Conjugates of magnetic particles and viral structures and their use in medical applications

Abstract

The present invention provides novel conjugates comprising a magnetic particle and a multitude of viral structures covalently bound to said particle and its manufacturing. These conjugates are suited for medical applications, both diagnostic and therapeutic.

Description

[0001] Conjugates of magnetic particles and viral structures and their use in medical applications

[0002] The present invention provides novel conj ugates comprising a magnetic particle and one or more viral structures covalently bound to said particle and its manufacturing . These conj ugates show good stabil ity, high magnetisation and high biological activity, making them suitable for a wide range of medical applications , both diagnostic and therapeutic .

[0003] Magnetic separation methods using nanoparticles are known and find applications in research .

[0004] Bloemen et al ( J Nanopart Res ( 2012 ) 14 : 1100 , DOI 10 . 1007 / s l l 051- 012- 1100-5 ) disclose superparamagnetic oleic acid-coated iron oxide nanoparticles and suggest their use in biomedical applications in aqueous environments . The particles disclosed are dispersible in various aqueous environments , including human serum and plasma and considered suitable for subsequent coupling reactions .

[0005] Further, the company Thermo Fisher Scienti fic Inc . published a product information entitled magnetic bead technology for better assay development for advertising its product line " Pierce Magnetic Beads" . These Particles contain a "double shell design" of non-magnetic core , first magnetite shell and second polymer shell activated by NHS . These particles are suited to covalently bind proteins and are intended for immunoprecipitation and custom created af finity resins . Although suitable for speci fic analytical purposes , their use is somehow restricted, particularly due to its low magnetic properties and sensitivity towards environment , given the above-described particle architecture .

[0006] There is an ever-existing need for improving such technologies and to expand them to new medical applications . These objectives are achieved by the conjugate as defined in claim 1 and the manufacturing as defined in claim 15. Further aspects of the invention are disclosed in the specification and independent claims, preferred embodiments are disclosed in the specification and the dependent claims.

[0007] The present invention will be described in more detail below. It is understood that the various embodiments, preferences, and ranges as provided / disclosed in this specification may be combined at will. Further, depending on the specific embodiment, selected definitions, embodiments or ranges may not apply.

[0008] Unless otherwise stated, the following definitions shall apply in this specification:

[0009] As used herein, the term "a", "an", "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0010] As used herein, the terms "including", "containing" and "comprising" are used herein in their open, non-limiting sense .

[0011] As used herein, the term "viral structures" includes viral capsids (VCs) and structural viral proteins (VPs) . The term viral capsid includes VCs with or without genetic material and VCs with or without a viral envelope (VE) . The term structural viral protein includes individual structural VPs and assemblies of structural VPs.

[0012] As used herein, the term "gene therapy" refers to the treatment, prevention, or delay of progression of a disease by introducing genetic material into cells to replace or complement defective or missing genes, to alter the genetic code of genes, or to modify gene expression. Particularly, the term includes methods of gene editing in a cell, in particular gene editing using CRISPR-Cas based systems, such as CRISPR-Cas9. In the context of the present application, the cells are part of a living organism in need of such treatment (a "subject in need") . In the context of the present invention, it is to be understood that the cell is a somatic cell (i.e. a cell of a mammalian body, including stem cells, excluding germline cells) Accordingly, human germline cells and human germline stem cells are excluded when referring to cells.

[0013] As used herein a "gene", refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0014] As used herein, a "subject in need" of a treatment refers to mammals, including human beings, livestock, companion animals and pets; in particular a human being in need of therapy, i.e. a "patient".

[0015] The present invention will be better understood by reference to the figures .

[0016] Figure 1. A schematic representation of the synthesis of conjugates comprising particles with VCs according to example 1. From left to right:

[0017] 1) Core-type or matrix-type particles with single core or multiple cores having soft magnetic properties; said particle covered by a first shell containing graphene layer .

[0018] 2) Said particle covered by a second shell of polyglycerol, NHS activated.

[0019] 3) Conjugate, comprising viral capsids covalently bond. Figure 2. Illustrates examples 2.2 and 2.3: Depletion of fluid (buffer, plasma, blood) from anti-viral capsid antibodies by incubation with conjugates and subsequent magnetic separation. From left to right

[0020] • Left: Inventive conjugate + blood containing anti-AAV antibodies and / or anti-AdV antibodies

[0021] • Right: purified blood (depleted in anti-AAV antibodies and / or anti-AdV antibodies) and antibody-conjugate- complex separated by applying a magnetic field.

[0022] Figure 3: Quantification of rAAV9 (rAAV9 with genetic material) and rAAV90(rAAV9 without genetic material) VCs bound to particles and antibody removal by conjugates in PBS .

[0023] • The percentage of rAAV9 VCs (black column) and rAAV90VCs (white column) bound to particles was quantified by measuring the VCs in the supernatant before and after coupling (A) .

[0024] • The antibody removal by conjugates with rAAV9 VCs (black column) or rAAV90VCs (white column) in ADK9-spiked (1 pg / mL) PBS was determined by measuring the amount of ADK9 before and after incubation with conjugates (B) .

[0025] The graphs represent n=2 conjugate batches, which were each measured in duplicate (A) or triplicate (B) . The mean ± SD is shown.

[0026] Figure 4. Conjugate treated human whole blood and plasma. Conjugate treatment (2 mg / mL, 1 treatment cycle, 50 minutes) was performed in whole blood and plasma generated from the respective whole blood.

[0027] The antibody levels before (left) and after treatment with inventive conjugate (right) were measured in an ELISA-based assay in triplicates (y-axis: fluorescence, a.u.] .

[0028] Figure 5. Performance of conjugates coupled to full versus empty capsids. The removal of anti-AAV9 antibodies in human plasma after conjugate treatment, with rAAV9 or rAAV90, treatment was measured for 3 donors. The plasma was treated for 3 cycles of 3 minutes and measured in either an ELISA- based assay [ (A) , antibody removal, fluorescence (a.u.) ] or in a virus neutralization assay [ (B) , transduction efficiency, (%) ] . The results from the 1:3 dilutions are depicted. The dotted horizontal line represents the average value of the low titer donors. The mean and SD are depicted. From left to right: untreated - treated with rAAV9 conjugate - treated with rAAV90conjugate.

[0029] Figure 6: illustrates the inventive method for removing antibodies against viral structures from blood in an ex vivo setting.

[0030] Figure 7: illustrates a Gene therapy treatment regime applying the inventive method to deplete antibodies against viral structures, conducting the gene therapy afterwards; followed by optional repeating these steps. Y-Axis: antibody titer; x-Axis: time (not scaled) .

[0031] Figure 8 illustrates an alternative embodiment of conjugates with polysiloxane layer replacing both the first graphene shell and the second polymer shell.

[0032] Figure 9 illustrates an alternative embodiment of conjugates with PMMA layer replacing the graphene shell.

[0033] Figure 10 illustrates various types of particles present in the inventive conjugate.

[0034] Type 1 particles are considered 'true' core shell particles, as a single core is covered by

[0035] • a first layer (functionalized graphene) ; or

[0036] • a second layer (polymer with functional end groups) or

[0037] • a first layer (functionalized graphene) covered by a second layer (polymer with functional end groups) ; or

[0038] • a first layer (non-functionalized graphene) covered by a second layer (polymer with functional end groups) .

