U.S. patent application number 16/376174 was filed with the patent office on 2019-11-28 for use of human cells of myeloid leukaemia origin for expression of antibodies.
The applicant listed for this patent is Glycotope GmbH. Invention is credited to Hans BAUMEISTER, Antje DANIELCZYK, Steffen GOLETZ, Anja LOEFFLER, Renate STAHN, Lars STOECKL.
Application Number | 20190359731 16/376174 |
Document ID | / |
Family ID | 39157600 |
Filed Date | 2019-11-28 |
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United States Patent
Application |
20190359731 |
Kind Code |
A1 |
GOLETZ; Steffen ; et
al. |
November 28, 2019 |
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.
Inventors: |
GOLETZ; Steffen; (Berlin,
DE) ; DANIELCZYK; Antje; (Berlin, DE) ;
BAUMEISTER; Hans; (Berlin, DE) ; STAHN; Renate;
(Berlin, DE) ; LOEFFLER; Anja; (Eichhorst, DE)
; STOECKL; Lars; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Glycotope GmbH |
Berlin |
|
DE |
|
|
Family ID: |
39157600 |
Appl. No.: |
16/376174 |
Filed: |
April 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14703498 |
May 4, 2015 |
10280230 |
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16376174 |
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12440562 |
May 14, 2009 |
9051356 |
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PCT/EP2007/007877 |
Sep 10, 2007 |
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14703498 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2863 20130101;
C07K 14/59 20130101; C07K 2317/41 20130101; C07K 16/461 20130101;
C07K 2317/72 20130101; C07K 16/3092 20130101; C07K 2317/734
20130101; C07K 16/00 20130101; C07K 2317/732 20130101; C07K 2317/24
20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/00 20060101 C07K016/00; C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; C07K 14/59 20060101
C07K014/59 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2006 |
EP |
06090162.6 |
Sep 18, 2006 |
EP |
06090171.7 |
Oct 13, 2006 |
EP |
06090190.7 |
May 4, 2007 |
EP |
07090094.9 |
Claims
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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/703,498, filed on May 4, 2015, which is a
continuation of U.S. patent application Ser. No. 12/440,562, filed
May 14, 2009, which is a national stage filing under 35 U.S.C.
.sctn. 371 of International Application No. PCT/EP2007/007877,
filed on Sep. 10, 2007, and claims the benefit of priority of
European Application No. 06090162.6, filed on Sep. 10, 2006;
European Application No. 06090171.7, filed on Sep. 18, 2006;
European Application No. 06090190.7, filed on Oct. 13, 2006; and
European Application No. 07090094.9, filed on May 4, 2007. All of
these applications, including International Application No.
PCT/EP2007/007877, are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr. 5, 2019, is named KNJ_015USCN2_SEQ.txt and is 24823 bytes
in size.
SUMMARY
[0003] The invention provides biotechnologically favourable methods
for the production of protein molecule compositions and in
particular antibody molecule compositions having increased activity
and/or increased yield and/or improved homogeneity and a human
glycosylation. It further provides novel host cells, nucleic acids,
and protein molecule compositions.
INTRODUCTION
[0004] A key feature and challenge for the industry is the
production of recombinant proteins and productivity, cost,
homogeneity, and protein activity are key issues which remain to be
optimised. Glycosylation is also a key issue in the production of
high yields of homogenous recombinant glycoproteins which poses a
series of critical problems for their production. Each current
production cell line offers a series of different challenges and
problems which are largely due to the complexity and species,
tissue and site specificity of the glycosylation. Therefore, the
optimisation of production systems in respect to glycosylation
remains one of the key aspects for optimisation. This particularly,
as differences in the glycosylation pattern often have a
considerable impact on activity, immugenicity, bioavailability and
half-life of the protein molecules. A detailed overview of
glycosylation properties of different cell lines derived from
different species and non-mammalian production systems is given in
Jenkins et al (Getting the glycosylation right: implications for
the biotechnology industry, Nat Biotechnology, 1996, 14:
975-981).
[0005] Antibodies are major tools for diagnosis and research, and
likely to become the largest family of therapeutics. Over ten
recombinant antibody therapeutics are on the market and hundreds in
clinical development. A key feature and challenge for the industry
is the production of recombinant antibodies ("rMAbs") and
productivity, cost, homogeneity, and antibody activity are key
issues which remain to be optimised. Nearly all therapeutic rMAbs
on the market have been produced in the rodent cell lines from
hamster (CHO) and mice (NS0 or Sp2/0). By far most of the rMABs in
development are produced in rodent cells, and others are under
development, however, none has been sufficient for optimising
productivity, cost, homogeneity, and antibody activity.
[0006] Glycosylation is also a key issue in the production of high
yields of homogenous and potent rMAbs which poses a series of
critical problems for the production of rMAbs. Each current
production cell line offers a series of different challenges and
problems which are largely due to the complexity and species,
tissue and site specificity of the glycosylation [Review: Royston
Jefferis, CCE IX: Glycosylation of Recombinant Antibody
Therapeutics; Biotechnol. Prog. 2005, 21, 11-16].
[0007] Therefore the optimisation of protein and in particular
antibody production systems in respect to glycosylation remains one
of the key aspects for optimisation.
[0008] The present invention provides new expression systems based
on immortalized human blood cells and in particular based on cells
of myeloid leukaemia origin. These cells surprisingly improve the
production of glycosylated proteins and in particular antibodies in
respect to activity, yield and/or homogeneity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The following tables 1 and 8 and FIGS. 1 to 17 illustrate
the present invention.
[0010] Table 1: Yield of chimaeric PankoMab expressed in CHOdhfr-
and NM-F9 cultured in medium supplemented with FCS.
[0011] Table 2: Yield of chimaeric PankoMab expressed in NM-H9D8
[DSM ACC2806] cultured in serum-free medium.
[0012] Table 3: Quantification of the sialic acid content in
PankoMab and CetuxiMab: The antibodies were produced by the
indicated cell line and quantified by integrating the peak area
obtained by reverse phase chromatography of the DMB labelled sialic
acid variants. NeuGc and NeuAc were differentiated by using a
sialic acid standard.
[0013] Table 4: Quantification of the differently charges
structures in Panko1 and PankoMab: The 2-AB labelled N-glycans were
subjected to anion exchange chromatography (Asahi-PAK-column) and
the peaks corresponding to the differently charges structures were
quantified by integration.
[0014] Table 5: The galactosylation degree of the antibodies Panko1
and PankoMab was determined by aminophase HPLC (Luna-NH2-column) of
the 2-AB labelled glycans. The peaks were quantified by integration
and underlying glycan structure was analysed by mass
spectrometry.
[0015] Table 6: Quantification of the triantennary and
biantennary+bisected structures in Panko1 and PankoMab The
potentially bisecting GlcNAc containing fractions from the
aminophase-HPLC were collected and subjected to a reverse phase
chromatography (RP18-column). By this triantennary and
biantennary+bisected structures can be distinguished. The
fucosylation degree of the antibodies was determined by aminophase
HPLC (Luna-NH2-column) of the 2-AB labelled glycans. The peaks were
quantified by integration and underlying glycan structure was
analysed by mass spectrometry. The fucosylated and non-fucosylated
structures were determined and the integrated peak areas were
quantified.
[0016] Table 7: Yield of hFSH expressed in CHOdhfr- and GT-2x
cultured in medium supplemented with FCS (CHOdhfr-) or serum free
medium.
[0017] Table 8: Yield of hFSH expressed in NM-H9D8 [DSM ACC2806]
cultured in serum-free medium.
[0018] Table 9: Suitable glycosylation and activity combinations
obtainable with the method according to the present invention.
[0019] Table 10: Obtained values of bisecGlcNAc and fucose in
different cell lines.
[0020] FIG. 1: fut8 mRNA expression of NM-F9, NM-D4, and NM-H9D8
[DSM ACC2806] cell. As a control HepG2 cells served.
[0021] FIG. 2: Europium release assay with Cetuximab isolated from
NM-H9D8-E6 cells, CHOdhfr- cells or SP2/0 cells against LS174T
cells as target cells. Assay was incubated for 4 h at an effector
to target cell ratio of 50:1 with antibody concentrations from 0 to
100 ng/ml.
[0022] FIG. 3: ADCC activity of chimaeric PankoMab isolated from
NM-F9 is .about.5 times higher than chimaeric PankoMab isolated
from the CHOdhfr- cells.
[0023] FIG. 4: Europium release assay with chimeric Panko1 isolated
from NM-H9D8-E6 cells, CHOdhfr- cells and NM-H9D8 cells against
ZR-75-1 cells as target cells.
[0024] Assay was incubated for 4 h at an effector to target cell
ratio of 80:1 with antibody concentrations from 0 to 1
.mu.g/ml.
[0025] FIG. 5: ADCC activity of chimeric Panko2 in an europium
release assay against ZR-75-1 cells after o.n. incubation with an
effector to target cell ratio of 50:1 and 5000 target cells per
well. Samples were incubated in triplicates.
[0026] FIG. 6: ADCC activity of chimeric Panko2 in an europium
release assay against ZR-75-1 cells after o.n. incubation with an
effector to target cell ratio of 50:1 and 10.000 target cells per
well. Samples were incubated in triplicates.
[0027] FIG. 7: CDC assay with chimeric PankoMab against
ZR-75-1.
[0028] FIG. 8: Binding activity of the chimeric PankoMab isolated
from NM-F9 to synthetic glycosylated MUC1 30-mer peptide is about
50% higher than the binding activity of the chimaeric PankoMab
isolated from the CHOdhfr- cells.
[0029] FIG. 9: Binding activity of the chimeric Panko2 isolated
from GT-2x and NM-H9D8 to synthetic glycosylated MUC1 30-mer
peptide is about 50% higher than the binding activity of the
chimeric Panko2 isolated from the CHOdhfr- cells.
[0030] FIG. 10: Western blot analysis was performed to identify the
differently sialylated heavy chain of antibody molecule
compositions expressed in CHOdhfr-, NM-F9, or NM-H9D8 [DSM
ACC2806]. Proteins were transferred to nitrocellulose and
visualized either by secondary anti-human IgG antibodies (A) or SNA
(B) which detects 2-6 sialylation.
[0031] FIG. 11: Western blot analysis was performed to identify the
differently sialylated heavy chain of antibody molecule
compositions expressed in CHOdhfr- or NM-H9D8. Proteins were
transferred to nitrocellulose and visualized by SNA which detects
2-6 sialylation.
[0032] FIG. 12: ELISA analysis was performed to identify the
differently sialylated antibody molecule compositions expressed in
CHOdhfr-, GT-2x, NM-H9D8, or NM-H9D8-E6.
[0033] FIGS. 13A and 13B: ELISA analysis was performed to identify
the differently sialylated antibody molecule compositions of
Cetuximab expressed in CHOdhfr-, NM-F9, or NM-H9D8. Sialylation was
analysed (FIG. 13A) by SNA which detects alpha2-6 sialylation with
or without neuraminidase treatment and (FIG. 13B) by MAL I which
detects alpha2-3 sialylation.
[0034] FIG. 14: Dot blots stained by SNA of the chimeric antibodies
Panko1 and Panko2 isolated from CHOdhfr-, NM-F9, NM-H9D8, or
NM-H9D8-E6 cells.
[0035] FIG. 15: The chimeric PankoMab isolated from NM-H9D8 cells
is longer available in the serum of nude mice than that isolated
from NM-F9.
[0036] FIG. 16: SDS-Page Analysis of hFSH produced in NM-H9D8 (lane
1) and GT-2x (lane 2). 5 .mu.g of purified hFSH was separated by
SDS-PAGE under reducing conditions per lane and stained by
Coomassie Brilliant Blue. The Marker indicates a range of 21-108
kD.
[0037] FIG. 17: Western blot analysis was performed to identify the
differentially sialylated hFSH molecule compositions. 1 .mu.g of
hFSH molecule composition from CHO (lane1), NM-H9D8 (lane 2) and
GT-2x (lane 3) were separated by SDS-Page in a 10% acrylamide gel
under reducing conditions. Proteins were transferred to
nitrocellulose and visualized SNA which detects 2-6
sialylation.
