Patent Description:
Infectious diseases continue to plague and decimate populations world-wide. Among the means at our disposal to counter this threat, vaccination has proven to be exceptionally powerful. Small-pox has been eradicated, measles, polio and tetanus constrained from the world by vaccination. Nonetheless, severe threats continue to challenge human health, notably also from emergent viruses that have adapted and emerged as new diseases or pathogenic strains with attributes facilitating pathogenicity.

Recent such examples are the severe threat posed by Chikungunya and Zika, insect-born viruses that is transmitted to humans by the bite of a mosquito. Both viruses are rapidly spreading to Asia and Europe, by means of its mosquito host, causing considerable alarm. Chikungunya and Zika disease can potentially incur severe costs for affected communities and economies, and a potent vaccination strategy to counter this emerging threat would be highly desirable. However, powerful vaccines are utterly lacking to date.

Ideally, a vaccine will be safe, non-replicative, efficient, and tunable. Moreover, it will be produced easily at industrial scale. Recombinant Virus Like Particles (VLPs) can be such ideal vaccines and therefore hold enormous promise. In this proposal, we will create such a VLP vaccine. We will make use of an amazingly versatile bio-similar multiprotein platform called ADDomer (Adenovirus dodecahedron derived multimer). ADDomer will serve to create vaccine candidates to combat infectious diseases caused by viruses (including but not limited to Chikungunya, Zika, others).

ADDomer is a synthetic scaffold derived from a virus-like particle (VLP) that in nature occurs during the human adenovirus serotype <NUM> (HAd3) replication cycle catalyzing internalization (<NPL>. ADDomer is a designed bio-similar derived from this natural VLP, retaining the aptitude to autonomously self-assemble into a dodecahedron. ADDomer is uniquely suited to display multiple peptide and protein epitopes by means of fully flexible, solvent exposed loops. Engineering these loops does not destroy the global architecture of ADDomer particles. These loops offer convenient options to insert, by using methods from synthetic biology, multiple copies of highly immunogenic peptide epitopes, for example from viral pathogens. ADDomer is not limited to vaccine development against infectious diseases. A wide range of applications will potentially benefit from the ADDomer technology, including also cancer therapy. Moreover, ADDomer can not only display peptide epitopes. Proteins or protein domains can be likewise exposed by ADDomer, significantly broadening the scope of its application.

<CIT> discloses a protein VLP vector which is a adenovirus dodecahedron (Dd) and can be used for delivering therapeutic substances belonging to classes of proteins, peptides, polysaccharides, nucleic acids, lipids, lipoproteins, or derivatives thereof, to mammalian cells.

<CIT> describes another VLP vector constituting an adenoviral dodecahedron which is intended for the delivery of therapeutic agents into specific mammalian tissues, especially low molecular weight agents, in particular low molecular weight anti-cancer drugs into cancer tissues.

<CIT> discloses a further adenoviral dodecahedral protein complex consisting of either <NUM> pentons each including at least one fibre and a penton base but no other element from an adenovirus genome, said fibre(s) and said penton base being derived from one or more adenoviruses, said pentons being bound by the penton bases and forming a proteolytic enzyme-stable dodecahedral structure, said complex having a molecular weight between <NUM> x <NUM><NUM> Da and <NUM> x <NUM><NUM> Da.

The present inventors have identified that certain regions in the penton base protomer are amenable to the introduction of heterologous peptide sequence without disrupting assembly of penton base protomers into penton subunits, which in turn can self-assemble into penton dodecamers forming virus like particles (VLPs) also referred to as ADDomers. The design is highly modular and enables rapid and flexible functionalization of extended loops for multipolypeptide display. The modularity is even further enhanced by using an adenovirus fibre protein fragment that specifically binds to the penton base protomer. The VLPs of the present invention are safe since they do not comprise genetic material. The penton base protomers can receive and display up to <NUM> foreign polypeptide motifs including antigens, neutralizing polypeptides, oncoepitope polypeptides, single chain antibodies, and nanobodies.

Disclosed herein is an engineered polypeptide comprising an adenovirus penton base protomer, wherein said penton base protomer comprises a first RGD-loop, a second RGD-loop, a variable loop (V loop), adenovirus fibre protein binding cleft and/or a N-terminal domain and comprises one or more of the following:.

As defined in claim <NUM>, the present invention relates to an a VLP comprising <NUM> pentamers each comprising five adenovirus penton base protomers and at least one engineered polypeptide, preferably <NUM> engineered polypeptides, comprising at least one adenovirus fibre protein N-terminal fragment specifically binding to an adenovirus fibre protein binding cleft of a penton base protomer and:.

wherein the adenovirus fibre protein N-terminal fragment comprises:.

In a further aspect the invention provides a method for producing a VLP of the invention as defined in claim <NUM>.

In a further aspect the present invention relates to a method for producing a VLP of the invention comprising disease and/or patient specific non-adenoviral polypeptides as defined in claim <NUM>.

In a further aspect the present invention relates to a method for producing a VLP of the invention comprising disease and/or patient specific non-adenoviral peptides as defined in claim <NUM>.

In a further aspect the present invention relates to a pharmaceutical composition comprising the VLP of the invention, and a pharmaceutically acceptable carrier and/or suitable excipient(s).

In a further aspect the present invention relates to the VLP of the invention for use in treating and/or preventing an infectious disease, an immune disease or cancer.

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Several documents are cited throughout the text of this specification.

To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf, e.g., <NPL>). 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.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or <NUM>'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the 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 e.g. RNA-DNA hybrids. The nucleic acids, can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, <NPL>). "Aptamers" are nucleic acids which bind with high affinity to a polypeptide. Aptamers can be isolated by selection methods such as SELEmirl46- a (see e.g. <NPL>; <NPL>; <CIT>) from a large pool of different single-stranded RNA molecules. Aptamers can also be synthesized and selected in their mirror-image form, for example as the L-ribonucleotide (<NPL>; <NPL>). Forms which have been isolated in this way enjoy the advantage that they are not degraded by naturally occurring ribonucleases and, therefore, possess greater stability.

The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bond-linked chain of amino acids, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the <NUM> naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility.

The term "penton base protein" or "penton base protomer" as used in the context of the present invention refers to an adenoviral protein that assembles into the so called "penton protein". Each penton protein comprises five penton base proteins. The penton protein is one of three proteins forming the adenoviruses coat. The other proteins are hexon and fibre. The structure of an assembled adenovirus is shown in <FIG> upper left corner. Penton base proteins that are used in the present invention originate from adenovirus specific to any mammalian species. Preferably the adenovirus is a human or non-human great ape adenovirus, preferably Chimpanzee (Pan), Gorilla (Gorilla) and orangutans (Pongo), more preferably Bonobo (Pan paniscus) and common Chimpanzee (Pan troglodytes). It is understood by the skilled person that the penton base proteins of different adenovirus will vary in their amino acid sequence all such naturally occurring variants are encompassed by the term "penton base protein". Additionally, the term encompasses artificial variants that comprise insertion, deletions and/or mutations of the naturally occurring penton base protein sequence. These mutations are in addition to the modifications of the N-terminal domain, V loop, first RGD, second RGD loop and/or sticky patch region described in more detail below. Any such artificial variants are comprised in as long as the artificially modified penton base protein assembles into penton subunits and <NUM> of these assemble into VLPs. Preferably, the artificial variants have at least <NUM>%, more preferably at least <NUM>%, more preferably at least <NUM>%, more preferably at least <NUM>%, more preferably at least <NUM>%, more preferably <NUM>%, more preferably <NUM>%, and more preferably <NUM>% sequence identity to a naturally occurring penton base protomer outside the N-terminal domain, the V loop, the first RGD, and the second RGD loop as defined below. Preferred penton base proteins are those indicated in SEQ ID NOs: <NUM> to <NUM>. The penton base proteins as defined above are the basis for the engineered penton base proteins of the present invention. Thus, the engineered penton base proteins differ in sequence from naturally occurring penton base proteins by amino acid insertions, deletions and mutations as outlined in more detail below.

The phrase that the "engineered polypeptide is capable of assembling into VLPs" or "assembles into a VLP" as used interchangeably in the context of the present invention refers to the ability of five penton base protomers to self-assemble into a penton protein and subsequently of twelve penton proteins to self-assemble into a small spherically shaped particle, i.e. a virus-like particle (VLP). The ability to assemble and to maintain the penton protein or preferably the VLP structure can be ascertained by methods known in the art and described herein, in particular by electron microscopy (EM). Preferred conditions at which the capability to assemble into VLPs is assessed is <NUM> and physiologic buffer conditions. In a further preferred embodiment the term encompasses engineered polypeptides that not only assemble into VLPs but maintain the spherical shape at temperatures above <NUM>, preferably at temperatures above <NUM>, preferably at temperatures above <NUM>, more preferably above <NUM> and even more preferably above <NUM>. The integrity of the spherical shape can be assessed by EM, preferably under physiological buffer conditions.

The term "first RGD-loop" as used in the context of the present invention refers to a polypeptide sequence of between <NUM> to <NUM> amino acids that is located N-terminally to the "RGD motif comprised in the penton protomer (see <FIG>). This polypeptide sequence is highly divergent between different adenoviruses. Accordingly, it cannot be defined by homology but can be defined by the sequence that is located N-terminally of its N-terminal end. Its C-terminal end within the penton protomer is determined by the RGD motif.