[0039] Type 2 particles are considered 'matrix - type' particles, as a multitude of cores is covered by

[0040] • a second layer (polymer with functional end groups) or

[0041] • a first layer (functionalized graphene) covered by a second layer (polymer with functional end groups) ; or

[0042] • a first layer (non-functionalized graphene) covered by a second layer (polymer with functional end groups) . In more general terms, in a first aspect, the invention relates to a conjugate comprising a particle and one or more viral structures, wherein said particle is micro- or nano-sized and shows soft magnetic properties, and said viral structures are covalently bound to said particle. More specifically, the invention provides for a conjugate comprising a particle and one or more viral structures, wherein (a) said particle (i) has a size of 1 nanometer to 100 micrometers and comprises (ii) one or more than one core, said core consists of a material having soft magnetic properties; (iii) each core optionally covered by a first shell, said first shell contains one or more graphene layers which are optionally functionalized; and / or (iv) said one or more cores are covered by a second shell, said second shell contains a polymer having functional end groups; with the proviso that, if no second shell is present, said one or more graphene layers are functionalized; and characterized in that none of the particle shells contains magnetic material; (b) said viral structures are selected from Viral capsids (VCs) with or without genetic material and with or without a viral envelope (VE) as well as individual structural viral proteins (VPs) or assemblies of structural viral proteins; characterized in that (c) said one or more viral structures are covalently bound to said particle shell.

[0043] This aspect of the invention, particularly the meaning of 'particle', 'viral structures' and 'covalent bonding' shall be explained in further detail below:

[0044] Particles :

[0045] Particles suitable for the present invention may be characterized by internal structure, size, materials of core, first, second shell as outlined in further detail below .

[0046] Particle Structure: Particles according to this invention are of the 'core- shell-type', i.e. an entity of a first material forming a core and one or more second materials fully covering said core and thereby forming one or more shells. Alternatively, particles according to this invention may be of the ' matrix-type ' , i.e. an entity where a first material forms a multitude of cores each core optionally being covered by a first shell and a second shell fully covering said multitude of cores.

[0047] In view of the above, a number of different types of particles, referred to as type 1 (core-shell type) and type 2 (matrix type) particles are encompassed:

[0048] Type la particles contain, particularly consist of:

[0049] • a single core, said core consists of a material having soft magnetic properties;

[0050] • covered by said first shell, which contains one or more graphene layers that are functionalized.

[0051] Type lb particles particles contain, particularly consist of :

[0052] • a single core, said core consists of a material having soft magnetic properties;

[0053] • covered by said second shell, said second shell contains a polymer having functional end groups.

[0054] Type 1c particles contain, particularly consist of:

[0055] • a single core, said core consists of a material having soft magnetic properties;

[0056] • covered by said first shell, which contains one or more graphene layers that are not functionalized; and

[0057] • covered by said second shell, said second shell contains a polymer having functional end groups.

[0058] In this embodiment, the viral structures are covalently bound to the functional end groups of the polymer of the second shell.

[0059] Type Id particles particles contain, particularly consist of :

[0060] • a single core, said core consists of a material having soft magnetic properties; • covered by said first shell , which contains one or more graphene layers that are functionali zed; and

[0061] • covered by said second shell , said second shell contains a polymer having functional end groups .

[0062] Type 2b particles particles contain, particularly consist of :

[0063] • a multitude of cores , said cores consists of a material having soft magnetic properties ;

[0064] • covered by said second shell and thereby embedding said multitude of cores , said second shell contains a polymer having functional end groups .

[0065] Type 2c particles particles contain, particularly consist of :

[0066] • a multitude of cores , said cores consists of a material having soft magnetic properties ;

[0067] • each of said cores being covered by said first shell , which contains one or more graphene layers that are not functionali zed; and

[0068] • all of said coated cores being covered by said second shell and thereby embedding said multitude of cores , said second shell contains a polymer having functional end groups .

[0069] Type 2d particles particles contain, particularly consist of :

[0070] • a multitude of cores , said cores consists of a material having soft magnetic properties ;

[0071] • each of said cores being covered by said first shell , which contains one or more graphene layers that are functionali zed; and

[0072] • covered by said second shell , said second shell contains a polymer having functional end groups .

[0073] In this embodiment , the viral structures are covalently bound to the functional end groups of the polymer of the second shell .

[0074] These particle types are visuali zed in fig . 10 . Depending on the synthesis , an assembly of conj ugate may comprise only core-shell-type particles , predominantly comprise core-shell-type particles, predominantly comprise matrixtype particles or exclusively comprise matrix-type particles .

[0075] Particle size depends on the amount of coating used and the particle type (core-shell or matrix) . Suitable ranges are 1 nm to 100 micrometers, preferably 5 nm to 5 micron, most preferably 5 nm to 0.6 micron. Particle size may be determined by Dynamic Light Scattering (DLS) .

[0076] Particle Core:

[0077] Magnetic Properties: A wide range of inorganic materials may be used as particle core. Materials particularly suitable in the context of this invention show soft magnetic properties, preferably with a coercive force below 30'000 A / m and a magnetic saturation above 30 emu / g. Preferably, the coercive force is below 16'000 A / m. In an ideal case, the coercive force is zero, resulting in a superparamagnetic material.

[0078] Materials: A wide range of iron-based materials may be used, preferably iron-based material which contains at least 50 wt% Fe . This includes iron alloys, iron oxides and iron carbides. As exemplary embodiments, Fe, Fe2Oa, FeaCg, Fe2<3, FeaC and FesC2 and are mentioned.

[0079] A wide range of cobalt-based materials may be used, preferably cobalt-based material which contains at least 50 wt% Co. This includes cobalt alloys, cobalt oxides, and cobalt carbides. As exemplary embodiments, Co, C02C, C03C, CoO and C03O4 are mentioned.

[0080] A wide range of nickel-based materials may be used, preferably nickel-based material which contains at least 50 wt% Ni . This includes nickel alloys, nickel oxides and nickel carbides. As exemplary embodiments, Ni, NiC3, NiO, N12O3 and N13O4 are mentioned

[0081] It is further comprised by the invention to use a material comprising at least two of the elements iron, cobalt, and nickel, where the total content of these three elements is at least 50% of the core mass. As exemplary embodiment, NiFe2O4 and Alnico (containing Al 8-12%, Ni 15-26%, Co 2- 24%, up to 6% Cu and optinally Ti with the remainder being Fe) is mentioned.

[0082] Core Size: The size of the particle core may vary over a broad range, including 1 nm to 500 nm, preferably 1 to 150 nm. This core size ranges are applicable for both core- shell-type particles and matrix-type particles.

[0083] Core size may be determined by Dynamic Light Scattering (DLS) or electron microscopy. It is preferred to have a narrow and uni-modal size distribution. Typically, more than 90 %, preferably more than 95 % (n / n) are within a defined range.

[0084] Particle Shell:

[0085] According to this invention, neither the first shell nor the second shell does contain magnetic material. It is considered beneficial to provide particles where the magnetic material is protected, not exposed to the environment (particularly blood) , and not exposed to viral structures .

[0086] First Shell:

[0087] The first shell comprises at least one, suitably 3 - 15 graphene layers. Choosing the term "graphene" indicates that the carbon atoms are predominantly (or almost exclusively) present in the sp2-hybridization state without additional atoms bound. Due to its size, such shells are sometimes also characterized as "Super- Buckminster-fullerenes" . Further advantageous embodiments of said shell are explained below.

[0088] Preferably, the graphene layer has a thickness between 0.3 and 10 nm, particular preferably 1 - 5 nm (as evaluated from transmission electron micrographs, corresponds to ~ 3 — 15 graphene layers) . This results in a carbon content (as measured by quantitative microanalysis using a LECO- thus prevent oxidative degradation of the particle core. It is thus preferred to implement graphene layers as a first shell where the material of the particle core is sensitive towards oxidation, such as iron, iron alloys and iron carbides.

[0089] In embodiments, the graphene layer is free of functional groups. This embodiment can be implemented in case a second shell is present.

