GLYCOSYLATED POLYPEPTIDES

Abstract

The present invention is directed to use of kifunensine for increasing sialylation of a glycosylated polypeptide, wherein a cell that produces the glycosylated polypeptide is contacted with kifunensine. Also provided are related methods for increasing sialylation of a glycosylated polypeptide and producing a glycosylated polypeptide, as well as glycosylated polypeptides and pharmaceutical compositions comprising the same, and their use in medicine.

Claims

1. Use of kifunensine for increasing sialylation of a glycosylated polypeptide, wherein a cell that produces the glycosylated polypeptide is contacted with kifunensine.

2. A method for increasing sialylation of a glycosylated polypeptide, the method comprising: a. providing a cell that produces the glycosylated polypeptide; and b. contacting the cell with kifunensine, thereby increasing sialylation of the glycosylated polypeptide produced by the cell.

3. A method for producing a glycosylated polypeptide having increased sialylation, the method comprising: a. providing a cell that produces the glycosylated polypeptide; and b. contacting the cell with kifunensine, thereby producing the glycosylated polypeptide having increased sialylation.

4. The method of claim 2, further comprising isolating the glycosylated polypeptide.

5. The use or method according to claim 2, wherein the cell is contacted with kifunensine prior to production of the glycosylated polypeptide by the cell, or wherein the cell is contacted with kifunensine during production of the glycosylated polypeptide by the cell.

6. The method according to claim 2, wherein the cell is contacted with a solution (e.g. culture medium) comprising kifunensine at a concentration of about 30-150 nM, 35-75 nM, or 40-60 nM, preferably wherein the cell is contacted with a solution (e.g. culture medium) comprising kifunensine at a concentration of about 50 nM.

7. The use or method according to claim 2, wherein the glycosylated polypeptide is characterised by increased mannosylation.

8. The use or method according to claim 2, wherein the glycosylated polypeptide is a recombinant glycosylated polypeptide, preferably wherein the glycosylated polypeptide is a human glycosylated polypeptide, and/or wherein the glycosylated polypeptide is an antibody, an antigen-binding portion of an antibody, a hormone, an Fc-fusion polypeptide, an albumin fusion polypeptide, an enzyme, or a cytokine.

9. The method according to claim 8, wherein the Fc-fusion polypeptide is abatacept, afilbercept, alefacept, belatacept, etarnecept or rilonacept, or wherein the hormone is erythropoietin, parathyroid hormone, growth hormone, insulin, glucagon, follicle stimulating hormone, luteinizing hormone or choriogonadotropin.

10. The method according to claim 2, wherein the glycosylated polypeptide is a monoclonal antibody or antigen-binding portion thereof, and/or wherein the glycosylated polypeptide is an IgG1 antibody or antigen-binding portion thereof, or an IgG2 antibody or antigen-binding portion thereof, preferably wherein the antibody or antigen-binding fragment thereof is adalimumab, abciximab, alemtuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, brodalumab, certolizumab, cetuximab, daratumumab, daclizumab, denosumab. dupilumab, durvalumab, eculizumab, efalizumab, gemtuzumab, golimumab, guselkumab, ibritumomab, infliximab, ixekizumab, muromonab-CD3, natalizumab, nivolumab, omalizumab, palivizumab; panitumumab, pembrolizumab, ranibizumab, risankizumab, rituximab, secukinumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab or vedolizumab.

11. The use or method according to claim 2, wherein the glycosylated polypeptide comprises at least one N-linked glycan, and/or wherein the glycosylated polypeptide is an antibody, and wherein the Fc portion thereof comprises at least one N-linked glycan, preferably wherein the N-linked glycan is a bi-antennary glycan.

12. The use or method according to claim 2, wherein the cell is a mammalian cell, preferably wherein the cell is a rodent cell, a human cell or a non-human primate cell, more preferably wherein the cell is a Chinese Hamster Ovary (CHO) cell or a murine myeloma cell (Sp2/0).

13. A glycosylated polypeptide obtainable by the method according to claim 2, optionally wherein the glycosylated polypeptide comprises increased sialylation and increased mannosylation.

14. A pharmaceutical composition comprising the glycosylated polypeptide according to claim 13 and a pharmaceutically acceptable carrier, excipient, adjuvant, and/or salt.

15. A glycosylated polypeptide according to claim 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0168] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0169] FIG. 1 shows percentage sialylation of IgG1 monoclonal antibodies produced in Sp2/0 cells supplemented with either 3, 6, 9 or 12 μg/L kifunensine.

