The field of the invention

The present invention relates to a composition comprising a solid carrier, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer. The present invention also relates to a method for the prevention, delay of progression or treatment of lung cancer in a subject using said composition and methods of producing said composition.

Background of the invention

Proteins such as enzymes are frequently needed, e.g. in industrial applications, diagnostics or for therapeutic use. In order to stabilize the proteins and/or to provide resistance to various types of stresses it has been suggested in the prior art to immobilize the proteins on the surface of a carrier and to protect them with a layer of protective material. Such an approach has been described e.g. in WO2015/014888 which discloses a biocatalytical composition comprising a solid carrier, an enzyme and a protective layer for protecting the enzyme by embedding the enzyme and a process to produce such biocatalytical composition. However biocatalytical compositions as described e.g in WO2015/014888 cannot be used in therapeutic application due to their lack of biocompability and bioavailability. Thus there is a need to provide biocatalytical compositions compatible and useful for therapeutic applications such as treatment of cancer, in particular treatment of lung cancer.

Summary of the invention

The present invention provides a composition comprising a solid carrier, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the particle size of the solid carrier is between 15 nm and 40 nm.

The present invention provides also a method of producing said composition comprising a solid carrier, wherein the particle size of the solid carrier is between 15 nm and 40 nm, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, the method comprising the following steps:

(a) providing a solid carrier, wherein the particle size of the solid carrier is between 15 nm and 40 nm;

(b) immobilizing an enzyme or a fragment thereof on the solid carrier;

(c) forming a protective layer on the surface of the solid carrier to protect the enzyme or the fragment thereof immobilized on the solid carrier;

(d) immobilizing a functional constituent on the surface of the protective layer.

It has been surprisingly found by the inventors of the present application that compositions as provided by the present invention if applied therapeutically have an unexpected tissue distribution resulting in a major accumulation in the lung, thus making them extremely promising for therapeutic use such as treatment of lung cancer.

Brief description of the figures

Figure 1) shows a schematic representation of the process for the production of the composition of the invention: a) an enzyme or fragment is immobilized on the solid carrier; b) and c) a protective layer grows around the immobilized enzyme or fragment thereof embedding the immobilized enzyme or fragment thereof; and d) a functional constituent is immobilized on the surface of the protective layer.

Figure 2) shows the biodistribution of functionalized nanoparticles in CD-I mice.

Functionalized NP-50 and Functionalized NP-30 were radiolabeled with 177-Lutetium and injected retro-orbitally to CD-I mice. (A) The comparative blood distribution of Functionalized NP-50 and Functionalized NP-30 is depicted. (B-C) The tissue biodistribution of Functionalized NP-50 (B) and Functionalized NP-30 (C) is shown at different time points (24, 120 and 168h). The radioactivity in blood and tissues was analyzed by the counting of tissue radioactivity in an automatic gamma counter and is expressed as percentage of the injected dose per gram of organ (%ID/g). Detailed description of the invention

The present invention relates to a composition comprising a solid carrier, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the particle size of the solid carrier is between 15 nm and 40 nm.

For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Features, integers, characteristics, compounds described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments.

The term “comprise” and variations thereof, such as, “comprises” and “comprising” is generally used in the sense of include, that is, as “including, but not limited to” , that is to say permitting the presence of one or more features or components.

The singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

The term "about" refers to a range of values ± 10% of a specified value. For example, the phrase "about 200" includes ± 10% of 200, or from 180 to 220.

The term “solid carrier” as used herein refers usually to a particle. Preferably the solid carrier is a monodisperse particle or a poly disperse particle, more preferably a monodisperse particle. The solid carrier usually comprises organic particles, inorganic particles, organic-inorganic particles, self-assembling organic particles, silica particles, gold particles, titanium particles and is preferably a silica particle, more preferably a silica nanoparticle (SNP).

The term “linker” or “cross-linker” which are used synonymously herein refers to any linking reagents containing groups, which are capable of binding to specific functional groups (e.g. primary amines, sulfhydryls, etc.). A linker in the context of the present invention usually connects the surface of the solid carrier with the enzyme. For example, a linker may be immobilized on the surface of the solid carrier e.g. on the silica surface as a carrier material and then the enzyme may be bound to an unoccupied binding-site of the linker. Alternatively, the linker may firstly bind to the enzyme and then the linker bound to the enzyme may bind with its unoccupied binding-site to the solid carrier. Various types of linkers are known in the art, including but not limited to straight or branched-chain carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers that are known in the art as tags.

The term “protective layer” as used herein refers to a layer for protecting the functional properties of the enzyme immobilized on the surface of the solid carrier. The protective layer of the present invention is usually built with building blocks at least part of which are monomers capable of interacting with both each other usually by covalent binding and the immobilized enzyme usually by non-covalent binding. The protective layers are usually homogeneous layers where at least 50%, preferably at least 70%, more preferably at least 90% of the enzyme or fragment therof are embedded in the protective layer.

The term, "enzyme or a fragment thereof' includes naturally occurring enzymes or a fragment thereof and also includes artificially engineered enzymes or a fragment thereof. Artificially engineered enzymes or a fragment thereof are e.g. variants or functionally active fragments of the enzyme. By “variants or functionally active fragments thereof’ in relation to the enzyme of the present invention is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of exercising the same physiological function as the enzyme. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least about 80% sequence identity more preferably at least about 90% sequence identity, even more preferably at least about 95% sequence identity, most preferably at least about 98% sequence identity to the relevant part of the enzyme.

The term “partially embedded enzyme” as used herein shall mean that the enzyme is not fully covered by the protective layer, thus, the enzyme is not fully embedded in the protective layer. In one embodiment less than 50% of the enzyme of interest are covered by the protective layer, though typically more at least 70% will be covered, thus improving protection of the enzyme. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% of the enzyme of interest is covered by the protective layer. In another preferred embodiment, around 70% to around 95%, more preferrably around 80% to around 95%, even more preferably around 90% to around 95%, most preferably around 90% to around 95, 96, 97, 98 or 99 % of the enzyme of interest are covered by the protective layer. In a particularly preferred embodiment, around 70%, particularly around 80%, more particularly around 90%, most particularly around 95% of the enzyme of interest is covered by the protective layer. In a more particularly preferred embodiment, around 70%, particularly around 80%, more particularly around 90%, most particularly around 95% of the enzyme of interest is covered by the protective layer, wherein the active site is not covered.

The term “fully embedded enzyme” as used herein shall mean that the enzyme of interest according to the invention is fully, i.e. 100% covered by the protective layer, i.e. that also the active site is covered.

The term “at least partially embedded enzyme” as used herein shall mean that the enzyme is at least partially embedded and may be fully embedded by the protective layer. Thus “at least partially embedded enzyme” means that the protective layer covers from about 30% and 100% of the enzyme or a fragment therof, preferably from about 50% to about 100%, more preferably from about 80% to about 100%, even more preferably from about 90% to about 100%, most preferably from about 95% to about 100 %, wherein the active site is preferably covered. The term “functional constitutenf ’ as used herein refers to a constituent which after being immobilized to the surface of the protective layer retains its characteristic, functional property. A functional constituent in the sense of the present invention can be e.g. an amphiphilic drug, an amino acid, a peptide, a protein or a fragment thereof, a silane copolymer or a combination of a protein or a fragment thereof and a silane copolymer, with the proviso that the protein or a fragment thereof is not the enzyme or a fragment thereof immobilized on the surface of the solid carrier.