The term "second RGD-loop" as used in the context of the present invention refers to a polypeptide sequence of between <NUM> to <NUM> amino acids that is located C-terminally to the "RGD motif comprised in the penton protomer (see <FIG>). This polypeptide sequence is highly divergent between different adenoviruses. Accordingly, it cannot be defined by sequence homology. Its N-terminal end within the penton protomer is determined by the RGD motif. Its C-terminal end within the protomer can be defined by the sequence that is located C-terminally of its C-terminal end, which is conserved among different adenoviruses.

The term "RGD motif as used in the context of the present invention refers to a three amino acid long polypeptide composed 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 protomers of the engineered polypeptides of the present invention may be intact or may be mutated in a way that the penton protomer does no longer bind to integrins.

The term "variable loop" as used in the context of the present invention corresponds to a sequence located between the beta sheet sheet b3 and the beta sheet b4 of the adenovirus penton base. Both the length and the aminoacids composition of this loop are are very variable amongst serotypes. The sequences corresponding to variable loops are highlighted in green in <FIG>. The term "N-terminal domain" as used in the context of the present invention refers to a highly conserved region in the N-terminus of the penton base protomer. This part of the protein comprises the al and a2 helices, the Bl and B2 sheets as well as the B and C domain (see <FIG>). It is involved in the interaction between penton base protomers and, thus suitable for the introduction of moieties, e.g. coupling residues that stabilize the interaction between the penton base protomers.

The term "adenovirus fibre protein binding cleft" as used in the context of the present invention refers to a fold of a penton base protomer forming the interaction surface with the adenovirus fibre protein. As can be seen in <FIG> the binding cleft is formed by several non-contiguous stretches of polypeptide sequence which are conserved among different adenoviruses.

The term "target specific binding domain" as used throughout the specification refers to a polypeptide which facilitates specific binding to a target. The binding of such a target specific binding domain is considered specific to a given target if it binds with the highest affinity to the respective target and only with lower affinity, e.g. at least <NUM>-fold lower, preferably at least <NUM>- fold lower affinity to other targets even to targets with a related amino acid sequence.

The term "target" as used in the present invention refers to a natural existing cellular or molecular structure towards which molecules have a certain binding affinity or to which molecules specifically bind. A target may comprise one or more epitopes. An antigen is a preferred example of a target.

The term "antigen" as used in the context of the present invention to refer to any structure recognized by molecules of the immune response, e.g. antibodies, T cell receptors (TCRs) and the like. An antigen may be foreign or toxic to the body or may be a cellular protein that is associated with a particular disease. Antigens are recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system and may elicit a humoral or cellular immune response. Antigens that elicit such a response are also referred to as immunogen. A fraction of the proteins inside cells, irrespective of whether they are foreign or cellular, are processed into smaller peptides and presented to by the major histocompatibility complex (MHC). A cellular immune response is elicited, if the small peptide fragment is bound by a T-cell receptor. Cell surface antigens can be selected from the group of cytokine receptors, integrins, cell adhesion molecules, cell type-specific cell surface antigen, tissue-specific cell surface antigen, cell surface-expressed tumor-associated antigen, cluster of differentiation antigens, or carbohydrates. The term "specific binding" as used in the context of the present invention to mean that a binding moiety (e.g. an antibody) binds stronger to a target, such as an epitope, for which it is specific compared to the binding to another target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Targets can be recognized by their ligands which bind with a certain affinity to their targets and thus, the ligand binding to its respective target results in a biological effect. Preferably, the binding is both specific and occurs with a high affinity, preferably with Kd of less than <NUM>"<NUM> , <NUM>" <NUM> , <NUM>"<NUM> , <NUM>"<NUM> or less. Such affinity is preferably measured at <NUM> Suitable assays include surface plasmon resonance measurements (e.g. Biacore), quartz crystal microbalance measurements (e.g. Attana), and competition assays.

The term "antibodies" as used in the context of the present invention are glycoproteins belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably. An antibody refers to a protein molecule produced by plasma cells and is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, its antigen.

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 within the term "antibody fragment" include a fragment antigen binding (Fab) fragment, a Fab' fragment, a F(ab')<NUM> fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and a fusion protein thereof.

The term "diabody" as used within this specification refers to a fusion protein or a bivalent antibody which can bind different antigens. A diabody is composed of two single protein chains which comprise fragments of an antibody, namely variable fragments. Diabodies comprise a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL, or VL-VH). By using a short peptide connecting the two variable domains, the domains are forced to pair with the complementary domain of another chain and thus, create two antigen-binding sites. Diabodies can target the same (monospecific) or different antigens (bispecific).

The term "single domain antibody" as used in the context of the present invention refers to antibody fragments consisting of a single, monomeric variable domain of an antibody. Simply, they only comprise the monomeric heavy chain variable regions of heavy chain antibodies produced by camelids or cartilaginous fish. Due to their different origins they are also referred to VHH or VNAR (variable new antigen receptor)-fragments. Alternatively, single-domain antibodies can be obtained by monomerization of variable domains of conventional mouse or human antibodies by the use of genetic engineering. They show a molecular mass of approximately <NUM>-<NUM> kDa and thus, are the smallest antibody fragments capable of antigen recognition. Further examples include nanobodies or nanoantibodies.

The term "antibody mimetic" as used within the context of the present specification refers to compounds which can specifically bind antigens, similar to an antibody, but are not structurally related to antibodies. Usually, antibody mimetics are artificial peptides or proteins with a molar mass of about <NUM> to <NUM> kDa which comprise one, two or more exposed domains specifically binding to an antigen. Examples include inter alia the LACI-D1 (lipoprotein-associated coagulation inhibitor); affilins, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10th type III domain of fibronectin; adnectins: knottins (cysteine knot miniproteins); atrimers; evibodies, e.g. CTLA4-based binders, affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; affilins; armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimetics as well (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs.

As used herein, the term "Kd" (usually measured in "mol/L", sometimes abbreviated as "M") is intended to refer to the dissociation equilibrium constant of the 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 determining Kd include, without limitation, ELISA and surface plasmon resonance assays.

The term an "epitope", also known as antigenic determinant, as used in the context of the present invention is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. As used herein, an "epitope" is the part of a macromolecule capable of binding to an antibody (e.g. an antibody or antigen-binding fragment thereof) as described herein. 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 former but not the latter is lost in the presence of denaturing solvents.

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 said macromolecule. In the context of the present application, a "conformational epitope" is a "discontinuous epitope", i.e. the conformational epitope on the macromolecule (e.g. a polypeptide) which is formed from at least two separate regions in the primary sequence of the macromolecule (e.g. the amino acid sequence of a polypeptide). In other words, an epitope is considered to be a "conformational epitope" in the context of the present invention, if the epitope consists of at least two separate regions in the primary sequence to which an antibody of the invention (or an antigen-binding fragment thereof) binds simultaneously, wherein these at least two separate regions are interrupted by one or more regions in the primary sequence to which an antibody of the invention (or an antigen-binding fragment thereof) does not bind. Preferably, such a "conformational epitope" is present on a polypeptide, and the two separate regions in the primary sequence are two separate amino acid sequences to which an antibody of the invention (or an antigen-binding fragment thereof) binds, wherein these at least two separate amino acid sequences are interrupted by one more amino acid sequences in the primary sequence to which an antibody of the invention (or an antigen-binding fragment thereof) does not bind. Preferably, the interrupting amino acid sequence is a contiguous amino acid sequence comprising two or more amino acids to which the antibody (or the antigen-binding fragment thereof) does not bind. The at least two separate amino acid sequences to which an antibody of the invention (or an antigen-binding fragment thereof) binds are not particularly limited with regard to their length. Such a separate amino acid sequence may consists of only one amino acid as long as the total number of amino acids within said at least two separate amino acid sequences is sufficiently large to effect specific binding between the antibody (or the antigen- binding fragment thereof) and the conformational epitope.

The term "adenovirus fibre protein" as used in the context of the present invention refers to an adenoviral protein that non-covalently binds to a penton protomer and aids in attachment of the adenovirus to the host cell.

The term "sequence identity" is used throughout the specification with regard to polypeptide and polynucleotide sequence comparisons. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID, if not specifically indicated otherwise. For example, a polypeptide sequence consisting of <NUM> amino acids compared to a reference <NUM> amino acid long polypeptide sequence may exhibit a maximum percentage of sequence identity of <NUM>% (<NUM>/<NUM>) while a sequence with a length of <NUM> amino acids may exhibit a maximum percentage of sequence identity of <NUM>%> (<NUM>/<NUM>). If <NUM> out of those <NUM> amino acids are different from the respective amino acids of the <NUM> amino acid long reference sequence, the level of sequence identity decreases to <NUM>%. The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (<NPL>), with hmmalign (HMMER package, http://hmmer. edu/) or with the CLUSTAL algorithm (<NPL>) available e.g. on http://www. uk/Tools/clustalw/ or on http://www. uk/Tools/clustalw2/index. html or on http://npsa-pbil. fr/cgi-bin/npsa_automat. pl?page=/NPSA/npsa_clustalw. Preferred parameters used are the default parameters as they are set on http://www. uk/Tools/clustalw/ or http://www. uk/Tools/clustalw2/index. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of <NPL>. BLAST polynucleotide searches are performed with the BLASTN program, score = <NUM>, word length = <NUM>. BLAST protein searches are performed with the BLASTP program, score = <NUM>, word length = <NUM>. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in <NPL>. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (<NPL>) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise. "Hybridization" can also be used as a measure of sequence identity or homology between two nucleic acid sequences. A nucleic acid sequence encoding F, N, or M2-<NUM>, or a portion of any of these can be used as a hybridization probe according to standard hybridization techniques. Hybridization conditions are known to those skilled in the art and can be found, for example, in<NPL>. "Moderate hybridization conditions" are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at <NUM>, followed by a wash in IX SSC, <NUM>% SDS at <NUM>. "Highly stringent conditions" are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at <NUM>, followed by a wash in <NUM> X SSC, <NUM> % SDS at <NUM>.

