Functionalized composition containing manipulated phenylalanine ammonia lyase (PAL)

Abstract

The present invention relates to a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or thiol group. The present invention also relates to a method for producing the composition and its use.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or thiol group. The present invention also relates to a method for producing the composition and its use. [Background technology]

[0002] Phenylketonuria (PKU) is a hereditary autosomal recessive disorder characterized by a deficiency of the intracellular hepatic enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine, and this enzyme activity is promoted by tetrahydrobiopterin (BH4). In PAH deficiency, the concentration of phenylalanine, which is toxic to the brain, becomes abnormally high. High phenylalanine levels in infancy and early childhood cause severe cognitive and developmental impairments, and poor control of blood phenylalanine levels in older children and adolescents is associated with learning disabilities, attention deficit hyperactivity disorder, and behavioral disorders. Inadequate control of blood phenylalanine levels in adulthood is associated with executive function impairment and a variety of behavioral and mental problems.

[0003] The basis of PKU treatment is a low-phenylalanine diet combined with phenylalanine-free L-amino acids. PAH deficiency in patients with serum phenylalanine levels exceeding 600 micromol / L can be corrected by enzyme substitution therapy using recombinant phenylalanine ammonia lyase (PAL) (Palynziq®), administered by subcutaneous injection up to three times daily. However, hypersensitivity reactions are common, and immune-mediated acute hypersensitivity reactions (type III) have been reported in patients maintained with pegylated derivatives of the enzyme phenylalanine ammonia lyase (pegvariase). Therefore, there is a need to provide suitable and effective treatments for phenylketonuria. [Overview of the project]

[0004] The present invention provides a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or thiol group.

[0005] The present invention also provides a method for producing a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, (b) A step of immobilizing the manipulated phenylalanine ammonia lyase or a fragment thereof onto the solid carrier, (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the manipulated phenylalanine ammonia lyase or fragment thereof immobilized on the solid carrier. (d) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group.

[0006] The inventors of this application have surprisingly found that the compositions provided by the present invention, when applied therapeutically, possess unexpectedly high in vivo enzymatic activity, exhibit low cytotoxicity, and do not disrupt the intestinal barrier when localized in the gastrointestinal tract, and are therefore extremely promising for therapeutic use, particularly for the prevention, delay of progression, or treatment of phenylketonuria. [Brief explanation of the drawing]

[0007] [Figure 1] A schematic representation of a method for producing the composition of the present invention is provided: a) immobilizing the processed PAL or fragment thereof onto a solid carrier; b) and c) growing a protective layer around the immobilized processed PAL or fragment thereof to embed it; and d) immobilizing the functional component onto the surface of the protective layer. [Figure 2] This shows the added value of the covalent bond between the enzyme surface and the protective layer. (A) Quantification of proteins performed on the reaction supernatant of NP-1(1), NP-1(2), and NP-1. (B) PAL loading per dry weight of SNPs. (C) SNP specific activity expressed as U / g SNPs. (D) PAL specific activity expressed as U / g PALs. [Figure 3] This shows the phenylalanine ammonia lyase (PAL) activity of nanoparticles. The biocatalytic activity of engineered PAL immobilized and protected on nanoparticles was quantified after exposure to phenylalanine (unit: U / g). [Figure 4A-B]It exhibits resistance to external stress. NP-1 and manipulated PAL were exposed to acidic conditions (pH 4), and their stability was evaluated by measuring PAL enzyme activity at different time points. [Figure 4C] This demonstrates PAL's resistance to external stress. NP-1 and engineered PAL were exposed to proteases, and their stability was evaluated by measuring PAL enzyme activity at different time points. [Figure 5] This shows the in vitro biocompatibility and efficacy of NP-1 in a model of the intestinal barrier. (A) In vitro evaluation of intestinal barrier integrity by measurement of transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to NP-1 (9.7 mU) for 6 hours in the presence or absence of pancreatin (30 mU), or in pancreatin (30 mU) alone. The graph shows the time course profile of averaged, normalized TEER data over 6 hours. The dashed line represents the untreated state. (B) In vitro metabolism of phenylalanine (Phe) in a model of the intestinal barrier. Differentiated Caco-2 / HT29-MTX-E12 cocultures cultured in cell medium containing 0.4 mM Phe were exposed to NP-1 (9.7 mU) or engineered PAL (9.7 mU) with or without pancreatin (30 mU) at the apical end of the barrier for 6 hours. Phe metabolism was assessed by quantification of trans-cinnamic acid (TCA) at the basal end of the barrier. The graph shows the time course profile of TCA accumulation over 6 hours. [Figure 6] This shows the quantification of trans cinnamic acid (TCA) in rat urine. Wistar rats were administered NP-1 (n=5) or NP-2 (n=5) intraduodenum, and d5-Phe was administered simultaneously via tube feeding. Urine was collected over 24 hours after administration and analyzed by LC-MS. The graph shows the d5-hypric acid concentration in the urine. **p<0.01 (by t-test). [Figure 7]This graph shows the plasma concentrations of Phe in BTBR-Pahenu2 / J mice. BTBR-Pahenu2 / J mice, which had free access to drinking water containing L-Phe, were administered NP-1 (0.581U; 7mg), NP-2 (7mg), or modified PAL (0.581U) intraduodenal twice daily for 12 days. Blood samples were collected on days 0, 4, 6, 8, 10, and 12, and plasma was extracted and analyzed by LC-MS. (A) Graph shows the plasma concentrations of Phe in BTBR-Pahenu2 / J mice. (B) Graph shows the normalized plasma concentrations of Phe in BTBR-Pahenu2 / J mice. [Figure 8] The absorbance of NP-1, NP-1(1), and NP-1(2) at a wavelength of 460 nm is shown. [Modes for carrying out the invention]

[0008] The present invention relates to a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or thiol group.

[0009] For the purposes of interpreting this specification, the following definitions apply, and wherever used in the singular, the plural form is also included, and vice versa. It should be understood that the terms used herein are intended solely to describe and not to limit specific embodiments.

[0010] Features, integers, properties, and compounds described in relation to specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, insofar as they do not contradict each other. All features and / or steps of methods or processes disclosed herein (including the claims, abstract, and drawings) can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of the embodiments described above.

[0011] The term "comprise," and its variations such as "comprises" and "comprising," are generally used to mean "include," that is, "to include, but not limited to," meaning to allow the presence of one or more features or components.

[0012] The singular forms "a," "an," and "the" include references to the plural form unless otherwise explicitly stated.

[0013] The term "approximately" refers to a range of values ​​that is ±10% of a given value. For example, the expression "approximately 200" includes ±10% of 200, i.e., from 180 to 220.

[0014] As used herein, the term “solid carrier” usually refers to particles. Preferably, the solid carrier is monodisperse or polydisperse particles, more preferably monodisperse particles. The solid carrier typically includes organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, and titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size of the solid carrier is typically 1 nm to 1000 μm, preferably 10 nm to 100 μm, and particularly about 50 nm.

[0015] As used synonymously herein, the terms "linker" or "crosslinker" refer to any linking reagent that includes a group capable of binding to a specific functional group (e.g., primary amine, sulfhydryl, etc.). Linkers relevant to the present invention typically connect the surface of a solid support to the engineered phenylalanine ammonia-lyase. For example, a linker can be immobilized on the surface of a solid support, such as a silica surface as the support material, and then the engineered phenylalanine ammonia-lyase can be bound to the unoccupied binding site of the linker. Alternatively, the linker can first be bound to the engineered phenylalanine ammonia-lyase, and then the linker bound to the engineered phenylalanine ammonia-lyase can bind its unoccupied binding site to the solid support. Various types of linkers are known in the art, including but not limited to straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as tags.

[0016] As used herein, the term "protective layer" refers to a layer formed on the surface of a solid support to protect the functional properties of the engineered phenylalanine ammonia-lyase or fragment immobilized thereon. The protective layer of the present invention is typically constructed of building blocks, at least some of which are monomers capable of interacting with each other, usually by covalent bonds, and usually with the engineered phenylalanine ammonia-lyase immobilized by non-covalent bonds. The protective layer is formed on the surface of the solid support to protect the engineered phenylalanine ammonia-lyase or fragment thereof immobilized on the solid support. The protective layer is typically a homogeneous layer in which at least 50%, preferably at least 70%, more preferably at least 90% of the engineered phenylalanine ammonia-lyase or fragment thereof is embedded in the protective layer.

