Recombinant neprosin polypeptides and methods of expression

Patent metadata

Field	Value
Publication number	US-2024026331-A1
Application number	US-202318315466-A
Country	US
Kind code	A1
Filing date	May 10, 2023
Priority date	May 10, 2022
Publication date	Jan 25, 2024
Grant date	—

Abstract

The present disclosure relates to methods of expressing recombinant neprosin polypeptide, host cells and expression vectors for expressing the recombinant neprosin polypeptide, and methods of using the expressed neprosin polypeptide for therapeutic and other purposes.

First claim

1 . A recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the pre-pro-polypeptide, pro-polypeptide, mature polypeptide, or polypeptide of SEQ ID NO: 1, wherein the amino acid sequence comprises at least at amino acid position 68, 145, 152, or 253, or combinations thereof, and equivalent positions thereof, a substitution with an amino acid other than asparagine (N), wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 1.

2 . The recombinant neprosin polypeptide of claim 1 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises at least a substitution at least at amino acid position 68 with an amino acid other than asparagine (N).

3 . The recombinant neprosin polypeptide of claim 1 wherein the amino acid sequence of the recombinant neprosin polypeptide comprises at least a substitution at least at amino acid position 152 with an amino acid other than asparagine (N).

4 . The recombinant neprosin polypeptide of claim 1 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises at least a substitution at least at amino acid position 253 with an amino acid other than asparagine (N).

5 . The recombinant neprosin polypeptide of claim 1 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises at least a substitution at least at amino acid position 145 with an amino acid other than asparagine (N).

6 . The recombinant neprosin polypeptide of claim 1 , wherein the signal sequence of the pre-pro-polypeptide is replaced with a signal sequence functional in a mammalian cell, insect cell, or fungal cell, or wherein the pro-polypeptide further comprises a signal sequence functional in a mammalian cell, insect cell, or fungal cell.

7 . A recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence comprises at least at amino acid position 44, 121, 128, or 229, or combinations thereof, a substitution with an amino acid other than asparagine (N), wherein the amino acid positions are relative to SEQ ID NO: 8.

8 . (canceled)

9 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises SEQ ID NO: 10.

10 . (canceled)

11 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 12, or comprises SEQ ID NO: 12.

12 . (canceled)

13 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 14, or comprises SEQ ID NO: 14.

14 . (canceled)

15 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 16, or comprises SEQ ID NO: 16.

16 . (canceled)

17 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 18, or comprises SEQ ID NO: 18.

18 . (canceled)

19 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 20, or comprises SEQ ID NO: 20.

20 . (canceled)

21 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 22, or comprises SEQ ID NO: 22.

22 . (canceled)

23 . The recombinant neprosin polypeptide of claim 7 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105 to 356 of SEQ ID NO: 32, or comprises SEQ ID NO: 32.

24 - 27 . (canceled)

28 . A recombinant neprosin polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to residues 105 to 356 of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8, wherein the amino acid sequence further comprises a signal sequence functional in a mammalian cell, insect cell, or fungal cell.

29 . The recombinant polynucleotide of claim 28 , wherein the amino acid sequence of the recombinant neprosin polypeptide comprises residues 105-356 of SEQ ID NO: 8, or comprises SEQ ID NO: 8.

30 . A recombinant polynucleotide comprising a polynucleotide sequence encoding a recombinant neprosin polypeptide of claim 1 .

31 - 33 . (canceled)

34 . An expression vector comprising a recombinant polynucleotide of claim 30 .

35 . (canceled)

36 . (canceled)

37 . A host cell comprising an expression vector of claim 34 , wherein the host cell is a eukaryotic cell.

38 - 42 . (canceled)

43 . A method of expressing recombinant neprosin, comprising culturing a host cell of claim 37 under suitable culture conditions such that the recombinant neprosin polypeptide is expressed.

44 - 66 . (canceled)

67 . A method of preparing mature neprosin polypeptide, comprising treating in vitro a neprosin pre-pro-polypeptide or pro-polypeptide of a recombinant neprosin polypeptide of claim 1 , to a pH of about 2 to about 6 under suitable conditions for formation of the mature neprosin polypeptide.

68 - 70 . (canceled)

71 . A pharmaceutical composition comprising a pro-polypeptide or mature neprosin polypeptide of a recombinant neprosin polypeptide of claim 1 .

72 . (canceled)

73 . A method of treating a disease or condition associated with gluten intolerance, comprising administering to a subject in need thereof an effective amount of a pro-polypeptide or proteolytically active mature neprosin polypeptide of a recombinant neprosin polypeptide of claim 1 .

74 - 78 . (canceled)

Assignees

• Codexis Inc

Inventors

• Celis Luna Arianna I
• Duan Da
• Jordan Paul Campion
• Molnar Kathleen Susan
• Silverman Adam P
• Windgassen Tricia

Classifications

• C12N9/6408

  Serine endopeptidases (3.4.21) · 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

• C12N9/63 Primary

  derived from plants · CPC title

• C12Y304/21026

  Prolyl oligopeptidase (3.4.21.26), i.e. proline-specific endopeptidase · CPC title

• A61P1/14

  Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents · CPC title