.
  • UV-visible spectrophotometry was utilized to quantify the amount of CPT encapsulated within the nanocages. Additionally, 500 ⁇ l of the sample or control was concentrated to circa 50 ⁇ l using Amicon Ultracentrifugal Filter with a cutoff of 10 kDa. This concentrated sample was used for Native-PAGE to confirm the presence of 24-meric nanocages. Prediction of the structure of PfEDUs. The structure of PfEDU-2 with an N-terminal Sumo was predicted using the Phyre server (Kelley, L. A.; Mezulis, S.; Yates, C.
  • the protocol for this process was as follows: Reagents: 100 mM Triethylammonium bicarbonate (TEAB; Sigma) – 500 ml 1 M TEAB in 4500 ml water 50 mM TEAB – 1 ml 100 mM TEAB added to 1 ml water. 10 mM Dithiothreitol (DTT; Sigma) – 15 mg in 1 ml 100 mM TEAB for 100 mM stock; dilute stock 10-fold. 55 mM Iodoacetamide (IAA; Sigma) – 10 mg in 1 ml 100 mM TEAB.
  • TEAB Triethylammonium bicarbonate
  • DTT Dithiothreitol
  • IAA Iodoacetamide
  • TEAB and ACN Volumes of TEAB and ACN (steps 2 to 8) are added in excess. 3. Add acetonitrile, decant after one round, then add same volume again to fully dehydrate the gel pieces (will turn white in appearance). Decant and dry in Speed Vac for 5 mins. Whilst samples are drying prepare DTT solution. 4. Rehydrate the gel with 10 mM DTT and heat at 56°C for 30 mins. 5. Decant DTT, add ACN (2x volume; see step 3), dehydrate and dry in Speed Vac (5 mins). Whilst samples are drying prepare 55 mM IAA solution. 6. Add 55 mM IAA; incubate at ambient temperature for 20 mins in the dark. 7.
  • Monoisotopic precursor ions were filtered using charge state (+2 to +7) with an intensity threshold set between 5.0e3 to 1.0e20 and a dynamic exclusion window of 35s ⁇ 10 ppm.
  • MS2 precursor ions were isolated in the quadrupole set to a mass width filter of 1.6 m/z.
  • Ion trap fragmentation spectra (ITMS2) were collected with an AGC target setting of 1.0e4 (100%) with a maximum injection time of 35 ms with CID collision energy set at 35%.
  • Database Searching Raw mass spectrometry data were processed into peak list files using Proteome Discoverer (ThermoScientific; v2.5).
  • the raw data file was processed and searched using the Sequest (Eng et al; PMID 24226387) search algorithm against the Uniprot All Taxonomy database (569,793 entries). Database searching was performed at a stringency of 1% FDR including a decoy search. Posttranslational modifications for carbamidomethylation (C; static) and oxidation (M; variable), were included in the database search.
  • a SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge.

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Abstract

The invention provides constructs, pharmaceutical compositions, methods of preparing a ferritin nanocage, ferritin nanocages, methods of treating or preventing a disease in a subject, and methods of raising an immune response against an antigen. Exemplary constructs include two ferritin subunits connected by a linker, wherein the linker includes a cleavage site, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage, and wherein cleavage of the linker at the cleavage site does not prevent the ferritin subunits from self-assembling into a ferritin nanocage.

Description

Control of Nanocage Self-Assembly Field of the Invention The present invention relates to constructs that can be used to produce ferritin nanocages and are specifically designed to control nanocage self-assembly. Also described herein are nucleic acid molecules, expression vectors, host cells, and pharmaceutical compositions. Also described herein are methods of preparing a ferritin nanocage, methods of treating or preventing a disease in a subject, and methods of raising an immune response against an antigen. Background to the Invention Natural protein nanocages are made of multiple subunits, which spontaneously self- assemble to form various structures with an internal cavity. Many protein nanocages have been described (1). A spherical-shape nanocage that is naturally produced by all kingdoms of life is the ferritin nanocage (2). Most ferritins consist of 24 subunits, but 12-mer ferritins are also known (e.g., Listeria innocua ferritin; Dps mini-ferritin). Ferritin is a universal intracellular protein that stores iron and releases it in a controlled fashion. Ferritin protein is produced by almost all living organisms, including archaea, bacteria, algae, higher plants, and animals. Ferritin is a globular protein complex consisting of 24 identical or similar protein subunits that form a hollow nanocage with multiple metal-protein interactions. The nanocage has an outer diameter of 12-13 nm, an interior cavity diameter of 7-8 nm, and a thickness of 2-2.5 nm. Every two subunits of ferritin form a group in antiparallel, and then these twelve pairs of subunits form an approximately regular dodecahedron, with 4-3-2 axial symmetry. The application of ferritin nanocages in medicine has found significant interest (3,4) because of several advantages of the ferritin nanocage over other systems. The ferritin nanocage is very stable, e.g., at 100°C for minutes to hours. Ferritin is naturally produced in humans and application of bacterial ferritin in animal models has been shown to be non-toxic (9,10). The ferritin nanocage has a spherical-shape structure with an internal cavity of 8 nm (see Figure 1A). Therefore, small molecules, peptides, or small RNAs can be encapsulated inside the ferritin nanocage. Human ferritin can target cancer cells and thus, can be used for targeted delivery of an anticancer drug (5,6). The N-terminal end of the ferritin subunit can be modified with a ligand which results in the ligand being expressed on the surface of the ferritin nanocage; this system can therefore be used to target specific cells, e.g., cancer cells (see Figure 1B). The N-terminus of each ferritin subunit is present on the surface of the ferritin nanocage at three-fold symmetry axis; thus viral surface glycoproteins can be added to the N-terminus of the ferritin subunits such that the assembled nanocages can be used to mimic viruses e.g., influenza (7), HIV-1 (8) or SARS-CoV2 (9,10), and be used as novel vaccines. These vaccines generate robust responses in preclinical studies (11). Ferritin protein can be lyophilized and thus, it offers a platform to make room- temperature stable therapeutics like vaccines. This will contribute to the reduction of CO2 emission from the cooling industry (about 10% of total global CO2 emission), since storage and distribution of available vaccines require a temperature between -20 to -80 °C. Despite so many advantages of ferritin nanocages as a drug delivery system or for making vaccines, a major bottleneck for commercial application is the nanocage itself. Like all other protein nanocages, ferritin nanocages spontaneously form when the protein subunit is overexpressed by E. coli or other hosts. Hence, to encapsulate a drug inside, or add multiple ligands to the N-terminus and create novel mosaic vaccines, first the nanocage must be disassembled. Current approaches to disassemble ferritin include: 1. Decreasing the pH to 2-3 (12,13). 2. Engineering the interface of two subunits and introducing metal binding sites (14,15). 3. Increasing temperature (~ 50-60 °C) (13,16). 4. Hydrostatic pressure (17). 5. Chaperna-mediated assembly (18). These known approaches are schematically shown in Figures 1C-E. One of the problems with these known approaches is that they are invasive and unsafe. The structure of ferritin or the attached ligands may be affected by these methods, causing degradation or mis-folding of ferritin and/or the ligands. Thus, they cannot be used with a large number of small molecules, peptides, or nucleic acid therapeutics. These methods may also generate unwanted molecules like protein-RNA complexes or excess levels of metals, which can be toxic. In addition, these methods cannot be used for controlling the self-assembly of ferritin inside the body in a safe manner. Being unable to control the self-assembly of ferritin nanocages in a simple and safe manner in vitro and in vivo has remained a major drawback for the commercialisation of ferritin nanocages as drug delivery systems and vaccines. Summary of the Invention Polypeptide In a first aspect, there is provided a construct comprising: two ferritin subunits connected by a linker, wherein the linker comprises a cleavage site, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage, and wherein cleavage of the linker at the cleavage site does not prevent the ferritin subunits from self-assembling into a ferritin nanocage. The inventors have developed a construct where two ferritin subunits are connected by a cleavable linker. When the linker is uncleaved, the two ferritin subunits are constrained and cannot self-assemble with other subunits into a ferritin nanocage. This provides the advantage that the ferritin subunits can be retained in a controlled form where they will not spontaneously self-assemble into a nanocage. Self-assembly of a ferritin nanocage can be induced by cleaving the linker, thus separating the ferritin subunits and allowing them to participate in the necessary interactions to form a ferritin nanocage. This provides the advantage that the timing of the assembly of a ferritin nanocage can be precisely controlled by the provision of a cleaving agent designed to cleave the linker. Accordingly, this technology can be used to control the assembly of ferritin nanocages to encapsulate a cargo molecule, such as a drug, and/or to control the assembly of ferritin nanocages with attached surface ligands, such as antigens, without exposing the ferritin to conditions that may result in degradation of the protein or ligands, or generate toxic molecules. Accordingly, this technology can be used in vitro and in vivo to generate ferritin nanocages. The term “construct” is used herein to refer to the molecule comprising the two ferritin subunits connected by a linker. In some embodiments, the construct is a polypeptide. For ease of explanation, the construct is referred to as having an N-terminal end and a C-terminal end, similar to a polypeptide. However, this may be substituted with a first end and a second end. The term "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The above term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are amino acids that are not naturally encoded. The term “naturally occurring amino acid molecule” generally refers to an amino acid molecule that occurs in nature. A naturally occurring amino acid molecule can be a proteinogenic or non-proteinogenic amino acid. The term “proteinogenic amino acid” as used herein refers to one of the twenty amino acids used for protein biosynthesis as well as other amino acids that can be incorporated into proteins during translation (including pyrrolysine and selenocysteine). The twenty proteinogenic amino acids include glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, serine, threonine, glutamine, asparagine, arginine, lysine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine and histidine. The term “non-proteinogenic amino acid” as used herein refers to an amino acid that is not encoded by the standard genetic code, or incorporated into proteins during translation. In some embodiments, the non- proteinogenic amino acid can result from posttranslational modification of proteins. Exemplary naturally occurring non-proteinogenic amino acids include, but are not limited to, hydroxyproline and selenomethionine. The term “non-naturally occurring amino acid molecule” as used herein refers to an amino acid that is not a naturally occurring amino acid molecule as defined herein. The term “non-naturally occurring amino acid molecule” can be used synonymously with the term “amino acid analog.” In some embodiments, the non-naturally occurring amino acid molecule is an amino acid formed by synthetic modification or manipulation of a naturally occurring amino acid. In some embodiments, a non-naturally occurring amino acid molecule can be a molecule which departs from the structure of the naturally occurring amino acids, but which have substantially the structure of an amino acid, such that they can be substituted within a polypeptide which retains its activity, e.g., ligand- binding activity. Thus, for example, in some embodiments amino acids can also include amino acids having side chain modifications or substitutions, and also include related organic acids, amides or the like. Examples of non-naturally occurring amino acid molecule include, but are not limited to, homocysteine; phosphoserine; phosphothreonine; phosphotyrosine; γ-carboxyglutamate; hippuric acid; octahydroindole-2-carboxylic acid; statine; 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid; penicillamine (3-mercapto-D-valine); ornithine (Orn); citruline; alpha-methyl- alanine; para-benzoylphenylalanine; para-aminophenylalanine; p-fluorophenylalanine; phenylglycine; propargylglycine; N-methylglycins (sarcosine, Sar); and tert- butylglycine; diaminobutyric acid; 7-hydroxy-tetrahydroisoquinoline carboxylic acid; naphthylalanine; biphenylalanine; cyclohexylalanine; amino-isobutyric acid (Aib); norvaline; norleucine (Nle); tert-leucine; tetrahydroisoquinoline carboxylic acid; pipecolic acid; phenylglycine; homophenylalanine; cyclohexylglycine; dehydroleucine; 2,2-diethylglycine; 1-amino-1-cyclopentanecarboxylic acid; 1-amino-1- cyclohexanecarboxylic acid; amino-benzoic acid; amino-naphthoic acid; gamma- aminobutyric acid; difluorophenylalanine; nipecotic acid; N-α-imidazole acetic acid (IMA); thienyl-alanine; t-butylglycine; desamino-Tyr; aminovaleric acid (Ava); pyroglutaminic acid (<Glu); α-aminoisobutyric acid (αAib); γ-aminobutyric acid (γAbu); α-aminobutyric acid (αAbu); αγ-aminobutyric acid (αγAbu); 3-pyridylalanine (Pal); Isopropyl-α-Nεlysine (ILys); Napthyalanine (Nal); α-napthyalanine (α-Nal); β- napthyalanine (β-Nal); Acetyl-β-napthyalanine (Ac-β-napthyalanine); α, β- napthyalanine; Nε-picoloyl-lysine (PicLys); 4-halo-Phenyl; 4-pyrolidylalanine; isonipecotic carboxylic acid (inip); beta-amino acids; and isomers, analogs and derivatives thereof. In some embodiments, a non-naturally occurring amino acid molecule can be a chemically modified amino acid. As used herein, the term “chemically modified amino acid” refers to an amino acid that has been treated with one or more reagents. In some embodiments, a non-naturally occurring amino acid molecule can be a beta- amino acid. Exemplary beta-amino acids include, but are not limited to, L-β- Homoproline hydrochloride; (±)-3-(Boc-amino)-4-(4-biphenylyl)butyric acid; (±)-3- (Fmoc-amino)-2-phenylpropionic acid; (1S,3R)-(+)-3-(Boc- amino)cyclopentanecarboxylic acid; (2R,3R)-3-(Boc-amino)-2-hydroxy-4- phenylbutyric acid; (2S,3R)-3-(Boc-amino)-2-hydroxy-4-phenylbutyric acid; (R)-2- [(Boc-amino)methyl]-3-phenylpropionic acid; (R)-3-(Boc-amino)-2-methylpropionic acid; (R)-3-(Boc-amino)-2-phenylpropionic acid; (R)-3-(Boc-amino)-4-(2- naphthyl)butyric acid; (R)-3-(Boc-amino)-5-phenylpentanoic acid; (R)-3-(Fmoc- amino)-4-(2-naphthyl)butyric acid; (R)-(−)-Pyrrolidine-3-carboxylic acid; (R)-Boc-3,4- dimethoxy-β-Phe-OH; (R)-Boc-3-(3-pyridyl)-β-Ala-OH; (R)-Boc-3-(trifluoromethyl)-β- Phe-OH; (R)-Boc-3-cyano-β-Phe-OH; (R)-Boc-3-methoxy-β-Phe-OH; (R)-Boc-3- methyl-β-Phe-OH; (R)-Boc-4-(4-pyridyl)-β-Homoala-OH; (R)-Boc-4-(trifluoromethyl)- β-Homophe-OH; (R)-Boc-4-(trifluoromethyl)-β-Phe-OH; (R)-Boc-4-bromo-β-Phe-OH; (R)-Boc-4-chloro-β-Homophe-OH; (R)-Boc-4-chloro-β-Phe-OH; (R)-Boc-4-cyano-β- Homophe-OH; (R)-Boc-4-cyano-β-Phe-OH; (R)-Boc-4-fluoro-β-Phe-OH; (R)-Boc-4- methoxy-β-Phe-OH; (R)-Boc-4-methyl-β-Phe-OH; (R)-Boc-β-Tyr-OH; (R)-Fmoc-4-(3- pyridyl)-β-Homoala-OH; (R)-Fmoc-4-fluoro-β-Homophe-OH; (S)-(+)-Pyrrolidine-3- carboxylic acid; (S)-3-(Boc-amino)-2-methylpropionic acid; (S)-3-(Boc-amino)-4-(2- naphthyl)butyric acid; (S)-3-(Boc-amino)-5-phenylpentanoic acid; (S)-3-(Fmoc-amino)- 2-methylpropionic acid; (S)-3-(Fmoc-amino)-4-(2-naphthyl)butyric acid; (S)-3-(Fmoc- amino)-5-hexenoic acid; (S)-3-(Fmoc-amino)-5-phenyl-pentanoic acid; (S)-3-(Fmoc- amino)-6-phenyl-5-hexenoic acid; (S)-Boc-2-(trifluoromethyl)-β-Homophe-OH; (S)- Boc-2-(trifluoromethyl)-3-Homophe-OH; (S)-Boc-2-(trifluoromethyl)-β-Phe-OH; (S)- Boc-2-cyano-β-Homophe-OH; (S)-Boc-2-methyl-β-Phe-OH; (S)-Boc-3,4-dimethoxy-β- Phe-OH; (S)-Boc-3-(trifluoromethyl)-β-Homophe-OH; (S)-Boc-3-(trifluoromethyl)-β- Phe-OH; (S)-Boc-3-methoxy-β-Phe-OH; (S)-Boc-3-methyl-β-Phe-OH; (S)-Boc-4-(4- pyridyl)-β-Homoala-OH; (S)-Boc-4-(trifluoromethyl)-β-Phe-OH; (S)-Boc-4-bromo-β- Phe-OH; (S)-Boc-4-chloro-β-Homophe-OH; (S)-Boc-4-chloro-β-Phe-OH; (S)-Boc-4- cyano-β-Homophe-OH; (S)-Boc-4-cyano-β-Phe-OH; (S)-Boc-4-fluoro-β-Phe-OH; (S)- Boc-4-iodo-3-Homophe-OH; (S)-Boc-4-methyl-β-Homophe-OH; (S)-Boc-4-methyl-β- Phe-OH; (S)-Boc-β-Tyr-OH; (S)-Boc-γ,γ-diphenyl-β-Homoala-OH; (S)-Fmoc-2- methyl-β-Homophe-OH; (S)-Fmoc-3,4-difluoro-β-Homophe-OH; (S)-Fmoc-3- (trifluoromethyl)-β-Homophe-OH; (S)-Fmoc-3-cyano-β-Homophe-OH; (S)-Fmoc-3- methyl-β-Homophe-OH; (S)-Fmoc-γ,γ-diphenyl-β-Homoala-OH; 2-(Boc- aminomethyl)phenylacetic acid; 3-Amino-3-(3-bromophenyl)propionic acid; 3-Amino- 4,4,4-trifluorobutyric acid; 3-Aminobutanoic acid; DL-3-Aminoisobutyric acid; DL-β- Aminoisobutyric acid puriss; DL-β-Homoleucine; DL-β-Homomethionine; DL-β- Homophenylalanine; DL-β-Leucine; DL-β-Phenylalanine; L-β-Homoalanine hydrochloride; L-β-Homoglutamic acid hydrochloride; L-β-Homoglutamine hydrochloride; L-β-Homohydroxyproline hydrochloride; L-β-Homoisoleucine hydrochloride; L-β-Homoleucine hydrochloride; L-β-Homolysine dihydrochloride; L-β- Homomethionine hydrochloride; L-β-Homophenylalanine allyl ester hydrochloride; L- β-Homophenylalanine hydrochloride; L-β-Homoserine; L-β-Homothreonine; L-β- Homotryptophan hydrochloride; L-β-Homotyrosine hydrochloride; L-β-Leucine hydrochloride; Boc-D-β-Leu-OH; Boc-D-β-Phe-OH; Boc-β3-Homopro-OH; Boc-β- Glu(OBzl)-OH; Boc-β-Homoarg(Tos)-OH; Boc-β-Homoglu(OBzl)-OH; Boc-β- Homohyp(Bzl)-OH (dicyclohexylammonium) salt technical; Boc-β-Homolys(Z)-OH; Boc-β-Homoser(Bzl)-OH; Boc-β-Homothr(Bzl)-OH; Boc-β-Homotyr(Bzl)-OH; Boc-β- Ala-OH; Boc-β-Gln-OH; Boc-β-Homoala-OAll; Boc-β-Homoala-OH; Boc-β-Homogln- OH; Boc-β-Homoile-OH; Boc-β-Homoleu-OH; Boc-β-Homomet-OH; Boc-β- Homophe-OH; Boc-β-Homotrp-OH; Boc-β-Homotrp-OMe; Boc-β-Leu-OH; Boc-β- Lys(Z)-OH (dicyclohexylammonium) salt; Boc-β-Phe-OH; Ethyl β- (benzylamino)propionate; Fmoc-D-β-Homophe-OH; Fmoc-L-β3-homoproline; Fmoc-β- D-Phe-OH; Fmoc-β-Gln(Trt)-OH; Fmoc-β-Glu(OtBu)-OH; Fmoc-β-Homoarg(Pmc)- OH; Fmoc-β-Homogln(Trt)-OH; Fmoc-β-Homoglu(OtBu)-OH; Fmoc-β- Homohyp(tBu)-OH; Fmoc-β-Homolys(Boc)-OH; Fmoc-β-Homoser(tBu)-OH; Fmoc-β- Homothr(tBu)-OH; Fmoc-β-Homotyr(tBu)-OH; Fmoc-β-Ala-OH; Fmoc-β-Gln-OH; Fmoc-β-Homoala-OH; Fmoc-β-Homogln-OH; Fmoc-β-Homoile-OH; Fmoc-β- Homoleu-OH; Fmoc-β-Homomet-OH; Fmoc-β-Homophe-OH; Fmoc-β-Homotrp-OH; Fmoc-β-Leu-OH; Fmoc-β-Phe-OH; N-Acetyl-DL-β-phenylalanine; Z-D-β-Dab(Boc)- OH; Z-D-β-Dab(Fmoc)-OH purum; Z-DL-β-Homoalanine; Z-β-D-Homoala-OH; Z-β- Glu(OtBu)-OH technical; Z-β-Homotrp(Boc)-OH; Z-β-Ala-OH purum; Z-β-Ala-ONp purum; Z-β-Dab(Boc)-OH; Z-β-Dab(Fmoc)-OH; Z-β-Homoala-OH; β-Alanine; β- Alanine BioXtra; β-Alanine ethyl ester hydrochloride; β-Alanine methyl ester hydrochloride; β-Glutamic acid hydrochloride; cis-2-Amino-3-cyclopentene-1- carboxylic acid hydrochloride; cis-3-(Boc-amino)cyclohexanecarboxylic acid; and cis- 3-(Fmoc-amino)cyclohexanecarboxylic acid. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 70% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 75% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 80% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 85% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 90% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 95% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 98% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1 or has at least 99% sequence identity thereto. In some embodiments, the construct comprises or consists of the amino acid sequence of SEQ ID NO: 1. As will be appreciated by one skilled in the art, the construct will have the functional requirements of (1) the linker being arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage when the linker is not cleaved, and (2) the ferritin subunits being arranged to self-assemble into a ferritin nanocage when the linker is cleaved. A subunit is a well understood term in molecular biology. The ferritin globular protein is typically formed of 24 subunits. The term “ferritin subunit” is understood to mean any subunit that can self-assemble into a multi-meric ferritin globular protein nanocage, including universal 24-meric ferritins, haem-containing 24-meric bacterioferritins and prokaryotic 12-meric Dps proteins. In certain embodiments, the ferritin subunit is a subunit that can self-assemble into a 24-meric ferritin globular protein. The ferritin subunits (i.e., ferritin monomers) may be ferritin subunits from any species. For example, the ferritin subunits may be selected from animal ferritin, plant ferritin, fungal ferritin, bacterial ferritin, archaeal ferritin, or combinations thereof. In a further example, the ferritin subunits may be selected from vertebrate ferritin, amphibian ferritin, plant ferritin, bacterial ferritin, archaea ferritin, gastropod ferritin, or combinations thereof. In some embodiments, the ferritin subunits are selected from human ferritin, bacterial ferritin, or combinations thereof. In some embodiments, the ferritin subunits are human ferritin subunits. In certain embodiments, the ferritin subunits are selected from heavy chain ferritin subunits (‘H’ chain, also referred to as ‘higher’), medium chain ferritin subunits (‘M’ chain), light chain (‘L’ chain, also referred to as ‘lower’) ferritin subunits, or a combination thereof. In various embodiments, the ferritin subunits are selected from heavy chain ferritin subunits or light chain ferritin subunits, or a combination thereof. In particular embodiments, the ferritin subunits are a combination of heavy chain ferritin subunits and light chain ferritin subunits. In certain embodiments, the two ferritin subunits are heavy chain ferritin subunits or the two ferritin subunits are light chain ferritin subunits. In certain embodiments, the ferritin subunits are identical. In particular embodiments, the ferritin subunits are selected from heavy chain human ferritin subunits or light chain human ferritin subunits, or a combination thereof. The term “self-assembly”, as used herein, is intended to mean that the ferritin subunits, when the linker is cleaved, spontaneously form a ferritin nanocage (e.g., a 24-meric ferritin nanocage), when exposed to nanocage-forming conditions. In some embodiments, the construct comprises more than two ferritin subunits connected by linkers, each linker comprising a cleavage site. For example, the construct may comprise three ferritin subunits connected by linkers in a linear chain, as schematically shown below (“N” and “C” refer to the N-terminus and the C-terminus of the molecule). N – FERRITIN – LINKER – FERRITIN – LINKER – FERRITIN – C In various embodiments, the ferritin subunits are connected by linkers in a closed loop. As a further example, the construct may comprise four ferritin subunits connected by linkers in a linear chain, as schematically shown below. N – FERRITIN – LINKER – FERRITIN – LINKER - FERRITIN – LINKER – FERRITIN – C The term “connected by a linker” is intended to mean that the two ferritin subunits are attached together via the linker when the cleavage site is uncleaved (i.e., the linker is intact). Typically, the linker has a first end and a second end, and the first end is connected to the first ferritin subunit and the second end is connected to the second ferritin subunit. Typically, the two ferritin subunits are connected in series (i.e., the C- terminus of the first subunit is connected to a first end of the linker, and the second end of the linker is connected to the N-terminus of the second subunit), as schematically shown below. N – FERRITIN – LINKER – FERRITIN – C The linker may be any suitable molecule that: (i) links the two ferritin subunits together and constrains their ability to form the necessary protein-protein interactions with other ferritin subunits such that the subunits cannot self-assemble into a nanocage; and (ii) can be cleaved at a cleavage site to remove the constraints on the ferritin subunits, thereby allowing the subunits to self-assemble into a nanocage (i.e., cleavage of the linker detaches the two ferritin subunits from each other, allowing them to interact with other ferritin subunits to form a ferritin nanocage). Accordingly, when a plurality of the constructs are in solution, a ferritin nanocage cannot form because the linker prevents the interaction of the ferritin subunits necessary for self-assembly (the subunits cannot form the necessary antiparallel side-by-side interactions). When the cleavage sites of the linkers are cleaved, the ferritin subunits are detached and are then able to interact with other ferritin subunits to self-assemble into a ferritin nanocage (the subunits are able to form the natural antiparallel side-by-side interactions with other subunits to induce formation of the nanocage). Ferritin nanocage self-assembly is well known in the art and has been reviewed in Zhang Y and Orner BP (Self-assembly in the ferritin nano-cage protein superfamily. Int J Mol Sci. 2011;12(8):5406-21), which is hereby incorporated by reference. The linker may be a peptide linker or a non-peptide linker (e.g., a PEG linker). Typically, the linker is a peptide linker comprising a cleavage site. Typically, the linker is a soluble peptide linker. The linker may comprise or consist of 5-1500 amino acid residues. In some embodiments, the linker comprises or consists of 10-1000 amino acid residues. In some embodiments, the linker comprises or consists of 15-500 amino acid residues. In some embodiments, the linker comprises or consists of 20-250 amino acid residues. In some embodiments, the linker comprises or consists of 25-150 amino acid residues. Typically, the linker comprises or consists of 5-100 amino acid residues. In some embodiments, the linker is a rigid linker. In other embodiments, the linker is a flexible linker. In some embodiments, the linker comprises an amino acid sequence to increase protein expression. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 70% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 75% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 80% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 85% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 90% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 95% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 98% sequence identity thereto. In various embodiments, the linker has the amino acid sequence of SEQ ID NO: 2 or has at least 99% sequence identity thereto. As will be appreciated by one skilled in the art, the linker will have the functional requirements of (1) the linker being arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage when the linker is not cleaved at the cleavage site, and (2) the linker being cleavable at the cleavage site to allow the ferritin subunits to self-assemble into a ferritin nanocage. In the description above, the term “identity” is used to refer to the similarity of two sequences. For the purpose of this invention, it is defined here that in order to determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second amino or nucleic acid sequence). The nucleotide/amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Generally, the two sequences are the same length. A sequence comparison is typically carried out over the entire length of the two sequences being compared. The skilled person will be aware of the fact that several different computer programs are available to determine the identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two nucleic acid sequences is determined using the sequence alignment software Clone Manager 9 (Sci-Ed software - www.scied.com) using global DNA alignment; parameters: both strands; scoring matrix: linear (mismatch 2, OpenGap 4, ExtGap 1). Alternatively, the percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. A further method to assess the percent identity between two amino acid or nucleic acid sequences can be to use the BLAST sequence comparison tool available on the National Center for Biotechnology Information (NCBI) website (www.blast.ncbi.nlm.nih.gov), for example using BLASTn for nucleotide sequences or BLASTp for amino acid sequences using the default parameters. The cleavage site may be any suitable cleavage site that can be specifically cleaved by a cleaving agent to allow the self-assembly of the ferritin subunits into a ferritin nanocage. The cleavage site and the cleaving agent should be selected such that the construct is not cleaved at any other site that would result in the nanocage not assembling. As an example, the cleaving agent should not cleave a site within the ferritin subunits. The cleavage site and the cleaving agent should be selected such that the effect of the cleaving agent is non-toxic. Examples of cleavable linkers include pH- sensitive linkers (e.g., hydrazone), photocleavable linkers, glutathione-sensitive (e.g., SPDB) and protease-sensitive linkers (e.g., valine-citrulline dipeptide). In some embodiments, the cleavage site is a protease cleavage site (i.e., the linker is a protease-sensitive linker). In various embodiments, the cleavage site is an enterokinase or thrombin cleavage site. Typically, the cleavage site is an enterokinase cleavage site. Exemplary recognition sequences for cleavage proteases are provided in the table below. In some embodiments, the linker comprises a recognition sequence selected from the sequences in the table below.
Figure imgf000014_0001
In some embodiments, the cleavage site is an extracellular matrix protease cleavage site, such as ACE2 or Thrombin. This provides the advantage that assembly of ferritin nanocages can be targeted to the extracellular matrix outside of cells, thus mimicking the viral budding process. In some embodiments, the cleavage site is a viral protease cleavage site, such as an HIV-1 protease cleavage site, an influenza virus protease cleavage site, or a SARS-CoV-2 protease cleavage site. This provides the advantage that ferritin nanocage self-assembly can be targeted to virus-infected cells, such that ferritin nanocages can be induced to form an active therapeutic at the infected cells. Nanocage self-assembly may be achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble (i.e., nanocage-forming conditions). In some embodiments, nanocage self-assembly is induced by exposing the cleaved construct to a buffer comprising 50 mM-10 M of a salt and a pH of 6-8.5. The buffer may be any suitable buffer that induces nanocage self-assembly. In some embodiments, the buffer is selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate-Bicarbonate buffer, and any combination thereof. In some embodiments, the buffer is Tris. The buffer may be present at any suitable concentration that facilitates the self-assembly of the ferritin subunits into a ferritin nanocage. In some embodiments, the buffer is present at a concentration of 25-200 mM. The buffer may be present at a concentration of 50-150 mM. The buffer may be present at a concentration of 75-125 mM. In particular embodiments, the buffer is present at a concentration of 25-75 mM. In particular embodiments, the buffer is present at a concentration of about 50 mM. In particular embodiments, the buffer is Tris at a concentration of about 50 mM. The salt may be any suitable salt that induces nanocage self-assembly. In some embodiments, the salt is selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, and any combination thereof. In some embodiments, the salt is NaCl. In some embodiments, the salt is present at a concentration of 75 mM – 5 M. In some embodiments, the salt is present at a concentration of 100 mM-2 M. In some embodiments, the salt is present at a concentration of 200 mM-1 M. In some embodiments, the salt is present at a concentration of 500-900 mM. In some embodiments, the salt is present at a concentration of 600-800 mM. In some embodiments, the pH is between 6.1 and 8.4. In some embodiments, the pH is between 6.2 and 8.3. In some embodiments, the pH is between 6.3 and 8.2. In some embodiments, the pH is between 6.4 and 8.1. In some embodiments, the pH is between 6.5 and 8.0. In some embodiments, the pH is between 6.6 and 7.9. In some embodiments, the pH is between 6.7 and 7.8. In some embodiments, the pH is between 6.8 and 7.7. In some embodiments, the pH is between 6.9 and 7.6. In some embodiments, the pH is between 7.0 and 7.5. In some embodiments, the pH is between about 7.5-8.2. In some embodiments, the buffer further comprises a solvent for solubilisation and encapsulation of a therapeutic agent. In some embodiments, the solvent is selected from the group consisting of DMSO, ethanol, acetonitrile, or any combination thereof. In some embodiments, the solvent is DMSO. In some embodiments, the solvent to construct ratio is from 0:1 to 0.5:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.1:1 to 0.4:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.2:1 to 0.3:1 (v/v). In some embodiments, the cleaved construct is exposed to suitable conditions which allow the nanocage to self-assemble for 0-4 h. In some embodiments, the cleaved construct is exposed to suitable conditions which allow the nanocage to self-assemble for 0.5-3 h. In some embodiments, the cleaved construct is exposed to suitable conditions which allow the nanocage to self-assemble for 1-2 h. In some embodiments, the cleaved construct is incubated at 20-30°C. In some embodiments, the cleaved construct is incubated at 22-28°C. In some embodiments, the cleaved construct is incubated at 24-26°C. In some embodiments, the cleaved construct is incubated at 25°C. In some embodiments, nanocage self-assembly is induced by exposing the cleaved construct to a buffer selected from the group consisting Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate- Bicarbonate buffer, and any combination thereof, wherein the buffer comprises 50 mM- 10 M of a salt selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, and any combination thereof, and a pH of 6-8.5. In some embodiments, nanocage self-assembly is induced by exposing the cleaved construct to a buffer selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate- Bicarbonate buffer, and any combination thereof, for 0-4 h at a temperature of 20-30°C, wherein the buffer comprises 50 mM-10 M of a salt selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, and a pH of 6-8.5. In some embodiments, nanocage self-assembly is induced by exposing the cleaved construct to a buffer selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate- Bicarbonate buffer, and any combination thereof, for 0-4 h at a temperature of 20-30°C, wherein the buffer comprises 50 mM-10 M of a salt selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, a solvent selected from the group consisting of DMSO, ethanol, acetonitrile, or combinations thereof, and a pH of 6-8.5. In some embodiments, nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble after the cleavage step. In some embodiments, nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self- assemble directly after the cleavage step. In some embodiments, nanocage self- assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble within 0-4 h of the cleavage step. In some embodiments, nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble within 0-2 h of the cleavage step. In some embodiments, nanocage self-assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self- assemble within 0-1 h of the cleavage step. In some embodiments, nanocage self- assembly is achieved by exposing the cleaved construct to any suitable conditions which allow the nanocage to self-assemble simultaneously with the cleavage step. The construct may comprise one or more further elements to enhance the function of the construct and/or aid the purification of the construct and/or enhance, modify or target the expression of the construct. In certain embodiments, the construct further comprises a purification tag. The purification tag may be any suitable tag that aids or facilitates the purification of the construct. For example, the purification tag may be a polyhistidine tag, a Glutathione-S transferase (GST) tag, a calmodulin binding protein tag, a maltose binding protein (MBP) tag, a myc tag, a human influenza hemagglutinin (HA) tag, a FLAG tag, a Spot- tag, a C-tag, a Strep-tag. The purification tag may be at the N-terminal end or the C- terminal end of the construct. In particular embodiments, the purification tag is a His tag, for example a 6-His tag or a 10-His tag. In various embodiments, the purification tag is an N-terminal His tag, for example, an N-terminal 6-His tag (i.e., the His tag is present at the N-terminal end of the construct). In particular embodiments, the purification tag is cleavable from the construct (e.g., the purification tag is positioned next to a protease cleavage motif, such that cleavage of the motif cleaves the tag from the construct). As will be appreciated by those skilled in the art, the cleavage motif may be the same as the cleavage site of the linker, or may differ therefrom. Accordingly, when the cleavage motif is the same as the cleavage site of the linker a single cleavage agent can be used to cleave both the purification tag and the linker. In some embodiments, the construct further comprises one or more therapeutic elements and/or one or more non-therapeutic elements. In particular embodiments, the construct comprises one or more therapeutic elements. The term “therapeutic element” is intended to have its broadest possible interpretation and refers to any element that when delivered to a bodily conduit of a living being produces a desired, usually beneficial, effect. More particularly, a therapeutic element relates to any element that can confer a therapeutic benefit on a subject and includes, without limitation, conventional drugs, gene therapy constructs, chemotherapeutic agents, antibiotics, macromolecules, protein bound drugs and antigens. In various embodiments, the construct comprises one or more non-therapeutic elements. The term “non-therapeutic element” is intended to cover any other element that is not a therapeutic element, i.e., an element that does not confer a therapeutic benefit on a subject. Any combination of therapeutic elements and/or non-therapeutic elements is envisaged. For example, the construct may comprise one therapeutic element, or one therapeutic element and one non-therapeutic element, or one non-therapeutic