[0017] As used herein, the term "phenylalanine ammonia-lyase or fragment thereof" or "PAL or fragment thereof" refers to the class of enzymes within the aromatic amino acid lyase family (EC 4.3.1.23, EC 4.3.1.24 and EC 4.3.1.25) that also includes histidine ammonia-lyase and tyrosine ammonia-lyase. Since PAL can use not only phenylalanine but also tyrosine as substrates, it is sometimes called phenylalanine / tyrosine ammonia-lyase. PAL catalyzes the conversion of L-phenylalanine to trans-cinnamic acid and ammonia. PAL activity refers to the enzymatic activity of the PAL polypeptide. PAL may also contain the cofactor 3,5-dihydro-5-methylene-4H-imidazol-4-one (MIO). This cofactor is required for catalytic activity and is formed by cyclization and dehydration of the conserved active site Ala167-Ser168-Gly169 tripeptide segment.

[0018] When used herein in reference to phenylalanine ammonia lyase or its fragments, the terms “manipulated” and “not naturally occurring” refer to phenylalanine ammonia lyase or its fragments that have been modified in a manner otherwise not found in nature, corresponding to the natural or native form of phenylalanine ammonia lyase or its fragments. The term “manipulated phenylalanine ammonia lyase or its fragments” does not include, nor does it encompass, “wild-type” and “naturally occurring” phenylalanine ammonia lyase or its fragments. As used herein, “wild-type” and “naturally occurring” refer to forms of phenylalanine ammonia lyase or its fragments that exist in nature. For example, wild-type phenylalanine ammonia lyase or its fragments are polypeptides present in living organisms that can be isolated from natural sources and have not been intentionally modified by artificial manipulation. Manipulated PAL or its fragments are, for example, variants or functionally active fragments of manipulated phenylalanine ammonia lyase. Accordingly, the terms “engineered phenylalanine ammonia lyase fragment,” “the fragment” in relation to the engineered phenylalanine ammonia lyase, and “functionally active fragment of the engineered phenylalanine ammonia lyase” are used synonymously herein. “Variant or functionally active fragment” in relation to the engineered phenylalanine ammonia lyase of the present invention means that the fragment or variant (such as an analog, derivative, or variant not found in nature) can exhibit the same or improved physiological function as wild-type phenylalanine ammonia lyase. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that such modification does not result in a loss of functional activity of the fragment or variant. The PAL fragment comprises a homotetrameric enzyme, where at least one monomer, preferably all four monomers, of the homotetrameric enzyme typically contains 100 to 550 amino acids, preferably 200 to 500 amino acids, more preferably 300 to 450 amino acids."Improved physiological function" or "improved enzymatic properties" refers to an engineered PAL that exhibits improvement in any enzymatic property compared to a reference PAL polypeptide, such as a wild-type PAL polypeptide. Improved properties include, but are not limited to, increased protein expression, increased thermal activity, increased heat tolerance, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity and / or affinity, increased specific activity, increased resistance to inhibition of substrates and / or end products, increased chemical stability, improved chemoselectivity, improved solvent stability, increased resistance to acidic pH, increased resistance to proteolytic activity (i.e., decreased sensitivity to proteolysis), reduced aggregation, increased solubility, decreased immunogenicity, and altered temperature profiles. A preferred engineered phenylalanine ammonia lyase or fragment thereof of the present invention is the engineered phenylalanine ammonia lyase described in International Publication No. 2018 / 148633.

[0019] As used herein, the term “partially embedded engineered phenylalanine ammonia lyase” means that the engineered phenylalanine ammonia lyase is not completely covered by the protective layer, and therefore, the engineered phenylalanine ammonia lyase is not completely embedded in the protective layer. In one embodiment, less than 50% of the engineered phenylalanine ammonia lyase of interest is covered by the protective layer, but typically more than that, at least 70%, is covered, thereby improving the protection of the engineered phenylalanine ammonia lyase. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the engineered phenylalanine ammonia lyase of interest is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, and most preferably about 90% to about 95, 96, 97, 98, or 99% of the engineered phenylalanine ammonia lyase of interest is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most specifically about 95% of the manipulated phenylalanine ammonia lyase of the target is covered by a protective layer. In a particularly more preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most specifically about 95% of the manipulated phenylalanine ammonia lyase of the target is covered by a protective layer, and the active site is not covered.

[0020] As used herein, the term “fully embedded engineered phenylalanine ammonia lyase” means that the engineered phenylalanine ammonia lyase of the present invention is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered. Preferably, the engineered phenylalanine ammonia lyase or fragment thereof according to the present invention is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered.

[0021] As used herein, the term “at least partially embedded engineered phenylalanine ammonia lyase” means that the engineered phenylalanine ammonia lyase is at least partially embedded and may be completely embedded by a protective layer. Thus, “at least partially embedded engineered phenylalanine ammonia lyase” means that the protective layer covers about 30% to 100%, preferably about 50% to 100%, more preferably about 80% to 100%, even more preferably about 90% to 100%, and most preferably about 95% to 100% of the engineered phenylalanine ammonia lyase or fragment thereof, and the active site is preferably covered.

[0022] As used herein, the term “functional component” refers to a component that, after being immobilized on the surface of a protective layer, retains its characteristic functional properties. In the sense of the present invention, a functional component is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0023] As used herein, the term “percent (%) sequence identity” refers to a comparative amount of polypeptide determined by comparing two optimally aligned sequences within a comparison window, where the portion of the polypeptide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence in order to optimally align the two sequences. The percentage can be calculated by determining the number of positions where identical amino acid residues exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the quotient by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where identical amino acid residues exist in both sequences or where amino acid residues align with gaps, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the quotient by 100 to obtain the percentage of sequence identity. Those skilled in the art will understand that there are numerous established algorithms available for aligning two sequences.

[0024] The term "reference sequence" is used herein to refer to a specific sequence used as the basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a full-length gene or a segment of a polypeptide sequence. Generally, a reference sequence is at least 20 amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the full length of a polypeptide.

[0025] As used herein, the term “comparison window” refers to a conceptual segment of at least approximately 20 adjacent amino acid residues, where a sequence can be compared to a reference sequence of at least 20 adjacent amino acids, and the portion of the sequence within the comparison window may contain no more than 20% additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) in order to optimally align the two sequences. The comparison window may be longer than 20 adjacent residues and may optionally include windows of 30, 40, 50, 100, or longer.

[0026] As used herein, the term “polymer comprising repeating units, each containing at least one amino group” refers to a polymer comprising a large number of repeating units (monomers), each of which contains at least one amino group. Preferred polymers comprise a large number of repeating units (monomers), each of which contains one amino group, in particular one primary amino group.

[0027] As used herein, the term “polymer comprising repeating units, each containing at least one thiol group” refers to a polymer comprising a large number of repeating units (monomers), each of which contains at least one thiol. A preferred polymer comprises a large number of repeating units (monomers), each of which contains one thiol group.

[0028] As used herein, the term “polycarbophil-cysteine ​​conjugate” refers to a conjugate containing cysteine ​​covalently bonded to polycarbophil. Such conjugates can be produced, for example, as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0029] As used herein, the term “polylysine” refers to α-polylysine and / or ε-polylysine (ε-poly-L-lysine, EPL), preferably ε-polylysine. α-Polylysine is a synthetic polymer and may consist of L-lysine or D-lysine. ε-Polylysine (ε-poly-L-lysine, EPL) is typically produced as a homopolypeptide of approximately 25 to 30 L-lysine residues.

[0030] As used herein, the term "polycysteine" may consist of L-cysteine ​​or D-cysteine, preferably L-cysteine, and preferably containing 2 to 30 cysteine ​​residues, more preferably 2 to 5 cysteine ​​residues.

[0031] As used herein, the term “polyglucosamine” refers to a linear aminopolysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamine contains a free amine (-NH2) group and is characterized by the ratio of N-acetyl-D-glucosamine units to D-glucosamine units, which is expressed as the degree of deacetylation (DDA) of fully acetylated polymer chitin. Preferred polyglucosamines of the present invention are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or their derivatives. Most preferred are chitosan or its derivatives.

[0032] As used herein, the term “chitosan or its derivatives” refers to chitosan or chitosan derivatives, including salts, having a molecular weight preferably of 2000 Da or more, preferably in the range of 25000 to 2000000 Da, more preferably in the range of about 50000 to 350000 Da, and most preferably in the range of about 50000 to 190000 Da or 190000 or 310000 Da. The term “derivatives” in relation to chitosan includes esters, ethers, or other derivatives formed by the reaction of an acyl group or alkyl group with an OH group. Examples of these are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Suitable derivatives are described, for example, in GAERoberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable salts of chitosan include nitrates, phosphates, sulfates, xanthogenic salts, hydrochlorides, glutamates, lactates, and acetates.

