Functionalized compositions comprising engineered phenylalanine ammonia lyase (PAL)

Abstract

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

Description

Technical Field

[0001] This invention relates to a composition comprising a solid carrier, an engineered phenylalanine aminolyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine aminolyase or the fragment thereof by encapsulating 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. The invention also relates to a method for producing the composition and its use. Background Technology

[0002] Phenylketonuria (PKU) is a genetic autosomal recessive disorder characterized by a deficiency of the intracellular liver enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine, and this enzyme activity can be promoted by tetrahydrobiopterin (BH4). PAH deficiency leads to abnormally high concentrations of phenylalanine, which is toxic to the brain. High phenylalanine levels in infancy and early childhood cause profound cognitive and developmental deficits, while poorly controlled blood phenylalanine levels in older children and adolescents are associated with learning difficulties, attention deficit hyperactivity disorder (ADHD), and behavioral problems. In adulthood, uncontrolled blood phenylalanine levels are associated with executive dysfunction and a variety of behavioral and psychiatric problems.

[0003] The basis of PKU treatment is a combination of a low-phenylalanine diet and a phenylalanine-free L-amino acid regimen. PAH deficiency in patients with blood phenylalanine levels greater than 600 μmol / L can be corrected with enzyme replacement therapy using recombinant phenylalanine ammonia lyase (PAL) (Palynziq®, administered via up to three subcutaneous injections daily). However, hypersensitivity reactions are common, and immune-mediated acute hypersensitivity reactions (type III) have been reported in patients maintained on a PEGylated derivative of the enzyme phenylalanine ammonia lyase (pegvaliase). Therefore, compatible and effective treatment for phenylketonuria is needed. Summary of the Invention

[0004] The present invention provides a composition comprising a solid carrier, an engineered phenylalanine aminolyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the engineered phenylalanine aminolyase or the fragment thereof by encapsulating the engineered phenylalanine aminolyase or the 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0005] The present invention also provides a method for producing the composition, the composition comprising: The method comprises the following steps: a solid carrier, an engineered phenylalanine aminolyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine aminolyase or a fragment thereof by encapsulating 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. (a) Provide a solid carrier; (b) Immobilize the engineered phenylalanine amino lyase or its fragments on the solid carrier; (c) A protective layer is formed on the surface of the solid carrier to protect the engineered phenylalanine aminolyase or its fragments fixed on the solid carrier. (d) A functional component is fixed to the surface of the protective layer, wherein the functional component fixed to the surface of the protective layer is a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0006] The inventors of this application have surprisingly discovered that the compositions provided by the present invention, when used therapeutically, have unexpectedly high in vivo enzyme activity, exhibit low cytotoxicity, and do not disrupt the intestinal barrier when localized in the gastrointestinal tract, thus making them extremely promising for therapeutic use, particularly for the prevention, delay of progression, or treatment of phenylketonuria. Attached Figure Description

[0007] Figure 1 The diagram shows a method for producing the composition of the present invention: a) immobilizing an engineered PAL or a fragment thereof on a solid carrier; b) and c) growing a protective layer around the immobilized engineered PAL or a fragment thereof, thereby encapsulating the immobilized engineered PAL or a fragment thereof; and d) immobilizing the functional component on the surface of the protective layer.

[0008] Figure 2 (A) Protein quantification of reaction supernatants of NP-1(1), NP-1(2), and NP-1. (B) PAL loading per unit dry weight of SNP. (C) SNP-specific activity expressed in U / g SNP. (D) PAL-specific activity expressed in U / g PAL.

[0009] Figure 3The activity of phenylalanine ammonia lyase (PAL) in nanoparticles was demonstrated. The biocatalytic activity (in U / g) of engineered PAL immobilized and protected on nanoparticles was quantified after exposure to phenylalanine.

[0010] Figure 4 The resistance to external stress was demonstrated. NP-1 and engineered PAL were exposed to (AB) acidic conditions (pH 4) or (C) protease, and their stability was assessed by measuring PAL enzyme activity at different time points.

[0011] Figure 5 shows the in vitro biocompatibility and potency of NP-1 in the intestinal barrier model. (A) In vitro assessment of intestinal barrier integrity by measuring transepithelial resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to NP-1 (9.7 mU) or pancreatic enzyme (30 mU) alone for 6 h with or without pancreatic enzyme (30 mU). The figure shows the time-course curve evolution of mean normalized TEER data over 6 h. Dashed lines indicate untreated conditions. (B) In vitro metabolism of phenylalanine (Phe) in the intestinal barrier model. Differentiated Caco-2 / HT29-MTX-E12 cocultures cultured in cell culture medium containing 0.4 mM Phe were exposed to NP-1 (9.7 mU) or engineered PAL (9.7 mU) (with or without pancreatic enzyme (30 mU)) on the top side of the barrier for 6 h. Phe metabolism was evaluated by quantifying trans-cinnamic acid (TCA) on the bottom side of the barrier. The figure shows the evolution of the TCA accumulation time curve over 6 hours.

[0012] Figure 6 The figure shows the quantification of trans-cinnamic acid (TCA) in rat urine. Wistar rats were administered NP-1 (n=5) or NP-2 (n=5) via duodenal administration, while simultaneously receiving d5-Phe via tube feeding. Urine was collected within 24 h post-administration and analyzed by LC-MS. The figure shows the concentration of D5-hippuric acid in urine. p<0.01, passing the t-test.

[0013] Figure 7 shows BTBR- Pah enu2 Plasma concentration of Phe in J mice. BTBR- mice with free access to drinking water containing L-Phe were compared twice daily over 12 days. Pah enu2 / J NP-1 (0.581U; 7mg), NP-2 (7mg), or engineered PAL (0.581U) were administered intraduodenally to mice. Blood samples were collected on days 0, 4, 6, 8, 10, and 12 for plasma extraction and LC-MS analysis. (A) Figure shows BTBR- Pah enu2 The plasma concentration of Phe in J mice. (B) Figure shows the BTBR- Pah enu2 Normalized plasma concentration of Phe in J mice.

[0014] Figure 8 The absorbance of nanoparticles NP-1, NP-1(1) and NP-1(2) at 460 nm is shown. Detailed Implementation

[0015] The present invention relates to a composition comprising a solid carrier, an engineered phenylalanine aminolyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer thereof protecting the engineered phenylalanine aminolyase or the fragment thereof by encapsulating the engineered phenylalanine aminolyase or the 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0016] For the purpose of interpreting this specification, the following definitions will be used, and where appropriate, terms used in the singular will also include the plural, and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] Features, integrals, properties, and compounds described in connection with specific aspects, embodiments, or examples of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments.

[0018] The term “comprise” and its variations, such as “comprises” and “comprising”, are generally used in the sense of inclusion, that is, “including but not limited to”, meaning that one or more features or components are allowed to be present.

[0019] Unless the context clearly indicates otherwise, the singular forms “a” and “the” include plural references.

[0020] The term “about” refers to a range of ±10% of a specified value. For example, the phrase “about 200” includes ±10% of 200, or 180 to 220.

[0021] As used herein, the term "solid support" generally refers to particles. Preferably, the solid support is monodisperse or polydisperse particles, more preferably monodisperse particles. Solid supports typically include organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, and are preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size of the solid support is typically from 1 nm to 1000 µm, preferably from 10 nm to 100 µm, and particularly about 50 nm.

[0022] As used herein, the terms "linker" or "cross-linker" refer to any linking agent containing a group capable of binding a specific functional group (e.g., primary amine, thiol, etc.). In the context of this invention, a linker typically links the surface of a solid support to an engineered phenylalanine aminolysin. For example, the linker can be immobilized on the surface of a solid support, such as as a carrier material on a silica surface, and then the engineered phenylalanine aminolysin can be bound to unoccupied binding sites on the linker. Alternatively, the linker can first bind the engineered phenylalanine aminolysin, and then the linker binding the engineered phenylalanine aminolysin can bind its unoccupied binding sites to the solid support. Various types of linkers are known in the art, including but not limited to linear or branched carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as labeled linkers.

[0023] As used herein, the term "protective layer" refers to a layer used to protect the functional properties of an engineered phenylalanine aminolysin or fragment immobilized on the surface of a solid support. The protective layer of the present invention is typically constructed with building blocks, at least a portion of which are monomers capable of interacting with each other typically via covalent bonding and with the immobilized engineered phenylalanine aminolysin typically via non-covalent bonding. A protective layer is formed on the surface of a solid support to protect the engineered phenylalanine aminolysin 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 aminolysin or fragment thereof is embedded.

[0024] As used herein, the terms "phenylalanine ammonia lyase or a fragment thereof" or "PAL or a fragment thereof" refer to a 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), which also includes histidine ammonia lyases and tyrosine ammonia lyases. PAL is sometimes also referred to as phenylalanine / tyrosine ammonia lyase because some PALs can use both tyrosine and phenylalanine as substrates. 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 may be required for catalytic activity and is formed through the cyclization and dehydration of the conserved active site Alal67-Serl68-Glyl69 tripeptide fragment.