element, or two therapeutic elements and one non-therapeutic element, etc. The therapeutic element(s) and/or non-therapeutic element(s) may be contained within the linker. For example, the linker may comprise a therapeutic peptide. In various embodiments, the therapeutic element(s) and/or non-therapeutic element(s) are contained within the linker and positioned adjacent to the N-terminus of the second ferritin subunit (i.e., the ferritin subunit attached to the linker at the N-terminal end of the subunit). The term “adjacent to” is intended to cover directly adjacent to (i.e., directly bonded to, with no intervening amino acid) and in proximity to (i.e., at least one intervening amino acid, and optionally between 1 and 100 intervening amino acids). Typically, the cleavage site is not located within the therapeutic element(s)/non- therapeutic element(s). For example, the linker may comprise a therapeutic peptide positioned either N- or C-terminal to the cleavage site. In some embodiments, the therapeutic element(s) and/or non-therapeutic element(s) may be located at the N-terminal end of the construct or in the N-terminal portion of the construct. In certain embodiments, the element(s) will be located on the surface of the ferritin nanocage once assembled. The term “N-terminal end” is intended to mean the element is located directly at the N-terminus (i.e., the N-terminal residue of the element forms the N-terminal end of the construct). The term “N-terminal portion” is intended to mean the region of the construct from (and including) the N-terminus of the construct up to but not including the N-terminal residue of the first ferritin subunit. For example, an antigen or therapeutic peptide may be located at the N-terminal end of the construct. As a further example, an antigen or therapeutic peptide may be located in the N- terminal portion of the construct, adjacent to a further element at the N-terminal end (e.g., a purification tag or a secretory signal). In some embodiments, a first therapeutic element or non-therapeutic element is positioned in the N-terminal portion of the construct and a second therapeutic element or non-therapeutic element is positioned adjacent to the N-terminus of the second ferritin subunit. This provides the advantage that when the linker is cleaved at the cleavage site, the N-terminus of the first ferritin subunit is attached to the first therapeutic element or non-therapeutic element, and the N-terminus of the second ferritin subunit at the N-terminus of the subunit is attached to the second therapeutic element or non-therapeutic element. In certain embodiments, the first and second therapeutic/non-therapeutic elements will be present on the surface of the nanocage once assembled. In various embodiments, the therapeutic element(s) and/or non-therapeutic element(s) may be located at the C-terminal end of the construct or in the C-terminal portion of the construct. The term “C-terminal end” is intended to mean the element is located directly at the C-terminus (i.e., the C-terminal residue of the element forms the C- terminal end of the construct). The term “C-terminal portion” is intended to mean the region of the construct from (and including) the C-terminus of the construct up to but not including the C-terminal residue of the ferritin subunit positioned closest to the C- terminus of the construct. In some embodiments, the construct further comprises one or more therapeutic elements selected from the group consisting of drugs, antibodies, proteins (including enzymes), peptides, genes, oligonucleotides, RNA therapeutics, antigens, other pharmaceutically active ingredients, or a combination thereof. In various embodiments, the therapeutic element is a therapeutic peptide. For example, the therapeutic peptide may be Exenatide, Liraglutide, Lixisenatide, Albiglutide, Dulaglutide, Semaglutide, Teduglutide, Linaclotide, Pramlintide, Abarelix, Degarelix, Carfilzomib, Mifamurtide, Aviptadil, Atosiban, Carbetocin, Taltirelin, Bremelanotide, Teriparatide, Abaloparatide, Plecanatide, Nesiritide, Angiotensin II, Icatibant, Enfuvirtide, Tesamorelin, Ziconotide, Romiplostim, Peginesatide, Lucinactant, Etelcalcetide, Afamelanotide, Pasireotide, Lutetium Lu 177 dotatate, Edotreotide gallium Ga-68, Setmelanotide, Taspoglutide, Somapacitan, Selepressin, Lenomorelin, Insulin peglispro, G17DT, Avexitide, Calcitonin gene-related peptide, Corticorelin, Leptin, Thymalfasin, Aclerastide, Thyrotropin, Somatropin pegol, Somatoprim, Pirnabine, Peptide YY, Pancreatic Polypeptide, Olcegepant, Deslorelin, Gastric inhibitory polypeptide, an antimicrobial peptide (AMP), or an antiviral peptide (AVP). Typically, the therapeutic peptide is 20 to 200 amino acids in length. The therapeutic element may be an antigen. The antigen may be selected such that when a nanocage is produced and administered, an immune response is raised against the antigen. In some embodiments, the antigen is a cancer antigen (e.g., a peptide expressed or overexpressed in a cancer), a self-antigen, or a microbial antigen (e.g., a bacterial or viral antigen). As an example, the antigen may be a tumour-associated antigen or a tumour-specific antigen. As a further example, the antigen may be a microbial antigen, such as a microbial peptide, where it is desired to raise an immune response against the microbe for vaccination. The microbial antigen may be a viral or bacterial protein or peptide. Typically, the antigen is a peptide antigen. The peptide antigen may be 20 to 1500 amino acids in length. In some embodiments, the therapeutic element is a plurality of antigens. In various embodiments, the construct comprises a plurality of antigens. The plurality of antigens may be the same antigen or different antigens. In some embodiments, the construct further comprises one or more non-therapeutic elements selected from the group consisting of secretory signals, binding molecules, targeting molecules, detectable moieties, or a combination thereof. The non-therapeutic element may be a secretory signal, such as a secretory signal peptide. The secretory signal may be selected based on the expression system, and various secretory signals are known in the art. Typically, the secretory signal is located at the C-terminal end of the construct. In some embodiments, the secretory signal is cleavable from the construct. The non-therapeutic element may be a binding molecule. The term “binding molecule” is intended to mean any molecule that can specifically bind and retain a separate molecule or molecules to the surface of the nanocage. This provides the advantage that the ferritin nanocage can be loaded with a variety of molecules on its surface, such as lipids, drugs etc, once the nanocage has been formed. In some embodiments, the binding molecule is biotin. This provides the advantage that a streptavidin-tagged molecule (such as an antigen or drug) can be attached to the surface of the ferritin nanocage through the biotin-streptavidin binding interaction. In various embodiments, the binding molecule is streptavidin. This provides the advantage that a biotin-tagged molecule (such as an antigen or drug) can be attached to the surface of the ferritin nanocage through the biotin-streptavidin binding interaction. In particular embodiments, the binding molecule is a click chemistry moiety. In some embodiments, the binding molecule is one or more non-naturally occurring amino acid molecules. The non-therapeutic element may be a targeting molecule. By “targeting molecule”, it is meant any molecule that targets the assembled nanocage to a particular location, environment or cell type. Various targeting molecules are known in the art. As examples, the targeting molecule may be a small molecule targeting ligand, an antibody/antibody-binding domain, a fragment antigen-binding (Fab) domain, a lectin, a protein receptor binding domain, or a targeting peptide. Folic acid is an example of a small molecule targeting ligand which is a high affinity ligand of endogenous folate receptor which is frequently upregulated in many types of cancers. Further examples of small molecule targeting ligands are monosaccharides such as glucose, mannose and galactose, urea derivatives such as glutamate urea and 2-[3-(1,3-dicarboxypropyl)- ureido] pentanedioic acid (DUPA), glycyrrhetinic acid (GA) derivatives, sulfonamide derivatives, benzamides, and phenyl boronic acid. An antibody or antibody-binding domain can be used to target a specific antigen, such as CD33, HER2, EGFR, or PSMA. Examples of targeting peptides include the RGD-4C peptide which targets tumours through RGD integrin αvβ3 interaction, octreotide, rabies virus glyocoprotein-29, angiopep-1, and iRGD. The non-therapeutic element may be a detectable moiety, for example a fluorescent tag or a non-fluorescent tag. Various fluorescent tags are known in the art. The fluorescent tag may be a fluorescent protein, such as GFP, EGFP, mNeonGreen, TurboGFP, RFP, mCherry, etc. The fluorescent tag may be an extrinsically fluorescent tag, such as a SNAP tag, CLIP tag, Halo tag, LOV, iLOV, etc. Examples of non-fluorescent tags include enzymatic labelling tags (e.g., Q-tag) In particular embodiments, the construct further comprises one or more therapeutic elements selected from the group consisting of a drug, an antibody, a protein, a peptide, a gene, an oligonucleotide, an RNA therapeutic, an antigen, another pharmaceutically active ingredient, and a combination thereof, and/or one or more non-therapeutic elements selected from the group consisting of a secretory signal, a binding molecule, a targeting molecule, a detectable moiety, and a combination thereof. In certain embodiments, the construct further comprises one or more of a therapeutic element selected from the group consisting of a therapeutic peptide, a peptide antigen, and a combination thereof, and/or one or more of a non-therapeutic element selected from the group consisting of a secretory signal peptide, a binding peptide, a targeting peptide, a fluorescent protein tag, and a combination thereof. In particular embodiments, the construct consists of or consists essentially of: two ferritin subunits connected by a linker, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage, and wherein cleavage of the linker at the cleavage site does not prevent the ferritin subunits from self-assembling into a ferritin nanocage. The term “consists essentially of” is intended to mean that other elements may be present in the polypeptide that do not materially affect the function of the polypeptide. In some embodiments, the construct consists of amino acids. In some embodiments, the construct consists of naturally-occurring amino acids. In a second aspect, there is provided a construct comprising: two ferritin subunits connected by a linker, wherein the linker comprises a cleavage site, wherein the linker is arranged to prevent the ferritin subunits from self-assembling into a ferritin nanocage when the linker is not cleaved at the cleavage site, and wherein the ferritin subunits are arranged to self-assemble into a ferritin nanocage when the linker is cleaved at the cleavage site. The description above in relation to the first aspect is equally applicable to this aspect. Nucleic acid molecule In a third aspect, there is provided a nucleic acid molecule encoding the construct described herein. The term "nucleic acid molecule" refers to either single- or double-stranded deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof. The above-mentioned term includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., RNA, mRNA). The above-mentioned term includes nucleic acids containing known analogs of naturally occurring nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Further, the above-mentioned term refers to oligonucleotide analogs including PNA (peptide nucleic acid) and DNA analogs (phosphorothioate, phosphoramidate, etc.) used in antisense techniques. Expression vector In a fourth aspect, there is provided an expression vector comprising the nucleic acid molecule described herein. The expression vector may be any suitable expression vector. For example, the expression vector may be a plasmid or a vector. For example, the vector may be an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector (such as a lentiviral vector), an alphaviral vector, a flaviviral vector, a herpes simplex viral vector, a rhabdoviral vector, a measles viral vector, a pox viral vector, a newcastle disease viral vector, a coxsackieviral vector, or a non-viral vector, such as a polyvalent cation, lipid nanoparticle, chitosan nanoparticle, PLGA dendrimer or other conjugate allowing cellular uptake. Preferably, the vector is a viral vector. More preferably, the vector is an AAV vector or a lentiviral vector. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 70% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 75% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 80% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 85% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 90% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 95% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 98% sequence identity thereto. In some embodiments, the expression vector has a nucleic acid sequence according to SEQ ID NO. 7 or has at least 99% sequence identity thereto. As will be appreciated by one skilled in the art, the expression vector will encode a functional construct (e.g., the construct does not self-assemble into a ferritin nanocage when uncleaved, but can be cleaved to result in ferritin nanocage self assembly). Host cell In a fifth aspect, there is provided a host cell comprising the expression vector described herein. The host cell may be any suitable host cell. As used herein, the term "host cell" refers to cells which harbour an expression vector of the invention, as well as cells that are suitable for use in expressing a recombinant gene or protein. It is not intended that the present invention be limited to any particular type of cell. Indeed, it is contemplated that any suitable cell will find use in the present invention as a host cell. A host cell according to the invention may permit the expression of a nucleic acid molecule of the invention. Thus, the host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell. The host cell may be any suitable eukaryotic cell into which the vector may be introduced. The host cell may be a mammalian cell or a plant cell. The host cell may be a human cell. The host cell may be in vivo, in vitro or ex vivo. Typical prokaryotic host cells include various bacterial cells such as Escherichia coli. Typical eukaryotic host cells include mammalian host cells, insect host cells, plant host cells, fungal host cells, eukaryotic algae host cells, nematode host cells, protozoan host cells, fish host cells, and the like. Mammalian host cells include Chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK293) cells, baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells. Typical fungal host cells include Aspergillus, Neurospora, Saccharomyces, Pichia, Hansenula, Schizosaculomyces. Pharmaceutical composition In a sixth aspect, there is provided a pharmaceutical composition comprising the construct described herein or the nucleic acid molecule described herein or the expression vector described herein or the host cell described herein and one or more pharmaceutically acceptable excipients. The one or more excipients include carriers, diluents and/or other medicinal agents, pharmaceutical agents or adjuvants, etc. Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the excipient, any suitable binder, lubricant, suspending agent, coating agent or solubilising agent. Preservatives, stabilizers and dyes may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. The pharmaceutical composition may be administered using any suitable administration method. For example, the pharmaceutical composition may be administered parenterally in which the composition is formulated in an injectable form, for delivery, by, for example, an intravenous, intradermal, intramuscular, subcutaneous or intraperitoneal route. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. Intradermal administration routes include any dermal-access means, for example, using microneedle-based injection and infusion systems (or other means to accurately target the intradermal space), needleless or needle-free ballistic injection of fluids or powders into the intradermal space, Mantoux-type intradermal injection, enhanced iontophoresis through microdevices, and direct deposition of fluid, solids, or other dosing forms into the skin, including the use of patches to deposit the composition onto the skin. The composition may also be formulated to be administered by oral or topical routes, including nasally, orally or epicutaneously. In various embodiments, the composition is formulated to be delivered by an oral or nasal route. Method of preparing a ferritin nanocage In a seventh aspect, there is provided a method of preparing a ferritin nanocage, the method comprising: contacting a plurality of construct described herein with a cleaving agent configured to cleave the linkers at the cleavage site, thereby cleaving the plurality of construct at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage. The description above in relation to the construct is equally applicable to the method of preparing a ferritin nanocage. In some embodiments, the method further comprises exposing the cleaved construct to nanocage-forming conditions (i.e., conditions which induce the self-assembly of the ferritin subunits into a ferritin nanocage). In some embodiments, the method further comprises incubating the cleaved constructs with a buffer comprising 50 mM-10 M of a salt and a pH of 6-8.5 to induce the self-assembly of the ferritin subunits into a ferritin nanocage. In some embodiments, the method further comprises adjusting the salt concentration and/or the pH to induce self-assembly of the ferritin subunits into a ferritin nanocage. The description above in relation to the conditions, for example the buffer (including all of the optional components of the buffer), that allow self-assembly of the nanocages is equally applicable to the method of preparing a ferritin nanocage. The plurality of constructs should be a sufficient number of constructs that, when cleaved, will self-assemble into a ferritin nanocage (either by themselves or with other ferritin subunits). In some embodiments, the plurality of constructs is at least 12 constructs. The method may be performed in vivo, in vitro or ex vivo. In some embodiments, the method is performed in vitro or ex vivo. The term “contacting” as used herein refers to bringing the plurality of constructs and the cleaving agent together in such a manner that the cleaving agent can cleave the cleavage site of the linker. The plurality of constructs may be contacted with a cleaving agent by any suitable method. In some embodiments, the plurality of constructs is mixed with a cleaving agent in solution. In other embodiments, the plurality of constructs is introduced into an environment where the cleaving agent is present and functional. For example, the plurality of constructs may be added to a cell culture where a cleaving agent is inherently present (e.g., an extracellular matrix protease) or where a cleaving agent has been added. The contacting step may be any