[0033] In a first embodiment, the present invention provides a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or thiol group.

[0034] The manipulated phenylalanine ammonia lyase or its fragments can be immobilized on the surface of a solid support by non-covalent or covalent bonds. Non-covalent bonds include electrostatic interactions such as pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and ionic interactions. Preferably, the manipulated phenylalanine ammonia lyase or its fragments are immobilized on the surface of a solid support by covalent bonds or by linker-mediated covalent bonds.

[0035] Solutions of manipulated phenylalanine ammonia lyase or its fragments typically contain the protein or its fragments in a buffer. Commonly usable buffers include phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. In addition, the solution may contain sugar alcohols or nonionic surfactants as described herein.

[0036] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, and titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size is usually measured by measuring the diameter of the particles and is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is monodisperse particles, its size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is polydisperse particles, its size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, and particularly 50 nm to 50 μm. In one embodiment, the composition comprises a solid support, the solid support comprising at least 15%, preferably at least 20%, particularly 15% to 25%, more specifically 15% to 20%, of immobilized manipulated phenylalanine ammonia lyase or fragments thereof, per dry weight of the solid support.

[0037] Typically, monodisperse particles or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in the present invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In a more preferred embodiment, the polydisperse particles are non-spherical polydisperse particles.

[0038] Solid carriers are usually provided in suspension. The suspension of solid carriers can be done, for example, in water, a buffer, or a nonionic surfactant, or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. Nonionic surfactants are typically ethoxylated sorbitan esters such as EG-40 diisostearate P-sorbitan, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated esters such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The material is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80).The buffers that can be used in the method of the present invention include phosphates, 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-[[1,3-dihydroxy-2-(hydroxymethyl)propane-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3-(N,N-bis[2-hydroxy These are ethyl]amino)-2-hydroxypropanesulfonic acid, N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, diglycine, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0039] In one embodiment, the surface of a solid support is modified to introduce molecules or functional chemical groups as anchor points, i.e., anchor points for the manipulated phenylalanine ammonia lyase, or for linkers connecting the manipulated phenylalanine ammonia lyase to the solid support. Preferably, the anchor points are amine functional chemical groups or moieties. In a non-limiting example, an amino-modified surface of a solid support, such as an amino-modified silica surface, can be used as a modified solid support. Such an amino-modified surface of a solid support can be obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support having a silica surface containing an amino-modified surface, and more preferably a solid support obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Such a modified support can form amide bonds between the manipulated phenylalanine ammonia lyase and amine groups on the surface of the support material, or amide bonds between linkers and amine groups on the surface of the support material. In one embodiment, the molecules or functional chemical groups introduced as anchor points are uniformly distributed on the surface of the solid support.

[0040] In some embodiments, the protective layer has a specified thickness of about 1 to about 200 nm, typically about 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, and particularly about 1 to about 15 nm. The most preferred specified thickness is about 1 to about 10 nm. In some embodiments, the layer has a specified 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, and particularly about 5 to about 15 nm. The most preferred specified thickness is about 5 to about 10 nm. The protective layer is usually porous, with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0041] In one embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof is partially embedded by a protective layer. In a preferred embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof is at least partially embedded by a protective layer. In a more preferred embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof is completely embedded by a protective layer.

[0042] In one embodiment, the protective layer is embedded with a solid carrier and an engineered phenylalanine ammonia lyase or fragment thereof, which is immobilized on the surface of the solid carrier. In one embodiment, a functional component immobilized on the surface of the protective layer is not embedded by the protective layer. Preferably, the protective layer is completely embedded with the solid carrier and an engineered phenylalanine ammonia lyase or fragment thereof, which is immobilized on the surface of the solid carrier. More preferably, the protective layer is completely embedded with the solid carrier and an engineered phenylalanine ammonia lyase or fragment thereof, which is immobilized on the surface of the solid carrier, and a functional component immobilized on the surface of the protective layer is not embedded by the protective layer. When the protective layer is completely embedded with the solid carrier and an engineered phenylalanine ammonia lyase or fragment thereof, which is immobilized on the surface of the solid carrier, the engineered phenylalanine ammonia lyase or fragment thereof is completely, i.e., 100%, covered by the protective layer, i.e., the active site is also covered, and the solid carrier is completely, i.e., 100%, covered by the protective layer.

[0043] In a preferred embodiment, the manipulated phenylalanine aminolyase or fragment thereof comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, or at least 97% sequence identity with respect to the sequence of SEQ ID NO: 1. In one embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof is SEQ ID NO: 2, 3, 4, or 5. In a particular preferred embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof comprises the polypeptide shown in SEQ ID NO: 5.

[0044] The thickness of the protective layer can be measured using a microscope such as a scanning electron microscope (SEM), transmission electron microscope (TEM), or scanning probe microscope (SPM), or by light scattering or ellipsometry.

[0045] The compositions of the present invention are typically produced in a reaction vessel such as a reactor. The formation of the protective layer is usually carried out by forming each protective layer with building blocks, which construct the protective layer through polycondensation reactions. Polycondensation can be carried out in different solvents, preferably aqueous solutions. Polycondensation can be easily controlled and stopped as needed, making it possible to achieve a specified thickness of the protective layer. The selection of building blocks that can be used to construct the protective layer may depend on the known structure of the manipulated phenylalanine ammonia lyase to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building blocks for the protective layer, structural building blocks and protective building blocks are typically used to construct the protective layer. A structural building block that can be used is, for example, tetraethyl orthosilicate (referred to herein as "TEOS" or "T"). The protective building blocks that can be used may be, for example, 3-aminopropyltriethoxysilane (referred to herein as "APTES" or "A"), propyltriethoxysilane (referred to herein as "PTES" or "P"), isobutyltriethoxysilane (referred to as "IBTES"), hydroxymethyltriethoxysilane (referred to herein as "HTMEOS" or "H"), benzyltriethoxysilane (referred to herein as "BTES"), ureidopropyltriethoxysilane (referred to as "UPTES"), or carboxyethyltriethoxysilane (referred to herein as "CETES"). The structural building blocks are typically precursors of inorganic silica and can form four covalent bonds in the resulting layer. The protective building blocks are typically organosilanes and retain an organic moiety that is well-suited to interacting with manipulated phenylalanine ammonia lyase. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes.More preferred structural building blocks are mixtures of tetravalent and trivalent silanes, particularly mixtures of tetra-alkoxysilanes and tri-alkoxysilanes. Even more preferred structural building blocks are selected from the group consisting of tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate.A more preferred protective building block is selected from the group consisting of carboxyethylsilanetriol, benzylsilane, propylsilane, isobutylsilane, n-octylsilane, hydroxysilane, bis(2-hydroxyethyl)-3-aminopropylsilane, aminopropylsilane, ureidopropylsilane, (N-acetylglycyl)-3-aminopropylsilane, and hydroxy(polyethyleneoxy)propyl]triethoxysilane, particularly benzyltriethoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, and (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimethoxysilane, and isobutyltrimethoxysilane. Selected from (isobutylimethoxysilane), n-octyltrimethoxysilane, hydroxylnetyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltriethoxysilane(N-acetylglycyl)-3-aminopropyltrimethoxysilane, or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureidopropyltrihydroxyethoxysilane(N-acetylglycyl)-3-aminopropyltrihydroxymethoxysilane.

[0046] Particularly preferred building blocks are TEOS as structural building blocks and APTES, BTES, and / or HTMEOS, preferably APTES, as protective building blocks. In particular, TEOS as structural building blocks and APTES as protective building blocks are used to construct protective layers.

[0047] The reaction time between the building blocks and the solid support depends on the length of the linker, if one is used, and the size of the manipulated phenylalanine ammonia lyase. The reaction usually takes place over a period of 0.5 to 10 hours, preferably 1 to 5 hours, more preferably 1 to 4 hours, and even more preferably 2 to 4 hours, preferably in an aqueous solution, preferably at room temperature at about 5 to about 25°C or about 20°C. The formation of the protective layer can be stopped by actively halting the polycondensation reaction, for example, by removing unreacted building blocks through a washing process, or by the self-termination of the polycondensation reaction caused by a limited amount of building blocks.

