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EP-4739349-A1 - FUNCTIONALIZED COMPOSITIONS COMPRISING AN ENGINEERED PHENYLALANINE AMMONIA LYASE (PAL)

EP4739349A1EP 4739349 A1EP4739349 A1EP 4739349A1EP-4739349-A1

Abstract

The present invention relates to a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase or a fragment thereof by embedding the engineered phenylalanine ammonia lyase or a fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group. The present invention also relates to methods of producing said composition and uses thereof.

Inventors

  • LAPRÉVOTTE, Emilie
  • BRIAND, Manon
  • DUDAL, Yves Victor René
  • SPECIOSO, Gabriele
  • SHAHGALDIAN, PATRICK
  • WATSON, John Ashleigh

Assignees

  • Perseo Pharma AG
  • Société des Produits Nestlé S.A.

Dates

Publication Date
20260513
Application Date
20240704

Claims (19)

  1. 1. A composition comprising a solid carrier, an engineered phenylalanine ammonia lyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof by embedding the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group.
  2. 2. The composition according to claim 1, wherein the polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group is a polyglucosamin selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or a derivative thereof.
  3. 3. The composition according to claim 1, wherein the polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group is chitosan or a derivative thereof.
  4. 4. The composition according to any one of claims 1-3, wherein the functional constituent is immobilized on the surface of the protective layer by non-covalent binding or by covalent binding.
  5. 5. The composition of any one of claims 1-4, wherein the protective layer embeds the solid carrier and embeds the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier.
  6. 6. The composition of any one of claims 1-5, wherein the functional constituent immobilized on the surface of the protective layer is not embedded by the protective layer.
  7. 7. The composition according to any one of claims 1-6, wherein the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof (a) comprises or consist 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. 8. The composition according to anyone of claims 1-6, wherein the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof comprises the polypeptide as shown in SEQ ID NO: 5.
  9. 9. The composition of any one of claims 1-8, for use as a medicament.
  10. 10. The composition of any one of claims 1-8, for use in a method for the prevention, delay of progression or treatment of phenylketonuria (PKU).
  11. I L A method of producing a composition, the composition comprising a solid carrier, an engineered phenylalanine ammonia lyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof by embedding the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units, wherein each repeat 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; (b) immobilizing an engineered phenylalanine ammonia lyase (PAL) or a fragment thereof on the solid carrier; (c) forming a protective layer on the surface of the solid carrier to protect the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof immobilized on the solid carrier; (d) immobilizing a functional constituent on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units, wherein each repeat unit comprises at least one amino group and/or at least one thiol group.
  12. 12. The method of claim 11, wherein in step (b), i) a linker is added to the solid carrier provided in step (a), and ii) the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof is added to the solid carrier and the linker, wherein the linker connects the solid carrier with the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof.
  13. 13. The method of claim 12, wherein the linker which has not connected the solid carrier with the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof in step (b), is present during formation of a protective layer on the surface of the solid carrier in step (c).
  14. 14. The method of claim 12, wherein there is no washing step between adding the linker to the solid carrier provided in step (a) in (i) and adding the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof to the solid carrier and the linker in ii).
  15. 15. The method of any one of claims 12-14, wherein there is no washing step between any of steps (a) to (c).
  16. 16. The method of any one of claims 12-15, wherein the linker which has not connected the solid carrier with the engineered phenylalanine ammonia lyase (PAL) or a fragment thereof in step (b), or a part therof, covalently binds the protective layer to the phenylalanine ammonia lyase (PAL) or the fragment thereof in step (c).
  17. 17. The method of any one of claims 12-16, wherein the linker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, l,5-difluoro-2,4-dinitrobenzene, BSOCOES (Bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone), DSP (Dithiobis[succinimidyl]propionate]), DTSSP (3,3 '- Dithiobis[sulfosuccinimidyl]propionate]), DTBP (Dimethyl 3,3 dithiobispropionimidate-2 HC1), DST (Disuccinimidyl tartarate), BMDB (1,4 bismaleimidyl-2,3-dihydroxybutane).
  18. 18. The method of any one of claims 12-16, wherein the linker is glutaraldehyde.
  19. 19. A composition comprising a solid carrier, an engineered phenylalanine ammonia lyase (PAL) or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof by embedding the engineered phenylalanine ammonia lyase (PAL) or the fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units, wherein each repeat 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.

Description

Functionalized compositions comprising an engineered phenylalanine ammonia lyase (PAL) The field of the invention The present invention relates to a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase or a fragment thereof by embedding the engineered phenylalanine ammonia lyase or a fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group. The present invention also relates to methods of producing said composition and uses thereof. Background of the invention Phenylketonuria (PKU) is an inherited, autosomal recessive disease characterized by a deficiency in the intracellular liver enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine, and this enzymatic activity is facilitated by tetrahydrobiopterin (BH4). PAH deficiency results in an abnormally elevated concentrations of phenylalanine, which is toxic to the brain. High phenylalanine levels during infancy and early childhood cause profound cognitive and developmental defects, and poorly controlled blood phenylalanine levels in older children and adolescents are associated with learning disabilities, attention deficit hyperactivity disorder, and behavioral. Uncontrolled blood phenylalanine levels in adulthood are associated with executive dysfunction, and a variety of behavioral and psychiatric problems. The cornerstone of PKU treatment is a low phenylalanine diet in combination with phenylalanine-free L-amino acid. The deficiency of PAH in patients with blood phenylalanine levels greater than 600 micromol/L can be corrected by an enzyme substitution therapy with recombinant phenylalanine ammonia lyase (PAL) (Palynziq®), administered daily by up to 3 subcutaneous injections). 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). Thus, there is a need to provide compatible and effective treatments for phenylketonuria. Summary of the invention The present invention provides a composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase or a fragment thereof by embedding the engineered phenylalanine ammonia lyase or a fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group. The present invention also provides a method of producing said composition comprising a solid carrier, an engineered phenylalanine ammonia lyase or a fragment thereof immobilized on the surface of the solid carrier, a protective layer to protect the engineered phenylalanine ammonia lyase or a fragment thereof by embedding the engineered phenylalanine ammonia lyase or a fragment thereof, and a functional constituent immobilized on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat 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; (b) immobilizing an engineered phenylalanine ammonia lyase or a fragment thereof on the solid carrier; (c) forming a protective layer on the surface of the solid carrier to protect the engineered phenylalanine ammonia lyase or a fragment thereof immobilized on the solid carrier; (d) immobilizing a functional constituent on the surface of the protective layer, wherein the functional constituent immobilized on the surface of the protective layer is a polymer comprising repeat units wherein each repeat unit comprises at least one amino group and/or at least one thiol group. It has been surprisingly found by the inventors of the present application that compositions as provided by the present invention if applied therapeutically have an unexpected high in vivo enzymatic activity, show low cytotoxicity, and do not disrupt the intestinal barrier if localized in the gastrointestinal tract, thus making them extremely promising for therapeutic use, in particular for prevention, delay of progression or treatment of phenylketonuria. Brief description of the figures Fig