suitable length such that the cleaving agent cleaves the linkers at the cleavage site. In some embodiments, the contacting step is 2-18 h. In some embodiments, the contacting step is 4-16 h. In some embodiments, the contacting step is 6-14 h. In some embodiments, the contacting step is 8-12 h. The contacting step may be carried out in any suitable conditions that allow the cleaving agent to cleave the linkers at the cleavage site. In some embodiments, the contacting step is carried out in a reaction buffer. In some embodiments, the reaction buffer is selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate-Bicarbonate buffer, and any combination thereof. In some embodiments, the reaction buffer comprises 1-500 mM salt. In some embodiments, the salt is selected from the group consisting of CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, and combinations thereof. In some embodiments, the contacting step is carried out at a pH between 6.0-8.5. In some embodiments, the contacting step is carried out at a pH between 6.1 and 8.4. In some embodiments, the contacting step is carried out at a pH between 6.2 and 8.3. In some embodiments, the contacting step is carried out at a pH between 6.3 and 8.2. In some embodiments, the contacting step is carried out at a pH between 6.4 and 8.1. In some embodiments, the contacting step is carried out at a pH between 6.5 and 8.0. In some embodiments, the contacting step is carried out at a pH between 6.6 and 7.9. In some embodiments, the contacting step is carried out at a pH between 6.7 and 7.8. In some embodiments, the contacting step is carried out at a pH between 6.8 and 7.7. In some embodiments, the contacting step is carried out at a pH between 6.9 and 7.6. In some embodiments, the contacting step is carried out at a pH between 7.0 and 7.5. In some embodiments, the contacting step is carried out at a pH between about 7.5-8.2. In some embodiments, the reaction buffer comprises a solvent for solubilisation and encapsulation of a therapeutic agent. In some embodiments, the solvent is selected from the group consisting of DMSO, ethanol, acetonitrile, and combinations thereof. In some embodiments, the solvent is DMSO. In some embodiments, the solvent to construct ratio is from 0:1 to 0.5:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.1:1 to 0.4:1 (v/v). In some embodiments, the solvent to construct ratio is from 0.2:1 to 0.3:1 (v/v). In some embodiments, the reaction buffer further comprises one or more metal ions. The one or more metal ions may be selected from the group consisting of iron, nickel, cobalt, copper, gold, manganese, palladium, platinum, molybdenum, tungsten and combinations thereof. In some embodiments, the contacting step is carried out in 25-75 mM reaction buffer comprising 50-150 mM salt at pH 6-8.5 for 2-16 h. In several embodiments, the plurality of constructs are provided by expressing the nucleic acid molecule described herein or the expression vector described herein. The cleaving agent may be any suitable cleaving agent that cleaves the cleavage site of the linker. Typically, the cleaving agent specifically cleaves the cleavage site and does not cleave any other site in the constructs. As an example, if the cleavage site is a protease cleavage site, the cleaving agent will be a protease that cleaves that cleavage site. As a further example, if the cleavage site is an enterokinase cleavage site or a thrombin cleavage site, the cleaving agent will be enterokinase or thrombin, respectively. The term “facilitating self-assembly” is intended to mean that the constraints on the ferritin subunits provided by the uncleaved linker are removed by the cleavage of the linker, thereby enabling the separated ferritin subunits to spontaneously form a ferritin nanocage (e.g., a 24-meric ferritin nanocage) when exposed to nanocage-forming conditions. Typically, the cleaved subunits are not subsequently purified following the cleavage step. For example, the method is intended to form ferritin nanocages, and not to separately purify the ferritin subunits. The ferritin nanocages may subsequently be purified following their self-assembly using any suitable purification technique. In some embodiments, the method further comprises contacting the plurality of constructs with a cargo molecule, such that the cargo molecule is encapsulated inside the ferritin nanocage during self-assembly of the ferritin subunits into the ferritin nanocage. The cargo molecule may be any molecule that is intended to be encapsulated inside the ferritin nanocage. The cargo molecule is extrinsic to the construct described herein. The term “encapsulated” is intended to mean that the cargo molecule is trapped inside the hollow cavity of the nanocage and is generally not covalently bound to the nanocage. The cargo molecule can be any biological molecule, chemical molecule, synthetic molecule, or any other molecule that can be encapsulated by the ferritin nanocage described herein. In some embodiments, the cargo molecule is a therapeutic element. For example, the therapeutic element may be a drug, an antibody, a protein, a peptide, a gene, an oligonucleotide, an RNA therapeutic, an antigen, another pharmaceutically active ingredient, or a combination thereof. The therapeutic element may be any other ingredient intended to have a therapeutic effect (e.g., metal nanoparticles). As a further example, the cargo molecule may be DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumour proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti- inflammatories, anti-histamines, anti-infectives, and/or chemotherapeutics. Other suitable cargo molecules include sensitizers (e.g., radiosensitizers) that can make a cell or subject more responsive (or sensitive) to a treatment or prevention and imaging or other diagnostic agents. The ferritin nanocages can be used as a monotherapy or in combination with other active agents for treatment or prevention of a disease. Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g., melatonin and thyroxine), small peptide hormones and protein hormones (e.g., thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eiconsanoids (e.g., arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydro testosteron cortisol). Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6- MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26 and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers). Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammants (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylae, and sodium salicaylate), paracetamol/acetaminophen, metamizole, nabumetone, phenazone, and quinine. Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotenergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepresents, and monoamine oxidase inhibitors), mebicar, afobazole, selank, bromantane, emoxypine, azapirones, barbituates, hyxdroxyzine, pregabalin, validol, and beta blockers. Suitable antipsychotics include, but are not limited to, benperidol, bromoperidol, droperidol, haloperidol, moperone, pipaperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dizyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, tiotixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzaprine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, befeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine. Suitable analgesics include, but are not limited to, paracetamol/acetaminophen, non- steroidal anti-inflammants (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupiretine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g., choline salicylate, magnesium salicylae, and sodium salicaylate). Suitable antispasmodics include, but are not limited to, mebeverine, papverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methodcarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable anti-inflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammants (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immune selective anti- inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives). Suitable anti-histamines include, but are not limited to, H1-receptor antagonists (e.g. acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromapheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebasine, embramine, fexofenadine, hydroxyzine, levocetirzine, loratadine, meclozine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2- receptor antagonists (e.g. cimetidine, famotidine, lafutidine, nizatidine, rafitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and β2-adrenergic agonists. Suitable anti-infectives include, but are not limited to, amebicides (e.g., nitazoxanide, paromomycin, metronidazole, tnidazole, chloroquine, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, abendazole, miltefosine, thiabendazole, oxamniquine), antifungals (e.g., azole antifungals (e.g., itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin, and amphotericin b), antimalarial agents (e.g., pyrimethamine/sulfadoxine, artemether/lumefantrine, atovaquone/proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g., aminosalicylates (e.g., aminosalicylic acid), isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethanmbutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g., amantadine, rimantadine, abacavir/lamivudine, emtricitabine/tenofovir, cobicistat/elvitegravir/emtricitabine/tenofovir, efavirenz/emtricitabine/tenofovir, avacavir/lamivudine/zidovudine, lamivudine/zidovudine, emtricitabine/tenofovir, emtricitabine/opinavir/ritonavir/tenofovir, interferon alfa-2v/ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpiviirine, delaviridine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, avacivr, zidovudine, stavudine, emtricitabine, xalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir/ritonavir, fosamprenvir, dranuavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, sawuinavir, ribavirin, valcyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g. doripenem, meropenem, ertapenem, and cilastatin/imipenem), cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceflaroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telvancin), glycylcyclines (e.g., tigecycline), leprostatics (e.g., clofazimine and thalidomide), lincomycin and derivatives thereof (e.g., clindamycin and lincomycin), macrolides and derivatives thereof (e.g., telithromycin, fidaxomicin, erthromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, beta lactam antibiotics (benzathine penicillin (benzatihine and benzylpenicillin), phenoxymethylpenicillin, cloxacillin, flucoxacillin, methicillin, temocillin, mecillinam, azlocillin, mezlocillin, piperacillin, amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, clavulanate/ticarcillin, penicillin, procaine penicillin, oxacillin, dicloxacillin, nafcillin, cefazolin, cephalexin, cephalosporin C, cephalothin, cefaclor, cefamandole, cefuroxime, cefotetan, cefoxitin, cefiximine, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, cefepime, cefpirome, ceftaroline, biapenem, doripenem, ertapenem, faropenem, imipenem, meropenem, panipenem, razupenem, tebipenem, thienamycin, azrewonam, tigemonam, nocardicin A, taboxinine, and beta-lactam), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (e.g., sulfamethoxazole/trimethoprim, sulfasalazine, and sulfasoxazole), tetracyclines (e.g., doxycycline, demeclocycline, minocycline, doxycycline/salicyclic acid, doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g. nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue). Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, aspargainase erwinia chyrsanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, all-trans retinoic acid, and other anti-cancer agents. Suitable sensitizing agents can include, but are not limited to, radiosensitizers, insulin sensitizers (e.g., metformin, thiazolidinediones,) and photosensitizers for photodynamic therapy (e.g., aminolevulinic acid (ALA), Silicon Phthalocyanine Pc 4, m- tetrahydroxyphenylchlorin (mTHPC) and mono-L-aspartyl chlorin e6 (NPe6)). Suitable imaging agents include but are not limited to, fluorescent molecules (e.g., Cy3, Cy5, and other commercially available fluorflores), paramagnetic ions, nanoparticles that can contain a paramagnetic ion, super-paramagnetic iron oxide molecules and nanoparticles thereof, 18F-fluorodeoxyglucose and other PET imaging agents, gadolinium containing contrast agents, radionuclides and compositions thereof. In particular embodiments, the method comprises binding a cargo molecule to the surface of the ferritin nanocage. The cargo molecule may be any suitable cargo molecule as discussed above; however, in this instance, the cargo molecule is intended to be bound to the surface of the nanocage rather than being encapsulated inside the nanocage. As an example, the cargo molecule may be a therapeutic element as defined above, such as a drug, peptide, protein, antigen, gene, oligonucleotide, or other pharmaceutically active ingredient. The cargo molecule may be any other ingredient intended to have a therapeutic effect (e.g., metal nanoparticles). The step of binding the cargo molecule (e.g., an antigen) to the binding molecule may comprise contacting the nanocage with the cargo molecule. In various embodiments, the cargo molecule may be cross-linked to the nanocage via transamidation of a glutamine tag in the construct using transglutaminase. In several embodiments, the cargo molecule is bound to the binding molecule via click chemistry. In some embodiments, the cargo molecule is bound to a non-naturally occurring amino acid molecule in the construct using chemical biology. As an example, the construct may include an azidoalanine residue at its N-terminus; a linker (e.g., a DBCO-NHS-ester linker) can be added to a lysine residue of a cargo molecule (e.g., an antibody) which can be linked to the azidoalanine residue via click chemistry. Ferritin nanocage In an eighth aspect, there is provided a ferritin nanocage produced by a method described herein. As will be appreciated by the skilled person, the assembled ferritin nanocage will differ from the ferritin nanocages of the prior art in that the ferritin subunits will remain connected to portions of the cleaved linker. Accordingly, the ferritin nanocage will comprise portions of the cleaved linker. In some embodiments, at least one of the ferritin subunits is connected to a portion of the cleaved linker. In various embodiments, each of the ferritin subunits is connected to a portion of the cleaved linker. Method of treatment and first medical use In a ninth aspect, there is provided a method of treating or preventing a disease in a subject, the method comprising administering a ferritin nanocage described herein to a subject, wherein the ferritin nanocage comprises a therapeutic element. In a tenth aspect, there is provided a method of treating or preventing a disease in a subject, the method comprising administering a construct described herein to a subject, wherein the construct comprises a therapeutic element. In an eleventh aspect, there is provided a method of treating or preventing a disease in a subject, the method comprising administering a nucleic acid molecule described herein or an expression vector described herein or a host cell described herein or a pharmaceutical composition described herein to a subject, wherein the nucleic acid molecule or the expression vector or the host cell or the pharmaceutical composition comprises a therapeutic element. The term “treating” a disease, as used herein, means the treatment of a disease in a subject, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development or progression; (b) relieving the disease, i.e., causing regression of the disease state; (c) relieving one or more symptoms of the disease; and (d) curing the disease. The term “preventing” a disease, as used herein, means the prevention of a disease in a subject, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; and (c) preventing or delaying the onset of at least one symptom of the disease. The disease may be any disease. Diseases that may be treated or prevented include cancers, neurological disorders, genetic disorders (e.g., protein deficiency), heart disease, stroke, arthritis, viral and bacterial infections, as well as immune system disorders, but are not limited to these. In some embodiments, the disease is selected from cancers or microbial infections. In various embodiments, the disease is cancer and the therapeutic element is an anti-cancer therapeutic (e.g., chemotherapeutic drug, cancer antigen, antibody, etc). In particular embodiments, the disease is a microbial infection and the therapeutic element is an antigen specific to the microbe. As used herein, the term “administering” refers to any method of providing the therapy (e.g., the ferritin nanocage, or the construct) to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, intracardiac administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. The ferritin nanocage may be administered to the subject via any suitable administration route. For example, the nanocage may be administered to the subject orally, intravenously, occularly, intraoccularly, intramuscularly, intravaginally, intraperitoneally, rectally, parenterally, topically, intranasally, or subcutaneously. As used herein, the term “subject” refers to the target of administration, e.g., an animal. Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. 