Adenoviral coat protein derived delivery vehicles

Abstract

To provide new adenoviral coat protein based delivery vehicles.SOLUTION: The present invention provides vehicles that are modified penton base protomers that assemble into virus-like particles (VLPs). Exposed areas of penton base proteins can specifically bind the VLP to any target and / or comprise any peptide epitope. An additional cargo can be attached to the VLP via EG fibre protein fragments. The present invention relates to such EG penton base protomers, EG proteins comprising an adenovirus fibre protein N-terminal fragment binding to an adenovirus fibre protein binding cleft of a penton base protomer, VLPs comprising the EG penton base protomers and optionally EG proteins comprising an adenovirus fibre protein N-terminal fragment of a penton base protomer, nucleic acids encoding the EG proteins, the VLPs as well as methods of producing the proteins and VLPs.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to novel delivery vehicles based on adenovirus coat proteins. The vehicles are modified penton base protomers that assemble into VLPs. The exposed portions of the penton base proteins can be modified to allow the VLPs to specifically bind any target and / or include any desired peptide epitope. Additional cargo, e.g., drugs, polypeptides, proteins, or nucleic acids, are reversibly or irreversibly attached to the VLPs via the EG fiber protein fragment. The present invention relates to the above-mentioned EG penton base protomers, EG proteins comprising fiber protein fragments capable of binding to the penton base protomers, VLPs comprising EG penton base protomers, and optionally EG proteins comprising fiber protein fragments, nucleic acids encoding EG proteins, VLPs, and methods of producing the proteins and VLPs. [Background technology]

[0002] Infectious diseases continue to afflict and kill people worldwide. Of the tools currently available to combat these threats, vaccination has proven to be particularly powerful: smallpox has been eradicated, and measles, polio, and tetanus have been controlled globally through vaccination. However, serious threats to human health continue to pose a threat, particularly from new viruses that have adapted and emerged as pathogenic species with attributes that promote virulence.

[0003] Recent examples of this include Chikungunya and Zika, two serious threats posed by insect-borne viruses transmitted to humans by mosquito bites. Both of these viruses, which use mosquitoes as hosts, have spread rapidly across Asia and Europe, raising serious alarms. Chikungunya and Zika could potentially cause great harm to affected communities and economies, making these threats a strong demand for a strong vaccination strategy. However, today, there is a total lack of strong vaccines.

[0004] Ideally, a vaccine would be safe, non-replicating, highly effective, and fine-tunable. Moreover, it would be easily produced on an industrial scale. Recombinant virus-like particles (VLPs) are a potential ideal vaccine and therefore hold great promise. In this proposal, we attempt to create a VLP vaccine. We utilize ADDomers (adenovirus dodecahedron-derived multimers) that have a remarkable diversity and biological similarity. ADDomers may help generate vaccine candidates to combat viral infectious diseases (chikungunya, Zika, and others).

[0005] ADDomer is a synthetic scaffold derived from virus-like particles (VLPs) that naturally arise during the human adenovirus serotype 3 (HAd3) replication cycle, catalyzing internalization (Fender, P., et al. (2012) J Virol 86, 5380-5385). The ADDomers have an engineered biological similarity derived from this natural VLP and retain the ability to autonomously self-assemble into a dodecahedron. The ADDomers are particularly suitable for displaying multiple peptide and protein epitopes due to the exposed loops that are sufficiently flexible to be engineered. Engineering these loops does not disrupt the overall structure of the ADDomer particle. These loops provide a convenient way to insert highly immunogenic peptide epitopes, for example from viral pathogens, using synthetic biology methods. ADDomers are not limited to vaccine development against infectious diseases. A wide range of potential applications for the ADDomr technology are anticipated, including cancer therapy. Furthermore, ADDomers cannot only display peptide epitopes. Proteins or protein domains can be exposed by ADDomers as well, significantly expanding their applications. Summary of the Invention

[0006] The inventors of the present invention have introduced heterologous peptide sequences into specific sites in the penton-based protomers without inhibiting the assembly of the penton-based protomers into penton subunits, which instead self-assemble into penton dodecahedrons (also called ADDomers) that form virus-like particles (VLPs). This design is truly modular, allowing for rapid and flexible functionalization of the extended loops for multi-peptide display. This modularity is further enhanced by using an adenovirus fiber protein fragment, which is an adenovirus fiber protein that specifically binds to the penton-based protomers. The VLPs of the present invention are safe because they do not contain genetic material. The penton-based protomers can receive and display up to 180 foreign peptide motifs, including antibodies, neutralizing polypeptides, oncoepitope polypeptides, single-chain antibodies, and nanobodies. Thus, in a first aspect, the invention relates to a modified polypeptide comprising an adenoviral penton base protomer, wherein the penton base protomer includes a first RGD loop, a second RGD loop, a variable loop (V loop), an adenoviral fiber protein binding cleft, and / or an N-term domain, and comprises one or more of the following: (i) at least one target-specific binding domain in the first, second, both the first and second RGD loops, and / or the V loop; and / or (ii) one or more non-adenoviral peptides in the first, second, both the first and second RGD loops, and / or the V loop; and / or (iii) a non-adenoviral polypeptide at the N- and / or C-terminus of the penton base protomer; and / or (iv) at least one heterologous coupling residue in the first, second, both the first and second RGD loops, the V loop, and / or in the N-terminal domain of the penton base protomer, where the N-terminus of the N-terminal domain in the penton base protomer t is defined as follows: X1-GRNSIR (SEQ ID NO: 44) And the C-terminus of the N-terminal domain within the penton base protomer is defined as follows: D-X2-RSRG (SEQ ID NO: 45), Here, X1 is selected from the group consisting of G and E, and X2 is selected from the group consisting of D and E; and / or (v) a drug or polypeptide covalently or non-covalently bound to one or more amino acids of the first, second, or both the first and second RGD loops, and / or one or more amino acids of the V loop of the penton base protomer; and / or (vi) at least one heterologous coupling residue in the adenovirus fiber protein binding cleft of the penton base protomer; Here, the modified polypeptides of the first embodiment are preferably capable of assembling into a VLP.

[0007] In a second aspect, the invention relates to a modified polypeptide having at least one adenoviral fiber protein N-terminal fragment that specifically binds to the adenoviral fiber protein binding cleft of a penton base protomer: (i) a non-adenoviral polypeptide, and / or (ii) Covalently or non-covalently linked to a drug or label. In a third aspect, the present invention relates to a nucleic acid encoding a modified polypeptide having an adenovirus penton base protomer of the invention and / or a modified polypeptide of the invention having an adenovirus penton base protomer that binds a fiber protein fragment.

[0008] In a fourth aspect, the present invention relates to an expression vector comprising the nucleic acid sequence of the invention.

[0009] In a fifth aspect, the present invention relates to a cloning vector encoding: (i) a polypeptide having an adenoviral penton base protomer, the penton base protomer containing a first RGD loop, a second RGD loop, a variable loop, and / or a binding site for an adenoviral fiber protein adapted to introduce a nucleic acid encoding a non-adenoviral polypeptide into a nucleic acid encoding the first RGD loop, the second RGD loop, and / or the variable loop; or (ii) A polypeptide having an adenoviral penton base protomer binding a fiber protein fragment adapted for introducing a nucleic acid encoding a non-adenoviral peptide at its C-terminus and / or N-terminus.

[0010] In a sixth aspect, the present invention relates to a recombinant host cell comprising an expression vector of the invention, or a cloning vector of the invention.

[0011] In a seventh aspect, the present invention relates to a pentamer having five modified polypeptides comprising the adenovirus penton base protomer of the invention.

[0012] In an eighth aspect, the present invention relates to a VLP comprising 12 pentamers of the invention.

[0013] In a ninth aspect, the present invention relates to a VLP having 12 pentamers, each of which has five adenoviral penton base protomers and at least one modified polypeptide of the invention comprising an adenoviral penton base protomer that binds a fiber protein fragment.

[0014] In a tenth aspect, the present invention relates to a modified polypeptide comprising an adenovirus penton base protomer of the invention and / or a method for producing a modified polypeptide comprising an adenovirus penton base protomer that binds a fiber protein, the method comprising the steps of: (a) providing a recombinant host cell of the invention; (b) expressing the modified polypeptide. (c) purifying the modified polypeptide.

[0015] In an eleventh aspect, there is provided a method for producing a VLP of the invention comprising the method of the tenth aspect of the invention, and the subsequent step of allowing a modified polypeptide to assemble into a VLP.

[0016] In a twelfth aspect, the present invention relates to a method for producing a disease and / or patient specific non-adenoviral polypeptide comprising the steps of: (a) providing a cloning vector of the invention; (b) determining the amino acid sequence of a disease- or patient-specific non-adenoviral polypeptide; (c) inserting at least one of the non-adenoviral polypeptides into the nucleic acid encoding the first RGD loop, the second RGD loop, and / or the variable loop of an adenoviral penton base protomer, and / or at a position in the nucleic acid immediately prior to or immediately following the nucleic acid encoding the N-terminus or C-terminus of a modified peptide containing an adenoviral penton base protomer that binds a fiber protein fragment. (d) expressing the modified adenoviral penton base protomer in a host cell, optionally together with a modified polypeptide that contains the adenoviral penton base protomer that binds the fiber protein. (e) purifying VLPs containing the adenovirus penton base protomers that selectively bind fiber protein or the modified polypeptides containing the adenovirus penton base protomers that bind fiber protein fragments.

[0017] In a thirteenth aspect, the present invention relates to a method for producing a VLP of the invention containing a disease and / or patient specific non-adenoviral polypeptide, comprising the steps of: (a) providing a cloning vector of the invention; (b) determining the nucleic acid sequence of a non-adenoviral polypeptide that is specific to the disease or patient; (c) at least one non-adenoviral polypeptide is inserted at a nucleic acid position immediately prior to or immediately following the nucleic acid encoding the N-terminus or C-terminus of an ETG polypeptide containing an adenoviral penton base promoter that binds a fiber protein fragment. (d) expressing in a host cell a modified polypeptide containing an adenoviral penton base protomer that binds a fiber protein, optionally together with the adenoviral penton base protomer. (e1) The modified polypeptide containing the adenovirus penton base protomer that binds the fiber protein fragment is purified and mixed with an adenovirus penton base protomer or an adenovirus penton base protomer of the invention. (e2) When the adenovirus penton base promoter is co-expressed, the VLPs are purified.

[0018] In a fourteenth aspect, the present invention relates to a method for producing a VLP of the invention containing a disease and / or patient specific non-adenoviral polypeptide, comprising the steps of: (a) determining the amino acid sequence of a non-adenoviral polypeptide that is specific to the disease or patient; (b) synthesizing a modified polypeptide of the invention containing an adenoviral penton base protomer that binds a fiber protein fragment and at least one of the non-adenoviral polypeptides. (c) mixing the modified polypeptide with an adenoviral penton base protomer, or mixing a modified adenoviral penton base protomer of the invention with a pentamer of the invention or a VLP of the invention.

[0019] In a fifteenth aspect, the present invention relates to a VLP producible by the method for producing a VLP of the invention.

[0020] In a sixteenth aspect, the present invention relates to a pharmaceutical composition comprising a modified polypeptide of the invention containing the adenoviral penton base protomer a of the invention and / or an adenoviral penton base protomer binding a fiber protein fragment, a nucleic acid encoding one or more of the modified proteins of the invention, an expression vector of the invention or a modified polypeptide containing a VLP of the invention, and which is pharma- ceutically acceptable as a carrier and / or suitable excipient.

[0021] In a seventeenth aspect, the present invention relates to modified polypeptides comprising the adenovirus penton base promoter of the invention, and / or modified polypeptides comprising the adenovirus penton base promoter binding a fiber protein fragment, one or more modified polypeptides of the invention, or VLPs of the invention for the treatment and / or prevention of an infectious disease, an immune disease or cancer. [Brief description of the drawings]

[0022] [Figure 1] 1 shows a synthetic self-assembling multimeric scaffold of the invention, also called a VLP. Five protomers form one penton subunit, and 12 pentons spontaneously self-assemble into a larger superstructure, also called a VLP or ADDomer. [Diagram 2] Side view of a penton. The penton protomer contains two highly heterogeneous RGD loops and one variable loop (V loop) of widely varying length and sequence that is not conserved. In this regard, the inventors have determined that they are similar to antibody CDRs and are suitable for introducing binding sites that allow the resulting VLP to bind any desired target. This makes it suitable for the display of diverse epitopes, and up to 180 epitopes can be displayed per VLP. [Diagram 3]The penton-based protomers contain a region (adhesive patch) that interacts with the adenoviral fiber protein. This adhesive patch can bind with sub-nanomolar affinity to fragments of the adenoviral fiber, also called "STICKER". These fiber fragments are preferably multimerized to increase the binding affinity. The STICKERn tag (n is preferably 2-4) can be fused to the C- and / or N-terminus of the protein to be attached to the VLP, or can be covalently or non-covalently attached to any other cargo. Thus, the STICKERn tag provides the ability to display on the surface of the VLP peptides, proteins, nucleic acids, liposomes, and any other cargo delivered by the VLP. One ADDomer has 12 sites for binding to STICKER-tagged cargo. In one embodiment, the sticky patch on the penton-based protomer is modified to include a coupling residue, preferably Cys, such that the Cys in the sticky patch is modified to include a Cys under non-reducing conditions and the STICKERn tag can form a covalent bond that is cleaved under reducing conditions. [Figure 4] The manufacturing process of ADDomers is shown diagrammatically. ADDomers are produced in high yield, are easy to purify, and are particularly stable. The pACEBac-ADDOMER vector has three regions encoding the first and second RGD loops and a V-loop that can be easily replaced with any peptide chain desired, e.g., a peptide that confers specific binding activity and / or an antigenic epitope. [Diagram 5] Penton-based protomers contain regions known as "strand swapping" that interact with adjacent protomers as they assemble into a dodecahedron. Mutation of the relevant amino acid residues to cysteines results in stabilization of the VLP superstructure through covalent disulfide bond formation, rendering the ADDomer thermostable. A schematic diagram depicting strand-swapping residues mutated to cysteines is shown. [Figure 6]The sequences shown are highly conserved across species. The alignment shows wild-type penton base sequences from different native serotypes. The sequences shown are aligned: subgroup C Ad2, accession no. PO3276; subgroup B Ad3, S41389; subgroup B Ad7, AAR89958; subgroup B Ad11, AAP49205; subgroup F Ad41, AAF14179; subgroup A Ad12, P36716; subgroup D Ad17, NP_049379; subgroup E Ad25, NP_478405; subgroup D Ad37, CAC82544. The V-loop is highlighted in a dark grey box. The RGD loop is highlighted in a light grey box. Those amino acid residues of the penton base protomer that bind to the fibre are highlighted in medium grey. They are all conserved among serotypes. [Figure 7] and [Figure 8] The structures of two preferred cloning vectors of the present invention are shown. The nucleic acid sequences of the cloning vectors are provided in SEQ ID NOs: 61 and 62, respectively. [Figure 9] Plug-and-play expression cassettes and baculovirus transfer plasmids. The ADDomer-encoding gene was designed to insert an "epitope of interest" at three different loci (shown in dark grey) flanked by unique restriction sites. The cassette was inserted at BamHI / HindIII of the pACEBac plasmid. BioBrick insertion of the epitope of interest using either ECoRI / RsrII, BssHII / SalI or SacI / XbaI can be easily performed in the construct pACEBac-ADDomer2.0. [Figure 10] Thermal stability of ADDomer 2.0. Purified ADDomer was stored at different temperatures followed by electron microscopy. Storage at room temperature or at 37°C resulted in full preservation of the particles. Dissociation of the building block (pentamer) was only observed at temperatures above 45°C. Thermal migration assays (TSA) confirmed stability up to 37°C, with a small onset of dissociation above 45°C and total denaturation only by incubation at 60°C. [Figure 11]Methodology for Chikungunya epitope insertion and epitope display. (a) The amino acid sequence incorporated into the display loop of ADDomer2.0 is shown above (SEQ ID NO:78). The major Chikungunya neutralizing epitope (highlighted in dark grey) was inserted. The N-terminus of the peptide contained extra amino acids encoding a TEV cleavage site (highlighted in light grey). (b) Schematic representation of the epitope display potential. ADDomer-TevCHIK was generated and both ends of the peptide were ligated to the ADDomer scaffold ("constrained epitope"). Incubation with TEV protease released the N-terminus of the peptide in a "native-like" configuration (relaxed epitope) and was sufficient to maintain the integrity of the ADDomer VLP (which at this point had been nicked). (c) Cleavage was monitored over time by SDS-PAGE analysis, which showed that the intact ADDomer (~60 kDa) was efficiently cleaved into two bands of 43 and 17 kDa, as expected (left). Despite cleavage, electron microscopy confirmed that the ADDomer scaffold was not destroyed (right). [Figure 12] ELISA for CHIK epitope recognition by mouse sera. Three groups of eight mice were treated at week 2 (w2) and week 4 (w4) with 10 mg of ADDomer scaffold alone (no antigenic epitope in the epitope-presenting loop), ADDomer-TevCHIKADomer-TevCHIKexp ("native-like" CHIK antigenic epitope exposed in the loop-presenting epitope, free N-terminus found in live Chikungunya free glycoprotein, C-terminus covalently attached to the scaffold) or ADDomer-TevCHIKconstr (CHIK antigenic epitope "constrained" in the epitope-presenting loop, N- and C-terminus attached to the ADDomer scaffold). Serum was collected every 2 weeks and tested for CHIK epitope recognition (dilution 1 / 100). ADDomer-TevCHIKexp with exposed native-like epitope efficiently elicits responses. [Figure 13]ADDomer with a large extended epitope-presenting loop. A linear epitope containing 200 amino acids was inserted into the epitope-presenting loop of the ADDomer scaffold and compared to the ADDomer scaffold alone (no insertion). SDS-PAGE gel shows the insertion as reflected by a shift to higher molecular weight (left). Analysis by mass spectrometry confirmed the molecular weight (63,573 Da for the ADDomer scaffold without the insertion; 81,179 Da for the "extended" ADD containing an extra 200 amino acid insertion in the epitope-presenting loop). [Figure 14] Covalent conjugation of the "STICKER" peptide to ADDomers with targeted cysteine ​​mutations. Wild-type ADDomers (wt) and ADDomers with one cysteine ​​mutation (K363C, Q476C or A477C, respectively) were incubated with STICKER peptides (C20 (SEQ ID NO: 77) and C9 (SEQ ID NO: 75), respectively). SDS-PAGE analysis was performed under reducing (+ bMeSH) and non-reducing (- bMeSH) conditions and transferred to a PVDF membrane. ADDomers (dark grey) and STICKER peptides (light grey) were visualized by conjugation of labeled antibodies and avidin binding, respectively, and the conjugation of STICKER to the cysteine ​​mutant ADDomers was demonstrated by specific disulfide bond formation (open circle mark in the lower panel). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, which may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and do not limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0024] The elements of the present invention are described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the embodiments explicitly described. The description should be understood to support and include embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described herein should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0025] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein shall be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0026] definition To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry and recombinant DNA technology are used, as described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual (2nd ed.), Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0027] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step or group of integers or steps but not the exclusion of other integers or steps or groups of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0028] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and are understood as polymeric or oligomeric macromolecules made from nucleotide monomers. A nucleotide monomer consists of a nucleic acid base, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose) and one to three phosphate groups. Typically, polynucleotides are formed via phosphodiester bonds between individual nucleotide monomers. In the context of the present invention referred to, nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids. Nucleic acids can be chemically synthesized, for example, according to the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). An "aptamer" is a nucleic acid that binds with high affinity to a polypeptide. Aptamers are isolated from a large pool of different single-stranded RNA molecules by selection methods such as SELEmir146-a (e.g., Jayasena (1999) Clin. Chem., 45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; U.S. Pat. No. 5,582,981). Aptamers can also be synthesized and selected in mirror-image forms, such as L-ribonucleotides (Nolte et al. (1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). The forms isolated in this way have the advantage that they are not degraded by naturally occurring ribonucleases and therefore have greater stability.

