LU102572B1 - An artificial protein-cage decorated with particular molecules on the exterior - Google Patents

Abstract

The present invention provides an artificial TRAP-cage decorated with particular molecules (proteins, peptides, small molecules, nucleic acids) on the exterior.

Description

An artificial Protein-cage decorated with particular molecules on the exterior LU102572

FIELD OF THE INVENTION The present invention falls within the biochemistry field. It is related to an artificial protein cage known as "TRAP-cage” decorated with particular molecules (proteins, peptides, small molecules, nucleic acids) on the exterior.

BACKGROUND Proteins that assemble into monodisperse cage-like structures are useful delivery/display vehicles for applications in biotechnology and medicine. Such protein cages exist in nature, e.g. viral capsids, but can also be designed and constructed in the laboratory. As such, inventors previously described that a cysteine-modified variant of the tryptophan RNA-binding attenuation protein from Geobacillus stearothermophilus, TRAPKSC.R45 can assemble into a hollow spherical structure composed of multiple ring-shape undecameric subunits via reaction with monovalent gold ions. The resulting protein cages exhibit an extremely high stability under many harsh conditions, but easily disassemble to the ring subunits in the presence of thiol- or phosphine- containing agents. Based on this appealing platform, development of a general methodology to modify the cage exterior is essential to expand the utility of the artificial protein cages in drug delivery and vaccination. The object of the invention is to provide chemical and enzymatic strategies to decorate the exterior surface of TRAP cage assemblies.

SUMMARY OF THE INVENTION The subject matter of the invention is an artificial TRAP-cage comprising a selected number of TRAP rings and a plurality of external decorations attached thereto. Preferably the external decorations are selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules, inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof. Preferably the external decoration is a viral, microbial or cancer antigen. Preferably the external decorations are the same or different from one another.

Preferably at least one of the external decorations comprises a cell penetrating agent LU102572 to promote intracellular delivery of the TRAP-cage.

Preferably the cell penetrating agent is PTD4. Preferably the TRAP-cage according to the invention further includes an intemal cargo encapsulated therein.

Preferably the number of TRAP rings in the TRAP-cage is between 6 to 60. Preferably number of TRAP rings in the TRAP-cage is 24. The subject matter of the invention is also use of the artificial TRAP-cage according to the invention as a delivery vehicle for delivery of its external decoration.

Preferably the delivery is for intracellular delivery.

Preferably the delivery is for extracellular delivery.

The subject matter of the invention is also use of the artificial TRAP-cage according to the invention as a vaccine.

The subject matter of the invention is also use of the artificial TRAP-cage according to the invention for the treatment of an illness or disease condition selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infection, auto-immune condition, neurological/neurodegenerative disease, arthritis and respiratory disease.

The subject matter of the invention is also a method of making an artificial TRAP-cage, the method comprising:

(I) obtaining TRAP ring units by expression of the TRAP ring units in a suitable expression system and purification of the said units from the expression system;

(in) conjugation of the TRAP ring units via at least one free thiol linkage with a molecular cross-linker;

(iii) formation of the TRAP-cage by self-assembly and modification of an external surface of the formed TRAP-cage to what is appropriate for the external decoration that is to be attached to the cage exterior surface;

{iv) decorating the external surface of the TRAP-cage with a moiety selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules, inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof: LU102572 and

(v) purification and isolation of the TRAP-cages.

Preferably the expression system in step (i) is selected from a cell-based expression system or other expression systems such as cell-free or plant expression systems.

Preferably purification of the said units from the expression system of step (i) by using FPLC-based purification employing appropriate columns such as a mixture a of affinity based and size exclusion columns.

Preferably the modification of step (ili) is selected from the group comprising: {D) chemical modification; (i) enzymatic coupling; (iii) bio-conjugation: (iv) genetic coupling; and (v) click chemistry.

Preferably the chemical modification is via cysteine maleimide-based conjugation.

Preferably the chemical modification is via lysine amide-based conjugation.

Preferably the enzymatic coupling is via a peptide ligase.

Preferably the peptide ligase is selected from the group comprising sortases, asparaginy! endoproteases, trypsin related enzymes and subtilisin-derived variants.

Preferably the bio-conjugation is via an azide-reactive side chain.

Preferably the azide-reactive side chain is DBCO.

Preferably the genetic coupling is via fusion to a C-terminus of TRAP.