The term "coupling residue" as used in the context of the present invention refers to a natural or non-naturally occurring amino acid that has a side chain, capable of forming a covalent bond. Coupling residues can be inserted into a polypeptide of the present invention. If the coupling residue is a naturally occurring amino acid that is encoded by DNA the insertion of a coupling residue merely requires the modification of the DNA that is directing expression of the polypeptide of the invention, e.g. insertion of a codon that encodes such amino acid or mutation of an existing codon. Preferred examples of naturally occurring amino acids that are coupling residues within the meaning of this term are Asp, Glu, Lys and Cys. Cys is particularly preferred since it will form a disulfide bond with another Cys depending on the redox-status of the environment. In particular the latter allows the formation of a stable interconnection between two separate polypeptides.

The term "label" as used in the context of the present invention refers to any kind of compound being suitable for diagnostic purposes. Preferred compounds are selected from a fluorescent dye, a radioisotope and a contrast agent. A contrast agent is a dye or other substance that helps to show abnormal areas inside the body. In one embodiment the term label refers to a compound that comprises a chelating agent which forms a complex with divalent or trivalent metal cations. Preferred radioisotopes/fluorescence emitting isotopes are selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescence emitting isotopes, such as18 F,<NUM> Cr,<NUM> Ga,<NUM> Ga,m In,<NUM> Tc,<NUM> La, <NUM> Yb,<NUM> Sm,<NUM> Ho,<NUM> Y,<NUM> Y,<NUM> Pm, <NUM> Lu,<NUM> Sc,<NUM> Pr,<NUM> Gd,<NUM> Bi,<NUM> As,<NUM> Se,<NUM> Ru, <NUM> Pd, <NUM> Rh,101ml5 Rh, <NUM> Sb,<NUM> Ba,<NUM><NUM>,<NUM><NUM>,<NUM> I,<NUM> Hg,<NUM> At, <NUM> Eu,<NUM> Pb,<NUM> Pb,<NUM> Cu,<NUM> Cu,<NUM> Re,<NUM> Re,<NUM> Au and199 Ag. Preferred fluorescent dyes are selected from the following classes of dyes: Xanthens (e.g. Fluorescein), Acridines (e.g. Acridine Yellow), Oxazines (e.g. Oxazine <NUM>), Cynines (e.g. Cy7 / Cy <NUM>), Styryl dyes (e.g. Dye- <NUM>), Coumarines (e.g. Alexa Fluor <NUM>), Porphines (e.g. Chlorophyll B), Metal-Ligand- Complexes (e.g. PtOEPK), Fluorescent proteins (e. g APC, R-Phycoerythrin), Nanocrystals (e. g QuantumDot <NUM>), Perylenes (e.g. Lumogen Red F300) and Phtalocyanines (e.g. IRDYE™700DX) as well as conjugates and combinations of these classes of dyes. Preferred contrast agents are selected from paramagnetic agents, e.g. Gd, Eu, W and Mn, preferably complexed with a chelating agent. Further options are supramagnetic iron (Fe) complexes and particles, compounds containing atoms of high atomic number, i.e. iodine for computer tomography (CT), microbubbles and carriers such as liposomes that contain these contrast agents.

The term "drug" is to be understood in the context of the present invention in its broadest sense to refer to any compound that elicits a prophylactic, therapeutic or palliative effect in a patient. Preferably, it is a small molecule, e.g. with a molecular size of below <NUM> D.

A "linker" in the context of the present invention refers to any chemical moiety that is flexible and sterically separates two chemical moieties, e.g. an engineered polypeptide comprised in the VLP of the invention from a drug or label. Preferred linkers are moieties wich have a length to width ratio of at least <NUM>: <NUM>, preferably of at least <NUM> : <NUM> , more preferably of at least <NUM>: <NUM>. Preferably, linkers are linear molecules. It is preferred that the two moieties linked by a linker are covalently or non-covalently, preferably covalently attached to the respective ends of the linker.

A "peptide linker" in the context of the present invention refers to an amino acid sequence, i.e. polypeptide, which sterically separates two parts within the engineered polypeptides of the present invention. Typically such linker consists of between <NUM> and <NUM>, preferably <NUM> to <NUM> more preferably <NUM> to <NUM> amino acids. Thus, such linkers have a minimum length of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> amino acids, and a maximum length of at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> amino acids or less. Peptide linkers may also provide flexibility among the two parts that are linked together. Such flexibility is generally increased, if the amino acids are small. Accordingly, flexible peptide linkers comprise an increased content of small amino acids, in particular of glycins and/or alanines, and/or hydrophilic amino acids such as serines, threonines, asparagines and glutamines. Preferably, more than <NUM>%, <NUM>%>, <NUM>%>, <NUM>%>, <NUM>%> or more of the amino acids of the peptide linker are small amino acids.

The terms "preparation" and "composition" are intended to include the formulation of the active compound, e.g. the VLPs of the present invention with a carrier and/or excipient.

"Pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier", as used herein, refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, surfactants, stabilizers, physiological buffer solutions or vehicles with which the therapeutically active ingredient is administered. Such pharmaceutical carriers can be liquid or solid. Liquid carrier include but are not limited to 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 and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.

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

"Surfactants" include anionic, cationic, and non-ionic surfactants such as but not limited to sodium deoxycholate, sodium dodecylsulfate, Triton X-<NUM>, and polysorbates such as polysorbate <NUM>, polysorbate <NUM>, polysorbate <NUM>, polysorbate <NUM> and polysorbate <NUM>.

"Stabilizers" include but are not limited to mannitol, sucrose, trehalose, albumin, as well as protease and/or nuclease antagonists.

"Physiological buffer solution" that may be used in the context of the present invention include but are not limited to sodium chloride solution, demineralized water, as well as suitable organic or inorganic buffer solutions such as but not limited to phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([<NUM> (<NUM> hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (<NUM> morpholino-<NUM> propanesulphonic acid). The choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer are suitable, for example, for injection and infusion solutions.

The term "adjuvant" refers to agents that augment, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at either the cellular or humoral level, e.g. immunologic adjuvants stimulate the response of the immune system to the actual antigen, but have no immunological effect themselves. Examples of such adjuvants include but are not limited to inorganic adjuvants (e.g. inorganic metal salts such as aluminium phosphate or aluminium hydroxide), organic adjuvants (e.g. saponins or squalene), oil-based adjuvants (e.g. Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g. IL-Iβ, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, GM-CFS, and INF-γ) particulate adjuvants (e.g. immuno- stimulatory complexes (ISCOMS), liposomes, or biodegradable microspheres), virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or muramyl peptides), synthetic adjuvants (e.g. non-ionic block copolymers, muramyl peptide analogues, or synthetic lipid A), or synthetic polynucleotides adjuvants (e. g polyarginine or polylysine).

An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent sufficient to achieve the 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 to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.

The present invention provides inter alia the following advantages over the prior art: (i) an easily modified scaffold for antigen and/or target specific binding domain insertion/presentation, which can be tailored to the need of a patient or easily adapted to the changing surface antigens of viruses, (ii) a stable composition that can be used for, e.g. vaccination even under adverse storage conditions, e.g. high heat, (iii) an extremely high density vehicle for presenting one or multiple antigens, (iv) the use of a fibre (STICKER) protein to add further antigens or other activities on the fly.

In one embodiment, the VLP as defined in claim <NUM> is further characterized in that the penton base protomers comprise a first RGD-loop, a second RGD-loop, a variable loop (V loop), adenovirus fibre protein binding cleft and/or a N-terminal domain, and comprises one or more of the following:.

and wherein the engineered polypeptide is preferably capable of assembling into VLPs.

If in one of above embodiments a residue or group of residues, e.g. a target specific binding domain, one or more non-adenoviral polypeptides or at least one heterologous coupling residue, is indicated to be comprised in a certain region of the penton base protein this residue or group of residues may be inserted within the respectively indicated region of the penton base protein, i.e. may be an addition, or it may be inserted and additional at least one or all of the amino acids forming the respectively indicated first RGD-loop, second RGD-loops and/or in V loop may be deleted without affecting the capability to assemble into VLPs.

In a another preferred embodiment of the VLP as defined in claim <NUM> is further characterized in that the penton base protomers comprise a first RGD-loop, a second RGD-loop, a variable loop (V loop), adenovirus fibre protein binding cleft and/or a N-terminal domain, and comprises one or more non-adenoviral polypeptides in the first, the second or both the first and the second RGD- loops and/or in the V loop; and optionally further comprises one or more of the following:.