[0029] The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bonded chain of amino acids, regardless of length or post-translational modification. Proteins (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) that can be used in the present invention can be further modified by chemical modification. This means that such chemically modified polypeptides contain other chemical groups other than the 20 naturally occurring amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of a polypeptide can provide advantageous properties compared to the parent polypeptide, such as, for example, one or more of improved stability, increased biological half-life, or increased water solubility.

[0030] The term "penton base protein" or "penton base protomer" as used in the context of the present invention refers to adenoviral proteins that are assembled into so-called "penton proteins". Each penton protein contains five penton base proteins. The penton protein is one of the three proteins that form the adenoviral coat. The other proteins are hexon and fiber. The structure of an assembled adenovirus is shown in the upper left corner of FIG. 1. The penton base protein used in the present invention is derived from an adenovirus specific to any mammalian species. The adenovirus is preferably a human or non-human large apelvirus, preferably chimpanzee (Pan), gorilla (Gorilla) and orangutan (Pongo), more preferably Bonobo (Pan paniscus) and common Chimpanzee (Pan troglodytes). The skilled artisan will understand that the penton base proteins of different adenoviruses will differ in their amino acid sequence, but all such naturally occurring variants are encompassed by the term "penton base protein". Furthermore, the term includes artificial variants that include insertions, deletions and / or mutations of the naturally occurring penton base protein sequence. These mutations are in addition to modifications of the N-terminal domain, V-loop, first RGD, second RGD loop and / or sticky patch region, as described in more detail below. Such artificial variants are included so long as the artificially modified penton base protein assembles into penton subunits, 12 of which are assembled into a VLP. Preferably, the artificial variant has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 92%, more preferably 94%, more preferably 96%, more preferably 98% sequence identity with the naturally occurring penton base protomers outside the N-terminal domain, V-loop, first RGD and second RGD loop, as defined below. Preferred penton base proteins are those depicted in SEQ ID NOs: 1-14.The penton based proteins defined above are the basis for the modified penton based proteins of the present invention, which thus differ in sequence from naturally occurring penton based proteins by amino acid insertions, deletions and mutations, as outlined in more detail below.

[0031] The phrases "modified polypeptides capable of assembling into VLPs" or "assemble into VLPs", used interchangeably in the context of the present invention, mean that the five penton base protomers self-assemble into penton proteins, and then the 12 penton proteins can self-assemble into small spherical particles, i.e., virus-like particles (VLPs). The ability to assemble and maintain penton proteins or preferably VLP structures can be confirmed by methods known in the art and described herein, in particular by electron microscopy (EM). The preferred conditions under which the ability to assemble into VLPs is evaluated are 20°C and physiological buffer conditions. In a further preferred embodiment, the term includes modified polypeptides that not only assemble into VLPs, but also maintain a spherical shape at temperatures above 20°C, preferably above 30°C, preferably above 40°C, more preferably above 45°C, and even more preferably at 50°C. The integrity of the spherical shape can be evaluated by EM, preferably under physiological buffer conditions.

[0032] The term "first RGD loop" as used in the context of the present invention refers to a polypeptide sequence of 10-40 amino acids located at the N-terminus of the "RGD motif" contained in the penton protomer (see Figure 6). This polypeptide sequence is highly divergent between different adenoviruses. It cannot therefore be defined by homology, but can be defined by the sequence located N-terminal to its N-terminus. Its C-terminus within the penton protomer is determined by the RGD motif.

[0033] The term "second RGD loop" as used in the context of the present invention refers to a polypeptide sequence of 10-35 amino acids located at the C-terminus of the "RGD motif" contained in the penton protomer (see Figure 6). This polypeptide sequence is highly divergent among different adenoviruses. Therefore, it cannot be defined by sequence homology. Its N-terminus within the penton protomer is determined by the RGD motif. Its C-terminus within the protomer can be defined by a sequence located at the C-terminal end of that C-terminus that is conserved among different adenoviruses.

[0034] The term "RGD motif" as used in the context of the present invention refers to a three amino acid long polypeptide consisting of arginine, glycine and aspartic acid. This motif was originally identified in fibronectin as mediating binding to integrins. The RGD motif is also present in many other receptors and mediates both cell-substrate and cell-cell interactions. The RGD motif in the penton protomer of the modified polypeptide of the present invention may be intact or may be mutated such that the penton protomer no longer binds to integrins.

[0035] The term "variable loop" as used in the context of the present invention corresponds to the sequence located between the β-sheets b3 and b4 of the adenovirus penton base. Both the length and amino acid composition of this loop are highly variable among serotypes. In Figure 6, the sequence corresponding to the variable loop is highlighted in green.

[0036] The term "N-terminal domain" as used in the context of the present invention refers to a highly conserved region at the N-terminus of the penton base protomer. This part of the protein contains the α1 and α2 helices, β1 and β2 sheets, and the B and C domains (see FIG. 6). It is involved in interactions between penton base protomers and is therefore suitable for the introduction of components such as coupling residues that stabilize interactions between penton base protomers. The term "adenovirus fiber protein binding cleft" as used in the context of the present invention refers to a fold of the penton base protomer that forms an interaction surface with the adenovirus fiber protein. As shown in Figure 6, the binding cleft is formed by several non-contiguous stretches of polypeptide sequence that are conserved among different adenoviruses.

[0037] The term "target-specific binding domain" as used throughout this specification refers to a polypeptide that promotes specific binding to a target. The binding of such a target-specific binding domain is considered to be specific for a given target if it binds with the highest affinity for the respective target and with a limited lower affinity, for example, 10-fold lower, preferably at least 100-fold lower, to a target having a related amino acid sequence.

[0038] The term "target" as used herein refers to a naturally occurring cellular or molecular structure to which a molecule has a certain binding affinity or to which a molecule specifically binds. A target may comprise one or more epitopes. An antigen is a preferred example of a target.

[0039] The term "antigen" as used in the context of the present invention refers to any structure recognized by a molecule of the immune response, such as an antibody, a T cell receptor (TCR), etc. Antigens can be foreign or toxic to the body, or they can be cellular proteins associated with a particular disease. Antigens are recognized by the highly variable antigen receptors of the adaptive immune system (B cell receptors or T cell receptors) and can induce a humoral or cellular immune response. Antigens that induce such a response are also called immunogens. Some of the proteins within the cells, whether they are foreign or cellular, are processed into smaller peptides and presented by the major histocompatibility complex (MHC). When the small peptide fragments are bound by the T cell receptor, a cellular immune response is elicited. The cell surface antigens can be selected from the group of cytokine receptors, integrins, cell adhesion molecules, cell type specific cell surface antigens, tissue specific cell surface antigens, cell surface expressed tumor associated antigens, clusters of differentiation antigens or carbohydrates.

[0040] The term "specific binding" as used in the context of the present invention means binding strongly to a target such as an epitope, where binding to a first target with a dissociation constant (Kd) lower than the dissociation constant of a second target makes it specific compared to binding to other ligands. Targets are recognized by their ligands that bind to the target with a certain affinity, and thus ligand binding to its respective target produces a biological effect. Preferably, the binding is specific and of high affinity, preferably with a Kd of 10-7, 10-8, 10-9, 10-10 M or less. Such affinity is preferably measured at 37°C. Suitable assays include surface plasmon resonance measurements (e.g., Biacore), quartz crystal microbalance measurements (e.g., Attana), and competitive assays.

[0041] The term "antibody" as used in the context of the present invention is a glycoprotein that belongs to the immunoglobulin superfamily, and the terms antibody and immunoglobulin are often used interchangeably. Antibodies refer to protein molecules produced by plasma cells and are used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Antibodies recognize a unique part of a foreign target, its antigen.

[0042] The term "antibody fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by this term include antigen fragment binding (Fab) fragments, Fab fragments, F(ab')2 fragments, heavy chain antibodies, single domain antibodies (sdAbs), single chain fragment variable (scFV), heavy chain fragments, fragment variable (Fv), VH domains, VL domains, single domain antibodies, nanobodies, IgNARs (immunoglobulin novel antigen receptors), di-scFvs, bispecific T-cells (BITEs), dual affinity retargeting (DART) molecules, triplets, bispecific antibodies, single chain bispecific antibodies, alternative scaffold proteins, and fusion proteins thereof.

[0043] The term "bispecific antibody" as used herein refers to a fusion protein or bivalent antibody that can bind to different antigens. Bispecific antibodies are composed of two single protein chains that contain antibody fragments, i.e., variable fragments. Bispecific antibodies contain a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL or VL-VH). By using a short peptide linking the two variable domains, the domains are forced to pair with complementary domains on another chain, thus creating two antigen-binding sites. Bispecific antibodies can target the same (monospecific) or different antigens (bispecific).

[0044] The term "single domain antibody" as used in the context of the present invention refers to an antibody fragment consisting of a single monomeric variable domain of an antibody. Simply, they contain only the monomeric heavy chain variable region of a heavy chain antibody produced by camelids or cartilaginous fish. Due to their different origin, they are also called VHH or VNAR (variable novel antigen receptor) fragments. Alternatively, single domain antibodies can be obtained by monomerizing the variable domains of conventional mouse or human antibodies by using genetic engineering. They exhibit a molecular weight of about 12-15 kDa and are therefore the smallest antibody fragments capable of antigen recognition. Further examples include nanobodies or nanobodies.

[0045] The term "antibody mimic" as used within the context of the present specification refers to a compound that can specifically bind to an antigen in a manner similar to an antibody, but is not structurally related to an antibody. Typically, an antibody mimic is an artificial peptide or protein with a molecular weight of about 3 to 20 kDa that contains one, two or more exposed domains that specifically bind to an antigen. For example, among others, LACI-D1 (lipoprotein-associated coagulation inhibitor); affilins, such as human gamma-B crystallins or human ubiquitin; cystatins; Sac7D from Sulfolobus acidocaldarius; lipocalins and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domains from Fyn; Kunits domains of protease inhibitors; monobodies, such as. 10th type III domain of fibronectin; adnectins: knottins (cysteine ​​knot miniproteins); atrimers, e.g. CTLA4-based binders; affibodies, e.g. three-helix bundles derived from the Z domain of protein A from Staphylococcus aureus; transbodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domains; e.g. trypsin inhibitor-II; affilins; armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimics (aptamers), but artificial antibodies, antibody fragments and fusion proteins composed of these are not considered. General advantages over antibodies are better solubility, tissue penetration, heat and enzymatic stability, and relatively low production costs.

[0046] As used herein, the term "Kd" (usually measured in "moles / L" and sometimes abbreviated as "M") refers to the dissociation equilibrium constant of a particular interaction between a binding moiety (e.g., an antibody or fragment thereof) and a target molecule (e.g., an antigen or epitope thereof). Methods for measuring Kd include, but are not limited to, ELISA and surface plasmon resonance assays. The term "epitope", also known as "antigenic determinant", as used in the context of the present invention, is a portion of a macromolecule that is recognized by the immune system, particularly antibodies, B cells, or T cells. As used herein, an "epitope" is a portion of a macromolecule that can bind to an antibody described herein (e.g., an antibody or an antigen-binding fragment thereof). Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes can be distinguished in that the binding to the latter but not the former is lost in the presence of denaturing solvents.

[0047] As used herein, a "conformational epitope" refers to an epitope of a linear macromolecule (e.g., a polypeptide) that is formed by the three-dimensional structure of the macromolecule. In the context of this application, a "conformational epitope" is a "discontinuous epitope", i.e., a conformational epitope on a macromolecule (e.g., a polypeptide) that is formed from at least two distinct regions in the primary sequence of the macromolecule (e.g., the amino acid sequence of the polypeptide). In other words, an epitope is considered to be a "conformational epitope" in the context of the present invention if it consists of at least two distinct regions in the primary sequence to which the antibody (or antigen-binding fragment thereof) of the present invention binds simultaneously. In this case, these at least two distinct regions are interrupted by one or more regions in the primary sequence to which the antibody (or antigen-binding fragment thereof) of the present invention does not bind. Preferably, such a "conformational epitope" is present on a polypeptide, and two distinct regions in the primary sequence are two distinct amino acid sequences to which the antibody (or antigen-binding fragment thereof) of the present invention binds, where the at least two distinct amino acid sequences are interrupted by another amino acid sequence in the primary sequence to which the antibody (or antigen-binding fragment thereof) of the present invention does not bind. Preferably, the interrupted amino acid sequence is a contiguous amino acid sequence comprising two or more amino acids to which the antibody (or antigen-binding fragment thereof) of the present invention does not bind. The at least two distinct amino acid sequences to which the antibody (or antigen-binding fragment thereof) of the present invention binds are not particularly limited in terms of their length. Such distinct amino acid sequences may consist of only one amino acid and a conformational epitope, as long as the total number of amino acids in the at least two distinct amino acid sequences is large enough to achieve specific binding between the antibody (or antigen-binding fragment thereof) and the antibody.

[0048] The term "adenovirus fiber protein" as used in the context of the present invention refers to an adenovirus protein that is non-covalently bound to the penton protomer and aids in the attachment of the adenovirus to the host cell.