[0025] When used to refer to phenylalanine aminolysin or fragments thereof as used herein, the terms “engineered” and “non-natural” refer to natural or naturally occurring forms of phenylalanine aminolysin or fragments thereof that are modified in a manner not found in nature. The term “engineered phenylalanine aminolysin or fragments thereof” does not include or encompass “wild-type” and “naturally occurring” phenylalanine aminolysin or fragments thereof. As used herein, “wild-type” and “naturally occurring” refer to forms of phenylalanine aminolysin or fragments thereof found in nature. For example, wild-type phenylalanine aminolysin or fragments thereof are polypeptides present in organisms that can be isolated from natural sources and are not intentionally modified by human intervention. Engineered PAL or fragments thereof are, for example, variants or functionally active fragments of engineered phenylalanine aminolysin. Therefore, the terms “fraction of engineered phenylalanine aminolysin,” “fraction thereof” in relation to engineered phenylalanine aminolysin, and “functionally active fragment of engineered phenylalanine aminolysin” are used synonymously herein. The term "variant or functionally active fragment thereof" in relation to the engineered phenylalanine aminolysin of the present invention refers to a fragment or variant (e.g., an analog, derivative, or mutant not found in nature) that performs the same or improved physiological functions as wild-type phenylalanine aminolysin. The addition, deletion, substitution, and derivatization of one or more amino acids are considered, provided that the modification does not result in a loss of functional activity of the fragment or variant. The PAL fragment comprises a homotetramerase, wherein at least one monomer of the homotetramerase, preferably all four monomers, typically contains 100 to 550 amino acids, preferably 200 to 500 amino acids, and more preferably 300 to 450 amino acids. "Improved physiological function" or "improved enzymatic properties" means that the engineered PAL exhibits any improved enzymatic properties compared to a reference PAL polypeptide such as a wild-type PAL polypeptide. Improved properties include, but are not limited to, the following: increased protein surface area; increased thermal activity; increased thermal stability; increased pH activity; increased stability; increased enzyme activity; increased substrate specificity and / or affinity; increased specific activity; increased resistance to substrate and / or end-product inhibition; increased chemical stability; improved chemoselectivity; improved solvent stability; increased tolerance to acidic pH; increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis); reduced aggregation; increased solubility; reduced immunogenicity; and altered temperature profile. The preferred engineered phenylalanine aminolyase or fragment thereof of the present invention is the engineered phenylalanine aminolyase described in WO2018 / 148633 A1.

[0026] As used herein, the term "partially embedded engineered phenylalanine aminolysin" means that the engineered phenylalanine aminolysin is not completely covered by the protective layer, and therefore, the engineered phenylalanine aminolysin is not completely embedded in the protective layer. In one embodiment, less than 50% of the intended engineered phenylalanine aminolysin is covered by the protective layer, although typically at least 70% is covered, thus improving the protection of the engineered phenylalanine aminolysin. 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 intended engineered phenylalanine aminolysin 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 intended engineered phenylalanine aminolysin is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the engineered phenylalanine aminolysin is covered by a protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the engineered phenylalanine aminolysin is covered by a protective layer in which the active site is not covered.

[0027] As used herein, the term "fully encapsulated engineered phenylalanine aminolyase" means that the engineered phenylalanine aminolyase according to the present invention is completely, i.e., 100% covered by the protective layer, meaning that the active site is also covered. Preferably, the engineered phenylalanine aminolyase or a fragment thereof according to the present invention is completely, i.e., 100% covered by the protective layer, meaning that the active site is also covered.

[0028] As used herein, the term "engineered phenylalanine ammonia lyase that is at least partially embedded" means that the engineered phenylalanine ammonia lyase is at least partially embedded and can be completely embedded by a protective layer. Therefore, "engineered phenylalanine ammonia lyase that is at least partially embedded" means that the protective layer covers about 30% to 100% of the engineered phenylalanine ammonia lyase or fragments thereof, preferably about 50% to about 100%, more preferably about 80% to about 100%, even more preferably about 90% to about 100%, and most preferably about 95% to about 100%, wherein the active site is preferably covered.

[0029] As used herein, the term "functional constitutent" refers to a component that retains its characteristic, functional properties after being fixed to the surface of a protective layer. In the context of this invention, a functional constitutent is a polymer comprising repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0030] As used herein, the term "sequence identity percentage (%)" refers to the amount of peptide being compared and is determined by comparing two optimally aligned sequences within a comparison window, where the portion of the peptide in the comparison window may contain additions or deletions (i.e., vacancies) to achieve optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions in both sequences where the same amino acid residues occur to arrive at the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same amino acid residues occur, or by comparing amino acid residues with vacancies to arrive at the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100. Those skilled in the art will understand that many established algorithms are available for aligning two sequences.

[0031] As used in this article, the term "reference sequence" refers to a qualified sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a fragment of a full-length gene or polypeptide sequence. Typically, 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.

[0032] As used herein, the term "comparison window" refers to a conceptual fragment of at least about 20 consecutive amino acid residues, wherein the sequence is comparable to a reference sequence of at least 20 consecutive amino acids, and wherein a portion of the sequence within the comparison window may contain 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which does not contain additions or deletions) to achieve optimal alignment of the two sequences. The comparison window may be longer than 20 consecutive residues and may optionally include windows of 30, 40, 50, 100, or longer.

[0033] As used herein, the term "polymer comprising repeating units, wherein each repeating unit contains at least one amino group" refers to a polymer comprising several repeating units (monomers), wherein each repeating unit contains at least one amino group. Preferred polymers comprise several repeating units (monomers), wherein each repeating unit contains one amino group, particularly a primary amino group.

[0034] As used herein, the term "polymer comprising repeating units, wherein each repeating unit contains at least one thiol group" refers to a polymer comprising several repeating units (monomers), wherein each repeating unit contains at least one thiol. Preferred polymers comprise several repeating units (monomers), wherein each repeating unit contains one thiol group.

[0035] As used herein, the term "polycarbophil-cysteine ​​conjugate" refers to a conjugate containing a cysteine ​​residue covalently linked to polycarbophil. Such conjugates can be generated as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0036] 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 that can 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.

[0037] As used herein, the term "polycysteine" may consist of L-cysteine ​​or D-cysteine, preferably L-cysteine, and preferably contains 2 to 30 cysteine ​​residues, more preferably 2 to 5 cysteine ​​residues.

[0038] As used herein, the term "polyglucosamine" refers to a linear amino-polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamine contains a free amine (-NH2) group and can be characterized by the ratio of N-acetyl-D-glucosamine units to D-glucosamine units, expressed as the degree of deacetylation (DDA) of a fully acetylated polymeric chitosan. Preferred polyglucosamines of this invention are selected from chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan, or derivatives thereof. Chitosan or its derivatives are most preferred.

[0039] As used herein, the term "chitosan or a derivative thereof" refers to chitosan or chitosan derivatives thereof, including its salts, preferably having a molecular weight of 2,000 Da or greater, more preferably in the range of 25,000 to 2,000,000 Da, more preferably about 50,000 to 350,000 Da, and most preferably about 50,000 to 190,000 Da or 190,000 to 310,000 Da. The term "derivative" in relation to chitosan includes esters, ethers, or other derivatives formed by the reaction of an acyl or alkyl group with an OH group. Examples are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Suitable derivatives are, for example, shown in GAE Roberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable salts of chitosan include nitrates, phosphates, sulfates, xanthates, hydrochlorides, glutamates, lactates, and acetates.

[0040] In a first aspect, the present invention provides a composition comprising a solid carrier, an engineered phenylalanine aminolyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine aminolyase or the fragment thereof by encapsulating the engineered phenylalanine aminolyase or the 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0041] Engineered phenylalanine aminolysin or fragments thereof can be immobilized on the surface of a solid support via non-covalent or covalent bonding. Non-covalent bonding includes pp (aromatic) interactions, van der Waals interactions, H-bonding interactions, and electrostatic interactions, such as ionic interactions. Preferably, engineered phenylalanine aminolysin or fragments thereof are immobilized on the surface of the solid support via covalent bonding or via a linker.

[0042] Solutions of engineered phenylalanine aminolysin or fragments thereof are typically contained in a buffer solution containing the protein or fragments thereof. Commonly used buffers include phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. The solution may additionally contain sugar alcohols or nonionic surfactants, as described herein.

[0043] In one embodiment, the solid support is selected from organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, and preferably silica particles, more preferably silica nanoparticles (SNPs). Particle size is typically measured by measuring the diameter of the particles and is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, particularly about 50 nm. When the solid support is a monodisperse particle, the particle size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, particularly about 50 nm. When the solid support is a polydisperse particle, the particle size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, particularly 50 nm to 50 μm. In one embodiment, the composition comprises a solid support, wherein the solid support comprises at least 15%, preferably at least 20%, particularly 15% to 25%, more particularly 15% to 20% immobilized engineered phenylalanine aminolyase or fragments / dry weight solid support thereof.