Preferably the N-terminus sequence of the external sequence is fused to a C- terminus sequence of TRAP that is available on the exterior of the TRAP-cage.

The subject matter of the invention is also a method of treatment of an individual in need of therapy suffering from a condition selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infection, cellular senescence auto-immune condition, neurological/Neurodegenerative disease, arthritis and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP-cage bearing one or more external decorations selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins,

peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, LU102572 signaling molecules, lipids, oligosaccharides, dye molecules , inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof.

The subject matter of the invention is also a method of vaccinating an individual suffering from a condition selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infection, cellular senescence, auto- immune conditions, neurological/neurodegenerative disease, arthritis and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP-cage bearing one or more external decorations selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules, inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof.

Preferably the TRAP-cage therapeutic is administered via intranasal inhalation or injection.

If no cysteine is present in the biomolecule, or they are present but not available for the reaction, -SH group, preferably as a group of cysteine, may be introduced into the biomolecule.

Introduction of cysteine can be carried out by any method known in the art.

For exampie, but not limited to, the introduction of the cysteine is performed by methods known in the art, such as commercial gene synthesis or PCR-based site-directed mutagenesis using modified DNA primers.

Above-mentioned methods are known by the persons skilled in the art and ready-to use kits with protocols are available commercially. -SH moiety may be introduced into the biomolecule also by modification of other amino acids in the biomolecule i.e. by site-directed mutagenesis or by solid phase peptide synthesis.

Reference herein to “exterior” refers to the outer surface of the TRAP-cage and the surface which, in vivo, is the surface presented to a host.

Accordingly, any exterior decoration is thus presented to a host and can illicit an appropriate response.

Reference herein to “aftached” refers to a physical or chemical bond of the exterior decoration to the exterior surface of the TRAP-cage.

Reference herein to "TRAP ring” is synonymous with a TRAP building block, a subunit of the TRAP-cage complex or a TRAP monomer assembly.

“Unit”, “subunit”, "molecule”, “biomolecule”. “monomer” are used alternatively in the LU102572 description and means one molecule which connects to another molecule for the complex formation. “Complex”, “assembly”, “aggregate”, are used alternatively in the description and means a superstructure constructed by the reaction between biomolecules. The amount of the units involved in the complex depends on the nature of the biomolecule. More specifically, it depends on the amount of the biomolecule and the amount of -SH groups present in the biomolecule. TRAP protein is a suitable biomolecule model for the method of the invention. This is likely due to its high intrinsic stability, toroid shape, lack of native cysteine residues (for easier control of the conjugation process) and availability of a residue that can be changed to cysteines with the resulting cysteine being in a suitable chemical and spatial environment suitable for proper bond formation. Nevertheless, a person skilled in the art would easily adapt the reaction conditions for other biomolecular monomers. Any biomolecular monomer that has free thiol(s) group(s) and/or its structure allows modification by introducing thiol group may be suitable for the method of conjugation of the biomolecules according to the invention.

DETAILED DESCRIPTION OF THE INVENTION TRAP cages are amenable to chemical modification. The Au(l)-mediated TRAP-cage assembly possesses 24 free cysteines per cage, four at each of the six at the 4-fold symmetrical pore regions. These cysteines have been used for labelling the cages with Alexa-647 fluorescent dye containing a maleimide moiety (Fig. 1a). The chemical modification was performed using the TRAP-cages that were loaded with a negatively supercharged variant of green fluorescent protein, GFP(-21) (Fig. 1a). The amount of Alexa-647-maleimide (which was equal to the number of TRAP cysteine groups) to be added has been optimized, where the TRAP-cage is readily labelled and no free dye is present in the sample, noting concentrations that were unsuccessful (Fig. 1b). TRAP naturally has three surface exposed lysines per monomer, corresponding to 792 lysines on the assembled cage, that are ready to react with many electrophile groups such as activated esters to form covalent bonds. To exploit this, the C-terminus of a model peptide PTD4 (YARAAARQARA), an optimised HIV TAT-based cell-penetrating peptide, has been converted to N-hydroxysuccinimide (NHS). This PTD4 derivative, Ac-YARAAARQARAG, has been attached to the amino groups on the surface- exposed lysines of TRAP-cages (Fig. 1a). The C-terminus of the peptide was activated with a sulfonated NHS using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

The assembled and purified TRAP cage was simply mixed with the peptide-NHS in LU102572 50mM HEPES, 150mM NaCl, pH 7.5 at room temperature for 2.5 hours.