[0170] FIG. 2 shows percentage sialylation of IgG2 monoclonal antibodies produced in CHO cells supplemented with 30, 40, 50 or 60 nM kifunensine.

TABLE-US-00002 SEQUENCE LISTING SEQ ID NO: 1-Golimumab Heavy Chain IgG1 (the sinqle glycosylation site at amino acid position 306 is shown in bold and underlined) QVQLVESGGG VVQPGRSLRL SCAASGFIFS SYAMHWVRQA PGNGLEWVAF MSYDGSNKKY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR GIAAGGNYYY YGMDVWGQGT TVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK SEQ ID NO: 2-Golimumab Liqht Chain EIVLTQSPAT LSLSPGERAT LSCRASQSVY SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPFTFG PGTKVDIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC SEQ ID NO: 3-Denosumab Heavy Chain IgG2 (the sinqle glycosylation site at amino acid position 298 is shown in bold and underlined) EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSG ITGSGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDP GTTVIMSWFD PWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK SEQID NO: 4-Denosumab Light Chain (kappa) EIVLTQSPGT LSLSPGERAT LSCRASQSVR GRYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVFYCQ QYGSSPRTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

EXAMPLES

Example 1

[0171] Effects of Kifunensine on the Sialylation of Human IgG1 Antibodies Produced in Sp2/0 Cells

[0172] Murine Sp2/0 cells transfected with expression vectors encoding SEQ ID NOs: 1 and 2 (which correspond to the heavy and light chains respectively of the human anti-TNFα IgG1 monoclonal antibody, golimumab) were cultured in perfusion bioreactors for 30 days under standard operating parameters. To examine the effect of kifunensine on the sialylation levels of the resulting IgG1, the perfusion cultures were supplemented with either 3, 6, 9, or 12 μg/L kifunensine (corresponding to 13 nM, 26 nM, 39 nM or 52 nM). A control culture was also maintained under the same conditions albeit in the absence of kifunensine. No significant impact on cell viability was observed in any of the cultures supplemented with kifunensine.

[0173] On culture day 18, samples were taken from the perfusion bioreactors and the percentage sialylation of the IgG1 antibodies was determined by glycan analysis. Briefly, antibodies were first purified using protein A chromatography, glycans were then enzymatically released from the antibody, fluorescently labelled with 2-aminobenzamide and analysed using hydrophilic interaction chromatography (HILIC) based methods. As shown in FIG. 1, an increase in sialylation compared to control was evident in cultures supplemented with 6 μg/L kifunensine and above, particularly 9 and 12 μg/L kifunensine. Peak sialylation levels were seen with 12 μg/L kifunensine (˜52 nM).

Example 2

[0174] Effects of Kifunensine on Fc-Glycan Sialylation of Human IgG2 Antibodies Produced in CHO Cells

[0175] CHO cells transfected with expression vectors encoding SEQ ID NOs: 3 and 4 (which correspond to the heavy and lights chains respectively of the human anti-RANKL IgG2 monoclonal antibody, denosumab) were cultured in bioreactors using standard fed-batch methods. To examine the effects of kifunensine on the Fc-glycan sialylation levels of the resulting IgG2, the cultures were supplemented with either 30, 40, 50 or 60 nM kifunensine on day 3 of the culture. A control culture was also maintained under the same conditions albeit in the absence of kifunensine. No significant impact on cell viability was observed in any of the cultures supplemented with kifunensine.

[0176] On day 20, samples were taken from bioreactors and the percentage Fc-glycan sialylation of the IgG2 antibodies was determined as described in Example 1. As shown in FIG. 2, an increase in Fc-glycan sialylation compared to control was evident when supplemented with >40 nM kifunensine. Peak Fc-glycan sialylation was observed in cultures supplemented with 60 nM kifunensine.

Example 3

[0177] Effects of Kifunensine on Sialylation of EPO Produced in CHO Cells

[0178] CHO cells transfected with expression vectors encoding recombinant human EPO (UniProt Accession No. P01588, Sequence Version 1, Entry Version 195) are cultured in perfusion bioreactors for 18 days under standard operating parameters. The culture medium is supplemented with 12 μg/L kifunensine from day 0.

[0179] Recombinant human EPO produced during the culture period is harvested throughout the production phase and at the end of the culture period, a sample is obtained to determine the glycosylation profile. The resultant recombinant human EPO has increased mannosylation and sialylation compared to that produced in cultures without kifunensine.