[0044] Typically, monodisperse or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in this invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In other preferred embodiments, the polydisperse particles are non-spherical polydisperse particles.

[0045] Solid carriers are typically provided in suspension form. The suspension of the solid carrier can be, for example, in water, a buffer solution, or a nonionic surfactant or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. The nonionic surfactant is typically selected from ethoxylated sorbitol esters such as PEG-40 sorbitol diisostearate, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); and block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 40. 7. Fatty acid ethoxylates such as PEG-5 oleate, PEG-8 stearate, polyoxyethylene 40 stearate, polyoxyethylene 15 hydroxy stearate; fatty alcohol ethoxylates such as stearyl alcohol polyether (steareth) 40; fatty acid esters such as palmitic acid ascorbate, beeswax, polyglycerol 3-oleate, propylene glycol monocaprylate, propylene glycol monolaurate; fatty alcohols such as cetearyl alcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol; glycerides; polyethylene glycol-modified triglycerides; sugar esters, preferably polysorbate, more preferably polysorbate 80 (PS80). The buffer solutions that can be used in the method of this invention are 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)propyl-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N,N-bis[2-hydroxyethyl]propanesulfonic acid, etc. [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, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0046] In one embodiment, the surface of the solid support is modified to introduce molecular or functional chemical groups as anchoring points, i.e., as anchoring points for the engineered phenylalanine aminolysin or as anchoring points for a linker connecting the engineered phenylalanine aminolysin to the solid support. Preferably, the anchoring points are amine functional chemical groups or portions. As a non-limiting example, an amino-modified surface of the 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 with a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support with a silica surface containing an amino-modified surface, more preferably a solid support obtained by reacting a solid support with a silica surface with an aminosilane (e.g., APTES). Such modified supports can form amide bonds between the engineered phenylalanine aminolysin and the amino groups on the surface of the support material, or between the linker and the amino groups on the surface of the support material. In one implementation, the introduced molecules or functional chemical groups are uniformly distributed on the surface of the solid carrier as anchoring points.

[0047] In some embodiments, the protective layer has a defined thickness of about 1 to about 200 nm, typically 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, particularly about 1 to about 15 nm. The most preferred defined thickness is about 1 to about 10 nm. In some embodiments, the layer has a defined thickness of about 5 to about 100 nm, preferably about 5 to about 50 nm, more preferably about 5 to about 25 nm, even more preferably about 5 to about 20 nm, particularly about 5 to about 15 nm. The most preferred defined thickness is about 5 to about 10 nm. The protective layer is typically porous with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0048] In one embodiment, the engineered phenylalanine ammonia lyase or a fragment thereof is partially embedded in a protective layer. In a preferred embodiment, the engineered phenylalanine ammonia lyase or a fragment thereof is at least partially embedded in a protective layer. In a more preferred embodiment, the engineered phenylalanine ammonia lyase or a fragment thereof is completely embedded in a protective layer.

[0049] In one embodiment, a protective layer encapsulates a solid carrier and encapsulates an engineered phenylalanine aminolysin or a fragment thereof immobilized on the surface of the solid carrier. In another embodiment, the functional component immobilized on the surface of the protective layer is not encapsulated by the protective layer. Preferably, the protective layer completely encapsulates the solid carrier and completely encapsulates the engineered phenylalanine aminolysin or a fragment thereof immobilized on the surface of the solid carrier. More preferably, the protective layer completely encapsulates the solid carrier and completely encapsulates the engineered phenylalanine aminolysin or a fragment thereof immobilized on the surface of the solid carrier, and the functional component immobilized on the surface of the protective layer is not encapsulated by the protective layer. If the protective layer completely encapsulates the solid carrier and completely encapsulates the engineered phenylalanine aminolysin or a fragment thereof immobilized on the surface of the solid carrier, then the engineered phenylalanine aminolysin or its fragment is completely, i.e., 100% covered by the protective layer, meaning the active site is also covered, and the solid carrier is completely, i.e., 100% covered by the protective layer.

[0050] In a preferred embodiment, the engineered phenylalanine ammonia lyase or a fragment thereof comprises or is composed of an amino acid sequence having at least 90%, at least 95%, at least 96%, or at least 97% sequence identity with the sequence of SEQ ID NO:1. In one embodiment, the engineered phenylalanine ammonia lyase or a fragment thereof is SEQ ID NO: 2, 3, 4, or 5. In a particularly preferred embodiment, the engineered phenylalanine ammonia lyase or a fragment thereof comprises a polypeptide as shown in SEQ ID NO: 5.

[0051] The thickness of the protective layer can be measured using microscopes such as scanning electron microscopes (SEM), transmission electron microscopes (TEM), scanning probe microscopes (SPM), light scattering methods, or elliptic polarization techniques.

[0052] The compositions of the present invention are typically produced in a reaction vessel such as a reactor. The formation of the protective layer is generally carried out by the formation of a corresponding protective layer from building units, wherein the building units construct the protective layer during a polycondensation reaction. Polycondensation can be carried out in various solvents, preferably in aqueous solutions. If suitable, polycondensation can be easily controlled and stopped, allowing for the achievement of a defined thickness of the protective layer. The selection of building units that can be used to construct the protective layer can depend on the known structure of the engineered phenylalanine aminolyase in order to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building units for the protective layer, both structural building units and protective building units are typically used to construct the protective layer. Structural building units that can be used are, for example, tetraethyl orthosilicate (referred to herein as “TEOS” or “T”). The protective building blocks that can be used are, for example, 3-aminopropyltriethoxysilane (referred to herein as “APTES” or “A”), propyltriethoxysilane (referred to herein as “PTES” or “P”), isobutyltriethoxysilane (referred to herein as “IBTES”), hydroxymethyltriethoxysilane (referred to herein as “HTMEOS” or “H”), benzyltriethoxysilane (referred to herein as “BTES”), ureopropyltriethoxysilane (referred to herein as “UPTES”), or carboxyethyltriethoxysilane (referred to herein as “CETES”). The structural building blocks are typically precursors of inorganic silica capable of forming four covalent bonds in the formed layers. The protective building blocks are typically organosilanes carrying an organic moiety conferred with the ability to interact with engineered phenylalanine aminolysin. Preferred building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes. More preferred building blocks are mixtures of tetravalent and trivalent silanes, particularly mixtures of tetraalkoxysilanes and trialkoxysilanes. Even more preferred building blocks are selected from tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate.Even more preferred protective building blocks are selected from carboxyethyl silanetriol, benzyl silane, propyl silane, isobutyl silane, n-octyl silane, hydroxy silane, bis(2-hydroxyethyl)-3-aminopropyl silane, aminopropyl silane, ureopropyl silane, and (N-acetylglycyl) silane. -3-aminopropylsilane, hydroxy(polyvinyloxy)propyl]triethoxysilane, especially selected from benzyltriethoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureopropyltriethoxysilane, (N-acetylglycyl)-3-aminopropyltriethoxysilane, or selected from benzyltrimethoxysilane, propyltrimethoxysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureopropyltrimethoxysilane, (N-acetylglycyl) -3-aminopropyltrimethoxysilane, or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureopropyltrihydroxyethoxysilane, (N-acetylglycyl)-3-aminopropyltrihydroxyethoxysilane.

[0053] The particularly preferred building blocks are TEOS as the structural building block, and APTES, PTES, and / or HTMEOS, with APTES being the preferred protective building block. Specifically, TEOS is used as the structural building block and APTES as the protective building block to construct the protective layer.

[0054] If a linker is used, the reaction time between the building blocks and the solid support depends on the length of the linker and the size of the engineered phenylalanine aminolysin. The reaction typically proceeds for 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 of about 5 to about 25°C or at 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, such as through a washing step; or by self-stopping the polycondensation reaction caused by a limited amount of building blocks.

[0055] In other preferred embodiments, the engineered phenylalanine aminolyase is immobilized on the solid support by introducing a molecule as described above for the engineered phenylalanine aminolyase as an anchoring point and by using a linker, preferably a crosslinking agent, that binds to the anchoring point and the engineered phenylalanine aminolyase to at least partially modify the surface of the solid support.

[0056] In one embodiment, the introduced molecules and / or binders serving as anchoring points are uniformly distributed on the surface of the solid carrier.

[0057] In a preferred embodiment, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl pimelimidate, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated thiol, thiol-reactive 2-pyridinedithiol, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[succinimide] propionate), DTBP (3,3'-dithiobispropionylimine dimethyl ester·2 HCl), DST (disuccinimidyl tartrate), sulfonyl-LC-SMPT (4-succinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoate), SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl-6-(3-[2-pyridyldithio]-propamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), BMDB (1,4-bismaleimide-2,3-dihydroxybutane). More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated thiol groups (e.g., thiol-reactive 2-pyridyl dithio). In a more preferred embodiment, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimide-oxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate]), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate]), DTBP (3,3'-dithiobispropionine dimethyl ester·2 HCl), DST (disuccinimide tartrate), and BMDB (1,4-bismaleimide-2,3-dihydroxybutane).More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated thiol groups (e.g., thiol-reactive 2-pyridyl dithiocarbamate). Glutaraldehyde is most preferred.