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. In one aspect, the subject is a patient. The therapeutic element may be any therapeutic element that is suitable for treating or preventing the disease in the subject. Examples of therapeutic elements are discussed above. There is also provided a ferritin nanocage described herein for use in therapy, wherein the ferritin nanocage comprises a therapeutic element. There is also provided a construct described herein or a nucleic acid molecule described herein or an expression vector described herein or a host cell described herein or a pharmaceutical composition described herein described herein for use in therapy, wherein the construct or the nucleic acid molecule or the expression vector or the host cell or the pharmaceutical composition comprises a therapeutic element. Methods of raising an immune response against an antigen In a twelth aspect, there is provided a method of raising an immune response against an antigen, the method comprising: administering a ferritin nanocage described herein to a subject, wherein the ferritin nanocage comprises an antigen. The ferritin nanocage comprising an antigen may be prepared by the methods as described above. As a first example, the nanocage may be prepared in a manner which results in antigen being present on the surface of the nanocage. The nanocage may be prepared by contacting a plurality of constructs described herein with a cleaving agent, wherein at least one of the plurality of constructs comprises an antigen, and wherein the cleaving agent is configured to cleave the cleavage site of each linker, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage. As a second example, the nanocage may be prepared in a manner which results in antigen being bound to binding molecules on the surface of the nanocage. The nanocage may be prepared by contacting a plurality of constructs described herein with a cleaving agent, wherein at least one of the plurality of constructs comprises a binding molecule, and wherein the cleaving agent is configured to cleave the cleavage site of each linker, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage, and binding an antigen to the binding molecule. As a third examples, the nanocage may be prepared in a manner which results in antigen being encapsulated inside the nanocage. The nanocage may be prepared by contacting a plurality of constructs described herein with a cleaving agent and a cargo molecule, wherein the cargo molecule is an antigen, and wherein the cleaving agent is configured to cleave the cleavage site of each linker, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self-assembly of the ferritin subunits into a ferritin nanocage and the encapsulation of the cargo molecule inside the ferritin nanocage. The description above in relation to the construct, the methods of preparing a ferritin nanocage and the methods of treatment is equally applicable to these aspects. As a particular example, the contacting step may be achieved in any suitable manner as described above, the cleaving agent may be any suitable cleaving agent as described above, the subject may be any subject as described above, and the administration step may be performed in any manner as discussed above. The antigen may be any antigen where it is intended to raise an immune response against that antigen. In some embodiments, the antigen is a cancer antigen (e.g., a peptide expressed or overexpressed in a cancer), a self-antigen, or a microbial antigen (e.g., a bacterial or viral antigen). As an example, the antigen may be a microbial antigen, such as a microbial peptide, where it is desired to raise an immune response against the microbe for vaccination. Typically, the antigen is a peptide antigen. The peptide antigen may be 20 to 1500 amino acids in length. In various embodiments, the plurality of constructs each comprise an antigen. The antigen may be the same antigen (i.e., all of the constructs contain the same single antigen) or different antigens (i.e., a first construct may contain a first antigen and a second construct may contain a second antigen), the latter resulting in nanocages presenting multiple antigens. Where the antigen is comprised within the construct, the antigen may be positioned at a suitable position within the construct as discussed above in relation to the polypeptide. Where the antigen is bound to the binding molecule on the nanocage, the binding molecule may be any suitable binding molecule as discussed above in relation to the construct. The step of binding the antigen to the binding molecule may comprise contacting the nanocage with the antigen. In various embodiments, the antigen may be cross-linked to the nanocage via transamidation of a glutamine tag in the construct using transglutaminase. In several embodiments, the antigen is bound to the binding molecule via click chemistry. In some embodiments, the antigen is bound to a non-naturally occurring amino acid molecule in the construct using chemical biology. According to further aspect, the present disclosure provides an ACE2-peptide mimetic comprising the sequence shown in SEQ ID NO:3. The ACE2-peptide mimetic was designed by combining the sequences of two polypeptides of the human ACE2 protein that interact with the SARS-CoV-2 receptor binding domain. A skilled person will appreciate that all aspects of the invention, whether they relate to, for example, the construct, the polypeptide, the nucleic acid molecule, the expression vector, the methods of raising an immune response, or the method of treatment for example, are equally applicable to all other aspects of the invention. In particular, aspects of the construct may have been described in greater detail than in some of the other aspects of the invention, for example, relating to the method of treatment. However, the skilled person will appreciate where more detailed information has been given for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The invention will now be described in detail by way of example only with reference to the figures. Brief Description of the Drawings Figure 1 shows various methods known in the prior art used for controlling ferritin self- assembly in biomedical applications. (A) Structure of 24-meric nanocage showing the internal cavity of 8 nm. (B) The N-terminus of each subunit is on the surface at 3-fold symmetry axis and can be used to add a ligand, such as an antigen. (C) Disassembly and assembly mediated by pH, temperature, and pressure. (D) Engineering the interface of subunits to introduce metal binding sites to control self-assembly. (E) Introducing an RNA binding protein at the N-terminus to create monomers upon RNA-binding and subsequent cleavage of RNA-binding protein to generate 24-meric nanocages. Figure 2 shows supporting data for the disclosure. (A) A schematic presentation of an exemplary embodiment of the invention combining two subunits of ferritin to generate PINCs. (B) SDS-Page analysis of purified protein. Lane (1) and (2): Nickel column was washed and a sample was run on the gel. Lane (3): Dimeric protein was eluted using 150 mM imidazole and a sample was run on the gel. (C) SDS-Page analysis of purified dimers in Lane (3) after cleavage by a site-specific protease (enterokinase) (Lane ((3)+P)). Lane (N): Native 24-meric ferritin. Lane ((3)+P): formation of 24-meric PINCs. (D) The process to generate PINCs can be used to encapsulate a red dye with an absorbance pick at 480 nm. Native 24-mer, engineered dimer alone, and engineered dimer + Protease (P) were incubated with phenol red dye overnight. Next, samples were washed with buffer (10-times of the volume of sample) using a 100 kDa Amicon Ultracentrifugal filter. Subsequently, UV-visible absorbance spectra were recorded. Concentration of dimer was 7 µM and that of assembled 24-mer was 0.6µM. Figure 3 shows various applications of the invention. (A) A drug delivery vehicle for various medicines. (B) Creating mosaic vaccines. (C) Creating multifunctional medicines. (D) Developing novel AAV-based vaccines mimicking a virus replication process to create efficacious and long-lasting immune response. In this process the AAV construct of ferritin-dimer carrying a viral antigen and a secretory signal is delivered to the cell. The dimer has a peptide sequence for cleavage by an extracellular protease. The dimer is secreted and subsequently cleaved by the protease forming 24- meric nanocages representing the virus antigen. This virus like nanocage will then activate the immune response. The process mimics the process of maturation and release of a virus particle. Figure 4 shows the designed Ferritin-ACE2-D construct. A 6xHis-tag polypeptide was added to the N-terminus of first subunit and the N-terminus of the second subunit (with an ACE2 peptide mimetic between the 6xHis-tag and the subunit in each case). The novel ACE2-peptide mimetic was designed from combining the sequences of two polypeptides of the human ACE2 protein interacting with the SARS-CoV-2 receptor binding domain. The recognition sequence for Enterokinase protease was added between the C-terminus of the first subunit and the second 6xHis-tag, thus, after cleavage with enterokinase, the two subunits will be identical. Figure 5 shows the exemplary construct in the original plasmid before subcloning into pBAD/His C. Figure 6 shows PINC technology can be used to decorate the surface of nanocages with multiple proteins. (a) PfEDU-2 with an N-terminus Sumo was cleaved using enterokinase to generate two subunits, i.e., Subunit-1 and Subunit-2. (b) Native-PAGE analysis of PfEDU-2 and PINCs formed due to spontaneous and random assembly of Subunit-1 and -2 with different ratios. Wild-type PfFtn (WT) (MW circa 480 kDa) was used as a control. Experiments were repeated at least five times using different batches of protein. (c) The predicted structure of PfEDU-2 showed that (d) the BC loops of Subunit-1 and -2 do not interact directly. (e) The structure showed that the enterokinase cleavage site is accessible. (f and g) Exemplary constructs according to the disclosure. Figure 7 shows PINC technology can be used to decorate the surface of nanocages and encapsulate both hydrophilic and hydrophobic drugs. (a) A schematic presentation of the encapsulation process using PINCs. (b) UV-visible absorbance spectrum of encapsulated DOX and (c) CPT. The encapsulation of DOX or CPT was performed by addition of either drug to PfEDU-2 and subsequent formation of PINCs as shown in scheme (a). In the control, DOX or CPT was added after formation of PINCs. Samples were subject to extensive dialysis to remove free drug before analysis using UV-visible spectrophotometry. Experiments were repeated at least three times. Figure 8 shows the amino acid sequence of the designed (A) PfEDU-2 construct and (B) HpEDU construct. Figure 9 shows analysis PfEDU-2 and PINCs using (a) size exclusion chromatography and (b) Native-PAGE. Analysis suggested PINCs generated from PfEDU-2 have a wide range of MW between 600-720 nm. NativePAGE™ 4-16% Bis-Tris Protein Gel was used. Figure 10 shows LC-MS/MS analysis of PINCs generated from PfEDUs, confirming decoration with Sumo protein. The list of peptides detected using LC-MS/MS. The Native-PAGE band associated with PINCs generated from PfEDUs were cut and subject to trypsin digestion and analysis. Figure 11 shows LC-MS/MS spectrum of the peptides assigned to Sumo protein. The band assigned to PINCs formed from PfEDU-2 was cut and subject to trypsin digestion and analysis using LC-MS/MS to identify SUMO protein. Figure 12 shows generation of PINCs using HpEDUs. (a) The HpEDUs with an N- terminus Sumo and insecticide protein were cleaved by enterokinase to generate two subunits. (b) Native gel analysis of HpEDUs and PINCs formed due to a spontaneous and random assembly of Subunit-1 and -2 with different ratios. Experiments were repeated at least three times using different batches of protein. Novex™ 4-12% Tris- Glycine Mini Gel was used. Figure 13 shows comparison of the initial rate of Fe(III) formation for PfEDU-2, PINCs, WT-PfFtn, and background oxidation of Fe(II) by molecular oxygen. Figure 14 shows kinetics of Fe(III) mineral core formation. Figure 15 shows Fe(III) oxidation and mineralization by HpEDUs and PINCs. (a) Raw data for oxidation of Fe(II) to Fe(III) and mineralization of Fe(III) product. Formation of Fe(III) was recorded after addition of Fe(II). Background oxidation of Fe(II) by molecular oxygen was characterized by exponential increase in the formation of Fe(III) as described previously (Honarmand Ebrahimi, K.; Hagedoorn, P.-L.; Jongejan, J. A.; Hagen, W. R. Catalysis of Iron Core Formation in Pyrococcus Furiosus Ferritin. JBIC J. Biol. Inorg. Chem. 2009, 14 (8), 1265–1274). However, in the presence of WT-PfFtn, there was a sudden jump in Fe(III) formation followed by linear increase. In the case of HpEDU and PINC the formation of Fe(III) was very similar to WT-PfFtn. Concentration of HpEDU was 4 µM, that of PINCs was 0.33 µM (24-mer), and that of WT-Pftn was 1.6 µM (24-mer). The final concentration of Fe(II) added was 0.4 mM. Buffer was MOPS 100 mM, NaCl 100 mM, pH 7.0. Figure 16 shows Native-PAGE analysis of PINCs encapsulating DOX. PfEDU-2 was used to generate PINC in the presence or absence (control) of DOX. To the control DOX was added after PINC formation. Both samples were subject to extensive dialysis and analyzed using Native-PAGE. NativePAGE™ 4-16% Bis-Tris Protein Gel was used. Figure 17 shows standard curve for DOX. (a) Absorbance of free DOX in solution was measured using UV-visible spectrophotometry. (b) The standard curve for DOX was obtained from the plot of maximum absorbance at 495 nm as the DOX concentrations (µM). Data are average of three measurements ± standard deviation. Figure 18 shows the effect of DMSO on PINCs formation. The PfEDU-2 were mixed with different ratios of DMSO (0-50%). Then, enterokinase was added to the solution after the formation of PINCs, samples were analyzed using Native-PAGE. The addition of more than 25% DMSO fully degrades PINCs. NativePAGE™ 4-16% Bis-Tris Protein Gel was used. Figure 19 shows Native-PAGE analysis of PINCs encapsulating CPT. The inventors compared PINCs after formation without dialysis with PINCs+CPT after extensive dialysis and with control. The control was PINCs incubated with the drug and extensively dialyzed to remove free CPT. NativePAGE™ 4-16% Bis-Tris Protein Gel was used. Figure 20 shows standard curve for CPT. Standard curve was measured by addition of CPT dissolved in DMSO to protein buffer to reach a DMSO concentration of 10% and desired concentration of CPT. Detailed Description of the Invention Ferritin nanocages are typically made of 24 (maxi-ferritin) or 12 (mini-ferritin) subunits. The subunits spontaneously self-assemble to form a spherical shape structure (Figure 1A) with an internal cavity of 8 nm. The protein has eight three-fold axes where the N- termini of the ferritin subunits are positioned towards the outside (Figure 1B). Due to narrow and uniform size distribution of nanocages, an internal cavity, and an N- terminus accessible for modification, there is a growing interest in using ferritin nanocages in drug delivery, antibody therapy, and vaccine development. Despite all the process in using the nanocage for developing therapeutics, a major bottleneck in the application of ferritin is the self-assembly of the protein. Since overexpression of subunits will spontaneously lead to formation of nanocages, encapsulation of various drugs or addition of multiple ligands to the surface cannot be achieved easily using a non-destructive method at neutral pH and room temperature/pressure. To date, harsh methods such as increasing or decreasing pH, increasing temperature, increasing pressure, extensive engineering of the protein interface to introduce metal binding sites, or protein modification to achieve RNA binding and aggregation, have been used to control self-assembly and access subunits or the internal cavity of the protein. To develop a non-destructive and easy to apply method to control self-assembly and access the surface of subunits for creating drug delivery systems, prophylactics, and mosaic therapeutics, linked two subunits of ferritin. The first step of spontaneous self- assembly of a ferritin nanocage requires that two subunits interact in an anti-parallel fashion via their interface. Therefore, the inventors hypothesised that if the C-terminus of one subunit is linked to the N-terminus of a second subunit (i.e., if the dimers are linked in series), the resulting protein dimers will not physically be able to come together in an anti-parallel fashion and initiate self-assembly. For the self-assembly to initiate, the linker between the dimers must be cleaved, and hence the inventors introduced the recognition sequence of a specific protease, Enterokinase, in the linker peptide between the two subunits. Example 1 As a proof of principle, the inventors aimed to design a prophylactic against SARS- CoV-2. Their aim was to create a nanocage furnished with ACE2-peptide mimetics for use as a novel inhaled prophylactic and decoy of SARS-CoV-2 to prevent infection of cells. Such an inhaled prophylactic could replace or supplement masks. Therefore, the inventors aimed to furnish the surface with a novel peptide sequence mimicking the key sequences of the human ACE2 receptor interacting with SARS-CoV-2 receptor binding domain (RBD). The ACE2-peptide mimetic was added to the N-terminus of the first subunit and the second subunit. To be able to purify the nanocage easily and demonstrate that dimers only form 24-meric nanocage in the presence of enterokinase, a 6xHis-tag polypeptide was introduced at the N-terminus of ACE2-peptide mimetic. For simplicity, a protease cleavage site for the 6xHis-tag polypeptide was not introduced. However, in other embodiments a protease cleavage site can be present at the C- terminus of each 6xHis-tag in order for the tags to be cleaved from the construct. Construct design Figure 2A shows an exemplary construct according to an embodiment of the disclosure. The construct includes two ferritin subunits linked by a linker containing a protease cleavage site. The N-terminus of the construct has a 6-His tag. The connection of the two subunits via the linker prevents the subunits from self-assembling into a ferritin nanocage. Cleavage of the linker at the cleavage site by a protease allows the self- assembly of the subunits (along with other monomeric subunits) into a ferritin nanocage (referred to as a Protease Induced Ferritin Nanocage; PINC). 