The at least one target specific binding domain in the first, the second or both the first and the second RGD-loops, and/or in the V loop provides the assembled VLP with the ability to specifically bind to a target structure, e.g. a cellular receptor on the surface of a cell. It is a surprising discovery of the present inventors that these parts of the penton base protomer can comprise a target specific binding domain of considerable length without disrupting penton or VLP formation. Additionally, the target specific binding domain comprised in these regions is free to interact with and bind to targets. The one or more target specific binding domains may be inserted at any point in the respective loops, i.e. without removing any of the loop amino acids. Alternatively, all or part of the respective loop amino acids may be replaced by the amino acids of the target specific binding domain. The target specific binding domain may be flanked N- and/or C-terminally by peptide linkers.

If the penton base protomer in the VLP of the invention as defined in claim <NUM> comprises more than one target specific binding domain, it is preferred that these are comprised in different loops of the penton base protomer, e.g. in the first and second RGD-loop, in the first RGD-loop and the V-loop, or the second RGD-loop and the V-loop. If the penton base protomer comprises more than one target specific binding domain it is also preferred that they bind to different targets, e.g. to a target on a first type of cell and to a different target on a second type of cell. Such dual or multiple specificities can be used to bring together cells that do not normally or not frequently enough interact with each other. Examples of such cells are tumor cells and cells of the immune system, in particular cytotoxic T cells.

In an alternative embodiment (ii) that can be combined with one or more of the other alternative embodiments outlined above the first, the second or both the first and the second RGD-loops and/or in the V loop comprise a non-adenoviral polypeptide. This embodiment is also based on the surprising observation that polypeptides inserted into one or more of these regions of the penton base protomer are sufficiently exposed to be recognized by cells of the immune system and, thus to elicit an immune response. The term "non-adenoviral" polypeptide refers to a polypeptide that has no sequence identity to any polypeptide present in an adenovirus, in particular in naturally occurring adenovirus penton base protomers over a length of at least <NUM> amino acids. Preferably, the non-adenoviral polypeptide has no sequence identity to any polypeptide present in adenovirus over a stretch of at least <NUM>, preferably at least <NUM> amino acids. The one or more non-adenoviral polypeptides may be inserted in each case independently at any point in the respective loops, i.e. without removing any of the loop amino acids. Alternatively, all or part of the respective loop amino acids may be replaced by the amino acids of the target specific binding domain. The non-adenoviral polypeptide comprised in one or more of the loops may be flanked N- and/or C-terminally by peptide linkers. This may be preferred to increase exposure of the non-adenoviral polypeptide on the surface of the VLP. If at least one non-adenoviral polypeptide is inserted into each loop than each penton base protomer comprises at least three identical or different non-adenoviral polypeptides on its surface. Once assembled into VLPs at least <NUM> non-adenoviral polypeptides may be displayed at the surface of the VLPs of the present invention.

The present inventors have surprisingly found that long amino acid sequences of <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more, <NUM> or more or <NUM> or more amino acids may be introduced into the first, the second or both the first and the second RGD-loops, and/or in the V loop without disrupting the penton base protomers ability to assemble into penton proteins and subsequently into VLPs. Thus, in the embodiment indicated under (i) and/or (ii) amino acid sequences of above indicated length may be inserted (with or without deletion of some of the amino acids with the respective loops).

If the alternative embodiments indicated under (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 present inventors have observed that polypeptides positioned at either N- and/or C-terminus of the penton base protomer do not interfere with penton and subsequently VLP assembly and are surface exposed in an assembled VLP. Thus, in a further alternative embodiment (iii) a non-adenoviral polypeptide may be linked with or without intervening peptide linker to the N- and/or C-terminus of the penton base protomer. Accordingly, if combined with the first and/or second embodiment a penton base protomer may comprise non- adenoviral polypeptides in one or more of the loops, preferably all three loops and at the N- terminus, C-terminus or N- and C-terminus. It is preferred that this alternative embodiment is combined with at least one of the other alternative embodiments (i), (iii), (iv), (v) and/or (vi).

The observations of the present inventors relating to the possibility to insert heterologous peptide sequence 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 respectively indicated loop) led to a further alternative embodiment (iv) that may be combined with one or more of the previously discussed alternative embodiments. In this embodiment at least one heterologous coupling residue is introduced in the first, the second, or both the first and the second RGD-loops and/or in the V loop. By the insertion of one or more coupling residues it becomes possible to covalently couple further molecules to the loops. It is, for example envisioned that a VLP is first assembled from the engineered polypeptide of the first aspect comprising one or more coupling residue in one or more of the loops and that subsequently a polypeptide also comprising a coupling residue is coupled covalently to the VLP. Using this strategy it is possible to "decorate" the surface of the VLP with polypeptides. Such VLPs may be used to elicit a humoral and/or cellular immune response against such polypeptides.

Furthermore, the present inventors have identified a region within the penton base protomer referred to as the "N-terminal domain of the penton base protomer". This domain is involved in the interaction between the penton base protomers within penton and also in the interaction among pentons forming a VLP. The insertion of coupling residues into this region allows the formation of covalent bonds between two or more penton base protomers within the same or separate pentons. The formation of such covalent bonds stabilizes the assembled VLP. The N-terminal domain is highly conserved among different adenovirus species. It is, therefore possible to further delineate the N-terminal and C-terminal end of this domain within the penton base protomer. Thus, it is preferred that one or more the coupling residues are comprised in the N-terminal domain. The coupling residue may replace an existing amino acid or may be inserted in addition to the amino acids forming the N-terminal domain. It is preferred that the one or more coupling residue replace a residue within the N-terminal domain. The N-terminus of the N-terminal domain within the penton base protomer is preferably defined as follows:
X<NUM>-G-R-N-S-I-R (SEQ ID NO: <NUM>)
and the C-terminus of the N-terminal domain within the penton base protomer is preferably defined as follows:
D-X<NUM>-R-S-R-G (SEQ ID NO: <NUM>), wherein.

Accordingly, in this alternative embodiment (iv) one or more coupling residues are comprised within the amino acid sequence of the penton base protomer comprised in the VLP of the present invention delimited by above N- and C-terminal region. It will be understood by the skilled person that it is also possible in this embodiment to replace one or more amino acid residues within SEQ ID NO: <NUM> or <NUM>. The coupling residue may be positioned anywhere within the N-terminal domain in as long as it does not interfere with assembly of penton or VLP.

A preferred protomer amino acid sequence that can be modified according to alternatives (i) to (vi) of the first embodiment is SEQ ID NO: <NUM> (the encoding nucleotide sequence is indicated in SEQ ID NO: <NUM>). It is preferred that the insertions 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 coupling residue according to embodiment (iv), and/or the covalent or non-covalent coupling of the drug or polypeptide to one or more amino acids of the first, the second or both the first and the second RGD-loops and/or one or more amino acids of the V loop according to embodiment (v) occurs in the first RGD-loop between amino acids <NUM> to <NUM> of SEQ ID NO: <NUM> and/or in the second RGD-loop between amino acids <NUM> to <NUM> of SEQ ID NO: <NUM> and/or in the V loop between amino acids <NUM> to <NUM> of SEQ ID NO: <NUM>.

Such insertion(s) may delete all or part of the respectively indicated amino acids belonging to the first and second RGD loop and the V loop.

Preferably, there has to be a PAIR of coupling residues, preferably mutations to cysteines to enable disulfide bond formation. The resulting stabilized VLP contains up to <NUM> disulfide bonds and is hyperstable at <NUM> and even at <NUM>, at least for several months. In a particularly preferred embodiment the coupling residues are located at amino acid position <NUM> and <NUM> with reference to SEQ ID NO: <NUM>, i.e. a preferred penton base protomer amino acid sequence based on human Ad B3 or at analogous positions of a penton base protomer of another adenovirus, or at amino acid position <NUM> and <NUM> with reference to SEQ ID NO: <NUM> or at analogous positions of a penton base protomer of another adenovirus.

It has been further discovered that a coupling residue at amino acid position <NUM> (with reference to SEQ ID NO: <NUM>) can form a covalent bond with a coupling residue at amino acid position <NUM> (with reference to SEQ ID NO: <NUM>) or at analogous positions of a penton base protomer of another adenovirus. The latter residue is outside the N-terminal domain. Thus, if a coupling residue is inserted at position <NUM> it is preferred that a second coupling residue is positioned at amino acid <NUM> with reference to SEQ ID NO: <NUM> or at analogous positions of a penton base protomer of another adenovirus. With reference to <FIG> and by including further penton base proteins in the alignment the skilled person can easily determine those residues in the respective penton base protomer that occupies an analogous position as amino acids. <NUM>, <NUM>, <NUM>, <NUM> and <NUM> of SEQ ID NO: <NUM>.

It is preferred in this embodiment that the penton base protomer comprises the following sequences:.

Particularly, preferred stabilized penton base protomers comprise or consist of the amino acid sequences according to SEQ ID NO: <NUM> to <NUM>. It is further preferred that these amino acid sequences comprise one or more of the modifications according to alternative embodiments (i), (ii), (iii), (iv) in as far as this alternative embodiment is not relating to the N-terminal domain, (v) or (vi) of the preferred embodiments of the invention described above.