Native-PAGE analysis of the resulting mixture showed a substantial mobility shift compared to unmodified TRAP cage, suggesting successful cage modification with the peptide (Fig. 1c). Negative stain TEM confirmed that modified cages remain intact (Fig. 1d). Efficient lysine modification in aqueous solution can be achieved with the compound containing an isothiocyanate moiety to yield a thiourea bond.

This possibility has been demonstrated through TRAP-cage modification with fluorescein isothiocyanate (FITC) (Fig. 2). Furthermore, TRAP modification with peptides can be achieved not only with NHS esters to form amide bonds with lysines, but also via cysteine-modification using maleimide-based conjugation.

As such, a PTD4 peptide derivative having a maleimide moiety at the N-terminus has been prepared.

Successful decoration with this maleimidyl peptide was confirmed by native-PAGE analysis using TRAP cages encapsulating guest mCherry proteins in the lumen via genetic fusion strategy (Fig.3). Moreover, TRAP cages are amenable to enzymatic modification with peptide/protein.

Despite the easiness of the modification using activated ester, this method is limited to peptides which do not contain any nucleophile amine and carboxylate in the sequence.

In order to overcome this issue, we next employed an enzymatic coupling system using a peptide ligase (Fig. 4). Sortase A (SrtA) is a bacterial transpeptidase that catalyzes the reaction of fusing the LPXTG protein motif to a N-terminal polyglycine chain, yielding a fusion LPXT(G),'. We equipped the TRAPX*C C-terminus, which exposed to the exterior when assembled to cages, with the polypeptide containing LPSTG, yielding TRAP"*C.srt (Fig. 4b). Like the parent protein, this modified variant can assemble into cage-like structures upon addition of Au(l), judged by negative-stain transmission electron microscopy (TEM) (Fig. 4c). As initial models to decorate the TRAP cage, we selected four fluorescent proteins, mCherry, td Tomato. dTomato and dsRed2. These proteins, although their monomer units have similar protein folding, have different sizes and quaternary states (Fig. 4d), allowing us to check the influence of these factors on the reaction with TRAP cages.

These model proteins were appended with a hexahistidine, a recognition sequence of the protease from tobacco etch virus (TEV protease), and a pentaglycine at the N-terminus {Fig. 4e), and recombinantly produced using E. coli cells.

After purification and cleavage of the tag using TEV protease, the resulting proteins possessing an N-terminal pentaglycine was mixed with cages composed of TRAP-srt in the presence of SrtA.

Analysis of the reaction mixtures using native-PAGE showed a substantial band shift compared to that of unmodified TRAP cages, suggesting successful modification of the protein cages with all the guest proteins (Fig. 3a). After isolation of the decorated LU102572 TRAP cages using size-exclusion chromatography, the samples were further analyzed by dynamic light scattering (DLS), showing an increase in the diameter of the modified cage in proportion to the size of the decoration protein; 24.38 nm, 29.71 nm, 32.38 nm,