[0049] Throughout this specification, the term "sequence identity" is used in relation to the comparison of polypeptide and polynucleotide sequences. If no reference sequence is specified to which two sequences are compared and the sequence identity percentage is calculated, the sequence identity is calculated based on the longer of the two sequences to be compared, unless otherwise indicated. If a reference sequence is indicated, the sequence identity is determined based on the full length of the reference sequence, as indicated by the SEQ ID, unless otherwise indicated. For example, a polypeptide sequence consisting of 200 amino acids compared to a reference 300 amino acid long polypeptide sequence has a maximum sequence identity percentage of 66.6% (200 / 300), and for a 150 amino acid long sequence, the maximum sequence identity percentage is 50% (150 / 300). If 15 amino acids out of the 150 amino acids differ from each amino acid of the 300 amino acid long reference sequence, the level of sequence identity drops to 45%. The similarity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined through sequence alignment. Such an alignment can be carried out using several known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), available at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html.The preferred parameters used are the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The degree of sequence identity (sequence match) can be calculated, for example, by BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990 J. Mol. Biol. 215:403-410). BLAST polynucleotide searches are performed using the BLASTN program (score = 100, word length = 12). BLAST protein searches are performed using the BLAST protein program (score = 50, word length = 3). To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Sequence match analysis may be complemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-162) or Markov Random Fields. When percentage sequence identity is referred to in this application, these percentages are calculated based on the full length of the longer sequence, unless otherwise indicated. "Hybridization" can also be used as a measure of sequence identity or homology between two nucleic acid sequences. Nucleic acid sequences encoding F, N, or M2-1, or portions of any of these, can be used as hybridization probes according to standard hybridization techniques. Hybridization conditions are known to those of skill in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 6.3.1-6.3.6, 1991. "Moderate hybridization conditions" are equivalent to hybridization in 2X sodium chloride / sodium citrate (SSC) at 30°C, followed by washing in 1X SSC, 0.1% SDS at 50°C. "High stringency conditions" are defined as equivalent to hybridization in 6X sodium chloride / sodium citrate (SSC) at 45°C, followed by washing in 0.2X SSC, 0.1% SDS at 65°C.

[0050] The term "coupling residue" as used in the context of the present invention refers to a natural or unnatural amino acid having a side chain capable of forming a covalent bond. The coupling residue can be inserted into the polypeptide of the present invention. If the coupling residue is a naturally occurring amino acid encoded by DNA, the insertion of the coupling residue only requires modification of the DNA leading to the expression of the polypeptide of the present invention, for example the insertion of a codon encoding such an amino acid or a mutation of an existing codon. Preferred examples of naturally occurring amino acids that are coupling residues in the sense of this term are Asp, Glu, Lys and Cys. Cys is particularly preferred since it forms disulfide bonds with other Cys depending on the redox state of the environment. In particular, the latter allows the formation of a stable interconnection between two separate polypeptides.

[0051] The term "label" as used in the context of the present invention refers to any type of compound suitable for diagnostic purposes. Preferred compounds are selected from fluorescent dyes, radioisotopes and imaging agents. Imaging agents are dyes or other substances that help to show abnormal areas in the body. In one embodiment, the term "label" refers to a compound that includes a chelating agent that forms a complex with a divalent or trivalent metal cation. Preferred radioisotopes / fluorescent emitting isotopes are: 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99 mTc, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y, 90 Y, 149 Pm, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m15 Rh, 119 Sb,128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 169 EU, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au and 199 The dye is selected from the group consisting of alpha emitting isotopes such as Ag, gamma emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, and fluorescent emitting isotopes. Preferred fluorescent dyes are selected from the following dye classes: xanthenes (e.g., fluorescein), acridines (e.g., acridine yellow), oxazines (e.g., oxazine 1), cynins (e.g., Cy7 / Cy3), styryl dyes (e.g., dye-28), coumarines (e.g., Alex Fluor 350), fluorescent proteins (e.g., APC, R-phycoerythrin), nanocrystals (e.g., QuantumDot 705), perylenes (e.g., Lumogen Red F300), Phtalocyanines (e.g., IRDYE™ 700DX), and conjugates and combinations of dyes from these classes. Preferred contrast agents are selected from paramagnetic agents such as Gd, Eu, W, and Mn, preferably complexed with a chelating agent. Other options are carriers such as hypervalent iron (Fe) complexes and particles, compounds containing atoms with high atomic numbers, i.e. iodine for computed tomography (CT), microbubbles and liposomes containing these contrast agents.

[0052] The term "drug" in the context of the present invention should be understood in its broadest sense to refer to any compound that induces a preventive, therapeutic or palliative effect in a patient. Preferably, it is a small molecule, e.g., with a molecular size of less than 500 D.

[0053] A "linker" in the context of the present invention refers to any chemical moiety that flexibly and sterically separates two chemical moieties, such as the modified polypeptide of the first aspect of the present invention of a drug or label. A preferred linker is a moiety that has a length to width ratio of at least 10:1, preferably at least 20:1, more preferably at least 50:1 or more. Preferably, the linker is a linear molecule. The two moieties connected by the linker are preferably covalently or non-covalently bonded, preferably covalently bonded to each end of the linker.

[0054] A "peptide linker" in the context of the present invention refers to an amino acid sequence, i.e., a polypeptide, that sterically separates two moieties in a modified polypeptide of the present invention. Typically, such a linker consists of 1 to 100, preferably 3 to 50, more preferably 5 to 20 amino acids. Thus, such linkers have a minimum length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and a maximum length of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 amino acids or less. Peptide linkers can also provide flexibility between the two linked moieties. Such flexibility is generally increased when the amino acids are small. Thus, the flexible peptide linker has an increased content of small amino acids, in particular glycine and / or alanine, and / or hydrophilic amino acids such as serine, threonine, asparagine and glutamine. Preferably, 20%, 30%, 40%, 50%, 60% or more of the amino acids in the peptide linker are small amino acids.

[0055] The terms "preparation" and "composition" include active compounds, such as VLPs of the invention, with carriers and / or excipients.

[0056] "Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government or approved in the United States Pharmacopeia or any other generally recognized pharmacopoeia for use in animals, especially humans.

[0057] The term "carrier" as used herein refers to a pharmacologically inert substance, such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer solution or vehicle, administered with a therapeutically active ingredient. Such pharmaceutical carriers may be liquid or solid. Liquid carriers include sterile liquids, such as saline solutions in water and oils, including, but not limited to, those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Saline solutions are the preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0058] Suitable pharmaceutical "excipients" include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0059] "Surfactants" include anionic, cationic and nonionic surfactants, such as, but not limited to, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, and polysorbates, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[0060] "Stabilizers" include, but are not limited to, mannitol, sucrose, trehalose, albumin, and protease and / or nuclease antagonists. "Physiological buffer solutions" that may be used in the context of the present invention include, but are not limited to, sodium chloride solutions, demineralized water, as well as phosphate buffers, citrate buffers, Tris buffers (tris(hydroxymethyl)aminomethane), HEPES buffers ([4(2hydroxyethyl)piperazino]ethanesulfonic acid) or MOPS buffers (3morpholino-1propanesulfonic acid), etc. The choice of the respective buffer generally depends on the desired buffer molarity. Phosphate buffers are suitable, for example, for injection and infusion solutions.

[0061] The term "adjuvant" refers to an agent that enhances, stimulates, activates, strengthens or modulates the immune response to an active ingredient of a composition, either at the cellular or humoral level. For example, an immune adjuvant stimulates the immune system's response to an actual antigen, but has no immunological effect by itself. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponin or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and INF-γ), particulate adjuvants (e.g., immune stimulating complexes (ISCOMS), liposomes or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl lipid A or muramyl peptides), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine).

[0062] An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve its intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal receiving the therapeutic agent, and the purpose of the administration. The effective amount in a particular case may be empirically determined by one of ordinary skill in the art according to established methods in the art. EXAMPLES

[0063] The present invention offers, inter alia, the following advantages over the prior art: (i) an easily modified scaffold for insertion / display of antigens and / or target specific binding domains, which can be tailored to the patient's needs and viral surface antigens; (ii) a composition that can be used, e.g., in vaccinations, even under adverse storage conditions, e.g., high heat; (iii) a very dense excipient for delivering one or more antigens; (iv) the use of fiber (STICKER) protein for adding further antigens or other activities in situ.

[0064] Thus, in a first aspect, the present invention relates to a modified polypeptide comprising, consisting essentially of or consisting of an adenovirus penton-based protomer, said penton-based protomer comprising a first RGD loop, a second RGD loop, a variable loop (V loop), an adenovirus fiber protein binding cleft and / or an N-terminal domain, and comprising two or more of the following: (i) at least one target-specific binding domain in the first, second or both the first and second RGD loop and / or V loop; and / or (ii) one or more non-adenoviral polypeptides of the first, second, or both the first and second RGD loop and / or V loop; and / or (iii) a non-adenoviral polypeptide at the N-terminus and / or C-terminus of the penton base protomer; and / or (iv) at least one heterologous binding residue in the first, second or both the first and second RGD loop, V loop and / or N-terminal domain of the penton base protomer, wherein the terminus of the N-terminal domain within the penton base protomer is defined as follows: X1-GRNSIR (SEQ ID NO: 44) Additionally, the C-terminus of the N-terminal domain within the penton base protomer is defined as follows: D-X2-RSRG (SEQ ID NO: 45), where X1 is selected from the group consisting of G and E, preferably E; X2 is selected from the group consisting of D and E, preferably E; and / or (v) a drug, label, and / or polypeptide covalently or non-covalently attached to two or more amino acids of the first, second, or both the first and second RGD loops and / or to two or more amino acids of the V loop of the penton base protomer; and / or (vi) at least one heterologous coupling moiety of the adenovirus fiber protein binding cleft of the penton base protomer, wherein the modified polypeptide is preferably capable of assembling into a VLP.

[0065] In one of the above embodiments, where a residue or group of residues, e.g., a target-specific binding domain, two or more non-adenoviral polypeptides, or at least one heterologous binding residue, is shown to be contained in a particular region of the penton base protein, this residue or group of residues may be inserted, i.e., added, to the respective indicated penton base protein, or may be inserted or added to one or all of the amino acids forming the respective indicated first RGD loop, second RGD loop, and / or V loop, and / or may be deleted without affecting the ability to assemble into a VLP.

[0066] A preferred embodiment of the modified polypeptide of the first aspect of the invention comprises, consists essentially of or consists of an adenoviral penton based protomer, wherein said penton based protomer comprises a first RGD loop, a second RGD loop, a variable loop (V loop), an adenoviral fiber protein binding cleft and / or an N-terminal domain and comprises one or more non-adenoviral polypeptides in the first, second and both the first and second RGD loops and / or V loops; optionally further comprises one or more of the following: (i) comprising at least one target-specific binding domain in the first, second or both the first and second RGD loop and / or V loop; and / or (ii) a non-adenoviral polypeptide at the N-terminus and / or C-terminus of the penton base protomer; and / or (iii) at least one heterologous binding residue in the first, second or both the first and second RGD loop, V loop and / or N-terminal domain of the penton base protomer, wherein the terminus of the N-terminal domain within the penton base protomer is defined as follows: X1-GRNSIR (SEQ ID NO: 44) Additionally, the C-terminus of the N-terminal domain within the penton base protomer is defined as follows: D-X2-RSRG (SEQ ID NO: 45), where X1 is selected from the group consisting of G and E, preferably E; X2 is selected from the group consisting of D and E, preferably E; and / or (iv) a drug, label, and / or polypeptide covalently or non-covalently attached to two or more amino acids of the first, second, or both the first and second RGD loops and / or to two or more amino acids of the V loop of the penton base protomer; and / or (v) at least one heterologous coupling moiety of the adenovirus fiber protein binding cleft of the penton base protomer, wherein the modified polypeptide is preferably capable of assembling into a VLP.

[0067] At least one target-specific binding domain of the first, second or both the first and second RGD loop and / or V-loop provides the penton-based protomer, and thus the assembled VLP, with the ability to specifically bind to a target structure, e.g., a cellular receptor on the cell surface. It is the inventors' surprising discovery that these portions of the penton-based protomer can contain a significant length of target-specific binding domain without disrupting penton or VLP formation. Moreover, the target-specific binding domain contained in these regions is free to interact with and bind to the target. The target-specific binding domain or domains can be inserted at any point in the respective loop, i.e., without removing any of the loop amino acids. Alternatively, all or part of the respective loop amino acids may be replaced with amino acids of the target-specific binding domain. The target-specific binding domain may be flanked at the N-terminus and / or C-terminus by a peptide linker.

[0068] When the penton-based protomer comprises two or more target-specific binding domains, it is preferred that these are contained in different loops of the penton-based protomer, for example, the first and second RGD loops, the first RGd loop and V loop, or the second RGD loop and V loop. When the penton-based protomer comprises two or more target-specific binding domains, it is also preferred that they bind to different targets, for example, a target on a first type of cell and a different target on a second type of cell. Such bi- or multispecificity can be used to bring together cells that do not normally or frequently interact with each other. Examples of such cells are tumor cells and cells of the immune system, in particular cytotoxic T cells.

[0069] In alternative embodiment (ii), which may be combined with two or more of the other alternative embodiments outlined above, the second RGD loop or both the first and second RGD loops and / or V loops comprise a non-adenoviral polypeptide. This embodiment is also based on the surprising observation that a polypeptide inserted into one or more of these regions of the penton-based protomer is recognized by cells of the immune system and is therefore sufficiently exposed to induce an immune response. The term "non-adenoviral" polypeptide refers to a polypeptide that does not have sequence identity with any polypeptide present in an adenovirus, in particular a naturally occurring adenoviral penton-based protomer over a length of at least 5 amino acids. Preferably, the non-adenoviral polypeptide does not have sequence identity with any polypeptide present in an adenovirus over a stretch of at least 10 amino acids, preferably at least 15 amino acids. One or more non-adenoviral polypeptides may be inserted independently at any point in each loop, i.e. without removing any of the loop amino acids. Alternatively, all or part of each loop amino acid may be replaced with amino acids of a target-specific binding domain. The non-adenoviral polypeptides contained in one or more loops may be flanked at the N-terminus and / or C-terminus by a peptide linker. This may be preferred to increase the exposure of the non-adenoviral polypeptides on the surface of the VLP. When at least one non-adenoviral polypeptide is inserted into each loop, each penton-based protomer contains at least three identical or different non-adenoviral polypeptides on its surface. Once assembled into a VLP, at least 180 non-adenoviral polypeptides can be displayed on the surface of the VLP of the present invention.

[0070] Surprisingly, the inventors have discovered that it may be possible to introduce longer amino acid sequences of 50 amino acids or more, 100 amino acids or more, 150 amino acids or more, 200 amino acids or more, 250 amino acids or more or 300 amino acids or more in the first, second or both the first and second RGD loop, and / or in the V loop, without disrupting the ability of the penton protein to subsequently assemble into a VLP. Thus, in embodiments (i) and / or (ii), amino acid sequences of the above lengths may be inserted (with or without deletion of some amino acids with the respective loops).

[0071] When the alternative embodiments depicted in (i) and (ii) are combined, it is further preferred that the non-adenoviral protein is inserted into a different loop than the target-specific binding domain. The inventors have observed that polypeptides located at either the N-terminus and / or C-terminus of the penton substrate protomer do not interfere with penton and subsequent VLP assembly and are surface-exposed in the assembled VLP. Thus, in a further alternative embodiment (iii), a non-adenoviral polypeptide may be linked to the N-terminus and / or C-terminus of the penton-based protomer, with or without an intervening peptide linker. Thus, in combination with the first and / or second embodiment, the penton-based protomer may comprise a non-adenoviral polypeptide in one or more loops, preferably in all three loops, as well as at the N-terminus, C-terminus or N- and C-terminus. This alternative embodiment is preferably combined with at least one of the other alternative embodiments (i), (iii), (iv), (v) and / or (vi).

[0072] The inventor's observations regarding the possibility of inserting heterologous peptide sequences into the V-loop, the first RGD-loop and / or the second RGD-loop (with or without concomitant deletion of all or part of the indicated loops, respectively) lead to yet another embodiment (iv), which can be combined with one or more of the alternative embodiments described above. In this embodiment, at least one heterologous binding residue is introduced into the first, the second or both the first and the second RGD-loop and / or the V-loop. The insertion of one or more binding residues allows further molecules to be covalently attached to the loops. For example, it is envisaged that a VLP is first assembled from a modified polypeptide of the first aspect, comprising one or more binding residues in one or more loops, and subsequently a polypeptide comprising a binding residue is covalently attached to the VLP. Using this strategy, it is possible to "decorate" the surface of the VLP with a polypeptide. Such a VLP can be used to induce a humoral and / or cellular immune response against such a polypeptide.