Clauses

[0180] 1. Use of kifunensine for increasing sialylation of a glycosylated polypeptide, wherein a cell that produces the glycosylated polypeptide is contacted with kifunensine. [0181] 2. A method for increasing sialylation of a glycosylated polypeptide, the method comprising: [0182] a. providing a cell that produces the glycosylated polypeptide; and [0183] b. contacting the cell with kifunensine, thereby increasing sialylation of the glycosylated polypeptide produced by the cell. [0184] 3. A method for producing a glycosylated polypeptide having increased sialylation, the method comprising: [0185] a. providing a cell that produces the glycosylated polypeptide; and [0186] b. contacting the cell with kifunensine, thereby producing the glycosylated polypeptide having increased sialylation. [0187] 4. The use according to clause 1 or the method according to clause 2 or 3, further comprising isolating the glycosylated polypeptide. [0188] 5. The use or method according to any one of the preceding clauses, wherein the cell is contacted with kifunensine prior to production of the glycosylated polypeptide by the cell. [0189] 6. The use or method according to any one of clauses 1-4, wherein the cell is contacted with kifunensine during production of the glycosylated polypeptide by the cell. [0190] 7. The use or method according to any one of the preceding clauses, wherein the cell is contacted with a solution (e.g. culture medium) comprising kifunensine at a concentration of about 30-150 nM. [0191] 8. The use or method according to any one of the preceding clauses, wherein the cell is contacted with a solution (e.g. culture medium) comprising kifunensine at a concentration of about 35-75 nM. [0192] 9. The use or method according to any one of the preceding clauses, wherein the cell is contacted with a solution (e.g. culture medium) comprising kifunensine at a concentration of about 40-60 nM. [0193] 10. The use or method according to any one of the preceding clauses, wherein the cell is contacted with a solution (e.g. culture medium) comprising kifunensine at a concentration of about 50 nM. [0194] 11. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is characterised by increased mannosylation. [0195] 12. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is a recombinant glycosylated polypeptide. [0196] 13. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is a human glycosylated polypeptide. [0197] 14. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is an antibody, an antigen-binding portion of an antibody, a hormone, an Fc-fusion polypeptide, an albumin fusion polypeptide, an enzyme, or a cytokine. [0198] 15. The use or method according to clause 14, wherein the Fc-fusion polypeptide is abatacept, afilbercept, alefacept, belatacept, etarnecept or rilonacept. [0199] 16. The use or method according to clause 14, wherein the hormone is erythropoietin, parathyroid hormone, growth hormone, insulin, glucagon, follicle stimulating hormone, luteinizing hormone or choriogonadotropin. [0200] 17. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is a monoclonal antibody or antigen-binding portion thereof. [0201] 18. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is an IgG1 antibody or antigen-binding portion thereof, or an IgG2 antibody or antigen-binding portion thereof. [0202] 19. The use or method according to clause 17 or 18, wherein the antibody or antigen-binding fragment thereof is adalimumab, abciximab, alemtuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, brodalumab, certolizumab, cetuximab, daratumumab, daclizumab, denosumab. dupilumab, durvalumab, eculizumab, efalizumab, gemtuzumab, golimumab, guselkumab, ibritumomab, infliximab, ixekizumab, muromonab-CD3, natalizumab, nivolumab, omalizumab, palivizumab; panitumumab, pembrolizumab, ranibizumab, risankizumab, rituximab, secukinumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab or vedolizumab. [0203] 20. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide comprises at least one N-linked glycan. [0204] 21. The use or method according to any one of the preceding clauses, wherein the glycosylated polypeptide is an antibody, and wherein the Fc portion thereof comprises at least one N-linked glycan. [0205] 22. The use or method according to clause 20 or 21, wherein the N-linked glycan is a bi-antennary glycan. [0206] 23. The use or method according to any one of the preceding clauses, wherein the cell is a mammalian cell. [0207] 24. The use or method according to any one of the preceding clauses, wherein the cell is a rodent cell, a human cell or a non-human primate cell. [0208] 25. The use or method according to any one of the preceding clauses, wherein the cell is a Chinese Hamster Ovary (CHO) cell or a murine myeloma cell (Sp2/0). [0209] 26. A glycosylated polypeptide obtainable by the method according to any one of clauses 2-25, optionally wherein the glycosylated polypeptide comprises increased sialylation and increased mannosylation. [0210] 27. A pharmaceutical composition comprising the glycosylated polypeptide according to clause 26 and a pharmaceutically acceptable carrier, excipient, adjuvant, and/or salt. [0211] 28. A glycosylated polypeptide according to clause 26 or the pharmaceutical composition according to clause 27 for use in medicine.

[0212] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.