[0058] After the protective layer is formed, a solid carrier containing the engineered phenylalanine aminolysin and the protective layer can be stored. Storage is typically accomplished, for example, by washing the formed composition with a buffer and suspending or dissolving it in the buffer for the required storage time. In a preferred embodiment, the solid carrier containing the engineered phenylalanine aminolysin and the protective layer is stored at a constant temperature of 2 to 25°C.

[0059] In other preferred embodiments, the solid carrier containing the engineered phenylalanine aminolysin and the protective layer is stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing the engineered phenylalanine aminolysin and the protective layer is stored at a constant temperature of 2 to 25°C, preferably at room temperature for 10 to 30 hours.

[0060] In one implementation, the functional ingredient binds to the mucus.

[0061] In one embodiment, a polymer comprising repeating units in which each repeating unit contains at least one amino group and / or at least one thiol group is a polymer comprising repeating units in which each repeating unit contains at least one amino group.

[0062] In one embodiment, a polymer comprising repeating units wherein each repeating unit contains at least one amino group and / or at least one thiol group is a polymer comprising repeating units wherein each repeating unit contains at least one thiol group.

[0063] In one embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, and amino-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a preferred embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, and polymeric APTES, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0064] In a more preferred embodiment, the polymer comprising repeating units is selected from polyglucosamines such as chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polymeric silanes-PEG-NH2; and amino-containing polymeric silanes, preferably polymeric APTES, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In an even more preferred embodiment, the polymer comprising repeating units is a polyglucosamine, preferably selected from polyglucosamines such as chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0065] The preferred polyglucosamine of this invention is selected from chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan, or derivatives thereof. Chitosan or its derivatives are most preferred. The preferred silane-PEG-NH2 is selected from silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. Preferred amino-containing polysilanes are selected from APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, especially APTES.

[0066] In other embodiments, the polymer comprising repeating units is selected from polyglucosamine, polysilane-PEG-NH2, amino-containing polysilanes, thiol-containing polysilanes, polycarbofibril-cysteine ​​conjugates, polysilane-PEG-thiols, and polycysteine, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In other more preferred embodiments, the polymer comprising repeating units is selected from polyglucosamine of chitosan, chitosan, polyglucosamine polysaccharide, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polysilane-PEG-NH2; thiol-containing polysilanes, preferably polyMPTS; polycarbofibril-cysteine ​​conjugates; polysilane-PEG-thiols; and polycysteine, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In even more preferred embodiments, the polymer comprising the repeating unit is polyglucosamine or a thiol-containing polymeric silane, preferably selected from chitosan, chitosan, polyglucosamine polysaccharide, chondroitin, heparin, keratin and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof or thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates and polymeric silane-PEG-thiol, preferably thiol-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0067] In a specific embodiment, the polymer containing repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polysilane-PEG-NH2 selected from polysilane-PEG4-NH2, polysilane-PEG2000-NH2, polysilane-PEG5000-NH2, amino-containing polysilanes, preferably polymeric APTES, and thiol-containing polysilanes, preferably polymeric MPTS, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0068] In one embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, amino-containing polymeric silanes, and thiol-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a preferred embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, polymeric APTES, and polymeric MPTS, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0069] In a more preferred embodiment, the polymer comprising repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, and dermatan, or polyglucosamine derivatives thereof; polymeric silane-PEG-NH2; amino-containing polymeric silanes, preferably polymeric APTES; and thiol-containing polymeric silanes, preferably polymeric MPTS, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a specific embodiment, the polymer comprising repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, dermatan, or derivatives thereof, most specifically chitosan or derivatives thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0070] In one embodiment, the polymer comprising the repeating unit is selected from thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates, polymeric silane-PEG-thiol, and polycysteine, and is preferably selected from thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates, and polymeric silane-PEG-thiol, and more preferably thiol-containing polymeric silanes, and most preferably polymeric MPTS, wherein each repeating unit comprises at least one thiol group. In one embodiment, the thiol-containing polymeric silane is preferably polymeric MPTS.

[0071] In one embodiment, 5% to 100%, preferably 10% to 100%, more preferably 50% to 100% of the surface of the protective layer is covered by a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0072] In one embodiment, the functional component is fixed to the surface of the protective layer by bonding, preferably covalent bonding. In a preferred embodiment, the functional component is fixed to the surface of the protective layer by non-covalent bonding, preferably by electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units is fixed to the surface of the protective layer by covalent bonding, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0073] In one embodiment, the functional component is immobilized on the surface of the protective layer using spacers that bind to both the protective layer surface and the functional component. Therefore, in one embodiment, the invention comprises a composition comprising a solid carrier, an engineered phenylalanine aminolyase or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine aminolyase or fragment thereof by encapsulating the engineered phenylalanine aminolyase 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, wherein 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 spacers with the functional component, such that the spacers bind to the functional component, and then reacting the functional component bound to the spacers with the surface of the protective layer.

[0074] The functional component is typically immobilized onto the surface of the protective layer in a reaction vessel, such as a reactor, by suspending a solid carrier, as described above, carrying an engineered phenylalanine aminolysin embedded in the protective layer, in, for example, water, a buffer solution, or a nonionic surfactant or a mixture thereof, preferably a mixture of water and nonionic surfactants. The nonionic surfactant is typically selected from ethoxylated sorbitol esters such as PEG-40 sorbitol diisostearate, 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 ethoxylates such as PEG-5 oleate and PEG-8 stearate. Esters, polyoxyethylene 40 stearate, polyoxyethylene 15 hydroxy stearate, fatty alcohol ethoxylates such as stearyl alcohol polyether (steareth) 40; fatty acid esters such as palmitic acid ascorbate, beeswax, polyglycerol 3-oleate, propylene glycol monocaprylate, propylene glycol monolaurate; fatty alcohols such as cetearyl alcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol; glycerides; polyethylene glycol-modified triglycerides; glycol esters, preferably polysorbate, more preferably polysorbate 80 (PS80). The functional component is then added to the suspension and reacts with the surface of the protective layer, usually under stirring, to immobilize the functional component on the surface of the protective layer. The resulting composition is usually washed and resuspended in water, buffer solution, or nonionic surfactant or mixtures thereof. Immobilization is carried out through non-covalent bonding, such as electrostatic bonding, or through covalent bonding of the functional component. Functional components can be immobilized by chemically modifying the protective layer and the surface of the functional component using methods such as "click chemistry" (e.g., copper-catalyzed azide-alkyne cycloaddition, see, for example, Kolb et al., (2001) Angew. Chem. 40(11) 2004-2021) or copper-free click chemistry (Wittig G, A Chem Ber, 1961, 94, 3260). For example, as described above, a solid carrier carrying an engineered phenylalanine aminolyase embedded in the protective layer is first reacted with a reactive compound such as an acetylene compound, and the functional component is modified by adding a reactive compound such as an azide residue. The two components are then reacted to immobilize the functional component on the surface of the protective layer.

[0075] In other respects, the present invention provides compositions as described above, which are used as pharmaceuticals.

[0076] In other aspects, the present invention provides compositions for use in methods of preventing, delaying the progression of, or treating phenylketonuria (PKU). Use of the compositions described herein in the preparation of medicaments for preventing, delaying the progression of, or treating PKU in an individual is also provided. Use of the compositions described herein for preventing, delaying the progression of, or treating PKU in an individual is also provided. A method for preventing, delaying the progression of, or treating PKU in an individual is also provided, the method comprising administering to the individual a therapeutically effective amount of the composition described herein. Preferably, when administered to an individual according to the method of the present invention, the composition degrades phenylalanine in the individual's intestine.

[0077] The compositions according to the invention are preferably pharmaceutical compositions and comprise a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions of the invention are suitable for oral administration to an individual. Unless otherwise stated, the pharmaceutical compositions of the invention are prepared in a manner known per se.

[0078] Compositions, such as the pharmaceutical compositions of the present invention, may be administered for one week or a portion thereof, for two weeks, for three weeks, for four weeks, for five weeks, or for six weeks, and then stopped for one week or a portion thereof, for two weeks, for three weeks, for four weeks, for five weeks, or for six weeks.

[0079] Compositions, such as the pharmaceutical compositions of the present invention, can be conveniently administered in unit dosage forms. Units of enzyme activity (“U”) can be described as the weight or mass of substrate hydrolyzed per unit time. Units (“U”) can be described as nmol of substrate converted per hour (or nmol / hr). In exemplary therapeutic regimens, the composition comprises 60 U to 1,000 U of engineered PAL.

[0080] As used herein, the term "effective amount" or "therapeutic effective amount" means an amount capable of causing one or more desired effects in an individual receiving a composition of the present invention. The determination of a therapeutically effective amount is entirely within the competence of those skilled in the art, especially based on the detailed disclosure provided herein.