It is preferred that the insertions 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 coupling residue according to embodiment (iv), and/or the covalent or non-covalent coupling of the drug or polypeptide to one or more amino acids of the first, the second or both the first and the second RGD-loops and/or one or more amino acids of the V loop according to embodiment (v) occurs in the first RGD-loop occurs between amino acids <NUM> to <NUM> of SEQ ID NO: <NUM> to <NUM> and/or that the insertion into the second RGD-loop occurs between amino acids <NUM> to <NUM> of SEQ ID NO: <NUM> to <NUM> and/or that the insertion into the V loop occurs between amino acids <NUM> to <NUM> of SEQ ID NO: <NUM> to <NUM>. Such insertion(s) may delete all or part of the respectively indicated amino acids belonging to the first and second RGD loop and the V loop.

The thermal stabilization of penton and VLPs is desirable also in the context of any of the other alternative embodiments of the present invention. Accordingly, the alternative embodiment mentioned under (iv) above in relation to the N-terminal domain is preferably combined with one or more alternative embodiments (i), (ii), (iii), (iv) in as far as this alternative embodiment is not relating to the N-terminal domain, (v) or (vi). It is also preferred that the alternative embodiments mentioned under (iv) and (vi) are present in the engineered penton base protomer and are combined with one or more of (i), (ii), (iii), (iv) in as far as this alternative embodiment is not relating to the N-terminal domain, or (v).

In a further alternative embodiment (v) of the invention that may be combined with one or more of the other alternative embodiments as outlined above a drug, label and/or polypeptide is covalently or non-covalently coupled to one or more amino acids of the first, the second or both the first and the second RGD-loops and/or one or more amino acids of the V loop of the penton base protomer. Again, this embodiment is based on the observation that the coupling of moieties to these regions does not interfere with penton and VLP assembly and leads to decoration of the VLP with these moieties. In a preferred embodiment the drug or label is attached to the penton base protomer through a linker, preferably a peptide linker, that is cleavable under physiologic condition, e.g. a protease thereby releasing the drug from the VLP at the site of action. In this preferred embodiment the linker, preferably peptide linker comprises an endopeptidase cleavage site.

In a preferred embodiment of the invention the N-terminal fragment of adenoviral fibre as defined in claim <NUM> is used to non-covalently attach a moiety, e.g. a polypeptide, drug, or label etc. to the assembled VLP. This interaction is mediated via the adenovirus fibre protein binding cleft of the penton base protomer that is present in the VLP of the invention. In another preferred embodiment described below the N-terminal fibre fragment comprises a heterologous coupling residue for covalent attachment of the fibre fragment to the penton base protomer. Since a coupling residue requires a counterpart, i.e. a residue with which it can form a covalent bond, it is a further preferred alternative embodiment (vi) of the of the invention that at least one heterologous coupling residue is comprised in the adenovirus fibre protein binding cleft of the penton base protomer. The coupling residue in the binding cleft and the fibre protein N-terminal fragment are positioned in such as to allow formation of a covalent bond once the fibre protein fragment is bound in the cleft of the penton base protomer.

Each penton base protomer interacts with one adenovirus fibre protein N-terminal fragment via the highly conserved region referred to herein as the "adenovirus fibre protein binding cleft of the penton base protomer". This interaction is used to indirectly attach a non-adenoviral peptide and/or a drug or label to the penton base protomer and to present upon assembly of <NUM> penton base protomers up to <NUM> further moieties on the surface of the assembled VLP of the present invention. Accordingly, as defined in claim <NUM> the present invention relates to VLP comprising <NUM> pentamers each comprising five adenovirus penton base protomers and at leat one engineered polypeptide comprising, essentially consisting or consisting of at least one adenovirus fibre protein N-terminal fragment specifically binding to an adenovirus fibre protein binding cleft of a penton base protomer and:.

wherein the the adenovirus fibre protein N-terminal fragment is further characterized as defined in claim <NUM>.

The at least one adenovirus fibre protein N-terminal fragment specifically binding to an adenovirus fibre protein binding cleft of a penton base protomer comprised in the engineered polypeptide of the VLP of the invention is throughout this specification also referred to as STICKER.

Surprisingly, a relatively small N-terminal fragment of adenovirus fibre protein as defined in claim <NUM> was sufficient to specifically bind to a penton base protomer. The smaller the fibre fragment the bigger the moiety that can be attached to the penton base protomer. Furthermore, the reduction of the length of the adenoviral fibre fragment reduces the likelihood that a new immune response is elicited against adenovirus fibre and/or that fibre bound to VLPs are cleared by preexisting anti-fibre antibody. It is, thus preferred that the fibre fragment has a length of <NUM> contiguous amino acids or less of N-terminal fibre sequence. It is more preferred that the length of the fragment is <NUM> amino acids or less, <NUM> amino acids or less, <NUM> amino acids or less, <NUM> amino acids or less or <NUM> amino acids or less. The minimal fibre amino acid sequence required for specific binding to the binding cleft of the penton base protomer is as defined in claim <NUM>. This minimal sequence is preferably flanked by other adenovirus fiber, preferably Ad3 amino acid sequence on both sides. This small fragment is used according to the invention for extending the versatility and/or the number or exposed epitope on the VLP surface. The addition of the STICKER tag to any protein or epitope sequence enables their binding to the VLP surface. This is done by in vitro incubation of the STICKER containing protein with the VLP or by co-expression of both components in a baculovirus system. It is preferred that the engineered polypeptide bound to the VLP of the invention does not comprise any further fibre amino acid sequence contiguous with STICKER. More preferably the polypeptide bound to the VLP of the invention of the invention does not comprise any other adenovirus proteins or polypeptides other than STICKER.

It has been surprisingly found that STICKER can be attached N- and/or C-terminally without interfering with its binding to a penton base protomer. Preferably, STICKER is attached to the N-terminus of a non-adenoviral polypeptide. This polypeptide can be any polypeptide for which it is desired to attach it to the surface of the VLPs of the present invention. The size of the polypeptide that is attached to STICKER is not particularly limited. It can be any size that still allows specific binding to the fibre protein binding cleft of a penton base protomer. The engineered polypeptide may 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 <NUM> of such polypeptides to bind via STICKER to the assembled VLP. A peptide linker may also be advantageous in situations in which the N- and/or C-terminus to which STICKER is attached is buried within the polypeptide.

The majority of the human population has been exposed to human Ad5 and have memory B cells capable of mounting an immune response against human Ad5. Accordingly, if human Ad5 based protomers and/or fibre are comprised in the VLP of the invention, the resulting VLPs are more likely to be cleared from the circulation by a pre-existing immunity. Thus, in a preferred embodiment the adenoviral proteins comprised in the VLP of the invention is based on adenovirus penton and fibre proteins, respectively, from human or non- human great ape adenoviruses, preferably from Chimpanzee (Pan) adenoviruses, Gorilla (Gorilla) adenoviruses and orangutans (Pongo) adenoviruses, more preferably Bonobo {Pan paniscus) and common Chimpanzee {Pan troglodytes).

It is particularly preferred that the VLP of the invention is based on penton and fibre proteins, respectively, of adenovirus selected from the group consisting of hAd3, hAd4, hAd5, hAd7, hAdl <NUM>, hAd26, hAd35 and hAd49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAdlO, ChAdl l, ChAdl6, ChAdl7, ChAdl9, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82, PanAdl, PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAdl46, and ChAdl47 described in <CIT> and <CIT>. The VLP of the invention is preferably based on the wild-type penton base protomer of SEQ ID NO: <NUM> to <NUM>, i.e. SEQ ID NO: <NUM> to <NUM> reflects the sequence of the protein prior to modification according to alternative embodiments (i) to (vi) outlined above. It will be understood by the skilled person that the insertion of a target specific binding domain into the V-loop, first and/or second RGD loop will alter the sequence in that part of SEQ ID NO: <NUM> to <NUM>. Similarly, the replacement of amino acids with coupling residues will also alter the amino acid sequence.

The modifications according to (i) and (ii) above require the modification of one or both of the RGD loops and/or the V loop. In a preferred embodiment of the VLP of the invention the region to be modified are defined by consensus sequences common to the majority of adenoviruses. Thus, these consensus sequences are based on the alignment of several preferred penton base protomer amino acid sequences from adenoviral species and are, suitable to determine the N- and C-terminus respectively, of the part of the penton base protein to modify according to embodiment (i) or (ii) noted above. Preferably, the N-terminus of the first RGD-loop within the penton base protomer is defined as follows:
X<NUM>-X<NUM>-X<NUM>-X<NUM>-X<NUM>-X<NUM>-X<NUM>-X<NUM>-X<NUM> (SEQ ID NO: <NUM>)
wherein.