[0038] FIG. 18: Shows an IgG antibody, wherein N-glycans are
covalently attached at a conserved Asn 297 residue in the C.sub.H2
domain of Fc. As indicated, there may be additional N-linked
oligosaccharides in the Fab domain, which can even influence the
binding activity of the antibody. The glycan structures only on one
half of the antibody are shown. The Fc carbohydrate is a branched
chain structure which lies mainly within the two C.sub.H2 domains,
with one arm/antenna of each oligosaccharide interacting with the
hydrophobic areas of the C.sub.H2 domains. Structural analysis of
polyclonal and myeloma human IgGs has demonstrated that the Fc
contains various amounts of a base biantennary core structure as is
demonstrated in FIG. 18. The meaning of the symbols is shown in the
corresponding table. Said core structure may contain none (G0), one
(G1) or two (G2) terminal galactose residues and/or a bisecting
GlcNAc and/or a fucose residue at the proximal GlcNAc. Further
fucose molecules may also be present at the other GlcNAc residues
or the galactose residues. The diversity is even increased, as the
terminal sialic acid may or may not be present depending on the
properties of the antibody, as sialic acid was reported to have a
negative impact on ADCC.
BACKGROUND ART
[0039] Proteins are a diverse group of which the function and
occurrence vary widely among each other. Among the proteins of
therapeutic potential most proteins are glycosylated, such as many
hormones (e.g. Growth hormone, Glycagon, FSH and LH), growth
factors (e.g. GM-CSF, G-CSF, VEGF and Erythopoietin), cytokines
(e.g. IL-2, IL-7, Interferon-alpha and -beta, TNF-alpha),
anti-coagulantia (e.g. Lepirudin, Desirudin), blood clotting
factors (e.g. factors VII, VIII and IX), vaccines (e.g. Hepatitis B
antigen) and antibodies. The established cellular production
systems are unable to produce proteins with the original human
glycosylation. Prokaryotic (e.g. bacteria) and most eukaryotic cell
systems (e.g. yeast, insect and plant cell) synthesize proteins
that lack glycosylation or carry glycans which largely differ from
human carbohydrate chains. Chinese hamster ovary (CHO) cells are a
commonly used production system that is able to glycosylate
proteins in a similar way as human cells. However, important
differences remain, such as in galactosylation, fucosylation,
particular glycosylation with N-acetylglucosamines, and especially
in various aspects of sialylation. These differences influence the
activity, the bioavailability, the immunogenicity and the
half-life.
[0040] At the time these production systems were established, it
was sufficient to produce therapeutic proteins that were at least
to some degree active. However, today large efforts concentrate to
improve the activity of a therapeutic protein with the aim (i) to
reduce the number and concentration of the applied doses of the
therapeutic proteins, (ii) to reduce the costs of a therapy, and
(iii) to reduce the side effects.
[0041] The major strategy to improve the bioactivity of proteins is
to elongate their serum-half life and hence their bioavailability.
This can be done e.g. by the process called PEGylation where
certain forms of polyethyleneglycol are added/linked chemically to
the produced protein. PEG increases the molecular weight and hence
the serum half-life. However, several problems are associated with
this process. For example, in nearly all cases PEGylation decreases
the activity of a protein by its cellular effector function,
repetitive administration in humans often results in an adverse
immune response as neutralizing antibodies, and/or the production
process needs additional chemical modification resulting in a
multistep process with additional costs, losses and time. Similar
carrier systems (e.g. HESylation or attachment of albumin) exist
which have comparable drawbacks.
[0042] Also the modification of the carbohydrate chains and thus
the glycosylation of proteins is in the focus in order to improve
the serum-half life of recombinantly expressed proteins. The
technologies thereby focus on the maximization of the sialylation
degree of a recombinant glycoprotein. Sialic acids are the most
prevalent terminal monosaccharides on the surface of eukaryotic
cells and it is generally believed that the more a glycoprotein is
sialylated the longer is its serum half-life during circulation.
This is based on the presence of certain receptors as the
asialoprotein-receptor in the liver which binds circulating
non-sialylated proteins and directs them into the cell for
degradation.
[0043] Various classes of antibodies are present in human and most
mammalian, namely IgG, IgM, IgE, IgA, IgD. While molecules of all
antibody classes are used in diagnostics or as research tools, the
majority of antibody based therapeutics on the market and under
development are IgG and to a lesser extend IgM. The human IgG class
is further classified into four subclasses, IgG1, IgG2, IgG3 and
IgG4. The basic structure of an IgG molecule consists of two light
chains and two heavy chains comprising two Fab regions each with
one variable domain comprising the antigen-binding site and one
constant domain, one Fc region comprising further constant domains
and the interaction sites for ligands, and the flexible hinge
region which connects Fab and Fc regions. Antibodies can exist as
whole molecules or as antibody fragments such as Fab fragments or
single chain Fv which comprise the two variable regions of the
heavy and light chain connected via a linker. FIG. 18. shows an IgG
antibody.
[0044] Recombinant antibodies for therapeutic use are most often
chimaeric, humanized or so-called fully human protein sequences in
order to reduce their immunogenicity. However, truly fully human
molecules has not be readily achieved since the posttranslational
modifications such as particularly the glycosylation is not human
due to the production in rodent or CHO cells and thus can differ
from those modifications found on human antibodies in human sera.
Differences in the modification, in particular the glycosylation
pattern, can have a serious impact on the activity and the
immunogenicity of the respectively produced antibody.
[0045] The activity of an antibody can be due to and influenced by
a combination of effects. On one hand side the antibody has a
certain specificity which is mediated by the variable region of the
antibody ("V region") located in the Fab region wherein certain
sequences, the CDR regions, of the V region. They play a key role
in determining the particular specificity and affinity of an
antibody. The V regions are thus decisive for the epitope binding
characteristics and vary from antibody to antibody. The affinity of
an antibody describes the strength and kinetics of the binding of a
single binding region of an antibody to its epitope. Since the
various classes and subclasses of antibodies carry between two and
ten identical variable regions in one antibody molecule, the
strength and kinetics of the binding of an antibody is influenced
by the number of binding sites available for binding to and the
accessibility of epitopes on the target antigen or cell and is
expressed in its avidity.
[0046] Another important factor for the quality of an antibody for
its use in diagnosis and especially in therapy is the number of
binding sites of an antibody on its target structure or cell as
well as its internalisation rate. These qualities influence the
effects and suitability of an antibody for its use especially in
therapy.
[0047] On the other hand, effector mechanisms relevant for
therapeutic use of an antibody are several fold whereby some of the
antibodies act via only one mechanism and others by a combination
of various effector mechanisms. One strategy is to couple
antibodies or antibody fragments to effector molecules which
mediate a therapeutic effect, such as coupling to an immune
effector molecule, for example interleukine 2 to recruit the immune
system, or coupling to toxins or radioisotopes in order to kill
target cells, for example for anti-tumor therapies. Radiolabelled
antibodies or antibody fragments are also valuable for in vivo
diagnosis.
[0048] The other strategy is to use non-labelled, so-called "naked"
antibodies which can have important advantages such as lower
toxicology, less complicated logistics, the use of natural immune
effector arms or triggering of therapeutic effects without the need
of additional effector molecules. A large number of studies have
been performed to elucidate the activity mechanisms and mode of
action of antibodies as well as develop antibodies using these
activities. Among the most important activities and effects are
antibody-dependent cell-mediated cytotoxicity activity (herein
referred to as "ADCC activity"), the complement-dependent
cytotoxicity activity (herein referred to as "CDC activity"), the
phagocytosis mediated cytotoxicity activity (herein referred to as
"phagocytosis activity"), a receptor mediated activity (herein
referred to as "receptor mediated activity") which comprises a
whole set of different effects and activities.
[0049] Receptor mediated activities are mainly based on the binding
of the antibody to a molecule or receptor on a target cell or by
the prevention of binding of certain molecules to a target cell
thereby inducing or inhibiting certain effects on these cells, such
as antagonistic or agonistic activity or receptor blockade of an
antibody, leading for example to the induction or inhibition of
apoptosis or proliferation of target cells or to the secretion or
inhibition of the secretion of certain molecules in a target cell,
or blocking or triggering certain other receptor mediated events
such as interactions between molecules and cells or between cells.
The ADCC activity, CDC activity and phagocytosis activity are
cytotoxic activities which are mediated by the Fc region of the
antibody (herein referred to as "Fc part mediated activities")
while the receptor mediated activities, affinity, avidity and
specificity of the antibody are mainly mediated via the binding
region of the antibody comprised in the Fab region.
[0050] The Fc part mediated activities are mediated via
immunological effector cells such as killer cells, natural killer
cells and activated macrophages, or various complement components
by binding to effector ligands such as Fc-gammaRI, Fc-gammaRII,
Fc-gammaRIII, C1q component of complement, the neonatal Fc receptor
(FcRn), etc. The human IgG1 subclass is reported to have the
highest ADCC and CDC activity among human IgG class molecules. For
IgM CDC activity is a dominant effector mechanism. The ADCC
activity and CDC activity involves the binding of the Fc region,
the constant region of the antibody, to Fc-receptors such as
Fc-gammaRI, Fc-gammaRII, Fc-gammaRIII of the effector cell or
complement components such as C1q. Important amino acid residues
are in the hinge region and in particular in the second domain of
the constant region ("Cgamma2 domain") A carbohydrate chain binding
to the Cgamma2 domains is important for the activity. The
carbohydrate chain is bound to the amino acid asparagine 297
(Asn-297) of Cgamma2 (N-glycoside linked carbohydrate chains). The
carbohydrate chain terminus which binds to asparagine is called the
reducing end and the opposite end is called a non-reducing end. The
Fc region of an IgG antibody has two carbohydrate binding sites.
FIG. 18 shows the structure of an IgG molecule and indicates the
position, where typically carbohydrate structures can be found.
Furthermore, the chemical structure and composition of these
carbohydrate structures is explained.
[0051] From the Fc part mediated activities, two are assumed to be
influenced by the glycosylation of the antibody: It has been shown
that the removal of the sugar chain in the Fc part of the antibody
results in the disappearance of the CDC and ADCC activity and also
a reduction of galactoses in these N-glycans causes a decrease in
CDC activity [Boyd et al., Molecular Immunol., 32, 1311, (1995);
U.S. Pat. No. 6,946,292]. Furthermore, it is described that
expression of antibodies in rat cells results in an increased ADCC
activity of certain antibodies expressed therein [Lifely et al.,
Glycobiology, 1995 December; 5(8):813-22; WO00/61739] when compared
to the same antibody expressed in hamster and rat cells. Both
reports assume that changes in the glycosylation cause these
differences in antibody ADCC activity, however, this is not clear.
Lifely et al. 1995 assumes that bisecting N-acetylglucosamine
("bisecGlcNAc") is responsible for an increased ADCC activity of
the antibody, while WO00/61739 assumes that a dramatic decrease of
fucose alpha1-6 linked to N-acetylglucosamine at the reducing end
of N-glycans ("core-fucose") is responsible for an increased ADCC
activity. Furthermore, it is described that recombinant expression
of the enzyme GnTIII in CHO cells which is responsible for
attaching the sugar bisecGlcNAc to N-glycans results in an increase
of the ADCC activity of certain antibodies when expressed in these
cells when compared to the same antibody expressed in CHO cells not
recombinantly transfected with GnTIII [U.S. Pat. No. 6,602,684,
Umana et al., Nature Biotechn 17: 176-180 (1999)]. Additionally, it
was described that the knock-out of the gene FUT8 in CHO cells
which results in the lack of core-fucose can increase the ADCC
activity of certain antibodies when expressed in such FUT8 KO CHO
cells [U.S. Pat. No. 6,946,292]. These results also demonstrate the
importance and complexity of the glycosylation pattern for the
activity of the antibody.
[0052] However, a "foreign" and thus non-optimized glycosylation
pattern may not only be detrimental for the activity, it may also
be immunogenic. Current expression and production systems for
proteins and in particular antibodies are mainly cell lines derived
from rodents and are based on cell lines from hamster, mice, or rat
such as CHO, BHK, NS0, Sp2/0 and YB2/0. It is known that rodent
cells can produce under non-optimal conditions a number of
abnormally glycosylated protein products that lack potency or are
immunogenic. In addition, rodent cells are known to express
carbohydrate structures with important differences to those
expressed in human cells comprising the presence of non-human
sugars and the lack of certain human sugar moieties which can
render proteins expressed in these cells for example immunogenic,
less effective, lower yield or suboptimal structural requirements
or folding. Most rodent cells express for example
N-glycolylneuraminic acid ("NeuGc") an alternative for
N-acetylneuraminic acid ("NeuNAc") not present in humans which is
immunogenic in humans and/or the immunogenic galactose alpha(1-3)
galactose modification ("Gal alpha1-3Gal"), and/or they lack
important carbohydrates such as the important alpha2-6 linked
NeuNAc or they lack bisecGlcNAc. There are also other known and
unknown differences. In addition, it is known from rodent
production that the glycoform profiles are mostly heterogeneous and
also the clone-specific glycoform profile which can vary widely
from clone to clone in CHO, NS0, or Sp2/0 cells and are dependent
on the mode of production and culture conditions.