[0081] As used herein, the term “treatment / treating” includes: (1) delaying the onset of clinical symptoms of a state, symptom, or condition in animals, particularly mammals, especially humans, who may have or be susceptible to the state, symptom, or condition but have not yet experienced or exhibited clinical or subclinical symptoms of the state, symptom, or condition; (2) suppressing a state, symptom, or condition (e.g., preventing, reducing, or delaying the development or recurrence of a disease, or at least one clinical or subclinical symptom thereof, in the case of maintenance treatment); and / or (3) alleviating a condition (i.e., causing the resolution of at least one of a state, symptom, or condition, or its clinical or subclinical symptoms). The benefit of treatment to a patient is statistically significant or at least perceptible to the patient or physician. However, it should be understood that when a patient is given medication to treat a disease, the outcome may not always be an effective treatment.

[0082] As used in this article, “delaying progression” means increasing the time to symptom onset or slowing the increase in symptom severity. Furthermore, “delaying progression” as used in this article includes reversing or inhibiting disease progression. “Inhibiting” an individual’s disease progression or complications means preventing or reducing an individual’s disease progression and / or complications.

[0083] Preventive therapy includes preventive treatment. In preventive use, the drug combination of the present invention is administered to an individual suspected of having the aforementioned disease or condition or at risk of developing the aforementioned disease or condition. In therapeutic use, the drug combination is administered to an individual in an amount sufficient to cure or at least partially stop the symptoms of the disease, such as a patient already suffering from the aforementioned disease or condition. The effective amount for this use will depend on the severity and course of the disease, previous treatment, the individual's health condition and response to the drug, and the judgment of the treating physician.

[0084] If the condition of any individual does not improve, The drug combination of the present invention can be administered for a prolonged period of time, including the entire duration of an individual's life, to improve or otherwise control or limit the symptoms of an individual's disease or condition.

[0085] In cases where an individual's condition has indeed improved, the drug combination can be administered continuously; alternatively, the dosage of the administered drugs can be temporarily reduced or temporarily suspended for a period of time (i.e., a "drug holiday"). Once the patient's condition has improved, a maintenance dose of the drug combination of the present invention is administered, if necessary. Subsequently, the dosage or frequency of administration, or both, is preferably reduced to a level that maintains the improvement in the disease, depending on the symptoms.

[0086] In other aspects, the present invention provides a method for producing the composition as described above, comprising, for example, a solid support, an engineered phenylalanine aminolysin or a fragment thereof immobilized on the surface of the solid support, a protective layer protecting the engineered phenylalanine aminolysin or the fragment thereof by encapsulating the engineered phenylalanine aminolysin or the 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group; the method

[0087] Includes the following steps: (a) Provide a solid carrier; (b) Immobilize the engineered phenylalanine amino lyase or its fragments on the solid carrier; (c) A protective layer is formed on the surface of the solid carrier to protect the engineered phenylalanine aminolyase or its fragments fixed on the solid carrier. (d) A functional component is fixed to the surface of the protective layer, wherein the functional component fixed to the surface of the protective layer is a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0088] Step (a) is typically performed by providing a solid support in a suspension of water, a nonionic surfactant, or a buffer solution, preferably a suspension of water and / or a nonionic surfactant, more preferably a suspension of water and / or a nonionic surfactant (wherein the suspension does not contain a buffer solution), even more preferably a suspension of a mixture of water and a nonionic surfactant, particularly a suspension of a mixture of water and a nonionic surfactant (wherein the suspension does not contain a buffer solution). In step (b) of the method of the invention, the engineered phenylalanine ammonia lyase is typically immobilized on the solid support by adding an engineered phenylalanine ammonia lyase solution to the suspension of the solid support. Preferably, a linker connecting the solid support and the engineered phenylalanine ammonia lyase is added to the suspension of the solid support before adding the engineered phenylalanine ammonia lyase solution to the suspension of the solid support. In a preferred embodiment, the engineered phenylalanine aminolysin (PAL) is immobilized on a solid support by providing a suspension of the solid support and adding a solution of the engineered PAL solution, wherein the suspension containing the engineered PAL solution is incubated to allow the enzyme to bind to the surface of the solid support. In a more preferred embodiment, the immobilization of the engineered PAL on the solid support in step b) is performed by: i) adding a ligand to the solid support provided in step (a), preferably adding the ligand to a suspension of the solid support provided in step a), and ii) adding the engineered PAL to the solid support and the ligand or a suspension containing the solid support and the ligand, preferably adding a solution of the engineered PAL to the solid support and the ligand or a suspension containing the solid support and the ligand, wherein the ligand connects the solid support to the engineered PAL. In one embodiment, before adding the engineered phenylalanine aminolysin or a fragment thereof to the solution, the building blocks of the protective layer, preferably monomers of the building blocks of the protective layer, more preferably organosilanes, even more preferably triethoxysilanes, particularly APTES, are added to the solid support and the linker or to a suspension containing the solid support and the linker. In a preferred embodiment, the surface of the solid support is at least partially modified to improve the immobilization of the engineered phenylalanine aminolysin on the solid support. Specifically, the surface of the solid support is at least partially modified before immobilizing the engineered phenylalanine aminolysin. As described above, the surface of the solid support can be at least partially modified by adding molecules that serve as anchoring sites for the engineered phenylalanine aminolysin.

[0089] Typically, after each of the above-described addition steps, a suspension containing a solid carrier is incubated to allow reactions, for example, between the solid carrier and the molecules serving as anchoring sites, between the solid carrier and the linker, and between the solid carrier containing the linker and the engineered phenylalanine aminolyase or a fragment thereof, respectively. This allows the engineered phenylalanine aminolyase or a fragment thereof to be linked to the solid carrier via the linker, preferably by covalent bonding, preferably the surface of the solid carrier, to the engineered phenylalanine aminolyase (PAL) or a fragment thereof, thereby immobilizing the engineered phenylalanine aminolyase or a fragment thereof on the solid carrier.

[0090] In one embodiment of step (b), the engineered phenylalanine aminolysin (PAL) or its fragments are immobilized on the solid support by linking the solid support to the engineered PAL or its fragments via a ligant, preferably by linking the solid support to the engineered PAL or its fragments via a ligant. The solid support is linked to the engineered PAL or its fragments via covalent bonding between the ligant and the solid support, and between the ligant and the engineered PAL or its fragments. Preferably, in step b), i) the ligant is added to the solid support provided in step (a), and ii) the engineered PAL or its fragments are added to the solid support and the ligant, wherein the ligant links the solid support to the engineered PAL or its fragments. The ligant used is as described above, and the surface of the solid support is linked to the engineered PAL or its fragments via preferred covalent bonding. More preferably, in step (b), the linker is added to the solid support in a molar excess relative to the engineered phenylalanine aminolysin (PAL) or its fragments. Preferably, in step (b), the linker is added to the solid support in a molar excess of 1 to 1000 times relative to the engineered phenylalanine aminolysin (PAL) or its fragments. More preferably, in step (b), the linker is added to the solid support in a molar excess of 2 to 300 times relative to the engineered phenylalanine aminolysin (PAL) or its fragments. Even more preferably, the linker is added to the solid support in a molar excess of 4 to 250 times relative to the engineered phenylalanine aminolysin (PAL) or its fragments. In particular, in step (b), the linker is added to the solid support in a molar excess of four times relative to the engineered phenylalanine aminolysin (PAL) or its fragments.

[0091] In a preferred embodiment, the linker that did not link the solid support to the engineered phenylalanine aminolysin (PAL) or its fragment in step (b) is present during step (c) when a protective layer is formed on the surface of the solid support. In a more preferred embodiment, the linker or a portion thereof that did not link the solid support to the engineered phenylalanine aminolysin (PAL) or its fragment in step (b) covalently binds the protective layer to the engineered phenylalanine aminolysin (PAL) or its fragment in step (c). In other preferred embodiments, the linker that did not link the solid support to the engineered phenylalanine aminolysin (PAL) or its fragment in step (b) is not removed in or between steps (b) and (c). In a specific embodiment, the ligand that did not link the solid support to the engineered phenylalanine ammonia lyase (PAL) or its fragment in step (b) is not removed in or between steps (b) and (c), and the ligand that did not link the solid support to the lipase or its fragment in step (b), or a portion thereof, covalently binds the protective layer to the engineered phenylalanine ammonia lyase (PAL) or its fragment in step (c). The amount of ligand that did not link the solid support to the engineered phenylalanine ammonia lyase (PAL) or its fragment in step (b) is typically 30% to 70% of the ligand dose added to the solid support in step (b), preferably 40% to 60%, more preferably about 50%. In one embodiment, there is no washing step between adding the ligand to the solid support provided in step (a) in (i) and adding the engineered phenylalanine ammonia lyase (PAL) or its fragment to the solid support and ligand in (ii). In one embodiment, there is no washing step between any of steps (a) to (c). In one embodiment, there is no washing step between adding the ligand to the solid carrier provided in step (A) in (i) and adding the engineered phenylalanine aminolyase (PAL) or a fragment thereof to the solid carrier and ligand in (ii), and there is no washing step between any of steps (a) to (C).