In a preferred embodiment of the VLP of the invention the amino acid sequence of X3 to X10 is independently of each other selected from the group consisting of DVTAYEES (SEQ ID NO: <NUM>), DVDAYENS (SEQ ID NO: <NUM>), DVAEYEKS (SEQ ID NO: <NUM>), DVEAYEKS (SEQ ID NO: <NUM>), DVDAYEKS (SEQ ID NO: <NUM>), DVSKYEAS (SEQ ID NO: <NUM>), NVKAYEDS (SEQ ID NO: <NUM>), DVKKYENS (SEQ ID NO: <NUM>), DVDAYQAS (SEQ ID NO: <NUM>), and DVDAYQAS (SEQ ID NO: <NUM>), the amino acid sequence of Xis to X24 is selected from the group consisting of IQPLEKD (SEQ ID NO: <NUM>), IQPVEKD (SEQ ID NO: <NUM>), IKPLEKD (SEQ ID NO: <NUM>), IVPLTKD (SEQ ID NO: <NUM>), IEPVETD (SEQ ID NO: <NUM>) and IKPLTED (SEQ ID NO: <NUM>), the amino acid sequence of X25 to X31 is selected from the group consisting of FKARVMV (SEQ ID NO: <NUM>), FRAKLMV (SEQ ID NO: <NUM>), and FRAKVMV (SEQ ID NO: <NUM>), the amino acid sequence of X33 to X39 is selected from the group consisting of YEWFEF (SEQ ID NO: <NUM>), YEWVEF (SEQ ID NO: <NUM>), and YEWAEF (SEQ ID NO: <NUM>).

It has been surprisingly found that large heterologous polypeptides can be inserted and or replace the first and/or second RGD loop without disrupting assembly of penton and subsequently the VLPs of the invention.

In a preferred embodiment of the VLP of the invention each of the target specific binding domain of the first RGD loop independently of each other has a length of between <NUM> to <NUM> amino acids, preferably between <NUM> to <NUM> amino acids; the target specific binding domain of the second RGD loop has a length of between <NUM> to <NUM> amino acids, preferably between <NUM> to <NUM> amino acid; and/or the target specific binding domain in the V loop has a length of between <NUM> to <NUM> amino acids, preferably between <NUM> to <NUM> amino acid.

In one alternative the target bound by the target specific binding domain is a moiety present on the surface of a cell or in the extracellular matrix. The specificity of the target specific binding domain is chosen, if the VLPs are targeted to a specific cell type in order to deliver its payload, e.g. drug or label. In an alternative preferred embodiment of the engineered polypeptide of the invention the at least one target specific binding domain is capable of specifically binding to an immunogenic peptide, pathogen neutralizing peptide, viral peptide, bacterial peptide, immune-modulating peptide, cancer peptide, to the surface of a cell, preferably a cellular receptor, a low molecular weight tag, preferably biotin or chitin. This provides an alternative and rapid way of binding various peptides to the surface of the VLPs.

In a preferred embodiment of the VLP the non-adenoviral polypeptide or the polypeptide inserted or attached is selected from the group consisting of immunogenic peptides, pathogen neutralizing peptides, viral peptides, bacterial peptides, immune-modulating peptides, and cancer peptides. Particularly preferred are viral peptides of Dengue HAK QDVVVLGSQEGAM (SEQ ID NO: <NUM>), Chikungunya STKDNFNVYKATRPYLAH (SEQ ID NO: <NUM>) and of Zika virus STKDNFNVYKATRPLAH (SEQ ID NO: <NUM>). Examples of the engineered polypeptide of the second aspect of the invention comprising STICKER and a Chikungunya peptide are AKRARLSTSFNPVPYEDESSTKDNFNVYKATRPYLAH (SEQ ID NO: <NUM>), AKRARLSTSFNP VP YEDEC S STKDNFNVYKATRPYLAH (SEQ ID NO: <NUM>) and AKRARLSTCFNPVPYEDESSTKDNFNVYKATRPYLAH (SEQ ID NO: <NUM>). The latter two examples comprise a coupling residue, i.e. Cys to form a covalent bond to a corresponding coupling residue in the binding cleft for fibre of the penton base protomer. 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 comprise a fragment antigen binding (Fab) fragment, a Fab' fragment, a F(ab')<NUM> fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and a fusion protein thereof.

If a non-adenoviral peptide or the peptide is inserted into the first RGD loop they preferably have a length of between <NUM> to <NUM> amino acids, preferably of between <NUM> to <NUM> amino acids. If the non-adenoviral peptide or the peptide is inserted into the second RGD loop they can have a length of between <NUM> to <NUM> amino acids, preferably between <NUM> to <NUM> amino acids. If the non-adenoviral peptide or the peptide is inserted into the second V loop they can have a length of between <NUM> to <NUM> amino acids, preferably between <NUM> to <NUM> amino acids.

In a preferred embodiment of the VLP of the invention the non-adenoviral polypeptide or the polypeptide 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: <NUM>). Such a cleavage site allows cleavage of the polypeptide of the first aspect of the invention once assembled into penton or VLP. Some antigens require exposure of the free N- and/or C- terminus to elicit an immune response. Thus, if penton proteins or VLPs have been assembled treatment of those with a protease will expose the N- and/or C-terminal sequence of such antigens, if the cleavage site is positioned at the N- and/or C-terminal end of such a non- adenoviral polypeptide. Quite surprisingly the present inventors have found such cleavage does not disrupt the assembled penton or VLP. This is extremely useful in situations in which a strong antigen specific immune response requires the exposure of free N- and/or C-termini of the antigen. Alternatively, the cleavage sit can be comprised in an engineered polypeptide of the first and/or second aspect of the invention to facilitate purification of the engineered polypeptide, e.g. it can be placed at the N- or C-terminus of the respective engineered polypeptide separating the penton or fibre comprising part of the engineered polypeptide from an affinity tag, e.g. biotin, chitin binding protein, Myc-taq. Such an affinity tag allows immobilization of the engineered polypeptide on a suitable affinity matrix and release of purified engineered polypeptide from the matrix. In a preferred embodiment of the VLP of the invention the coupling residue is selected from the group comprising Lys, Cys, Asp, and Glu, preferably Cys.

In a preferred embodiment of the VLP of the invention the drug is selected from the group of chemotherapeutic drug, antipathogenic drug, immune modulating drug, and anti-inflammatory drug.

According to the invention the fibre protein N-terminal fragment comprises, consists essentially of or consists of:
X<NUM>-F-N-P-V-Y-P-Y-X<NUM> (SEQ ID NO: <NUM>)
wherein.

Preferably, the fibre protein fragment has a length between <NUM> to <NUM> contiguous amino acids of fibre.

It is further preferred that the engineered polypeptide contained in the in the VLP of the invention comprises <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> repeats of the fragment of the fibre protein. The multimerization increases binding affinity. It has been observed by the present inventors that <NUM> or <NUM>, preferably consecutive repeats are suitable to mediate binding to the fibre binding cleft on the penton base protomer with subnanomolar affinities. Preferably, the multimers are arranged in a head-to-tail orientation.

As has been set out above, it is preferred according to alternative embodiment (vi) that one or more coupling residues are comprised in the binding cleft of the penton base protomer. To facilitate formation of covalent bonds between these one or more coupling residues in the penton base protomer and coupling residues comprised in the engineered polypeptide contained in the VLP of the invention. In a preferred embodiment of the engineered polypeptide contained in the VLP of the invention the at least one coupling residue is inserted into and/or positioned at the N- and/or C-terminus of the fibre protein fragment, preferably inserted into and/or positioned at the N- and/or C-terminus of SEQ ID NO: <NUM> or attached to an amino acid of the fibre protein fragment. As has been set out above coupling residues have to form covalent bonds with corresponding coupling residues. Once two polypeptides interact it is preferred that the coupling residue of the one polypeptide is sterically close to the coupling residue in the other polypeptide. In a preferred embodiment of alternative embodiment (vi) the coupling residue is positioned in the penton base protomer comprised in the VLP of the invention at amino acid position <NUM> and/or <NUM> (with reference to the amino acid sequence of SEQ ID NO: <NUM>) or at an analogous amino acid position of a penton base protomer of another adenovirus. The analogous position in another adenovirus penton base protomer can be determined by aligning the sequence according to SEQ ID NO: <NUM> with other adenoviral penton base protomer sequences with a standard alignment tool, like, e.g. CLUSTAL. The skilled person can easily determine the amino acid that occupies an analogous position to amino acids <NUM> or <NUM> in another adenovirus penton protein. It is preferred that the engineered protein according to the first aspect of the invention and according to alternative embodiment (iv) comprises the sequence
KSFX<NUM>NXc1Xc2 AVY (SEQ ID NO: <NUM>)
wherein.

If the coupling residue in the penton base protomer is positioned at either amino acid position <NUM> and/or <NUM> or at an analogous amino acid position of a penton base protomer of another adenovirus than it is preferred that a corresponding coupling reside of the engineered polypeptide of the second aspect of the invention comprises a coupling residue at the C-terminus of the STICKER part of the polypeptide. It is preferred that the coupling residue is located in STICKER as shown in below sequence:.

Thus, the engineered protein contained in the VLP of the invention comprises in a preferred embodiment a STICKER polypeptide according to SEQ ID NO: <NUM>. A particularly preferred STICKER polypeptide that may be comprised in the VLP of the invention is.

In a particularly preferred embodiment the STICKER polypeptide comprises the coupling residue Cys at position <NUM> and consists of the following amino acid sequence:
A-K-R-A-R-L-S-T-S-F-N-P-V-Y-P-Y-E-D-E-C (SEQ ID NO: <NUM>).

