RECOMBINANT ORGANISMS AND METHODS FOR PRODUCING GLYCOMOLECULES WITH LOW SULFATION
The invention provides a recombinant Labyrinthulomycetes cell for the production of a low sulfate glycomolecule. The cell comprises a nucleic acid encoding a heterologous glycomolecule, and a sequence encoding a heterologous oligosaccharyltransferase. The cell produces the heterologous glycomolecule having fewer sulfated glycans compared to the same heterologous glycomolecule produced by a corresponding cell not comprising the heterologous oligosaccharyltransferase. The cells advantageously produce and, optionally secrete, the heterologous glycomolecule. Thus, the invention provides recombinant organisms that provide glycomolecules having a glycosylation profile that is more similar to the glycosylation profile produced in mammalian cell.
1 . A recombinant cell of the family Thraustochytriaceae for the production of a glycomolecule having low sulfation, comprising
a nucleic acid sequence encoding a heterologous glycomolecule;
a nucleic acid sequence encoding a heterologous oligosaccharyltransferase;
wherein the recombinant cell produces the heterologous glycomolecule having fewer sulfated glycans compared to the same heterologous glycomolecule produced by a corresponding cell not comprising the heterologous oligosaccharyltransferase.
2 . The recombinant cell of claim 1 wherein the glycomolecule is a glycoprotein or glycopeptide, and wherein the recombinant cell further comprises a genetic modification in a mannosyl transferase gene.
3 . The recombinant cell of claim 2 wherein the mannosyl transferase gene is alg3.
4 . The recombinant cell of claim 3 wherein the heterologous oligosaccharyltransferase is from a protozoa, and further comprises a protozoal promoter that regulates the sequence encoding the heterologous oligosaccharyltransferase.
5 . The recombinant cell of claim 4 wherein the heterologous oligosaccharyltransferase is a single protein enzyme.
6 . The recombinant cell of claim 3 wherein the oligosaccharyltransferase is from a protozoa of the Family Trypanosomatidae.
7 . The recombinant cell of claim 5 wherein the protozoa is a trypanosome.
8 . The recombinant cell of claim 5 wherein the protozoa is of the genus Leishmania.
9 . The recombinant cell of claim 5 wherein the protozoal gene comprises a protozoal promoter that regulates the sequence encoding the heterologous oligosaccharyl-transferase.
10 . The recombinant cell of claim 8 wherein the heterologous oligosaccharyltransferase comprises the Stt3 subunit of a protozoal oligosaccharyltransferase.
11 . The recombinant cell of claim 10 wherein the heterologous oligosaccharyltransferase is a protozoal enzyme encoded by a gene selected from the group consisting of: TbStt3A, TbStt3B, LmStt3D, LbStt3_1, and LbStt3 3.
12 . The recombinant cell of claim 11 wherein the gene is under the control of a promoter from an organism of the family Thraustochytriaceae.
13 . The recombinant cell of claim 3 wherein the heterologous glycoprotein or glycopeptide comprises less than 65% sulfated glycans.
14 . The recombinant cell of claim 13 wherein the heterologous glycoprotein or glycopeptide comprises less than 50% sulfated glycans.
15 . The recombinant cell of claim 3 wherein the cell produces and secretes the heterologous glycoprotein or glycopeptide molecule or functional portion thereof.
16 . The recombinant cell of claim 15 wherein the heterologous glycoprotein or glycopeptide is an antibody molecule, or functional portion thereof.
17 . The recombinant cell of claim 3 wherein the glycans are N-glycans and comprise Man3-5GlcNAc2.
18 . The recombinant cell of claim 3 wherein the heterologous glycoprotein or glycopeptide comprises a ratio of S-Man3-5/(S-Man3-5+Man3-5) of less than 60%.
19 . The recombinant cell of claim 3 wherein the heterologous glycoprotein or glycopeptide molecule is an antibody molecule, or portion thereof.
20 . The recombinant cell of claim 3 wherein the Thraustochytriaceae cell is of a genus selected from the group consisting of: Japanochytrium, Oblongichytrium, Thraustochytrium, Aurantiochytrium , and Schizochytrium.
21 . The recombinant cell of claim 20 wherein the Thraustochytriaceae cell is of the genus Aurantiochytrium or Schizochytrium.
22 . The recombinant cell of claim 3 wherein the heterologous glycoprotein or glycopeptide is selected from the group consisting of: trastuzumab, eculizumab, natalizumab, cetuximab, omalizumab, usteinumab, panitumumab, and adalimumab, or a functional fragment of any of them.
23 . The recombinant cell of claim 4 wherein the heterologous glycoprotein or glycopeptide comprises less than 65% sulfated glycans.
24 . The recombinant cell of claim 23 wherein the heterologous glycoprotein or glycopeptide comprises less than 50% sulfated glycans.
25 . A composition comprising the heterologous glycoprotein or glycopeptide produced by the recombinant cell of claim 4 .
26 . The composition of claim 25 wherein the heterologous glycoprotein or glycopeptide is an immunoglobulin.
27 . The composition of claim 25 wherein the heterologous glycoprotein or glycopeptide is selected from the group consisting of: trastuzumab, eculizumab, natalizurnab, cetuximab, omalizumab, usteinumab, paniturnumab, and adalimumab, or a functional fragment of any of them.
28 . The composition of claim 25 comprised in a pharmaceutically acceptable carrier.
29 . A method of producing a glycomolecule having a glycosylation profile with low glycan sulfation, comprising
providing a recombinant Thraustochytriaceae cell comprising a nucleic acid encoding a heterologous glycomolecule;
a sequence encoding a heterologous oligosaccharyltransferase; and
wherein the recombinant cell produces the heterologous glycomolecule having fewer sulfated glycans compared to the same heterologous glycomolecule produced by a corresponding cell not comprising the heterologous oligosaccharyltransferase.
30 . The recombinant cell of claim 29 further comprising a genetic modification in a mannosyl transferase gene.
31 . The recombinant cell of claim 30 wherein the mannosyl transferase gene is alg3.
32 . The recombinant cell of claim 30 wherein the heterologous glycomolecule has a glycan profile having less than 65% sulfated glycans.
33 . The recombinant cell of claim 30 wherein the heterologous glycoprotein or glycopeptide comprises a ratio of S-Man 3-5 /(S-Man 3-5 +Man 3-5 ) of less than 60%.
34 . The recombinant cell of claim 32 wherein the oligosaccharyltransferase is from a trypanosome.
35 . The recombinant cell of claim 34 wherein the heterologous glycoprotein or glycopeptide molecule is an antibody molecule, or portion thereof.
36 . The recombinant cell of claim 35 wherein the heterologous glycoprotein or glycopeptide is selected from the group consisting of: trastuzumab, eculizumab, natalizumab, cetuximab, omalizumab, usteinumab, paniturnumab, and adalimurnab, or a functional fragment of any of them.
37 . The recombinant cell of claim 34 wherein the heterologous oligosaccharyltransferase is a protozoal enzyme encoded by a gene selected from the group consisting of: TbStt3A, TbStt3B, LmStt3D, LbStt3 1, and LbStt3 3.
38 . The recombinant cell of claim 37 wherein the gene is under the control of a promoter from an organism of the family Thraustochytriaceae.