24 ferritin subunits are required to form the ferritin nanocage. Construction of ACE2-ferritin dimer (ACE2-Fr-D) A construct for expression of a Pyrococcus furiosus ferritin dimer containing an ACE2 peptide-mimetic at each terminus of the subunit was designed (see Figure 4). The construct had an amino acid sequence according to SEQ ID NO: 1. Each ferritin subunit (SEQ ID NO:4) has a poly histidine peptide tag (SEQ ID NO: 5) and an ACE2- peptide mimetic (SEQ ID NO:3) at the N-terminus of each subunit. The two subunits are linked by a peptide sequence (SEQ ID NO:2) cleaved specifically by Enterokinase enzyme. The construct is referred to as ACE2-Fr-D. The codon optimised gene for expression of ACE2-Fr-D in E. coli was obtained from GeneArt (ThermoFisher Scientific; SEQ ID NO: 6) (see Figure 5). For cloning of the gene into a bacterial expression system the sequences for two restriction enzymes were added. The sequence for NcoI restriction enzyme at the 5′ end the sequence for EcoRI restriction enzyme at the 3′ end. This designed gene was then cloned into PBAD/His C expression plasmid using NcoI and EcoRI restriction sites. The vector sequence had a nucleic acid sequence according to SEQ ID NO: 7. The resulting construct was then transformed into E. coli Top10 cells. Overexpression A singly colony of Top10 cells carrying the ACE2-Fr-D plasmid were inoculated into LB media (typically 10-50 ml) and grown overnight in a shaker, 37 °C with 200 rpm. After 12-16 hours the overnight culture was inoculated into Terrific Broth (TB) media (100-500 ml). Cells were grown for approximately 2 hours until the Optical Density at 600 nm reached 0.5-0.8. Then expression of protein was induced by addition of arabinose (final concentration of 0.04% (W/V)). Cells were grown for another 5-6 hours and then collected using centrifugation (5000 rpm, 4 °C). Purification To purify the protein, cells were resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 1 mM PMSF and 0.1mM DNase, pH 8.0. The buffer contained 0.02 w/v TritonX- 100 and 0.5 mM dithiothreitol (DTT) to reach maximum cell lysis efficiency using sonification. Cells were sonicated for 5 minutes (30 seconds each cycle and 10 cycles in total) by using a probe sonicator (Branson Sonifier). The % amplitude of sonication from 30%. Protein concentration was measured by using Implen NanoPhotometer. Sonication was performed on ice. After sonification, the cell-debris were removed by centrifugation at 5000 rpm for 20 min. The cell-free extract was used for purification of the protein using his-tag affinity purification using cOmpleteTM His-tag purification resin (Roche). The resin was equilibrated with buffer A containing 50 mM NaH2PO4, pH 7.6, 300 mM NaCl, 0.02 w/v TritonX-100 and 0.5mM DTT and 10 mM Imidazole. Next, the cell-free extract obtain from E. coli was incubated with the cOmpleteTM His- tag purification resin and incubated in cold room (4 °C) for 2-4 hours with continuous shaking. Next, the solution was transferred to a gravity purification column (ThermoFisher Scientific). The resin was washed with wash buffer A (approximately 10 ml) and protein was eluted using buffer A containing 150 mM Imidazole. The concentration of protein was measured using BCA assay or alternatively by measuring the absorbance at 260 nm using a Nano spectrophotometer. The purified protein was flash-frozen in liquid nitrogen and kept in -80 °C freezer for future experiments. Analysis of protein assembly using SDS-PAGE Gel electrophoresis The gel electrophoresis apparatus, pre-casted polyacrylamide gels (Any kDTM) and running buffers were purchased from Bio-Rad Laboratories. The protein ladder Precision Plus ProteinTM All Blue Standard 10-250kDa (Bio-Rad Laboratories) was used. The protein loading dye 4xLaemmli sample buffer (Bio-Rad Laboratories) was used for running SDS-PAGE. The native gel protein loading buffer (4x) was made of 2.5x Tris-Borate-EDTA buffer, 50% glycerol, 0.1% bromophenol blue and ddH2O. Gel electrophoresis was run at 120V for 30 minutes. The polyacrylamide gel was immersed in the Coomassie blue dye PageBlue Protein Staining Solution (Thermo Scientific) and incubated for 4 to 12 hours on a shaker. Polyacrylamide gel was destained by water for 1 hour on a shaker before visualized in the Gel Image System. Figure 2B shows SDS- PAGE gel analysis of the purified construct, clearly showing purification of the 58 kDa dimer in lane (3). Enterokinase cleavage of ACE2-Fr-D Enterokinase were purchased from Sigma (100 U/mg). The lyophilised protein was dissolved in 400 µl milli-Q water. The ACE2-Fr-D sample was washed multiple times (20 times of its volume) using Amicon Ultracentrifugation filter (10 kDa) to exchange the buffer to 50 mM Tris, pH 8.0 (a buffer which facilitates enterokinase cleavage and nanocage self-assembly). To cleave ACE2-Fr-D an Enterokinase:ACE2-Fr-D ratio of 1:20 or 1:200 (mg/ml) was used. After addition of Enterokinase the sample was incubated at 25 °C for 16 hr. Subsequently, the reaction was stopped by addition of SDS denaturing solution and heating the sample at 70 C for 10 min. The resulting material was analysed using SDS-PAGE electrophoresis or SEC-MALS. Figure 2C shows SDS- PAGE gel analysis of the constructs following enterokinase cleavage, clearly showing purification of PINCs in lane (3)+P at the expected theoretical molecular weight of 696 kDa. SEC-MALS analysis The actual molecular weight (MW) of ACE2-Fr 24-mer nanocage was detected by SEC- MALS (Size-Exclusion Chromatography Multi-Angle Light Scattering). SEC-MALS measured the UV absorbance at the wavelength (280 nm and 230nm) for four protein samples including bovine serum albumin standard (BSA standard, theoretical MW: 66.4 kDa), natural P. furiosus ferritin (theoretical MW: 480 kDa), ACE2-Fr-D (theoretical MW: 58 kDa), and ACE2-Fr 24-meric nanocage (theoretical MW: 696 kDa). The estimation of MW corresponding to defined peaks were conducted in the software (Astra). An aliquot of 120 µL of each protein was used and the concentration of each protein was 2 mg/mL. Each sample was transferred into clear glass snap-top microvials (Thermo Scientific). The protein buffer and running buffer was 50 mM Tris-HCl, pH 8.0. The SEC analytical column was WTC-MP030S5. The results are provided in Table 1. The measured molecular weight of the ACE2-Fr 24-mer nanocage (692.00 ± 52.72 kDa) was similar to the theoretical molecular weight (696kDa). It was not possible to accurately measure the molecular weight of ACE2-Fr-D because the protein is not globular and is flexible due to the presence of the linker; accordingly a very broad band with multiple small peaks was observed, reflecting the various shapes of the dimer in solution. Table 1. Measurement of molecular weight (MW) of ACE2-Ferritin Nanocage using SEC-MALS. Protein Samples BSA ACE2-Ferritin Nanocage Estimated MW (kDa) 68.95±0.26 692.00 ± 52.72 Encapsulation analysis Native 24-mer, ACE2-Fr-D (engineered dimer) alone, and ACE2-Fr-D + Protease (P) (PINC) were incubated with phenol red dye overnight. Next, samples were washed with buffer (10-times of the volume of sample) using a 100 kDa Amicon Ultracentrifugal filter. This washing will result in complete removal of phenol red dye that is free in the solution (and not encapsulated within the nanocage). Subsequently, UV-visible absorbance spectra were recorded at 480 nm to record absorbance of phenol red. Concentration of dimer was 7 µM (14 µM in monomer) and that of assembled 24-mer was 0.6µM (14.4 µM in monomer). Native 24-meric ferritin and ACE2-Fr-D did not show the absorbance at 480 nm (Figure 2D). This observation suggested that the phenol red was not encapsulated in native 24-meric ferritin and ACE2-Fr-D (engineered dimer). Figure 2D shows that PINCs had greater absorbance at 480 nm compared to the engineered dimer and native 24-mer, indicating that phenol red dye must have been successfully encapsulated inside the PINCs. Example 2 The inventors used PINC technology to decorate the surface of nanocages with a protein. The inventors created engineered dimer units (EDUs) using the hyperthermophilic ferritin from Pyrococcus furiosus (Pf) and an enterokinase cleavage site at the linker peptide between the two subunits. The inventors added the gene encoding the Sumo protein to the 5’-end of the gene encoding PfEDU (Figure 6a). The resulting construct (PfEDU-2) was cloned into a pBAD vector or pET28a vector, and PfEDU-2 was overexpressed in E. coli. The inventors used His-tagged affinity chromatography and showed that they could purify the PfEDU-2 (Figure 6a). Native- PAGE and size exclusion chromatography showed that in the solution potentially PfEDU-2 can form dimers with a MW of circa 138 kDa (Figure 6). After the reaction with enterokinase and induction of PINCs formation, samples were analyzed using native gel electrophoresis (Figure 6b). The results were consistent with the PfEDU-2 (predicted MW circa 58 kDa) conversion to PINCs due to enterokinase cleavage. The activity of enterokinase will generate two units (Figure 6a), subunit-1 (circa 35 kDa) with an N-terminus Sumo and subunit-2 (circa 23 kDa). Theoretically, the largest MW PINC (circa 840 kDa) will have only subunit-1, and the smallest PINC (circa 550 kDa) will have only subunit-2. However, in reality, these subunits are expected to combine randomly to generate PINCs with different ratios of subunit-1 and subunit-2. This prediction was confirmed by native gel electrophoresis. The band of PINCs on the gel was poorly defined and showed smearing (Figure 6b). Because of this randomness, the analysis of samples with size-exclusion chromatography resulted in the broadening of pick beyond detection (Figure 9). However, Native-PAGE analysis, using a high molecular weight protein marker (Figure 9), was consistent with the formation of PINCs with molecular weight ranging from circa 600 kDa to 720 kDa. These values are within the theoretical maximum and minimum size of expected nanocages. Mass spectrometry-based proteomics confirmed that these PINCs were decorated with Sumo protein (Figures 10-11). The inventors also generated PINCs using H. pylori ferritin (HpFtn), largely used to create vaccines. These PINCs were decorated with Sumo and an insecticide protein (Figure 12). To understand why EDUs cannot self- assemble spontaneously and visualize the accessibility of the protease cleavage site, the inventors used computational methods to predict and optimize the structure of PfEDU-2 (Figure 6c). The model predicted that in the EDUs, the ferritin monomer’s BC loop, whose residues play a vital role in the spontaneous self-assembly of wild-type ferritin monomers, cannot interact (Figure 6d). This lack of interaction is likely why the EDUs don’t spontaneously self-assemble. Additionally, analysis of the modelled structure showed that the enterokinase cleavage site (DDDDK) was accessible from the solvent (Figure 6e). The inventors then characterized the Fe(II) oxidation and Fe(III) mineralisation activity of EDUs and the resulting PINCs. They determined the initial rates of Fe(III) core formation for both PfEDU-2 and PINCs. They compared the results with Fe(II) oxidation by WT-PfFtn and the background oxidation of Fe(II) by molecular oxygen (Figure 13). The results of the initial rate measurement show that PfEDUs have the highest activity (Figure 13). It is suggested that in natural ferritin nanocages, the Fe(II) ions must transfer through the pores at 3-fold symmetry to reach the ferroxidase centre, where they are oxidized. Without wishing to be bound by theory, the inventors suggest that PfEDUs have the highest activity because Fe(II) ions readily access the ferroxidase centre from the solution. Next, the inventors measured the kinetics of Fe(III) mineral core formation (Figure 14). The results showed that Fe(III) mineralisation in PfEDU-2, PINCs and WT-PfFtn was similar. They all showed an initial rapid phase followed by a gradual increase in Fe(III) mineral core absorbance. Thus, they could generate a mineral core similar to WT-PfFtn. Additionally, the results suggested that PfEDU-2 potentially forms assembled soluble aggregates to keep the insoluble Fe(III) as a soluble mineral core similar to Fe(III) mineralization by a monomeric ferritin-like protein from Pyrococcus furiosus ferritin or frataxin. Similar results were obtained using HpEDU and their resulting PINCs (Figure 15). After establishing the formation of PINCs decorated with one or two proteins, the inventors aimed to create nanocages decorated with a protein on the surface and carrying a drug as cargo inside the cavity. The inventors decided to encapsulate a hydrophilic and a hydrophobic drug covering a wide range of therapeutic small molecules used to treat different diseases. As an example of a hydrophilic drug, the inventors used the anticancer drug doxorubicin (DOX). As an example of a hydrophobic drug, the inventors used the anticancer drug camptothecin (CPT). The inventors mixed PfEDU-2 with DOX and then, added enterokinase to form PINCs and encapsulate DOX (Figure 7a). As a control, the inventors first generated PINCs and then added DOX to the nanocages. Both samples were subject to extensive dialysis using dialysis tubes with a molecular weight cutoff of 300 kDa. Subsequently samples were analysed using Native-PAGE to confirm the presence of PINCs (Figure 16). The inventors used UV- visible spectrophotometry to measure encapsulated DOX with a maximum absorbance peak at 490 nm (Figure 7b). While in the control group the peak at 490 nm completely disappeared (Figure 7b), in the reaction to which DOX was added to PfEDU-2 before enterokinase addition, the absorbance peak of DOX was observed (Figure 7b). These results established that the inventors were able to successfully encapsulate DOX inside PINCs and that the encapsulated DOX could not be removed using extensive dialysis. Further quantification using a standard curve (Figure 17) confirmed that each nanocage approximately stored 400 ± 20 DOX (circa 15 DOX per subunit). This amount is 5-10 times higher than the previously reported values of 33 or 92 DOX per ferritin nanocage. Next, the inventors encapsulated CPT, which is not soluble in water. To solubilize CPT, the inventors used DMSO. Therefore, the inventors first tested the effect of DMSO on PINCs formation (Figure 18). The results confirmed a DMSO concentration of up to 20- 30% did not significantly affect the PINCs formation. Consequently, the inventors used DMSO to dissolve CPT, and the mixture was added to PfEDU-2 to a final concentration of 10% DMSO. After the formation of PINCs, the solution was subject to extensive dialysis and the presence of encapsulated CPT was measured using UV-visible spectrophotometry (Figure 7c). The results were consistent with those obtained using DOX. Native-PAGE analysis confirmed formation of PINCs (Figure 19). Quantification using a standard curve (Figure 20) confirmed encapsulation of 930 ± 200 CPT per 24-meric PINC. In summary, the inventors demonstrated a simple approach mimicking viral maturation process to control self-assembly of protein nanocages such as ferritin. This technology enabled the inventors to simultaneously decorate the surface of nanocages with proteins and efficiently encapsulate both hydrophobic and hydrophilic drugs, which has not been achieved using previous strategies. Therefore, PINC technology unlocks new opportunities for delivery of a wide range of drugs and developing advanced multifunctional therapeutics to tackle diseases such as viral infection and cancer. Chemicals and Reagents. All chemicals were reagent grades and were purchased from Merck or Fisher Scientific. Enterokinase from the porcine intestine (40 UN) was from Merck. The lyophilized power of enterokinase was dissolved in 400 µl 50mM Tris 100mM NaCl pH 8.0, and the solution was divided into small aliquots, flash frozen using liquid nitrogen, and stored at -80 °C freezer for future use. Protein constructs. The inventors designed three constructs (Figure 6(f)), two engineered dimer units (EDUs) using Pyrococcus furiosus (Pf) ferritin subunits and one construct using H. pylori (Hp) ferritin subunits. The first PfEDU construct (PfEDU-1) had an N-terminus peptide, and the second construct (PfEDU-2) had an N-terminus Sumo protein. The HpEDU had Sumo protein and an insecticide protein at the N- terminus of subunit-1 and the pesticide protein at the N-terminus of subunit-2. The E. coli codon-optimized gene encoding PfEDU-2 was obtained from GeneArt (Thermo Fisher) in pBAD/His A plasmid. This gene was then subcloned into the Escherichia coli (E. coli) expression vector pET-28a using the SacI and HindIII restriction sites. The E. coli codon-optimized gene encoding PfEDU-1, and HpEDU was obtained from GeneArt in pMA vector. These genes were subsequently subcloned into pBAD/His C vector using KpnI and EcoRI restriction sites. The presence of the correct insert was confirmed by double digestion and agarose gel electrophoresis. Therefore, after cloning, all constructs had a N-terminus His-tag (Figure 6(g)). Overexpression and Purification of EDUs. To overexpress PfEDU-2, the pET28a plasmid encoding the PfEDU-2 was transformed into chemically competent BL21 cells (ThermoFisher). To overexpress PfEDU-1 and HpEDU, the pBAD/His C plasmid encoding PfEDU-1 or HpEDU protein was transformed into chemically competent TOP10 cells (ThermoFisher). To overexpress EDUs, the E. coli cells were cultured overnight in 50 ml Luria-Bertani (LB) medium supplemented with 100µg/mL Kanamycin (PfEDU-2) or 100 µg/ml ampicillin (PfEDU-1 or HpEDU). The flasks were incubated in a shaker at 200 rpm and 37°C. After the overnight incubation, the cells were inoculated into 500 ml of terrific broth (TB) medium, with a ratio of LB:TB as 1:10. The flasks were incubated in a shaker at 200 rpm and 37°C. When OD@600 nm reached a value of 0.5-0.8, 1 mM (final concentration) IPTG was added to induce the expression of PfEDUs or 0.04% (final concentration) of L-arabinose was added to induce the expression of HpEDUs. Subsequently, the cells were incubated at 37°C and 200 rpm for another 8 hours to allow protein overproduction. Following this period, the cells were collected using centrifugation at 3750 rpm for 20 minutes and suspended in 2 ml lysis buffer (50 mM Tris, 300 mM NaCl, Ph 8.0 containing 2% triton, 0.5 mg PMSF, 0.05 mg DNase, and 1 mM DTT, 10mM Imidazole 0.1mg/ml lysozyme). These cells were stored at -80°C freezer for the subsequent purification step. After thawing cells, they were fully resuspended in the lysis buffer containing (50 ml of 50 mM Tris, 300 mM NaCl, pH 8.0 containing 2% triton, 0.5 mg PMSF, 0.05 mg DNase, and 1 mM DTT, 10mM Imidazole 0.1mg/ml lysozyme). Cells were subject to sonication (10 cycles of 30 seconds with 10-15 seconds off intervals with 50% amplitude). A total of 25 ml of cell lysate was processed each time. Next, the lysed cell mixture was subjected to ultracentrifugation at 4°C and 20,000 rpm for 20 minutes to remove cell debris. The resulting supernatant, containing soluble proteins, was collected and added to Ni2+ resin (250 µl of resin per 500 ml of bacterial growth culture) (HisPur™ Ni-NTA Resin, ThermoFisher). This solution was then incubated at 4 °C for 1 hour. After that, the Ni2+ resin, now containing the bound EDUs, was loaded onto a gravity column. The resins were washed 5 times (each time 2-time of the volume of the Ni2+ resin) using the wash buffer (50 mM Tris, 300 mM NaCl, pH 7.5, 20 mM Imidazole, 0.2% Triton, 1 mM DTT). Subsequently, EDUs were eluted using the elution buffer (50 mM Tris, 300 mM NaCl, pH 7.5, 500 mM Imidazole, 0.2% Triton, 1 mM DTT). After purification, PD10 desalting column was used to exchange the buffer to 50mM Tris, 300mM NaCl pH 8. The concentration of EDUs was determined using the BCA assay. The protein solution was subsequently diluted to a final concentration of 1.5 µM and divided into aliquots, and flash-frozen using liquid nitrogen. The samples were stored at -80 °C freezer for further analysis. Overexpression and Purification of WT-PfFtn. The construct for expression of WT- PfFtn is previously described (Honarmand Ebrahimi, K.; Bill, E.; Hagedoorn, P.-L.; Hagen, W. R. The Catalytic Center of Ferritin Regulates Iron Storage via Fe(II)-Fe(III) Displacement. Nat. Chem. Biol. 2012, 8, 941–948). The overexpression and purification of WT-PfFtn using a heat step was performed as used previously (Honarmand Ebrahimi, K.; Bill, E.; Hagedoorn, P.-L.; Hagen, W. R. The Catalytic Center of Ferritin Regulates Iron Storage via Fe(II)-Fe(III) Displacement. Nat. Chem. Biol. 2012, 8, 941– 948; Honarmand Ebrahimi, K.; Hagedoorn, P.-L.; Jongejan, J. A.; Hagen, W. R. Catalysis of Iron Core Formation in Pyrococcus Furiosus Ferritin. JBIC J. Biol. Inorg. Chem. 2009, 14 (8), 1265–1274). Protein concentration was measured using BCA assay. Purified protein was divided into aliquots, flash frozen using liquid nitrogen, and stored at -80 °C. Formation of PINCs. The EDUs were incubated with enterokinase (10µl enterokinase into 1ml PfFtn) at 37°C overnight. Following this enzymatic digestion, an inducer buffer composed of 100ul of 1M MOPS and 1M NaCl at pH 7 was added to the reaction mixture and incubated for an additional 1 hour at 37°C. The protein solution was then concentrated using an Amicon ultracentrifugal filter with a 10kDa cutoff. The concentration of protein was determined using polyacrylamide gel electrophoresis (PAGE). Polyacrylamide gel electrophoresis (PAGE). Each protein was mixed with 4x Native sample buffer (Invitrogen) (2.5 µl). Then, 10 µl of each sample or the standard was loaded into each well. Novex™ 4-12% Tris-Glycine Mini Gels, or NativePAGE™ 4- 16% Bis-Tris Protein Gels were used. The NativeMark™ Unstained Protein Standard (ThermoFisher) (5 µl) mixed with 2.5 µl and 2.5 µl Milli-Q water was used for NativePAGE. 7 µl of the PageRuler™ Plus Prestained Protein Ladder (Fisher Scientific) was used with Novex™ 4-12% Tris-Glycine Mini Gels. The Mini Gel Tank (Life Technologies) was used for running gel with Anode and Cathode buffer (Invitrogen) at 150V for 100 mins. After running, the gel was washed with MiliQ water 3 times, each 5 minutes. Then the gels were stained with SimplyBlue SafeStain (novex, life technologies) for 1 hr. Finally, MiliQ water was used to destain the gel for 1 hr. The UVp geldoc system was used for imaging the gels. Fe(II) oxidation and Fe(III) mineralization activity. Iron oxidation was performed as described previously (Honarmand Ebrahimi, K.; Hagedoorn, P.