Production of proteins in labyrinthulomycetes

Patent metadata

Field	Value
Publication number	US-10584347-B2
Application number	US-201715460038-A
Country	US
Kind code	B2
Filing date	Mar 15, 2017
Priority date	Mar 16, 2016
Publication date	Mar 10, 2020
Grant date	Mar 10, 2020

Abstract

The present invention provides recombinant host cells that produce proteins or therapeutic proteins, and nucleic acid constructs for producing the cells. The cells have nucleic acid constructs that encode a heterologous protein, for example an antibody. The nucleic acid constructs also can have a functional signal sequence that directs the secretion of the protein from the cell. The signal sequence can be any functional signal sequence, and various signal sequences are disclosed herein. The invention also provides methods of producing the proteins.

First claim

The invention claimed is:

1. A recombinant Labyrinthulomycetes cell comprising an expression cassette that encodes a heterologous protein and a functional signal sequence that directs the secretion of the heterologous protein from the cell, wherein
a. the signal sequence has at least 90% sequence identity to a sequence of at least 10 contiguous amino acids in the amino acid sequence selected from any one of SEQ ID NOs: 19, 72, 75, and 76;
b. the signal sequence has at least 95% sequence identity to a sequence of at least 10 contiguous amino acids in the amino acid sequence of SEQ ID NO: 20, or the signal sequence has at least 90% sequence identity to a sequence of at least 15 contiguous amino acids in the amino acid sequence of SEQ ID NOs: 20, or the signal sequence has at least 90% sequence identity to a sequence of at least 20 contiguous amino acids in the amino acid sequence of SEQ ID NO: 20; or
c. the signal sequence is encoded by a nucleic acid sequence having at least 90% sequence homology to any one of SEQ ID NOs: 31, 32, 88, 91, and 92.

2. The cell of claim 1 wherein the heterologous protein is a therapeutic protein.

3. The cell of claim 1 wherein the heterologous protein is a functional antibody.

4. The cell of claim 3 wherein the functional antibody comprises at least one heavy chain and at least one light chain.

5. The cell of claim 3 wherein the antibody is selected from the group consisting of: eculizumab, natalizumab, cetuximab, omalizumab, usteinumab, panitumumab, denosumab, trastuzumab and adalimumab.

6. The cell of claim 1 wherein the heterologous protein comprises a bispecific T cell engager.

7. The cell of claim 1 wherein the heterologous protein comprises a binding molecule that binds to an antigen selected from the group consisting of: anti-CD19, anti-CD3, anti-CD38, and prostate-specific membrane antigen (PSMA).

8. The cell of claim 1 wherein the expression cassette encodes a promoter active in a Labyrinthulomycetes and that regulates transcription of the heterologous protein.

9. The cell of claim 8 wherein the promoter is selected from the group consisting of: tubulinα promoter (tubαp) or translation elongation factor (TEFp) promoter.

10. The cell of claim 8 wherein the expression cassette further encodes a terminator selected from pgk1t or eno2t.

11. The cell of claim 3 wherein the antibody has at least 90% sequence identity to an amino acid sequence of at least 100 contiguous amino acids selected from the group consisting of SEQ ID NO: 5-8.

12. An Aurantiochytrium cell comprising an expression cassette that encodes a heterologous protein and a functional signal sequence that directs the secretion of the heterologous protein from the cell, wherein
a. the signal sequence has at least 90% sequence identity to a sequence of at least 10 contiguous amino acids in the amino acid sequence selected from any one of SEQ ID NOs: 19, 72, 75, and 76;
b. the signal sequence has at least 95% sequence identity to a sequence of at least 10 contiguous amino acids in the amino acid sequence of SEQ ID NO: 20, or the signal sequence has at least 90% sequence identity to a sequence of at least 15 contiguous amino acids in the amino acid sequence of SEQ ID NOs: 20, or the signal sequence has at least 90% sequence identity to a sequence of at least 20 contiguous amino acids in the amino acid sequence of SEQ ID NO: 20; or
c. the signal sequence is encoded by a nucleic acid sequence having at least 90% sequence homology to any one of SEQ ID NOs: 31, 32, 88, 91, and 92.

13. The cell of claim 12 wherein the heterologous protein is a functional antibody.

14. The cell of claim 13 wherein the antibody has a light chain and a heavy chain.

15. The cell of claim 12 wherein
the signal sequence has at least 90% sequence identity to at least 20 contiguous amino acids in the amino acid sequence selected from any one of SEQ ID NOs: 19, 20, 72, 75, and 76.

16. The cell of claim 12 wherein the signal sequence is encoded by a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence of at least 120 contiguous nucleotides selected from any one of SEQ ID NOs: 31, 32, 88, 91, and 92.

17. A therapeutic protein comprising an antibody molecule linked to a signal sequence selected from the group consisting of SEQ ID NOs: 19, 20, 72, 75, and 76.

18. The therapeutic protein of claim 17 wherein the antibody is anti-TNF-alpha or anti-HER2.

19. The cell of claim 1 , wherein:
a. the signal sequence is selected from any one of SEQ ID NOs: 19, 20, 72, 75, and 76; or
b. the signal sequence is encoded by the nucleic acid sequence selected from any one of SEQ ID NOs: 31, 32, 88, 91, and 92.

20. The cell of claim 12 , wherein:
a. the signal sequence is selected from any one of SEQ ID NOs: 19, 20, 72, 75, and 76; or
b. the signal sequence is encoded by the nucleic acid sequence selected from any one of SEQ ID NOs: 31, 32, 88, 91, and 92.

21. The cell of claim 12 wherein the expression cassette further comprises a promoter active in Aurantiochytrium operably linked to the coding sequence of the heterologous protein, and a terminator sequence active in Aurantiochytrium.

Assignees

• Conagen Inc

Inventors

• Caiazza Nicky C
• Urano Jun
• Win Maung N
• Boles Kent S

Classifications

• C07K16/241

  Tumor Necrosis Factors · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K2317/515

  Complete light chain, i.e. VL + CL · CPC title

• C07K2319/036

  targeting to the medium outside of the cell, e.g. type III secretion · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title