The term “peptide” as used herein designates a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues and have usually an amino acid sequence comprising between at least 10 amino acids and not more than 100 amino acids.

The term “protein or fragment thereof’ as used herein contains usually between 100 and 1500 amino acids, preferably between 100 and 800 amino acids, more preferably between 100 and 500 amino acids. A fragment of a protein as defined herein does usually have the same functional properties as the protein from which it is derived.

"Immunoglobulins" (Ig), also synonymously called "antibodies" herein, are generally comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain, single domain antibodies (dAbs) which can be either be derived from a heavy or light chain); including full length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized and fully human antibodies, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multi specific, and dual variable domain immunoglobulins; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) and allotype.

An "immunoglobulin fragment", as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length, including, but not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of a Fab(Fa) fragment, which consists of the VHand CHI domains; (iv) a variable fragment (Fv) fragment, which consists of the VLand VHdomains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain FvFragment (scFv); (viii) a diabody, which is a bivalent, bispecific antibody in which VHand VLdomains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites; and (ix) a linear antibody, which comprises a pair of tandem Fvsegments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (x) a nanobody and (xi) other non-full length portions of immunoglobulin heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

The term “lung cancer” also synonymously called “lung carcinoma” as used herein refers to a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung. The term “lung cancer” as used herein comprises primary and secondary cancers of the lung. Primary cancers of the lung refer to a cancer which starts growing in the lung. Secondary cancers of the lung also synonymously called “metastatic tumors in the lung” refer to a cancer that spreads from its original site to the lung as distant part of the body. Almost any cancer can spread to the lung, the most commun types are: bladder cancer, breast cancer, colon cancer, kidney cancer, melanoma, salivary gland cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, stomach cancer, thyroid cancer, testicular cancer, uterine cancer and sarcoma. Lung cancers are classified according to histological type. Lung cancers are carcinomas - malignancies that arise from epithelial cells. Lung carcinomas are normally categorized by the size and appearance of the malignant cells seen by a histopathologist under a microscope. For therapeutic purposes, two classes are distinguished: non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC) such as adenocarcinoma in situ, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma and sarcomatoid carcinoma. In a first aspect the present invention provides a composition comprising a solid carrier, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the particle size of the solid carrier is between 15 nm and 40 nm.

The enzyme can be immobilized on the surface of the solid carrier by non-covalent binding or covalent binding. Non-covalent binding includes p-p (aromatic) interactions, van der Waals interactions, H-bonding interactions, and ionic interactions. Preferably the enzyme is immobilized on the surface of the solid carrier by covalent binding or by covalent binding via a linker.

In one embodiment the solid carrier is selected from the group of organic particles, inorganic particles, organic-inorganic particles, self-assembling organic particles, silica particles, gold particles, titanium particles and is preferably a silica particle, more preferably a silica nanoparticle (SNP). The particle size of the solid carier is usually measured by measuring the diameter of the solid carrier and is usually between 15 nm and 45 nm, preferably between 15 nm and 40 nm, more preferably between 20 nm and 40 nm, even more preferably between 25 nm and 35 nm, particularly about 30 nm. The particle size of the solid carrier is measured by measuring the diameter of the solid carrier prior to immobilizing an enzyme or a fragment thereof on the solid carrier i.e. the particle size of the solid carrier is measured by measuring the diameter of the solid carrier per se. The particle size of the solid carrier is usually measured by using a microscope such as scanning electron microscope (SEM).

Usually monodisperse particles or polydisperse particles, preferably monodisperse particles are used as solid carrier in the present invention. In a preferred embodiment the monodisperse particles are spherical monodisperse particles.

The solid carrier is usually provided in suspension. Suspension of the solid carrier can be e.g. in water, buffer or non-ionic surfactants or mixtures thereof, preferably in mixtures of water and non-ionic surfactants. Buffers which can be used in the method of the present invention are phosphate, piperazine-N,N'-bis(2-ethanesulfonic acid), 2-Hydroxy-3- morpholinopropanesulfonic acid, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), (3-(N- morpholino)propanesulfonic acid), 2-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid, 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid), 3-(N,N- Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid, N, N-Bis(2-hy droxy ethyl)-3-amino-

2-hydroxypropanesulfonic acid, N-[Tris(hydroxymethyl)methyl]glycine, Diglycine, 4-(2- Hy droxy ethyl)- 1 -piperazinepropanesulfonic acid, N,N-Bis(2-hy droxy ethyl)gly cine, N- [Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, N-(l, l-Dimethyl-2-hy droxy ethyl)-

3-amino-2-hydroxypropanesulfonic acid.

In one embodiment the surface of the solid carrier is modified to introduce a molecule or functional chemical group as anchoring point i.e. as anchoring point for the enzyme or for the linker connecting the enzyme to the solid carrier. Preferably, said anchoring point is an amine functional chemical group or moiety. As a non-limiting example, an amino-modified surface of the solid carrier e.g. an amino-modified silica surface may be used as modified solid carrier. Such an amino-modified surface of the solid carrier may be obtained by reacting a solid carrier having a silica surface with an amino silane, e.g. with APTES. Thus in a preferred embodiment, the solid carrier is a solid carrier having a silica surface with an amino-modified surface, more preferably a solid carrier obtained by reacting the solid carrier having a silica surface with an amino silane, e.g. with APTES. Such a modified carrier may form an amide linkage between the enzyme and the amine group at the surface of the carrier material or an amide linkage between the linker and the amine group at the surface of the carrier material. In one embodiment the introduced molecule or functional chemical group as anchoring point is homogeneously distributed on the surface of the solid carrier. Preferably the surface of the solid carrier is only partially amino-modified. Thus in a preferred embodiment, the solid carrier is a solid carrier having a silica surface with an amino-modified surface, more preferably a solid carrier obtained by reacting the solid carrier having a silica surface with an amino silane, e.g. with APTES, even more preferably a solid carrier obtained by reacting the solid carrier having a silica surface partially with an amino silane, e.g. with APTES.

In some embodiments the protective layer has a defined thickness of about 1 to about 200 nm, usually 1 to about 100 nm, preferably about 1 to about 50 nm, more preferably about 1 to about 25 nm, even more preferably about 1 to about 20 nm, in particular about 1 to about 15 nm. The most preferred defined thickness is about 1 to about 15 nm. In some embodiments the layer has a defined thickness of about 5 to about 100 nm, preferably about 5 to about 50 nm, more preferably about 5 to about 25 nm, even more preferably about 5 to about 20 nm, in particular about 5 to about 15 nm. The most preferred defined thickness is about 5 to about 15 nm. The protective layer is usually porous and the pore size is between 1 and 100 nm, preferably between 1 and 20 nm.