[0073] Furthermore, the inventors have identified a region in the penton-based protomer called the "N-terminal domain of the penton-based protomer". This domain is involved in the interaction of the penton-based protomer in the penton and the interaction between the pentons forming the VLP. Insertion of binding residues in this region allows the formation of covalent bonds between two or more penton-based protomers in the same or separate pentons. The formation of such covalent bonds stabilizes the penton and the assembled VLP. The N-terminal domain is highly conserved among different adenovirus species. It is therefore possible to further delineate the N-terminus and C-terminus of this domain in the penton-based protomer. Thus, it is preferred that one or more binding residues are included in the N-terminal domain. The binding residues may replace existing amino acids or may be inserted in addition to the amino acids forming the N-terminal domain. It is preferred that one or more binding residues replace residues in the N-terminal domain. The N-terminus of the N-terminal domain in the penton-based protomer is preferably defined as follows: X1-GRNSIR (SEQ ID NO: 44) The C-terminus of the N-terminal domain within the penton base protomer is preferably defined as follows: D-X2-RSRG (SEQ ID NO: 45), where X1 is selected from the group consisting of G and E, preferably E; and X2 is selected from the group consisting of D and E.

[0074] Thus, in this alternative embodiment (iv), one or more binding residues are included within the amino acid sequence of the penton base protomer contained in the modified polypeptide of the invention defined by the N- and C-terminal regions described above. The skilled artisan will appreciate that in this embodiment, it is also possible to substitute one or more amino acid residues in SEQ ID NO: 44 or 45. The binding residue may be located anywhere within the N-terminal domain, as long as it does not interfere with the assembly of the penton or VLP.

[0075] A preferred protomer amino acid sequence that can be modified according to options (i) to (vi) of the first embodiment is SEQ ID NO: 64 (encoding nucleotide sequence is shown in SEQ ID NO: 63). The insertion of at least one target-specific binding domain according to embodiment (i), and / or the insertion of one or more non-adenoviral peptides according to embodiment (ii), and / or the insertion of one or more heterologous binding residues according to embodiment (iv), and / or the covalent or non-covalent attachment of a drug or polypeptide to one or more amino acids of the first, second or both the first and second RGD loops and / or V-loops according to embodiment (v) occurs in the first RGD loop between amino acids 312 to 339 of SEQ ID NO: 64 and / or the second RGD loop between amino acids 343 to 367 of SEQ ID NO: 64 and / or the V-loop between amino acids 150 to 178 of SEQ ID NO: 64. Such insertions can delete all or part of the indicated amino acids belonging to the first and second RGD loops and V-loops, respectively.

[0076] Preferably, there should be a mutation of the linking residues PAIR, preferably to cysteine, to allow disulfide bond formation. The resulting stabilized VLPs contain up to 120 disulfide bonds and are ultrastable for at least several months at 37° C., and sometimes even at 42° C. In a particularly preferred embodiment, the linking residues are located at amino acid positions 51 and 54 relative to SEQ ID NO: 64, i.e., the penton base protomer amino acid based on human Ad B3, or analogous positions in another adenoviral penton base protomer, or amino acid positions 54 and 113 relative to SEQ ID NO: 64, or analogous positions in another adenoviral penton base protomer.

[0077] It has further been discovered that the binding residue at amino acid position 53 (relative to SEQ ID NO:1) can form a covalent bond with a binding residue at amino acid position 543 (relative to SEQ ID NO:64) or at a similar position in another adenoviral penton base protomer. The latter residue is outside the N-terminal domain. Thus, if a binding residue is inserted at position 53, it is preferred that a second binding residue is located at amino acid 541 relative to SEQ ID NO:64 or at a similar position in another adenoviral penton base protomer. With reference to FIG. 6 and by further including a penton base protein in the alignment, one skilled in the art can readily determine the residues in the respective penton base protomers that occupy the analogous amino acid positions, 51, 53, 54, 114 and 541 of SEQ ID NO:64.

[0078] In this embodiment of the modified polypeptide of the invention, it is preferred that the penton base protomer comprises the following sequence: PT-X1-X c -RNX c -IR (sequence number: 50). PT-X1-GRX c -SIR (SEQ ID NO: 51) and TQTINX 60 -X c -X 61 (SEQ ID NO: 52) or PT-X1-GRNX c -IR (SEQ ID NO: 53) and TCPX c -VX 62 -KALG (SEQ ID NO: 54) where X1 is selected from the group consisting of G and E, preferably E X c is in each case a binding residue, preferably C; D, E and K, most preferably C; X 60 is selected from the group consisting of F, I and L, preferably F and L, most preferably F; X 61 is selected from the group consisting of D and E, preferably E; and X 62 is selected from the group consisting of H and Y, preferably Y.

[0079] In particular, preferred stabilized penton base protomers comprise or consist of the amino acid sequences according to SEQ ID NOs: 65 to 67. Furthermore, these amino acid sequences preferably comprise one or more modifications according to alternative embodiment (i), (ii), (iii), (iv) or (v) or (vi) of the first aspect of the invention above, insofar as this alternative embodiment does not concern the N-terminal domain. It is preferred that the insertion of at least one target-specific binding domain according to embodiment (i), and / or the insertion of one or more non-adenoviral peptides according to embodiment (ii), and / or the insertion of at least one heterologous binding residue according to embodiment (iv), and / or the covalent or non-covalent attachment of a drug or polypeptide to one or more amino acids of the first, second and both the first and second RGD loops and to one or more amino acids of the V loop according to embodiment (v) occurs between amino acids 312 to 339 of SEQ ID NO: 65-67 and / or that the insertion into the second RGD loop occurs between amino acids 150 to 178 of SEQ ID NO: 65-67 and / or that the insertion into the V loop occurs between amino acids 65 to 67 of SEQ ID NO: 65-67. Such insertions may delete all or part of the indicated amino acids belonging to the first and second TGD loops and the V loop, respectively. Thermostabilization of pentons and VLPs formed by the engineered proteins of the invention is also desirable in conjunction with any of the other alternative embodiments of the engineered proteins of the invention. Thus, the alternative embodiment mentioned in (iv) above in relation to the N-terminal domain is preferably combined with one or more of the alternative embodiments (i), (ii), (iii), (v) or (vi), insofar as this alternative (v), (vi) does not relate to the N-terminal domain. The alternative embodiments mentioned in (iv) and (vi) are present in the engineered penton base protomers and are preferably combined with one or more of (i), (ii), (iii), (iv), insofar as this alternative embodiment, i.e. (v), does not relate to the N-terminal domain.

[0080] In a further alternative embodiment (v) of the first aspect of the invention, which may be combined with one or more of the other alternative embodiments of the first aspect of the invention, the drug, label and / or polypeptide is covalently or non-covalently attached to one or more amino acids of the first, second and both the first and second RGD loops and / or the V loop of the penton-based protomer. One or more amino acids and / or one or more amino acids of the V loop of the penton-based protomer may be attached. Again, this embodiment is based on the observation that moiety attachment to these regions does not interfere with penton and VLP assembly, but decorates the VJP with these moieties. In a preferred embodiment, the drug or label is attached to the penton-based protomer via a linker, e.g. a peptide linker, cleavable under physiological conditions by a protease, whereby the protease releases the drug from the VLP at the site of action. In this preferred embodiment, the linker, preferably a peptide linker, comprises an endopeptidase cleavage site. In a preferred embodiment, a fragment of an adenovirus fiber is used to non-covalently attach a moiety, such as a polypeptide, a drug, or a label, to the penton based protomer, assembled pentons, and / or assembled VLPs. This interaction is mediated via the adenovirus fiber protein binding cleft of the penton based protomer present in the modified polypeptide of the first aspect of the invention. In a preferred embodiment described below, the fiber fragment comprises a heterologous binding residue for covalent attachment of the fiber fragment to the penton based protomer. Since a binding residue requires a counterpart, i.e. a residue capable of forming a covalent bond, at least one heterologous binding residue contained in the adenovirus fiber protein binding cleft of the penton based protomer is a further preferred alternative embodiment (vi) of the modified protein of the first aspect of the invention. The binding cleft and the binding residue of the fiber protein fragment are positioned to allow for the formation of a covalent bond upon binding of the fiber protein fragment to the cleft of the penton based protomer.

[0081] Each penton based protomer interacts with one adenovirus fiber protein through a highly conserved region referred to herein as the "adenovirus fiber protein binding cleft of the penton based protomer". This interaction allows for the indirect attachment of additional moieties, preferably polypeptides, drugs or labels, to the penton based protomer and, upon assembly of 60 penton based protomers of the invention, displays 60 or more moieties on the surface of the assembled VLP. Thus, in a second aspect, the invention provides a method for the preparation of a penton based protomer comprising the steps of: (i) associated with a non-adenoviral peptide; and / or (ii) is covalently or non-covalently attached to a drug or label; At least one adenoviral fiber protein N-terminal fragment that specifically binds to the adenoviral fiber protein binding cleft of the penton base protomer contained in the modified polypeptide of the second aspect of the invention is also referred to as a "STICKER" throughout this specification.

[0082] Surprisingly, a relatively small N-terminal fragment of the adenoviral fiber protein was sufficient to specifically bind to the penton-based protomer. The smaller the fiber fragment, the larger the portion that can bind to the penton-based protomer. Furthermore, shortening the length of the adenoviral fiber fragment reduces the possibility of eliciting a new immune response against the adenoviral fiber and / or of the fiber bound to the VLP being removed by pre-existing anti-fiber antibodies. Thus, it is preferred that the fiber fragment has a length of 50 or less contiguous amino acids of the N-terminal fiber sequence. More preferably, the fragment is 40 or less, 35 or less, 30 or less, 25 or less, or 20 or less amino acids in length. The minimal fiber amino acid sequence required for specific binding to the binding cleft of the penton-based protomer is FNPVYPY. This minimal sequence is preferably flanked by other adenoviral fibers, preferably Ad3 amino acid sequences on both sides. This small fragment can be used to increase the versatility and / or the number of exposed epitopes on the VLP surface. Alternatively, the addition of the STICKER tag to any protein or epitope sequence allows for their attachment to the surface of the VLP, either by in vitro incubation of STICKER containing proteins with VLPs or by co-expression of both components in the baculovirus system.

[0083] Preferably the modified polypeptide does not contain any further fibre amino acid sequence adjacent to STICKER, More preferably the polypeptide of the second aspect of the invention does not contain other adenoviral proteins or polypeptides other than STICKER.

[0084] Surprisingly, it has been found that STICKER can be attached to the N- and / or C-terminus without interfering with the binding to the penton base protomer. Preferably, STICKER is attached to the N-terminus of the non-adenoviral polypeptide. This polypeptide can be any polypeptide that is desired to be attached to the surface of the VLP of the present invention. The size of the polypeptide attached to STICKER is not particularly limited. It can be any size that still allows specific binding to the fiber protein binding cleft of the penton base protomer. The modified polypeptide can further comprise a peptide linker between the non-adenoviral polypeptide and STICKER. This may be required if the non-adenoviral polypeptide has a size that prevents 60 of the polypeptide from binding to the assembled VLP via STICKER. A peptide linker may also be advantageous in situations where the N-terminus and / or C-terminus to which STICKER is attached is embedded within the polypeptide.

[0085] A large proportion of the human population has been exposed to human Ad5 and has memory B cells that mount an immune response against human Ad5. Thus, when a human Ad5-based protomer and / or fiber is comprised in the modified protein of the first and second aspects of the invention, the resulting VLPs are more likely to be cleared from the circulation by pre-existing immunity. Thus, in a preferred embodiment, the adenoviral proteins comprised in the modified polypeptide according to the first and / or second aspects of the invention are based on adenoviral penton and fiber proteins from human or non-human large apeithoviruses, preferably chimpanzee (Pan) adenovirus, gorilla (Gorilla) adenovirus and orangutan (Pongo) adenovirus, more preferably from Bonobo (Pan paniscus) and common Chimpanzee (Pan troglodytes).

[0086] Preferably, the modified polypeptides comprising an adenoviral penton base protomer and the modified polypeptides comprising at least one adenoviral penton base protomer-binding fiber protein fragment are based on the penton and fiber proteins, respectively, of an adenovirus selected from the group comprising hAd3, hAd4, hAd5, hAd7, hAd11, hAd26, hAd35 and hAd49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and as described in WO 2005 / 071093 and WO 2010 / 086189.

[0087] The modified polypeptides of the first aspect of the invention are preferably based on the wild-type penton base protomers of SEQ ID NOs: 1-14, i.e. SEQ ID NOs: 1-14 reflect the sequence of the protein before modification, in accordance with substitutions (i)-(vi) above. A person skilled in the art will appreciate that inserting a target-specific binding domain into the V-loop, the first and / or the second RGD loop will modify the sequence of that portion of SEQ ID NOs: 1-14. Similarly, replacing an amino acid with a binding residue will also modify the amino acid sequence.

[0088] The modifications according to (i) and (ii) above require modification of one or both of the RGD loop and / or V loop. In a preferred embodiment of the modified polypeptide of the present invention, the region to be modified is defined by consensus sequences common to most adenoviruses. These consensus sequences are therefore based on the alignment of several preferred penton base protomer amino acid sequences from adenovirus species, and are suitable for determining the N-terminus and C-terminus, respectively, of the portion of the penton base protein to be modified according to the embodiment, as described in either (i) or (ii). Preferably, the N-terminus of the first RGD loop in the penton base protomer is defined as follows: X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:15) where X3 is selected from the group consisting of D, E and N, preferably D; X4 is selected from the group consisting of V, L and I, preferably V; X5 is any amino acid, preferably selected from the group consisting of A, D, E, K, S and T, more preferably T; X6 is any amino acid, preferably selected from the group consisting of A, D, E and K, more preferably A; X7 is selected from the group consisting of F, Y and W, preferably Y; X8 is selected from the group consisting of A, D, E, N and Q, preferably E or Q, more preferably E; X9 is preferably any amino acid selected from the group consisting of A, D, E, N and K, more preferably E; X 10 is selected from the group consisting of S or T, preferably S; and X 11 is any amino acid and constitutes the N-terminal amino acid of the first RGD loop. and / or The following sequence defines the C-terminus of the first RGD loop and simultaneously defines the N-terminus of the second RGD loop within the penton base protomer: X 12 -X 13 -X 14 -X 15 -X 16 (SEQ ID NO:16) where X 12 is any amino acid and constitutes the C-terminal amino acid of the first RGD loop. X 13 is R; X 14 is G; X 15 is D; and X 16 is any amino acid of the second RGD loop and the N-terminal amino acid; and / or The following sequence constitutes the C-terminus of the second RGD loop in the penton base protomer. X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X 24 (Sequence number 17). where X 17 is any amino acid and constitutes the C-terminal amino acid of the second RGD loop. X 18 is selected from the group consisting of I, L and V, preferably I; X 19 is selected from the group consisting of D, E, K, N, Q and V, preferably Q or K, more preferably Q; X 20 is selected from the group consisting of C, G and P, preferably P; X 21 is selected from the group consisting of I, L and V, preferably L or V, more preferably L; X 22is selected from the group consisting of D, E, S and T, preferably E or T, more preferably E; X 23 is selected from the group consisting of D, E, K, S and T, preferably E, K or T, more preferably K; and X 24 is selected from the group consisting of D and E, preferably D. Similarly, the following sequence defines the N-terminus of the V loop of the penton base protomer: X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 (Sequence number 18). where X 25 is selected from the group consisting of F, Y and W, preferably F; X 26 is selected from the group consisting of H, K and R, preferably K; X 27 is selected from the group consisting of A, V, I and L, preferably A; X 28 is selected from the group consisting of H, K and R, preferably R; X 29 is selected from the group consisting of A, V, I and L, preferably V; X 30 is selected from the group consisting of A, V, I, L and M, preferably M; X 31 is selected from the group consisting of A, V, I and L, preferably V; and X 32 is any amino acid and constitutes the N-terminal amino acid of the V-loop. and / or The following sequence defines the C-terminus of the V-loop: X 33 -X 34 -X 35 -X 36 -X 37 -X38 -X 39 (SEQ ID NO:19) where X 33 is any amino acid and constitutes the C-terminal amino acid of the V-loop. X 34 is selected from the group consisting of F, Y and W, preferably Y; X 35 is selected from the group consisting of D, E, S and T, preferably E or T, more preferably E; X 36 is selected from the group consisting of F, Y and W, preferably W; X 37 is selected from the group consisting of A, F, V, Y and W, preferably F or V, more preferably F; X 38 is selected from the group consisting of D, E, S and T, preferably D or E, more preferably E; and X 39 is selected from the group consisting of F, Y and W, preferably F; and / or one or more of the following non-contiguous peptides within the penton base protomer form the adenovirus fiber protein binding cleft (amino acids in bold directly interact with the fiber): X 32 is the N-terminal amino acid, and X 33 is the C-terminal amino acid of the V loop. One or more or all of the amino acids of the V loop may be replaced by an inserted target-specific binding domain and / or a non-adenoviral polypeptide.