[0053] Furthermore, expression systems exist for the production of
proteins which incorporate human cells, such as e.g. Hek 293 cells,
which are derived from human embryonal kidney cells or the cell
system PerC6.RTM. which is derived from a single, human
retina-derived cell, which was purposely immortalized using
recombinant DNA technology. However, these cell lines even though
often preferred over non-human cell systems, still have some
drawbacks. They have a unique glycosylation pattern which is
attributable to the glycosylation machinery of the respective
cells. However, depending on the protein to be produced, they might
not deliver an optimised glycosylation profile e.g. regarding
activity and/or serum half-life of the protein. In particular a
certain degree and pattern of sialylation is difficult to achieve
with these cells. E.g. the cell systems known in the state of the
art are often not capable of providing a detectable alpha 2-6
linked sialylation, which, however, is important for the serum
half-life.
[0054] From these facts it becomes clear that there is still a need
for expression and production systems which can further optimise
the productivity, homogeneity, and/or antibody activity by
providing alternatives and/or improved expression systems.
Furthermore, from these facts it also becomes clear that it can not
be said which carbohydrate structures and especially which human
carbohydrate structures are optimal to improve the activity or
homogeneity of proteins and in particular antibodies and what kind
of glycosylation pattern a cell line and especially a human cell
line has to provide in order to optimise the activity of a protein,
in particular an antibody. Therefore, there is a need for
expression systems, providing products with a diverging
glycosylation pattern compared to the products obtained with the
expression systems known in the state of the art.
[0055] The present invention provides solutions to these problems
by providing new expression systems based on human immortal blood
cell lines and in particular cells of myeloid leukaemia origin.
Using immortalized human blood cells is advantageous compared to
the system known in the prior art because these cells provide a
different glycosylation profile than other known human cell systems
derived from different tissue (e.g. kidney or retina). These
differences may be advantageous regarding activity, homogeneity and
product yield. Furthermore, these cells can be transfected and
grown in suspension under serum-free conditions.
[0056] Hence, according to a first embodiment, a method for
producing a protein composition is provided, comprising the
following steps: [0057] (a) introducing in a host cells which is an
immortalized human blood cell at least one nucleic acid encoding at
least a part of said protein; and [0058] (b) culturing said host
cell under conditions which permit the production of said protein
composition; and [0059] (c) isolating said protein composition.
[0060] This method utilizing immortalized human blood cells
improves the production of proteins and in particular antibodies in
respect to activity, yield and/or homogeneity. This is surprising
since so far it was not shown that immortalized human blood cells
and in particular myeloid leukaemia cells are suitable for the
production of proteins and particularly lead to antibodies with
respectively improved properties. While a certain rat leukaemia
cell was used for expression of antibodies the glycosylation
machinery between human and rat are critically different whereby
the latter can express carbohydrate moieties which can be
immunogenic in human such as NeuGc and Gal alpha1-3 Gal. The rat
YB2/0 leukaemia cell was described to yield antibodies with higher
ADCC activity due to higher presence of bisecGlcNAc or due to
drastic downregulation of core-fucose.
[0061] It was even more surprising that the problem could be solved
by this invention using immortalized human blood cells and in
particular myeloid cells since it was previously reported that the
cell line K562--a myeloid leukaemia cell--does not express the
enzyme for bisecGlcNAc, a finding that was described and shown by
gene hybridisation of GnTIII [Yoshimura M et al., Cancer Res.
56(2): 412-8 (1996)] and does express the gene FUT8 which expresses
the fucosyltransferase which causes addition of core-fucose as
shown by RT-PCR [example 1]. It was surprising because K562 is not
resistant to lectin treatment since it is bound strongly by the
lectin LCA, the basis for cells negative or strongly down-regulated
for core-fucosylation. Due to this information on the glycosylation
profile of K 562 cells, it could not be expected that those cells
can improve the activity of antibodies expressed therein as it was
assumed that the glycosylation machinery necessary for a favourably
activity pattern was not present. It was also surprising that
proteins and in particular antibodies with increased binding
activity, improved homogeneity and/or higher yields can be
generated and achieved with the production method of the present
invention compared to cells of current expression systems.
[0062] The strategy of the invention is to generate suitable human
cell lines in order to achieve a system, which can provide protein
products with a whole set of posttranslational modifications as
near as possible to the human system and hence non- or less
immunogenic properties and/or improved activity in the human
system. The aim is to provide a tailor made glycosylation pattern
for each protein/antibody to be expressed.
[0063] Due to the fact that carbohydrate structures are very
complex, that the glycosylation machinery of cells comprise several
hundred enzymes which are involved in their synthesis and that
those enzymes are mainly species specific and tissue specific
expressed, the major strategy of the inventors to achieve a human
glycosylation is to provide suitable biotechnologically human
expression systems to express proteins and in particular
antibodies, with an optimised glycosylation pattern. As the
optimised glycosylation pattern may differ from protein to protein
and from antibody to antibody, the invention provides cell lines
producing different glycosylation patterns, thereby allowing to
select the cell line for production which produces a glycosylation
pattern optimised for the respective protein/antibody product.
[0064] Hence, the invention provides biotechnologically favourable
methods for the production of protein compositions and in
particular antibody molecule compositions having increased activity
and/or increased yield and/or improved homogeneity and fully human
glycosylation. It further provides novel host cells, nucleic acids,
and molecule compositions.
[0065] Hence, a method for producing a protein composition,
preferably an antibody molecule composition, having a defined
glycosylation pattern is provided, comprising the following steps:
[0066] (a) introducing in an immortalized human blood cell as host
cell at least one nucleic acid encoding a protein or at least one
part thereof; [0067] (b) culturing said host cell under conditions
which permits the production of said protein composition; and
[0068] (c) isolating said protein composition having the intended
glycosylation characteristics.
[0069] In order to obtain a protein composition and particularly an
antibody composition having improved properties according to the
present invention, the host cell is selected to produce a
protein/antibody composition having at least one of the following
glycosylation characteristics: [0070] (i) It comprises no
detectable NeuGc.
[0071] As was outlined above, a NeuGc glycosylation may have
immunogenic properties in humans. Hence, it is desirable to avoid a
respective glycosylation as far as possible. A respective
glycosylation is avoided by using immortalized human blood cells
and in particular by using a host cell of human myeloid leukaemia
origin. [0072] (ii) It has a galactosylation degree, that is 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 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.
[0073] The galactose residues are found mainly beta 1-4 linked to
the GlcNAc residues on the antennas of the complex type N-glycan of
antibodies, but also beta-1,3 linkages have been found. However,
they usually occur in triantennary structures. The influence of the
degree of galactosylation on the activity is in particular
regarding antibodies remarkable. It has been demonstrated that
depletion of galactose leads to a reduced CDC activity. Hence, it
may be preferred to have a high degree of galactosylation.
Galactosylation may also play an important role for other
proteins.
[0074] When referring to the total carbohydrate structure of a
protein molecule, all glycosylations of the protein molecule are
considered. In case the carbohydrate structures at one particular
glycosylation site of the protein molecule is analysed, the focus
lies on a specific carbohydrate structure(s), such as e.g. the
carbohydrate structure(s) attached to the Asn 297 of the Fc part of
an antibody molecule (please also refer to FIG. 18). In case a
respective specific structure is evaluated, the content/composition
of this specific structure is determined. One could also refer to
total carbohydrate units and particular carbohydrate chains for
defining said characteristics (these are synonyms). [0075] (iii) 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 the 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.
[0076] In particular, in case an antibody is produced according to
the methods of the present invention, a high amount of G2
structures is beneficial. A "G2 structure" defines a glycosylation
pattern wherein galactose is found at both ends of the biantennary
structure bound to the Fc region in case of an antibody (please
also refer to FIG. 18). If one galactose molecule is found, it is
called a G1 structure, if there is no galactose, a G0 structure. A
G2 glycosylation pattern was often found to improve the CDC of
antibodies. Hence, it is preferred that at least 10%, 20%, 30%,
40%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 95% or even more than 100%
higher amount of G2 structures are present in the protein/antibody
composition produced. Suitable cell lines achieving a respective
high G2 glycosylation pattern are described herein.
[0077] As a high overall galactosylation degree is often beneficial
for the CDC of antibodies, it is often preferred to obtain 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95% or even more than 95% of G2
and/or G1 structures on the total carbohydrate structures or on the
carbohydrate structures at one particular glycosylation site of the
protein, in particular an antibody molecule.
[0078] According to a further embodiment, more than 35% (40%, 45%,
50%, 55%, 60%, 65% or more than 70%) G2 structures are present 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 composition. [0079] (iv) 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 the 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.
[0080] As outlined above, a high degree of galactosylation is
usually advantageous. Hence, the cell lines are preferably selected
such that G0 structures are avoided. The amount of G0 structures is
preferably lower than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90% or the amount is even lower.
[0081] According to a further embodiment, less than 22% (20%, 18%,
15%, 12%, 10%, 9%, 8%, 7%, 6%, less than 5%) G0 structures are
present 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 composition.
[0082] (v) It comprises no detectable terminal Galalpha1-3Gal.
[0083] As was outlined above, a Galalpha1-3Gal glycosylation may be
immunogenic in humans. This glycosylation characterises a pattern,
wherein a second galactose residue is linked in alpha 1,3 position
to the first galactose residue, resulting in the highly immunogenic
Galalpha 1-3 Gal disaccharide. By using immortalized human blood
cells and in particular a host cell of human myeloid leukaemia
origin, a respective disadvantageous glycosylation is avoided.
[0084] (vi) 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 the
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.
[0085] Fucose residues are found on different sites within the
N-glycan tree so particularly: [0086] alpha 1,6 linked to the
GlcNAc residue proximal to the amino acid strain; [0087] alpha 1,3
and alpha 1,4 linked to the antennary located GlcNAc residue;
[0088] alpha 1,2 linked to antennary located Gal residue.
[0089] On antibody attached N-glycans the vast majority of fucose
residues is found 1,6 linked to the proximal GlcNAc residue (so
called "core fucose"). It has been found that the absence of core
fucose on the reducing end of the N-glycan attached to antibodies
enhances the ADCC activity of antibodies by the factor 25 to 100.
Due to this beneficial effect on ADCC, it is preferred that the
amount of fucose is at least 10%, 15%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%,
1500% or more than 2000% 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. The present invention also
provides specially engineered cell lines which achieve a respective
low overall fucosylation.
[0090] According to a further embodiment, said host cell is
selected to produce a glycoprotein, comprising at least 10% (15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more
than 80%) carbohydrates of the total carbohydrate structures or of
at least one particular carbohydrate structure at a particular
glycosylation site of the protein molecule of said protein
molecules in said protein molecule composition, which lack fucose.
Regarding antibodies it is particularly preferred that the
N-glycoside linked carbohydrate chains bound to the Fc region
comprises a reducing end comprising GlcNAc, wherein the
carbohydrate chains do not contain fucose bound to the 6 position
of the GlcNAc in the reducing end of the carbohydrate chain. [0091]
(vii) It comprises at least one carbohydrate structure containing
bisecting GlcNAc.
[0092] Bisecting N-Acetylglucosamine (bisGlcNAc) is often found
beta 1,4 attached to the central mannose residue of the
tri-mannosyl core structure of the N-glycans found in antibodies.
The presence of bisecting GlcNAc at the central mannose residue of
the antibody Fc-N-glycan increases the ADCC activity of
antibodies.
[0093] According to a further embodiment, said host cell is
selected such that said protein produced comprises more
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 containing no fucose
and no bisecting GlcNAc than those respective carbohydrate
structures which contain bisecting GlcNAc and no fucose.
[0094] According to a further embodiment, said host cell is
selected such that said protein produced comprises more
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 containing more
bisecting GlcNAc and fucose than those respective carbohydrate
structures which contain bisecting GlcNAc and no fucose. [0095]
(viii) 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.
[0096] The influence of the sialylation degree/pattern on the
activity, half-live and bioavailability differs between different
proteins/antibodies. Hence, it is beneficial to determine for each
protein/antibody molecule the optimised sialylation pattern in
advance by using the screening method according to the present
invention, before establishing the production with the most
suitable host cell, providing the desired glycosylation pattern.