[0048] In a more preferred embodiment, the manipulated phenylalanine ammonia lyase is immobilized on a solid support by introducing the molecule as an anchor point for the manipulated phenylalanine ammonia lyase, and by at least partially modifying the surface of the solid support using a linker, preferably a crosslinker that binds to the anchor point and the manipulated phenylalanine ammonia lyase.

[0049] In one embodiment, the molecules and / or linkers introduced as anchor points are uniformly distributed on the surface of the solid support.

[0050] In preferred embodiments, the crosslinker may be glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) Selected from the group consisting of HCl), DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated sulfhydrils (e.g., sulfurhydryl-reactive 2-pyridyldithio).In a more preferred embodiment, the crosslinker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2HCl), DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated sulfhydrils (e.g., sulfurhydryl-reactive 2-pyridyldithio). Most preferably, glutaraldehyde is selected.

[0051] After the protective layer is formed, the solid carrier containing the manipulated phenylalanine ammonia lyase and the protective layer can be stored. Storage is usually achieved, for example, by washing the formed composition with a buffer and storing it suspended or dissolved in the buffer for a desired period of time. In a preferred embodiment, the solid carrier containing the manipulated phenylalanine ammonia lyase and the protective layer are stored at a constant temperature of 2 to 25°C. In a more preferred embodiment, the solid carrier containing the manipulated phenylalanine ammonia lyase and the protective layer are stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing the manipulated phenylalanine ammonia lyase and the protective layer are stored for 10 to 30 hours at a constant temperature between 2 and 25°C, preferably at room temperature.

[0052] In one embodiment, the functional component binds to the mucus.

[0053] In one embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a polymer comprising repeating units, each containing at least one amino group.

[0054] In one embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a polymer comprising repeating units, each containing at least one thiol group.

[0055] In one embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, and polymerized silane containing an amino group. In a preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, and polymerized APTES.

[0056] In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; and polymerized silanes containing an amino group, preferably polymerized APTES. In an even more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is polyglucosamine, preferably polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof, and more preferably chitosan or a derivative thereof.

[0057] The preferred polyglucosamine of the present invention is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or their derivatives. The most preferred is chitosan or its derivatives. The preferred polymerized silane-PEG-NH2 is selected from the group consisting of silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. The preferred polymerized silane containing an amino group is selected from the group consisting of APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, with APTES being particularly preferred.

[0058] In further embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silane containing an amino group, polymerized silane containing a thiol group, polycarbophil-cysteine ​​conjugate, polymerized silane-PEG-thiol, and polycysteine. In more preferred further embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof, and are selected from the group consisting of polyglucosamine; polymerized silane-PEG-NH2; polymerized silane containing a thiol group, preferably polymerized MPTS; polycarbophil-cysteine ​​conjugate; polymerized silane-PEG-thiol; and polycysteine. In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a thiol-containing polyglucosamine or polymerized silane, preferably a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof, more preferably chitosan or a derivative thereof, or a thiol-containing polymerized silane, polycarbophil-cysteine ​​conjugate, and polymerized silane-PEG-thiol, preferably a thiol-containing polymerized silane.

[0059] In certain embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polymerized silane-PEG-NH2, polymerized silane-PEG2000-NH2, polymerized silane-PEG5000-NH2, polymerized silanes containing amino groups, preferably polymerized APTES, and polymerized silanes containing thiol groups, preferably polymerized MPTS.

[0060] In one embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silane containing an amino group, and polymerized silane containing a thiol group. In a preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized APTES, and polymerized MPTS.

[0061] In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; polymerized silane containing an amino group, preferably polymerized APTES; and polymerized silane containing a thiol group, preferably polymerized MPTS. In a particular embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, dermatan or derivatives thereof, most specifically chitosan or a derivative thereof.

[0062] In one embodiment, the polymer comprising repeating units, each containing at least one thiol group, is selected from the group consisting of polymerized silanes containing thiol groups, polycarbophil-cysteine ​​conjugates, polymerized silane-PEG-thiols, and polycysteine, preferably selected from the group consisting of polymerized silanes containing thiol groups, polycarbophil-cysteine ​​conjugates, and polymerized silane-PEG-thiols, more preferably polymerized silanes containing thiol groups, and most preferably polymerized MPTS. In one embodiment, the polymerized silane containing thiol groups is preferably polymerized MPTS.

[0063] In one embodiment, 5% to 100%, preferably 10% to 100%, and more preferably 50% to 100% of the surface of the protective layer is covered with a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0064] In one embodiment, the functional component is immobilized on the surface of the protective layer by bonding, preferably covalent bonding. In a preferred embodiment, the functional component is immobilized on the surface of the protective layer by non-covalent bonding, preferably by electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is immobilized on the surface of the protective layer by covalent bonding.

[0065] In one embodiment, the functional component is immobilized on the surface of a protective layer using spacers that bond to the surface of the protective layer and the functional component. Thus, in one embodiment, the present invention comprises a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase or fragment thereof by embedding the engineered phenylalanine ammonia lyase or fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, and the functional component is immobilized on the surface of the protective layer by spacers. Examples of such spacers include polyethylene such as PEG4, PEG2000, and PEG5000. The functional component immobilized on the surface of the protective layer by spacers is typically produced by first reacting the spacer and the functional component so that the spacer bonds to the functional component, and then the functional component bonded to the spacer reacts with the surface of the protective layer.

[0066] The immobilization of functional components onto the surface of the protective layer is typically carried out by suspending a solid support carrying the manipulated phenylalanine ammonia lyase embedded in the aforementioned protective layer in a reaction vessel such as a reactor, for example, in water, a buffer, a nonionic surfactant, or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. Nonionic surfactants are typically ethoxylated sorbitan esters such as EG-40 diisostearate P-sorbitan, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated surfactants such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The functional component is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80). The functional component is then added to the suspension and reacted with the surface of the protective layer, usually under stirring, to immobilize the functional component on the surface of the protective layer. Typically, such compositions obtained are washed and resuspended in water, buffer, or a nonionic surfactant or a mixture thereof. Immobilization is achieved by non-covalent bonds, such as electrostatic bonds, or by covalent bonds of the functional component.Functional components can be immobilized by chemically modifying the surface of the protective layer and the functional components using "click chemistry" such as copper-catalyzed click chemistry (Copper-catalyzed azide-alkyne cycloaddition, see eg. Kolb et al. (2001) Angew. Chem. 40(11) 2004-2021), or by using click chemistry that does not use copper (Wittig G, A Chem Ber, 1961, 94, 3260), for example, by first reacting a solid support carrying the manipulated phenylalanine ammonia lyase embedded in the aforementioned protective layer with a reactive compound such as an ethynyl compound, modifying the functional components by adding a reactive compound, such as an azide residue, and then reacting the two components to immobilize the functional components on the surface of the protective layer.

[0067] In a further embodiment, the present invention provides the aforementioned compositions for use as pharmaceuticals.

[0068] In further embodiments, the present invention provides compositions for use in methods for the prevention, delay of progression, or treatment of phenylketonuria (PKU). Furthermore, the use of the compositions described herein for the manufacture of a pharmacopoeia for the prevention, delay of progression, or treatment of phenylketonuria (PKU) in a subject is provided. Furthermore, the use of the compositions described herein for the prevention, delay of progression, or treatment of phenylketonuria (PKU) in a subject is provided. Furthermore, a method for the prevention, delay of progression, or treatment of phenylketonuria (PKU) in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject. Preferably, when administered to a subject in the method of the present invention, the composition breaks down phenylalanine in the intestines of the subject.

[0069] The compositions according to the present invention are preferably pharmaceutical compositions comprising a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions according to the present invention are suitable for oral administration to a subject. Unless otherwise indicated, the pharmaceutical compositions according to the present invention are prepared by known methods.

[0070] The composition of the present invention, for example, a pharmaceutical composition, may be appropriately administered over a continuous period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks, and then discontinued for a period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks.

[0071] The compositions of the present invention, such as pharmaceutical compositions, can be conveniently administered in unit dose form. The unit of enzyme activity ("U") can be expressed as the weight or mass of substrate hydrolyzed per unit time. The unit ("U") can be described in nmols (or nmol / hr) of substrate converted per hour. In an exemplary therapeutic regimen, the composition contains 60 U to 1,000 U of manipulated PAL.

