Engineered phenylalanine ammonia lyase polypeptides

Patent metadata

Field	Value
Publication number	US-2017191050-A1
Application number	US-201715431491-A
Country	US
Kind code	A1
Filing date	Feb 13, 2017
Priority date	Apr 18, 2013
Publication date	Jul 6, 2017
Grant date	—

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 with at least 90% sequence identity to reference sequence SEQ ID NO:24.

2 . The engineered polypeptide of claim 1 , wherein said engineered polypeptide comprises at least one substitution at a position in SEQ ID NO:24, and wherein said positions are numbered with reference to SEQ ID NO:24.

3 . The engineered polypeptide of claim 1 , wherein said engineered polypeptide exhibits at least one improved property selected from enhanced catalytic activity, reduced sensitivity to proteolysis, increased tolerance to acidic pH, as compared to the reference sequence SEQ ID NO:24.

4 . The engineered polypeptide of claim 1 wherein the engineered polynucleotide comprises at least one amino acid residue differences as compared to SEQ ID NO:24, wherein the amino acid residue differences are at one or more amino acid positions selected from 20, 24, 27, 39, 43, 45, 47, 54, 58, 59, 62, 70, 73, 80, 82, 91, 94, 98, 104, 105, 110, 112, 115, 117, 118, 119, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 153, 154, 156, 157, 158, 159, 172, 174, 175, 176, 177, 178, 180, 187, 191, 195, 199, 205, 206, 210, 212, 213, 214, 232, 240, 243, 245, 247, 248, 250, 256, 257, 266, 270, 275, 278, 279, 285, 286, 289, 290, 292, 304, 305, 307, 308, 309, 319, 321, 326, 331, 332, 334, 349, 353, 355, 364, 365, 369, 370, 371, 372, 374, 375, 377, 378, 379, 381, 382, 383, 384, 385, 387, 389, 394, 396, 399, 400, 403, 404, 407, 417, 418, 425, 431, 432, 433, 434, 435, 436, 437, 438, 439, 443, 446, 447, 453, 456, 459, 460, 461, 463, 471, 472, 473, 474, 475, 476, 477, 478, 479, 482, 483, 503, 507, 509, 521, 522, 524, 525, 528, 538, 546, 547, 551, 558, 560, 564, 565, and/or any combinations thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO:24.

5 . The engineered polypeptide of claim 4 , wherein the amino acid residue difference as compared to SEQ ID NO:24 is selected from one or more of the following substitutions 112C, 134Q, 158H, 180A, 195E, 240R/W, 243I/L, 245L, 256G, 257W/A, 270K, 304H, 307Q/M, 308Q, 326F, 349M, 353A/N, 364Q, 394V, 399N, 400K, 404A, 443H, 453G, 459F, 460G, 463N, 474Q, 509L, 521S, 522Y/F/N, 528L, 546R, and 564 G/L/M, when optimally aligned with the polypeptide of SEQ ID NO:24.

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

7 . The engineered polypeptide of claim 6 , wherein the 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.

8 . The engineered polypeptide of claim 1 , wherein said engineered polypeptide is deimmunized.

9 . The engineered polypeptide of claim 1 , wherein said engineered polypeptide has phenylalanine ammonia lyase activity.

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

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

12 . The polynucleotide sequence of claim 11 , wherein said polynucleotide sequence is operably linked to a control sequence.

13 . The polynucleotide sequence of claim 11 , wherein said polynucleotide sequence is codon-optimized.

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

15 . The expression vector of claim 14 , wherein said control sequence is a promoter.

16 . A host cell transformed with at least expression vector of claim 15 .

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

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

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

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

21 . The composition of claim 20 , wherein said composition is a pharmaceutical composition.

22 . The composition of claim 21 , wherein said composition is suitable for the treatment of phenylketonuria.

23 . The pharmaceutical composition of claim 21 , wherein said composition is suitable for oral administration to a human.

24 . The pharmaceutical composition of claim 23 , wherein said composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion.

25 . The pharmaceutical composition of claim 24 , wherein said pill, tablet, capsule, or gelcap further comprises an enteric coating.

26 . The pharmaceutical composition of claim 21 , wherein said composition is suitable for parenteral injection into a human.

27 . The pharmaceutical composition of claim 21 , wherein said composition is coadministered with at least one additional therapeutically effective compound.

28 . 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 21 , and providing said pharmaceutical composition to said subject.

29 . The method of claim 28 , wherein said symptoms of phenylketonuria are ameliorated.

30 . The method of claim 29 , 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 (PAL) activity.

31 . The method of claim 30 , 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

• A61P43/00

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

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title

• C12Y403/01024

  Phenylalanine ammonia-lyase (4.3.1.24) · CPC title

• C12N9/88 Primary

  Lyases (4.) · CPC title