E.g. several publications exist reporting a negative impact of
sialic acid residues present on the Fc glycan of antibodies on
downstream effects, i.e. CDC and ADCC. However, it was found that a
high silalylation prolongs the half-life of the sialylated
molecules. Hence, depending on the protein/antibody produced, a
different sialylation pattern could be advantageous and the present
invention provides immortalized human blood cell lines having
different sialylation activities thereby allowing to obtain
proteins/antibodies depicting an optimized glycosylation
pattern.
[0097] According to one embodiment, the host cell is selected such
that it produces a protein, having a decreased sialylation degree
with at least a 10% (preferably 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, >95%) lower amount
of sialic acids 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 than 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.
According to one embodiment, the product comprises even no
detectable NeuNAc. Depending on the protein/antibody produced, the
presence of sialic acids and particularly NeuNAc may not contribute
to the activity of the protein/antibody. In these cases, it may be
favourable to avoid sialic acids glycosylation in order make the
product more homogeneous. This, as the NeuNAc glycosylation pattern
can also vary in the resulting protein composition. This can cause
difficulties in the regulatory approval of the product because the
product is due to the varying NeuNAc content less homogeneous.
[0098] For proteins/antibodies which do not rely on the presence of
a NeuNAc glycosylation for their activity, an avoidance of a NeuNAc
glycosylation can be beneficial in order to increase homogeneity.
However, "no detectable NeuNAc" does not necessarily mean that
there is absolutely no NeuNAc present. Conversely, also embodiments
are encompassed, which have a rather low degree of NeuNAc (e.g. 1
to 10%).
[0099] According to one embodiment, the product has a decreased
sialylation degree with a at least 15% (20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%,
>500%) lower 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 than the same amount of
protein molecules of at least one protein molecule composition of
the same protein molecule isolated from CHOdhfr- [ATCC No.
CRL-9096] when expressed therein. This embodiment is beneficial in
case a protein/antibody is supposed to be expressed, wherein the
sialylation has a negative effect on the activity of the
protein/antibody.
[0100] A respective glycosylation (absence or very low degree of
sialic acid or particularly NeuNAc) can be achieved by using
sialylation deficient cells such as NM-F9 and NM-D4 in a serum-free
medium.
[0101] According to a further embodiment, the product 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 a 15% (20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%, 250%, 300%, 350%, 400%, 450% or more than 500%) 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.
[0102] As was outlined above, a respectively increased degree of
sialylation may provide a positive effect on the serum half-life of
the protein by prolonging it. In these cases it is preferred to use
a cell line which provides a higher degree of sialylation than is
reached in CHOdhfr- cells [ATCC No. CRL-9096] and which also
provides a higher degree of sialylation than is reached in
silalylation deficient cells (such as e.g. NM-F9 and NM-D4),
wherein a precursor needs to be added in order to allow sialylation
to occur. However, even if a respective precursor is added when
growing these sialylation deficient cells, these cells usually only
reach about 50 to 60% of the sialylation degree that is obtained
with immortalized human blood cells having no genetic
mutation/defect in the glycosylation machinery necessary for
sialylation. Hence, for embodiments, wherein a higher degree of
sialylation is aimed at, it is preferred to use cell lines capable
of providing a respective high sialylation degree and not to use
NM-F9 and NM-D4.
[0103] According to a further embodiment, the product comprises
alpha2-6 linked NeuNAc. Additionally, alpha2-3 linked NeuNAc may be
present to some extent. Regarding some proteins/antibodies the
presence of a NeuNAc glycosylation is beneficial in particular
regarding the half-life of the protein/antibody. To provide an
alpha 2-6 linked NeuNAc is beneficial, because this glycosylation
pattern resembles a human glycosylation pattern. Rodent cells
usually provide an alpha2-3 linked NeuNAc. Also other existing
human cell lines are not capable to provide a sufficient alpha 2-6
linked NeuNAc glycosylation.
[0104] Suitable cell lines to provide a respective glycosylation
pattern are e.g. NM-H9D8 and NM-H9D8-E6.
[0105] According to a further embodiment, a host cell is used,
which produces a protein comprising 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 said
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.
[0106] The charge profile of a carbohydrate chain may also
influence the properties and should thus be considered. Chemical
groups which charge carbohydrate chains are e.g, sulphur groups or
sialic acid.
[0107] According to an alternative embodiment, said product
comprises at least 20% less 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 said 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.
[0108] According to a further embodiment, said host cell is
selected to produce a glycoprotein, comprising at least 2% (5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, or more than 45%) 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 contain bisecting GlcNAc.
[0109] According to one embodiment, a host cell is used for the
production of the protein, which depicts the following properties
[0110] it has an increased activity, increased yield and/or
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]; and/or [0111] 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]; and/or [0112] it has an improved homogeneity,
which is an improved glycosylation homogeneity wherein said
antibody molecule composition has a lower sialylation degree than
sialylation degree 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 [0113] 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 50% 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].
[0114] As was outlined above, the respective properties can be
obtained by optimising the glycosylation of the protein as
described herein. It was surprising to see that also the binding
profile can be altered and improved with some antibodies based on
the glycosylation profile. Suitable host cells are also described
herein.
[0115] According to a further embodiment, the improved homogeneity
of said protein molecule composition is an improved glycosylation
homogeneity of said protein molecule composition comprising at
least one of the following characteristics: [0116] no detectable
NeuGc; [0117] no detectable NeuNAc; [0118] more NeuNAc than a
protein molecule composition from the same protein molecule when
expressed in the cell line CHOdhfr-[ATCC No. CRL-9096]; [0119]
detectable alpha2-6 linked NeuNAc.
[0120] According to a further embodiment, said host cell is
selected to produce a protein composition comprising protein
molecules having one of the following characteristic glycosylation
pattern:
[0121] (a) [0122] it comprises no detectable NeuGc [0123] it
comprises no detectable Galalpha1-3Gal [0124] it comprises a
galactosylation pattern as defined in claim 2 [0125] it has a
fucose content as defined in claim 2 [0126] it comprises
bisecGlcNAc [0127] 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 a sialylation deficient cell line such as NM-F9 and NM-D4.
[0128] (b) [0129] it comprises no detectable NeuGc [0130] it
comprises no detectable Galalpha1-3Gal [0131] it comprises a
galactosylation pattern as defined in claim 2 [0132] it has a
fucose content as defined in claim 2 [0133] it comprises
bisecGlcNAc [0134] it comprises an decreased 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].
[0135] (c) [0136] it comprises no detectable NeuGc [0137] it
comprises no detectable Galalpha1-3Gal [0138] it comprises a
galactosylation pattern as defined in claim 2 [0139] it has a
fucose content as defined in claim 2 [0140] it comprises
bisecGlcNAc [0141] it comprises 2-6 NeuNAc.
[0142] Further suitable characteristic combinations leading to
improved characteristics are described in Table 9.
[0143] According to the present invention the term "protein
molecule" means protein of interest or active fragments and/or
mutants thereof whereby any protein can be used, preferably any
glycoprotein of human origin. The term protein molecule means any
polypeptide molecule or a part thereof. It can be encoded by one or
several nucleic acids. It can be produced in a secretory fashion or
a fraction thereof or a fusion protein with a fusion partner.
Preferably, the protein is secreted into the supernatant. This
embodiment is in particular beneficial regarding the overall
production process, as e.g. shedding steps (e.g. with phorbol
esters) can be avoided.
[0144] Examples of mammalian glycoproteins include molecules such
as cytokines and their receptors, for instance the tumor necrosis
factors TNF-alpha and TNF-beta; renin; human growth hormone and
bovine growth hormone; growth hormone releasing factor; parathyroid
hormone; thyroid stimulating hormone; lipoproteins;
alpha-1-antitrypsin; insulin A-chain and B-chain; gonadotrophins,
e.g. follicle stimulating hormone (FSH), luteinizing hormone (LH),
thyrotrophin, and human chorionic gonadotrophin (hCG); calcitonin;
glucagon; clotting factors such as factor VIIIC, factor IX, factor
VII, tissue factor and von Willebrands factor; anti-clotting
factors such as protein C; atrial natriuretic factor; lung
surfactant; plasminogen activators, such as urokinase, human urine
and tissue-type plasminogen activator; bombesin; thrombin;
hemopoietic growth factor; enkephalinase; human macrophage
inflammatory protein; a serum albumin such as 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 such as bone-derived neurotrophic factor,
neurotrophin-3, -4, -5, -6 and nerve growth factor-beta;
platelet-derived growth factor; fibroblast growth factors;
epidermal growth factor; transforming growth factor such as
TGF-alpha and TGF-beta; insulin-like growth factor-I and -II;
insulin-like growth factor binding proteins; CD proteins such as
CD-3, CD-4, CD-8 and CD-19; erythropoietin (EPO); osteoinductive
factors; immunotoxins; a bone morphogenetic protein; an interferon
such as interferon-alpha, -beta, and -gamma; colony stimulating
factors (CSF's), e.g. M-CSF, GM-CSF and G-CSF; interleukins (IL's),
e.g. IL-1 to IL-12; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; antibodies and
immunoadhesins; Glycophorin A; MUC1.
[0145] Many of the aforementioned glycoproteins belong to the
cytokines herein referring to the general class of hormones
occurring in cells of the immune system, both lymphokines and
monokines, and others. The definition is meant to include, but is
not limited to, those hormones that act locally and do not
circulate in the blood, and which, when used in accord with the
present invention, will result in an alteration of an individual's
immune response. Examples of further suitable immunomodulatory
cytokines include, but is not limited to, interferons (e.g.
IFN-alpha, IFN-beta and IFN-gamma), interleukins (e.g. IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 and IL-12), tumor
necrosis factors (e.g. TNF-alpha and TNF-beta), erythropoietin
(EPO), FLT-3 ligand, macrophage colony stimulating factor (M-CSF),
granulocyte colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), CD2 and
ICAM. Taking erythropoietin, the molecule is believed to cause
progenitor cells to mature into erythrocytes whereas thrombopoietin
is thought to drive progenitor cells along the thrombocytic
pathway. CSF refers to a family of lymphoicines which induce
progenitor cells found in the bone marrow to differentiate into
specific types of mature blood cells. The particular type of mature
blood cell that results from a progenitor cell depends upon the
type of CSF present. Similarly, granulocyte-macrophage colony
formation is dependent on the presence of GM-CSF. Additionally,
cytokines of other mammals with substantial homology to the human
forms of IL-2, GM-CSF, TNF-alpha and others, will be useful in the
invention when demonstrated to exhibit similar activity on the
immune system. Adhesion or accessory molecules or combinations
thereof may be employed alone or in combination with the
cytokines.
[0146] Similarly, proteins that are substantially analogous to any
particular protein, but have relatively minor changes of protein
sequence, will also find use in the present invention. It is well
known that some small alterations in the amino acid sequence in
protein sequence may often be possible without disturbing the
functional abilities of the protein molecule, and thus proteins can
be made that function as the parental protein in the present
invention but differ slightly from current known sequences.
Respective variants maintaining the biological function are thus
also comprised.
[0147] Preferred glycoproteins are selected from the group
comprising Glycophorin A, EPO, G-CSF, GM-CSF, FSH, hCG, LH,
interferons, interleukins, antibodies and/or fragments thereof.
[0148] All protein molecules mentioned above can be fused to other
peptide or polypeptide sequences such as but not limited to linker,
activating molecules or toxins.
[0149] In a preferred embodiment of the invention the nucleic acid
encodes a secretory form of the protein or a fragment hereof. In a
preferred embodiment the secretory form lacks transmembrane
domains.
[0150] In accordance with the present invention the term "protein
molecule composition" means the molecules of any protein molecule
expressed according to the methods of the present invention and in
particular in a host cell of the invention which can be isolated.
Said protein molecule composition comprises at least one protein
molecule. Said protein composition comprises at least one glycoform
of a protein molecule. Said glycoform of a protein molecule means a
protein molecule which carries a particular glycosylation or
carbohydrate chain which is different in at least one sugar
building block, for example but not limited to an additional
galactose, sialic acid, bisecGlcNAc, fucose, or another sugar
modification such as but not limited to acetylation or sulfatation
from another glycoform of the same protein molecule. In another
embodiment of the invention the protein molecule composition can
comprise molecules of more than one protein molecule expressed in a
host cell. In a preferred embodiment the protein molecule
composition of the invention comprises more molecules in percent of
such glycoform or such glycoforms of the protein molecule which
mediate a higher activity than a protein molecule composition of
the same protein molecule obtained from at least one of the cell
lines CHO, or CHOdhfr-, or BHK, or NS0, or SP2/0, or PerC.6 or
mouse hybridoma, preferably CHOdhfr- [ATCC No. CRL-9096], when
expressed therein. In a further preferred embodiment the protein
molecule composition of the invention comprises more molecules of
such glycoform or glycoforms of the protein molecule in percent
which mediate a higher activity than the protein molecule
composition of the same protein molecule obtained from the cell
lines CHO, or CHOdhfr-, or BHK, or NS0, or SP2/0, or PerC.6 or
mouse hybridoma, preferably CHOdhfr- [ATCC No. CRL-9096], when
expressed therein.