-L.; Jongejan, J. A.; Hagen, W. R. Catalysis of Iron Core Formation in Pyrococcus Furiosus Ferritin. JBIC J. Biol. Inorg. Chem. 2009, 14 (8), 1265–1274). A stock solution of PfEDU-2 (8.3 µM), its PINC (0.7 µM on 24-mer basis), or WT-PfFtn (8.3 µM) was used for Fe(II) oxidation studies. The Fe(II) stock (24mM) was prepared from Fe(NH4)2(SO4)2 in MOPS buffer (100mM MOPS 100mM NaCl pH 7) under anaerobic conditions. For each measurement, 300 µl of protein (WT-PfFtn, PINCs, or PfEDU-2) was mixed with 695 µl of MOPS buffer. The mixture was added to a plastic cuvette and placed in the UV-visible spectrophotometer (PerkinElmer Lambda 365). Absorbance was recorded at 340 nm as a function of time. After circa two minutes, if the absorbance stayed stable, 5µl Fe(II) stock was added to the reaction and mixed well, and absorbance continued to be measured for another 5-8 minutes. To measure the background oxidation of Fe(II), 300 µl of buffer was used instead of protein. The Fe(II) to 24-meric nanocage ratio was 4,800:1. In the case of HpEDU and its PINCs, each reaction was set up as follow: 990 µl buffer (MOPS 100 mM, NaCl 100 mM, pH 7.0) was mixed with 10 µl of each protein. The mixture was added to a plastic cuvette, and absorbance at 340 nm was recorded. After circa 5 min, if the absorbance was stable, the cuvette was removed, and 0.5 µl of Fe(II) solution was added. The solution was mixed quickly and returned to the spectrometer to continue recording the absorbance. As a result there is a 5-10 s delay in measuring Fe(III) formation due to mixing time. A molar extinction coefficient of 1950 M-1 cm-1 at 340 nm was used for Fe(III) core formation (Baaghil, S.; Lewin, A.; Moore, G. R.; Le Brun, N. E. Core Formation in Escherichia Coli Bacterioferritin Requires a Functional Ferroxidase Center. Biochemistry 2003, 42, 14047–14056). Size Exclusion Chromatography-Multiangle Light Scattering (SEC-MALS). BSA, WT-PfFtn, PfEDU-2, and PINCs were characterized using SEC-MALS (Wyatt Technology). The buffer was 5 mM Mops, 300 mM NaCl, pH 7. PINCs were generated as explained above and subject to dialysis using a dialysis tube with a MW cutoff of 300 kDa to exchange the buffer to 5 mM Mops, 300 mM NaCl, pH 7.0. Dialysis was done with continuous stirring at 4℃. On the next day, BSA, WT-PfFtn, PfEDU-2, and PINC were prepared and concentrated to 200 μl (circa 2 mg/ml). 100 µl of each sample was injected. The column was WTC- 030S5. Absorbance was recorded for 25 min at 280 nm. Encapsulation of Doxorubicin (DOX). Two reactions were set up for encapsulating DOX into PfEDU-2: (a) Sample: 380 µl of 0.8mM DOX solution in 50 mM Tris and 300mM NaCl at pH 8 was added to 1 ml of 1.5 µM PfEDU-2 in 50 mM Tris 300 mM NaCl pH 8, followed by the addition of 10 µl of enterokinase. The mixture was then incubated at 37°C overnight. Subsequently, 100 µl of an inducer buffer composed of 1 M MOPS and 1 M NaCl at pH 7 was added and the reaction was kept at 37°C for another 1 hour. (b) Control: 10 µl of enterokinase was added directly to 1 ml of 1.5 µM PfEDU-2 in the absence of any drug, and the mixture was incubated at 37°C overnight. Following the overnight incubation, 100 µl inducer buffer was added to the reaction and the sample was incubated at 37°C for 1 hour to generate PINCs. Then 380 µl of 0.8 mM DOX solution in 50 mM Tris and 300 mM NaCl at pH 8 was added to the PINCs and the control was incubated at 37°C for 1 hour. Both sample (a) and control (b) were subject to extensive dialysis (Float-A-Lyzer™ G2 Dialysis Devices, Spectrum), to remove free DOX. The dialysis process was repeated three times, each with 3 L of fresh dialysis buffer 5 mM MOPS and 300 mM NaCl at pH 7. The length of dialysis steps was 6 hours, overnight, and 20 hours. All dialysis steps were done at 4°C. After completing the dialysis, UV-visible spectrophotometry was utilized to quantify the amount of DOX encapsulated within the nanocages. For UV-visible spectroscopy, a quartz cuvette was used. Additionally, 500 µl of the sample or control was concentrated to circa 50 µl using Amicon Ultracentrifugal Filter with a cutoff of 10 kDa. This concentrated sample was used for Native-PAGE to confirm the presence of 24-meric nanocages. Encapsulation of Camptothecin (CPT). A solution of 5 mM CPT was prepared in DMSO. Two separate reactions were set up for encapsulating CPT into PfEDU-2. (a) Sample: 130 µl of mM CPT solution in DMSO was added to 1 ml of 1.5µM PfEDU-2 in 50 mM Tris 300 mM NaCl pH 8, followed by the addition of 10 µl of enterokinase (the final concentration of CPT was 575µM). The mixture was then incubated at 37°C overnight. Subsequently, 100 µl of an inducer buffer composed of 1 M MOPS and 1 M NaCl at pH 7 was added, and the reaction was kept at 37°C for another 1 hour. (b) Control: 10 µl of enterokinase was added directly to 1 ml of 1.5 µM PfEDU-2 without any drug, and the mixture was incubated at 37°C overnight. Following the overnight incubation, 100 µl inducer buffer was added to the reaction and the sample was incubated at 37°C for 1 hour to generate PINCs. Then 130 µl of mM CPT solution in 50 mM Tris and 300 mM NaCl at pH 8 was added to the PINCs, and the control was incubated at 37°C for 1 hour. Both sample (a) and control (b) were subject to extensive dialysis (300kD Float-A-Lyzer G2 Dialysis Device, Spectrum Laboratories) to remove free CPT. The dialysis process was repeated three times, each time with 3 L of fresh dialysis buffer 5 mM MOPS and 300 mM NaCl at pH 7. The length of dialysis steps was 6 hours, overnight, and 20 hours. All dialysis steps were done at 4°C. After completing the dialysis, UV-visible spectrophotometry was utilized to quantify the amount of CPT encapsulated within the nanocages. Additionally, 500 µl of the sample or control was concentrated to circa 50 µl using Amicon Ultracentrifugal Filter with a cutoff of 10 kDa. This concentrated sample was used for Native-PAGE to confirm the presence of 24-meric nanocages. Prediction of the structure of PfEDUs. The structure of PfEDU-2 with an N-terminal Sumo was predicted using the Phyre server (Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, M. N.; Sternberg, M. J. E. The Phyre2 Web Portal for Protein Modeling, Prediction and Analysis. Nat. Protoc. 2015, 10, 845–858.). Subsequently, the model was energy-optimized using MM2 calculations by Chem3D software. PyMol software was used to visualize the structure and prepare the pictures. LC-MS/MS analysis of PINCs. Enzymatic Digestion The gel bands were prepared for enzymatic digestion. Cysteine residues were reduced with dithiothreitol and derivatized by treatment with iodoacetamide to form stable carbamidomethyl derivatives. Trypsin digestion was conducted overnight at room temperature after initial incubation at 37°C for 2 hours. The protocol for this process was as follows: Reagents: 100 mM Triethylammonium bicarbonate (TEAB; Sigma) – 500 ml 1 M TEAB in 4500 ml water 50 mM TEAB – 1 ml 100 mM TEAB added to 1 ml water. 10 mM Dithiothreitol (DTT; Sigma) – 15 mg in 1 ml 100 mM TEAB for 100 mM stock; dilute stock 10-fold. 55 mM Iodoacetamide (IAA; Sigma) – 10 mg in 1 ml 100 mM TEAB. 13 ng/ml Trypsin – add 250 ml 0.1% TFA to a 25 mg vial (Bovine, Cat. No. 000000011047841001; Sigma) and remove 30 ml to a new tube. Add 200 ml 50 mM TEAB to achieve 13 ng/ml. Add 50 ml to each sample for a total of 650 ng trypsin. 1. Cut the gel band into ~2 mm2 pieces and transfer into a fresh 1.5 ml centrifuge tube. 2. Decant storage liquid and wash the gel cubes with 100 mM TEAB for 5 mins (decant off water first if gel pieces have been stored) and decant. Volumes of TEAB and ACN (steps 2 to 8) are added in excess. 3. Add acetonitrile, decant after one round, then add same volume again to fully dehydrate the gel pieces (will turn white in appearance). Decant and dry in Speed Vac for 5 mins. Whilst samples are drying prepare DTT solution. 4. Rehydrate the gel with 10 mM DTT and heat at 56°C for 30 mins. 5. Decant DTT, add ACN (2x volume; see step 3), dehydrate and dry in Speed Vac (5 mins). Whilst samples are drying prepare 55 mM IAA solution. 6. Add 55 mM IAA; incubate at ambient temperature for 20 mins in the dark. 7. Discard the supernatant, wash briefly with 100 mM TEAB buffer then replace and wash for a further 5 mins and discard the buffer. 8. Decant the liquid, dehydrate once again with acetonitrile as in step 3 and dry off in a Speed Vac for 5 mins. 9. Rehydrate the gel pieces in 50 µl trypsin solution (650 ng total enzyme) at 4°C for 20 mins. Remove unabsorbed trypsin and add 50 µl of 50 mM TEAB to cover the gel pieces and keep them wet during enzyme cleavage. Incubate at 37°C for 2 hours then overnight at room temperature. Peptide extraction 10. Decant supernatant from gel pieces and collect into a new centrifuge tube. 11. Wash gel pieces in 50 µl 50 mM TEAB for 5 mins at 37°C, decant and pool into tube from step 11. 12. Dehydrate gel pieces with 50 µl ACN for 10 mins at 37oC, decant and pool supernatant into tube from step 11. 13. Repeat steps 12 and 13. 14. Dry down the pooled supernatants to completion in a Speed Vac. Stored at - 80°C until required. LC-MS/MS The dried extracted peptide samples were resuspended in 20 µl of resuspension buffer (2% ACN in 0.05% TFA), 6 µl of which was injected to be analyzed by LC-MS/MS. Chromatographic separation was performed using a U3000 UHPLC NanoLC system (ThermoFisherScientific, UK). Peptides were resolved by reversed phase chromatography on a 75 µm C18 Pepmap column (50 cm length) using a three-step linear gradient of 80% acetonitrile in 0.1% formic acid. The gradient was delivered to elute the peptides at a flow rate of 250 nl/min over 60 min starting at 5% B (0-5 minutes) and increasing solvent to 40% B (5-40 minutes) prior to a wash step at 99% B (40-45 minutes) followed by an equilibration step at 5% B (45-60 minutes). The eluate was ionized by electrospray ionization using an Orbitrap Fusion Lumos (ThermoFisherScientific, UK) operating under Xcalibur v4.3. The instrument was first programmed to acquire using an Orbitrap-Ion Trap method by defining a 3 s cycle time between a full MS scan and MS/MS fragmentation by collision induced dissociation. Orbitrap spectra (FTMS1) were collected at a resolution of 120,000 over a scan range of m/z 375-1600 with an automatic gain control (AGC) setting of 4.0e5 (100%) with a maximum injection time of 35 ms. Monoisotopic precursor ions were filtered using charge state (+2 to +7) with an intensity threshold set between 5.0e3 to 1.0e20 and a dynamic exclusion window of 35s ± 10 ppm. MS2 precursor ions were isolated in the quadrupole set to a mass width filter of 1.6 m/z. Ion trap fragmentation spectra (ITMS2) were collected with an AGC target setting of 1.0e4 (100%) with a maximum injection time of 35 ms with CID collision energy set at 35%. Database Searching Raw mass spectrometry data were processed into peak list files using Proteome Discoverer (ThermoScientific; v2.5). The raw data file was processed and searched using the Sequest (Eng et al; PMID 24226387) search algorithm against the Uniprot All Taxonomy database (569,793 entries). Database searching was performed at a stringency of 1% FDR including a decoy search. Posttranslational modifications for carbamidomethylation (C; static) and oxidation (M; variable), were included in the database search. Sequences SEQ ID NO:1 (exemplary construct amino acid sequence): MGGSHHHHHHGMASMTGGQQMQSTTEELAKTFLEKFNHEAEELSYQSSLASP ATWDLGKGDFRIKMCTKVMLSERMLKALNDQLNRELYSAYLYFAMAAYFED LGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKA FEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKL KFAKDSPQILFMLDKELSARAPKLPGLLMQGGEDLYDDDDKDRWGSEMGGSH HHHHHGMASMTGGQQMQSTTEELAKTFLEKFNHEAEELSYQSSLASPATWDL GKGDFRIKMCTKVMLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGF ANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYE HEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDS PQILFMLDKELSARAPKLPGLLMQGGE SEQ ID NO: 2 (linker amino acid sequence): DLYDDDDKDRWGSEMGGSHHHHHHGMASMTGGQQMQSTTEELAKTFLEKF NHEAEELSYQSSLASPATWDLGKGDFRIKMCTKV SEQ ID NO:3 (ACE2 peptide mimetic amino acid sequence designed based on the ACE2 interacting with SARS-CoV-2 receptor binding domain): QSTTEELAKTFLEKFNHEAEELSYQSSLASPATWDLGKGDFRIKMCTKV SEQ ID NO:4 (Pyrococcus furiosus ferritin subunit amino acid sequence): MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEI GHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAA LAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSA RAPKLPGLLMQGGE SEQ ID NO:5 (polyhistidine peptide amino acid sequence): MGGSHHHHHHGMASMTGGQQM SEQ ID NO:6 (Ferritin-ACE2-D gene vector nucleic acid sequence): AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTA CTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTG TAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTA TAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGC TATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTT ATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTGGGCTAACAGGAGGA ATTAACCATGGGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATG ACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGAT CGATGGATCCGACCTCGAGATCTGCAGATGGTACCATATGGGAATTCGAAG CTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAAT CAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCG CGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCG CCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCAT CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTT GTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATT TGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCA TAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTT TTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAG ATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCA ACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGAT GAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTT GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAAC ATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAAC AATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCT CGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCG TGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGT ATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCA GACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAA GGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGA TAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG AACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTAT CTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTG ATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTT TTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTT ATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC GGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCA CACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA GCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGA CACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCAT CCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGT TTTCACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGA AGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTC TGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAA TTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGG AACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAG AGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGG GTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGC TTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACG GCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTG CCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGG TGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCA AGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGT TAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGG GCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATG CCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGC CTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAAT ATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGA CCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCG ATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACC AGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCA GCCATACTTTTCATACTCCCGCCATTCAGAG SEQ ID NO. 7 (pBAD/His C vector nucleic acid sequence): CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAA TCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATC AAAAGAATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATT CGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTT CGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTT GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AGCGCGACGTAATACGACTCACTATAGGGCGAATTGGCGGAAGGCCGTCAA GGCCGCATCATATGCCCATGGGTGGTAGCCATCACCATCATCATCATGGTAT GGCAAGCATGACCGGTGGTCAGCAGATGCAGAGCACCACCGAAGAACTGG CAAAAACCTTTCTGGAAAAATTCAACCATGAAGCGGAAGAACTGAGCTATC AGAGCAGCCTGGCAAGTCCGGCAACCTGGGATTTAGGTAAAGGTGATTTTC GCATTAAGATGTGCACCAAAGTTATGCTGAGCGAACGTATGCTGAAAGCAC TGAATGATCAGCTGAATCGTGAACTGTATAGCGCCTATCTGTATTTTGCAAT GGCAGCCTATTTTGAAGATCTGGGTTTAGAAGGTTTTGCCAATTGGATGAAA GCACAGGCCGAAGAAGAAATTGGTCATGCACTGCGCTTTTACAACTATATC TATGATCGTAATGGTCGCGTGGAACTGGATGAAATTCCGAAACCGCCTAAA GAATGGGAAAGTCCGCTGAAAGCCTTTGAAGCAGCCTATGAACATGAGAAA TTTATCAGCAAAAGCATCTATGAACTGGCAGCACTGGCAGAAGAGGAAAAA GATTATAGCACCCGTGCATTTCTGGAATGGTTTATTAACGAACAGGTTGAAG AAGAAGCCAGCGTTAAAAAGATTCTGGACAAACTGAAATTCGCCAAAGATA GTCCGCAGATTCTGTTCATGCTGGATAAAGAACTGTCAGCACGTGCACCGA AACTGCCTGGTCTGCTGATGCAAGGTGGCGAAGATCTGTATGATGACGATG ATAAAGATCGTTGGGGTAGCGAAATGGGTGGTTCACATCATCACCACCATC ACGGTATGGCCTCAATGACAGGCGGACAGCAAATGCAGTCAACAACCGAA GAATTAGCCAAGACATTCCTTGAGAAATTCAATCACGAGGCAGAGGAACTG TCATATCAGTCAAGCCTGGCATCACCAGCCACCTGGGATCTTGGCAAAGGC GACTTCCGTATCAAAATGTGTACAAAAGTGATGCTGTCAGAGCGCATGTTA AAAGCCCTGAACGATCAATTAAACCGCGAACTGTATTCTGCGTATTTGTACT TCGCTATGGCTGCGTACTTTGAAGATTTAGGTCTGGAAGGCTTCGCAAATTG GATGAAGGCCCAAGCAGAGGAAGAGATTGGCCACGCGCTGCGTTTCTATAA TTACATTTATGATCGCAACGGTCGTGTTGAGTTAGATGAAATCCCTAAGCCT CCGAAAGAGTGGGAATCACCTCTGAAAGCATTCGAAGCCGCATACGAACAC GAAAAGTTTATCTCGAAAAGCATTTACGAATTAGCAGCCCTGGCGGAAGAA GAGAAGGATTACTCAACGCGTGCCTTTTTAGAATGGTTCATCAATGAGCAA GTGGAAGAGGAAGCATCCGTTAAGAAAATCCTGGATAAGCTGAAGTTTGCG AAAGATTCACCTCAGATCCTGTTTATGTTAGACAAAGAGCTGAGCGCTCGC GCACCGAAATTACCGGGACTGTTAATGCAAGGCGGTGAATAAGAATTCAAG CTTCTGGGCCTCATGGGCCTTCCGCTCACTGCCCGCTTTCCAGTCGGGAAAC CTGTCGTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGG CGCTCTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAA GCCTGGGGTGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA AAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCC TTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAA CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCT GACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTAT TTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACG CTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGA GCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGT TGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAA GTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTC AATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAG ATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCA AAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCC CCGAAAAGTGCCAC SEQ ID NO. 8 (Enterokinase recognition site) DDDDKX