[0072] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of the composition of the present invention that is capable of producing one or more desired effects in a subject to which it is administered. Determining the therapeutic effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0073] As used herein, the terms “treatment” and “to treat” include: (1) delaying the onset of clinical symptoms of a condition, disorder, or pathology in animals, particularly mammals, particularly humans, that are suffering from or susceptible to a condition, disorder, or pathology but have not yet experienced or manifested clinical or subclinical symptoms of the condition, disorder, or pathology; (2) inhibiting a condition, disorder, or pathology (e.g., stopping, reducing, or delaying the onset of the disease with respect to at least one clinical or subclinical symptom, or stopping, reducing, or delaying its recurrence in the case of maintenance treatment); and / or (3) reducing a pathology (i.e., causing a regression of the condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms). The benefit to the patient being treated is statistically significant or at least perceptible to the patient or physician. However, it will be understood that when a patient is given medicine to treat a disease, the result is not always an effective treatment.

[0074] As used herein, “delay in progression” means extending the time to the onset of symptoms or delaying the increase in the severity of symptoms. Furthermore, as used herein, “delay in progression” includes setback or inhibition of disease progression. “Inhibition” of disease progression or disease complications in a subject means preventing or mitigating disease progression and / or disease complications in a subject.

[0075] Preventive measures include prophylactic treatment. For preventive use, the drug combination of the present invention is administered to subjects suspected of having or at risk of developing the above-mentioned disease or disorder. For therapeutic use, the drug combination is administered to subjects, for example, patients already suffering from the above-mentioned disease or disorder, in an amount sufficient to cure or at least partially cessate the symptoms of the disease. The effective dose for such use will depend on the severity and course of the disease, previous treatments, the subject's health status and response to the drug, and the judgment of the attending physician.

[0076] If the target condition does not improve, the pharmaceutical combination of the present invention may be administered chronically, i.e., over a long period including the subject's entire life, to improve, or otherwise control or limit the symptoms of the target disease or condition.

[0077] If the patient's condition improves, the drug combination can be administered continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily stopped over a specific period (i.e., a “drug-free period”). Once the patient’s condition improves, a maintenance dose of the drug combination of the present invention may be administered as needed. Thereafter, the dose, or the frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved disease is maintained.

[0078] In a further embodiment, the present invention provides a method for producing a composition comprising the aforementioned composition, for example, a solid carrier, an engineered phenylalanine ammonia lyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, (b) A step of immobilizing the manipulated phenylalanine ammonia lyase or a fragment thereof onto the solid carrier, (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the manipulated phenylalanine ammonia lyase or fragment thereof immobilized on the solid carrier. (d) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group.

[0079] Step (a) is usually carried out by providing the solid carrier suspended in water, a nonionic surfactant, a buffer, or a mixture thereof, preferably suspended in water and / or a nonionic surfactant, more preferably suspended in water and / or a nonionic surfactant without a buffer in the suspension, even more preferably suspended in a mixture of water and a nonionic surfactant, particularly suspended in a mixture of water and a nonionic surfactant without a buffer in the suspension. The immobilization of the manipulated phenylalanine ammonia lyase onto the solid carrier in step b) of this method is usually carried out by adding a solution of the manipulated phenylalanine ammonia lyase to the suspension of the solid carrier. Preferably, a linker connecting the solid carrier and the manipulated phenylalanine ammonia lyase is added to the suspension of the solid carrier before adding the solution of the manipulated phenylalanine ammonia lyase to the suspension of the solid carrier. In a preferred embodiment, the immobilization of the manipulated phenylalanine ammonia lyase onto a solid carrier is carried out by providing a suspension of the solid carrier and adding a solution of the manipulated phenylalanine ammonia lyase, and the suspension to which the solution of the manipulated phenylalanine ammonia lyase has been added is incubated so that the enzyme can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of the manipulated phenylalanine ammonia lyase or fragment thereof onto a solid carrier in step b) is carried out by i) adding a linker to the solid carrier provided in step (a), preferably adding a linker to the suspension of the solid carrier provided in step a), and ii) adding the manipulated phenylalanine ammonia lyase or fragment thereof, preferably adding a solution of the manipulated phenylalanine ammonia lyase or fragment thereof to the solid carrier and linker, or to a suspension containing the solid carrier and linker, the linker linking the solid carrier to the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof.In one embodiment, a protective layer building block, preferably a monomer of the protective layer building block, more preferably an organosilane, even more preferably a triethoxysilane, particularly APTES, is added to the solid carrier and linker, or to a suspension containing the solid carrier and linker, before adding a solution of the manipulated phenylalanine ammonia lyase or fragment thereof. In a preferred embodiment, the surface of the solid carrier is at least partially modified to improve the immobilization of the manipulated phenylalanine ammonia lyase to the solid carrier. In particular, the surface of the solid carrier is at least partially modified before immobilizing the manipulated phenylalanine ammonia lyase. The surface of the solid carrier can be at least partially modified by adding molecules as anchor points for the manipulated phenylalanine ammonia lyase to the surface of the solid carrier as described above.

[0080] The suspension containing the solid support is typically incubated after each of the above addition steps so that the manipulated phenylalanine ammonia lyase or fragment thereof is linked to the solid support, preferably the surface of the solid support, and to the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof via a linker, preferably by covalent bond, thereby immobilizing the manipulated phenylalanine ammonia lyase or fragment thereof on the solid support, for example, between the solid support and the molecule as an anchor point, between the solid support and the linker, and between the solid support containing the linker and the manipulated phenylalanine ammonia lyase or fragment thereof.

[0081] In one embodiment, in step (b), the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof is immobilized on a solid carrier by linking the solid carrier and the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof via a linker, preferably by linking the solid carrier and the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof via a linker, and the solid carrier is linked to the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof by covalent bonds between the linker and the solid carrier, and between the linker and the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof. Preferably in step b), i) a linker is added to the solid carrier provided in step (a), and ii) the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof is added to the solid carrier and the linker, and the linker links the solid carrier and the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof. The linker used is as described above, and preferably connects the surface of the solid carrier to the manipulated phenylalanine ammonia lyase by covalent bonds. More preferably, the linker is added to the solid carrier in step (b) in a molar excess relative to the manipulated phenylalanine ammonia lyase (PAL) or its fragments; preferably, the linker is added to the solid carrier in step (b) in a molar excess of 1 to 1000 times relative to the manipulated phenylalanine ammonia lyase (PAL) or its fragments; more preferably, the linker is added to the solid carrier in step (b) in a molar excess of 2 to 300 times relative to the manipulated phenylalanine ammonia lyase (PAL) or its fragments; even more preferably, the linker is added to the solid carrier in step (b) in a molar excess of 4 to 250 times relative to the manipulated phenylalanine ammonia lyase (PAL) or its fragments; in particular, the linker is added to the solid carrier in step (b) in a molar excess of 4 times relative to the manipulated phenylalanine ammonia lyase (PAL) or its fragments.

[0082] In a preferred embodiment, linkers that did not link the phenylalanine ammonia lyase (PAL) or its fragments, which were operated with the solid carrier in step (b), are present while a protective layer is formed on the surface of the solid carrier in step (c). In a more preferred embodiment, linkers, or a portion thereof, that did not link the phenylalanine ammonia lyase (PAL) or its fragments, which were operated with the solid carrier in step (b), covalently bond the protective layer and the operated phenylalanine ammonia lyase (PAL) or its fragments in step (c). In an even more preferred embodiment, linkers that did not link the phenylalanine ammonia lyase (PAL) or its fragments, which were operated with the solid carrier in step (b), are not removed in step (b) or step (c), or between steps (b) and (c). In certain embodiments, linkers that did not link the phenylalanine ammonia lyase (PAL) or its fragments, which were operated with the solid carrier in step (b), are not removed in step (b) or step (c), or between steps (b) and (c). Linkers that did not link the phenylalanine ammonia lyase (PAL) or its fragments, which were operated with the solid carrier in step (b), or a portion thereof, are covalently bonded in step (c) to the protective layer and the operated phenylalanine ammonia lyase (PAL) or its fragments. The amount of linkers that did not link the phenylalanine ammonia lyase (PAL) or its fragments, which were operated with the solid carrier in step (b), is usually 30% to 70%, preferably 40% to 60%, and more preferably about 50% of the amount of linker added to the solid carrier in step (b). In one embodiment, there is no washing step between (i) adding the linker to the solid carrier provided in step (a) and (ii) adding the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof to the solid carrier and the linker. In one embodiment, there is no washing step between any of steps (a) to (c).In one embodiment, there is no washing step between (i) adding the linker to the solid carrier provided in step (a) and ii) adding the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof to the solid carrier and the linker, and there is no washing step between any of steps (a) to (c).