[0090] In embodiments, the graphene layer contains functional groups on its outermost layer. Such functional groups, also termed linkers, improve adhesion of the second shell or allow covalent bonding to the viral structures. Thus, such functional group (I) serves as a linker. Suitable linkers are of formula (I) ( I ) , wherein n is an integer between 1 and 5; m is an integer between 0 and 20;

[0091] Z represents a spacer selected from the group of alkyl, alkenyl, alkyloxy, amino acids, and saccharides;

[0092] FG represents independent from each other OH, OR, N3, NO2, NH2, NHR, NR2, OHO, COR, COOH, COOR, SO2, SR, SOR, SO2R, halogen, P(O)OH, P(O)OR, P(O)2OH, P(O)2OR, l,2,3-triazol-4-R, l,2,3-triazol-5-R

[0093] R represents Ci-C4alkyl, Ci-C4alkoxy, C3-Ciocyclo-alkyl , C4-Cio aryl, or a 5-membered heterocycle containing at least one atom selected from the group of N, 0, and S, or a 6-membered heterocycle containing at least one atom selected from the group of N, 0, and S.

[0094] Establishing such functional groups is known in the field and involves reagents and catalysts commercially available. If FG represents 1 , 2 , 3-triazol-4-R, 1,2,3- triazol-5-R, such moiety is available via azide-alkyne Huisgen cycloaddition.

[0095] It is understood that the functional group FG will react with either the viral structure or the polymer of the second shell, forming a covalent bond. The structures shown herein are prior to such reaction.

[0096] In embodiments, m = 0, i.e. Z is absent, and FG is directly bond to the phenyl group in formula (I) .

[0097] Second Shell:

[0098] If present, a broad range of polymers is suited as a second shell. In view of the intended use in pharmacy, suitable polymers are preferably compatible with blood. Further, said polymer contains functional groups allowing, directly or after activation, covalent bonding to viral structures. Accordingly, as used herein, a polymer having functional end groups refers to a polymer comprising accessible functional groups which allow covalent coupling chemistry. This includes polymers functionalized with polar groups [such as hydroxyl (-OH) , carboxyl (-COOH) , amino (-NH2) or thiol (-SH) groups or derivatives thereof] , polymers functionalized with halogens (such as chlorine) , polymers functionalized with azides and derivatives thereof (such as 1 , 2 , 3-triazoles as discussed above) .

[0099] In embodiments, the polymer is selected from the group consisting of Polyglycerol, Polyethylene glycol (PEG) , dextran, Hydroxyethyl starch, Polylactic acid (PLA) , Styrene-maleic acid (SMA) copolymers, Polystyrene (PS) , Poly ( ethyleneimine ) (PEI) , Chitosan, and Polymethylmethacrylate (PMMA) .

[0100] In embodiments, the polymer's functional end groups are selected from OH, OR, N3, NO2, NH2, NHR, NR2, CHO, COR, COOH, COOR, SO2, SOR, SO2R, halogen, P(O)OH, P(O)OR, P(O)2OH, P(O)2OR, 1 , 2 , 3-triazol-4-R, and 1 , 2 , 3-triazol-5-R where R represents a group selected from C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkyloxy, an amino acid, a dipeptide, a tripeptide, a mono saccharide, and a disaccharide. A preferred polymer is polyglycerol, particularly hyperbranched polyglycerol.

[0101] A preferred functional end group of said second shell may be selected from thioethers -SR' ' and amides -C(O)NHR' ' where R' ' represents of C1-C4 alkyl, C1-C4 alkyloxy, or an amino acid.

[0102] The amount of functional end groups may vary. At least one functional end group per viral structure is required. Accordingly, the amount of functional end groups is at least one per particle.

[0103] Depending on the type of particle, the polymer of the second shell is either covalently bound to the linker of formula (I) (type 1c, 2c particles) , or without covalent bond to the first shell (type Id, 2d particles) , or in direct contact with the core, with or without bonds (type lb, 2b particles) . If a second shell is present, the viral structures are covalently bound to said second shell.

[0104] In embodiments, the polymer accounts for 10 wt% - 60 wt% of the particle including second shell.

[0105] Viral Structures:

[0106] The term viral structure is defined above. The term shall include both, wild type, and genetically engineered viral structures. The term "genetically engineered" excludes natural serotypes (wild type) . Further, the term "genetically engineered" is chosen to cover both, recombinant viral structures obtainable by classical genetic engineering methods (e.g. recombinant AdV or AAV) and viral structures obtainable by gene editing methods (e.g. Crispr / Cas9) .

[0107] In embodiments of the invention, the viral structures are selected from viral capsids. The term is used, as conventional in the field, to describe the protein shell of a virus. The viral capsids may or may not contain genetic material, e.g., DNA or RNA, and may or may not be enclosed by a viral envelope, i.e., a protecting membrane consisting out of lipids and proteins that protect the viral capsid.

[0108] In embodiments of the invention, the viral structures are selected from structural viral proteins, i.e., proteins that have the function to maintain the shape and structural integrity of the virus, e.g., capsid proteins.

[0109] In embodiments, viral structures are selected from adenoviruses (AdV) . AdV shall include wild type AdV and genetically engineered adenoviruses (AdV) . In embodiments of the invention, the viral structure is based on recombinant adenoviruses (rAdV) , which includes all serotypes of adenoviruses.

[0110] In embodiments, viral structures are selected from adeno- associated-viruses (AAV) . AAV shall include wild type AAV and genetically engineered adeno-associated-viruses (AAV) . In embodiments of the invention, the viral structure is based on recombinant adeno-associated-viruses (rAAV) , which includes all serotypes of AAV.

[0111] In embodiments, viral structures are selected from modified vaccinia Ankara virus (MVA) . MVA shall include MVA itself and genetically engineered viruses thereof. In embodiments of the invention, the viral structure is based on recombinant MVA, which includes all serotypes of MVA.

[0112] In embodiments, viral structures are selected from dengue virus. Dengue virus shall include wild type and genetically engineered viruses. In embodiments of the invention, the viral structure is based on recombinant dengue virus, which includes all serotypes of dengue viruses.

[0113] In further embodiments, viral structures are selected from the group consisting of Poxviruses (i.e. vaccinia virus or myxoma virus) and Flaviviruses (i.e. Dengue virus) . Covalent Bonding / Conjugate

[0114] The term "conjugate" is chosen to describe an entity comprising a coated particulate structure and one or more viral structures, where said viral structures are covalently bound to the coating of said particulate structure. Such conjugate is schematically shown in fig.l, right side.

[0115] One or more viral structures are covalently bound. The number of viral structures depends on the size of the particle, the number of binding sites available and the size of the viral structure. For example, a coated particle having 2 microns diameter and a spherical shape, approx. 40'000 viral structures may covalently bind (assuming 20 nm diameter of circular viral structure) . This number may substantially increase in case of porous / non-even polymer surface. It is preferred to have 2 - 1'000'000 viral structures covalently bound to the particles surface, preferably 4 - 40'000 viral structures. This may be quantified by the concentration determination of unbound viral structures that remain in the liquid phase after the coupling reaction.

[0116] One or more covalent bonds may be present to link a viral structure to the particles surface. It is considered beneficial that already one single covalent bond allows a stable and reliable linkage of viral structure to particle surface. Without being bound to theory, it is believed this covalent bonding does not, or almost not, influence the 3D viral structure and thus enables the beneficial applications discussed below.

[0117] In embodiments, the conjugate has a zeta potential < 0 mV.