[0151] In accordance with the present invention the term "antibody
molecule" means any whole antibody or antibody fragment or a
molecule comprising an antibody fragment. Said whole antibody can
be any antibody or immunoglobulin molecule of any class or subclass
or any molecule comprising at least one immunoglobulin domain known
to those skilled in the art comprising but not limited to IgG,
IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD of animal origin such as but
not limited to human, simian, rodent, mouse, rat, hamster, rabbit,
camel, avian, chicken, or shark origin, and can also be a molecule
which comprises protein sequences from antibodies originating from
various animals such as chimaeric or humanized antibodies where
various percentages of for example murine and human sequences are
combined to whole antibodies and/or are mutated for example to
decrease immunogenicity or increase affinity as known to those
skilled in the art. In another embodiment of the invention said
whole antibody can also be the afore described whole antibody with
at least one additional amino acid or polypeptide sequence.
[0152] In a preferred embodiment said whole antibody is a human,
humanized or chimaeric IgG, IgG1, IgG2, IgG3, IgG4, or IgM which
comprises a human Fc region. In an even further preferred
embodiment of the invention said whole antibody is a human,
humanized or chimaeric IgG1, IgG4, or IgM with a human Fc region.
Said human Fc region comprises at least one 20 amino acid sequence,
more preferably at least 100 amino acids of a constant domain of
the Fc region of a human antibody, preferably it comprises at least
one Fc domain of a human antibody, more preferably it comprises the
human Cgamma2 domain, and more preferably it comprises all constant
domains of the Fc region of a human antibody of a certain class or
subclass. Said human Fc region can also comprise human sequences
from which at least one amino acid was mutated.
[0153] In the most preferred embodiment of the invention the whole
antibody molecule is either a (i) fully human antibody generated
for example from a human antibody producing blood cell or cells or
from a transgenic mouse in which the mouse antibody gene locus is
at least partially exchanged by human antibody sequences, or (ii) a
humanized whole antibody in which at least parts of the variable
regions of a murine or rat antibody, such as the framework regions
or at least one amino acid of a framework, were exchanged to human
sequences or mutated to be less immunogenic in humans which
comprise human constant domains, or (iii) a chimaeric whole
antibody in which the variable region is murine or rat and
comprises human constant domains.
[0154] Said fully human antibodies, humanized whole antibodies, and
chimaeric whole antibodies or parts thereof as well as the methods
to construct, identify, test, optimise, and select these antibody
molecules with or without suitable additional sequences as well as
methods to construct, identify, test and select most suitable
nucleic acids encoding these antibody molecules are known to those
skilled in the art.
[0155] Said antibody fragment is any fragment of an antibody
comprising at least 20 amino acids from said whole antibody,
preferably at least 100 amino acids. In a preferred embodiment the
antibody fragment comprises the binding region of the antibody such
as a Fab, a F(ab)2, multibodies comprising multiple binding domains
such as diabodies, triabodies or tetrabodies, single domain
antibody or affibodies. In another preferred embodiment the
antibody fragment comprises the Fc region with all or parts of its
constant domains, preferably comprising the second domain (Cgamma2
domain). In another embodiment the antibody fragment is a truncated
whole antibody where at least one amino acid, polypeptide stretches
or whole domains are deleted. Those molecules can be combined with
additional sequences for stabilization or for improving the binding
of the molecules such as linkers.
[0156] Said molecule comprising an antibody fragment is any
molecule which comprises any of said antibody fragments or other
immunoglobulin domains of at least 20 amino acids. In a preferred
embodiment said molecule comprising an antibody fragment are fusion
molecules where an antibody fragment is fused to other protein
sequences such as effector sequences, for example cytokines,
co-stimulatory factors, toxins, or antibody fragments from other
antibodies in order to generate molecules with multiple binding
specificities such as bi- or tri-specific antibodies, or
multimerisation sequences such as MBP (mannan binding protein)
domains for resulting in the multimerisation of binding domains, or
sequences for detection, purification, secretion or stabilization
such as tags, localization signals or linkers, or the like. In
another preferred embodiment said molecule comprising an antibody
fragment are fusion molecules comprising the Fc region of a whole
antibody or parts thereof, preferably comprising the second
constant domain (Cgamma2 domain). Said fusion molecules are fused
by genetic means, where the molecules are encoded by one nucleic
acid or are fused by co-expression of at least two nucleic acids
whereby the fusion is caused by non-covalent or covalent protein
interactions or are fused by a combination of both. The genetic
fusion between an antibody fragment and another polypeptide
sequence or protein molecule can be achieved by genetic engineering
where both parts are encoded by a single nucleic acid with or
without additional amino acids in between. In a further preferred
embodiment said fusion molecules comprise at least one binding
region from an antibody fragment such as a single domain antibody,
or Fab or a binding sequence not derived from antibodies, such as a
lectin domain, and a Fc region or parts thereof comprising the
second domain (Cgamma domain). In another preferred embodiment, the
fusion molecules comprise IL-2, IL-12, IL-15, GM-CSF, a peptide
toxin, or parts thereof. They are e.g. fused by genetic means,
where the molecules are encoded by one nucleic acid or are fused by
co-expression of at least two nucleic acids whereby the fusion is
caused by non-covalent or covalent protein interactions or are
fused by a comion from an antibody fragment such as a single domain
antibody, Fab or Fab which is linked to multimerisation sequence of
MBP.
[0157] All those antibody molecules or parts thereof as well as the
methods to construct, identify, test and select these antibody
molecules with or without suitable additional sequences as well as
methods to construct, identify, test and select most suitable
nucleic acids encoding these antibody molecules are known to those
skilled in the art.
[0158] In accordance with the present invention the term "antibody
molecule composition" means the molecules of any antibody molecule
expressed in a host cell of the invention which can be isolated.
Said antibody molecule composition comprises at least one antibody
molecule. Said antibody composition comprises at least one
glycoform of an antibody molecule. Said glycoform of an antibody
molecule means an antibody molecule which carries a particular
glycosylation or carbohydrate chain which is different in at least
one sugar building block, for example but not limited to an
additional galactose, sialic acid, bisecGlcNAc, fucose, or another
sugar modification such as but not limited to acetylation or
sulfatation from another glycoform of the same antibody molecule.
In another embodiment of the invention the antibody molecule
composition can comprise molecules of more than one antibody
molecule expressed in a host cell. In a preferred embodiment the
antibody molecule composition of the invention comprises more
molecules in percent of such glycoform or such glycoforms of the
antibody molecule which mediate a higher Fc-mediated cellular
cytotoxicity and/or an improved binding than an antibody molecule
composition of the same antibody molecule obtained from at least
one of the cell lines CHO, or CHOdhfr-, or BHK, or NS0, or SP2/0,
or PerC.6 or mouse hybridoma, preferably CHOdhfr- [ATCC No.
CRL-9096], when expressed therein. In a further preferred
embodiment the antibody molecule composition of the invention
comprises more molecules of such glycoform or glycoforms of the
antibody molecule in percent which mediate a higher Fc-mediated
cellular cytotoxicity and/or an improved binding than the antibody
molecule composition of the same antibody molecule obtained from
the cell lines CHO, or CHOdhfr-, or BHK, or NS0, or SP2/0, or
PerC.6 or mouse hybridoma, preferably CHOdhfr- [ATCC No. CRL-9096],
when expressed therein.
[0159] In accordance with the present invention the term "host cell
of human myeloid leukaemia origin" or equivalent formulations means
any cell or cell line of human myeloid leukaemia origin, or any
human myeloid or myeloid precursor cell or cell line which can be
obtained from a leukaemia patient, or any myeloid or myeloid
precursor cell or cell line which can be obtained from a human
donor, or a cell or cell line derived from anyone of said host
cells, or a mixture of cells or cell lines comprising at least one
of those aforementioned cells.
[0160] In another embodiment of the invention said host cell of
human myeloid leukaemia origin or said immortalized human blood
cell of the invention also comprise such cells or cell lines which
were obtained by fusing at least one of aforementioned host cells
in particular those of myeloid leukaemia origin with another cell
of human or animal origin, such as but not limited to B cells, CHO
cells. Those skilled in the art are able to identify and use
suitable sources and methods to obtain, generate and/or immortalize
suitable cells and cell lines from humans for suitable host cells
of human myeloid leukaemia origin.
[0161] The term cell or cell line derived from said host cell means
any cell or cell line which can be obtained by any means of
cultivation and cloning with or without prior mutation or genetic
engineering of said host cell of myeloid leukaemia origin and
comprises selection of those cells or cell lines derived from said
host cell with the desired properties. Said cultivation and cloning
is based on the fact that cell clones with differing properties can
be obtained from primary cell cultures, cultures of cells and even
cultures of cell clones by multiple rounds of passaging and cloning
of the cells using preferably single cell cloning methods such as
limited dilution or flowcytometry based cell sorting. In a
preferred embodiment said cell or cell line derived from said host
cells is selected by binding to a lectin or carbohydrate-binding
antibody. Said mutation can be performed by treatment known to
those skilled in the art with physical, chemical or biological
mutagens, such as but not limited to radiation, alkylating agents
or EMS (ethylmethanesulfonate), proteins such as lectins, or virus
particles. Said genetic engineering can be performed by methods
known to those skilled in the art such as knock-out of genes via
site specific homologous recombination, use of transposons,
site-specific mutagenesis, transfection of certain nucleic acids,
or silencing of genes, or gene products. Methods for said
cultivation and cloning, said mutation and mutagens and said
genetic engineering are known to those skilled in the art and some
examples are described in detail in WO2005/017130 A2, US
2003/0115614 A1, or are described herein. Those skilled in the art
are able to select and/or adopt and/or modify a suitable method or
combination of methods for generation of a suitable cell or cell
line derived from said host cell of the invention.
[0162] Said cell or cell lines derived from said host cell are
selected due to properties of those cells which are advantageous
when compared to their parent cell or cell line such as but not
limited to shorter doubling times, faster growth, possibility to
grow under higher densities, can produce more, are growing under
serum free conditions and/or in protein free media, higher cloning
efficiencies, higher transfection efficiencies for DNA, higher
expression rates of antibody molecule compositions, higher
activities for an antibody molecule composition expressed therein,
higher homogeneities of an antibody molecule composition expressed
herein, and/or higher robustness to scaling up. Methods for
selecting those cells with advantageous properties are known to
those skilled in the art or described herein. The invention
provides a method for generating a host cell of the invention,
comprising (a) incubating a human myeloid leukaemia cell with a
lectin or an antibody recognizing a desialylated epitope or an
epitope lacking a sialic acid but which binds not or significantly
less the sialylated form of the epitope, and (b) isolating cells
bound to said lectin or antibody, and (c) culturing the isolated
cells for a suitable time, and (d) select a cell, cells or a cell
clone which strongly binds to a lectin or an antibody which binds
to an epitope with sialic acid.
[0163] More preferred is the method as described above, wherein
said lectin or said antibody recognizing a desialylated epitope or
an epitope lacking a sialic acid is Nemod-TF1, Nemod-TF2, A78-G/A7,
or PNA and wherein said human myeloid leukaemia cell is the cell
line K562, KG-1, MUTZ-3, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605],
NM-H9, H9, NM-H10.
[0164] The method for generating a host cell of the invention with
high sialylation and favourable biotechnological properties such as
rapid cell growth comprising (i) incubating a human myeloid
leukaemia cell of the invention as originator cell, preferably
K562, with a lectin or preferably an antibody recognizing a
desialylated epitope or an epitope lacking a sialic acid but which
binds not or less the sialylated form of the epitope, such as but
not limited to Nemod-TF1, Nemod-TF2, A78-G/A7, or PNA (lectin from
Arachis hypogaea, peanut agglutinin), preferably bound to magnetic
beads, and (ii) isolating cells bound to said lectin or antibody,
and (iii) culturing the isolated cells for a suitable time, and
(iv) select a cell, cells or a cell clone, preferably after single
cell cloning, which strongly binds to a lectin or an antibody which
binds to an epitope with sialic acid, such as SNA (Sambucus nigra
agglutinin) or MAL (Maackia amurensis lectin), MAL I (Maackia
amurensis lectin I), preferably SNA.