29.55nm, and 50.96 nm for TRAP***C-sit cages, ones modified with mCherry, tdTomato, dTomato, and DsRed2, respectively. The exterior decoration and intact cage structure upon enzymatic was also confirmed by TEM (Fig. 3b). The differences between the model proteins turned out not to have a significant impact on the modification process, suggesting the possibility of using this method to obtain a wide spectrum of modifications with various proteins. To further demonstrate the utility of the srtA-mediated decoration of TRAP cages, we chose nanobodies (Nbs), an isolated, binding portion of an antibody originally sourced from camelid single domain antibodies as next models (Muyldermans S. Nanobodies: natural single-domain antibodies. Annu Rev Biochem. 2013:82:775-797. dot:10.1146/annurev-biochem-063011-092449). Nbs are currently of great interest due to their high stability, easy expression in bacterial systems, small size and excellent binding affinity. However, their small size leads to quick filtration in the kidney, a marked disadvantage in the potential medical usage. We hypothesized that Modification on the protein cage exterior can extend the lifetime of Nbs in blood stream. Additionally, multiple nanobodies displayed on a single particle may increase the avidity of binding to target. Nanobodies displayed on the exterior of protein cages could conceivably be used to localise cages and their therapeutic cargoes specifically at sites of interest e.g. receptors overexpressed on cancer cells. A GFP-binding Nb was used to facilitate the functional evaluation upon modification on the TRAP cage exterior. SDS- and native-PAGE analysis of the reaction with TRAP-srt cages in the presence of SrtA suggested successful exterior decoration with Nbs via covalent bond formation (Fig. 6a). Samples were further analyzed by DLS, showing an increase in the diameter of the modified cage; 26.21 nm; 30.65 nm, ones modified with Nbs and Nbs with further addition of GFP, respectively. Furthermore, upon the modification, the anti-GFP Nbs still retained the ability to bond with GFP (Fig. 6b). Summarizing, TRAP cages are amenable to both chemical and enzymatic modification with peptide/protein. Likewise, many other molecules/materials such as DNAs, lipids, oligosaccharides, synthetic polymers and metal nanoparticles could be attached on TRAP cage exterior by introducing either NHS ester or polyglycine units in the structure for ester bond or sortase-mediated attachment respectively. Such robust and general exterior decoration strategies will contribute largely to drug carrier and vaccine LU102572 development based on artificial protein cages.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1. TRAP cage decoration via cysteine and lysine modification. (a) Scheme of TRAP-cage encapsulation with GFP(-21) and external modifications with Alexa-647 dye and PTD4 peptide. (b) Native PAGE gels showing TRAP-cage carrying GFP(-21) after titration of Alexa-647 in the conjugation reaction. Gels were analysed by fluorescence detection of Alexa-647 (left panel, exct. 647) and stained for proteins (right panel). Arrows show optimal decoration conditions used in further experiments.{c) Native PAGE gels showing encapsulation of GFP({-21) by unmodified TRAP-cage or TRAP-cage externally modified by Alexa-647 and PTD4. Lane 1: TRAP- cage with GFP{-21); 2: TRAP-cage with GFP(-21) decorated with Alexa-547; 3: TRAP- cage with GFP(-21) decorated with Alexa-647 and PTD4; 4: molecular weight marker for native PAGE. Gels were stained for protein (upper panel) and analysed by fluorescence detection of GFP (middle panel, exct. 488 nm) and Alexa-647 (bottom panel, exct. 647). (d) Negative stain transmission electron microscopy of TRAP-cage with GFP(-21) (left panel); TRAP-cage with GFP(-21) decorated with Alexa-647 (middle panel); TRAP-cage with GFP(-21) decorated with Alexa-647 and PTD4 (right panel). Fig. 2. External decoration of TRAP-cage with FITC dye. (a) Schemeatic for reaction of TRAP-cage with FITC. (b) Native PAGE gels showing TRAP-cage after conjugation with FITC dye. Gels were stained for proteins (left panel) and analysed by fluorescence detection of FITC (right panel, exct. 488). Lanes: 1: molecular weight marker for Native PAGE electrophoresis; 2: TRAP-cage with FITC in PBS buffer; 3: TRAP-cage with FITC in carbonate-biscarbonate buffer; 4: TRAP-cage with FITC in DMEM. Fig. 3. External decoration of TRAP-cage filled with mCherry with 6- maleimidehexanoic-PTD4 peptides. (a) Schematic representation of cysteine- mediated TRAP-cage decoration. (b) Native PAGE gels showing TRAP-cage carrying mCherry after titration of PTD4 in the conjugation reaction. TRAP-cage after conjugation with PTD4 peptides. Fig. 4. Concept and strategy of sortase-mediated TRAP cage decoration. (a) Schematic representation of sortase-mediated TRAP cage decoration with guest molecules. (b) The construct of TRAPK?*¢ possessing C-terminal sortase recognition sequence, TRAP**“.srt. (c) TEM images of Au(l)-induced assemblies composed of TRAP**C-grt and srtA-mediated decoration at the exterior. The samples were stained with 2% uranyl acetate. (d) Quaternary states of four red fluorescent proteins used in LU102572 this study. (e) The construct design of model red fluorescent protein possessing a pentaglycine at the N-terminus. Fig. 5. Sortase-mediated TRAP cage decoration with model fluorescent proteins. Native-PAGE analysis (a) and {b) TEM imaging of TRAPK®*C-srt cages modified with mCherry (mCh), tdTomato (tdT), dTomato (dT) dsRed2 (dsR2), Nanobodies (Nbs) and Nanobodies with further addition of GFP (Nbs-GFP). The protein bands on the gel was visualized using Instant Blue staining. For TEM, the samples were stained with 2% uranyl acetate. Fig. 6. Sortase-mediated TRAP cage decoration with nanobodies. (a,b) SDS-PAGE (a) and nativePAGE (b) analysis of sortase mediated conjugation between TRAPKC. srt (9.5 kDa) cage and anti-GFP Nbs possessing an N-terminal pentaglycine (G5- Nbs®"F, 13.2 kDa). Theoretical molecular mass of conjugated product is 22.65 kDa. The ability of the resulting TRAP cage displaying Nbs to bind with GFP was also confirmed (b, rightmost lane). These gels were visualized by Instant Blue staining.