[0118] In embodiments, the invention relates to conjugates as described herein that are sterilized. It was found that sterilisation in the course of the manufacturing process does not negatively impact performance of the conjugates. Specific Embodiments:

[0119] In an embodiment, the invention relates to conjugates as described herein, where the particle is iron-based and of type 1c or 2c and the particle comprises as first shell with a linker of formula (I) discussed above; a second shell consisting of a hyperbranched polyglycerol which is covalently bound to the linker of formula (I) and which contains functional groups selected from thioethers -SR' ' and amides -C(O)NHR' ' where R' ' represents C1-C4 alkyl, C1-C4 alkyloxy, or an amino acid, thereby allowing covalent bonding to the viral structures.

[0120] In a further embodiment, the invention relates to conjugates as described herein, where the graphene layers are absent and the second layer is a poly (methacrylic acid) coating ("PMMA coating") . In this embodiment, the conjugate comprises a particle and a multitude of viral structures wherein said particle has a size of 1 nanometer to 100 micrometers and comprises:

[0121] • one or more than one core, said core consists of a metal oxide having soft magnetic properties;

[0122] • said one or more core are covered by a shell, said shell contains a PMMA coating which is optionally functionalized; and with the proviso that the particle shell does not contain magnetic material; and wherein said viral structures are as described above and characterized in that said one or more viral structures are covalently bound to said PMMA coating .

[0123] Architectures according to this embodiment are of type (lb) and (2b) , as discussed above and shown in fig. 10.

[0124] This embodiment is particularly suited in case the metal core is a metal oxide having soft magnetic properties, due to the chemical bonding of carboxy-groups to the surface of the metal oxide. The preferred metal oxide is iron oxide. PMMA coated ferrimagnetic iron oxide nanoparticles are known and described in Yu et al ( J .Mater . Chem. , 2004, 14, 2781-2786) . In this embodiment, a PMMA coating is preferably formed by emulsion polymerisation of MMA in the presence of the metal oxide particle under acidic conditions. As a consequence of abundantly present OH-groups at the metal oxide surface, acid-base reaction with MMA occurs to form chelates comprising Metal-O-C species with the elimination of water. Further MMA present in the system polymerises to yield said PMMA coating.

[0125] Such coating has reactive COO- or COOH end groups. This is illustrated in fig. 9. These end-groups may be directly used to form covalent bonds to the viral structures. Alternatively, such COO- or COOH end groups may be further modified or activated ('functionalized' ) to ensure that covalent bonding to the viral structures occurs.

[0126] In an alternative embodiment, the invention relates to conjugates as described herein, where the graphene layers are replaced by a covalently bonded silane layer ("silicone coating") . In this embodiment, the conjugate comprises a particle and a multitude of viral structures wherein said particle has a size of 1 nanometer to 100 micrometer and comprises :

[0127] • one or more than one core, said core consists of a metal oxide having soft magnetic properties;

[0128] • said one or more than one core is covered by a first shell, said shell contains a polycondensated silane layer which is optionally functionalized; and

[0129] • said silane layer is optionally covered by a second shell, said second shell contains a polymer having functional end groups; with the proviso that, if no second shell is present, said one or more polycondensated silane layers are functionalized; with the proviso that the particle shell does not contain magnetic material; and wherein said viral structures are as described above and characterized in that said one or more viral structures are covalently bound to outer particle shell (i.e. functionalized silane layer if no second shell being present or functionalized polymer shell when being present. Architectures according to this embodiment are of type (la) , (1c) , (Id) and (2c) or (2d) , as discussed above and shown in fig. 10.

[0130] This embodiment is particularly suited in case the metal core is a metal oxide having soft magnetic properties, due to the chemical bonding by a siloxy group to the surface of the metal oxide. The preferred metal oxide is iron oxide. Silicon coated superparamagnetic iron oxide nanoparticles are known and described in Bloemen et al ( J . Nanopart . Res . (2012) 14:1100) .

[0131] In this embodiment, a polycondensated silane layer is formed by reacting the metal oxide particle with a trialkoxysilane in the presence of water and using ultrasound. As a consequence of abundantly present OH- groups at the metal oxide surface, covalent bonding and polycondensation occurs, resulting in a polycondensated silane layer fully covering the metal oxide core (thereby forming said "silicone coating") . This is illustrated in fig. 8. Depending on the trialkoxy silane chosen, a nonfunctionalized first shell if formed requiring a second shell, or a functionalized first shell is formed, making a second polymer shell optional.

[0132] Formulations

[0133] Suitably, the inventive conjugates are handled, applied, or administered in the form of a liquid formulation. Such formulations contain the inventive conjugates and a dispersion medium.

[0134] Suitably, the dispersion medium is selected from pharmaceutically acceptable aqueous media, preferably buffered aqueous media. Such dispersion media are known to the skilled person and include balanced salt solutions, such as PBS buffer.

[0135] Suitably, the concentration of the inventive conjugates is in the range of 1 - 1000 mg / mL, preferably 20 - 200 mg / mL. Suitably, the pH of the formulation is in the range of 4 - 10, preferably 5 - 9.

[0136] Suitably, the osmolality of the formulation is in the range of 200 - 400 mOsm / kg. In embodiments, the formulation contains one single type of conjugates. In this embodiment, all conjugates contain the same particle and the same viral structure on its surface. This is beneficial if it is known that the patient has antibodies against a specific viral vector (e.g., a serotype of AAV, such as AAV9) .

[0137] In embodiments, the formulation contains two or more types of conjugates (a "cocktail") . In this embodiment, a 1st group of conjugates contains a 1st group of viral structures, a 2nd group of conjugates contains a 2nd group of viral structures, and a nth group of conjugates contains a nth group of viral structures on its surface. This is beneficial since the conjugate cocktail could be adjusted to the viral vectors to be used for the gene therapy if more than one is used (e.g., different serotypes of AAV or AdV) , cross-reactive antibodies that could interfere with the gene therapy could be removed, and finally a cocktail of conjugates could be used as one treatment that fits all intended gene therapies and all patients.

[0138] In embodiments, the formulation described herein is a sterile formulation. It was found that sterilisation does not negatively impact performance of the formulations described herein.

[0139] In a second aspect, the invention relates to a process for manufacturing conjugates as described herein, comprising the step of forming at least one covalent bond between particle and viral structure (both as described herein) and to a process for manufacturing formulations as described herein. This aspect of the invention shall be explained in further detail below:

[0140] In embodiments, the invention provides for a process for manufacturing a conjugate as described herein, 1staspect, the process comprising the steps of: a) providing a first suspension comprising particles as described herein; b ) providing a second suspension ( or solution) comprising viral structures as described herein; c ) combining said first and second suspension ( or solution) to allow reacting the viral structures with the particles to form a covalent bond, optionally in the presence of an auxiliary reagent , optionally by applying heat or ultrasound, optionally by applying a catalyst , to thereby obtain a suspension comprising the conj ugate described herein; d) optionally further treatment of the suspension obtained in step c ) .

[0141] It is considered beneficial that the inventive process is applicable to a wide range of coated particles and to a wide range of viral structures by applying coupling chemistry known to the skilled person . The inventive process shall be explained in further detail below, referring to the steps a ) ... d)

[0142] Step a) : Suitably, the particles are surface-activated with suitable functional groups to allow or facilitate covalent bonding . Suspending particles in a dispersing medium is known in the field . To improve reaction in step c ) and / or to de-agglomerate particles , the suspension of step a ) may be subj ected to ultrasonication or vortexing . Suitable diluents include aqueous solutions optionally containing pH adj usting agents , surface-active agents , viscosity modi fiers and / or complexing agents . Suitable are , for example , PBS buf fer containing EDTA.