[0165] In a preferred embodiment the invention provides a method
for generating a host cell of the invention with high sialylation
and favourable biotechnological properties such as rapid cell
growth comprising (i) incubating K562 with Nemod-TF1, Nemod-TF2,
A78-G/A7, or PNA bound to magnetic beads, and (ii) isolating cells
bound to said lectin or antibody, and (iii) culturing the isolated
cells for a suitable time of about one to 6 weeks, and (iv) select
a cell clone after single cell cloning, which strongly binds to
SNA. In a preferred embodiment those cell bind stronger to SNA than
the originator cell, and in another preferred embodiment they grow
faster than the originator cell.
[0166] In a preferred embodiment of the invention the originator
cell is treated with ethylmethanesulfonate prior to step (i).
[0167] Those skilled in the art are able to select suitable
conditions and methods and optimise them in order to generate these
host cells in sense of the invention. More details for some of the
steps can be found under WO2005/017130 A2 and WO2005/080585 A1.
[0168] According to one embodiment, immortalised human blood cells
are used as host cells, which are selected from the following
groups 1 to 4: [0169] (a) group 1, comprising host cells having a
high sialylation activity such as K562; [0170] (b) group 2,
comprising host cells having due to a genetic deficiency or
expression inhibition means (e.g. RNAi) a low or no sialylation;
activity comparable to and including NM-F9 [DSM ACC2606], NM-D4
[DSM ACC2605] and GTX-2; [0171] (c) group 3, comprising host cells
having a higher sialylation degree than K562 such as NM-H9 and
NM-H9D8; [0172] (d) group 4, comprising host cells having a low or
even no fucosylation activity such as NM-H9D8-E6 and NM
H9D8-E6Q12.
[0173] In a preferred embodiment, said the cell line generated is
NM-H9D8. As the most preferred cell clone generated by the method
described above, NM-H9D8 was selected and deposited under DSM ACC
2806 at the "DSMZ-Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH" in Braunschweig (Germany), by Glycotope GmbH,
Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Sep. 15, 2006.
Other cell clones such as NM-E-2F9, NM-C-2F5, or NM-H9D8-E6 (DSM
ACC 2807) were selected by incubation of the parental cells with
one or more lectins and following single cell cloning using flow
cytometry cell sorting whereby a positive selection procedure or a
combination of negative and positive selection was performed. To
get cell clones with stable characteristics obtained cell clones
were recloned at least once by limited dilution as described
above.
[0174] In a preferred embodiment of the invention said immortalized
human blood cell which is preferably a host cell of myeloid
leukaemia origin, grows and produces the protein/antibody molecule
composition of the invention under serum-free conditions. In an
even further preferred embodiment said immortalized human blood
cell which is preferably a host cell of myeloid leukaemia origin
grows under serum-free conditions. Furthermore, also the nucleic
acid encoding the protein/antibody molecule can be introduced in
these cells and the protein/antibody molecule composition can also
be isolated under serum-free conditions. To be able to work under
serum-free conditions is particularly important when preparing
therapeutic proteins, as serum contaminations are unacceptable in
the regulatory process.
[0175] In a preferred embodiment of the invention the host cell of
human myeloid leukaemia origin of the invention is the cell or cell
line K562, KG1, MUTZ-3, NM-F9 [deposited under DSM ACC 2606 at the
"DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH"
in Braunschweig (Germany), by Nemod Biotherapeutics GmbH &
Co.KG, Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Aug. 14,
2003], NM-D4 [deposited under DSM ACC 2605 at the "DSMZ-Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH" in Braunschweig
(Germany), by Nemod Biotherapeutics GmbH & Co.KG,
Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Aug. 14, 2003] or
a cell or cell line derived from any one of said host cells, or a
mixture of cells or cell lines comprising at least one of those
aforementioned cells. The host cell is preferably selected from the
group consisting of NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605],
NM-E-2F9, NM-C-2F5, NM-H9D8 [deposited under DSM ACC 2806 at the
"DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH"
in Braunschweig (Germany), by Glycotope GmbH, Robert-Rossle-Str.
10, 13125 Berlin (Germany) on Sep. 15, 2006], or NM-H9D8-E6
[deposited under DSM ACC 2807 at the "DSMZ-Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH" in Braunschweig (Germany),
by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin (Germany) on
Oct. 5, 2006], or NM H9D8-E6Q12 [deposited under DSM ACC 2856 at
the "DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH" in Braunschweig (Germany), by Glycotope GmbH,
Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Aug. 8, 2007],
GT-2X [deposited under DSM ACC 2858 at the "DSMZ-Deutsche Sammlung
von Mikroorganismen and Zellkulturen GmbH" in Braunschweig
(Germany), by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin
(Germany) on Sep. 7, 2007] or a cell or cell line derived from any
of these cell lines.
[0176] The cells NM-F9 [DSM ACC2606] and NM-D4 [DSM ACC2605] were
deposited by Nemod Biotherapeutics GmbH & Co.KG,
Robert-Rossle-Str. 10, 13125 Berlin, Germany, who authorised the
applicant to refer to the deposited biological material described
herein.
[0177] In a further preferred embodiment of the invention the host
cell of human myeloid leukaemia origin of the invention is the cell
or cell line K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], or a
cell or cell line derived from anyone of said host cells.
[0178] In a preferred embodiment of the invention the host cell of
human myeloid leukaemia origin of the invention is the cell or cell
line K562, such as K562 [ATCC CCL-243], or a cell or cell line
derived from said host cell.
[0179] In a further preferred embodiment of the invention the host
cell of human myeloid leukaemia origin of the invention is the cell
or cell line K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC 2605],
NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6, or GT-2X, or NM
H9D8-E6Q12 or a cell or cell line derived from anyone of said host
cells.
[0180] In a preferred embodiment of the invention the host cell of
human myeloid leukaemia origin of the invention is the cell or cell
line K562, KG1, MUTZ-3, NM-F9 [deposited under DSM ACC 2606 at the
"DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH"
in Braunschweig (Germany), by Nemod Biotherapeutics GmbH &
Co.KG, Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Aug. 14,
2003], NM-D4 [deposited under DSM ACC 2605 at the "DSMZ-Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH" in Braunschweig
(Germany), by Nemod Biotherapeutics GmbH & Co.KG,
Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Aug. 14, 2003] or
a cell or cell line derived from any one of said host cells, or a
mixture of cells or cell lines comprising at least one of those
aforementioned cells. The host cell is preferably selected from the
group consisting of NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605],
NM-E-2F9, NM-C-2F5, NM-H9D8 [deposited under DSM ACC 2806 at the
"DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH"
in Braunschweig (Germany), by Glycotope GmbH, Robert-Rossle-Str.
10, 13125 Berlin (Germany) on Sep. 15, 2006], or NM-H9D8-E6
[deposited under DSM ACC 2807 at the "DSMZ-Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH" in Braunschweig (Germany),
by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin (Germany) on
Oct. 5, 2006], or NM H9D8-E6Q12 [deposited under DSM ACC 2856 at
the "DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH" in Braunschweig (Germany), by Glycotope GmbH,
Robert-Rossle-Str. 10, 13125 Berlin (Germany) on Aug. 8, 2007],
GT-2X [deposited under DSM ACC 2858 at the "DSMZ-Deutsche Sammlung
von Mikroorganismen and Zellkulturen GmbH" in Braunschweig
(Germany), by Glycotope GmbH, Robert-Rossle-Str. 10, 13125 Berlin
(Germany) on Sep. 7, 2007] or a cell or cell line derived from any
of these cell lines.
[0181] According to one embodiment, the immortalized human blood
cell and the host cell of human myeloid leukaemia origin is not one
of the sialylation deficient cell lines NM-F9 and NM-D4 or a cell
or cell line derived from anyone of said host cells having the same
properties. This embodiment is beneficial in case a high degree of
sialylation is aimed at. For these embodiments, the host cell is
preferably selected from the group consisting of K562, NM H9D8, NM
H9D8-E6, NM H9D8-E6Q12 and host cells derived from any of these
host cells.
[0182] The cell lines described in conjunction with the present
invention have a doubling time from 14 to 24 hours which is very
fast compared to other mammalian expression system, depending on
the cell line of the invention.
[0183] Furthermore no general suitable high expression vector
system is known for high yield antibody expression in human cell
lines since human cells normally do not lack the DHFR gene and
hence do not allow the use of the dhfr/methotrexate amplification
system. Hence, according to a further embodiment of the present
invention an embodiment is provided, which allows to increase the
product yield.
[0184] According to this embodiment, additionally a nucleic acid is
introduced in the host cell, encoding an antifolate resistant
DHFR-variant. The dihydrofolate reductase, or DHFR, reduces
dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron
donor, which can be converted to the kinds of tetrahydrofolate
cofactors used in 1-carbon transfer chemistry. Antifolates inhibit
the DHFR enzyme, leading to cell death. To provide a nucleic acid
encoding an antifolate-resistant DHFR variant, a tool is provided
to select cells which were transfected with the nucleic acid and
furthermore, allows an amplification of the nucleic acids to be
expressed in the host cells.
[0185] The nucleic acid encoding said antifolate resistant
DHFR-variant can e.g. be introduced via a separate vector than the
nucleic acid encoding the protein/antibody to be expressed in the
host cell. It is preferred to transfect the vector encoding the
antifolate resistant DHFR-variant basically at the same time as the
vector comprising the nucleic acid encoding the protein/antibody.
This embodiment encourages that the nucleic acid encoding said
protein/antibody to be expressed is integrated in the genome of the
host cell at the same genetic site as the nucleic acid encoding the
antifolate resistant DHFR-variant which is beneficial in case an
amplification of the nucleic acid encoding said protein/antibody is
desired.
[0186] Alternatively, a vector system may be used which comprises
the nucleic acid encoding at least a part of said protein to be
expressed as well as the nucleic acid encoding the antifolate
resistant DHFR-variant.
[0187] The host cells are then cultured with said antifolate. This
has the effect that those host cells, which were successfully
transfected with the nucleic acid encoding said antifolate
resistant DHFR-variant can grow despite the presence of the
antifolate. Thereby successfully transfected cells can be
selected.
[0188] According to a further embodiment, the nucleic acid sequence
encoding at least part of said protein/antibody is amplified by
stepwise increasing the antifolate concentration in the culture.
The increase of the antifolate concentration in the culture medium
leads to an increase of the copies of the antifolate resistant
DHFR-variant in the genome. It is assumed that this is achieved by
recombination events in the cells. Thereby, also the copy number of
the nucleic acid encoding at least part of the protein to be
expressed is also increased if the nucleic acid encoding said
protein is located near the antifolate resistant DHFR variant in
the genome what can be promoted by either transfecting separate
vectors simultaneous or by using one vector comprising both nucleic
acid sequences. By this mechanism host cells are obtained, which
express the protein/antibody at a higher yield.
[0189] Preferably, the antifolate is methotrexate.
[0190] The nucleic acid sequence encoding said protein, preferably
an antibody molecule or a part thereof, is preferably amplified by
culturing said host cell with at least two successive rounds of
antifolate, preferably methotrexate, whereby the concentration of
said antifolate, preferably methotrexate, is increased by at least
100% in each successive round.
[0191] A suitable nucleic acid for providing said antifolate
resistant DHFR-variant encodes a polypeptide of the group of
sequence ID No. 1 to 9, preferably sequence ID No 1.
[0192] Further details and embodiments on this amplification system
are also described in further detail below.
[0193] The invention also provides a method for producing a
protein, preferably an antibody molecule composition comprising:
[0194] (a) introducing in a host cell of human myeloid leukaemia
origin at least one nucleic acid encoding a protein and preferably
an antibody 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 of the group of sequence #1 to sequence
#9, preferably sequence #1; and [0195] (b) amplifying the nucleic
acid sequence encoding said protein, preferably an antibody
molecule or at least one part thereof by culturing said host cell
with methotrexate, preferably by culturing said host cell with at
least two successive rounds of methotrexate whereby the
concentration of methotrexate is preferably increased by at least
about 50%, more preferably by at least about 100%, in each
successive round; and [0196] (c) culturing said host cell under
conditions which permits the production of said protein, preferably
an antibody molecule composition, and [0197] (d) isolating said
protein, which preferably is an antibody molecule composition.
[0198] The invention also provides a method for producing a protein
composition, preferably an antibody molecule composition having
increased activity and/or increased yield and/or improved
homogeneity comprising: [0199] (a) introducing in a host cell of
human myeloid leukaemia origin at least one nucleic acid encoding
protein, preferably an antibody molecule or at least one part
thereof; and [0200] (b) culturing said host cell under conditions
which permits the production of said protein composition, which
preferably is an antibody molecule composition; and [0201] (c)
isolating said protein composition, which preferably is an antibody
molecule composition having increased activity and/or increased
yield and/or improved homogeneity.