[0083] In one embodiment, the linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) A selection is made from the group consisting of HCl, DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), preferably glutaldehyde.

[0084] In preferred embodiments, the linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2 It is selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane), and is preferably glutaldehyde.

[0085] The formation of the protective layer in step (c) of this method is usually carried out by forming each protective layer using building blocks, and the building blocks construct the protective layer by the polycondensation reaction described above. The immobilization of the functional components on the surface of the protective layer in step (d) of this method is usually carried out as described above.

[0086] In one embodiment, the protective layer is formed by building blocks, where structural building blocks and protective building blocks are used to form the protective layer, the structural building blocks being inorganic silica precursors capable of forming four covalent bonds in the layer being formed, and the protective building blocks being the aforementioned organic silanes.

[0087] In one embodiment, the protective layer contains approximately 30% to 100% of the manipulated phenylalanine ammonia lyase.

[0088] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles, and titanium particles, preferably silica particles, and more preferably silica nanoparticles (SNPs).

[0089] A preferred method of the present invention is a method for producing a composition comprising a solid carrier, an operated phenylalanine ammonia lyase (PAL) or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the operated phenylalanine ammonia lyase (PAL) or fragment thereof by embedding the operated phenylalanine ammonia lyase (PAL) or fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspended state, preferably in a suspended state in water and / or a nonionic surfactant, and more preferably in a suspended state in a mixture of water and a nonionic surfactant. (b) A step of immobilizing manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof onto a solid carrier, preferably, the surface of the solid carrier is at least partially modified before the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof is immobilized onto the solid carrier, i) a linker is added to the suspension of the solid carrier, or i) the linker is added to the suspension of the solid carrier after at least partially modification of the surface of the solid carrier, and ii) a solution of the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof, preferably the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof, is added to the suspension of the solid carrier and the linker, the linker linking the solid carrier to the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof. (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof immobilized on the solid carrier, wherein the linker, or a portion thereof, that did not connect the solid carrier and the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof in step (b), covalently bonds the protective layer with the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof. (d) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, A method that includes this.

[0090] Furthermore, a composition is provided comprising a solid carrier, an engineered phenylalanine ammonia lyase (PAL) or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase (PAL) or fragment thereof by embedding the engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, and the composition can be obtained by the present invention, particularly by the preferred method of the present invention described above. [Examples]

[0091] Materials and methods reagent: - 99% tetraethyl orthosilicate (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), polysorbate 80, acetic acid, bovine serum albumin (BSA), Tris buffer, L-phenylalanine, pancreatin, and pronase were purchased from Sigma-Aldrich. BSA, pancreatin, and pronase were dissolved in water to reconstitute the stock buffer. - Chitosan 95 / 500 P was purchased from Heppe Medical Chitosan GmbH. - Manipulated PAL (SEQ ID NO: 5) was supplied by Nestle Health Science at a concentration of 90 mg / mL in 25 mM sodium phosphate, 250 mM sodium chloride, 5% D-mannitol, 0.2% poloxamer 188, pH 7.5. - Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Authenticated Cell Cultures (ECACC). - ThinCert TM The cell culture insert plates (1.0 μm membrane) were purchased from Greiner bio-one. - Fetal bovine serum, penicillin / streptomycin (10,000 U / ml penicillin / 10,000 μg / ml streptomycin), L-glutamine 200 mM (100x), Dulbecco phosphate-buffered saline (DPBS) (1x), 0.25% trypsin-EDTA (1x), DMEM, and white DMEM were purchased from Gibco. - Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, was purchased from Corning. - The animal feed Altromin 1324 was purchased from Altromin International. - I purchased the phenylalanine-free 5LF2 animal feed from LabDiet. - The catheter was purchased from Instech Laboratories.

[0092] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20,000 g for 20 minutes and washed sequentially with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with image analysis software Olympus Stream Motion.

[0093] Generation of NP-1: SNPs (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.9 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Processed PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized processed PAL using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20°C.

[0094] Generation of NP-2: SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a water bath at 20°C.

[0095] Generation of NP-1 variants: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and enzyme activity.

[0096] In the initial experiment, silica nanoparticles (NP-1(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L) was mixed with APTES (3.9 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. / Washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). The manipulated PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized manipulated PAL using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then subjected to H2O / The particles were washed three times in PS80 (8 mg / L) (by centrifugation at 20,000 rcf for 5 minutes) and resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a water bath at 20°C.

[0097] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and nanoparticles (NP-1(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 59 nm) and PS80 (8 mg / L) were added to phosphate buffer (25 mM, pH 7.5), to which APTES (3.9 mM) was added. The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The manipulated PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized manipulated PAL using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-1(2) was cured overnight in a water bath at 20°C.

[0098] In the third experiment, nanoparticles (NP-1) were generated in H2O / PS80 (8 mg / L) according to the section titled "Generation of NP-1" above. The nanoparticles were not washed between each chemical step in order to retain the excess glutaraldehyde in the reaction mixture, which did not bind the solid support to the manipulated PAL. Therefore, glutaraldehyde remained present during layer growth, causing covalent bonding between the protective layer and the manipulated PAL. This covalent bonding between the protective layer and the manipulated PAL can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of imine bonds by the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine amino acids in the manipulated PAL and organosilica layers. The absorbance of nanoparticles NP-1(1), NP-1(2), and NP-1 at 460 nm was measured after the formation of the organic silica layer and after the final particle washing, i.e., after the organic silica layer was formed and the particles were washed three times in H2O / PS80 and resuspended in H2O / PS80 as described in the section titled "NP-1 Generation". NP-1 showed a much higher absorbance than NP-1(1) and NP-1(2) (see Figure 8). NP-1(1) and NP-1(2) still showed some absorbance at this wavelength, which is because imine bonds are formed during enzyme immobilization. However, the absorbance of NP-1 was significantly higher than the others, indicating that further imine bonds were formed by covalent bonding between the protective layer and the manipulated PAL.

[0099] NP-1 activity assay: In a 96-well plate, NP-1 (20 μL, 1.75 mg / mL) in Tris buffer (0.1 M, pH 7.5) was mixed with L-Phe solution (180 μL, 50 mM). The pharmacokinetics of PAL were monitored with a spectrophotometer at 37°C for 30 minutes at λ = 290 nm.

[0100] Resilience to external stress: - Tolerance to acidic conditions The engineered PAL or NP-1 was incubated at pH 4 for 24 hours. Enzyme activity was evaluated at 0, 1, 3, 6, and 24 hours as described in the "Activity assay of NP-1".

[0101] - Resistance to protease The engineered PAL or NP-1 was treated with pancreatin (30 mU) or pronase (0.8 U) and incubated at 37 °C with shaking at 300 rpm for 4 hours. Enzyme activity was evaluated at 0, 0.1, 0.25, 0.5, 1, 2, and 4 hours as described in the "Activity assay of NP-1".

[0102] Cell culture: In all experiments, cells were cultured at 37 °C and 5% CO2.

[0103] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin. For the development of the intestinal barrier model, cells were seeded at a density of 2.6×10 5 cells / cm 2 onto Transwell PET inserts (pore size 1 μm). All cell models were used in experiments on day 21. For co-culture, Caco-2 cells and HT-29-MTX-E12 cells were used at a ratio of 75% - 25%.

[0104] Trans-epithelial electrical resistance The integrity of the cell barrier was evaluated by measuring trans-epithelial electrical resistance (TEER) using a CellZscope system (NanoAnalytics). After refreshing the cell medium and treating with nanoparticles, TEER was automatically measured every 15 minutes for up to 24 hours in the range from 1 Hz to 100,000 Hz.

[0105] In vitro metabolism of Phe Differentiated Caco-2 / HT29-MTX-E12 cells were co-cultured in white DMEM (hereinafter referred to as "cell medium") supplemented with 1% thermo-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0106] The intestinal barrier was exposed to NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 hours at the apical end of the barrier in the presence of pancreatin (30 mU). At each time point, a 150 μL aliquot was withdrawn from the basal end of the intestinal barrier and replaced with an equal volume of pre-warmed cell medium. The barrier was further incubated at 37°C. The absorbance of the withdrawn aliquot samples was measured at 290 nm to quantify the level of trans-cinnamic acid (TCA).

[0107] animal: All animal experiments were conducted under a license approved by the National Animal Testing Service of the Danish Ministry of Food, Agriculture and Fisheries.

[0108] - Wisteria rat: This study was conducted on male Wistar rats (8 weeks old) from Janvier, France.