[0143] Step b) : Depending on the viral structure and the diluent , the material provided may be in the form of a suspension or in the form of a solution . To reflect this situation, the term ' second suspension ( or solution) is chosen . Suitable diluents are as described above , step a ) . Suitable are , for example PBS buf fer containing Glycerol and nonionic surfactants . Step c) : Viral structures contain free amine groups on their surface. These groups are capable in forming of a covalent bond with a reactive group present on the coated particle. Such reactions are known per se in organic chemistry, but not yet applied to the present starting materials (i.e. Viral structures and coated particles as described herein) . Further, the viral structures are optionally activated with suitable functional groups and / or linkers to allow or facilitate covalent bonding to the particle surface. Further, step c) may take place in the presence of an auxiliary reagent, and / or by applying heat or ultrasound, and / or in the presence of a catalyst.

[0144] Step d) , if applied, involves one or more of the following steps : dl) separating the liquid media of the suspension from the obtained conjugates, d2 ) lyophilizing the thus obtained conjugate, d3) purifying the thus obtained conjugate.

[0145] In embodiments, sterilization takes place after step a) and / or b) , and / or c) . In a preferred embodiment, sterilization takes place after step a) (i.e. before coupling) , followed by aseptically processing the remaining mandatory steps b) ,c) and optional steps d) , e) .

[0146] In embodiments, the invention provides for manufacturing a liquid formulation as described herein, the method comprising the step of f) combining conjugates as described herein (e.g. the composition obtained in step c) or d) or isolated ('dry' or 'lyophilized' ) conjugates) with a diluent compatible with blood, e.g. a pharmaceutically acceptable aqueous solution.

[0147] In a third aspect, the invention relates to new uses / applications of the inventive conjugates described herein. Generally speaking, the inventive conjugates are suited to remove antibodies present in a fluid, specifically blood (thereby enabling blood purification and analytical applications, see fig. 6) and as adjuvant therapy (thereby enabling or improving gene therapies and vaccination, see fig. 7) . This aspect of the invention shall be explained in further detail below:

[0148] In embodiments, the invention provides for conjugates and formulation as described herein for use in the separation of antibodies from a fluid, preferably in the removal of antibodies from a body fluid wherein said antibodies are capable of binding to the viral structures of said conj ugate .

[0149] In embodiments, the invention provides for conjugates and formulation as described herein for use in the detection of antibodies, preferably in the detection antibodies in a body fluid wherein said antibodies are capable of binding to the viral structures of said conjugate.

[0150] It is considered a substantial benefit of the inventive uses / applications that the conjugates contacting the fluid (e.g. blood) are capable of unrestricted movement within the fluid (e.g. blood) to be treated. Further, due to the small size of the conjugates used, they possess a large accessible specific surface area. Without being bound to theory, it is believed that these two attributes synergistically interact and therefore positively enhance mass transfer rates and augment the adsorption capacity of the antibodies relative to conventional methodologies described above. Still further, due to its magnetic properties, safe removal conjugates and complexes is achieved without contaminating the patient. Finally, the conjugates and formulations described herein may be sterilized without affecting its beneficial properties, thereby providing an additional layer of safety when applied to a subject in need. Overall, the method provides improved removal efficiency, reduced treatment times, and enhanced safety for the patient benefit when compared to the conventional treatments described above. In embodiments, the invention provides for conjugates as described herein for use in the removal of antibodies from blood. For this purpose, a sample of blood is taken from a patient's circulation and treated before it is returned to the circulation. This is illustrated in fig.6.

[0151] In embodiments, the invention relates to a method for removing antibodies from a fluid, particularly a body fluid, or detecting antibodies in a fluid, particularly in a body fluid, the method comprising: a. contacting the fluid with a conjugate as described herein; b. mixing the thus obtained composition to thereby effect binding of said antibodies to the conjugate to obtain an antibody-conjugate-complex; c. separating the antibody-conjugate-complex from the fluid by means of a magnetic material; and wherein said antibodies are capable of binding to the viral structures of said conjugate.

[0152] In embodiments, the antibody is selected from an anti-AAV antibody, an anti-AdV antibody, an anti-dengue virus antibody, an anti-vaccinia virus antibody, an anti-myxoma virus antibody, an anti-modified vaccinia Ankara virus antibody .

[0153] In embodiments, said fluid is selected from whole blood, blood serum, blood plasma, urine, spinal fluid, vitreous humor, and sputum; particularly whole blood.

[0154] In embodiments, the invention relates to a conjugate as described herein for use in virus-vector-based Gene therapy. This use is illustrated in fig.7.

[0155] In embodiments, the invention relates to a conjugate as described herein for use in AdV or AAV or MVA-vector based vaccination . In embodiments, the invention relates to a conjugate as described herein for use in the prevention and / or treatment of dengue fever.

[0156] To further illustrate the invention, the following examples are provided. These examples are provided with no intent to limit the scope of the invention.

[0157] Example 1.1 Synthesis of type Id particles conjugated to rAAV9 VCs via amide bond

[0158] Stocks of viral capsids (VC) of recombinant Adeno- Associated Virus Serotype 9 (AA V9) with genetic material (rAAV9, "full" capsid) and without genetic material (rAA V90, "empty" capsid) were provided in PBS pH 7.2 + 0.001% Pluronic F68 + 5% glycerol at 3.78E13 vgc / mL rAAV9 (equivalent to 1.0E14 - 1.3E14 VC / mL) and 3.0E14 vgc / mL rAA V90( equivalent to 2.24E14 VC / ml) . The VC stocks were diluted to 1.0E13 VCs / mL in PBS.

[0159] Magnetic graphene-coated iron-carbide core-shell particles coated with a hyperbranched polyglycerol were synthesized as described in [1] , [2] and [3] . The polymer -OH groups were then reacted to -COOH groups via succinylation as described in [4] . The carboxy groups were then activated using a one-pot reaction with EDC / sulfo-NHS as described in [5] .

[0160] The NHS-activated particles with semi-stable aminereactive NHS-esters on the surface were washed and resuspended in PBS buffer (containing 1 mM EDTA) at a particle concentration of 2 mg / mL. Vortexing and ultrasonication for 5 min were applied to homogeneously disperse the particles. The NHS-activated particle suspension was then immediately added to the previously prepared solution of rAAV9 at a particle concentration of 1 mg / mL. After ultrasonication for 1 min, this reaction mixture was then shaken for at least 3 h at 900 rpm at room temperature. The NHS-activated particles react with free amines on the VCs forming a covalent amide bond. The resulting particles-VC conjugates were sonicated for 30 sec and subsequently magnetically separated. A small sample of the supernatant was removed to determine the concentration of unreacted VC (see below section "Quantification of VC bound to particles") . The conjugates were again resuspended using ultrasonication (1 min) and a quenching solution of Tris buffer (20 mM) was added to the suspension. After mixing for 10 minutes at 500 rpm, the conjugates were washed three times with PBS containing 1 mM EDTA and then resuspended in PBS at concentrations of 5 mg / mL .

[0161] Example 1.2 Synthesis of type Id particles conjugated to rAAV9o VCs via amide bond

[0162] Example 1.1 was repeated, but the following was changed: Instead of binding rAAV9 VCs (VCs with genetic material) to the -COOH groups of the particle surface, rAA V90VCs (VCs without genetic material) were employed. The synthetic protocol is unchanged.

[0163] Example 1.3 Synthesis of type Id particles conjugated to rAAV2 or rAAV2o VCs via amide bond

[0164] Example 1.1 is repeated, but the following is changed: Instead of binding rAAV9 VCs to the particle surface, rAAV2 (with genetic material) or rAAV20(without genetic material) VCs are employed. As the EDC / sulfo-NHS activated particle surface binds to -NH2groups which are exhibited on different types of AAV serotypes, the covalent bonding via amide group is applicable different serotypes.

