Multiple Protease Deficient Filamentous Fungal Cells and Methods of Use Thereof

Patent metadata

Field	Value
Publication number	US-2019309338-A1
Application number	US-201916297851-A
Country	US
Kind code	A1
Filing date	Mar 11, 2019
Priority date	Jul 10, 2013
Publication date	Oct 10, 2019
Grant date	—

Abstract

The present disclosure relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells.

First claim

1 - 15 . (canceled)

16 . A Myceliophthora thermophila filamentous fungal cell comprising at least one endogenous sep protease having no protease activity and a recombinant polynucleotide encoding a mammalian polypeptide, wherein the gene encoding endogenous sep protease of SEQ ID NO: 792, 794 or 797 comprises a mutation that eliminates the activity of the endogenous sep protease.

17 . The Myceliophthora thermophila cell of claim 34 comprising at least three, four, five, six, seven or eight endogenous proteases having eliminated activity.

18 . The Myceliophthora thermophila cell of claim 35 , wherein the proteases are selected from the group consisting of aspartic proteases, trypsin-like serine proteases, subtilisin proteases, glutamic proteases, and metalloproteases.

19 . The Myceliophthora thermophila cell of claim 36 , wherein the Myceliophthora thermophila cell further has no detectable protease activity of pep4 of SEQ ID NO: 499, slp2 of SEQ ID NO: 540, and slp3 of SEQ ID NO: 546.

20 . The Myceliophthora thermophila cell of claim 34 , wherein the mammalian polypeptide is selected from the group consisting of an antibody, a growth factor, an interferon, a cytokine, and an interleukin.

21 . The Myceliophthora thermophila cell of claim 34 , wherein the Myceliophthora thermophila cell further comprises ALG3 having eliminated activity.

22 . The Myceliophthora thermophila cell of claim 34 , further comprising
a) an N-acetylglucosaminyltransferase I catalytic domain, and, optionally, b) an N-acetylglucosaminyltransferase II catalytic domain.

23 . A method of improving stability of a mammalian polypeptide, comprising
a) providing the Myceliophthora thermophila cell of claim 34 , and b) culturing the Myceliophthora thermophila cell such that the mammalian polypeptide is expressed,
wherein the mammalian polypeptide exhibits increased stability compared to the mammalian polypeptide produced in a corresponding parental Myceliophthora thermophila cell in which the proteases do not have reduced activity.

24 . A method of making a mammalian polypeptide, comprising
a) providing the Myceliophthora thermophila cell of claim 34 ; b) culturing the Myceliophthora thermophila cell such that the mammalian polypeptide is expressed; and, c) purifying the mammalian polypeptide.

Assignees

• Glykos Finland Oy
• Novartis Ag

Inventors

• Landowski Christopher
• Huuskonen Anne
• Westerholm-Parvinen Ann
• Saloheimo Markku
• Kanerva Anne
• Hiltunen Jukka

Classifications

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• C12Y304/00

  Hydrolases acting on peptide bonds, i.e. peptidases (3.4) · CPC title

• C12N15/80

  for fungi · CPC title

• C12Y304/21

  Serine endopeptidases (3.4.21) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title