In one embodiment, the enzyme or a fragment thereof is partially embedded by the protective layer. In another embodiment the enzyme or a fragment thereof is fully embedded by the protective layer. In a preferred embodiment the enzyme or a fragment thereof is at least partially embedded by the protective layer.

In one embodiment, the protective layer embeds the solid carrier and embeds the enzyme or a fragment thereof immobilized on the surface of the solid carrier. In one embodiment, the functional constituent immobilized on the surface of the protective layer is not embedded by the protective layer. Preferably, the protective layer fully embeds the solid carrier and fully embeds the enzyme or a fragment thereof immobilized on the surface of the solid carrier. More preferably, the protective layer fully embeds the solid carrier and fully embeds the enzyme or a fragment thereof immobilized on the surface of the solid carrier, wherein the functional constituent immobilized on the surface of the protective layer is not embedded by the protective layer. If the protective layer fully embeds the solid carrier and fully embeds the enzyme or a fragment thereof immobilized on the surface of the solid carrier, the enzyme is fully, i.e. 100% covered by the protective layer, so that also the active site is covered and the solid carrier is fully, i.e. 100% covered by the protective layer.

In one embodiment, the present invention comprises a composition comprising a solid carrier, wherein the particle size of the solid carrier is between 15 nm and 40 nm, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is different from the enzyme or the fragment thereof immobilized on the surface of the solid carrier. In one embodiment, the present invention comprises a composition comprising a solid carrier, wherein the particle size of the solid carrier is between 15 nm and 40 nm, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is not the enzyme or the fragment thereof immobilized on the surface of the solid carrier.

In one embodiment the enzyme or a fragment thereof is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, transpeptidases, or ligases, or a fragment thereof and mixtures thereof. Particular preferred is a hydrolase or a fragment thereof, more particular a hydrolase or a fragment thereof selected from the group consisting of a deaminase or a fragment thereof, a glucuronidase or a fragment thereof and a peptidase or a fragment thereof, even more particular a peptidase or a fragment thereof, preferably a peptidase or a fragment thereof selected from the group consisting of cysteinase, methioninase arginase and asparaginase, or a fragment therof, most particular an asparaginase or a fragment thereof.

The protective layer thickness can be measured, by using a microscope such as scanning electron microscope (SEM), transmission electron microscopy (TEM), scanning probe microscopy (SPM), light scattering methods or by ellipsometry.

The composition of the present invention is usually produced in a reaction vessel like a reactor. The formation of the protective layer is usually carried out by forming the respective protective layer by building blocks, wherein the building blocks build the protective layer in a polycondensation reaction. The polycondensation can be effected in different solvents, preferably in aqueous solution. Polycondensation can be easily controlled and stopped if appropriate, allowing achievement of a defined thickness of the protective layer. The choice of the building blocks, which can be used to build the protective layer, may depend on the known structure of the enzyme in order to adapt the affinity of the protective layer according to optimal and/or desired parameters. As building blocks for the protective layer usually structural building blocks and protective building blocks are used to build the protective layer. Structural building blocks which can be used are e.g. tetraethylorthosilicate (designated herein as “TEOS” or “T”). Protective building blocks which can be used are e.g. 3-Aminopropyltriethoxysilane (designated herein as “APTES” or “A”), Propyltriethyoxysilane (designated herein as “PTES” or P”), Isobutyltriethoxysilane (designated as “IBTES”), Hydroxymethyltriethoxysilane (designated herein as “HTMEOS” or H), Benzyltriethoxysilane (designated herein as “BEES”), Ureidopropyltriethoxysilane (designated as “UPTES”), or Carboxyethyltriethoxysilane (designated herein as “CETES”). Structural building blocks are usually precursors of inorganic silica, capable of forming 4 covalent bonds in the layer formed. Protective building blocks are usually organosilanes, bearing an organic moiety endowed with the ability to interact with the enzymes (e.g., enzyme). Preferred structural building blocks are tetravalent silanes, in particular tetra-alkoxy-silanes. Preferred protective building blocks are trivalent silanes, in particular tri-alkoxy-silanes. More preferred structural building blocks are mixtures of tetravalent silanes and trivalent silanes, in particular mixtures of tetra-alkoxy-silanes and tri- alkoxy-silanes. Even more preferred structural building blocks are selected from the group consisting of tetraethylorthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethylorthosilicate. Even more preferred protective building blocks are selected from the group consisting of carboxyethylsilanetriol, benzyl silanes, propylsilanes, isobutyl silanes, n-octylsilanes, hydroxysilanes, bis(2-hydroxyethyl)-3 -aminopropylsilanes, aminopropylsilanes, urei dopropyl sil anes, (N - Acetylgly cyl)-3 -aminopropyl silanes, hydroxy(polyethyleneoxy)propyl]triethoxysilanes, in particular selected from benzyltriethoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltri ethoxy silane, bis(2-hydroxyethyl)-3 -aminopropyltriethoxy silane, 3- Aminopropyltriethoxy silane, ureidopropyltriethoxysilane, (N- Acetylgly cyl)-3 - aminopropyltriethoxy silane, or selected from benzyltrimethoxysflane, propyltrimethoxysilane, isobutylimethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2- hydroxyethyl)-3 -aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltrimethoxysilane (N-Acetylglycyl)-3 -aminopropyltrimethoxysilane or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxy ethoxysilane, n-octyltrihydroxy ethoxysilane, hydroxymefilyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3 - aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, Ureidopropyltrihydroxy ethoxysilane (N-Acetylglycyl)-3-aminopropyltrihydroxymethoxysilane. Particular preferred building blocks are TEOS as structural building block and APTES, PTES, and/or HTMEOS, preferably APTES as protective building block. In particular TEOS as structural building block and APTES as protective building block are used to build the protective layer.

The reaction time of the building blocks with the solid carrier depends on the length of the linker, if a linker is used, and the size of the enzyme. The reaction is usually carried out for a time period of between 0.5 to 10 hours, preferably between 1 and 5 hours, more preferably between 1 and 4 hours, even more preferably between 2 and 4 hours, preferably in aqueous solution and preferably at room temperature of about 5 to about 25 °C or at about 20 °C. The formation of the protective layer can be stopped by actively stopping the polycondensation reaction e.g by removing the non-reacted building blocks e.g. by a washing step or by selfstopping of the polycondensation reaction caused by a limited amount of buidling blocks.

In a further more preferred embodiment the enzyme is immobilized on the solid carrier by at least partly modifying the surface of the solid carrier by introducing a molecule as anchoring point as described supra for the enzyme and by using a linker, preferably a cross-linker binding to the anchoring point and the enzyme.

In one embodiment the introduced molecule as anchoring point and/or the linker are homogeneously distributed on the surface of the solid carrier.