[0089] As noted above, the portion of the penton based protomer that specifically binds to STICKER is a non-contiguous epitope. Thus, preferably, one or more of the following non-contiguous peptides within the penton based protomer form the adenovirus fiber protein binding cleft (amino acids in bold directly interact with the fiber): MTIDLMNNAIX 40 -X 41 -X 42 -YLX 43 -X 44 -GRQX 45 - GVLES (SEQ ID NO: 20). WDPX 46 -TX 47 -X 48 -PG (SEQ ID NO: 46); X 49 -VX 50 -X 51 -YX 52 -X 53 (sequence number); X 54 -X 55 -RSY (SEQ ID NO: 48); and / or LTX 56 -VFNRFPX 57 (SEQ ID NO:49) where X 40 is selected from the group consisting of V, I and L; X 41 is selected from the group consisting of E and D; X 42 is selected from the group consisting of H, N and Q, preferably H and N; X 43 is selected from the group consisting of K, E, R, Q and A; X 44 is selected from the group consisting of V, L and I, preferably V and I; X 45 is selected from the group consisting of H, N and Q, preferably H and N; X 46 is selected from the group consisting of V, I, L, E or D, preferably V and E; X 47 is selected from the group consisting of V, L and I, preferably V and I; X 48 is selected from the group consisting of M, T and S, preferably M and T; X 49is selected from the group consisting of D, E, N and Q, preferably D and N; X 50 is any amino acid preferably selected from the group consisting of A, D, P, K and T; X 51 is selected from the group consisting of A, D, E, K and R, preferably A, E and K; X 52 is selected from the group consisting of D, E, L, I, Q and N, preferably E, L and Q; X 53 is selected from the group consisting of A, D, E, K, N, Q and R, preferably A, E, N and K; X 54 is selected from the group consisting of K, R, S and T, preferably K, S and T; X 55 is selected from the group consisting of A, D, E, G, K, N, Q, R, S and T, preferably D, G, K, N and S. X 56 is selected from the group consisting of H, K and R, preferably H and R; and X 57 is selected from the group consisting of D and E.

[0090] In each preferred embodiment of the modified polypeptide of the present invention, X 10 is independently selected from the group consisting of DVTAYEES (SEQ ID NO: 21), DVDAYENS (SEQ ID NO: 22), DVAEYEKS (SEQ ID NO: 23), DVEAYEKS (SEQ ID NO: 24), DVDAYEKS (SEQ ID NO: 25), DVSKYEAS (SEQ ID NO: 26), NVKAYEDS (SEQ ID NO: 27), DVKKYENS (SEQ ID NO: 28), DVDAYQAS (SEQ ID NO: 29) and DVDAYQAS (SEQ ID NO: 30); 10 ~X 24is independently selected from the group consisting of DVTAYEES (SEQ ID NO: 21), DVDAYENS (SEQ ID NO: 22), DVAEYEKS (SEQ ID NO: 22), DVEAYEKS (SEQ ID NO: 24), DVDAYEKS (SEQ ID NO: 25), DVSKYEAS (SEQ ID NO: 26), NVKAYEDS (SEQ ID NO: 27), DVKKYENS (SEQ ID NO: 28), DVDAYQAS (SEQ ID NO: 29), and DVDAYQAS (SEQ ID NO: 30); 25 ~X 31 is independently selected from the group consisting of FKARVMV (SEQ ID NO: 37), FRAKLMV (SEQ ID NO: 38) and FRAKVMV (SEQ ID NO: 39); 33 ~X 39 is uniquely selected from the group including YEWFEF (SEQ ID NO: 40), YEWVEF (SEQ ID NO: 41), and YEWAEF (SEQ ID NO: 42). Surprisingly, it has been found that large heterologous polypeptides can be inserted into and / or substituted for the first and / or second RGD loop without disrupting the assembly of the penton and subsequent VLP of the invention.

[0091] In a preferred embodiment of the modified polypeptide of the invention, each of the target-specific binding domains of the first RGD loop has, independently of one another, a length of 5 to 300 amino acids, preferably 6 to 200 amino acids, the target-specific binding domain of the second RGD loop has a length of 5 to 300 amino acids, preferably 10 to 200 amino acids, and / or the target-specific binding domain in the V loop has a length of 5 to 300 amino acids, preferably 10 to 200 amino acids.

[0092] In one alternative, the target that is bound by the target-specific binding domain is a moiety present on the surface of a cell or in the extracellular matrix. When the VLP is targeted to a specific cell type to deliver its payload, such as a drug or label, the specificity of the target-specific binding domain is selected. In another preferred embodiment of the modified polypeptide of the present invention, at least one target-specific binding domain can specifically bind to an immunogenic peptide, a pathogen-neutralizing peptide, a viral peptide, a bacterial peptide, an immunomodulatory peptide, a cancer peptide, to the cell surface, preferably to a cell receptor, a low molecular weight tag, preferably to biotin or chitin. This provides an alternative and rapid way to bind various peptides to the surface of the VLP. In a preferred embodiment of the modified polypeptide of the first or second aspect of the invention, the non-adenoviral polypeptide or the inserted or attached polypeptide is selected from the group consisting of immunogenic peptides, pathogen neutralizing peptides, viral peptides, bacterial peptides, immunomodulatory peptides and cancer peptides. The viral peptides Dengue HAKKQDVVVLGSQEGAM (SEQ ID NO: 55), C Chikungunya STKDNFNVYKATRPYLAH (SEQ ID NO: 56) and Z Zika STKDNFNVYKATRPLAH (SEQ ID NO: 57) are particularly preferred. Examples of modified polypeptides according to the second aspect of the invention comprising STICKER and chikungunya peptides are AKRARLSTSFNPVPYEDESSTKDNFNVYKATRPYLAH (SEQ ID NO: 58), AKRARLSTSFNPVPYEDECSSTKDNFNVYKATRPYLAH (SEQ ID NO: 59) and AKRARLSTCFNPVPYEDESSTKDNFNVYKATRPYLAH (SEQ ID NO: 60). The latter two examples include a binding residue, i.e. a Cys, for forming a covalent bond with the corresponding binding residue in the binding cleft of the fiber of the penton base protomer.

[0093] Preferred examples of target-specific binding domains are antibodies, single chain antibodies, antibody fragments, nanobodies, light or heavy chains, variable light or variable heavy chains, diabodies or antibody mimetics. Preferred antibody fragments include fragment antigen binding (Fab) fragments, Fab' fragments, F(ab')2 fragments, heavy chain antibodies, single domain antibodies (sdAb), single chain fragment variable (scFv) fragment variable (Fv), VH domains, VL domains, single domain antibodies, nanobodies, IgNARs (immunoglobulin novel antigen receptors), di-scFv, bispecific T cell engagers (BITEs), dual specific antibody (DART) molecules, triplet, bispecific antibodies, single chain bispecific antibodies, alternative scaffold proteins, and fusion proteins thereof.

[0094] When non-adenoviral peptides or peptides are inserted into the first RGD loop, they preferably have a length of 5 to 60 amino acids, preferably 6 to 45 amino acids. When non-adenoviral peptides or peptides are inserted into the second RGD loop, they can have a length of 5 to 50 amino acids, preferably 10 to 36 amino acids. When non-adenoviral peptides or peptides are inserted into the second V loop, they can have a length of 5 to 30 amino acids, preferably 10 to 21 amino acids.

[0095] In a preferred embodiment of the modified polypeptide of the first or second aspect, the non-adenoviral polypeptide or polypeptides comprises a protease cleavage site, preferably a sequence-specific endopeptidase cleavage site, more preferably a TEV cleavage site. A preferred example of such a TEV cleavage site is ENLYFQG (SEQ ID NO: 60). Such a cleavage site allows cleavage of the polypeptide of the first aspect of the invention once assembled into a penton or VLP. Some antigens require exposure of free N- and / or C-termini to elicit an immune response. Thus, when penton proteins or VLPs are assembled, their treatment with a protease will expose the N- and / or C-terminal sequences of such antigens if the cleavage site is located at the N- and / or C-terminus of the non-adenoid polypeptide. Surprisingly, the inventors have found that such cleavage does not destroy the assembled penton or VLP. This is extremely useful in situations where a strong antigen-specific immune response requires exposure of free N- and / or C-termini of the antigen. Alternatively, a cleavage locus may be included in the modified polypeptides of the first and / or second aspect of the invention to facilitate purification of the modified polypeptide. For example, it may be placed at the N- or C-terminus of each modified polypeptide to separate the penton or fibre comprising part of the modified polypeptide from an affinity tag, for example an affinity tag such as biotin, chitin binding protein, Myc-taq etc. Such an affinity tag allows immobilisation of the modified polypeptide on a suitable affinity matrix and release of the purified modified polypeptide from the matrix.

[0096] In a preferred embodiment of the modified polypeptide of the first or second aspect of the invention the linking residue is selected from the group comprising Lys, Cys, Asp and Glu, preferably Cys.

[0097] In a preferred embodiment of the modified polypeptide of the first or second aspect of the invention, the drug is selected from the group of chemotherapeutic drugs, anti-pathogenic drugs, immunomodulatory drugs and anti-inflammatory drugs.

[0098] In a preferred embodiment of the modified polypeptide of the second aspect of the invention, the fiber protein fragment comprises or consists essentially of: X 58 -FNPVYPYX 59 (SEQ ID NO: 43) where X 58 is selected from the group consisting of S, D and T, preferably S or D, more preferably S; and X 59 is selected from the group consisting of E, D and G, preferably E or D, more preferably E. Preferably, the fiber protein fragment has a length of between 9 and 20 consecutive amino acids of the fiber.

[0099] It is further preferred that the modified polypeptide of the second aspect of the invention comprises 2, 3, 4, 5, 6, 7 or 8 repeats of a fragment of the fiber protein. Multimerization increases binding affinity. It has been observed by the inventors that 2 or 3, preferably consecutive repeats, are suitable to mediate binding to the fiber binding cleft on the penton base protomer with sub-nanomolar affinity. Preferably, the multimers are arranged in a head to tail orientation.

[0100] As indicated above, according to alternative embodiment (vi), it is preferred that one or more binding residues are included in the binding cleft of the penton base protomer to facilitate the formation of a covalent bond between these one or more binding residues in the penton base protomer and the binding residues included in the modified polypeptide of the second aspect of the invention. In a preferred embodiment of the modified polypeptide of the second aspect of the invention, at least one binding residue is inserted and / or positioned at the N- and / or C-terminus of the fiber protein fragment, preferably inserted and / or located at the N- and / or C-terminus of SEQ ID NO: 43, or inserted and / or attached to the N- and / or C-terminus of the fiber protein fragment. As stated above, the binding residue must form a covalent bond with the corresponding binding residue. When the two polypeptides interact, it is preferred that the binding residue of one polypeptide is sterically closer to the binding residue of the other polypeptide.

[0101] In a preferred embodiment of alternative embodiment (vi), the binding residues are present in the penton base protomer comprised in the modified polypeptide of the first aspect of the invention at amino acid positions 476 and / or 477 (with reference to the amino acid sequence of SEQ ID NO: 64) or at analogous amino acid positions in another adenoviral penton base protomer. Analogous positions in other adenoviral penton base protomers can be determined by aligning the sequence according to SEQ ID NO: 64 with other adenoviral penton base protomer sequences, for example with standard alignment tools such as CLUSTAL. The skilled artisan can readily determine an amino acid occupying an analogous position to amino acid 476 or 477 in another adenoviral penton protein. The modified protein according to the first aspect and alternative embodiment (iv) of the invention comprises the following sequence: KSFX 64 NX c 1X c2 AVY (SEQ ID NO: 68) where X 64is selected from the group consisting of Y and F, preferably Y; X c1 is selected from the group consisting of D, E and a linking residue, preferably Cys; X c2 is selected from the group consisting of L, Q and a linking residue, preferably Cys; Also, here X c1 and X c2 At least one, and preferably both, of these are binding residues, preferably Cys. Where the binding residue in the penton base protomer is located at amino acid positions 476 and / or 477, or a similar amino acid position in a penton base protomer of another adenovirus, it is preferred that the corresponding binding residue in the modified polypeptide of the second aspect of the invention comprises a binding residue at the C-terminus of the STICKER portion of the polypeptide. The binding residue is preferably located within STICKER, as shown in the sequence below. X 58 -FNPVYPYX 59 -(X 63 )n-Xc (SEQ ID NO: 69) where X 58 is selected from the group consisting of S, D and T, preferably S or D, more preferably S; and X 59 is selected from the group consisting of E, D and G, preferably E or D, more preferably E; X 63 is independently in each instance any amino acid, preferably an amino acid that naturally occurs in fiber protein at this or these positions; Xc is a linking residue, preferably C, D, E, and K, most preferably C; and n is an integer of 0 to 10, that is, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1 to 5, and more preferably 2. Thus, the modified protein of the second aspect of the invention in a preferred form comprises the STICKER polypeptide of SEQ ID NO: 69. Particularly preferred STICKER polypeptides which may be included in the modified protein of the second aspect of the invention are as follows: AKRARLSTX 58 -FNPVYPYX 59 -DE-Xc (SEQ ID NO: 76) where X 58 is selected from the group consisting of S, D and T, preferably S or D, more preferably S; X 59 is selected from the group consisting of E, D and G, preferably E or D, more preferably E; and Xc is a linking residue, preferably C, D, E and K, most preferably C.

[0102] In a particularly preferred embodiment, the STICKER polypeptide comprises a binding residue Cys at position 20 and consists of the following amino acid sequence: AKRARLSTSFNPVYPYEDEC (SEQ ID NO: 77)

[0103] In another preferred embodiment of alternative embodiment (vi), the binding residue is positioned in the penton base protomer comprised in the modified polypeptide of the first aspect of the invention at Lys376 of the penton base protomer according to SEQ ID NO: 64 or at a similar position in a penton base protomer of another adenovirus. The modified protein of the first aspect preferably comprises the following sequence: Xc-X 65 -RSYN (SEQ ID NO: 73) where Xc is a linking residue, preferably C, D, E, and K, most preferably C; and X 65is any amino acid, preferably selected from the group consisting of D, E, G, K, N or S, more preferably S or N. When a linking residue is included at this position, the modified polypeptide of the second aspect of the invention preferably comprises the corresponding linking residue shown in the following amino acid sequence: Xc-FNPVYPYX 59 (SEQ ID NO: 70) where X 59 is selected from the group consisting of E, D and G, preferably E or D, more preferably E; and / or Xc is a binding residue, preferably C, D, E, and K, and most preferably C.

[0104] Thus, the modified protein of the second aspect of the invention in a preferred form comprises the STICKER polypeptide of SEQ ID NO: 70. Particularly preferred STICKER polypeptides which may be comprised in the modified protein of the second aspect of the invention are as follows: AKRARLST-XC-FNPVYPYX 59 -DES (SEQ ID NO: 74) where Xc is a linking residue, preferably C, D, E, and K, most preferably C; and X 59 is selected from the group consisting of E, D and G, preferably E or D, more preferably E. In a particularly preferred embodiment, the STICKER polypeptide comprises a binding residue Cys at position 9 and consists of the following amino acid sequence: AKRARLSTCFNPVYPYEDES (SEQ ID NO: 75)

[0105] In some embodiments of the modified polypeptides of the first aspect of the invention, the RGD motif located between the first and second RGD loops remains intact and binds the penton substrate protomer, penton or VLP to specific cellular and extracellular structures present in a patient, or, if such targeting is not desired for a particular application of the modified polypeptides of the first aspect of the invention, the RGD motif is mutated so as to lose its ability to bind integrins.