In an alternative preferred embodiment of alternative embodiment (vi) the coupling residue is positioned in the penton base protomer comprised in the VLP of the invention at Lys376 of the penton base protomer according to SEQ ID NO: <NUM> or an analogous position of a penton base protomer of another adenovirus. The engineered adenovirus penton base protein comprised in the VLP of the invention preferably comprises the following sequence:
Xc-X<NUM>-R-S-Y-N (SEQ ID NO: <NUM>)
wherein
Xc is a coupling residue, preferably C, D, E, and K, most preferably C; and X<NUM> is any amino acid, is preferably selected from the group consisting of D, E, G, K, N, or S, more preferably S or N.

If the coupling residue is comprised at this position it is preferred that the engineered polypeptide comprised in the VLPof the present invention comprises a corresponding coupling residue as indicated in below amino acid sequence:.

Accordingly, the engineered protein comprised in the VLP of the invention comprises in a preferred embodiment a STICKER polypeptide according to SEQ ID NO: <NUM>. A particularly preferred STICKER polypeptide that may be comprised in the engineered protein contained in the VLP of the invention is.

In a particularly preferred embodiment, the STICKER polypeptide comprises the coupling residue Cys at position <NUM> and consists of the following amino acid sequence:
A-K-R-A-R-L-S-T-C-F-N-P-V-Y-P-Y-E-D-E-S (SEQ ID NO: <NUM>).

In some embodiments of the VLP of the invention it is desired that the RGD motif positioned between the first and second RGD loop is intact to facilitate binding of the penton base protomer, penton or the VLP to certain cellular and extracellular structures present in a patient. Alternatively, if such targeting is not desired in a particular application of the VLP of the invention, the RGD motif may be mutated in such that it loses its ability to bind to integrin.

Disclosed herein are nucleic acids encoding the engineered adenovirus penton base protomer polypeptides and/or the engineered polypeptide contained in the VLP of the invention.

Also disclosed herein is an expression vector comprising the disclosed nucleic acid. Expression vectors comprise plasmids as well as viral vectors and contain a coding sequence encoding the engineered adenovirus penton base proteins and/or the engineered polypeptide comprised in the VLP of the invention and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. It has been realized in the art that immunization against diseases that rapidly change their antigenic epitopes, e.g. influenza, or that are characterized by a patient specific epitope mixture require rapid adaption or individualization of the vaccine. The VLPs of the present invention can be rapidly adapted to present the respectively desired antigens. Accordingly, a cloning vector amenable to rapid insertion of nucleic acid segments into the first and/or second RGD-loop or V-loop, which encode one or more desired antigen. A cloning vector may comprise:.

The adaptation may comprise one or more restriction enzyme sites, preferably BamHI, Kpnl, Kasl, Narl, Sfdl, EcoRI and RsrII, Pfol, BssHII, Sail, Sacl, Xbal, BstEII, and Hindlll. The nucleic acid sequence of preferred examples of such a cloning vector is provided in SEQ ID NO: <NUM> and <NUM>. The structure of these vectors termed pACEBac-ADDOmerl. O and pACEBac-ADDOmer2. <NUM> is provided in <FIG> and <FIG>. The sequence of the cloning vector pACEBac-ADDOmer2. <NUM> comprising the preferred Chikungunya virus antigenic epitope is provided in SEQ ID NO: <NUM> and <NUM>.

Also disclosed is a recombinant host cell comprising an expression vector or a cloning vector as described above. An expression vector or a cloning vector may be found inside the host cell (i) freely dispersed as such, or (ii) integrated into the host cell genome or mitochondrial DNA. The recombinant host cell is used for expression of the engineered polypeptides comprised in the VLP. The term "recombinant host cell" includes the progeny of the original cell which has been transformed, transfected, or infected with the polynucleotide or the recombinant vector of the invention. A recombinant host cell may be a bacterial cell such as an E. coli cell, a yeast cell such as Saccharomyces cerevisiae or Pichia pastoris, a plant cell, an insect cell such as SF9 or Hi5 cells, 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, and the like.

The engineered adenovirus penton base polypeptides contained in VLP of the present invention are capable in spite of their modification according to alternative embodiments (i) to (vi) in comparison to the respective wild-type penton base protomers to assemble into a penton subunit, i.e. to form a pentamer. Whether a given engineered adenovirus penton base protein assembles into pentamers can be easily assessed by methods well known to the skilled person that comprise non-denaturing polyacrylamide gel electrophoresis, size exclusion chromatography, mass spectroscopy or the like.

The pentamers assembled from the engineered adenovirus penton base polypeptides are capable of assembly into a VLP of the invention. As defined in claim <NUM> the present invention relates to a virus-like particle (VLP) comprising <NUM> pentamers. The VLP is stable and 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 coupling residues in the penton base protomers.

The VLP of the present invention comprises at least one engineered polypeptide as defined in claim <NUM>, preferably up to <NUM> engineered polypeptide as defined in claim <NUM>. In the latter embodiment all fibre protein binding clefts of the engineered penton base proteins are occupied by the engineered polypeptide comprising an adenovirus fibre protein N-terminal fragment as defined in claim <NUM>. It is preferred that the VLPs of the present invention comprise an engineered adenovirus penton base protein comprising the modification according to alternative (vi), preferably combined with a modification according to alternative (i), (ii), (iii), (iv), and/or (v) and an engineered polypeptide as defined in claim 1comprising at least one coupling residue, preferably the at least one coupling residue in inserted into and/or positioned at the N- and/or C-terminus of the fibre protein fragment, preferably inserted into and/or positioned at the N- and/or C-terminus of SEQ ID NO: <NUM> or attached to an amino acid of the fibre protein fragment. Preferably, the engineered protein of the first aspect of the invention comprises KSFX<NUM>NXc1Xc2 AVY (SEQ ID NO: <NUM>), wherein X<NUM>, Xc1, and Xc2 have the meaning outlined above and the engineered protein of the second aspect of the invention comprises X<NUM>- F-N-P-V-Y-P-Y-X<NUM>-(X<NUM>)n-Xc (SEQ ID NO: <NUM>), wherein X<NUM>, X<NUM>, X<NUM>, Xc, and n have the meaning outlined above. In an alternative preferred embodiment the engineered adenovirus penton base protein contained in the VLP of the invention comprises the following sequence Xc -X<NUM>-R-S-Y-N (SEQ ID NO: <NUM>), wherein Xc and X<NUM> have the meaning outlined above and the engineered adenovirus fibre protein N-terminal fragment comprises Xc-F-N-P-V-Y-P-Y-X<NUM> (SEQ ID NO: <NUM>), wherein Xc and X<NUM> have the meaning outlined above.

Preferably, the VLPs which comprise coupling residues to covalently couple the engineered adenovirus penton base polypeptide to the engineered polypeptide comprising the adenovirus fibre protein N-terminal fragment are assembled and/or stored under non-reducing conditions to allow the formation of covalent bonds between coupling residues in the penton base protomers and the STICKER polypeptide comprised in the VLP of the invention.

It is also envisioned that VLPs consisting of or comprising wild-type penton base protomers are used to provide a vehicle and that these are decorated with different non- adenoviral polypeptides by using the engineered polypeptide comprising the adenovirus fibre protein N-terminal fragment as defined in claim <NUM>. Due to the short length that the engineered polypeptide comprising the adenovirus fibre protein N-terminal fragment as defined in claim 1has in preferred embodiments, it can be synthesized, e.g. by solid state chemistry within less than a day. Thus, according to claim <NUM>, the present invention relates to a VLP comprising <NUM> pentamers each comprising five adenovirus penton base protomers and at least one engineered polypeptide comprising the adenovirus fibre protein N-terminal fragment. It is preferred that all fibre protein binding clefts of the penton base protomers are occupied and, thus that these VLPs comprise <NUM> engineered polypeptides comprising the adenovirus fibre protein N-terminal fragment as defined in claim <NUM>.

As defined in claim <NUM>, the present invention relates to a method for producing a VLP of the invention comprising disease and/or patient specific non-adenoviral peptides, comprising the steps of:.

In a further aspect the present invention relates to a pharmaceutical composition comprising the VLP of the invention, and a pharmaceutically acceptable carrier and/or suitable excipient(s). Preferably, such composition is a pharmaceutical composition. In preferred embodiments the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient and optionally one or more additional active substances. Preferably, the composition contains a therapeutically effective amount of the VLP, preferably in purified form, together with a suitable amount of carrier and/or excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.

For preparing pharmaceutical compositions of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form compositions include powders, tablets, pills, capsules, lozenges, cachets, suppositories, and dispersible granules. A solid excipient can be one or more substances, which may also act as diluents, flavouring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the excipient is preferably a finely divided solid, which is in a mixture with the finely divided inhibitor of the present invention. In tablets, the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

Liquid form compositions include solutions, suspensions, and emulsions, for example, water, saline solutions, aqueous dextrose, glycerol solutions or water/propylene glycol solutions. For parenteral injections (e.g. intravenous, intraarterial, intraosseous infusion, intramuscular, subcutaneous, intraperitoneal, intradermal, and intrathecal injections), liquid preparations can be formulated in solution in, e.g. aqueous polyethylene glycol solution. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously.

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

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

Furthermore, such pharmaceutical composition may also comprise other pharmacologically active substance 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 polynucleotides adjuvants.

The present invention also relates the VLP of the invention for use in treating and/or preventing an infectious disease, an immune disease or cancer.