In a preferred embodiment the cross-linker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, l,5-difluoro-2,4-dinitrobenzene, activated sulfhydrils, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (Bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone), DSP (Dithiobis[succinimidyl]propionate]), DTSSP (3,3 '-Dithiobis[sulfosuccinimidyl]propionate]), DTBP (Dimethyl 3,3 '- dithiobispropionimidate-2 HC1), DST (Disuccinimidyl tartarate), Sulfo-LC-SMPT (4- Sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexa noate)), SPDP (N-Succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (Succinimidyl 6-(3-[2-pyridyl dithio]- propionamido)hexanoate), SMPT (4-Succinimidyloxycarbonyl-methyl-a-[2- pyridyldithio]toluene), DPDPB (l,4-Di-[3'-(2'-pyridyldithio)-propionamido]butane), DTME (Dithio-bismaleimidoethane), BMDB (1,4 bi smaleimidyl-2, 3 -dihydroxybutane), Dibenzocy cl ooctyne-mal eimide (DBCO-mal eimide) and Azido PEG Mai eimide (N3-PEG- maleimide). More preferably said cross-linker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bisfsulfosuccinimidyl] suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, l,5-difhioro-2,4-dinitrobenzene, activated sulfhydrils (e.g. suflhydryl-reactive 2-pyridyldithio), Dibenzocy clooctyne-maleimide (DBCO- maleimide) and Azido PEG Maleimide (N3-PEG-maleimide). Paticular preferred is glutaraldehyde, Dibenzocy clooctyne-maleimide (DBCO-maleimide) and Azido PEG Maleimide (N3-PEG-maleimide). Most preferred is glutaraldehyde. Some compounds of the above mentioned cross-linkers, like e.g. Dibenzocy clooctyne-maleimide (DBCO-maleimide) and Azido PEG Maleimide (N3-PEG-mal eimide), respectively, form the cross linker by reacting with each other e.g. in situ e.g. in a “click chemistry” reaction (Copper-catalysed azide-alkyne cycloaddition, see e.g. Kolb et al. (2001) Angew. Chem. 40(11)2004-2021), e.g.

Dibenzocy clooctyne-maleimide (DBCO-maleimide) can be bound to the surface of the protective layer and Azido PEG Maleimide (N3-PEG-mal eimide) can be bound to the functionlisation constituent e.g. to an immunoglobulin, and the Dibenzocyclooctyne-maleimide (DBCO-maleimide) bound to the surface of the protective layer and the Azido PEG Maleimide (N3-PEG-mal eimide) bound to the functionlisation constituent are reacted by click chemistry in situ to form a composition comprising a functional constituent immobilized on the surface of the protective layer via a cross-linker, wherein the cross-linker is composed of Dibenzocyclooctyne-maleimide (DBCO-maleimide) reacted with Azido PEG Maleimide (N3- PEG-maleimide). Thus in a preferred embodiment the cross-linker is composed of two compounds reacted by click chemistry, more peferably the cross-linker is composed of Dibenzocyclooctyne-maleimide (DBCO-maleimide) reacted with Azido PEG Maleimide (N3- PEG-mal eimide).

After the protective layer has been formed, the solid carrier comprising the enzyme and the protective layer can be stored. Storing is usually accomplished e.g. by washing the composition formed e.g. with a buffer and storing it suspended or solved in that buffer for a desired time period. In a preferred embodiment the solid carrier comprising the enzyme and the protective layer is stored at a constant temperature between 2 to 25 °C. In a further preferred embodiment, the solid carrier comprising the enzyme and the protective layer is stored 5 to 48 hours, preferably 10 to 30 hours. More preferably the solid carrier comprising the enzyme and the protective layer is stored at a constant temperature between 2 to 25 °C, preferably at room temperature for 10 to 30 hours.

In one embodiment, the functional constituent i) reduces phagocytosis of the composition; ii) increases the circulation time of the composition, and/or iii) targets a tumor and/or promotes the internalization of the composition into tumor cells; when the composition of the present invention is administered to a subject, preferably the functional constituent reduces phagocytosis of the composition and/or increases the circulation time of the composition; or targets a tumor and/or promotes the internalization of the composition into tumor cells.

In one embodiment the functional constituent is selected from the group consisting of an amphiphilic drug, an amino acid, a peptide, a protein or a fragment therof, a silane copolymer, and a combination of a protein or a fragment therof and a silane copolymer, with the proviso that the protein or a fragment thereof is not the enzyme or a fragment thereof immobilized on the surface of the solid carrier. An amphiphilic drug is preferably a cationic amphiphilic drug. Characteristically, cationic amphiphilic drugs contain a hydrophobic part consisting of a nonpolar ring system and a hydrophilic group with one or more nitrogen groups which can bear a net positive charge at physiological pH. More preferably an amphiphilic drug is a cationic amphiphilic drug selected from the group consisting of Fluoxetine, Thirodazine, Promazine, Maprotiline, Loratadine, Imipramine, Doxepine, Desipramine, Clozapine, Clomipramine, Chlopromazine, Chloroquine, Labetalol, Dapoxetine, Fluvoxamine, Indalpine, Paroxetine, Zimelidine, Sertaline and Propanolol and salts, metabolites and prodrugs thereof. Further examples of suitable cationic amphiphilic drugs include the drugs stated above like Fluphenazine, Haloperidol (Haldol, Serenace), Prochlorperazine, Mesoridazine, Loxapine, Molindone (Moban), Perphenazine (Trilifon) , Thiothixene (Navane), Trifluoperazine (Stelazine), Fluphenazine (Prolixin), Droperidol, Zuclopenthixol (Clopixol), Periciazine, Triflupromazine, Olanzapine, Quetiapine, Asenapine, Sulpiride, Amisulpiride, Remoxipride, Melperone, lloperidone, Paliperidone, Risperidone, Perospirone, Ziprasidone, Sertindole, Aripiprazole, Fluvoxamine (Luvox), Paroxetine (Paxil), Sertraline (Zoloft), Desvenlafaxine (Pristiq), Duloxetine (Cymbalta), Milnacipram (Ixel), Venlafaxine (Effexor), Mianserin (Tolvon), Mirtazapine, Atomoxetine (Strattera), Mazindol (Mazanor, Sanorex), Reboxetine (Edronax), Viloxazine (Vivalan), Bupropion, Tianeptine, Agomelatine, Amitriptyline (Elavil, Endep), Clomipramine (Anafranil), Doxepin (Adapin, Sinequan), Imipramine (Tofranil), Trimipramine (Surmontil) , Nortriptyline (Pamelor, Aventyl) , Protriptyline (Vivactil) , Moclobemide (Aurorix, Manerix), Tranylcypromine (Parnate), Buspirone (Buspar), Gepirone (Ariza), Nefazodone (Serzone), Tandospirone (Sediel), Trazodone (Desyrel), Dosulepin, Etoperidone, Femoxetine, Lofepramine, Mazindol, Milnacipran, Nefazodone, Nisoxetine, Nomifensine, Oxaprotiline, Protryptiline, Viloxazine, Diphenhydramine, Loratadine, Desloratadine, Meclizine, Quetiapine, Fexofenadine Pheniramine, Cetirizine, Promethazine, Chlorpheniramine, Levocetirizine, Cimetidine, Famotidine, Ranitidine, Nizatidine, Roxatidine, Lafutidine, A-349,821 , ABT-239, Ciproxifan Clobenpropit, Thioperamide, Thioperamide, JNJ 7777120, VUF-6002, Alprenolol, Bucindolol, Carteolol, Carvedilol, Labetalol, Nadolol, Penbutolol, Pindolol, Timolol, Acebutolol, Atenolol, Betaxolol, Bisoprolol, Celiprolol, Esmolol, Metoprolol, Nebivolol, and Butaxamine and salts, metabolites and prodrugs thereof. Even more preferably the amphiphilic drug is Chloroquine or Chlorpromazine. The amino acid is usually selected from the group consisting of alanine, arginine, asparagine aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, and is preferably lysine. The peptide is usually selected from the group consisting of cell-penetrating peptides like Penetratin (DOI: 10.1038/ncommsl952: Kondo E et al., Nat commun, 2012; Derossi D et al., J. Biol. Chem. 1994;269: 10444-10450) and RGD (tripeptide consisting of arginine, glycine and aspartate) and is preferably RGD. The protein or a fragment therof is usually selected from the group consisting of serum albumin or a fragment therof and an immunoglobulin or a fragment thereof and is preferably serum albumin or a fragment therof, more preferably human serum albumin or a fragment therof. The immunoglobulin or a fragment thereof is usually selected from the group consisiting of a Fc or Fab fragment of an immunoglobulin, a Fc or Fab fragment of an immunoglobulin and a cross-linker, a monoclonal antibody and a nanobody and is preferably a Fc fragment of an immunoglobulin or a Fc fragment of an immunoglobulin and a cross-linker. The silane copolymer is usually selected from the group consisting of polyethylene glycol/silane copolymers, and polysorbate/silane copolymer and is preferably a silane PEG (PEG-Si), more preferably mSilane-PEG 2kDa or a mSilane-PEG 5kDa, most preferably a mSilane-PEG 5kDa.