[0092] In one embodiment, the linker is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl pimelimidate, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated thiol, thiol-reactive 2-pyridinedithiol, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[succinimide] propionate), DTBP (3,3'-dithiobispropionylimine dimethyl ester·2 HCl), DST (disuccinimidyl tartrate), sulfonyl-LC-SMPT (4-succinimidyl-6-methyl-α-(2-pyridyldithio)toluamide]hexanoate), SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl-6-(3-[2-pyridyldithio]-propamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-2'-pyridyldithio)-propionamido]butane), DTME (dithio-bismaleimide ethane), BMDB (1,4-bismaleimide-2,3-dihydroxybutane), and preferably glutaraldehyde.

[0093] In a preferred embodiment, the binder is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimide-oxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate), DTBP (3,3'-dithiobispropionine dimethyl ester·2HCl), DST (disuccinimide tartrate), BMDB (1,4-bismaleimide-2,3-dihydroxybutane), and glutaraldehyde is preferred.

[0094] The formation of the protective layer in step (C) of the method according to the invention is generally carried out by forming the corresponding protective layer with building units, wherein the building units construct the protective layer in a polycondensation reaction as described above. The immobilization of the functional components on the surface of the protective layer in step (d) of the method according to the invention is generally carried out as described above.

[0095] In one embodiment, the protective layer is formed by building units, wherein structural building units and protective building units are used to form the protective layer, wherein the structural building units are precursors of inorganic silica capable of forming four covalent bonds in the formed layer, and the protective building units are organosilanes as described above.

[0096] In one embodiment, the protective layer encapsulates approximately 30% to approximately 100% of the engineered phenylalanine aminolysin.

[0097] In one embodiment, the solid support is selected from organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles, and titanium particles, and is preferably silica particles, more preferably silica nanoparticles (SNPs).

[0098] A preferred method of the present invention is a method for producing a composition comprising a solid support, an engineered phenylalanine aminolysin (PAL) or a fragment thereof immobilized on the surface of the solid support, a protective layer protecting the engineered PAL or the fragment thereof by encapsulating the engineered PAL or the 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension, preferably wherein the solid carrier is provided in the form of a suspension in water and / or a nonionic surfactant, more preferably wherein the solid carrier is provided in the form of a suspension in a mixture of water and a nonionic surfactant; (b) Immobilizing engineered phenylalanine aminolysin (PAL) or a fragment thereof onto a solid support, wherein preferably the surface of the solid support is at least partially modified before immobilizing the engineered phenylalanine aminolysin or a fragment thereof onto the solid support, wherein i) a linker is added to a suspension of the solid support or i) a linker is added to a suspension of the solid support after at least partially modifying the surface of the solid support, and ii) adding a solution of engineered phenylalanine aminolysin (PAL) or a fragment thereof, preferably engineered phenylalanine aminolysin (PAL) or a fragment thereof, to a suspension of the solid support and the linker, wherein the linker links the solid support to the engineered phenylalanine aminolysin (PAL) or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the engineered phenylalanine aminolyase (PAL) or a fragment thereof fixed on the solid support, wherein the linker or a portion thereof that did not connect the solid support to the engineered phenylalanine aminolyase (PAL) or a fragment thereof in step (b) covalently binds the protective layer to the phenylalanine aminolyase (PAL) or a fragment thereof. (d) Fixing a functional component onto the surface of a protective layer, wherein the functional component fixed onto the surface of the protective layer is a polymer comprising repeating units, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0099] A composition is also provided comprising a solid carrier, an engineered phenylalanine aminolyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the engineered phenylalanine aminolyase (PAL) or the fragment thereof by encapsulating 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition can be obtained by the method described herein, particularly by the preferred method of the present invention as described above.

[0100] Example

[0101] Materials and methods:

[0102] Reagents:

[0103] - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% aqueous solution), polysorbate 80, acetic acid, bovine serum albumin (BSA), Tris buffer, L-phenylalanine, trypsin, streptomycin, purchased from Sigma-Aldrich. BSA, trypsin, and streptomycin were dissolved in water to reconstitute the stock buffer.

[0104] Chitosan 95 / 500P, purchased from Heppe Medical Chitosan GmbH

[0105] - The engineered PAL (SEQ ID NO: 5) was provided by Nestlé Health Science at a concentration of 90 mg / mL in 25 mM sodium phosphate, 250 mM sodium chloride, 5% D-mannitol, and 0.2% poloxamer 188 at pH 7.5.

[0106] -Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line), European Collection of Cell Cultures (ECACC).

[0107] -ThinCert™ cell culture inserts (1.0 μm membrane), purchased from Greiner bio-one.

[0108] - Fetal bovine serum, penicillin / streptomycin (10,000 U / ml penicillin / 10,000 μg / ml streptomycin), L-glutamine 200 mM (100X), Dulbecco's phosphate-buffered saline DPBS (1X), 0.25% trypsin-EDTA (1X), DMEM, white DMEM, purchased from Gibco.

[0109] -Matrigel® Low Growth Factor (GFR) Basement Membrane Matrix, LDEV-free, purchased from Corning.

[0110] - Animal diet Altromin 1324, purchased from Altromin International.

[0111] - Animal diet phenylalanine-free 5LF2, purchased from LabDiet.

[0112] - Catheter, purchased from Instech Laboratories

[0113] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stöber method described in WO2015 / 014888A1. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm for 22 h at 20 °C. The solution was then centrifuged at 20,000 g for 20 min and washed successively with ethanol and water. Particle size was measured using SEM micrographs obtained at 150,000x magnification using the Olympus flow motion image analysis software.

[0114] Generation of NP-1: APTES (3.9 mM) was added to SNPs (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. Engineered PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized engineered PAL surface using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a 20°C water bath.

[0115] Generation of NP-2: APTES (3.8 mM) was added to the SNP (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. BSA solution was added to bring the final BSA concentration to 1.42 mg / mL, and the reaction mixture was reacted at 20°C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized BSA surface using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20°C water bath.

[0116] The generation of NP-1 variants: The following experiments investigated the effects of covalently linking the enzyme to the protective layer on enzyme stability and enzyme activity.

[0117] In the first experiment, nanoparticles (NP-1(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.9 mM) was added to the SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. The particles were washed three times in H2O / 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 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Engineered PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized engineered PAL surface using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 min. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a water bath at 20°C.

[0118] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to WO2015 / 014888A1 to generate nanoparticles (NP-1(2)) in buffer. The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.9 mM) was added to SNP (10 mg / mL, 59 nm) in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. 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 min. 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). Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. 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). Engineered PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized engineered PAL surface 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 resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 min. 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). NP-1(2) was cured overnight in a water bath at 20°C.

[0119] 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. To maintain an excess of glutaraldehyde in the reaction mixture that did not bind the solid support to the engineered PAL, the nanoparticles were not washed between each chemical step. Therefore, the glutaraldehyde remained during layer growth, resulting in a covalent bond between the protective layer and the engineered PAL. This covalent bond between the protective layer and the engineered PAL can be observed as a yellow / orange appearance with maximum absorption at 460 nm. This color is due to the formation of imine bonds between the aldehyde functional groups of the glutaraldehyde linker and the primary amines of the amino acids in the engineered PAL and the organosilicon layer. After the formation of the silicone layer and final particle washing, i.e., after the formation of the silicone layer, the absorbance of nanoparticles NP-1(1), NP-1(2), and NP-1 was measured at 460 nm. The particles were washed three times in H2O / PS80 and then resuspended in H2O / PS80, as described in the section titled "NP-1 Generation" above. This showed that the absorbance of NP-1 at 460 nm was much higher than that of NP-1(1) and NP-1(2) (see [link]). Figure 8 Since imine bonds are also formed during enzyme immobilization, NP-1(1) and NP-1(2) still show a certain degree of absorbance at this wavelength. However, the absorbance of NP-1 is significantly higher, indicating that the covalent bonding between the protective layer and the engineered PAL leads to the formation of additional imine bonds.

[0120] 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). PAL kinetics were monitored for 30 min at 37 °C with a spectrophotometer at λ = 290 nm.

[0121] Resistance to external stress: - Resistance to acidic conditions Engineered PAL or NP-1 were cultured at pH 4 for 24 hours. Enzyme activity was assessed at 0, 1, 3, 6, and 24 h as described in the "NP-1 Activity Assay".

[0122] - Resistance to proteases

[0123] Engineered PAL or NP-1 cells were treated with trypsin (30 mU) or streptomycin (0.8 U) and cultured at 37°C with shaking at 300 rpm for 4 h. Enzyme activity was assessed at 0, 0.1, 0.25, 0.5, 1, 2, and 4 h as described in the "NP-1 Activity Assay" section.

[0124] Cell culture: For all experiments, cells were cultured at 37°C and 5% CO2.