[0109] 〇Food and drinking water: The rats were given free access to a pellet-type complete diet called "Altromin 1324." The animals also had free access to drinking water.

[0110] Duodenal catheterization The animals were anesthetized with isoflurane (2-4%) in the induction chamber and then transferred to a nose cone containing isoflurane for surgery. The catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the mesenteric duodenum, near the biliary and pancreatic duct opening. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's neck. The abdominal and cervical incisions were then closed with sutures. The animals were kept warm throughout the procedure and carefully monitored until they had fully recovered from anesthesia.

[0111] Evaluation of the effectiveness of NP-1 in rats Prior to drug administration and d5-L-Phe administration, the animals were starved for 4 hours. Then, rats were administered NP-1 (0.85 U) or NP-2 (8.5 mg) intraduodenally, followed immediately by enteral nutrition of 3.6 mg of d5-L-Phe. The rats were housed in metabolic cages for 24 hours.

[0112] ○ Urine collection, metabolic cage Urine was collected over 24 hours in a metabolic cage. Total excretion volume was obtained, and the urine was collected in Eppendorf tubes and stored at -80°C until shipment for analysis.

[0113] Measurement of d5-hypric acid in rat urine: Quantification of the target analyte was performed using an LC system: Thermo Vanquish Horizon Binary Pump and mass spectrometer: Thermo Q Exactive. Urine samples were prepared as follows: 50 μL of urine sample was spiked into 5 μL of ISTD (100 μM 13C6-HIP, final concentration 2 μM in each sample), 200 μL of 100% methanol was added, and the mixture was vortexed. After being placed on ice for 20 minutes, the samples were centrifuged at 16000 g at 4°C for 10 minutes. The supernatant was transferred to a fully recovered MS glass vial and used for analysis.

[0114] The injection volume used was 2.5 μL, and the runtime was 4.8 minutes at a flow rate of 1 mL / min. Mobile phase A was H2O and formic acid (0.1%), and mobile phase B was methanol and formic acid (0.1%). Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) (gradient: 10% B to 100% B).

[0115] Mass spectrometry (MS) was performed using a Thermo Q Exactive mass spectrometer in DDA top5 capture mode. The MS parameters were as follows: MS1 resolution: 70,000 and MS2 resolution: 17,500. HCD fragmentation was performed at normalized stepwise collision energies of 10, 20, and 30. Data analysis was performed using Thermo quan Browser software.

[0116] - BTBR-Pah enu2 / J: This study was conducted at Jackson Laboratory in the United States, using stock male and female BTBR- Pahenu The experiment was conducted using 2 / J mice (8 weeks old).

[0117] 〇Food and drinking water: The mice were given free access to phenylalanine-free feed (5LF2, LabDiet). The animals were also free to drink water.

[0118] Duodenal catheterization The animals were anesthetized with isoflurane (2-4%). ​​An incision was made in the abdominal cavity through the linea alba, and a catheter (C19PB-MGI1923, Instech Laboratories) was placed in the duodenum from the opposite side of the mesentery. The tip of the catheter was advanced and positioned near the opening of the biliary and pancreatic ducts. The catheter was ligated to the intestinal wall, exposed through a subcutaneous tunnel dug in the animal's neck, and then closed. The abdominal and cervical incisions were then sutured closed. The animals were kept on a heated bed throughout the procedure and carefully monitored until they had fully recovered from anesthesia.

[0119] Evaluation of the effectiveness of NP-1 in mice Before use in the experiment, mice were kept on a phenylalanine-free diet for at least 3 days, then their drinking water was supplemented with a low concentration (0.03 g / L) of L-Phe for 3 days, and subsequently the L-Phe concentration in the drinking water was increased to 0.5 g / L. The mice were free to drink the L-Phe-supplemented water at night.

[0120] Mice were administered NP-1 (0.581U, 7mg), NP-2 (7mg), or modified PAL (0.581U) intraduodenally twice daily for 12 days. Blood samples were collected by EDTA on days 0, 4, 6, 8, 10, and 12. The blood samples were then centrifuged (10 min, 4°C, 2000×g), and at least 20uL of plasma was transferred to an Eppendorf tube and stored at -80°C until analysis of Phe content.

[0121] Measurement of Phe in mouse plasma: Quantification of the target analyte was performed using an LC system: Thermo Vanquish Horizon Binary Pump and mass spectrometer: Thermo TSQ Quantiva.

[0122] Plasma samples were prepared as follows: 20 μL of plasma sample was centrifuged at 13.2 krpm at 4°C for 10 minutes. 10 microliters of the supernatant was added to 10 μL of ISTD (1000 μM D5-Phe) and 80 μL of 100% MeOH. The sample was then vortexed and centrifuged at 13.2 krpm at 4°C for 10 minutes, after which 50 μL of the supernatant was dried at 30°C under a gentle flow of nitrogen. 500 microliters of 0.1% (v / v) formic acid in water were added, and the sample was shaken at 900 rpm for 10 minutes at 15°C before centrifugation (13.2 krpm, 10 minutes, 4°C). Finally, 350 μL of the supernatant was transferred to a total recovery glass vial for analysis.

[0123] The injection volume used was 2 μL, and the runtime was 5 minutes at a flow rate of 1 mL / min. Mobile phase A was H2O and formic acid (0.1%), and mobile phase B was methanol and formic acid (0.1%). Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) (gradient: 100% A to 100% B).

[0124] MS was performed using a Thermo TSQ Quantiva mass spectrometer in capture mode: selective reaction monitoring.

[0125] The MS parameters were as follows: Q§1 resolution: 0.7; Q3 resolution: 0.7; Fragmentation: CID fragmentation in argon (1.5 mTorr). The analyte concentration was calculated from the peak area ratio of Phe and the internal standard d5-Phe. Data analysis was performed using Thermo Quan Browser software.

[0126] result: Example 1: Improvement of enzyme stability and load by covalent bonding to a protective layer In the first experiment, nanoparticles NP-1(1) were generated under unbuffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, nanoparticles NP-1(2) were generated under buffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the third experiment, nanoparticles NP-1 were generated under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0127] To determine the PAL immobilization yield on the surface of NP-1(1), NP-1(2), and NP-1, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield doubled under conditions where the presence of glutaraldehyde was maintained (NP-1) (Figure 2A), and consequently, the enzyme load per dry weight of SNP doubled compared to buffer conditions where glutaraldehyde was removed by washing (NP-1(2)) (Figure 2B). Similarly, enzyme immobilization under conditions where the presence of glutaraldehyde was maintained (NP-1) doubled the enzyme load per dry weight of SNP compared to buffer conditions where glutaraldehyde was removed by washing (NP-1(1.5)) (Figure 2B).

[0128] The biocatalytic activity of NP-1(1), NP-1(2), and PAL immobilized and protected on NP-1 was evaluated. Even more surprising than the increased load due to enzyme immobilization while maintaining the presence of glutaraldehyde was the threefold increase in nanoparticle specific activity compared to buffered conditions where glutaraldehyde was removed by a washing step, and the twofold increase compared to unbuffered conditions where glutaraldehyde was removed by a washing step (Figure 2C). This extremely surprising fact of a threefold increase in nanoparticle specific activity is closely related to the specific activity (units per gram of PAL) of the enzyme immobilized under unbuffered conditions where the presence of glutaraldehyde was maintained, which is comparable to the specific activity of the enzyme immobilized under buffered conditions. This result is completely unexpected, as enzymes are thought to have significantly higher activity in the presence of buffers (Figure 2D). In summary, covalent bonding of a protective layer to the enzyme surface unexpectedly improves its load and stability compared to enzymes protected by an organic silica layer via electrostatic interactions only.

[0129] Example 2: Phenylalanine ammonia lyase (PAL) activity of NP-1 The biocatalytic activity of immobilized, protected, and manipulated PAL was evaluated. The results shown in Figure 3 report PAL activity at NP-1. This demonstrates the ability to access and convert L-Phe of shielded, functionalized SNPs despite shielding and functionalization. Verification of biocatalytic activity on NP-1 confirms the potential for using nanoparticles for therapeutic purposes.

[0130] Example 3: Resistance to external stress NP-1 is a nanoparticle developed for use in the gastrointestinal tract. Due to the physiological characteristics of the gastrointestinal tract, NP-1 is subjected to various stresses. To ensure the sustained activity of NP-1 in the gastrointestinal tract, the protection of immobilized engineered PAL was evaluated. First, NP-1 or engineered PAL was placed under acidic conditions (pH 4). Monitoring of PAL activity over 24 hours revealed that enzyme activity was sustained on NP-1 (Figure 4B), while the free form of engineered PAL lost its activity over time (Figure 4A). These data demonstrate that engineered PAL immobilized and protected on NP-1 is protected in an acidic environment.