EXAMPLES Example 1. Chemical modification of TRAP cages. TRAP-cage carrying GFP labeling with Alexa-647 and decorated with cell-penetrating peptide Alexa Fluor-647 C2 maleimide fluorescent dye (Alexa-647, Thermo Fisher Scientific) and cell-penetrating PTD4 peptide were conjugated to the TRAP-cage filled with GFP via a crosslinking reactions with cysteines and lysines present in the TRAP protein (Fig. 1a). To achieve fluorescent labelling, TRAP-cage carrying GFP was mixed with a Alexa- 647 C2 maleimide dye, the reaction was conducted in 50 mM HEPES with 150 mM NaCl pH 7.5 for 2.5 h at room temperature with continuous stirring at 450 rom. The optimal interaction ratio of maleimide-conjugated Alexa-647 to TRAP-cage was assessed by titration (Fig. 1b). Briefly, aliquots of TRAP-cage loaded with GFP(-21) were mixed with maleimide-conjugated Alexa-647 ranging from 0.1 pM to 100 uM. Samples were then separated by native gel electrophoresis and visualized by fluorescence detection in a Chemidoc, with excitation at 647 nm. Reactions where no free Alexa-647 is present in the sample, were considered as optimal decoration conditions.

For the cell-penetrating peptide decoration, the peptide chain was constructed on resin LU102572 using standard Fmoc-based solid phase peptide synthesis (SPPS) using a N,N"- diisopropylcarbodiimide (DIC)/Oxyma coupling system and the N-terminus was capped using acetic anhydride.

After cleavage from the resin and deprotection, the peptide was purified by reverse-phase high performance liquid chromatography (RP- HPLC). Purified PTD4 peptide was mixed with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 10 pl, 83 mM) and N-hydroxysuccinimide (NHS, 10 ui, 435 mM), all reagents dissolved in ddH:O.

Subsequently, the excess of activated PTD4 peptides were added to TRAP-cage filled with GFP(-21) and labelled with Alexa- 647 and incubated for next 2.5h at room temperature, with continuous stirring at 450 rpm.

The reaction was stopped by addition of 5 ul of 200 mM Tris-HCI pH 7.5. The conjugation efficiency was verified by native PAGE and fluorescent gel imaging.

A change in molar weight of the decorated TRAP-cage results in a band shift observed in native PAGE (Fig. 1c). Negative stain transmission electron microscopy (TEM) confirmed that the modified TRAP-cages retained their characteristic shape (Fig. 1d). TRAP-cage labeling with FITC (fluorescein isothiocyanate) dye FITC (fluorescein isothiocyanate) fluorescent dye (FITC, Sigma) was conjugated to the TRAP-cage via reactions with lysines present in the TRAP protein.

To achieve fluorescent labelling, TRAP-cage (200 pl, 0.5 mg/ml nM) was mixed with a FITC dye (50 ul, 0.25 mg/ml), the reaction was conducted in 0.1 M sodium carbonate- bicarbonate buffer, pH 9.0, for overnight, 4 °C with gentle stirring.

Excess of FITC dye was removed using the Sephadex G-25M column following the manufacturer's recommended protocol.

Samples were subsequently analyzed by native PAGE followed by Instant blue gel staining and visualized by fluorescence detection in a Chemidoc, with excitation at 488 nm (Fig. 2b). TRAP-cage with mCherry decoration with cell-penetrating peptides À maleimide moiety was introduced at the N-terminus of the peptide on resin using 6- maleimide hexanoic acid and a DIC/Oxyma coupling protocol.

