Claims for Patent: 10,280,230

Summary for Patent: 10,280,230

Title:	Use of human cells of myeloid leukemia origin for expression of antibodies
Abstract:	The invention relates to a method for producing a protein molecule composition having a defined glycosylation pattern, comprising (a) introducing in a host cell which is an immortalized human blood cell at least one nucleic acid encoding at least a part of said protein; and (b) culturing said host cell under conditions which permit the production of said protein molecule composition; and (c) isolating said protein molecule composition.
Inventor(s):	Goletz; Steffen (Berlin, DE), Danielczyk; Antje (Berlin, DE), Baumeister; Hans (Berlin, DE), Stahn; Renate (Berlin, DE), Loeffler; Anja (Eichhorst, DE), Stoeckl; Lars (Berlin, DE)
Assignee:	Glycotope GmbH (Berlin, DE)
Application Number:	14/703,498
Patent Claims:	1. A method for producing a protein molecule composition, comprising (a) selecting an immortalized human blood host cell which has been identified as producing a protein molecule composition which (i) comprises no detectable NeuGc, (ii) comprises alpha2-6 linked NeuNAc, and (iii) has an increased sialylation degree with an amount of NeuNAc on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 15% higher compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; (b) introducing in the host cell at least one nucleic acid encoding at least a part of said protein; (c) culturing said host cell under conditions which permit the production of said protein molecule composition; and (d) isolating said protein molecule composition. 2. The method according to claim 1, wherein said host cell is selected to produce a protein molecule composition having at least one of the following characteristics: (i) it comprises no detectable terminal Galalpha1-3Gal; (ii) it comprises at least 2% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of a protein molecule of the protein molecules in said protein molecule composition which contains bisecting GlcNAc; (iii) it comprises at least 5% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of a protein molecule of the protein molecules in said protein molecule composition which contains bisecting GlcNAc; (iv) it has an increased sialylation degree with an amount of NeuNAc on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 20% higher compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; (v) it comprises at least 50% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of a protein molecule of the protein molecules in said protein molecule composition, lacking fucose; and (vi) it has a higher degree of sialylation than is reached in NM-F9 [DSM ACC2606] or NM-D4 [DSM ACC2605] cells; wherein the NM-F9 [DSM ACC2606] and NM-D4 [DSM ACC2605] cells only reach about 50 to 60% of the sialylation degree that is obtained with said host cell. 3. The method according to claim 1 for producing a protein molecule composition, wherein said host cell is selected to produce a protein composition having at least one of the following glycosylation characteristics: (i) it has a galactosylation degree on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of the protein molecules in said protein molecule composition, that is increased compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; (ii) it has an amount of G2 structures on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 5% higher compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; (iii) it has an amount of G0 structures on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 5% lower compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; (iv) it comprises an amount of fucose on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 5% less compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; and (v) it has a sialylation pattern which is altered compared to the sialylation pattern of at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein, and wherein the protein molecules have at least one of the glycosylation characteristics (i) to (v). 4. The method according to claim 3, wherein said sialylation pattern is characterised by at least one of the following characteristics: it has an increased sialylation degree with an amount of NeuNAc on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of the protein molecules in said protein molecule composition which is at least 20% higher compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein, and/or it comprises at least 20% more charged N-glycosidically linked carbohydrate chains of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of the protein molecule of the protein molecules in said protein molecule composition compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein. 5. The method according to claim 1, wherein said host cell is selected to produce a glycoprotein, comprising at least 10% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of the protein molecule of the protein molecules in said protein molecule composition, lacking fucose; and/or at least 5% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of the protein molecule of the protein molecules in said protein molecule composition which contains bisecting GlcNAc; and/or more than 35% G2 structures on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule in said protein molecule composition; and/or less than 22% G0 structures on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule in said protein molecule composition. 