[0165] Example 1.4 Synthesis of type Id particles conjugated to rAAV9 VCs via thioether

[0166] Example 1.1 is repeated, but the following is changed: Instead of transforming the polymer -OH groups of the hyperbranched polyglycerol on the particle surface into - COOH groups, they are transformed into -NH2 groups as described in [6] . Free thiol groups are introduced to the surface of the particles via one of the two different methods : SPDP

[0167] The heterobifunctional, crossslinker succinimidyl 3- (2- pyridyldithio ) propionate (SPDP) allows to covalently link two target groups. The succinimidyl ester chemically reacts with the primary amines on the particles. Immediately before the subsequent conjugation reaction, mild reduction of the dithiol is performed, e.g. with DTT, (Dithiothreitol ) , where pyridine 2-thione is cleaved off and particles with free -SH groups are obtained.

[0168] SATA

[0169] N-Succinimidyl-S-acetylthioacetate (SATA) allows the introduction of thiol-groups in a protected form. SATA reacts with the NH2groups on the particles upon formation of a stable amide bond and release of NHS . The acetyl- protected thiol is stable for storage and deprotection to the free thiol with a 0.5 M solution of hydroxylamine is performed immediately before the subsequent conjugation reaction .

[0170] Subsequent conjugation to VCs

[0171] In order to form a covalent bond to the VCs, the amine groups on the VCs are first activated by introducing maleimide functionalities using the bifunctional linker sulfo-SMCC [7] . The maleimide-activated VCs are then added to the thiolated particles obtained above according to a) or b) , with the formation of a covalent thioether in a thia-Michael "click" addition.

[0172] Example 1.5 Synthesis of type la particles conjugated to rAAV9 VCs via amide bond

[0173] Example 1.1 is repeated, but the following is changed: Magnetic graphene-coated iron-carbide core-shell particles are synthesized as described in [1] and [2] . Instead of coating the particles with a hyperbranched polyglycerol as in Example 1.1, the -OH groups on the graphene shell are then directly reacted to -COOH groups via succinylation as described in [4] . The carboxy groups are then activated using a one-pot reaction with EDC / sulfo-NHS as described in [5] , and the coupling to VCs is performed as described in Example 1.1.

[0174] Example 1.6 Synthesis of type 2b particles with dextran conjugated to rAAV9 VCs

[0175] Example 1.1 is repeated but instead of using graphene- coated iron carbide particles, commercially available magnetic multi-core iron oxide particles with dextran as polymer shell functionalized with -COOH groups are employed (e.g., from micromod Partikeltechnologie GmbH, Nanomag-D, diameter = 130 nm, cat. no. 09-02-132) .

[0176] The carboxy groups are then activated using a one-pot reaction with EDC / sulfo-NHS and the coupling to rAAV9 VCs is performed as described in Example 1.1.

[0177] Reference list to examples 1.1 - 1.6

[0178] [1] R. N. Grass, et al, Carbon coated magnetic nanoparticles and their use in separation processes, EP2086687 (WO 2008 / 055371 (15.05.2008 Gazette 2008 / 20) )

[0179] [2] I. K. Herrmann, R. N. Grass, D. Mazunin and W. J. Stark, Synthesis and Covalent Surface Functionalization of Nonoxidic Iron Core-Shell Nanomagnets, Chem. Mater. 2009, 21, 3275-3281.

[0180] [3] S. Doswald et al, Low molecular weight glycerol derived coatings on magnetic nanoparticles: role of initiator, temperature, rate of monomer addition, enhanced biocompatibility and stability, RSC Adv., 2021, 11, 40140- 40147.

[0181] [4] Yang, Hee-Man; et al, Succinate Functionalization of Hyperbranched Polyglycerol-Coated Magnetic Nanoparticles as a Draw Solute During Forward Osmosis, Journal of Nanoscience and Nanotechnology, Volume 15, Number 10, October 2015, pp . 8279-8284 (6) .

[0182] [5] D. Bartczak and A. G. Kanaras, Preparation of Peptide- Functionalized Gold Nanoparticles Using One Pot EDC / Sulfo- NHS Coupling, Langmuir 2011, 27, 10119-10123.

[0183] [6] K. Paczyhska, et al, The Effect of Modifying Canadian Goldenrod (Solidago canadensis) Biomass with Ammonia and Epichlorohydrin on the Sorption Efficiency of Anionic Dyes from Water Solutions, Materials 2023, 16, 4586.

[0184] [7] M. Peacey, et al, Versatile RHDV Virus-Like Particles: Incorporation of Antigens by Genetic Modification and Chemical Conjugation, Biotechnology and Bioengineering, Vol. 98, No. 5, December 1, 2007.

[0185] Analysis :

[0186] The analytical data and results disclosed below are based on examples 1.1 and 1.2. Similar results are expected for examples 1.3 - 1.6.

[0187] DLS

[0188] Aqueous suspensions of conjugates were analyzed with dynamic light scattering (DLS) (Malvern Zetasizer) at final concentrations of 0.02 mg conjugates / mL diluted in water. Suspension homogeneity was assured by ultrasonication for 10 min in an ultrasonication bath during sample preparation and prior to analysis with DLS.

[0189] Zeta -potent! al

[0190] The surface charges of conjugates were evaluated using zeta potential measurements (Malvern Zetasizer) , where the velocity of the particles in an electrical field is recorded using laser Doppler electrophoresis. Aqueous suspensions of conjugates were diluted in 1 mM KC1 to a final concentration of 0.02 mg / mL. Suspension homogeneity was assured by ultrasonication for 10 min in an ultrasonication bath during sample preparation and prior to analysis with DLS.

[0191] AAV9 VC quantification assay

[0192] To quantify the numbers of rAAV9 or rAAV90VCs that were covalently bound to the surface of the particles, a sample of the supernatant of the coupling suspension was taken at the end of the reaction (but before addition of the quencher molecule) and analyzed with an AAV9 quantification ELISA kit (Progen, Cat. No. PRAAV9XP) . To generate the supernatant, the conjugates were magnetically separated. Subtracting the number of capsids in the supernatant from capsids offered to the particles gives the number of capsids bound to the particle surface.

[0193] # (VCs bound to particle surface) =

[0194] # (VCs offered in coupling reaction) - # (VCs left in supernatant after coupling reaction)

[0195] Results :

[0196] Physico-chemical characterization of conjugates with DLS and Zeta potential measurements

[0197] Two different size fractions were found by DLS: The average hydrodynamic diameter of the first fraction was in the range of 110-143 nm, that of the second fraction remained in the area of 400 to 450 nm.

[0198] Due to the carboxy-groups on the surface of the particles the zeta potential is typically negative, which was confirmed with the result of -37.89 mV. The zeta potential values of the conjugates are in the range between -39 and -33 mV. This is not surprising, as the carboxy groups of the precursor Particle Polymer typically give a negative surface charge, and AAV9 capsids also were reported to exhibit negative zeta potential values. Furthermore, the remaining activated groups on the particles surface where no capsid has bound (due to steric reasons) are quenched with a small molecule which also exhibits carboxy groups.

[0199] Quantification of VCs covalently bound to particles Using the AAV9 VC quantification assay, on average 80% of the VCs were bound per mg particles (Figure 3A) , which corresponds on average to 40% of the offered VCs.