The 6- maleimidehexanoic-PTD4 peptide ranging from 0.1 uM to 0.5 mM was mixed with TRAP-cage filled with mCherry (100 pl, 0.3 mg/ml) and incubated overnight at room temperature, with continuous stirring at 450 rpm.

The conjugation efficiency was verified by native PAGE and fluorescent gel imaging.

A change in molar weight of the decorated TRAP-cage results in a band shift observed in native PAGE (Fig 3b). Example 2. Enzymatic modification of TRAP cages.

Protein design, production and purification

The TRAP cages were obtained as described previously (Malay, Ali D., et al. "An ultra- LU102572 stable gold-coordinated protein cage displaying reversible assembly." Nature

569.7756 (2019). 438-442.), with the TRAP variant having K35C mutation and the appended amino acid sequence of GTGGSLPSTG at the C-terminus. SrtA gene was ordered from commercial vendor (BioCat), already subcloned into pET30b(+) plasmid. E. coli strain BL21 (DE3) cells were transformed with the plasmid and precultured in LB medium at 37°C until the OD600 value reached to ~0.6 at which point protein expression was induced by addition of isopropyl B-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM, followed by further cell culture at 25°C overnight. After cell lysis by sonication, these proteins were purified by Ni-NTA affinity chromatography and size-exclusion chromatography using a Superdex 200 Increase 10/300 column (GE Healthcare). The genes of the fluorescent proteins (mCherry, tdTomato, dTomato, dsRed2) and nanobodies (anti-GFP nanobodies) were modified with genes encoding a 6xHis tag at the N-terminus linked to the ENLYFQG sequence recognized by TEV protease and a pentaglycine. The modified fluorescent protein genes were prepared in the laboratory and cloned into the pET28 plasmid, while the pET28 plasmid containing the nanobodies sequence was obtained from a commercial vendor, BioCat GmbH. E. cofi strain BL21 (DE3) cells were transformed with the plasmid and precultured in LB medium at 37°C until the OD600 value reached to ~0.6 at which point protein expression was induced by addition of IPTG to a fina! concentration of 0.3 mM, followed by further cell culture at 25°C overnight. After cell lysis by sonication, these proteins were purified by Ni-NTA affinity chromatography and size-exclusion chromatography using a Superdex 75 increase 10/300 column (GE Healthcare). Sortase-mediated modification and cage characterization Conjugation of the TRAP cages with fluorescent proteins was performed in a PBS buffer Proteins were mixed in the reaction buffer to final concentration of 40 uM TRAP with respect to monomer, 10 uM fluorescent proteins, and 3 uM sortase A (SrtA). The reaction was carried out for 2 hours at room temperature. Part of the reaction mixtures were analyzed by native-PAGE (Fig. 5a) The resulting cages were then purified by size-exclusion chromatography using a Superose& increase 10/300 column (GE Healthcare) in 2xPBS buffer. Isolated cages were subsequently analyzed by negative- stain transmission electron microscope (TEM) (Fig. 5b) and dynamic light scattering (DLS). For TEM, the protein samples were diluted to 0.04 mg/mL. Copper grids (FC400Cu100, Lab Soft) were glow-discharged (Leica EM Ace200, Leica Microsystems), and then 4 uL of the protein samples were applied to them and left for

1 minute.

The grids were then dried using a filter paper, and 4 uL of 2% uranyl acetate LU102572 was applied to the grids, and dried immediately with the same method.

Then, 4 uL of 2% uranyl acetate were transferred to the grid for 15s, and dried.

Samples were then visualized on a JOEL-1230 electron microscope with 80 kV operation.

The DLS measurement was performed on a Malvern ZetaSizer Nano S.

An analogous protocol was used for decoration with nanobodies.

For the binding of Nbs®P GFP was mixed in PBS with the modified TRAP cages, to a final concentration of 13 uM of TRAP and 2 uM of GFP, and kept at room temperature for 30 minutes.

The resulting reaction mixtures were analyzed by SDS- and native-PAGE (Fig. 6). Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires.

In particular, where the indefinite article is Used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings). and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

The invention is not restricted to the details of any foregoing embodiments.

The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings). or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims (1)

1. CLAIMS LU102572

   1. An artificial TRAP-cage comprising a selected number of TRAP rings and a plurality of external decorations attached thereto.

