Plant production of immunoglobulins with reduced fucosylation
This invention provides for the plant production of immunoglobulins, wherein at least a portion of the glycans attached to the immunoglobulins lack fucose. The invention also provides the constructs; plasmids; vectors; transformed plant cells, transformed plant calli; transformed plant tissues (e.g., leaves, seeds, tubers, etc.); transformed whole plants used to produce such immunoglobulins; methods of producing the immunoglobulins; the immunoglobulins produced by the disclosed methods; and the use of such immunoglobulins.
1. A plant-produced heavy chain (HC) or light chain (LC) of an immunoglobulin, wherein the immunoglobulin is an IgA molecule specific to herpes simplex virus; and wherein the HC or LC has a glycopeptide profile comprising at least one glycopeptide which lacks fucose, the at least one glycopeptide containing a glycan that is 2Man, 2GlcNAc, 1Xyl, in which Man=Mannose, GlcNAc=N-acetylglucosamine, and Xyl=xylose, wherein 2Man, 2GlcNAc, 1Xyl has the structure set forth in FIG. 12 , item 2.
2. The HC of claim 1 , wherein the at least one glycopeptide comprises an asparagine (Asn) residue in a CH2 region of the plant-produced heavy chain.
3. A plant-produced immunoglobulin, wherein the immunoglobulin has a free glycan profile comprising at least one glycan which lacks fucose, the glycan being 2Man, 2GlcNAc, 1Xyl, in which Man=Mannose, GlcNAc=N-acetylglucosamine, and Xyl=xylose, wherein 2Man, 2GlcNAc, 1Xyl has the structure set forth in FIG. 12 , item 2, and wherein the immunoglobulin is an IgA molecule specific to herpes simplex virus.
4. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin comprises an asparagine (Asn) residue.
5. The immunoglobulin of claim 3 , wherein the free glycan profile is the same as or substantially the same as that provided in FIG. 12 .
6. The immunoglobulin of claim 3 , wherein the free glycan profile further comprises a glycan selected from the group consisting of 3Man, 2GlcNAc, 1Xyl having the structure set forth in FIG. 12 , item 1; 3Man, 3GlcNAc, 1Xyl having the structure set forth in FIG. 12 , item 5; 3Man, 2GlcNAc having the structure set forth in FIG. 12 , item 6; 3Man, 3GlcNAc having the structure set forth in FIG. 12 , item 7; 4Man, 2GlcNAc having the structure set forth in FIG. 12 , item 8; 5 Man, 2GlcNAc having the structure set forth in FIG. 12 , item 9; and 6Man, 2GlcNAc having the structure set forth in FIG. 12 , item 10, wherein Man=Mannose, GlcNAc=N-acetylglucosamine and Xyl=xylose.
7. The immunoglobulin of claim 3 , wherein the free glycan profile is the same as or substantially the same as one of the glycan profiles provided in FIG. 16 .
8. The immunoglobulin of claim 3 , wherein the free glycan profile further comprises 3Man, 2GlcNAc having the structure set forth in FIG. 12 , item 6; and 3Man, 2GlcNAc, 1 Xyl having the structure set forth in FIG. 12 , item 1, wherein Man=Mannose, GlcNAc=N-acetylglucosamine and Xyl=xylose.
9. The immunoglobulin of claim 3 , wherein the free glycan profile further comprises 8Man, 2GlcNAc; 3Man, 2GlcNAc, 1Xyl; 4Man, 2GlcNAc, 1Xyl; 5Man, 2GlcNAc; 6Man, 2GlcNAc; 7Man, 2GlcNAc; 3Man, 3GlcNAc, 1Xyl; or 4Man, 2GlcNAc, wherein Man=Mannose, GlcNAc=N-acetylglucosamine and Xyl=xylose, and wherein 3Man, 2GlcNAc, 1Xyl has the structure set forth in FIG. 12 , item 1, 5Man, 2GlcNAc has the structure set forth in FIG. 12 , item 9, 6Man, 2GlcNAc has the structure set forth in FIG. 12 , item 10, 3Man, 3GlcNAc, 1 Xyl has the structure set forth in FIG. 12 , item 5, and 4Man, 2GlcNAc has the structure set forth in FIG. 12 , item 8.
10. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin comprises an asparagine (Asn) residue in its CH2 region.
11. The immunoglobulin of claim 3 , wherein its free glycan profile is determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-Tof MS) analysis of free N-linked glycans enzymatically-released from the immunoglobulin.
12. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin is a human immunoglobulin.
13. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin comprises a heavy chain lacking a tailpiece.
14. The plant-produced immunoglobulin heavy chain (HC) or light chain (LC) of claim 1 , wherein the heavy chain of the immunoglobulin lacks a tailpiece.
15. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin is isolated from the plant used to produce the immunoglobulin.
16. The immunoglobulin of claim 3 , comprising an amino acid fragment lacking an attached glycan with fucose, wherein the plant-produced immunoglobulin has an attached glycan with fucose on the same amino acid fragment or on substantially the same amino acid fragment when the immunoglobulin is mammalian-produced.
17. The immunoglobulin of claim 3 , comprising an amino acid fragment having an attached glycan lacking fucose, wherein the plant-produced immunoglobulin also lacks an attached glycan with fucose on the same amino acid fragment or on substantially the same amino acid fragment when the immunoglobulin is mammalian-produced.
18. The immunoglobulin of claim 16 , wherein the plant-produced immunoglobulin is produced in a maize cell.
19. A method of producing an isolated a monomeric anti-herpes simplex virus antibody comprising: (i) introducing into a plant cell nucleic acids having either SEQ ID NO: 1 or either SEQ ID NO: 5 and SEQ ID NO: 9 or SEQ ID NO: 13, each of which is operably-linked to a promoter, to produce a transformed plant cell; (ii) culturing the transformed plant cell to express the introduced nucleic acids; and (iii) isolating the monomeric anti-herpes simplex virus antibody produced by the plant cell.
20. The method of claim 19 further comprising regenerating a transformed plant from the transformed plant cell.
21. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 6.
22. The immunoglobulin of claim 3 , wherein the plant-produced immunoglobulin comprises a light chain comprising the amino acid sequence of SEQ ID NO: 14.