In a further embodiment the functional constituent is selected from the group consisting of serum albumin or a fragment therof; serum albumin or a fragment therof and a polyethylene glycol/silane copolymer; a polyethylene glycol/silane copolymer; a Fc fragment of an immunoglobulin; and a Fc fragment of an immunoglobulin and a cross-linker.

In a further embodiment the functional constituent is selected from the group consisting of serum albumin or a fragment therof, wherein the serum albumin or a fragment therof binds to the surface of the protective layer; serum albumin or a fragment therof and a polyethylene glycol/silane copolymer, wherein one part (end) of the polyethylene glycol/silane copolymer binds to the surface of the protective layer and the other part (end) to the serum albumin or the fragment therof; a polyethylene glycol/silane copolymer wherein the polyethylene glycol/silane copolymer binds to the surface of the protective layer; a Fc or Fab fragment of an immunoglobulin; and a Fc or Fab fragment of an immunoglobulin and a cross-linker, wherein one part (end) of the cross-linker binds to the surface of the protective layer and the other part (end) to the Fc or Fab fragment of an immunoglobulin.

In a further embodiment the functional constituent is a polyethylene glycol/silane copolymer, preferably a mSilane-PEG 2kDa or a mSilane-PEG 5kDa, wherein the polyethylene glycol/silane copolymer binds to the surface of the protective layer.

In a further embodiment the functional constituent is selected from the group consisting of serum albumin or a fragment therof; serum albumin or a fragment therof and a polyethylene glycol/silane copolymer; a polyethylene glycol/silane copolymer; a Fc fragment of an immunoglobulin; and a Fc fragment of an immunoglobulin and a cross-linker, wherein the functional constituent reduces phagocytosis of the composition and/or increases the circulation time of the composition.

In a further embodiment the functional constituent is selected from the group consisting of serum albumin or a fragment therof, preferably serum albumin or a fragment therof, wherein the serum albumin or a fragment therof binds to the surface of the protective layer; serum albumin or a fragment therof and a polyethylene glycol/silane copolymer, wherein one part (end) of the polyethylene glycol/silane copolymer binds to the surface of the protective layer and the other part (end) to the serum albumin or the fragment therof; a polyethylene glycol/silane copolymer wherein the polyethylene glycol/silane copolymer binds to the surface of the protective layer; a Fc or Fab fragment of an immunoglobulin; and a Fc or Fab fragment of an immunoglobulin and a cross-linker, wherein one part (end) of the cross-linker binds to the surface of the protective layer and the other part (end) to the Fc or Fab fragment of an immunoglobulin, wherein the functional constituent immobilized on the surface of the protective layer targets a tumor and/or promotes the internalization of the composition into tumor cells, when the composition is administered to a subject.

In a further embodiment the functional constituent is serum albumin or a fragment therof, preferably serum albumin. Preferably, the serum albumin or a fragment therof binds to the surface of the protective layer. The serum albumin or a fragment therof used as functional constituent herein is preferably human and/or recombinant serum albumin or a fragment therof, more preferably human serum albumin or a fragment therof.

In a further embodiment the functional constituent is serum albumin or a fragment therof, preferably serum albumin, wherein the functional constituent reduces phagocytosis of the composition and/or increases the circulation time of the composition.

In a further embodiment the functional constituent is a polyethylene glycol/silane copolymer, preferably a mSilane-PEG 2kDa or a mSilane-PEG 5kDa, wherein the polyethylene glycol/silane copolymer binds to the surface of the protective layer.

In a preferred embodiment the functional constituent is a polyethylene glycol/silane copolymer, preferably a mSilane-PEG 2kDa or a mSilane-PEG 5kDa, wherein the polyethylene glycol/silane copolymer binds to the surface of the protective layer, wherein the functional constituent reduces phagocytosis of the composition and/or increases the circulation time of the composition. In one embodiment the functional constituent is selected from the group consisting of a peptide; a peptide and a cross-linker; an immunoglobulin or a fragment thereof; and an immunoglobulin or a fragment thereof and a cross-linker.

In a further embodiment the functional constituent is selected from the group consisting of a peptide wherein the peptide binds to the surface of the protective layer; a peptide and a crosslinker wherein one part (end) of the cross-linker binds to the surface of the protective layer and the other part (end) to the peptide; an immunoglobulin or a fragment thereof wherein the immunoglobulin or a fragment thereof binds to the surface of the protective layer; and an immunoglobulin or a fragment thereof and a cross-linker wherein one part (end) of the crosslinker binds to the surface of the protective layer and the other part (end) to the immunoglobulin or a fragment thereof.

In a further embodiment the functional constituent is selected from the group consisting of a peptide wherein the peptide binds to the surface of the protective layer; a peptide and a crosslinker wherein one part (end) of the cross-linker binds to the surface of the protective layer and the other part (end) to the peptide; an immunoglobulin or a fragment thereof wherein the immunoglobulin or a fragment thereof binds to the surface of the protective layer; and an immunoglobulin or a fragment thereof and a cross-linker wherein one part (end) of the crosslinker binds to the surface of the protective layer and the other part (end) to the immunoglobulin or a fragment thereof, wherein the peptide and the immunoglobulin or a fragment thereof targets a tumor and/or promotes the internalization of the composition into tumor cells.