   2. The cage according to claim 1 wherein the external decorations are selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules , inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof.

   3. The cage according to either claim 1 or 2 wherein the external decoration is a viral, microbial or cancer antigen. 4 The cage according to any preceding wherein the external decorations are the same or different ffom one another.

   5. The cage according to any preceding claim wherein at least one of the external decorations comprises a cell penetrating agent to promote intracellular delivery of the cage. 6, The cage according to claim 5 wherein the cell penetrating agent is PTD4.

   7. The cage according to any preceding claim that further includes an internal cargo encapsulated therein.

   8. The cage according to any preceding claim wherein the number of TRAP rings in the TRAP-cage is between 6 to 60.

   9. The cage according to claim 8 wherein the number of TRAP rings in the TRAP-cage is 24.

   10. Use of the artificial TRAP-cage according to any preceding claim as a delivery vehicle for delivery of its external decoration.

   11. Use according to claim 10 wherein the delivery is for intracellular delivery.

   12. Use of the artificial TRAP-cage according to any one of claims 1 to 9 as a vaccine.

   13. Use of the artificial TRAP-cage according to any one of claims 1 to 9 for the treatment of an illness or disease condition selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infection, auto-immune condition, neurological/neurodegenerative disease, arthritis and respiratory disease.

   14. A method of making an artificial TRAP-cage, the method comprising: {i) obtaining TRAP ring units by expression of the TRAP ring units in a suitable expression system and purification of the said units from the expression system; (i) conjugation of the TRAP ring units via at least one free thiol linkage with a molecular cross-linker; (i) formation of the TRAP-cage by self-assembly and modification of an external surface of the formed TRAP-cage to what is appropriate for the external decoration that is to be attached to the cage exterior surface; (iv) decorating the external surface of the TRAP-cage with a moiety selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules , inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof: and (v} purification and isolation of the TRAP-cages.

   15. The method of claim 14 wherein step (i) the expression system is from a cell- LU102572 based expression system or other expression systems such as cell-free or plant expression systems.

   16. The method of either of claims 14 or 15 wherein purification of the said units from the expression system of step (i} by using FPLC-based purification employing appropriate columns such as a mixture of affinity based and size exclusion columns.

   17. The method of any of claims 14 to 16 wherein the modification of step (iii) is selected from the group comprising: (1) chemical modification; {ii enzymatic coupling; (iii) bio-conjugation; (iv) genetic coupling; and (v) click chemistry.

   18. The method of claim 17 wherein the chemical modification is via cysteine maleimide-based conjugation.

   19. The method of claim 17 wherein the chemical modification is via lysine amide- based conjugation.

   20. The method of claim 17 wherein the enzymatic coupling is via a peptide ligase.

   21. The method according to claim 20 wherein the peptide ligase is selected from the group comprising sortases, asparaginyl endoproteases, trypsin related enzymes and subtilisin-derived variants.

   22. The method according to claim 17 wherein the bio-conjugation is via an azide- reactive side chain.

   23. The method according to claim 22 wherein the azide-reactive side chain is LU102572 DBCO.

   24. The method according to claim 17 wherein the genetic coupling is via fusion to a C-terminus of TRAP.

   25. The method according to claim 245 wherein the N-terminus sequence of the external sequence is fused to a C- terminus sequence of TRAP that is available on the exterior of the TRAP-cage.

   27. A method of treatment of an individual in need of therapy suffering from a condition selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infection, cellular senescence, auto-immune condition, neurological/neurodegenerative disease, arthritis and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP-cage bearing one or more external decorations selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules , inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof.

   28. A method of vaccinating an individual suffering from a condition selected from the group comprising cancer, vascular disease, cardiovascular disease, diabetes, infection, cellular senescence, auto-immune condition, neurological/neurodegenerative disease, arthritis and respiratory disease, the method comprising administering a therapeutically effective amount of an artificial TRAP-cage bearing one or more external decorations selected from the group comprising nanobodies, antibodies, epitopes, antigens, proteins, peptides, cell penetrating peptides, antigenic peptides, polypeptides, nucleic acids, signaling molecules, lipids, oligosaccharides, dye molecules, inorganic nanoparticles, specific ligands and small molecule therapeutics or fragments thereof.

   29. The methods of either claims 27 or 28 wherein the TRAP-cage therapeutic is LU102572 administered via intranasal inhalation or injection.