[0106] In a third aspect, the present invention relates to a nucleic acid encoding a modified polypeptide according to the first aspect of the invention and / or a modified polypeptide according to the second aspect of the invention. In a fourth aspect, the invention relates to an expression vector comprising a nucleic acid of the invention. Expression vectors include plasmids and viral vectors and contain a coding sequence encoding a modified protein of the first and / or second aspect of the invention and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammalian) or in vitro expression system. Cloning vectors are generally used to modify and amplify some desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment. It is recognized in the art that immunization against diseases that rapidly change antigenic epitopes, such as influenza, or diseases characterized by patient-specific epitope mixtures, requires rapid adaptation or individualization of the cutin. The VLPs of the invention can be rapidly adapted to display each desired antigen. Thus, in a fifth aspect, the invention relates to a cloning vector suitable for the rapid insertion of nucleic acid segments into the first and / or second RGD loop or V loop, encoding one or more desired antigens. The cloning vector of this form of the invention comprises: (i) a polypeptide comprising an adenoviral penton base protomer, the penton base protomer comprising a binding site of an adenoviral fiber protein suitable for inserting a nucleic acid encoding a first RGD loop, a second RGD loop, a variable loop, and / or a non-adenoviral peptide into the nucleic acid encoding the first RGD loop, the second RGD loop, the variable loop; or (ii) A polypeptide comprising an adenoviral penton base protomer-binding fiber protein fragment suitable for introducing into its C- and / or N-terminus a nucleic acid encoding a non-adenoviral peptide.

[0107] In the cloning vector of the preferred embodiment of the present invention, the indication comprises one or more restriction enzyme sites, preferably BamHI, KpnI, KasI, NarI, SfdI, EcoRI and RsrII, PfoI, BssHII, SalI, SacI, XbaI, BstEII and HindIII. The nucleic acid sequences of preferred examples of such cloning vectors are shown in SEQ ID NOs: 61 and 62. The structures of these vectors, called pACEBac-ADD Omer1.0 and pACEBac-ADD Omer2.0, are illustrated in Figures 7 and 8. The nucleotide sequence of the cloning vector pACEBac-ADD Omer2.0 containing preferred chikungunya virus antigenic epitopes is shown in SEQ ID NOs: 71 and 72.

[0108] In a sixth aspect, the present invention relates to a recombinant host cell comprising an expression vector of the present invention or a cloning vector of the present invention. The expression vector of the present invention or the cloning vector of the present invention may be integrated into a host cell (i) that is distributed freely or in its entirety or into the host cell genome or mitochondrial DNA. The recombinant host cell is used for the expression of the modified polypeptide of the present invention. The term "recombinant host cell" includes the progeny of an original cell that has been transformed, transfected or infected with a polynucleotide or a recombinant vector of the present invention. The recombinant host cell may be a bacterial cell, such as an E. coli cell, a yeast cell, such as a Saccharomyces cerevisiae or Pichia pastoris cell, a plant cell, an insect cell, such as an SF9 or Hi5 cell, or a mammalian cell. Preferred examples of mammalian cells are Chinese Hamster Ovary (CHO) cells, Green African Monkey Kidney (COS) cells, Human Embryonic Kidney (HEK293) cells, HELA cells, etc.

[0109] The modified polypeptides of the first aspect of the invention, despite their modifications according to alternative embodiments (i) to (vi), can assemble into penton subunits, i.e. pentamers, as compared to the respective wild-type penton base protomers. Whether a given modified protein of the first aspect of the invention assembles into a pentamer can be readily assessed by methods well known to those skilled in the art, including non-denaturing polyacrylamide gel electrophoresis, size exclusion chromatography, mass spectrometry, and the like. Thus, in a seventh aspect, the invention relates to a pentamer comprising five modified polypeptides comprising an adenoviral penton base protomer of the invention. This pentamer can additionally be included between one and the modified polypeptides of the second aspect of the invention.

[0110] The pentamers assembled from the modified polypeptides of the first aspect of the invention can be further assembled into VLPs. Thus, in an eighth aspect, the invention relates to a virus-like particle (VLP) comprising a dodecamer of the invention. The VLP is stable and is the most suitable composition for administration to a patient. Preferably, the VLP is assembled and / or stored under non-reducing conditions to allow the formation of covalent bonds between the binding residues in the penton base protomers.

[0111] In a preferred embodiment, the VLP of the invention further comprises at least one modified polypeptide of the second aspect of the invention, preferably up to 60 modified polypeptides of the second aspect of the invention. In the latter embodiment, all fiber protein binding clefts of the penton base protein of a modified polypeptide of the first aspect of the invention are occupied by a modified protein of the second aspect of the invention. The VLP of the invention preferably comprises a modified protein of the first aspect of the invention, preferably comprising a modification according to alternative aspect (vi), preferably in combination with modifications according to alternative aspects (i), (ii), (iii), (iv) and / or (v), and at least one linking residue, preferably inserted and / or located at the N- and C-terminus of the fiber protein fragment, preferably at least one linking residue inserted and / or located at the N-terminus and / or C-terminus of SEQ ID NO: 43 or attached to an amino acid of the fiber protein fragment. Preferably, the modified protein of the first aspect of the invention comprises KSFX64NXc1Xc2AVY (SEQ ID NO: 68), where X64, Xc1, and Xc2 have the meanings outlined above. Also, the engineered protein of the second aspect of the invention comprises X58-FNPVYPY-X59-(X63)n-Xc (SEQ ID NO: 69), where X58, X59, X63, Xc, and n have the meanings outlined above. In another preferred embodiment, the engineered protein of the first aspect of the invention comprises the following sequence Xc-X65-RSYN (SEQ ID NO: 73), where Xc and X65 have the meanings outlined above, and the second aspect of the invention comprises Xc-FNPVYPY-X59 (SEQ ID NO: 70), where Xc and X59 have the meanings outlined above.

[0112] Preferably, the VLP comprising a linking residue for covalently linking a modified polypeptide of the first aspect of the invention to a modified polypeptide of the second aspect of the invention is assembled and / or stored under non-reducing conditions to allow covalent linkage between the linking residue of the penton base protomer and a STICKER polypeptide comprised in the modified polypeptide of the second aspect of the invention. It is also envisaged that VLPs consisting of or containing wild-type penton base protomers are used to provide excipients, and these VLPs are modified with different non-adenoviral polypeptides by using modified polypeptides of the second aspect of the invention. Since the modified polypeptides of the second aspect of the invention are in preferred embodiments short, they can be synthesized, for example, by solid-state chemistry, within one day. This is achieved. Thus, in a ninth aspect, the invention relates to a VLP comprising 12-mers each comprising five adenoviral penton base protomers and at least one modified polypeptide of the second aspect of the invention. It is preferred that all fibre protein binding clefts of the penton base protomers are occupied, and thus these VLPs comprise 60 modified polypeptides of the second aspect of the invention.

[0113] In a tenth aspect, the present invention provides a method for producing a modified polypeptide of the first or second aspect of the invention, comprising the steps of: (a) providing a recombinant host cell of the invention; (b) expressing the modified polypeptide; and (c) purifying the modified polypeptide.

[0114] In an eleventh aspect, there is provided a method of producing a VLP of the invention comprising the steps of the method of the tenth aspect of the invention and the further step of assembling modified polypeptides into a VLP.

[0115] The method of the tenth aspect of the invention further comprises the step of incubating the VLPs with a protease, preferably a sequence specific endopeptidase cleavage site, more preferably TEV.

[0116] In a twelfth aspect, the present invention relates to a method for producing a VLP of the present invention comprising a disease and / or patient specific non-adenoviral peptide, comprising the steps of: (a) providing a cloning vector of the invention; (b) determining the amino acid sequence of a disease- or patient-specific non-adenoviral peptide; (c) inserting a nucleic acid encoding at least one of the non-adenoviral peptides into a nucleic acid encoding the first RGD loop of an adenoviral penton base, the second RGD loop, and / or the variable loop of an adenoviral penton base protomer, and / or at a nucleic acid position before or after a nucleic acid encoding the N- or C-terminus of a modified polypeptide comprising an adenoviral penton base protomer that binds a fiber protein fragment. (d) expressing a modified adenoviral penton base protomer in a host cell, preferably together with a modified polypeptide comprising the adenoviral penton base protomer that binds a fiber protein fragment; and (e) purifying the VLPs comprising the adenovirus penton base protomers that bind the fiber protein fragment, or the modified polypeptides comprising the adenovirus penton base protomers that bind the fiber protein fragment.

[0117] In a thirteenth aspect, the present invention provides a method for producing a VLP of the present invention comprising a disease- and / or patient-specific non-adenoviral peptide, comprising the steps of: (a) providing a cloning vector of the invention; (b) determining the amino acid sequence of a disease- or patient-specific non-adenoviral peptide; (c) a nucleic acid encoding at least one of said non-adenoviral polypeptides is inserted at a nucleic acid position before or after the nucleic acid encoding the N-terminus or C-terminus of a modified polypeptide that includes a fiber protein fragment. (d) optionally expressing a modified polypeptide comprising the adenoviral penton base protomer that binds the fiber protein fragment in a host cell together with the adenoviral penton base protomer; (e1) purifying the modified polypeptide comprising a fiber protein fragment-bound adenoviral penton base protomer and mixing it with an adenoviral penton base protomer or a modified adenoviral penton base protomer of the invention; or (e2) Purifying the VLPs when an adenovirus penton base protomer is co-expressed.

[0118] In a fourteenth aspect, the present invention provides a method for producing a VLP of the present invention comprising a disease and / or patient-specific non-adenoviral peptide, comprising the steps of: (a) determining the amino acid sequence of a disease- or patient-specific non-adenoviral peptide; (b) synthesizing a modified polypeptide of the invention comprising an adenoviral penton base protomer that binds a fiber protein fragment and at least one of said non-adenoviral peptides; or (c) mixing the modified polypeptide with an adenoviral penton base protomer or the modified adenoviral penton base protomer of the invention with a pentamer of the invention or a VLP of the invention.

[0119] In a fifteenth aspect, the present invention relates to a VLP producible by the method for producing a VLP of the present invention.

[0120] In a sixteenth aspect, the invention relates to a pharmaceutical composition, comprising a modified polypeptide comprising an adenoviral penton base protomer of the invention, and / or an adenoviral penton base protomer binding a fiber protein fragment, a nucleic acid encoding one or more modified proteins of the invention, an expression vector of the invention, or a VLP of the invention, and a pharma- ceutically acceptable carrier and / or suitable excipient. Preferably, such a composition is a pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient, and optionally one or more additional active substances. Preferably, the composition of the fifth aspect comprises a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of a carrier and / or excipient to provide a form for proper administration to a patient. The formulation should be compatible with the mode of administration.

[0121] The pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations, etc. The pharmaceutical compositions can be formulated as suppositories, with traditional binders and carriers such as triglycerides.

[0122] For preparing the pharmaceutical composition of the present invention, the pharma- ceutically acceptable carrier can be either solid or liquid. The solid form of the composition includes powders, tablets, pills, capsules, lozenges, cachets, suppositories and dispersible granules. The solid excipient can be one or more substances that can also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials. In powders, the excipient is preferably a finely divided solid, which is mixed with the finely divided inhibitor of the present invention. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in the appropriate proportions and compressed into the desired shape and size. Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. To prepare suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active ingredient is dispersed homogeneously therein as by stirring.The molten homogeneous mixture is then poured into convenient sized molds and allowed to cool and solidify.Tablets, powders, capsules, pills, cachets and lozenges can be used as solid dosage forms suitable for oral administration.

[0123] Liquid form compositions include solutions, suspensions, and emulsions, for example, water, saline, aqueous dextrose, glycerol solutions, or water / propylene glycol solutions. For parenteral injection (e.g., intravenous, intraarterial, intraosseous, intramuscular, subcutaneous, intraperitoneal, intradermal, and intrathecal injection), liquid preparations are made up in a solution, for example, in an aqueous polyethylene glycol solution. Saline is a preferred carrier when the pharmaceutical composition is administered intravenously.

[0124] Preferably, the pharmaceutical composition is in unit dosage form.In such form, the composition may be subdivided into unit doses containing appropriate amounts of active ingredients.The unit dosage form may be a packaged composition, the package having discrete quantities of the composition, such as packaged tablets, capsules, and powders in vials or ampoules.Also, the unit dosage form may be a capsule, injection vial, tablet, cachet, or lozenge itself, or the appropriate number of any of these in packaged form.

[0125] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. In addition, such pharmaceutical compositions may contain other pharmacologically active substances, such as, but not limited to, adjuvants and / or additional active ingredients. Adjuvants in the context of the present invention include, but are not limited to, inorganic adjuvants, organic adjuvants, oil-based adjuvants, cytokines, particulate adjuvants, virosomes, bacterial adjuvants, synthetic adjuvants, or synthetic polynucleotide adjuvants. In a seventeenth aspect, the present invention relates to a modified polypeptide comprising an adenoviral penton base protomer of the invention and / or a modified polypeptide of the invention comprising a fiber protein fragment-bound adenoviral penton base protomer, a nucleic acid encoding one or more modified proteins of the invention, an expression vector of the invention or a VLP of the invention for treating and / or preventing an infectious disease, an immune disease or cancer.

[0126] Application Examples ADDomers were designed and manufactured with very high yields (tens of grams per liter of expression culture). A general three-step protocol was established to purify ADDomers to homogeneity (see below). In a proof-of-concept project, highly immunogenic epitopes were experimentally determined to be inserted into the functionalized loops of ADDomers without perturbing particle formation or significantly reducing yield. ADDomers containing chikungunya epitopes were purified to homogeneity and cell-based and animal testing was initiated to determine their efficacy as vaccine candidates. ADDomers were confirmed by a variety of techniques including electron microscopy, homogeneously structured discrete multimers (dodecahedrons). Cysteine-disulfide chemistry was implemented to further enhance the already remarkable thermostability of ADDomers (elimination of cold chain requirement). Furthermore, we are preparing ADDomers, which contain not only peptide epitopes but also whole proteins with protein domains and high affinity binders (nanobodies, DARPins, antibody fragments), and are establishing efficient protocols for their large-scale production. ADDomer-based Zika vaccine candidates, triggered by the recent emergence of the Zika virus, have also been designed, and ADDomers have also been designed to combat multiple diseases simultaneously (combo vaccines). Cell-based and animal studies to validate these are being performed. The following describes the experiments and protocols for generating and validating ADDomer VLPs and ADDomer VLP vaccines:

[0127] 1.ADDomer Design The atomic structures of naturally occurring 12-mer species (e.g., from the adenovirus Ad3 serotype) have been determined by X-ray crystallography (Szolajska E et al., PLoS One. 2012; 7(9):e46075 and Zubieta C et al., Mol Cell 2005:17(1):121-35). Careful inspection of the atomic structures revealed the presence of extended loop structures. More precisely, one variable loop (called the V-loop) and two regions in the so-called RGD-loop of the wild-type 12-mer were identified as potential sites of functionalization. Comparison of multiple 12-mer protomers revealed a wide variability of the V-loop and the two RGD-loop regions across species, both in length and sequence composition, highlighting their potential. Using this information, we have de novo designed DNA sequences encoding synthetic designer 12-mer promoters. BioBrick design (Shetty et al. J. Biol. End. 2008) was applied by introducing DNA sequences representing endonuclease cleavage sites, facilitating designed mutations (including randomization) of the amino acids representing the V-loop and the two RGDs (RGDloop2, RGDloop2). Iterative optimization of the protomer design was performed until a protomer was identified that yielded a recombinant 12-mer (ADDomer) featuring the perfect BioBrick design of the loop regions while maintaining the high solubility and structural integrity of the wild-type human Ad3 serotype 12-mer.

[0128] 2. Design of Ultra-Stable ADDomer The ADDomer has already been shown to be highly thermostable and can be stored for long periods at 37 °C, without the need for a cold chain in remote locations with poor infrastructure. Inspection of the crystal coordinates of native Ad3 particles revealed so-called "strand-swapping" regions, where segments of a protomer extend into the vicinity of an adjacent protomer, resulting in juxtaposition of amino acids that are within a distance that allows for the formation of covalent bonds. We genetically replaced these amino acids in the ADDomer with cysteines, such that two cysteines from different protomers were within the distance required for disulfide bond formation.