In a further embodiment the functional constituent is selected from the group consisting of a protein or a fragment thereof, preferably serum albumin or a fragment therof; a silane copolymer, preferably a polyethylene glycol/silane copolymer; an immunoglobulin or a fragment thereof, preferably an antibody or a fragment thereof; and an immunoglobulin or a fragment thereof and a cross-linker, preferably an antibody or a fragment thereof and a crosslinker, preferably an antibody or a fragment thereof and a cross-linker composed of two compounds reacted by click chemistry. In a further embodiment the functional constituent is selected from the group consisting of a protein or a fragment thereof, preferably serum albumin or a fragment therof; a silane copolymer, preferably a polyethylene glycol/ silane copolymer; an immunoglobulin or a fragment thereof, preferably an antibody or a fragment thereof; and an immunoglobulin or a fragment thereof and a cross-linker, preferably an antibody or a fragment thereof and a crosslinker, preferably an antibody or a fragment thereof and a cross-linker composed of two compounds reacted by click chemistry, wherein the functional constituent targets a tumor and/or promotes the internalization of the composition into tumor cells.

In one embodiment the surface of the protective layer is only partially covered by the immobilized functional constituent. Preferably, between about 0.1% and about 100% of the surface of the protective layer are covered by the immobilized functional constituent. More preferably between about 5% and about 80%, even more preferably between about 10% and about 50%, most preferably about 20% of the surface of the protective layer are covered by the immobilized functional constituent.

In one embodiment the functional constituent is immobilized on the surface of the protective layer by binding, preferably covalent binding.

The immobilization of the functional constituent to the surface of the protective layer is usually carried in a reaction vessel like a reactor by suspending the solid carrier carrying the enzyme embedded in a protective layer as described supra in e.g. in water, buffer or non-ionic surfactants or mixtures thereof, preferably in mixtures of water and non-ionic surfactants. The functional component is then added to the suspension to react usually under stirring with the surface of the protetctive layer to immobilize the functional constitutent on the surface of the protective layer. Ususally such obtained composition is washed and resuspended into water, buffer or non-ionic surfactants or mixtures thereof. Depending on the functional constituent used immobilization takes place by polycondensation e.g. in case a PEG silane is used as functional constituent or by covalent binding e.g. in case a protein is used as functional constituent. The functional constituent may also be immobilized by chemically modifying the surface of the protective layer and the functional constituent using e.g. “click chemistry” (Copper-catalysed azide-alkyne cycloaddition, see e.g. Kolb et al. (2001) Angew. Chem. 40(11)2004-2021), whereas the solid carrier carrying the enzyme embedded in a protetctive layer as described supra is first reacted with a reactive compound like an ethynyl compound and the functional constituent is modified by adding a reactive compound e.g.an azide residue and then both components are reacted to immobilize the functional constituent on the surface of the protective layer.

In a further embodiment the composition further comprises a chelating agent, wherein the chelating agent optionally comprises a radioactive or luminescent label. Preferably a chelating agent is selected from the group consisting of DOTA, DTP A, NOTA, TETA, AAZTA, TRAP, NOPO and HEHA. More preferably DOTA or HEHA are used. Even more preferably a chelating agent which comprises a radioactive or luminescent label is used, in particular p- SCN-Bn-DOTA or Lutetium- 177-radiolabeled-DOTA is used. If the composition further comprises a chelating agent, the solid carrier carrying the enzyme embedded in a protetctive layer is usually pretreated with a chelating agent and a different chelating agent which comprises a radioactive or luminescent label is added to the such pretreated composition. Preferably a radioactive label is used, more preferably a compound of the lanthanides family, even more preferably Gadolinium, Lutetium, or Europium.

In a further aspect the present invention provides the composition as described supra for use as a medicament.

In a further aspect the present invention provides the composition for use in a method for the prevention, delay of progression or treatment of cancer, preferably for use in a method for the delay of progression or treatment of cancer, wherein the cancer is preferably lung cancer, in a subject, the method comprising administering to the subject said composition, wherein the composition is administered in an amount that is sufficient to treat the subject. Also provided is the use of the composition as described herein for the manufacture of a medicament for the prevention, delay of progression or treatment of cancer, preferably lung cancer in a subject. Also provided is the use of the composition as described herein for the prevention, delay of progression or treatment of cancer, preferably lung cancer in a subject. Also provided is a method for the prevention, delay of progression or treatment of cancer, preferably lung cancer, in a subject, comprising administering to said subject a therapeutically effective amount of the composition as described herein. In the method for the prevention, delay of progression or treatment cancer, preferably lung cancer in a subject, the functional constituent immobilized on the surface of the protective layer in the composition of the invention is preferably selected from the group consisting of an amphiphilic drug, an amino acid, a peptide, a protein or a fragment thereof, a silane copolymer, and a combination of a protein or a fragment thereof and a silane copolymer, with the proviso that the protein or the fragment thereof is not the enzyme or the fragment thereof immobilized on the surface of the solid carrier; more preferably selected from the group consisting of serum albumin or a fragment therof, wherein the serum albumin or a fragment therof binds to the surface of the protective layer; serum albumin or a fragment therof and a polyethylene glycol/silane copolymer, wherein one part (end) of the polyethylene glycol/silane copolymer binds to the surface of the protective layer and the other part (end) to the serum albumin or the fragment therof; a polyethylene glycol/silane copolymer wherein the polyethylene glycol/silane copolymer binds to the surface of the protective layer; a Fc or Fab fragment of an immunoglobulin; and a Fc or Fab fragment of an immunoglobulin and a cross-linker, wherein one part (end) of the cross-linker binds to the surface of the protective layer and the other part (end) to the Fc or Fab fragment of an immunoglobulin; and an even more preferably serum albumin or a fragment therof or a polyethylene glycol/silane copolymer , in particular serum albumin or a mSilane-PEG 2kDa or a mSilane-PEG 5kDa, more particular human and/or recombinant serum albumin or a fragment therof or a mSilane-PEG 2kDa or a mSilane-PEG 5kDa, even more particular a mSilane-PEG 2kDa or a mSilane-PEG 5kDa.

The terms “treatment’ ’/’’treating” as used herein includes: (1) delaying the appearance of clinical symptoms of the state, disorder or condition developing in an animal, particularly a mammal and especially a human, that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g. arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); and/or (3) relieving the condition (i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms). The benefit to a patient to be treated is either statistically significant or at least perceptible to the patient or to the physician. However, it will be appreciated that when a medicament is administered to a patient to treat a disease, the outcome may not always be effective treatment.

As used herein, "delay of progression" means increasing the time to appearance of a symptom of a cancer or a mark associated with a cancer or slowing the increase in severity of a symptom of a cancer. Further, "delay of progression" as used herein includes reversing or inhibition of disease progression. "Inhibition" of disease progression or disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.

Preventive treatments comprise prophylactic treatments. In preventive applications, the pharmaceutical combination of the invention is administered to a subject suspected of having, or at risk for developing cancer. In therapeutic applications, the pharmaceutical combination is administered to a subject such as a patient already suffering from cancer, in an amount sufficient to cure or at least partially arrest the symptoms of the disease. Amounts effective for this use will depend on the severity and course of the disease, previous therapy, the subject's health status and response to the drugs, and the judgment of the treating physician.