[0200] Example 2: Anti-AAV9 antibody removal

[0201] Example 2.1: Anti-AAV9 antibody removal from PBS using conjugates with rAAV9 VCs

[0202] Example 2.2: Anti-AAV9 antibody removal from PBS using conjugates with rAAV90VCs

[0203] Materials and Methods:

[0204] Anti-AAV9 antibody binding assay A high binding black 384-well microtiter plate (Greiner, 781077) was coated with 1E11 vgc / mL rAAV9 in CMF-DPBS overnight at 4 °C. The plate was washed manually three times with CMF-DPBS, blocked with assay buffer (3 Sigma-Aldrich, Cat. No. A9576) in 1% Casein blocker (Thermo Fisher Scientific, Cat. No. 37528) for 3 hours at RT and washed again 3 times. The samples were added to the plate, incubated for 2 hours at RT, and washed 5 times with PBS. An HRP-conj ugated anti-human Fc detection antibody (Southern Biotech, 9040-05, 0.167 nM) for detection of human anti-AAV9 antibodies, or anti-mouse Fc detection antibody (Jackson ImmunoResearch, 115-035-071, 0.25 nM) for detection of ADK9 was added and incubated for 1 hour at RT in the dark. A QuantaBlu kit (ThermoFisher, 15189) was used as detection method by adding QuantaBlu substrate (1:10) to each well, incubating for 30 minutes at RT in the dark, and lastly adding STOP solution to each well. The plate fluorescent signal was measured in a Tecan Infinite F200 Fluorescence Reader within 30 minutes.

[0205] ADK9 removal assay

[0206] 1 mg / mL conjugates were added to PBS spiked with mouse ADK9 (anti-AAV9 monoclonal antibody, Progen, 610178) at 1 pg / mL. The mixture was incubated for 50 minutes at room temperature under continuous agitation, after which the magnetic conjugates were magnetically separated and the remaining ADK9 was quantified with the "Anti-AAV9 antibody binding assay", an ELISA-based method (see above) . The obtained values were compared to control samples (ADK9- spiked PBS without exposure to conjugates) to determine the relative removal of ADK9 by conjugates.

[0207] Results :

[0208] Determination of ADK9 anti-AAV9 antibody removal capacity of conjugates with rAAV9 or rAAV90

[0209] The goal of this experiment was to determine the anti-AAV9 antibody removal capacity of conjugates (1) in a controlled setting in PBS independent from biological varieties in human plasma and (2) using ADK9, a commercially available anti-AAV9 monoclonal antibody, allowing a quantitative evaluation of the removal capacity of manufactured conjugates using the ADK9 removal assay.

[0210] The average removal of ADK9 from PBS by conjugates with rAAV9 VCs was in average 43% (SD ± 26) and for rAAV90VCs was 57% (SD ± 9) (Figure 3B) .

[0211] Example 2.3: Anti-AAV9 antibody removal from human plasma using conjugates with rAAV9 VCs

[0212] Example 2.4: Anti-AAV9 antibody removal from human whole blood using conjugates with rAAV9 VCs

[0213] Materials and Methods:

[0214] Conjugate treatment of human plasma or blood

[0215] The conjugates were applied to remove VC-specific antibodies from blood in an application as described in WO 2021 / 063708 Al. These experiments were performed in a setup like the clinical application (Figure 2) : Conjugates were sonicated and vortexed for at least 30 seconds prior to diluting them in PBS to reach the required concentration used for treating the human plasma. The conjugates with VCs with or without genomic material were then added to the human plasma samples reaching a final concentration of either 2.0 or 0.5 mg / mL. The duration of one treatment cycle was either 3 minutes or 50 minutes at 37°C under continuous agitation in a HulaMixer Sample Mixer. In some experiments multiple treatment cycles of 3 minutes were performed by magnetically separating the conjugates from the human plasma and adding fresh conjugates for each consecutive treatment cycle. After the last treatment cycle the conjugates were magnetically separated. Both the treated and untreated human plasma was diluted at least 1:3 and the anti-AAV9 antibodies were measured in an ELISA- based method (see above "Anti-AAV9 antibody binding assay") . The values were normalized by correcting for the background the ratio was determined by dividing the measured value by the value of the positive control.

[0216] Results :

[0217] In the clinical setting the conjugate treatment will be performed in whole blood. To ensure that this was feasible, the treatment was performed with EDTA-anticoagulated blood from one donor (cat# 92000, Interregionale Blutspende SRK) containing anti-AAV9 antibodies. The results of the whole blood treatment were comparable to the treatment of human plasma to demonstrate that the treatment in either matrix is comparable. There were no differences in between the conjugate treatment in whole blood versus plasma, proving that the treatment works in both matrices (Figure 4) .

[0218] Example 3: Improvement of gene transduction efficiency after treating human plasma with conjugates

[0219] Example 3.1: Treatment with particle-rAAV9 conjugates Example 3.2: Treatment with particle-rAAV90conjugates

[0220] Material and Methods :

[0221] Transduction assay

[0222] In the virus neutralization assay the neutralizing antibodies are detected by measuring the transduction efficiency of AAV9 after exposure to anti-AAV antibodies. To determine the virus neutralizing capacities of the antibodies in human plasma before and after conjugate treatment, a virus neutralization assay was performed. HEK293T / 17 HG2 wells were seeded in a tissue culture treated 384-well plate at 8000 cells / well and allowed to attach for 6 hours 37°C + 5% CO2. In the meantime, an 11- point 3-fold serial dilution was prepared with the human plasma, ADK9, and isotype control. A recombinant AAV (GMO RA generic@ACl) containing luciferase was prepared at 4E9 genome copies / mL (multiplicity of infection 5000 on 8000 cells) and the virus was added to the plasma / IgG dilution series. Directly after, the virus / IgG mixture was added to the cells and the plate was incubated for 48 hours at 37°C + 5% CO2 • The amount of virus in the cells was determined with Steady-Gio.

[0223] Transduction efficiency after conjugate treatment of human plasma

[0224] To assess if the conjugate treatment would restore transduction efficiency, human plasma was treated with the conjugate and the binding antibodies and transduction ef ficiency were measured . The treatment was performed with conj ugates containing VCs with or without genetic material . This treatment protocol was tested for 3 donors with veri fied anti-AAV titers . Serum from 3 donors with low titers were used to represent the "gene therapy treatable range" , defined as the average value ± SD of these donors in the assay . Treatment with both conj ugates led to a reproducible depletion of antibodies that resulted in titers in the same range as the low titer donor samples ( Figure 5A) . These depletions correlated with an increased transduction ef ficiency that was similar for both treatments as well ( Figure 5B ) . Based on these results it can be concluded that both full and empty capsids can be used as binding agents to deplete anti-AAV antibodies .

Claims

Claims1. A conjugate comprising a particle and one or more viral structures, wherein said particle has a size of 1 nanometer to100 micrometers and comprises:• one or more than one core, said core consists of a material having soft magnetic properties;• each core optionally covered by a first shell, said shell contains one or more graphene layers which are optionally functionalized; and / or• said one or more cores are covered by a second shell, said second shell contains a polymer having functional end groups; with the proviso that, if no second shell is present, said one or more graphene layers are functionalized; with the proviso that the particle shell does not contain magnetic material; and wherein said viral structures are selected from the group consisting of Viral capsids (VCs) , individual structural viral proteins (VPs) , and assemblies of structural viral proteins; characterized in that said one or more viral structures are covalently bound to said particle shell .

