Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-11136571-B2
Application number	US-201916601228-A
Country	US
Kind code	B2
Filing date	Oct 14, 2019
Priority date	Apr 18, 2013
Publication date	Oct 5, 2021
Grant date	Oct 5, 2021

Abstract

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.

First claim

What is claimed is:

1. An engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to reference sequence SEQ ID NO:10, wherein said amino acid sequence comprises an amino acid residue selected from leucine, methionine, and glutamine at position 305, and wherein said engineered polypeptide further comprises at least one or more of the following substitution sets selected from 32/54/73/305/503/521/565, 54/59/73/305/503/521/565, and 54/59/305/503/521/565, wherein said positions in said amino acid sequence are in reference to SEQ ID NO:10.

2. The engineered polypeptide of claim 1 , wherein said engineered polypeptide exhibits an improved property selected from reduced sensitivity to proteolysis, increased tolerance to acidic pH, or a combination thereof, as compared to the reference sequence SEQ ID NO:10.

3. The engineered polypeptide of claim 2 , wherein the improved property is selected from reduced sensitivity to proteolysis or increased tolerance to acidic pH.

4. The engineered polypeptide of claim 1 , wherein said engineered polypeptide is resistant to proteolysis and is acid stable.

5. The engineered polypeptide of claim 4 , wherein said engineered polypeptide is resistant to proteolysis by at least one digestive tract enzyme, wherein said engineered polypeptide is resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases.

6. The engineered polypeptide of claim 1 , wherein said polypeptide is purified.

7. A polynucleotide sequence encoding at least one engineered polypeptide of claim 1 .

8. The polynucleotide sequence of claim 7 , wherein said polynucleotide sequence is operably linked to a control sequence.

9. The polynucleotide sequence of claim 7 , wherein said polynucleotide sequence is codon-optimized.

10. An expression vector comprising at least one polynucleotide sequence of claim 7 , and at least one control sequence.

11. An expression vector comprising at least one polynucleotide sequence of claim 9 , and at least one control sequence.

12. The expression vector of claim 10 , wherein said control sequence is a promoter.

13. The expression vector of claim 11 , wherein said control sequence is a promoter.

14. A host cell transformed with the expression vector of claim 10 .

15. A host cell transformed with the expression vector of claim 11 .

16. A method of producing an engineered polypeptide in a host cell comprising culturing the host cell of claim 14 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

17. A method of producing an engineered polypeptide in a host cell comprising culturing the host cell of claim 15 , under suitable culture conditions, such that at least one engineered polypeptide is produced.

18. The method of claim 16 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

19. The method of claim 17 , further comprising recovering at least one engineered polypeptide from the culture and/or host cells.

20. The method of claim 18 , further comprising the step of purifying said at least one engineered polypeptide.

21. The method of claim 19 , further comprising the step of purifying said at least one engineered polypeptide.

22. A composition comprising at least one engineered polypeptide of claim claim 1 .

23. The composition of claim 22 , wherein said composition is a pharmaceutical composition.

24. The pharmaceutical composition of claim 23 , wherein said composition is suitable for the treatment of phenylketonuria.

25. The pharmaceutical composition of claim 23 , wherein said composition is suitable for oral administration to a human.

26. The pharmaceutical composition of claim 25 , wherein said composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion.

27. The pharmaceutical composition of claim 26 , wherein said pill, tablet, capsule, or gelcap further comprises an enteric coating.

28. The pharmaceutical composition of claim 23 , wherein said composition is suitable for parenteral injection into a human.

29. The pharmaceutical composition of claim 23 , wherein said composition is coadministered with at least one additional therapeutically effective compound.

30. A method for treating and/or preventing the symptoms of phenylketonuria in a subject, comprising providing a subject having phenylketonuria and the pharmaceutical composition of claim 24 , and providing said pharmaceutical composition to said subject.

31. The method of claim 30 , wherein said symptoms of phenylketonuria are ameliorated.

32. The method of claim 30 , wherein said subject is able to eat a diet that is less restricted in its methionine, phenylalanine and/or tyrosine content than diets required by subjects who have not been provided at least one pharmaceutical composition comprising at least one engineered polypeptide having phenylalanine ammonia lyase activity.

33. The method of claim 32 , wherein said subject is an infant, child, young adult or adult human.

Assignees

• Codexis Inc

Inventors

• Huisman Gjalt W
• Agard Nicholas J
• Mijts Benjamin
• Vroom Jonathan
• Zhang Xiyun

Classifications

• C12N9/88 Primary

  Lyases (4.) · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · CPC title