In the case wherein the subject's condition does not improve, the pharmaceutical combination of the invention may be administered chronically, which is, for an extended period of time, including throughout the duration of the subject's life in order to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.

In the case wherein the subject's status does improve, the pharmaceutical combination may be administered continuously; alternatively, the dose of drugs being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). Once improvement of the patient's condition has occurred, a maintenance dose of the pharmaceutical combination of the invention is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is optionally reduced, as a function of the symptoms, to a level at which the improved disease is retained.

The expression “effective amount” or “therapeutically effective amount” as used herein refers to an amount capable of invoking one or more of the following effects in a subject receiving the combination of the present invention: (i) inhibition or arrest of tumor growth, including, reducing the rate of tumor growth or causing complete growth arrest; (ii) complete tumour regression; (iii) reduction in tumor size; (iv) reduction in tumor number; (v) inhibition of metastasis (i.e., reduction, slowing down or complete stopping) of tumor cell infiltration into peripheral organs; (vi) enhancement of antitumor immune response, which may, but does not have to, result in the regression or elimination of the tumor; (vii) relief, to some extent, of one or more symptoms associated with cancer; (viii) increase in progression-free survival (PFS) and/or; overall survival (OS) of the subject receiving the combination.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In some embodiments, a therapeutically effective amount may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent, and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) delay occurrence and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In various embodiments, the amount is sufficient to ameliorate, palliate, lessen, and/or delay one or more of symptoms of cancer.

In one embodiment the cancer is lung cancer, preferably a lung cancer selected from the group consisting of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), more preferably selected from the group consisting of small-cell lung carcinoma (SCLC), adenocarcinoma in situ, squamous cell carcinoma, large cell carcinoma, adenosquamous carcinoma and sarcomatoid carcinoma.

In one embodiment the cancer is lung cancer, preferably a solid tumor of a primary or secondary cancer in the lung, more preferably a secondary cancer in the lung, wherein the secondary cancer in the lung originates from a cancer selected from the group consisting of bladder cancer, breast cancer, colon cancer, kidney cancer, salivary gland cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, stomach cancer, thyroid cancer, testicular cancer, uterine cancer and sarcoma.

In a further aspect the present invention provides a method of producing a composition as described supra, e.g. a composition a solid carrier, wherein the particle size of the solid carrier is between 15 nm and 45 nm, an enzyme or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the enzyme or the fragment thereof by embedding the enzyme or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer.; wherein the method comprises the following steps:

(a) providing a solid carrier, wherein the particle size of the solid carrier is between 15 nm and 45 nm;

(b) immobilizing an enzyme or a fragment thereof on the solid carrier;

(c) forming a protective layer on the surface of the solid carrier to protect the enzyme or the fragment thereof immobilized on the solid carrier;

(d) immobilizing a functional constituent on the surface of the protective layer.

Step (a) is usually carried out by providingthe solid carrier in suspension in water or a buffer. The suspension can be stirred e.g at 400 rpm, 20°C for 30 min. The immobilization of the enzyme on the solid carrier in step b) of the present method is usually carried out by adding a solution of the enzyme to the suspension of the solid carrier. In a preferred embodiment the immobilization of theenzyme on the solid carrier is carried out by providing a suspension of the solid carrier and adding a solution of the enzyme, wherein the suspension with the added solution of the enzyme is incubated to allow the enzyme to bind on the surface of the solid carrier. In a preferred embodiment the surface of the solid carrier is at least partly modified to improve immobilization of the enzyme on the solid carrier. In particular, the surface of the solid carrier is at least partly modified before the enzyme is immobilized. The surface of the solid carrier can be at least partly modified by introducing a molecule as anchoring point for the enzyme to the surface of the solid carrier as described supra. The formation of the protective layer according to step (c) of the present method is usually carried out by forming the respective protective layer with building blocks, wherein the building blocks build the protective layer in a polycondensation reaction as described supa. The immobilization of a functional constituent on the surface of the protective layer according to step (d) of the present method is usually carried out as described supra.

In one embodiment the protective layer is formed by building blocks, wherein as building blocks structural building blocks and protective building blocks are used to form the protective layer, wherein the structural building blocks are precursors of inorganic silica, capable of forming 4 covalent bonds in the layer formed and the protective building blocks are organosilanes.

In one embodiment the protective layer embeds fom about 30% to about 100% of the enzyme.

In one embodiment the solid carrier is selected from the group of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles and titanium particles. The particle size of the solid carier is usually between 15 nm and 45 nm, preferably between 15 nm and 40 nm, more preferably between 20 nm and 40 nm, particularly about 30 nm.

Examples

Materials and Methods:

Reagents:

Tetraethyl orthosilicate 99%(TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), Chelex® sodium form, asparaginase (EC3.5.1.1), MES buffer and sodium chloride were purchased from Sigma-Aldrich.p-SCN-Bn-DOTA was purchased from Macrocyclics. Methoxy PEG silane (5000Da) was purchased from Nanocs.

Synthesis of silica nanoparticles.

Silica nanoparticles 50nm:

Silica nanoparticles of 50 nm particle size have been synthetized following the original Stober process as described in WO2015/014888 Al. Briefly, ethanol, distilled water (6M) and ammonium hydroxide (0.13M) were mixed and stirred at 400rpm for Ih. TEOS (0.28M) was added and the solution was stirred at 400rpm at 20°C for 22h. The solution was then centrifuged at 20 000g for 20min and washed successively with ethanol and water. Particle size measurement was carried out on SEM micrographs acquired at a magnification of 150 OOOx using the image analysis software Olympus stream motion.

These nanoparticles which have been produced as described in WO2015/014888 Al are further referred herein as “core nanoparticles 50” or “core NP-50”.

Silica nanoparticles 30nm:

Silica nanoparticles of 30 nm particle size have been synthetized following the original Stober process as described in WO2015/014888 Al. Briefly, ethanol, distilled water (3M) and ammonium hydroxide (0.055M) were mixed and stirred at 400rpm for Ih. TEOS (0.28M) was added and the solution was stirred at 400rpm at 20°C for 22h. The solution was then centrifuged at 20 000g for 20min and washed successively with ethanol and water. Particle size measurement was carried out on SEM micrographs acquired at a magnification of 150 OOOx using the image analysis software Olympus stream motion.

These nanoparticles which have been produced as described in WO2015/014888 Al are further referred herein as “core nanoparticles 30” or “core NP-30”. Enzyme shielding:

Shielded nanoparticles 50nm:

Core nanoparticles 50 produced according to section “Synthesis of silica nanoparticles” above in water-polysorbate 80 (8mg/L) were reacted with APTES (2.75mM) for 30min at 20°C under stirring (400rpm). Unreacted reagents were removed from the nanoparticles suspension using the ami con stirred cells with 300kDa NMWL, Biomax 10 polyethersulfone ultrafiltration discs, and the nanoparticles suspension were sonicated at 62.5 Watts for 5min on ice (hereafter called “washing step”). Nanoparticles were then incubated with 0.1%(v/v) of aqueous glutaraldehyde solution for 30min at 20°C under stirring (400rpm). After a washing step, the nanoparticles were resuspended in MES buffer (lOrnM, pH 6.2) with polysorbate 80 (8mg/L) and reacted with asparaginase (20ug/mL) for Ih at 20°C under stirring (400rpm). The nanoparticles were washed and TEOS (42.5mM) was added and allowed to react for Ih at 20°C under stirring (400rpm). Subsequently, APTES (4.5mM) was added to the reaction mixture. The silane polycondensation was stopped after 21h by washing the nanoparticles suspension. The silica nanoparticles obtained after silane polycondensation comprise the enzyme asparaginase immobilized on the surface of the silica particle which is fully embedded by the protective layer comprising polycondensed silanes. Particle size measurement was carried out on SEM micrographs acquired at a magnification of 150 OOOx using the image analysis software Olympus stream motion.