[0202] Furthermore, the invention provides a method for producing
protein composition, preferably an antibody molecule composition
having increased activity and/or increased yield and/or improved
homogeneity comprising: [0203] (a) introducing in a host cell of
human myeloid leukaemia origin at least one nucleic acid encoding a
protein, preferably an antibody 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 of the
group of sequence #1 to sequence #9, preferably sequence #1; and
[0204] (b) amplifying the nucleic acid sequence encoding said
antibody molecule or at least one part thereof by culturing said
host cell with methotrexate, preferably by culturing said host cell
with at least two successive rounds of methotrexate whereby the
concentration of methotrexate is preferably increased by at least
about 50%, more preferably by at least about 100%, in each
successive round; and [0205] (c) culturing said host cell under
conditions which permits the production of said protein
composition, which preferably is an antibody molecule composition;
and [0206] (d) isolating said protein composition, which preferably
is an antibody molecule composition having increased activity
and/or increased yield and/or improved homogeneity.
[0207] Said nucleic acid encoding an antibody molecule or at least
one part of it and said nucleic acid comprising at least one
nucleic acid sequence encoding at least one polypeptide of the
group of sequence #1 to sequence #9, preferably sequence #1, can be
one nucleic acid or two separate nucleic acids.
[0208] In order to select a suitable host cell for obtaining a
protein having an optimised glycosylation profile, it is
advantageous to perform the screening/selection method according to
the present invention. After a suitable host cell is determined by
the selection method according to the present invention, said host
cell is then used for producing the protein as defined in claims 1
to 20.
[0209] Hence, the invention also provides a method for selecting a
host cell for producing a protein having at least one of the
following glycosylation characteristics: [0210] (i) it comprises no
detectable NeuGc; and/or [0211] (ii) 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; and/or [0212] (iii) 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; and/or [0213] (iv) 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; and/or [0214]
(v) it comprises no detectable terminal Galalpha1-3Gal; and/or
[0215] (vi) 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/or [0216] (vii) it comprises at least one carbohydrate
structure containing bisecting GlcNAc; and/or [0217] (viii) 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; by the following steps [0218] (a) introducing in
at least two different immortalized human blood cells as host cells
at least one nucleic acid encoding a protein or at least one part
thereof; and [0219] (b) culturing said at least two different host
cells, wherein each different host cell produces a protein
composition having a glycosylation pattern diverging from the
glycosylation pattern produced by the other host cell; [0220] (c)
isolating said expressed protein compositions carrying a different
glycosylation pattern from the at least two different host cells;
and [0221] (d) selecting said host cell producing a protein
composition which as at least one of the glycosylation
characteristics defined in (i) to (viii).
[0222] The details regarding the glycosylation pattern and suitable
cell lines for obtaining said pattern are described above and are
also applicable to and suitable for the screening method for
selecting a suitable host cell according to the present
invention.
[0223] To perform a respective screening step prior to establishing
the production method as described in claims 1 to 20 is
advantageous as this embodiment allows the selection of the most
suitable host cell for producing the protein/antibody molecule
composition having an optimised glycosylation pattern. According to
the basic idea of this screening system, the protein of interest is
expressed in at least two different cell lines which have a
diverging glycosylation pattern. E.g. one cell line may depict a
high degree of sialylation and the other one may depict a low
degree of sialylation (or fucosylation and/or galactosylation) or
even unknown glycosylation characteristics. The products obtained
from the different cell lines accordingly carry a glycosylation
pattern characteristic for the respective cell line.
[0224] The characteristics of the proteins produced in the
different cell lines e.g. with respect to their activity (e.g. ADCC
and CDC in antibodies), affinity, serum half-life and other
important characteristics can then be determined. The results allow
to choose the cell line, which has the best glycosylation machinery
in order to obtain a protein which is optimised regarding its
glycosylation pattern. As the decisive characteristics (e.g.
affinity, serum half-life etc.) vary, this method is particularly
advantageous.
[0225] Preferably, said protein to be produced depicts at least one
of the following characteristics: [0226] (a) in case it is an
antibody molecule composition 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]; [0227] and/or [0228] (b) in
case it is an antibody molecule composition it has an increased
antigen mediated or Fc-mediated binding which is at least 50%
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]; [0229] and/or [0230] (c) it
has an increased average or maximum yield of said protein molecule
composition 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].
[0231] According to one embodiment, at least one of said host cells
is an immortalised human blood cell and preferably a cell of human
myeloid leukaemia origin such as K562, NM-F9 [DSM ACC2606], NM-D4
[DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived therefrom.
[0232] Said at least one host cell of human myeloid leukaemia
origin can be selected from one of the following groups 1 to 4:
[0233] (a) group 1, comprising host cells having a high sialylation
activity such as K562 or a cell or cell lined derived therefrom,
[0234] (b) group 2, comprising host cells due to a genetic
deficiency or expression suppression means (e.g. RNAi) a low or no
sialylation such as NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605] and
GTX-2 or a cell or cell lined derived therefrom, [0235] (c) group
3, comprising host cells having a higher sialylation degree than
K562 such as NM-H9 and NM-H9D8 or a cell or cell lined derived
therefrom, [0236] (d) group 4, comprising host cells having a low
or no fucosylation activity such as NM-H9D8-E6 or a cell or cell
lined derived therefrom.
[0237] Preferably, at least two or three host cells used in the
screening process are selected from the above groups. However, it
is also possible to include other host cells derived from another
origin (e.g. Hek 293 cells) in the screening system according to
the present invention in order to further broaden the different
glycosylations patterns analysed.
[0238] The host cell obtained preferably produces a protein, in
particular an antibody depicting at least one of the glycosylation
patterns shown in Table 9.
[0239] According to the invention the term introducing a nucleic
acid means any method known to those skilled in the art to
introduce a nucleic acid, or two or more nucleic acids into a
mammalian host cell or cells by methods such as but not limited to
electroporation, transfection using cationic lipids, calcium
phosphate, DEAE-dextran, or infection by virus particles such as
adenoviruses or retroviruses or a combination thereof. Those
skilled in the art are able to select and optimise a suitable
method for introduction of one or more nucleic acids of the
invention.
[0240] In accordance with the present invention the nucleic acid
encoding an antibody molecule is any nucleic acid which encodes the
antibody molecule or at least one part thereof. The antibody
molecule of the invention can thereby be encoded by a single or by
multiple nucleic acid molecules.
[0241] Said part of an antibody molecule encoded by said nucleic
acid comprises at least one 20 amino acid sequence, more preferably
at least 100 amino acids of an antibody molecule or of a constant
and/or variable domain of the antibody molecule. The comprised
sequence encoding the antibody molecule or at least one part
thereof can be separated by at least one other sequence, such as
e.g. an intron. The sequence of a domain can comprise at least one
amino acid mutation.
[0242] In accordance with the present invention the nucleic acid
encoding a protein molecule is any nucleic acid which encodes the
protein molecule or at least one part thereof. The protein molecule
of the invention can thereby be encoded by a single or by multiple
nucleic acid molecules. The sequence encoding the protein molecule
or at least one part thereof can be separated by at least one other
sequence, such as e.g. an intron. The sequence of the protein
molecule can comprise at least one amino acid mutation.
[0243] In a preferred embodiment the nucleic acid encoding an
antibody molecule or at least one part of it comprises at least one
variable and/or at least one constant domain of the antibody
molecule, more preferably both, more preferably such which
comprises a human sequence at least in the part of the constant
domain or domains, and even more preferred such which comprises the
human Cgamma2 domain, and most preferably it comprises all constant
domains of the Fc region of a human antibody of a certain class or
subclass and the variable domain.
[0244] According to one embodiment the protein and in particular
antibody molecule is encoded by a single nucleic acid. In another
preferred embodiment protein, in particular an antibody molecule is
encoded by two nucleic acids or by three nucleic acids.
[0245] In a further preferred embodiment one nucleic acid encodes
one part of the antibody molecule which encodes for the variable
and/or the constant domain of the light chain and another nucleic
acid encodes for another part of the antibody molecule which
encodes for the variable and/or at least one constant domain of the
heavy chain.
[0246] In accordance with the present invention the nucleic acid
comprising at least one nucleic acid sequence encoding at least one
polypeptide of the group of sequence #1 to sequence #9 means that
said nucleic acid encodes for at least one polypeptide of the group
of sequence #1 to sequence #9, preferably sequence #1. Any number
of these sequences is suitable, as long as it can be introduced
successfully into the host cell of the invention. In a preferred
embodiment said nucleic acid encodes for one, two or three
polypeptides of the group of sequence #1 to sequence #9, more
preferably for one polypeptide, and most preferably for the
polypeptide of sequence #1. In sense of the invention said nucleic
acid can also encode for a polypeptide of the group of sequence #1
to sequence #9, preferably sequence #1, which has at least one
amino acid mutation as long as this mutation allows the
amplification of the nucleic acid encoding an antibody molecule or
at least one part thereof by methotrexate as described elsewhere
herein.
[0247] The nucleic acid sequence encoding at least one polypeptide
of the group of sequence #1 to sequence #9, preferably sequence #1,
can be part of the same nucleic acid molecule as the nucleic acid
encoding an antibody molecule or at least one part thereof as
described elsewhere herein or on separate nucleic acid
molecules.
[0248] In another preferred embodiment of the invention a separate
nucleic acid comprising a nucleic acid sequence encoding a sequence
selected from the group of sequence #1 to sequence #9, most
preferably sequence #1, can be introduced into the host cell of the
invention separately from said nucleic acid or nucleic acids
encoding the antibody molecule or parts thereof of the invention.
This can be done either by introducing said separate nucleic acid
comprising a nucleic acid sequence encoding a sequence selected
from the group of sequence #1 to sequence #9, together, before or
after introducing said nucleic acid or nucleic acids encoding the
antibody molecule or parts thereof of the invention. In a preferred
embodiment this is performed in parallel and in another preferred
embodiment the host cell of the invention already comprises said
separate nucleic acid comprising a nucleic acid sequence encoding a
sequence selected from the group of sequence #1 to sequence #9,
most preferably sequence #1.
[0249] Further preferred embodiments are described in the
examples.
[0250] Amplification of stably introduced said nucleic acid or
several copies of said nucleic acid encoding the protein, which is
preferably an antibody molecule or fraction thereof can be
performed as described elsewhere herein and in the examples using
methotrexate.
[0251] In a preferred embodiment the nucleic acid encoding a
protein, which is preferably an antibody molecule or at least one
part thereof comprise at least one other genetic element which
allow the selection of those cells in which the nucleic acid was
successfully introduced, such as but not limited to antibiotic
resistance genes, such as but not limited to the genetic element
coding for a puromycin or neomycin resistance. Furthermore these
nucleic acids comprise one or several genetic elements such as
promoters, enhancers, polyadenylation sites, and/or introns, for
expression of the antibody molecule in the host cells of the
invention, and genetic elements such as bacterial origin of
replication, promoters and elements for selection of transfected
bacteria such as antibiotic resistance genes for multiplying the
nucleic acid in a bacteria.
[0252] Suitable promoters include the promoter of IE (immediate
early) gene of cytomegalovirus (CMV), SV40 early promoter, the
promoter of a retrovirus, metallothionein promoter, heat shock
promoter, SR alpha promoter, EF-1alpha promoter, etc. The enhancer
of IE gene of human CMV may be used in combination with the
promoter.
[0253] Those and further genetic elements are known to those
skilled in the art and can be selected, combined, optimised and
introduced into said nucleic acid encoding a protein, which is
preferably antibody molecule or at least one part thereof by those
skilled in the art. Preferred embodiments of said nucleic acid
encoding a protein, which is preferably an antibody molecule or at
least one part thereof or a combination of nucleic acids are
described herein and in the examples as well as preferred genetic
elements for use and combination, however, the invention is not
restricted to the use of those and can be combined or further
optimised by those skilled in the art.
[0254] Those skilled in the art are able to select and/or combine
the suitable genetic element, construct the according nucleic acid
vectors or elements to introduce one or more nucleic acids of the
invention into a cell line according to the invention.