[0125] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) 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.

[0126] To develop an intestinal barrier model, cells were stored at a density of 2.6 × 10⁻⁶. 5 cells / cm 2 Cells were seeded at a density in Transwell PET inserts (1 μm pores). All cell models were used for experiments on day 21. For co-culture, Caco-2 and HT-29-MTX-E12 cells were used at a ratio of 75%–25%.

[0127] Transepithelial resistance

[0128] Cell barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). TEER was automatically measured every 15 minutes for 24 h, in the range of 1 Hz to 100,000 Hz, after cell culture medium renewal and treatment with nanoparticles.

[0129] Phe's in vitro metabolism

[0130] Caco-2 / HT29-MTX-E12 cells were co-cultured in white DMEM (hereinafter referred to as “cell culture medium”) supplemented with 1% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids and 100 U / mL penicillin / streptomycin.

[0131] On the apical side of the intestinal barrier, the intestinal barrier was exposed to NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 h in the presence of trypsin (30 mU). At each time point, a 150 μL aliquot was removed from the basal side of the intestinal barrier and replaced with the same volume of preheated cell culture medium. The barrier was further incubated at 37 °C. The absorbance of the removed aliquots was measured at 290 nm to quantify the level of trans-cinnamic acid (TCA).

[0132] animal:

[0133] All animal experiments were conducted with permission from the National Animal Experiments Inspectorate under the Ministry of Food, Agriculture and Fisheries of Denmark.

[0134] - Wistar rats: The study was conducted on male Wistar rats (8 weeks old) of the original stock from Janvier, France.

[0135] o Food and drinking water: Rats were fed a complete pelleted diet, "Altromin 1324," with free access to food and drinking water.

[0136] o Duodenal catheter insertion

[0137] The animal was placed in the induction chamber and anesthetized with isoflurane (2-4%), then transferred to a nasal cannula with isoflurane for surgery. A catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the duodenal mesentery, near the opening of the bile and pancreatic ducts. The catheter was ligated and secured to the intestinal wall and then tunneled through a subcutaneous tunnel to the animal's neck, exposing it. The abdominal and cervical incisions were then sutured shut. Throughout the procedure, the animal was kept warm and closely monitored until fully recovered from anesthesia.

[0138] o Evaluating the potency of NP-1 in rats

[0139] Animals were starved for 4 h before administration of d5-L-Phe. Rats were then administered NP-1 (0.85 U) or NP-2 (8.5 mg) via the duodenum, followed immediately by tube feeding of 3.6 mg of d5-L-Phe. Rats were then housed in metabolic cages for 24 h.

[0140] o Urine sampling, metabolic cage

[0141] Urine was collected in a metabolic cage for 24 h. The total urine output was obtained, and urine samples were taken into Eppendorf tubes and stored at -80°C until transport for analysis.

[0142] o Measuring D5-hippuric acid in rat urine: Quantification of the target analyte was performed using an LC system: a Thermo Vanquish Horizon binary pump and a Thermo Q Exactive mass spectrometer. Urine samples were prepared as follows: 50 μL of urine sample was mixed with 5 μL of ISTD (100 μM 13C6-HIP, final concentration 2 μM per sample), and 200 μL of 100% methanol was added before vortexing. After incubating on ice for 20 min, the sample was centrifuged at 16000 g for 10 min at 4 °C. The supernatant was transferred to a total recovery MS vial for analysis.

[0143] The injection volume used was 2.5 μL, the run time was 4.8 min, and the flow rate was 1 mL / min. Mobile phase A was H₂O and formic acid (0.1%); 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) with a gradient of 10% B to 100% B.

[0144] MS was performed using a Thermo Q Exactive mass spectrometer in DDA top5 mode. MS parameters were as follows: MS1 resolution: 70,000 Å and MS2 resolution: 17,500 Å. HCD fragmentation was performed using normalized step collision energies of 10, 20, and 30 Å. Data analysis was performed using Thermo Q Exactive Browser software.

[0145] - BTBR-Pah enu2 / J:

[0146] This study involved male and female BTBR-Pah, the original species, at Jackson Laboratory in the United States. enu2 The study was conducted in J mice (8 weeks old).

[0147] o Food and drinking water: Mice were fed a phenylalanine-free diet (5LF2, LabDiet) with free access to food and drinking water.

[0148] o Duodenal catheter insertion

[0149] Anesthetize the animal with isoflurane (2-4%). ​​Make an incision along the linea alba and insert a catheter (C19PB-MGI1923, Instech Laboratories) into the duodenum via the contralateral mesentery. Advance the catheter tip close to the opening of the bile and pancreatic ducts. Ligate the catheter to the intestinal wall and tunnel it through a subcutaneous tunnel to the animal's neck, exposing and closing it. Then suture the abdominal wall and neck incisions. Throughout the procedure, keep the animal on a warm bed and monitor closely until fully recovered from anesthesia.

[0150] o Evaluating the potency of NP-1 in mice

[0151] Prior to the study, mice were kept on a phenylalanine-free diet for at least 3 days, then supplemented with a low concentration (0.03 g / L) of L-Phe in their drinking water for 3 days, after which the concentration of L-Phe in the drinking water was increased to 0.5 g / L. Mice were allowed free access to the L-Phe-supplemented drinking water at night.

[0152] Mice were administered NP-1 (0.581 U, 7 mg), NP-2 (7 mg), or engineered PAL (0.581 U) twice daily via the duodenum over a 12-day period. Blood samples were collected in 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 20 μL of plasma was transferred to Eppendorf tubes and stored at –80°C until Phe content was analyzed.

[0153] o Measurement of mouse plasma Phe: Quantification of target analytes using an LC system: Thermo Vanquish Horizon binary pump and Thermo TSQ Quantiva mass spectrometer.

[0154] Plasma samples were prepared as follows: 20 μL of plasma sample was centrifuged at 13.2 krpm for 10 min at 4 °C. 10 μL 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 for 10 min at 4 °C. 50 μL of the supernatant was then dried at 30 °C under a gentle nitrogen stream. 500 μL of 0.1% (v / v) formic acid aqueous solution was added, and the sample was shaken at 900 rpm for 10 min at 15 °C (or 13.2 krpm for 10 min at 4 °C), followed by centrifugation. Finally, 350 μL of the supernatant was transferred to a total recovery vial for analysis.

[0155] The injection volume was 2 μL, the run time was 5 min, and the flow rate was 1 mL / min. Mobile phase A consisted of H₂O and formic acid (0.1%); mobile phase B consisted of methanol and formic acid (0.1%). Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) with a gradient of 100% A to 100% B.

[0156] MS was performed using a Thermo TSQ Quantiva mass spectrometer, in selective reaction monitoring mode.

[0157] MS parameters were as follows: Q§1 resolution: 0.7; Q3 resolution: 0.7; fragmentation: CID fragmentation using argon (1.5 mTorr). Analyte concentrations were calculated from the peak area ratio of Phe to the internal standard d5-Phe. Data analysis was performed using Thermo Quan Browser software.

[0158] result: Example 1: Enzyme stability and loading can be enhanced by covalently linking to the protective layer. In the first experiment, nanoparticles NP-1(1) were generated under unbuffered conditions, with washing (i.e., removal of glutaraldehyde before layer growth) included after each chemical step. In the second experiment, nanoparticles NP-1(2) were generated under buffered conditions, with washing (i.e., removal of glutaraldehyde before layer growth) included after each chemical step. In the third experiment, nanoparticles NP-1 were prepared under unbuffered conditions without any intermediate washing steps (i.e., unreacted glutaraldehyde remained in the reaction mixture during layer growth).

[0159] Protein quantification was performed on the reaction supernatant to determine the PAL immobilization yield on the surfaces of NP-1(1), NP-1(2), and NP-1. Surprisingly, enzyme immobilization under conditions maintaining glutaraldehyde (NP-1) increased the enzyme immobilization yield by two-fold. Figure 2 A), resulting in a 2-fold increase in SNP enzyme loading per unit dry weight compared to buffer conditions (NP-1(2)) where glutaraldehyde is removed via a washing step. Figure 2 B). Similarly, enzyme immobilization under conditions maintaining glutaraldehyde (NP-1) resulted in an enzyme loading per unit dry weight of SNP ( Figure 2 B) is 1.5 times higher than the unbuffered conditions (NP-1(1)) in which glutaraldehyde is removed by a washing step.

[0160] The biocatalytic activity of PAL immobilized and protected on NP-1(1), NP-1(2), and NP-1 was evaluated. Even more surprisingly than the increase in enzyme immobilization load while maintaining glutaraldehyde, the specific activity of the nanoparticles increased threefold compared to buffered conditions where glutaraldehyde was removed via a washing step, and twofold compared to unbuffered conditions where glutaraldehyde was removed via a washing step. Figure 2 C). This astonishing three-fold increase in the specific activity of the nanoparticles is closely correlated with the specific activity (units / g PAL) of the enzyme immobilized under unbuffered conditions maintaining glutaraldehyde, which is comparable to that of the enzyme immobilized under buffered conditions. This result is completely unexpected, as it was presumed that the enzyme would have much higher activity in the presence of buffer ( Figure 2 D). In summary, compared with enzymes protected by an organosilicon layer solely through electrostatic interactions, the covalent connection between the protective layer and the enzyme surface unexpectedly enhanced their loading capacity and stability.