6. The method according to claim 1, wherein said protein molecule composition produced has at least one of the following characteristics: it has an increased activity and/or increased yield compared to at least one protein molecule composition of the same protein molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]; it has an improved homogeneity compared to at least one protein molecule composition of the same protein molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]; it has an increased average or maximum yield which is at least 10% higher than the yield of at least one protein molecule composition from the same protein molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]; it has an improved homogeneity, which is an improved glycosylation homogeneity compared to at least one protein molecule composition of the same protein molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]; it has an increased activity compared to at least one protein molecule composition of the same protein molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]; in case said protein molecule is an antibody molecule, it has an increased Fc-mediated cellular cytotoxicity which is at least 2 times higher than the Fc-mediated cellular cytotoxicity of at least one antibody molecule composition from the same antibody molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]; and/or in case said protein molecule is an antibody molecule, it has an increased antigen mediated or Fc-mediated binding which is at least 15% higher than the binding of at least one antibody molecule composition from the same antibody molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096]. 7. The method according to claim 1, wherein a host cell is used, which is selected from the group consisting of: (a) host cells having a higher sialylation degree than K562; (b) host cells having a low or no fucosylation activity; (c) host cells of human myeloid leukaemia origin; (d) host cells derived from the cell line K562; and (e) host cells selected from the group consisting of NM-H9D8 [DSM ACC2806], NM-H9D8-E6 [DSM ACC2807], and NM-H9D8-E6Q12 [DSM ACC2856]. 8. The method according to claim 1, wherein a nucleic acid is introduced in the host cell, encoding an antifolate resistant DHFR-variant; and wherein the host cell is cultured with said antifolate. 9. The method according to claim 8, wherein at least one of the following characteristics is fulfilled (a) the antifolate is methotrexate (b) the nucleic acid encoding said antifolate resistant DHFR variant encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 1 to 9. 10. The method according to claim 1, wherein the protein is selected from the group consisting of the group of cytokines, cytokine receptors, renin; human growth hormone, bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin; gonadotrophins calcitonin; glucagon; clotting factors, von Willebrands factor; anti-clotting factors, atrial natriuretic factor; lung surfactant; plasminogen activators, bombesin; hemopoietic growth factor; enkephalinase; a serum albumin; mullerian-inhibiting substance; relaxin; prorelaxin; mouse gonadotropin-associated peptide; vascular endothelial growth factor; protein A and D; neurotrophic factors, platelet-derived growth factor; fibroblast growth factors; epidermal growth factor; insulin-like growth factor-I and -II; insulin-like growth factor binding proteins; osteoinductive factors; superoxide dismutase; T-cell receptors; surface membrane proteins; antibodies, immunoadhesins; and MUC1. 11. The method according to claim 1, wherein said protein is an antibody or a fragment thereof. 12. The method according to claim 11, wherein the antibody is selected from the group consisting of antibodies against ganglioside GD3, antibodies against human interleukin-5 receptor alpha-chain, antibodies against HER2, antibodies against CC chemokine receptor 4, antibodies against CD20, antibodies against CD22, antibodies against neuroblastoma, antibodies against MUC1, antibodies against epidermal growth factor receptor. 13. The method according to claim 11, wherein the antibody is selected from the group consisting of Pankomab, Muromomab, Daclizumab, Basiliximab, Abciximab, Rituximab, Trastuzumab, Gemtuzumab, Alemtuzumab, Ibritumomab, Cetuximab, Bevacizumab, Tositumomab, Pavlizumab, Infliximab, Eculizumab, Epratuzumab, Omalizumab, Efalizumab, Adalimumab, Alemtuzumab, Edrecolomab Basiliximab, Ruplizumab, OKT3, Declizumab, Abciximab, Palivizumab, Tuvirumab, Sevirumab, Oregovomab, Etaracizumab, anti-CC chemokine receptor 4 antibody KM2160, and anti-neuroblastoma antibody chCE7. 14. The method according to claim 11, wherein the antibody is selected from the group consisting of an antibody against MUC1; an antibody against HER2 and an antibody against EGFR. 15. The method according to claim 14, wherein the antibody is selected from the group consisting of PankoMab, Panko 1, Panko 2, Trastuzumab and CetuxiMab. 16. The method according to claim 10, wherein the cytokine is selected from the group consisting of TNF-alpha, TNF-beta, interferon, interferon-alpha, interferon-beta, interferon-gamma, M-CSF, GM-CSF, G-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, TGF-alpha, and TGF-beta. 17. The method according to claim 10, wherein the clotting factor is selected from the group consisting of factor VIIIC, factor IX, factor VII, tissue factor, and thrombin. 18. The method according to claim 10, wherein the anti-clotting factor is protein C. 19. The method according to claim 10, wherein the plasminogen activator is urokinase or human urine and tissue type plasminogen activator. 20. The method according to claim 10, wherein the serum albumin is human serum albumin. 21. The method according to claim 10, wherein the antibody is a rheumatoid factor. 22. The method according to claim 10, wherein the neurotrophic factor is selected from the group consisting of neutrophin-3, neutrophin-43, neutrophin-5, neutrophin-6, and nerve growth factor beta. 23. The method according to claim 10, wherein the gonadotrophin is selected from the group consisting of a follicle stimulating hormone, luteinizing hormone, and human chorionic gonadotrophin. 24. The method according to claim 10, wherein the osteoinductive factor is a bone morphogenic protein. 25. The method according to claim 10, wherein the surface membrane protein is selected from the group consisting of CD3, CD4, CD8, CD19, CD55, glycophorin A, and integrin. 