[0129] 3. Expression of MultiBac-based ADDomers The ADDomer was then expressed using the MultiBac system. The gene encoding the ADDomer was synthesized from scratch (S SEQ ID NO:63:63 and the encoded ADDomer is provided in SEQ ID NO:64) and inserted into pACEBac, the transfer plasmid of the MultiBac system, by classical cloning methods (restriction / ligation). Synthetic MultiBac containing the ADDomer gene was specifically developed by one of the inventors (Berger) for the production of complex biologics such as ADDomer. Synthetic MultiBac virus containing the ADDomer gene was prepared (see Figures 7 and 8) and infected insect cell cultures were prepared using previously described protocols (Berger Iet al., J Vis Exp. (2013) 77: e50159 and Fitzgerald DJ et al. Nat Methods (2006) 3 (12): 1021). ADDomer protein-containing cell pellet was prepared by centrifugation as described. The cell pellet was stored at -80 °C. Expression of ADDomers inserted with peptide or protein epitopes, as well as expression of the ultrastable ADDomer, all gave rise to comparable, very high yields and uniformly structured dodecahedral particles.

[0130] 3. Neutralizing epitopes As an example, an ADDomer-CHIKADDomer-based VLP vaccine candidate was constructed that displays multiple copies of the major neutralizing Chikungunya immune epitope HAKKQDVVVLGSQEGAM (SEQ ID NO: 55). The major neutralizing immune epitope is a linear peptide epitope located at the extreme N-terminus, part of the Chikungunya envelope protein (Kam YWet al., EMBO Mol Med. (2012); 4(4):330-4). In the vast majority of patient sera, antibodies reacting with this linear peptide epitope are found. ADDomers provide a means to display linear epitopes while maintaining the structural integrity of the ADDomer scaffold, either in a restricted manner (N- and C-termini covalently linked to the ADDomer scaffold), or in an unrestricted manner (N-termini released by cleavage with a specific protease), or in a combined restricted and unrestricted manner. The preferred configurations used are as follows: AKRARLSTSFNPVPYEDESSTKDNFNVYKATRPYLAH (SEQ ID NO:58), AKRARLSTSFNPVPYEDECSSTKDNFNVYKATRPYLAH (SEQ ID NO:59) and AKRARLSTCFNPVPYEDESSTKDNFNVYKATRPYLAH (SEQ ID NO:60). Native-like display of the major neutralizing epitopes of Chikungunya was achieved by TEV protease-mediated cleavage of ADDomers, each of which contains a specific TEV cleavage site preceding the neutralizing epitope sequence, retaining the native N-terminus and displaying multiple copies of the epitope. A similar approach can be utilized for any epitope or peptide or protein domain displayed by an ADDomer.

[0131] 4. Purification of ADDomers and Mutants Spodoptera frugiperda Sf21 insect cell pellets were lysed by freeze-thawing. Lysates were clarified by centrifugation following standard protocols for insect cells (Berger I et al., J Vis Exp. (2013)(77):e50159 and Fitzgerald DJ et al., Nat Methods, 2006 3(12):1021). The clarified supernatant was loaded onto a 15-40% sucrose gradient and centrifuged overnight using a Beckman SW41 rotor. Fractions of 1.1 mL were collected from the top of the gradient and loaded onto denaturing SDS-polyacrylamide electrophoresis (SDS-PAGE) to analyze protein content and pooled size exclusion chromatography (SEC) and / or ion exchange (IEX) were performed after sucrose dialysis.

[0132] 5. Validation of ADDomer by Electron Microscopy Purified ADDomers and ADDomer variants were visualized by negative stain electron microscopy (EM) to assess their assembly state and their structural integrity. A standard mica-carbon preparation was utilized with ADDomer at a concentration of approximately 0.1 mg / ml prior to deposition onto the carrier material. Samples were stained with 1% (wt / vol) sodium silicotungstate (pH 7.0) and visualized at 100 kV with a JEOL electron microscope. Images were recorded and analyzed using software provided by Gatan. For thermal stability experiments, ADD monomers were frozen, stored at 4°C, room temperature (RT) or 37°C for 1 week. Electron microscopy showed that storage at room temperature or 37°C resulted in correctly self-assembled particles exhibiting thermal stability. Incubation of ADDomer (SEQ ID NO: 64) for 2 hours at 45°C resulted in reversible particle disassembly, which reassembled when returned to room temperature. This reversible dissociation was also observed by heat transfer assay (Figure 10, see arrows), although irreversible dissociation was only observed at temperatures above 50°C.

[0133] 6. Design of animal (murine) studies to evaluate ADDomer immunogenicity ADDomer-CHIK For murine immune analysis of the chikungunya ADDomer VLP vaccine candidate, 6-week-old BALB / c female mice were used. Four groups of 8 mice were used (e.g., for the chikungunya VLP vaccine candidate: i) ADDomer, ii) ADDomer CHIK unrestricted epitope, iii) ADDomer CHIK restricted epitope, iv) isolated CHIK major neutralizing peptide epitope cross-linked to KLH as a positive control). Each animal was injected with 10 μg of ADDomer and ADDomer variants at 2-week intervals. IgA, IgM, total IgG, IgG1 IgG2a and anti-CHIK antibodies were titered from mouse sera by ELISA. Immune analysis of other ADDomer VLP vaccine candidates was designed in a similar way. Two types of epitope display on the ADDomer surface were tested (restricted and relaxed, see item 7 below). Time-dependent responses were observed (weeks 0 to 6). The superior ability of the relaxed form of the epitope over the restricted form to elicit an anti-Chik epitope response was demonstrated (FIG. 12).

[0134] 7. Exposing the epitope of interest on the ADDomer surface Addition of a TEV cleavage site upstream of the epitope of interest allows its display in two different forms: constrained or relaxed. Upon purification, the epitope is naturally constrained in the ADDomer loop. By adding TEV (Tobacco Etch Virus) protease (1 / 100 w:w) for 2 hours at room temperature, the epitope can be relaxed and linearly displayed on the scaffold surface. Cleavage efficiency can be easily monitored by SDS-PAGE. Of note, the entire ADDomer scaffold is unaffected by this cleavage (Figure 11).

[0135] 8. Extension of epitope insertion capacity in ADDomer The ability of the ADDomer to carry a large epitope sequence was evaluated. For this purpose, a 200 amino acid long artificial epitope was inserted into the ADDomer (named extended ADDomer). This resulted in a correctly autoassembled ADDomer. The insertion was confirmed by both SDS-PAGE analysis and mass spectrometry, as shown in Figure 13.

[0136] 9. Covalent attachment of epitopes to the ADDomer surface To broaden the capabilities of ADDomers, a system was developed that allows for the addition of additional epitopes on ADDomers. A single cysteine ​​was inserted at a specific position in the ADDomer sequence (either K363C, Q476C, or A477C). K363C was designed to form a covalent disulfide bridge with a 20 amino acid long fiber protein fragment (peptide C20 (SEQ ID NO:77) derived from SEQ ID NO:59), while Q476C or A477C were designed to act similarly with another fiber protein fragment (peptide C9 (SEQ ID NO:75) derived from SEQ ID NO:60). Covalent interaction of peptides with the corresponding Cys-modified ADDomers was confirmed by incubating the particles with the corresponding peptides under oxidizing (i.e., in the absence of β-mercaptoethanol) or reducing conditions (addition of β-mercaptoethanol). The complexes were run on SDS-PAGE, the ADDomer was detected by a specific antibody and a Cy3-labeled secondary antibody, and the biotinylated peptide was detected with Alexa488-labeled avidin. When the right Cys-ADDomer / peptide was used, the presence of the peptide was detected at the ADDomer band size (circle in Figure 14). This interaction was specific to the disulfide bridge formed between the Cys-ADDomer and the peptide, since it was prevented under reducing conditions.

[0137] The present invention relates to the following aspects. 1. A modified polypeptide comprising an adenovirus penton-based protomer comprising a first RGD loop, a second RGD loop, a variable loop (V loop), an adenovirus fiber protein binding cleft and / or an N-terminal domain, comprising one or more of the following: (i) at least one target-specific binding domain in the first, second or both of the first and second RGD loops, and / or the V loop; and / or (ii) one or more non-adenoviral peptides of the first, second or both of the first and second RGD loops and / or the V loop; and / or (iii) a non-adenoviral peptide at the N-terminus and / or C-terminus of the penton base protomer; and / or (iv) at least one heterologous coupling residue in the first, second or both of the V loop and / or the first and second RGD loop in the N-terminal domain of the penton based protomer, the termini of the N-terminal domain in the penton based protomer being defined as follows: X1-GRNSIR (SEQ ID NO: 44) The C-terminus of the N-terminal domain within the penton base protomer is defined as follows: D-X2-RSRG (SEQ ID NO: 45) Here, X1 is selected from the group consisting of G and E; X2 is selected from the group consisting of D and E; and / or (v) a drug, label, or polypeptide penton base protomer covalently or non-covalently attached to one or more amino acids of the first and second RGD loops and / or one or more V loops; and / or (vi) At least one heterologous coupling residue in the adenovirus fiber protein binding cleft of the penton base protomer, the modified polypeptide is preferably capable of being incorporated into a VLP. 2. A modified polypeptide comprising at least one adenovirus fiber protein N-terminal fragment and specifically binding to the adenovirus fiber protein binding cleft of a penton base protomer, (i) a non-adenoviral peptide and / or (ii) is covalently or non-covalently attached to a drug or label. 3. The modified polypeptide according to item 1 or 2 is an adenovirus of a human or non-human ape, preferably an adenovirus of a chimpanzee (Pan), a gorilla (Gorilla) or an orangutan (Pongo), more preferably an adenovirus of a bonobo (Pan paniscus) or a common chimpanzee. 4. The modified polypeptide according to paragraph 3, wherein the adenovirus is selected from the group consisting of: hAd3, hAd4, hAd5, hAd7, hAd11, hAd26, hAd35, and hAd49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, and ChAd5 PanAd1, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, and ChAd147 5. The modified polypeptide according to item 1 or item 3 or item 4, wherein the sequence of the wild-type penton base protomer is selected from the group consisting of SEQ ID NOs: 1 to 14. 6. The modified polypeptide of paragraph 1 or paragraphs 3 to 5, wherein the following sequence defines the N-terminus of the first RGD loop in the penton base protomer: X3-X4-X5-X6-X7-X8-X9-X 10 -X 11 (SEQ ID NO:15) where X3 is selected from the group consisting of D, E and N, preferably D; X4 is selected from the group consisting of V, L and I, preferably V; X5 is any amino acid, preferably selected from the group consisting of A, D, E, K, S and T, more preferably T; X6 is any amino acid, preferably selected from the group consisting of A, D, E and K, more preferably A; X7 is selected from the group consisting of F, Y and W, preferably Y; X8 is selected from the group consisting of A, D, E, N and Q, preferably E or Q, more preferably E; X9 is preferably any amino acid selected from the group consisting of A, D, E, N and K, more preferably E; X 10 is selected from the group consisting of S or T, preferably S; and X 11 is any amino acid and constitutes the N-terminal amino acid of the first RGD loop. The following sequences define the C-terminus of the first RGD loop and the N-terminus of the second RGD loop within the penton base protomer: X 12- X 13 -X 14 -X 15 -X 16 (SEQ ID NO:16) Here, X 12 is any amino acid and constitutes the C-terminal amino acid of the first RGD loop; X 13 is R; X 14 is G; X 15 is D; and X 16 is any amino acid and constitutes the N-terminal amino acid of the second RGD loop; and / or The following sequence defines the C-terminus of the second RGD loop in the penton base protomer: X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -X24 (SEQ ID NO:17) where X 17 is any amino acid and constitutes the C-terminal amino acid of the second RGD loop; X 18 is selected from the group consisting of I, L and V, preferably I; X 19 is selected from the group consisting of D, E, K, N, Q and V, preferably Q or K, more preferably Q; X 20 is selected from the group consisting of C, G and P, preferably P; X 21 is selected from the group consisting of I, L and V, preferably L or V, more preferably L; X 22 is selected from the group consisting of D, E, S and T, preferably E or T, more preferably E; X 23 is selected from the group consisting of D, E, K, S and T, preferably E, K or T, more preferably K; and X 24 is selected from the group consisting of D and E, preferably D; and / or The following sequence defines the N-terminus of the V-loop: X 25 -X 26 -X 27 -X 28 -X 29 -X 30 -X 31 -X 32 (Sequence number 18). where X 25 is selected from the group consisting of F, Y and W, preferably F; X 26 is selected from the group consisting of H, K and R, preferably K; X 27 is selected from the group consisting of A, V, I and L, preferably A; X28 is selected from the group consisting of H, K and R, preferably R; X 29 is selected from the group consisting of A, V, I and L, preferably V; X 30 is selected from the group consisting of A, V, I, L and M, preferably M; X 31 is selected from the group consisting of A, V, I and L, preferably V; and X 32 is any amino acid and constitutes the N-terminal amino acid of the V-loop. and / or The following sequence defines the C-terminus of the V loop: X 33 -X 34 -X 35 -X 36 -X 37 -X 38 -X 39 (SEQ ID NO:19) where X 33 is any amino acid and constitutes the C-terminal amino acid of the V-loop; X 34 is selected from the group consisting of F, Y and W, preferably Y; X 35 is selected from the group consisting of D, E, S and T, preferably E or T, more preferably E; X 36 is selected from the group consisting of F, Y and W, preferably W; X 37 is selected from the group consisting of A, F, V, Y and W, preferably F or V, more preferably F; X 38 is selected from the group consisting of D, E, S and T, preferably D or E, more preferably E; and X 39 is selected from the group consisting of F, Y and W, preferably F; and / or one or more of the following non-contiguous peptides within the penton base protomer form the adenovirus fiber protein binding cleft (amino acids in bold directly interact with the fiber): MTIDLMNNAIX 40 -X 41 -X 42 -YLX 43 -X 44 -GRQX 45 - GVLES (SEQ ID NO: 20). WDPX 46 -TX 47 -X 48 -PG (SEQ ID NO: 46); X 49 -VX 50 -X 51 -YX 52 -X 53 (sequence number); X 54 -X 55 -RSY (SEQ ID NO: 48); and / or LTX 56 -VFNRFPX 57 (SEQ ID NO:49) where X 40 is selected from the group consisting of V, I and L; X 41 is selected from the group consisting of E and D; X 42 is selected from the group consisting of H, N and Q, preferably H and N; X 43 is selected from the group consisting of K, E, R, Q and A; X 44 is selected from the group consisting of V, L and I, preferably V and I; X 45 is selected from the group consisting of H, N and Q, preferably H and N; X 46 is selected from the group consisting of V, I, L, E or D, preferably V and E; X 47is selected from the group consisting of V, L and I, preferably V and I; X 48 is selected from the group consisting of M, T and S, preferably M and T; X 49 is selected from the group consisting of D, E, N and Q, preferably D and N; X 50 is any amino acid preferably selected from the group consisting of A, D, P, K and T; X 51 is selected from the group consisting of A, D, E, K and R, preferably A, E and K; X 52 is selected from the group consisting of D, E, L, I, Q and N, preferably E, L and Q; X 53 is selected from the group consisting of A, D, E, K, N, Q and R, preferably A, E, N and K; X 54 is selected from the group consisting of K, R, S and T, preferably K, S and T; X 55 is selected from the group consisting of A, D, E, G, K, N, Q, R, S and T, preferably D, G, K, N and S. X 56 is selected from the group consisting of H, K and R, preferably H and R; and X 57 is selected from the group consisting of D and E. 7. Independently from each other, the amino acid sequences of X3 to X10 are selected from the group consisting of DVTAYEES (SEQ ID NO: 21), DVDAYENS (SEQ ID NO: 22), DVAEYEKS (SEQ ID NO: 22), DVEAYEKS (SEQ ID NO: 24), DVDAYEKS (SEQ ID NO: 25), DVSKYEAS (SEQ ID NO: 26), NVKAYEDS (SEQ ID NO: 27), DVKKYENS (SEQ ID NO: 28), DVDAYQAS (SEQ ID NO: 29), and DVDAYQAS (SEQ ID NO: 30); and the amino acid sequences of X18 to X24 are selected from the group consisting of IQPLEKD (SEQ ID NO: 31), IQPVEKD (SEQ ID NO: 32), IKPLEKD (SEQ ID NO: 33), IVPLTKD (SEQ ID NO: 34), IEPVETD (SEQ ID NO: 35), and IKPLTED (SEQ ID NO: 36), and The amino acid sequences of X33 to X39 are selected from the group consisting of FKARVMV (SEQ ID NO: 37), FRAKLMV (SEQ ID NO: 38), and FRAKVMV (SEQ ID NO: 39), and include YEWFEF (SEQ ID NO: 40), YEWVEF (SEQ ID NO: 41), and YEWAEF (SEQ ID NO: 42). 8. The modified polypeptide according to any one of items 1 or 3 to 7, wherein the target-specific binding domains of the first RGD loop have, independently of each other, a length of 5 to 300 amino acids, preferably 6 to 200 amino acids. The target-specific binding domain of the second RGD loop has a length of 5 to 300 amino acids, preferably 10 to 200 amino acids. And / or the target-specific binding domain in the V loop has a length of 5 to 300 amino acids, preferably 10 to 200 amino acids. 9. The modified polypeptide according to any of items 1 or 3 to 8, wherein at least one of the target-specific binding domains is capable of specifically binding to an immunogenic peptide, a pathogen-neutralizing peptide, a viral peptide, a bacterial peptide, an immunomodulatory peptide, preferably to the surface of a cellular receptor, a low molecular weight tag, preferably biotin or chitin. 10. The modified polypeptide according to any of items 1 to 9, wherein the non-adenoviral polypeptide or polypeptides are selected from the group consisting of immunogenic peptides, pathogen-neutralizing peptides, viral peptides, bacterial peptides, immunomodulatory peptides and cancer peptides. 11. The modified polypeptide according to any of items 10, wherein the non-adenoviral peptide or peptide comprises a protease cleavage site, preferably a sequence-specific endopeptidase cleavage site, more preferably a Tobacco Etch Virus NIa protease (TEV) protease. 12. The modified polypeptide according to any of items 1 to 11, wherein the coupling residue is selected from the group comprising Lys, Cys, Asp, and Glu, preferably Cys. 13. The modified polypeptide of any one of claims 1 to 12, wherein the drug is selected from the group consisting of chemotherapeutic drugs, antipathogenic drugs, immunomodulatory drugs, and anti-inflammatory drugs. 14. The modified polypeptide of item 2, wherein the fiber protein fragment comprises: X 58 -FNPVYPYX 59 (SEQ ID NO:43) where X 58 is selected from the group consisting of S, D and T, preferably S or D, more preferably S; and X 59 is selected from the group consisting of E, D and G, preferably E or D, and more preferably E. 15. The modified polypeptide of claim 14, wherein the fiber protein fragment has a length of 9 to 20 amino acids. 16. The modified polypeptide according to items 2 and 14 or 15, wherein at least one coupling residue is preferably inserted and / or located at the N-terminus and / or C-terminus of the fiber protein fragment, either at the N-terminus and / or C-terminus of SEQ ID NO: 43 or attached to an amino acid of the fiber protein fragment. 17. A nucleic acid encoding a polypeptide according to any one of items 1 to 16. 18. An expression vector comprising the nucleic acid of item 17. 19. A cloning vector encoding: (i) a polypeptide comprising an adenoviral penton base protomer, the penton base protomer having a binding site for an adenoviral fiber protein adapted for introducing a nucleic acid encoding a non-adenoviral peptide into the amino acids encoding the first RGD loop, the second loop, the variable rule, and / or the first RGD loop, the second RGD loop, the variable loop; or (ii) A polypeptide comprising an N-terminal fragment of an adenoviral fiber protein that specifically binds to the adenoviral fiber protein binding cleft of a penton base promoter adapted for introducing a nucleic acid encoding a non-adenoviral peptide into the C- and / or N-terminus. 20. The cloning vector of item 19, comprising one or more enzyme sites for compatibility, preferably BamHI, KpnI, KasI, NarI, SfdI, EcoRI and RsrII, PfoI, BssHII, SalI, SacI, XbaI, BstEII and HindIII. 21. A recombinant host cell comprising the expression vector of item 18 or the cloning vector of item 19 or 20. 22. A pentamer comprising five modified polypeptides comprising the adenovirus penton base promoter according to items 1, 3 to 13. 23. A virus-like particle (VLP) comprising 12 pentamers according to item 22. 24. The VLP according to item 23, comprising at least one modified polypeptide comprising an N-terminal fragment of adenovirus fiber protein that specifically binds to the adenovirus fiber protein binding cleft of the penton base promoter according to claims 2 to 4 and 10 to 16. 25. The VLP according to item 24, further comprising at least one mutation at Cys of amino acid residue, such as G51C, S555C, G53C, Y64C, S54C, D114C. 26. A pentamer 10 to 16 comprising at least one modified polypeptide comprising an adenovirus fiber protein N-terminal fragment that specifically binds to the adenovirus fiber protein binding cleft described in any one of claims 2 to 4 and 12 pentamer comprising five adenovirus penton base protomers. 27. A method for producing a modified polypeptide according to any one of claims 1 to 16, comprising the steps of: (a) providing a recombinant host cell of item 21; (b) expressing the modified polypeptide; and (c) purifying the modified polypeptide. 28. A method for producing a VLP according to any one of items 23 to 26, comprising the method according to item 27 and further comprising a step of assembling the modified polypeptide into a VLP. 29. The method according to item 28, further comprising incubating the VLPs with a protease, preferably a sequence-specific endopeptidase cleavage site, more preferably TEV. 30. A method for producing VLPs according to any one of items 23 to 26, comprising a disease- and / or patient-specific non-adenoviral peptide, comprising: (a) providing a cloning vector according to items 19 and / or 20; (b) determining the amino acid sequence of the disease- or patient-specific non-adenoviral peptide; (c) inserting a nucleic acid encoding at least one of said non-adenoviral peptides into a nucleic acid encoding the first RGD loop, the second RGD loop, and / or the variable loop of an adenoviral penton base protomer, and / or before or after a nucleic acid encoding the N-terminus or C-terminus of a modified polypeptide comprising an adenoviral fiber protein N-terminal fragment that specifically binds to the adenoviral fiber protein binding cleft of the nucleic acid penton base protomer; (d) expressing the engineered adenovirus penton base protomer in a host cell, optionally together with a modified polypeptide comprising an adenovirus fiber protein N-terminal fragment that specifically binds to the adenovirus fiber protein binding cleft of the penton base protomer; and (e) optionally purifying the VLP comprising the adenovirus penton base protomer-bound fiber protein fragment or the modified polypeptide comprising the adenovirus penton base protomer-bound fiber protein fragment. 31. A method for producing a VLP according to any one of items 23 to 26, comprising a disease- and / or patient-specific non-adenovirus peptide, comprising: (a) providing a cloning vector according to item 20; (b) determining the amino acid sequence of the disease- or patient-specific non-adenoviral peptide; (c) inserting a nucleic acid encoding at least one of the non-adenoviral peptides at a nucleic acid position before or after a nucleic acid encoding the N-terminus or C-terminus of the modified polypeptide; the adenoviral fiber protein binding cleft of the penton base protomer; (d) expressing, optionally together with an adenovirus penton base protomer, a modified polypeptide comprising an adenovirus fiber protein N-terminal fragment that specifically binds to the adenovirus fiber protein binding cleft of the penton base protomer in the host cell; and (e1) purifying the modified polypeptide comprising an N-terminal fragment of an adenoviral fiber protein and mixing it with an adenoviral penton-based protomer or an engineered adenoviral penton-based protomer at the binding site of the adenoviral fiber protein; (e2) purifying the VLPs when the adenovirus penton base protomer is co-expressed. 32. A method for producing VLPs according to any one of items 23 to 26, comprising a disease- and / or patient-specific non-adenoviral peptide, comprising: (a) determining the amino acid sequence of a disease- or patient-specific non-adenoviral peptide; (b) synthesizing a modified polypeptide according to any one of items 2 to 4 and 10 to 16, comprising an adenovirus fiber protein N-terminal fragment that specifically binds to the adenovirus fiber protein binding cleft of a penton base protomer and at least one of the non-natural amino acid sequences, an adenovirus peptide; and (c) mixing the modified polypeptide with the adenovirus penton base protomer or the adenovirus penton base protomer and the pentamer of the VLP of item 19 of item 18 of item 19. 33. A VLP producible by the method according to any one of items 28 to 32. 34. A pharmaceutical composition comprising the modified polypeptide according to any one of items 1 to 16, the nucleic acid according to item 17, the expression vector according to item 18 or the VLP according to any one of items 23 to 26 or 33, and a pharma- ceutically acceptable carrier and / or suitable excipient. 35. A modified polypeptide according to any one of claims 1 to 16, a nucleic acid according to item 17, an expression vector according to item 18 or a VLP according to any one of items 23 to 26 or 33, a polypeptide for the treatment and / or prevention of an infectious disease, an immune disease or cancer.

