Use of human cells of myeloid leukaemia 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.

1 . 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.   
     
     
         2 - 25 . (canceled) 
     
     
         26 . A protein or protein molecule composition obtainable by the production method according to  claim 1 , wherein the protein molecule composition has the following glycosylation characteristics:
 (i) it comprises no detectable NeuGc;   (ii) it comprises alpha2-6 linked NeuNAc; and   (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.   
     
     
         27 - 28 . (canceled) 
     
     
         29 . The protein or protein molecule composition according to  claim 26 , wherein said composition comprises at least one antibody molecule, binding a MUC1 epitope, comprising the amino acid sequence DTR of the extracellular tandem repeat region. 
     
     
         30 . The protein or protein molecule composition according to  claim 29 , wherein said antibody binds the TA-MUC1 epitope, comprising the amino acid sequence DTR, wherein the T is glycosylated. 
     
     
         31 . (canceled) 
     
     
         32 . The protein or protein molecule composition according to  claim 30 , wherein said antibody is selected from
 the antibody PankoMab or a variant thereof, competitively binding the same TA MUC-1 epitope as PankoMab;   the antibody Panko 1 or a variant thereof, competitively binding the same TA MUC-1 epitope as Panko 1;   the antibody Panko 2 or a variant thereof, competitively binding the same TA MUC-1 epitope as Panko 2.   
     
     
         33 . The protein or protein molecule composition according to  claim 26 , wherein said protein is an antibody and has at least one of the following glycosylation characteristics:
 (a) it has an increased sialylation degree with at least a 15% higher amount of N-acetylneuraminic acid on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the antibody molecule of the antibody molecules in said antibody molecule composition than the same amount of antibody molecules of at least one antibody molecule composition of the same antibody molecule isolated from CHOdhfr− [ATCC No. CRL-9096] when expressed therein;   (b) it has a higher galactosylation degree with at least a 5% higher amount of G2 structures on the total carbohydrate structures or on the carbohydrate structures at one particular glycosylation site of the antibody molecule of the antibody molecules in said antibody molecule composition than the same amount of antibody molecules of at least one antibody molecule composition of the same antibody molecule isolated from CHOdhfr− [ATCC No. CRL-9096] when expressed therein;   (c) it comprises bisecGlcNAc.   
     
     
         34 - 47 . (canceled) 
     
     
         48 . The protein or protein molecule composition according to  claim 26 , having at least one of the following characteristics:
 (i) it comprises no detectable terminal Galalpha 1-3Gal;   (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/or   (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.   
     
     
         49 . The protein or protein molecule composition according to  claim 26 , wherein the protein is selected from the group consisting of the group of cytokines and their receptors, the tumor necrosis factors TNF-alpha and TNF-beta; renin; human growth hormone, bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain and B-chain; gonadotrophins, follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotrophin, human chorionic gonadotrophin (hCG); calcitonin; glucagon; clotting factors, factor VIIIC, factor IX, factor VII, tissue factor, von Willebrands factor; anti-clotting factors, protein C; atrial natriuretic factor; lung surfactant; plasminogen activators, urokinase, human urine and tissue-type plasminogen activator; bombesin; thrombin; hemopoietic growth factor; enkephalinase; human macrophage inflammatory protein; a serum albumin, human serum albumin; mullerian-inhibiting substance; relaxin A-chain and B-chain; prorelaxin; mouse gonadotropin-associated peptide; vascular endothelial growth factor; receptors for hormones or growth factors; integrin; protein A and D; rheumatoid factors; neurotrophic factors, bone-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, neurotrophin-6, nerve growth factor-beta; platelet-derived growth factor; fibroblast growth factors; epidermal growth factor; transforming growth factor, TGF-alpha, TGF-beta; insulin-like growth factor-I and -II; insulin-like growth factor binding proteins; CD proteins, CD-3, CD-4, CD-8, CD-19; erythropoietin (EPO); osteoinductive factors; immunotoxins; bone morphogenetic proteins; interferons, interferon-alpha, interferon-beta, interferon-gamma; colony stimulating factors (CSF's), M-CSF, GM-CSF, G-CSF; interleukins, IL-1 to IL-12; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; antibodies, immunoadhesins; glycophorin A; and MUC 1. 
     