26. The method according to claim 1, wherein said protein (i) is fused to another peptide or polypeptide sequence selected from the group consisting of a linker, an activating molecule and a toxin; or (ii) is an antibody fragment fused to another protein sequence selected from the group consisting of cytokines, co-stimulatory factors, toxins, antibody fragments from other antibodies, multimerisation sequences, and sequences for detection, purification, secretion or stabilization; or (iii) is an antibody or antibody fragment coupled to an effector molecule which mediates a therapeutic effect selected from the group consisting of an immune effector molecule, a toxin and a radioisotope. 27. The method according to claim 1, wherein the protein is selected from the group consisting of erythropoietin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, M-CSF, GM-CSF, and G-CSF. 28. The method according to claim 1, wherein the protein is selected from the group consisting of CD proteins; immunotoxins; colony stimulating factors, transforming growth factors interferons and interleukins. 29. The method according to claim 28, wherein the transforming growth factor is TGF-alpha or TGF-beta. 30. The method according to claim 28, wherein the interferon is selected from the group consisting of interferon-alpha, interferon-beta, and interferon-gamma. 31. The method according to claim 28, wherein the interleukin is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12. 32. The method according to claim 28, wherein the CD protein is selected from the group consisting of CD3, CD4, CD8, CD19, and CD55. 33. A method for producing a protein molecule composition, comprising (a) introducing in a host cell which is an immortalized human blood cell at least one nucleic acid encoding at least a part of said protein; and (b) culturing said host cell under conditions which permit the production of said protein molecule composition; and (c) isolating said protein molecule composition; wherein the host cell is selected to produce a protein composition comprising protein molecules having the following characteristic glycosylation combinations: (a) it comprises no detectable NeuGc it comprises no detectable Galalpha1-3Gal it comprises a galactosylation pattern as defined in claim 3 it has a fucose content as defined in claim 3 it comprises bisecGlcNAc it comprises an increased amount of sialic acid compared to a protein composition of the same protein molecule when expressed in the cell line CHOdhfr- [ATCC No. CRL-9096] or compared to the sialylation deficient cell line NM-F9 [DSM ACC2606] or NM-D4 [DSM ACC2605]; or (b) it comprises no detectable NeuGc it comprises no detectable Galalpha1-3Gal it comprises a galactosylation pattern as defined in claim 3 it has a fucose content as defined in claim 3 it comprises bisecGlcNAc it comprises 2-6 NeuNAc. 34. An immortalized human blood cell, capable of producing a protein molecule composition having the following glycosylation characteristics: (i) it comprises no detectable NeuGc; (ii) it comprises alpha2-6 linked NeuNAc; (iii) it has an increased sialylation degree with an amount of NeuNAc on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 15% higher compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; and (iv) it comprises at least 2% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of a protein molecule of the protein molecules in said protein molecule composition which contains bisecting GlcNAc. 35. The cell according to claim 34, wherein said protein molecule composition further has at least one of the following characteristics: (i) it comprises no detectable terminal Galalpha1-3Gal; (ii) it comprises at least 5% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of a protein molecule of the protein molecules in said protein molecule composition which contains bisecting GlcNAc; (iii) it has an increased sialylation degree with an amount of NeuNAc on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the protein molecule of said protein molecules in said protein molecule composition which is at least 20% higher compared to the same amount of protein molecules in at least one protein molecule composition of the same protein molecule isolated from CHOdhfr- [ATCC No. CRL-9096] when expressed therein; (iv) it comprises at least 50% carbohydrate structures of the total carbohydrate units or of at least one particular carbohydrate chain at a particular glycosylation site of a protein molecule of the protein molecules in said protein molecule composition, lacking fucose; and/or (v) it has a higher degree of sialylation than is reached in NM-F9 [DSM ACC2606] or NM-D4 [DSM ACC2605] cells; wherein the NM-F9 [DSM ACC2606] and NM-D4 [DSM ACC2605] cells only reach about 50 to 60% of the sialylation degree that is obtained with said host cell. 36. The isolated cell according to claim 34, selected from the group consisting of NM-H9D8 [DSM ACC 2806], NM-H9D8-E6 [DSM ACC 2807], and NM-H9D8-E6Q12 [DSM ACC 2856]. 37. The cell according to claim 34, comprising at least one nucleic acid encoding a protein molecule or at least one part thereof, and at least one nucleic acid comprising at least one nucleic acid sequence encoding at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No: 1 to 9.

Details for Patent 10,280,230

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2026-09-10
Grifols Therapeutics Llc	ALBUKED, PLASBUMIN-20, PLASBUMIN-25, PLASBUMIN-5	albumin (human)	For Injection	101138	10/21/1942		2026-09-10
Baxalta Us Inc.	BUMINATE, FLEXBUMIN	albumin (human)	Injection	101452	03/03/1954		2026-09-10
Csl Behring Ag	ALBURX	albumin (human)	Injection	102366	07/23/1976		2026-09-10
Grifols Biologicals Llc	ALBUTEIN	albumin (human)	Injection	102478	08/15/1978		2026-09-10
Instituto Grifols, S.a.	HUMAN ALBUMIN GRIFOLS	albumin (human)	Injection	103352	02/17/1995		2026-09-10
Instituto Grifols, S.a.	HUMAN ALBUMIN GRIFOLS	albumin (human)	Injection	103352	06/11/2003		2026-09-10