ADDomers were designed and produced in very high yields (tens of grams per liter expression culture). A generic three-step protocol was established to purify ADDomer to homogeneity (see below). In a proof-of-concept project, it was experimentally established that highly immunogenic Chikungunya epitopes can be inserted into the functionalized loops of ADDomer, without perturbing particle formation or decreasing yield noticeably. ADDomers containing Chikungunya epitopes were purified to homogeneity and cell-based and animal studies were initiated to establish their potency as vaccine candidates. The ADDomers were validated with a range of techniques including electron microscopy, evidencing homogenously structured, discrete multimers (dodecahedra). Cysteine-disulfide chemistry was implemented to even further increase the already remarkable thermostability of ADDomer (elimination of cold chain requirement). Moreover, ADDomers were preparaed which contain not only peptide epitopes but also protein domains and entire proteins including high-affiny binders (nanobodies DARPins, antibody fragments) and are establishing efficient protocols to manufacture these in large scale. Triggered by the recent emergence of Zika virus, ADDomer-based Zika vaccine candidates were designed, and also ADDomers to potentially combat more than one disease simultaneously (combo vaccine). Cell-based and animal experiments to validate these are performed.

In the following, experiments and protocols are described to produce and validate ADDomer VLPs and ADDomer VLP vaccines.

The atomic structure of naturally occurring dodecamer species (e.g. derived from Adenovirus Ad3 serotype) has been determined by X-ray crystallography (<NPL> and <NPL>). Careful inspection of the atomic structures revealed the presence of extended loop structures. More precisely, one variable loop (denominated V-loop) and two regions in the so-called RGD-loop of the wild-type dodecahedron were indentified as potential sites of functionalization. Comparison of a number of dodecahedron protomers revealed a wide variability of the V-loop and the two RGD-loop regions throughout the species, both in length and in sequence composition, underscoring their potential. Using this information, we designed de novo a DNA sequence encoding for a synthetic designer dodecahedron promoter. BioBrick design (<NPL>) was applied by introducing DNA sequences representing endo-nuclease cleavage sites, to facilitate designed variations (and even randomization) of the amino acids representing the V-loop and the two RGD-loop regions (RGDloop2, RGDloop2). The protomer design was optimized iteratively until a protomer was identified that would give rise to recombinant dodecahedra (ADDomer) characterized by complete BioBrick design of the above described loop regions, while maintaining the high solubility and structure integrity of wild- type human Ad3 serotype dodecahedron.

ADDomer is already remarkably thermostable and can be stored at <NUM> for protracted times, indicative of not requiring a cold-chain in remote areas with poor infrastructure. Inspection of the crystal coordinates of native Ad3 particles revealed a so-called 'strand- swapping' region, where segments of the protomers extended to the vicinity of the adjacent protomers resulting in juxtaposition of amino acids that were within a distance that could allow covalent bond formation. We genetically substituted these amino acids in ADDomer with cysteines, such that two cysteines each coming from distinct protomers were within the distance required for disulfide bond formation.

Next, ADDomers were expressed by using the MultiBac system. The gene encoding for ADDomer was synthesized from scratch (SEQ ID NO: <NUM> and the encoded ADDomer is provided in SEQ ID NO: <NUM>) and inserted by classical cloning methods (restriction/ligation) into pACEBac, a transfer plasmid of the MultiBac system. MultiBac was developed by one of the inventors (Berger) specifically for the production of complex biologies such as ADDomer. Composite MultiBac virus containing the ADDomer gene was prepared (see <FIG> and <FIG>) and insect cell cultures infected following previously described protocols (<NPL> and <NPL>). ADDomer protein-containing cell pellets were prepared by centrifugation as described. Cell pellets were stored at -<NUM> degrees. Expression of ADDomer with peptide or protein epitopes inserted, and also expression of hyperstable ADDomer, all gave rise to comparable, very high yields and homogeneously structured dodecahedral particles.

Example ADDomer-CHIKADDomer-based VLP vaccine candidate presenting multiple copies of the major neutralizing Chikungunya immune epitope HAKKQDVVVLGSQEGAM (SEQ ID NO: <NUM>) were constructed. The major neutralizing immune epitope is part of a Chikungunya envelope protein, and is a linear peptide epitope located at the extreme N-terminus (<NPL>). In an overwhelming majority of patient sera, antibodies are found which react with this linear peptide epitope. ADDomer provides the means to present linear epitopes either in a constrained fashion (bot N- and C- termini covalently linked to ADDomer scaffold), or in an uncontrained fashion (N-termini liberated by cleavage with a specific protease) or a combination of constrained or unconstrained epitopes, all the while preserving structural integrity of the ADDomer scaffold.

Preferred arrangements used were the following:.

Purification of ADDomer and variants Spodoptera frugiperda Sf21 insect cells pellets were lysed by freeze-thawing. Lysate was cleared by centrifugation following standard protocols for insect cells (<NPL> and <NPL>). Cleared supernatant was loaded on a <NUM> to <NUM>% sucrose gradient and centrifuged utilizing a Beckman SW41 rotor overnight. Fractions of <NUM> were collected from the top of the gradient and loaded on denaturing SDS polyacrylamide gels (SDS-PAGE) for analyzing protein content and identifying fractions containing ADDomers for pooling Size exclusion chromatography (SEC) and/or ion-exchange (IEX) was then carried out after dialyzing away the sucrose.

Validation of ADDomer by electron microscopy Purified ADDomer and ADDomer variants were visualized by negative-stain electron microscopy (EM) to assess their assembly status and their structural integrity. Standard mica- carbon preparation was utilized with ADDomer at approximately <NUM>/ml concentration prior to deposition on the support material. Samples were stained using <NUM>% (wt/vol) sodium silicotungstate (H <NUM>) and visualized on a JEOL electron microscope at <NUM> kV. Images were recorded and analysed was carried out using software supplied by Gatan.

For thermostability experiments, ADDomers were stored either frozen, at <NUM>, at room temperature (RT) or at <NUM> for one week. Electron microscopy showed that storage at RT or at <NUM> resulted in correctly auto-assembled particles demonstrating their thermostability. Incubation of ADDomer (SEQ ID NO: <NUM>) for <NUM> at <NUM> resulted in reversible particle disassembling which reassemble when turned back at RT. This reversible dissociation was also observed by Thermal Shift Assay (<FIG>, see arrow) but irreversible dissociation was seen only for temperature above <NUM>.

For murine immune analysis of ADDomer-CHIK Chikungunya ADDomer VLP vaccine candidates, six weeks old BALB/c female mice were used. Four groups of eight mice were designated per immunization with ADDomer species (e. g, in case of Chikungunya VLP vaccine candidate: i) ADDomer, ii) ADDomer CHIK unconstrained epitope, iii) ADDomer CHIK constrained epitope, iv) isolated CHIK major neutralizing peptide epitope cross-linked to KLH as positive control). Each animal was injected with <NUM>µg of ADDomer and ADDomer variants at <NUM> week intervals. IgA, IgM, total IgG, IgGl IgG2a and anti-CHIK antibodies were titered from mouse sera by ELISA. Immune analysis of other ADDomer VLP vaccine candidates were designed in an analogous fashion. Two kind of epitope display at the ADDomer surface were tested (Constrained and Relaxed, see below: point <NUM>). A time dependent response was observed (Week <NUM> to <NUM>). The superior potential of the relaxed form of the epitope over the constrained form to trigger an anti-Chik epitope response was shown (<FIG>). Exposition of the epitope of interest at the ADDomer surface.

The addition of a TEV cleavage site upstream the epitope of interest enables its display under two different configurations: constrained or relaxed. Upon purification, the epitopes are naturally constrained in the ADDomer loops. By addition of the TEV (Tobacco Etch Virus) protease (<NUM>/<NUM> w:w) for <NUM> at RT the epitope can be relaxed and displayed in a linear form at the scaffold surface. The cleavage efficiency is easily monitored by SDS-PAGE. Of note, the overall ADDomer scaffold is not affected by this cleavage (Fig.l <NUM>).

Extension of the epitope insertion capacity in ADDomer The capacity of ADDomer to carry large epitope sequence was assessed. To this end, a <NUM> amino acid in length artificial epitope was inserted in ADDomer (named extended ADDomer). This resulted in correctly auto-assembled ADDomer. The insertion was confirmed by both SDS-PAGE analysis and mass spectroscopy as shown in <FIG> <FIG>.

Claim 1:
A VLP comprising <NUM> pentamers each comprising five adenovirus penton base protomers and at least one engineered polypeptide, preferably <NUM> engineered polypeptides, comprising at least one adenovirus fibre protein N-terminal fragment specifically binding to an adenovirus fibre protein binding cleft of a penton base protomer and:
(i) a non-adenoviral peptide, optionally linked to the fibre protein fragment via peptide linker, and/or
(ii) is covalently or non-covalently coupled to a drug or label;
wherein the adenovirus fibre protein N-terminal fragment comprises:
X<NUM>-F-N-P-V-Y-P-Y-X<NUM> (SEQ ID NO: <NUM>) wherein
X<NUM> is selected from the group consisting of S, D and T, preferably S or D, and is more preferably S; and
X<NUM> is selected from the group consisting of E, D and G, is preferably E or D, and is more preferably E.