     
         50 . The protein or protein molecule composition according to  claim 26 , wherein the protein is an antibody or a fragment thereof. 
     
     
         51 . The protein or protein molecule composition according to  claim 50 , wherein the antibody is of the IgG class, and/or a human, humanized or chimeric IgG which comprises a human Fc region. 
     
     
         52 . The protein or protein molecule composition according to  claim 50 , comprising at least one glycoform of an antibody molecule with at least one carbohydrate chain attached to another glycosylation site of the antibody molecule than the amino acid Asn-297 in the second domain of the Fc region. 
     
     
         53 . The protein or protein molecule composition according to  claim 50 , wherein the antibody has at least one N-glycosylation site having the amino acid sequence Asn-Xaa-Ser/Thr, wherein Xaa can be any amino acid except Pro, and/or at least one O-glycosylation site in the sequence of the Fab region. 
     
     
         54 . The protein or protein molecule composition according to  claim 50 , comprising
 (i) an antibody molecule which comprises at least one carbohydrate chain attached to another glycosylation site of the antibody molecule than the amino acid Asn-297 in the second domain of the Fc part, wherein the antibody composition has an extended serum-half life and/or bioavailability, when measured in at least one mammal, than the antibody molecule composition of the same antibody molecule isolated from at least one of the cell lines CHO, CHOdhfr−, BHK, NS0, SP2/0, NM-F9, NM-D4, PerC.6 or mouse hybridoma when expressed therein; or   (ii) an antibody molecule which comprises at least one carbohydrate chain attached to at least one N-glycosylation site and/or at least one O-glycosylation site in a sequence of the Fab region of the antibody molecule, wherein the antibody composition has an extended serum-half life and/or bioavailability, when measured in at least one mammal, than the antibody molecule composition of the same antibody molecule isolated from at least one of the cell lines CHO, CHOdhfr−, BHK, NS0, SP2/0, PerC.6 or mouse hybridoma when expressed therein.   
     
     
         55 . The protein or protein molecule composition according to  claim 50 , wherein the antibody
 (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 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.   
     
     
         56 . The protein or protein molecule composition according to  claim 50 , 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, and antibodies against epidermal growth factor receptor. 
     
     
         57 . The protein or protein molecule composition according to  claim 50 , wherein the antibody is selected from the group consisting of Pankomab, Muromomab, Daclizumab, Basiliximab, Abciximab, Rituximab, Herceptin, Gemtuzumab, Alemtuzumab, Ibritumomab, Cetuximab, Bevacizumab, Tositumomab, Pavlizumab, Infliximab, Eculizumab, Epratuzumab, Omalizumab, Efalizumab, Adalimumab, Campath-1H, C2B8, Panorex, BrevaRex, Simulect, Antova, OKT3, Zenapax, ReoPro, Synagis, Ostavir, Protovir, OvaRex, Vitaxin, anti-CC chemokine receptor 4 antibody KM2160, and anti-neuroblastoma antibody chCE7. 
     
     
         58 . The protein or protein molecule composition according to  claim 26 , which is selected from the group consisting of
 (i) an antibody against epidermal growth factor receptor (EGFR);   (ii) an antibody against HER2;   (iii) an antibody against CD20;   (iv) gonadotrophins; and   (v) clotting factors.   
     
     
         59 . The protein or protein molecule composition according to  claim 26 , which is selected from the group consisting of
 (i) the antibody cetuximab or a variant thereof, binding the same epitope as cetuximab;   (ii) the antibody herceptin;   (iii) the antibody Rituximab;   (iv) the gonadotrophins follicle stimulating hormone (FSH), luteinizing hormone (LH), thyrotrophin, and human chorionic gonadotrophin (hCG); and   (v) the clotting factors factor VIIIC, factor IX, factor VII, tissue factor and van Willebrand factor.   
     
     
         60 . A method of therapeutic or prophylactic treatment of a disease in a patient in need thereof, comprising administering a therapeutically effective amount of the protein or protein molecule composition according to  claim 26  to said patient. 
     
     
         61 . The method according to  claim 60 , wherein the disease to be treated is selected from the group consisting of leukemia, neutropenia, cytopenia, cancer, bone marrow transplantation, diseases of the hematopoietic system, infertility and autoimmune diseases.