[0131] During digestion, pancreatic enzymes, particularly proteases that can affect therapeutic enzymes in the gastrointestinal tract, are released. Therefore, NP-1 and engineered PAL were exposed to various proteases at 37°C. First, to mimic physiological digestive conditions, NP-1 or engineered PAL was incubated with pancreatin (30 mU), a mixture of pancreatic enzymes extracted from porcine pancreas, and then PAL activity was evaluated. After 4 hours, both NP-1 and engineered PAL exhibited sustained PAL activity (Figure 4C). Next, to further evaluate the benefits of the protective shield under harsh conditions, NP-1 or engineered PAL was exposed to pronase (0.88 U), a cocktail of purified proteases. The results shown in Figure 4C indicate that 80% of the PAL activity on NP-1 was maintained after 4 hours of exposure, while the enzymatic activity of engineered PAL was lost after 4 hours. In summary, these data demonstrate the added value of immobilizing and protecting manipulated PALs on nanoparticles, highlighting the potential of NP-1 for therapeutic applications of the gastrointestinal tract.

[0132] Example 4: In vitro biocompatibility and efficacy of NP-1 Maintaining the integrity of the intestinal barrier is fundamental to preventing unwanted lumen contents, such as pathogens or food allergens, from entering the body. To evaluate the biocompatibility of nanoparticles, a monolayer of Caco2-HT29-MTX-E12 cells was exposed to NP-1 in the presence or absence of pancreatin to mimic digestive conditions, and transepithelial electrical resistance (TEER) was measured. The data shown in Figure 5A report that the integrity of the intestinal epithelial barrier was maintained after 6 hours of contact with NP-1, with or without pancreatin. This result demonstrates the in vitro biocompatibility of NP-1 for gastrointestinal application.

[0133] NP-1 was developed to metabolize Phe in the intestinal lumen. To evaluate the in vitro efficacy of NP-1, a monolayer of Caco2-HT29-MTX-E12 cells cultured in cell medium containing 0.4 mM L-Phe was exposed at its apical end to NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 hours in or without pancreatin (30 mU). Quantification of Phe metabolites at the basal side of the barrier is shown in Figure 5B. The graph reports that TCA accumulated at the basal extrabasal side of the barrier under all conditions. This result demonstrates the in vitro efficacy of NP-1 in the digestive environment and suggests its use for therapeutic purposes.

[0134] Example 5: In vivo activity of NP-1 in rats To evaluate the transition from in vitro to in vivo applications, the efficacy of NP-1 was assessed in rats. Rats were administered either NP-1 (nanoparticles containing engineered PAL) or NP-2 (nanoparticles containing bovine serum albumin (BSA) for which Phe is not a substrate) intraduodenally before enteral nutrition of d5-Phe. In vivo, TCA, a metabolite of Phe, is rapidly metabolized to hypric acid. The in vivo activity of NP-1 was then evaluated by quantifying d5-hypric acid in rat urine collected over 24 hours post-administration. As shown in Figure 6, the significant increase in d5-hypric acid in rats administered with NP-1 compared to NP-2 indicates that NP-1 has the ability to digest Phe within the intestinal lumen, suggesting the use of NP-1 for therapeutic purposes.

[0135] Example 6: In vivo therapeutic efficacy of NP-1 in mice Phenylketonuria (PKU) is characterized by a deficiency in the intracellular hepatic enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine. In PAH deficiency, the concentration of phenylalanine, which is toxic to the brain, becomes abnormally high. The basis of PKU treatment is a low-phenylalanine diet combined with phenylalanine-free L-amino acids. Currently, enzyme replacement therapy using recombinant phenylalanine ammonia lyase is available via subcutaneous injection, but this treatment can induce hypersensitivity reactions and immune-mediated acute hypersensitivity reactions.

[0136] NP-1 was developed to exhibit sustained PAL activity in a gastrointestinal environment (acidic and exposed to proteases). Our method for controlling patients' Phe levels involves breaking down Phe in the intestines (from food intake) to avoid its absorption and accumulation in the blood.

[0137] This treatment strategy is based on BTBR-, a representative animal model of the disease in which the gene encoding PAH is mutated. Pahenu The evaluation was performed using a 2 / J mouse.

[0138] Mice that could ingest L-Phe supplemented drinking water were administered NP-1, NP-2, or engineered PAL twice daily for 12 days. Impressively, mice administered NP-1 showed a steady decrease in plasma Phe concentration throughout the study period, while mice administered engineered PAL showed unstable plasma concentrations (Figure 7). As shown in Figure 7B, normalization of plasma Phe concentrations revealed a 30% decrease in plasma Phe concentration in mice at the end of NP-1 treatment.

[0139] The interaction between nanoparticles and intestinal mucus leads to the transient engraftment of NP-1 and sustained PAL activity in the intestinal wall, while the free form of the manipulated PAL is washed away in the intestine. These results confirm and establish a strategy involving the degradation of Phe in the intestine, supporting the remarkable therapeutic efficacy of NP-1 against PKU.

Claims

1. A composition comprising a solid carrier, manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier, a protective layer that protects the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof by embedding the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each containing at least one amino group and / or thiol group.

2. The composition according to claim 1, wherein the polymer comprising repeating units, each having at least one amino group and / or at least one thiol group, is a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof.

3. The composition according to claim 1, wherein the polymer comprising repeating units, each each having at least one amino group and / or at least one thiol group, is chitosan or a derivative thereof.

4. The composition according to any one of claims 1 to 3, wherein the functional component is immobilized on the surface of the protective layer by non-covalent or covalent bonds.

5. The composition according to any one of claims 1 to 4, wherein a solid carrier is embedded in the protective layer, and manipulated phenylalanine ammonia lyase or a fragment thereof is embedded on the surface of the solid carrier.

6. The composition according to any one of claims 1 to 5, wherein the functional component immobilized on the surface of the protective layer is not embedded by the protective layer.

7. Manipulated phenylalanine ammonia lyase (PAL) or fragment thereof (a) an amino acid sequence having at least 95%, at least 96%, or at least 97% sequence identity with the sequence of Sequence ID No. 1, or consisting of such an amino acid sequence, (b) Sequence IDs 2, 3, 4, or 5, The composition according to any one of claims 1 to 6.

8. The composition according to any one of claims 1 to 6, wherein the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof comprises the polypeptide shown in SEQ ID NO:

5.

9. A composition according to any one of claims 1 to 8 for use as a pharmaceutical.

10. A composition according to any one of claims 1 to 8, for use in a method for the prevention, delaying the progression of, or treating phenylketonuria (PKU).

11. A method for producing a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase (PAL) or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine ammonia lyase (PAL) or fragment thereof by embedding it, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, and the method is: (a) A step of providing a solid carrier, (b) A step of immobilizing the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof onto a solid carrier. (c) A step of forming a protective layer on the surface of a solid carrier in order to protect the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof immobilized on the solid carrier. (d) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer containing repeating units, and each repeating unit contains at least one amino group and / or at least one thiol group. A method that includes this.

12. The method according to claim 11, wherein in step b), i) a linker is added to the solid carrier provided in step (a), and ii) manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof is added to the solid carrier and the linker, and the linker connects the solid carrier to the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof.

13. The method according to claim 12, wherein a linker that did not link the solid carrier to the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof in step (b) is present during the formation of a protective layer on the surface of the solid carrier in step (c).

14. The method according to claim 12, wherein there is no washing step between (i) adding a linker to the solid carrier provided in step (a) and (ii) adding manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof to the solid carrier and the linker.

15. The method according to any one of claims 12 to 14, wherein there is no washing step between any of steps (a) to (c).

16. The method according to any one of claims 12 to 15, wherein a linker, or a portion thereof, that did not link the solid carrier with the manipulated phenylalanine ammonia lyase (PAL) or fragment thereof in step (b) covalently bonds the protective layer with the phenylalanine ammonia lyase (PAL) or fragment thereof in step (c).

17. The linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) The method according to any one of claims 12 to 16, selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane).

18. The method according to any one of claims 12 to 16, wherein the linker is glutaraldehyde.

19. A composition comprising a solid carrier, manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof by embedding the manipulated phenylalanine ammonia lyase (PAL) or a fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, a composition that can be obtained by the method of any one of claims 13 to 18.

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