SEQ ID NO. 9 (Thrombin recognition site) LVPRGS SEQ ID NO. 10 (HRV3C Protease recognition site) LEVLFQGP SEQ ID NO. 11 (TEV protease recognition site) ENLYFQX SEQ ID NO. 12 (PfFtn-Sumo exemplary construct) MHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGGA TYMLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEE EIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYEL AALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKEL SARAPKLPGLLMQGGESMTGGQQMGRDLYDDDDKDRWGSEMGGSHHHHHH GMASMTGGQQMMLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFA NWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEH EKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDKLKFAKDSP QILFMLDKELSARAPKLPGLLMQGGE SEQ ID NO. 13 (His-Sumo sequence) MHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGGA TY SEQ ID NO. 14 (Linker peptide) SMTGGQQMGRDLYDDDDKDRWGSEMGGSHHHHHHGMASMTGGQQM SEQ ID NO. 15 (PfEDU-2 DNA sequence) ATGCTAGCATGCATCATCACCATCATCATGGTAGCCTGCAGGATAGCGAAG TTAATCAAGAAGCAAAACCGGAAGTTAAGCCGGAAGTGAAACCTGAAACA CATATTAACCTGAAAGTGAGTGATGGTAGCAGCGAGATCTTTTTCAAAATC AAAAAGACCACACCGCTGCGTCGTCTGATGGAAGCATTTGCAAAACGTCAG GGTAAAGAAATGGATAGCCTGACCTTTCTGTATGATGGCATTGAAATTCAG GCAGATCAGACACCGGAAGATCTGGATATGGAAGATAACGATATTATCGAA GCACATCGTGAGCAGATTGGTGGTGCAACCTATATGCTGAGCGAACGTATG CTGAAAGCACTGAATGATCAGCTGAATCGTGAACTGTATAGCGCCTATCTGT ATTTTGCAATGGCAGCCTATTTTGAAGATTTAGGTCTGGAAGGTTTTGCCAA TTGGATGAAAGCACAGGCCGAAGAAGAAATTGGTCATGCACTGCGCTTTTA CAACTATATCTATGATCGTAATGGTCGCGTGGAACTGGATGAAATTCCGAA ACCGCCTAAAGAATGGGAAAGTCCGCTGAAAGCCTTTGAAGCAGCATATGA ACATGAGAAGTTTATCAGCAAAAGCATCTATGAACTGGCAGCACTGGCAGA AGAGGAAAAAGATTATAGCACCCGTGCATTTCTGGAATGGTTTATTAACGA ACAGGTTGAAGAAGAAGCCAGCGTTAAAAAGATTCTGGACAAACTGAAATT CGCCAAAGATAGTCCGCAGATTCTGTTCATGCTGGATAAAGAACTGAGCGC ACGTGCACCGAAACTGCCTGGTCTGCTGATGCAAGGTGGTGAAAGCATGAC CGGTGGTCAGCAGATGGGTCGTGATCTGTATGACGATGATGATAAAGATCG TTGGGGTAGCGAAATGGGTGGTAGCCATCACCACCACCATCACGGCATGGC AAGTATGACAGGCGGACAGCAGATGATGCTGTCAGAGCGTATGTTAAAAGC CTTAAACGATCAACTGAACCGCGAGCTGTATTCAGCATATTTGTACTTCGCT ATGGCTGCGTATTTTGAGGATCTGGGTTTAGAAGGCTTCGCAAATTGGATGA AGGCCCAAGCAGAGGAAGAGATTGGCCACGCGCTGCGTTTCTATAATTACA TTTATGATCGCAACGGTCGTGTTGAGTTAGATGAAATCCCTAAGCCTCCGAA AGAGTGGGAATCACCTCTGAAAGCATTCGAGGCTGCCTATGAACACGAAAA ATTCATTAGCAAGAGCATTTATGAGTTAGCAGCCCTGGCGGAAGAAGAGAA GGATTACTCAACGCGTGCCTTTTTAGAATGGTTCATCAATGAGCAAGTGGAA GAGGAAGCATCCGTTAAGAAAATCCTGGATAAGCTGAAGTTTGCGAAAGAT TCACCTCAGATCCTGTTTATGTTAGACAAAGAGCTGTCAGCTCGCGCACCGA AATTACCGGGACTGTTAATGCAAGGCGGAGAATAA SEQ ID NO. 16 (pET28a plasmid sequence) ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGA GGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTC AGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGG TGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGCG ACCCATTTGCTGTCCACCAGTCATGCTAGCCATATGGCTGCCGCGCGGCACC AGGCCGCTGCTGTGATGATGATGATGATGGCTGCTGCCCATGGTATATCTCC TTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTCACA ATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTC TACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCT GGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTC GGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCC GGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGG TGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCA TAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGCGCAAAACCTTTC GCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATG TGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCA GACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCG GGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGT GGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTC CAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGC GCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGC GTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGT GGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAA GCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACAC CCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGA GCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTA AGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTC GCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGT CCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGC GATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTAC CGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGA TACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGA TTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAG GGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGA AAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAG TGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATC TCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCG CGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAAC TCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCT TTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTT GCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGC GAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGATC GTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTT AGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCA AAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTC GTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGAT CTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTA ACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCC ATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATC ATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACC CCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAA AAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTC TGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAAT CGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGG TGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGC TTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGC GGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAG CGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGA GTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAAT ACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT ATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCC AGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAA CCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC TAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAG CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAAACTGT CTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGG AAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATA TGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTA TCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAA AGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCT GACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGAT GATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTA TTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGT TCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGAT CGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTG ATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCT GGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCA TGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGT TGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCC ATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTT TTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTT GATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGA ATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAAT TTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCC CTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTT GGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGA AAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCA AGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGG AGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAA GGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAG CGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTAC AGGGCGCGTCCCATTCGCCA SEQ ID NO. 17 (HpFtn – Sumo – insecticide (HpEDU)) MHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGGA TYNLVPRGSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCDMLSKDIIKL LNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFL NENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATF NFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSRDLY DDDDKDRSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCDMLSKDIIKLL NEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLN ENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFN FLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS SEQ ID NO. 18 (Insecticide protein (Omega-hexatoxin-Hv1a)) SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD SEQ ID NO. 19 (H. pylori ferritin monomer) MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEH AKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAI KGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIA KSRKS SEQ ID NO. 20 (Linker peptide) RDLYDDDDKDR SEQ ID NO. 21 (HpFtn – Sumo-insecticide (HpEDU) DNA sequence) ATGCATCATCACCATCATCATGGTAGCCTGCAGGATAGCGAAGTTAATCAA GAAGCAAAACCGGAAGTTAAGCCGGAAGTGAAACCTGAAACACATATTAA CCTGAAAGTGAGTGATGGTAGCAGCGAGATCTTTTTCAAAATCAAAAAGAC CACACCGCTGCGTCGTCTGATGGAAGCATTTGCAAAACGTCAGGGTAAAGA AATGGATAGCCTGACCTTTCTGTATGATGGCATTGAAATTCAGGCAGATCAG ACACCGGAAGATCTGGATATGGAAGATAACGATATTATCGAAGCACATCGT GAGCAGATTGGTGGTGCAACCTATAATCTGGTTCCGCGTGGTAGCAGTCCG ACCTGTATTCCGAGCGGTCAGCCGTGTCCGTATAATGAAAATTGTTGTAGCC AGAGCTGCACCTTCAAAGAAAATGAAAATGGCAATACCGTGAAACGCTGTG ATATGCTGAGCAAAGATATTATCAAACTGCTGAACGAACAGGTGAACAAAG AAATGAACAGCAGCAATCTGTATATGAGCATGAGCAGCTGGTGTTATACCC ATAGCCTGGATGGTGCAGGTCTGTTTCTGTTTGATCATGCAGCCGAAGAATA TGAACATGCCAAAAAGCTGATTGTGTTCCTGAATGAAAATAATGTTCCGGTT CAGCTGACCAGCATTAGCGCTCCGGAACATAAATTTGAAGGTCTGACCCAG ATTTTCCAGAAAGCATATGAACACGAACAGCACATTAGCGAAAGCATTAAC AACATTGTGGATCATGCCATCAAAGGCAAAGATCATGCAACCTTTAACTTTC TGCAGTGGTATGTGAGCGAACAGCATGAAGAAGAAGTGCTGTTTAAAGACA TCCTGGATAAAATTGAACTGATCGGCAATGAAAACCATGGTCTGTATCTGG CGGATCAGTATGTTAAAGGTATTGCAAAAAGCCGCAAAAGCCGTGATCTGT ATGACGATGATGATAAAGATCGTAGCCCGACATGCATTCCGTCAGGCCAGC CTTGTCCTTATAACGAGAACTGTTGCTCACAGTCTTGCACGTTTAAAGAAAA CGAGAACGGTAACACCGTTAAACGTTGCGACATGCTGTCAAAAGACATCAT TAAGCTGCTGAATGAGCAAGTCAATAAAGAGATGAATAGTTCCAACCTGTA TATGTCCATGTCAAGTTGGTGCTATACACATTCACTGGACGGTGCCGGTTTA TTCCTGTTCGACCATGCCGCAGAGGAATATGAGCACGCAAAGAAACTGATC GTTTTTCTGAACGAAAACAACGTGCCTGTGCAGCTGACCTCAATTAGTGCAC CTGAACACAAATTCGAAGGCTTAACACAGATCTTTCAAAAAGCCTACGAAC ATGAGCAACATATCAGCGAATCCATCAATAACATTGTTGACCACGCCATTA AGGGGAAAGATCACGCCACATTCAATTTCTTACAGTGGTACGTTTCAGAAC AGCACGAGGAAGAGGTTCTTTTTAAGGATATTCTGGACAAGATCGAGCTGA TTGGCAACGAAAATCACGGACTGTACTTAGCAGATCAGTACGTGAAAGGCA TAGCCAAATCACGTAAAAGCTAA References 1. Aumiller, W. M., Uchida, M. & Douglas, T. Protein cage assembly across multiple length scales. Chem. Soc. Rev. 47, 3433–3469 (2018). 2. Honarmand Ebrahimi, K., Hagedoorn, P.-L. & Hagen, W. R. Unity in the biochemistry of the iron-storage proteins ferritin and bacterioferritin. Chem. Rev. 115, 295–326 (2014). 3. Truffi, M. et al. Ferritin nanocages: A biological platform for drug delivery, imaging and theranostics in cancer. Pharmacol. Res. 107, 57–65 (2016). 4. Honarmand Ebrahimi, K. Ferritin as a Platform for Creating Antiviral Mosaic Nanocages: Prospects for Treating COVID-19. ChemBioChem 22, 1371–1378 (2021). 5. Fan, K., Gao, L. & Yan, X. Human ferritin for tumor detection and therapy. WIREs Nanomedicine and Nanobiotechnology 5, 287–298 (2013). 6. Wang, C. et al. Ferritin-based targeted delivery of arsenic to diverse leukaemia types confers strong anti-leukaemia therapeutic effects. Nat. Nanotechnol. 16, 1413–1423 (2021). 7. Kanekiyo, M. et al. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 499, 102–106 (2013). 8. Sliepen, K. et al. Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicity. Retrovirology 12, 82 (2015). 9. Joyce, M. G. et al. A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates. Sci. Transl. Med. 0, eabi5735. 10. Carmen, J. M. et al. SARS-CoV-2 ferritin nanoparticle vaccine induces robust innate immune activity driving polyfunctional spike-specific T cell responses. npj Vaccines 6, 151 (2021). 11. Wuertz, K. M. et al. A SARS-CoV-2 spike ferritin nanoparticle vaccine protects hamsters against Alpha and Beta virus variant challenge. npj Vaccines 6, 1–11 (2021). 12. Kim, M. et al. pH-Dependent Structures of Ferritin and Apoferritin in Solution: Disassembly and Reassembly. Biomacromolecules 12, 1629–1640 (2011). 13. Zhang, J. et al. Cargo loading within ferritin nanocages in preparation for tumor- targeted delivery. Nat. Protoc. 16, 4878–4896 (2021). 14. Haurd, D. J. E., Kane, K. M. & Tezcan, F. A. Re-engineering protein interfaces yields copper-inducible ferritin cage assembly. Nat. Chem. Biol. 9, 169–176 (2013). 15. Sana, B., Johnson, E. & Lim, S. The unique self-assembly/disassembly property of Archaeoglobus fulgidus ferritin and its implications on molecular release from the protein cage. Biochim. Biophys. Acta - Gen. Subj. 1850, 2544–2551 (2015). 16. Yin, S. et al. Development of purification process for dual‐function recombinant human heavy‐chain ferritin by the investigation of genetic modification impact on conformation. Eng. Life Sci. 21, 630–642 (2021). 17. Le Vay, K. et al. Controlling Protein Nanocage Assembly with Hydrostatic Pressure. J. Am. Chem. Soc. 142, 20640–20650 (2020). 18. Kim, Y.-S. et al. Chaperna-mediated assembly of ferritin-based Middle East respiratory syndrome-coronavirus nanoparticles. Front. Immunol. 9, 1093 (2018).

Claims

Claims 1. A construct comprising: two ferritin subunits connected by a linker, wherein the linker comprises a cleavage site, wherein the linker is arranged to prevent the ferritin subunits from self- assembling into a ferritin nanocage, and wherein cleavage of the linker at the cleavage site does not prevent the ferritin subunits from self-assembling into a ferritin nanocage.
2. A construct according to claim 1, wherein the cleavage site is a protease cleavage site, optionally wherein the cleavage site is an enterokinase or thrombin cleavage site.
3. A construct according to any preceding claim, wherein the ferritin subunits are human ferritin subunits.
4. A construct according to any preceding claim, wherein the ferritin subunits are selected from heavy chain ferritin subunits or light chain ferritin subunits, or a combination thereof.
5. A construct according to any preceding claim, wherein the construct further comprises a purification tag, optionally wherein the purification tag is a His tag, optionally an N-terminal His tag, optionally an N-terminal 6-His tag.
6. A construct according to any preceding claim, wherein the linker comprises 5-100 amino acid residues.
7. A construct according to any preceding claim, wherein the construct further comprises one or more therapeutic elements and/or one or more non-therapeutic elements.
8. A construct according to claim 7, wherein the one or more therapeutic elements and/or the one or more non-therapeutic elements is located at the N-terminal end of the construct or in the N-terminal portion of the construct.
9. A construct according to claim 7 or 8, wherein the construct further comprises one or more therapeutic elements selected from the group consisting of drugs, antibodies, proteins, peptides, genes, oligonucleotides, RNA therapeutics, antigens, other pharmaceutically active ingredients, or a combination thereof.
10. A construct according to claim 7 or 8, wherein the construct further comprises one or more non-therapeutic elements selected from the group consisting of secretory signals, binding molecules, targeting molecules, detectable moieties, or a combination thereof.
11. A nucleic acid molecule encoding the construct of any one of claims 1 to 10.
12. An expression vector comprising the nucleic acid molecule according to claim 11.
13. A host cell comprising the expression vector according to claim 12.
14. A pharmaceutical composition comprising the construct according to any one of claims 1 to 10 or the nucleic acid molecule according to claim 11 or the expression vector according to claim 12 or the host cell according to claim 13 and one or more pharmaceutically acceptable excipients.
15. A method of preparing a ferritin nanocage, the method comprising: contacting a plurality of constructs according to any one of claims 1 to 10 with a cleaving agent configured to cleave the linkers at the cleavage site, thereby cleaving the plurality of constructs at the cleavage sites and facilitating the self- assembly of the ferritin subunits into a ferritin nanocage.
16. A method of preparing a ferritin nanocage according to claim 15, further comprising incubating the cleaved constructs with a buffer comprising 50 mM-10 M of a salt and a pH of 6-8.5 to induce the self-assembly of the ferritin subunits into a ferritin nanocage.
17. A method of preparing a ferritin nanocage according to claim 16, wherein: (i) the buffer is selected from the group consisting of Tris, MOPS, MES, ADA, PIPES, ACES, MOPSO, Cholamine chloride, BES, TES, HEPES, DIPSO, TAPSO, Acetamidoglycine, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Glycylglycine, Bicine, TAPS, AMPSO, CHES, CAPSO, CAPS, CABS, Phosphate buffer, Carbonate-Bicarbonate buffer, and any combination thereof; (ii) the buffer comprises a solvent selected from DMSO, ethanol, acetonitrile or any combination thereof, optionally wherein the solvent to construct ratio is from 0:1 to 0.5:1 (v/v); (iii) the salt is selected from the group consisting of NaCl, CaCl2, MgCl2, MnCl2, KCl, CaI2, NaI, KI, MgI2, and any combination thereof; and/or (iv) wherein the cleaved constructs are incubated with the buffer for 0-4 h.
18. A method of preparing a ferritin nanocage according to any one of claims 15 to 17, further comprising adjusting the salt concentration and/or the pH to induce self-assembly of the ferritin subunits into a ferritin nanocage.
19. A method of preparing a ferritin nanocage according to any one of claims 15-18, the method further comprising: contacting the plurality of constructs with a cargo molecule, such that the cargo molecule is encapsulated inside the ferritin nanocage during self-assembly of the ferritin subunits into the ferritin nanocage.
20. A method of preparing a ferritin nanocage according to claim 19, wherein the cargo molecule is a therapeutic element.
21. A method of preparing a ferritin nanocage according to any one of claims 15 to 20, wherein the plurality of constructs are provided by expressing a nucleic acid molecule according to claim 11 or an expression vector according to claim 12.
22. A method of preparing a ferritin nanocage according to any one of claims 15 to 21, the method further comprising: binding a cargo molecule to the surface of the ferritin nanocage, optionally wherein the cargo molecule is a targeting molecule or a therapeutic element.
23. A ferritin nanocage produced by the method of any one of claims 15 to 22.
24. A method of treating or preventing a disease in a subject, the method comprising: (i) administering a ferritin nanocage according to claim 23 to a subject, wherein the ferritin nanocage comprises a therapeutic element; and/or (ii) administering a construct according to any one of claims 1 to 10 to a subject, wherein the construct comprises a therapeutic element.
25. A method of raising an immune response against an antigen, the method comprising: administering a ferritin nanocage according to claim 23 to a subject, wherein the ferritin nanocage comprises an antigen.
PCT/GB2023/053057 2022-11-24 2023-11-23 Control of nanocage self-assembly WO2024110757A1 (en)

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