These nanoparticles which have been produced as described in WO2015/014888 Al are further referred herein as “shielded nanoparticles 50” or “Shielded NP-50”.

Shielded nanoparticles 30nm:

Core nanoparticles 30 produced according to section “Synthesis of silica nanoparticles” above in water-polysorbate 80 (8mg/L) were reacted with APTES (6.5mM) for 30min at 20°C under stirring (400rpm). Unreacted reagents were removed from the nanoparticles suspension using the amicon stirred cells with 300kDa NMWL, Biomax 10 polyethersulfone ultrafiltration discs, and the nanoparticles suspension were sonicated at 62.5 Watts for 5min on ice (hereafter called “washing step”). Nanoparticles were then incubated with 0.1%(v/v) of aqueous glutaraldehyde solution for 30min at 20°C under stirring (400rpm). After a washing step, the nanoparticles were resuspended in MES buffer (lOmM, pH 6.2) with polysorbate 80 (8mg/L) and reacted with asparaginase (5ug/mL) for Ih at 20°C under stirring (400rpm). The nanoparticles were washed and TEOS (63,7mM) was added and allowed to react for Ih at 20°C under stirring (400rpm). Subsequently, APTES (6.5mM) was added to the reaction mixture. The silane polycondensation was stopped after 21h by washing the nanoparticles suspension. The silica nanoparticles obtained after silane polycondensation comprise the enzyme asparaginase immobilized on the surface of the silica particle which is fully embedded by the protective layer comprising polycondensed silanes. Particle size measurement was carried out on SEM micrographs acquired at a magnification of 150 OOOx using the image analysis software Olympus stream motion.

These nanoparticles which have been produced as described in WO2015/014888 Al are further referred herein as “shielded nanoparticles 30” or “Shielded NP-30”.

Functionalization of nanoparticles with PEG.

Shielded NP-50 and Shielded NP30 produced according to section “Enzyme shielding” above in MES buffer (lOrnM, pH 6,2) with polysorbate 80 (8mg/L), were reacted with PEG silane 5000 (Img/mL) for Ih at 20°C under strirring (400rpm). After a washing step, the nanoparticles were resuspended in MES buffer (lOmM, pH 6.2) with polysorbate 80 (8mg/L). The silica nanoparticles obtained after functionalization with PEG comprise the enzyme asparaginase immobilized on the surface of the silica particle which is fully embedded by the protective layer comprising polycondensed silanes and further comprises PEG silane as functional constituent immobilized on the surface of the protective layer.

The shielded nanoparticles 50 functionalized with PEG silane 5000 are further referred herein as “Functionalized nanoparticles 50” or “Functionalized NP-50”.

The shielded nanoparticles 30 functionalized with PEG silane 5000 are further referred herein as “Functionalized nanoparticles 30” or “Functionalized NP-30”.

Labeling of nanoparticles .

Functionalized nanoparticles produced according to section “Functionalization of nanoparticles with PEG” above were resuspended in phosphate buffer (0.1M, pH 7.4) with polysorbate 80 (8mg/L), pretreated with Chelex®, and p-SCN-Bn-DOTA (Img/mL) was added and allowed to react for Ih at 20°C under stirring (400rpm). After a washing step, the nanoparticles were resuspended in MES buffer (lOmM, pH 6.2) with polysorbate 80 (8mg/L). These particles are incubated with 2800uCi of 177-Lutetium for 12 hours at 45°C, and further washed prior to injection.

Biodistribution:

CD-I mice were injected retro-orbitally with 50mg/kg of 177-Lutetium-labeled-Functionalized NP-50 or of 177-Lutetium-labeled-Functionalized NP-30. At terminal time points, the animals were anesthetized by intraperitoneal injection of a mixture of ketamine hydrochloride (50mg/kg) and xylazine hydrochloride (10 mg/kg), and then they were rapidly sacrificed by exsanguination via intracardiac puncture. The organs of interest were excised, rinsed in physiological serum and weighed using a precision balance. The counting of tissue radioactivity was performed in an automatic gamma counter (Wallace Wizard 2470 - Perkin Elmer) calibrated for Lutetium-177 radionuclide (efficiency: 13.8%; LLOQ: 500 cpm). The radioactivity in sampled tissues was expressed as percentage of the ID per gram of tissue (%ID/g).

Results:

Example 1:

To evaluate the impact of the size of the nanoparticles on their biodistribution, two different target sizes of nanoparticles were produced (50 nm and 30nm, respectively). The size measurement of core NP-50 by SEM shows monodisperse nanoparticles with an average diameter of 51 ± 4nm. Functionalized NP-50 (69 ± 5nm) have an increased diameter compared to the reference core NP-50 with a homogeneous size distribution. The size measurement of core NP-30 by SEM shows a homogeneous size distribution of monodisperse core NP-30 with an average diameter of 28 ± 4nm. The enzyme shielding and functionalization of core NP-30 led to an increase of their diameter (41 ± 4nm). The resulting Functionalized NP-30 suspension shows a monodisperse distribution.

Example 2:

To evaluate the impact of the size of nanoparticles on tissues biodistribution, Lutetium-177- radiolabeled-Functionalized NP-50 (Fig. 2 A and B) and Lutetium- 177-radiolabeled- Functionalized NP-30 (Fig. 2 A and C) were injected at 50mg/kg in animals. Tissues and blood radioactivity were performed at different time points (0 to 168h). Results show that the profile of clearance from the bloodstream of both nanoparticles was similar, showing that the size of the nanoparticles has no impact on their blood half-life circulation time (Fig. 2A). However, the tissue distribution of Functionalized NP-50 and Functionalized NP-30 surprisingly exhibits clear differences. While the liver and the spleen were the major sites of accumulation of Functionalized NP-50 (Fig. 2B), a major accumulation of Functionalized NP-30 was surprisingly noticed at 24 hours in the lung. In fact, the %ID/g of both formulations of nanoparticles shows that the retention of functionalized NP-30 in the lung is much higher than Functionalized NP-50 (36,3 vs 6,67 respectively) (Fig 2B and C). Importantly, the tissue biodistribution over the time of Functionalized NP-30 points out that the nanoparticles do not accumulates but are cleared from the lung gradually (Fig 2C). Altogether, these in vivo data demonstrate the possibility of using Functionalized NP-30 for targeting the lung for specific therapeutic applications such as lung cancer.