Claims

1. A modified polypeptide comprising at least one adenovirus fiber protein N-terminal fragment that specifically binds to the adenovirus fiber protein binding cleft of a penton base protomer, The adenovirus fiber protein N-terminal fragment is (i) specifically binds to a non-adenoviral polypeptide; and / or (ii) Covalently or non-covalently bound to a drug or label Modified peptides.

2. 2. The modified polypeptide of claim 1, wherein the fiber protein fragment comprises the sequence: X 58 -FNPVYPYX 59 (SEQ ID NO:43) where X 58 is selected from the group consisting of S, D and T, preferably S or D, more preferably S; and X 59 is selected from the group consisting of E, D and G, preferably E or D, more preferably E.

3. The modified polypeptide of claim 1, wherein the non-adenoviral polypeptide is selected from the group consisting of immunogenic peptides, pathogen-neutralizing peptides, viral peptides, bacterial peptides, immunomodulatory peptides, and cancer peptides.

4. The modified polypeptide of claim 3, wherein the viral peptide is selected from the group consisting of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:

60.

5. 2. The modified polypeptide of claim 1, comprising a protease cleavage site.

6. 6. The modified polypeptide of claim 5, wherein the protease cleavage site is a sequence-specific endopeptidase cleavage site.

7. 7. The modified polypeptide of claim 6, wherein the sequence-specific endopeptidase cleavage site is a TEV cleavage site.

8. 2. The modified polypeptide of claim 1, wherein the drug is selected from the group consisting of chemotherapeutic drugs, anti-pathogenic drugs, immunomodulatory drugs, and anti-inflammatory drugs.

9. 2. The modified polypeptide of claim 1, comprising 2, 3, 4, 5, 6, 7 or 8 repeats of an N-terminal fragment of an adenovirus fiber protein.

10. 10. A modified polypeptide as described in claim 9, comprising two or three consecutive repeats of an N-terminal fragment of an adenovirus fiber protein.

11. 10. The modified polypeptide of claim 9, wherein the repeats of the adenovirus fiber protein N-terminal fragment are arranged in a head-to-tail orientation.

12. 2. The modified polypeptide of claim 1, wherein the adenovirus fiber protein N-terminal fragment has a length of 50 or less contiguous amino acids of the N-terminal fiber sequence.

13. 13. The modified polypeptide of claim 12, wherein the adenovirus fiber protein N-terminal fragment has a length of no more than 20 contiguous amino acids of the N-terminal fiber sequence.

14. 2. The modified polypeptide of claim 1, wherein an adenoviral fiber protein N-terminal fragment is positioned at the N-terminus of a non-adenoviral polypeptide.

15. 2. The modified polypeptide of claim 1, comprising a peptide linker between the non-adenoviral polypeptide and the adenoviral fiber protein N-terminal fragment.

16. 2. The modified polypeptide of claim 1, comprising a binding residue.

17. 17. A modified polypeptide as described in claim 16, wherein binding residues are inserted and / or positioned at the N- and / or C-terminus of the adenoviral fiber protein N-terminal fragment.

18. 20. The modified polypeptide of claim 17, wherein the adenovirus fiber protein N-terminal fragment has the sequence: X 58 -FNPVYPY-X59-(X63)n-Xc (SEQ ID NO: 69) where X 58 is selected from the group consisting of S, D and T; and X 5９ is selected from the group consisting of E, D and G; X ６３ is any amino acid occurring independently in each case; Xc is a binding residue; and n is an integer from 0 to 10.

19. 20. The modified polypeptide of claim 17, wherein the adenovirus fiber protein N-terminal fragment has the sequence: AKRARLST-X58-FNPVYPY-X59-DE-Xc (SEQ ID NO: 76) where X 58 is selected from the group consisting of S, D and T, preferably S or D, more preferably S; X 5９ is selected from the group consisting of E, D and G, preferably E or D, more preferably E; and Xc is a binding residue.

20. 18. The modified polypeptide of claim 17, wherein the adenovirus fiber protein N-terminal fragment has the sequence of SEQ ID NO:

77.

21. 17. The modified polypeptide of claim 16, wherein the linking residue is selected from the group consisting of Lys, Cys, Asp and Glu.

22. A nucleic acid encoding the polypeptide of claim 1.

23. An expression vector comprising a nucleic acid encoding the modified polypeptide of claim 1.

24. A cloning vector encoding a polypeptide, comprising: A cloning vector comprising an adenoviral fiber protein N-terminal fragment, the polypeptide specifically binding to the adenoviral fiber protein binding cleft of a penton base promoter adapted for introducing a nucleic acid encoding a non-adenoviral peptide into the C- and / or N-terminus.

25. A recombinant host cell comprising an expression vector encoding the modified polypeptide of claim 1.

26. A recombinant host cell comprising a cloning vector encoding a polypeptide comprising an N-terminal fragment of adenoviral fiber protein that specifically binds to the adenoviral fiber protein binding cleft of a penton base promoter adapted for introducing a nucleic acid encoding a non-adenoviral peptide into the C- and / or N-terminus.

27. A virus-like particle (VLP) comprising 12 pentamers comprising five adenovirus penton base promoters and at least one modified polypeptide of claim 1 that binds to the adenovirus fiber protein binding cleft.

28. 28. The virus-like particle (VLP) of claim 27, comprising up to 60 modified polypeptides of claim 1, each of which binds to the adenovirus fiber-binding cleft of an adenovirus penton base promoter.

29. 28. The virus-like particle (VLP) of claim 27, wherein each adenovirus penton based protomer is a modified polypeptide comprising a first RGD loop, a second RGD loop, a variable loop (V loop), an adenovirus fiber protein binding cleft and / or an N-terminal domain, and further wherein each adenovirus penton based protomer comprises one or more non-adenovirus peptides in the first RGD loop or the second RGD loop or both the first and second RGD loops and / or V loops, and can be assembled into modified VLPs.

30. 30. The virus-like particle (VLP) of claim 29, wherein the modified adenovirus penton base promoter comprises at least one target-specific binding domain in the first, second, both the first and second RGD loops, and / or the V loop.

31. 30. The virus-like particle (VLP) of claim 29, wherein the modified adenovirus penton base protomer comprises a non-adenovirus protein at the N- or C-terminus of the penton base protomer.

32. 30. The virus-like particle (VLP) of claim 29, wherein the modified adenovirus penton base protomer comprises heterologous binding residues in the first, second, both the first and second RGD loops, and / or the V loop, and / or the N-terminal domain of the penton base protomer, and wherein the N-terminus of the N-terminal domain in the penton base protomer is defined by the sequence X 1 -GRNSIR (SEQ ID NO: 44) The C-terminus of the N-terminal domain within the penton base protomer is defined as follows: DX 2 -RSRG (SEQ ID NO: 45) Here, X 1 is selected from the group consisting of G and E; X 2 is selected from the group consisting of D and E Virus-like particles (VLPs).

33. 30. The virus-like particle (VLP) of claim 29, wherein the modified adenovirus penton base protomer comprises a drug, label and / or polypeptide covalently or non-covalently bound to two or more amino acids of the first, second or both the first and second RGD loops of the penton base protomer and / or to two or more amino acids of the V loop of the penton base protomer.

34. 30. The virus-like particle (VLP) of claim 29, wherein the modified adenovirus penton base protomer comprises at least one heterologous binding residue in the adenovirus fiber protein binding cleft of the penton base protomer.

35. A method for producing the modified polypeptide according to claim 1, comprising the steps of: (a) providing a recombinant host cell comprising an expression vector that includes a nucleic acid encoding the modified polypeptide of claim 1. (b) expressing the modified polypeptide, and (c) purifying the modified polypeptide.

36. 28. A method for producing the VLP of claim 27, comprising the step of mixing the modified polypeptide of claim 1 with an adenovirus penton base promoter.

37. 28. A method for producing a VLP according to claim 27 comprising a disease and / or patient specific non-adenoviral peptide, comprising the steps of: (a) determining the amino acid sequence of a disease- or patient-specific non-adenoviral peptide; (b) synthesizing a modified polypeptide comprising the adenoviral fiber protein binding cleft of a penton base protomer and at least one of said non-adenoviral peptides; and (c) mixing the modified polypeptide with an adenovirus penton base protomer.

38. A pharmaceutical composition comprising the modified polypeptide of claim 1.

39. 28. A pharmaceutical composition comprising the VLP of claim 27.

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