[0161] Example 2: NP-1 phenylalanine aminolyase (PAL) activity

[0162] The biocatalytic activity of immobilized and protected engineered PALs was evaluated. Figure 3 The results shown reported PAL activity on NP-1. This indicates that, despite protection and functionalization, the protected functionalized SNP still possesses the ability to approach and transform L-Phe. Validation of the biocatalytic activity of NP-1 confirms the potential for using nanoparticles for therapeutic purposes.

[0163] Example 3: Resistance to external stress

[0164] NP-1 is a nanoparticle developed for gastrointestinal applications. Due to the physiological characteristics of the gastrointestinal tract, NP-1 will be subjected to various stresses. To ensure the sustained activity of NP-1 in the gastrointestinal tract, the protection of immobilized PAL was evaluated. First, NP-1 or engineered PAL was subjected to acidic conditions (pH 4). Monitoring of PAL activity over 24 h revealed sustained enzymatic activity against NP-1. Figure 4 B), while the free-form PAL of engineering modifications loses its activity over time. Figure 4 A). These data demonstrate the protection of engineered PALs immobilized and protected on NP-1 in an acidic environment.

[0165] During digestion, pancreatic enzymes are released, particularly proteases that may affect therapeutic enzymes in the gastrointestinal tract. Therefore, NP-1 and engineered PAL were exposed to various proteases at 37°C. First, to simulate physiological digestive conditions, NP-1 or engineered PAL was incubated with pancreatic enzymes (30 mU) (a mixture of pancreatic enzymes extracted from porcine pancreas), and then their PAL activity was evaluated. After 4 h, both NP-1 and engineered PAL exhibited sustained PAL activity (…). Figure 4 C). Then, to further evaluate the benefits of the protective shield under harsh conditions, NP-1 or engineered PAL was exposed to streptomycin (0.88 U), a mixture of purified proteases. Figure 4 The results shown by C indicate that 80% of the PAL retained its activity against NP-1 after 4 hours of exposure, while the engineered PAL lost its enzymatic activity after 4 hours. In summary, these data demonstrate the added value of engineered PAL for immobilization and protection on nanoparticles and highlight the potential use of NP-1 in gastrointestinal therapeutic applications.

[0166] Example 4: In vitro biocompatibility and potency of NP-1

[0167] Maintaining the integrity of the intestinal barrier is crucial for preventing unwanted lumen contents, such as pathogens or food allergens, from entering the body. To evaluate the biocompatibility of the nanoparticles, Caco2-HT29-MTX-E12 cell monolayers were exposed to NP-1 in the presence or absence of pancreatic enzymes to simulate digestive conditions, and transepithelial electrical resistance (TEER) was measured. Figure 5A The data presented showed that, with or without pancreatic enzymes, the integrity of the intestinal epithelial barrier was maintained after 6 h of exposure to NP-1. These results demonstrate the in vitro biocompatibility of NP-1 in gastrointestinal applications.

[0168] NP-1 has been developed for metabolizing Phe in the intestinal lumen. To evaluate the in vitro potency of NP-1, a monolayer of Caco2-HT29-MTX-E12 cells cultured in cell culture medium containing 0.4 mM L-phenylalanine was exposed at its apical side to NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 h in the presence or absence of trypsin (30 mU). Figure 5B The figure shows the quantification of Phe metabolites on the basal side of the barrier. The graph reports the accumulation of TCA on the basal side of the barrier under all conditions. These results demonstrate the in vitro potency of NP-1 in the digestive environment and suggest its potential use in therapeutic applications.

[0169] Example 5: NP-1 activity in rats

[0170] To evaluate the transition from in vitro to in vivo application, the potency of NP-1 was assessed in rats. Rats were administered NP-1 (nanoparticles containing engineered PAL) or NP-2 (nanoparticles containing bovine serum albumin (BSA), where Phe was not a substrate) via the duodenum, followed by gavage administration of d5-Phe. In vivo, the Phe metabolite TCA is rapidly metabolized to hippuric acid. The in vivo activity of NP-1 was then evaluated by measuring d5-hippuric acid in rat urine collected 24 h after administration. Figure 6 The results showed that rats receiving NP-1 had significantly increased levels of d5-hippuric acid compared to rats receiving NP-2, indicating that NP-1 can digest Phe in the intestinal lumen and suggesting that NP-1 can be used for therapeutic applications.

[0171] Example 6: Therapeutic efficacy of NP-1 in mice

[0172] Phenylketonuria (PKU) is characterized by a deficiency of the intracellular liver enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine. PAH deficiency leads to abnormally high phenylalanine concentrations, which are toxic to the brain. The basis of PKU treatment is a combination of a low-phenylalanine diet and a phenylalanine-free L-amino acid regimen. Currently, Enzyme replacement therapy using recombinant phenylalanine aminolysin can be administered subcutaneously, but this treatment can cause allergic reactions and immune-mediated acute hypersensitivity reactions.

[0173] NP-1 has been developed to exhibit sustained PAL activity in the gastrointestinal environment (acidic and exposed to proteases). Our approach to controlling Phe levels in patients involves degrading Phe (from dietary intake) in the intestine to prevent its absorption and accumulation in the blood.

[0174] Using a representative animal model of the disease—BTBR- Pah enu2 / J mice—the mice in which the gene encoding PAH was mutated—were used to evaluate this treatment strategy.

[0175] During the 12-day period, Mice given drinking water supplemented with L-phenylalanine were injected twice daily with NP-1, NP-2, or genetically engineered PAL. Impressively, during the study period, plasma Phe levels in mice given NP-1 showed a stable decrease, while plasma concentrations in mice given modified PAL were unstable (Figure 7). Figure 7B As shown, the normalization of Phe plasma concentration highlights that at the end of NP-1 treatment, Phe plasma concentration in mice decreased by 30%.

[0176] The interaction between nanoparticles and intestinal mucus leads to the temporary attachment of NP-1 to the intestinal wall, enabling PAL to remain active on the intestinal wall, while the free form of engineered PAL is flushed downstream. These results validate and reinforce the strategy of degrading phenylalanine in the intestine and support the significant therapeutic effect of NP-1 on PKU.

Claims

1. A composition comprising a solid support, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof immobilized on the surface of the solid support, a protective layer protecting the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof by embedding the engineered 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

2. The composition according to claim 1, wherein the polymer comprising repeating units is a polyglucosamine selected from chitin, chitosan, a polyglucosamine glycan, a chondroitin, a heparin, a keratin, and a dermal, or a derivative thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

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

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 binding or by covalent binding.

5. The composition according to any one of claims 1 to 4, wherein the protective layer embeds the solid support and embeds the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof immobilized on the surface of the solid support.

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. The composition according to any one of claims 1 to 6, wherein the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof (a) comprises or consists of an amino acid sequence having at least 95%, at least 96%, or at least 97% sequence identity to the sequence of SEQ ID NO: 1; or (b) is SEQ ID NO: 2, 3, 4, or 5.

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

5.

9. The composition according to any one of claims 1 to 8 for use as a medicament.

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

11. A method of producing a composition comprising a solid support, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof immobilized on the surface of the solid support, a protective layer formed by embedding the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof to protect the engineered 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the steps of: (a) providing a solid support; (b) immobilizing an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof on the solid support; (c) forming a protective layer on the surface of the solid support to protect the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof immobilized on the solid support; (d) immobilizing a functional component 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

12. The method of claim 11, wherein in step (b) i) a linker is added to the solid support provided in step (a), and ii) the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof is added to the solid support and the linker, wherein the linker links the solid support to the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof.

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

14. The method of claim 12, wherein there is no washing step between i) the addition of the linker to the solid support provided in step (a) and ii) the addition of the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof to the solid support and the linker.

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

16. The method of any one of claims 12-15, wherein the linker or a portion thereof that does not link the solid support to the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof in step (b) covalently binds the protective layer to the phenylalanine ammonia-lyase (PAL) or a fragment thereof in step (c).

17. The method according to any one of claims 12-16, wherein the linker is selected from glutaraldehyde, dithiosuccinimidyl tartrate, bis[sulfosuccinimidyl]suberate, ethyleneglycol bis(sulfosuccinimidylsuccinate), dimethyl adipimidate, heptanediamide, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimidooxy carbonyloxyethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'- dithiobis[sulfosuccinimidyl]propionate), DTBP (3,3'-dithiobispropionimidate.2 HC1), DST (dithiosuccinimidyl tartrate), BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane).

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

19. A composition comprising a solid support, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof immobilized on the surface of the solid support, a protective layer protecting the engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof by embedding the engineered 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition is obtainable by the method of any one of claims 13-18.

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