2. The conjugate of claim 1, wherein said particle consists of:Either ('type la particles' )• a single core, said core consists of a material having soft magnetic properties;• covered by said first shell, which contains one or more graphene layers that are functionalized;OR ('type lb particles' )• a single core, said core consists of a material having soft magnetic properties;• covered by said second shell, said second shell contains a polymer having functional end groups;OR ('type 1c particles' )• a single core, said core consists of a material having soft magnetic properties;• covered by said first shell, which contains one or more graphene layers that are not functionalized; and• covered by said second shell, said second shell contains a polymer having functional end groups; OR ('type Id particles' )• a single core, said core consists of a material having soft magnetic properties;• covered by said first shell, which contains one or more graphene layers that are functionalized; and• covered by said second shell, said second shell contains a polymer having functional end groups;OR ('type 2b particles' )• a multitude of cores, said cores consists of a material having soft magnetic properties;• covered by said second shell and thereby embedding said multitude of cores, said second shell contains a polymer having functional end groups;OR ('type 2c particles' )• a multitude of cores, said cores consist of a material having soft magnetic properties;• each of said cores being covered by said first shell, which contains one or more graphene layers that are not functionalized; and• all of said coated cores being covered by said second shell and thereby embedding said multitude of cores, said second shell contains a polymer having functional end groups;OR ('type 2d particles' )• a multitude of cores, said cores consists of a material having soft magnetic properties;• each of said cores being covered by said first shell, which contains one or more graphene layers that are functionalized; and• covered by said second shell, said second shell contains a polymer having functional end groups.

3. The conjugate of claim 1 or 2 wherein the core• is an iron-based material, which preferably contains at least 50wt% Fe; or• is a cobalt-based material, which preferably contains at least 50% Co; or• is a nickel-based material, which preferably contains at least 50% Ni; and / or• is a material comprising at least two of the elements iron, cobalt and nickel, where the total content of these three elements is at least 50% of the core mass; or• has a size of 1 nm - 500 nm; or• has soft magnetic properties with a coercive force below 30'000 A / m.

4. The conjugate according to any of claims 1 - 3, wherein the first shell• Comprises 3 - 15 graphene layers; and / or• Comprises linkers of formula (I)wherein n is an integer between 1 and 5; m is an integer between 0 and 20;Z represents a spacer selected from the group of alkyl, alkenyl, alkyloxy, amino acids, and saccharides ;FG represents independent from each other OH, OR, N3, NO2, NH2, NHR, NR2, CHO, COR, COOH, COOR, SO2SR, SOR, SO2R, halogen, P(O)OH, P(O)OR, P(O)2OH, P(O)2OR, 1 , 2 , 3-triazol-4-R, 1 , 2 , 3-triazol-5-RR represents Ci-C4alkyl, Ci-C4alkoxy, C3-Ci0cyclo- alkyl, C4-Cio aryl, or a 5-membered heterocycle containing at least one atom selected from the group of N, 0, and S, or a 6-membered heterocycle containing at least one atom selected from the group of N, 0, and S.

5. The conjugate according to any of claims 1 - 4, wherein the second shell comprises or consists of a polymer having functional end groups, wherein• the polymer is selected from the group consisting of Polyglycerol, Polyethylene-glycol (PEG) , Dextran, Hydroxyethyl starch, Polylactic acid, Styrenemaleic acid (SMA) copolymers, Polystyrene,Poly ( ethylene imine) , Chitosan, and Polymethylmethacrylate; and• the functional end groups are selected from OH, OR, N3, NO2, NH2, NHR, NR2, OHO, COR, COOH, COOR, S02, SOR, SO2R, halogen, P(O)OH, P(O)OR, P(O)2OH, P(O)2OR, 1 , 2 , 3-triazol-4-R, and 1 , 2 , 3-triazol-5-R• R represents a group selected from C1-C4 alkyl, C2- C4 alkenyl, C1-C4 alkyloxy, an amino acid, a dipeptide, a tripeptide, a monosaccharide, and a disaccharide; and / or• the amount of functional end groups is at least one per particle.

6. The conjugate according to any of claims 1 - 5, wherein• the viral structures are based on adenoviruses(AdV) or adeno-associated-viruses (AAV) or modified vaccinia Ankara virus (MVA) or dengue viruses; and / or• the viral structures are selected from the group consisting of o VCs with genetic material and with a viral envelope (VE) ; o VCs with genetic material but without a viral envelope (VE) ; o VCs without genetic material but with a viral envelope (VE) ; and o VCs without genetic material and without a viral envelope (VE) .

7. The conjugate according to any of claims 1 - 6, in the form of a bulk (dry) material or in the form of a suspension, which is sterile.

8. The conjugate according to any of claims 1 - 7, wherein• said particle has a hydrodynamic diameter of 5 - 2000 nm and• 2 to 1'000'000, preferably 4 to 40'000, viral structures are covalently bound to the surface of said particle.

9. A liquid formulation comprising conjugates according to any of claims 1 - 8 and a dispersion medium, the formulation complying with one or more of the following :• the dispersion medium being selected from pharmaceutically acceptable aqueous media;• the concentration of said conjugates being in the range of 2 - 200 mg / mL;• the formulation having a pH in the range of 4 - 10;• the formulation having an osmolality in the range of 200 - 400 mOsm / kg; and / or• the formulation is sterile.

10. The liquid formulation according to claim 9, comprising either• a multitude of one type of conjugates; or• a multitude of two or more types of conjugates.

11. The conjugate according to any of claims 1 - 8 or the liquid formulation according to any of claims 9 - 10, for use• in the separation of antibodies from a fluid, preferably in the removal of antibodies from a body fluid; or• in the detection of antibodies, preferably in the detection antibodies in a body fluid; wherein said antibodies are capable of binding to the viral structures of said conjugate.

12. A method for removing antibodies from a fluid, particularly a body fluid, or detecting antibodies ina fluid, particularly in a body fluid, the method comprising : a. contacting the fluid with a conjugate according to claim 1 - 8 or a formulation according to claims 9 - 10; b. mixing the thus obtained composition to thereby effect binding of said antibodies to the conjugate to obtain an antibody-conjugate- complex; c. separating the antibody-conjugate-complex from the fluid by means of a magnetic material; and wherein said antibodies are capable of binding to the viral structures of said conjugate.

13. The method according to claim 12, or the conjugate for use according to claim 11, wherein• the antibody is selected from an anti-AAV antibody, an anti-AdV antibody, an anti-dengue virus antibody, an anti-modified vaccinia Ankara virus antibody; and / or• said fluid is selected from whole blood, blood serum, blood plasma, urine, spinal fluid, vitreous humor, and sputum.

14. The conjugate of claim 1 - 8 for use• in AdV- or AAV based Gene therapy; or• in AdV or AAV or MVA-vector based vaccination; or• in the prevention and / or treatment of dengue fever.

15. A process for manufacturing a conjugate according to any of claims 1 - 8, the process comprising the steps of : a) providing a first suspension comprising particles as defined in claim 1; b) providing a second suspension or solution comprising viral structures as defined in claim 1; c) combining said first and second suspension or solution to allow reacting the viral structures with the particles to form a covalent bond, optionally in the presence of an auxiliaryreagent, optionally by applying heat or ultrasound, optionally by applying a catalyst, to thereby obtain a suspension comprising the conjugate according to any of claims 1 - 8; d) optionally further treatment of the suspension obtained in step c) .

16. The process of claim 15, wherein in step a) the particles are surface-activated with suitable functional groups to allow covalent bonding; and / or in step b) the viral structures are optionally activated with suitable functional groups and / or linkers to allow covalent bonding to the particle surface; and / or step d) takes place and involves one or more of the following steps: dl) separating the liquid media of the suspension from the obtained conjugates, d2 ) lyophilizing the thus obtained conjugate, d3) purifying the thus obtained conjugate.

17. The process of claim 15 or 16, wherein sterilization takes place after step a) and / or b) , and / or c) .

18. A method for manufacturing a liquid formulation according to claim 9 or 10, the method comprising the steps of: e) providing an aqueous solution with buffers and / or excipients, and f) combining the conjugate suspension of claim 15 step c) or d) with the solution from step d) .

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