[0255] Said nucleic acid or combination of nucleic acids encoding
the protein, which is preferably an antibody molecule or at least
one part thereof, as well as said additional genetic elements, as
well as the methods to introduce them such as transfecting them in
the host cells of the invention for expression of the antibody
molecule composition or into bacteria for multiplication are known
to those skilled in the art as well as the methods to construct,
identify, test, optimise, select and combine these nucleic acid or
acids and combine them with suitable additional sequences for
selection of those cells which are successfully transfected as well
as methods to construct, identify, test and select most suitable
nucleic acids encoding these antibody molecules are known to those
skilled in the art.
[0256] Preferred embodiments of the invention are described in
detail in the examples.
[0257] In a preferred embodiment of the invention the nucleic acid
encoding a protein, which is preferably an antibody molecule or at
least one part thereof comprises a genetic element for selection of
those host cells of the invention in which the nucleic acid is
successfully introduced such as but not limited to neomycin or
puromycin, more preferably it comprises in addition a promoter such
as EF-1alpha or CMV promoter, more preferably it comprises in
addition a CMV enhancer, more preferably it comprises in addition a
genetic element which allows the selection of bacteria which are
transfected with the nucleic acid and a genetic element for
replication such as the ColE1 replication origin for multiplication
of said nucleic acid in bacteria, and even more preferably it
comprises in addition a genetic element for multiplication of said
the nucleic acid in COS cells such as the SV40 origin. These
elements are known to those skilled in the art and can be selected,
combined, optimised and used by those skilled in the art.
[0258] In an even further preferred embodiment of the invention
said above described nucleic acid encoding a protein molecule or at
least one part thereof comprises one nucleic acid sequence.
Preferably said nucleic acid encoding protein molecule or at least
one part thereof encodes additionally for the genetic element
encoding puromycin resistance, or neomycin resistance, or a nucleic
acid sequence encoding at least one sequence selected from the
group of sequence #1 to sequence #9, most preferably sequence
#1.
[0259] In an even further preferred embodiment of the invention
said above described nucleic acid encoding an antibody molecule or
at least one part thereof comprises at least one sequence encoding
the variable domain and at least one constant domain of the heavy
and/or the light chain of the antibody molecule.
[0260] In an even further preferred embodiment of the invention
said above described nucleic acid encoding an antibody molecule or
at least one part thereof comprises at least one sequence encoding
the variable domain and constant domain of the light chain of the
antibody molecule or the variable domain and all constant domains
of the heavy chain of the whole antibody molecule.
[0261] In an even further preferred embodiment of the invention one
of said above described nucleic acid encodes at least one part of
the antibody molecule comprising at least one sequence encoding the
variable domain and the constant domain of the light chain of the
antibody molecule and a second of said above described nucleic acid
encodes at least one other part of the antibody molecule comprising
at least one sequence encoding the variable domain and at least one
constant domain, preferably all constant domains of the heavy chain
of the antibody molecule, and both nucleic acids are introduced
into the same host cell of the invention.
[0262] Preferably one of said nucleic acids encodes additionally
for the genetic element encoding puromycin resistance and the other
said nucleic acid encodes additionally for the genetic element
encoding neomycin resistance.
[0263] In an even further preferred embodiment of the invention
said above described nucleic acid encoding a protein molecule or at
least one part thereof comprises at least two nucleic acid
sequences encoding two amino acid sequences of the protein molecule
or at least one part thereof.
[0264] Preferably one of said nucleic acids encodes additionally
for the genetic element encoding puromycin resistance, one other
said nucleic acid encodes additionally for the genetic element
encoding neomycin resistance. More preferably one of said nucleic
acids encodes additionally for the genetic element encoding
puromycin or neomycin resistance, preferably puromycin resistance,
and one other said nucleic acid comprises in addition a nucleic
acid sequence encoding at least one sequence selected from the
group of sequence #1 to sequence #9, most preferably sequence
#1.
[0265] In an even further preferred embodiment of the invention
said above described nucleic acid encoding a protein molecule or at
least one part thereof comprises three nucleic acid sequences
encoding three amino acid sequences of the protein molecule or at
least one part thereof.
[0266] Preferably one of said nucleic acids encodes additionally
for the genetic element encoding puromycin resistance, one other
said nucleic acid encodes additionally for the genetic element
encoding neomycin resistance, and one other said nucleic acid
comprises in addition a nucleic acid sequence encoding at least one
sequence selected from the group of sequence #1 to sequence #9,
most preferably sequence #1.
[0267] In an even more preferred embodiment one of said nucleic
acids encodes additionally for the genetic element encoding a
puromycin or neomycin resistance and the other said nucleic acid
comprises in addition a nucleic acid sequence encoding at least one
sequence selected from the group of sequence #1 to sequence #9,
most preferably sequence #1. In a even more preferred embodiment
one of said nucleic acids comprising sequences encoding the
variable domain and the constant domain of the light chain and
comprises in addition a nucleic acid sequence encoding at least one
sequence selected from the group of sequence #1 to sequence #9,
most preferably sequence #1, and the other said nucleic acid
comprises sequences encoding the variable domain and at least one
constant domain, preferably all constant domains, of the heavy
chain of the antibody molecule and comprises in addition a nucleic
acid sequence encoding for a puromycin or neomycin resistance,
preferably puromycin resistance.
[0268] In an even further preferred embodiment of the invention
said nucleic acid encoding the antibody molecule or at least one
part thereof comprises at least one sequence encoding the variable
domain and the constant domain of the light chain of the antibody
molecule and at least one sequence encoding the variable domain and
at least one constant domain, preferably all constant domains of
the heavy chain of the antibody molecule.
[0269] In a preferred embodiment of the invention the constant
domains encoded by above or elsewhere described nucleic acids of
the invention are human constant domains of IgG or IgM, preferably,
human IgG1, IgG4 or IgM, or one domain or a combination of domains
thereof, whereby preferably genomic sequences or sequences derived
from genomic sequences comprising at least one intron are used,
which can be selected, constructed and optimised by those skilled
in the art.
[0270] In a further preferred embodiment of the invention said
nucleic acid encoding a protein, which is preferably an antibody
molecule or at least one part thereof of the invention further
comprises the EF-1alpha promoter/CMV enhancer or a CMV promoter
derived promoter, preferably CMV-E.
[0271] In a further preferred embodiment of the invention said
nucleic acid encoding a protein, which is preferably an antibody
molecule or at least one part thereof of the invention further
comprises a secretion signal peptide, preferably the T cell
receptor secretion signal.
[0272] In an even further preferred embodiment of the invention
said nucleic acid encoding a protein, which is preferably an
antibody molecule or at least one part thereof of the invention
further comprises a secretion signal peptide, preferably the
sequence #10.
[0273] Said nucleic acid or combination of nucleic acids encoding a
protein, which is preferably an antibody molecule or at least one
part thereof, as well as said additional genetic elements, as well
as the methods to introduce them are known to those skilled in the
art as well as the methods to construct, identify, test, optimise,
select and combine these nucleic acid or acids and combine them
with suitable additional sequences for selection of those cells
which are successfully transfected as well as methods to construct,
identify, test and select most suitable nucleic acids encoding
these antibody molecules are known to those skilled in the art.
[0274] Preferred embodiments of the invention are described in
detail in the examples.
[0275] The introduction of the nucleic acid can be either transient
or stable.
[0276] In accordance with the present invention the term amplifying
the nucleic acid sequence encoding a protein or antibody molecule
or at least one part thereof by culturing said host cell with an
antifolate, in particular methotrexate means that a host cell of
the invention described elsewhere herein in which at least one
nucleic acid encoding the protein to be expressed such as e.g. an
antibody molecule or at least one part thereof, and at least one
nucleic acid comprising at least one nucleic acid sequence encoding
an antifolate resistant DHFR variant, preferably at least one
polypeptide of the group of sequence #1 to sequence #9, preferably
sequence #1, was introduced an is cultivated by at least one
antifolate, preferably methotrexate concentration. A typical
cultivation time is between one to two weeks for each round, at
typical concentrations from about 20 nM bis 3000 nM, preferably
between about 50 nM to 2000 nM, more preferably between about 100
nM to 2000 nM. The duration of an antifolate/methotrexate
amplification treatment as well as the concentration and the
according vectors of nucleic acids of the invention can be
optimised by those skilled in the art and is also described in a
preferred embodiment in the examples. Optimal amplification
conditions can differ between different proteins/antibody molecules
encoded and usage of different nucleic acids or nucleic acid
constructs or combinations thereof described afore. Those skilled
in the art are able to select and optimise the most suitable
conditions and nucleic acids of the invention. Said amplification
leads to an integration of more nucleic acid copies encoding the
protein/antibody molecule or at least one part thereof into the
genome of the host cell than without antifolate/methotrexate
cultivation or than without the introduction of the nucleic acid
sequence encoding at least one antifolate resistant DHFR variant,
in particular a polypeptide of the group of sequence #1 to sequence
#9 and/or it leads to an increased production of the
protein/antibody molecule composition.
[0277] In a preferred embodiment of the invention said host cell
for amplification of the nucleic acid or nucleic acids encoding a
protein/antibody molecule or at least one part thereof, which was
introduced in said host cell in which at least one nucleic acid
comprising at least one nucleic acid sequence encoding at least one
polypeptide of the group of sequence #1 to sequence #9, preferably
sequence #1, was introduced, as described elsewhere herein
including its preferred embodiments, are a human myeloid leukaemia
origin, preferably the cell or cell line KG1, MUTZ-3, K562, NM-F9
[DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line derived
from anyone of said host cells, more preferably the cell or cell
line K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9,
NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or
cell line derived from anyone of said host cells, and even more
preferably the cell or cell line NM-F9 [DSM ACC2606], NM-D4 [DSM
ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived from anyone of
said host cells.
[0278] In a preferred embodiment of the invention said host cell is
cultivated with at least two successive rounds of
antifolate/methotrexate whereby the concentration of
antifolate/methotrexate is preferably increased by at least about
50%, more preferably by at least about 100%, in each successive
round. In an even further preferred embodiment of the invention
said host cell is cultivated with at least three, more preferably
with four, more preferably with 5, and even more preferably with 6
successive rounds of antifolate/methotrexate, whereby the
concentration of antifolate/methotrexate is preferably increased by
at least about 50%, more preferably by at least about 100%, in each
successive round. Even more preferred are concentrations of
antifolate/methotrexate between about 20 nM and 3000 nM, more
preferred between about 50 nM to 2000 nM, more preferably between
about 100 nM to 2000 nM, and even more preferred of about 100 nM,
200 nM, 500 nM, 1000 nM, 2000 nM which are in the preferred
embodiment are used in successive rounds starting from 100 nM.
[0279] It is surprising that the introduction of a nucleic acid
sequence encoding an antifolate resistant DHFR-variant and
preferably at least one polypeptide of the group of sequence #1 to
sequence #9 allows the amplification of the nucleic acid sequence
encoding the said protein/antibody molecule or at least one part
thereof in the host cell of human myeloid leukaemia origin of the
invention, and especially in K562, NM-F9 [DSM ACC2606], NM-D4 [DSM
ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived from anyone of
said host cells, by culturing with antifolate/methotrexate.
[0280] It is especially surprising that the introduction of a
nucleic acid sequence encoding at least one polypeptide of sequence
#1 allows the amplification of the nucleic acid sequence encoding
the said antibody molecule or at least one part thereof in the host
cell of the invention, and especially in K562, NM-F9 [DSM ACC2606],
NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived from anyone of
said host cells, by culturing with methotrexate.
[0281] It is even more surprising that the introduction of a
nucleic acid sequence encoding at least one polypeptide of the
group of sequence #1 to sequence #9 allows an even further
amplification of the nucleic acid sequence encoding the said
antibody molecule or at least one part thereof in the host cell of
the invention, and especially in K562, NM-F9 [DSM ACC2606], NM-D4
[DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived from anyone of
said host cells, by culturing said host cell with at least two
successive rounds of methotrexate whereby the concentration of
methotrexate is increased by at least about 50%, more preferably by
at least about 100%, in each successive round.
[0282] It is even more surprising that the introduction of a
nucleic acid sequence encoding at least one polypeptide of sequence
#1 allows an even further amplification of the nucleic acid
sequence encoding the said antibody molecule or at least one part
thereof in the host cell of the invention, and especially in K562,
NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5,
NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line
derived from anyone of said host cells, by culturing said host cell
with at least two successive rounds of methotrexate whereby the
concentration of methotrexate is increased by at least about 50%,
more preferably by at least about 100%, in each successive
round.
[0283] In a preferred embodiment, the term that amplification is
stable means that it leads to the production of high yields of the
antibody molecule composition over at least 35 generations of host
cell division cycles. Amplification of stably introduced said
nucleic acid or nucleic acids encoding the protein/antibody
molecule or fraction thereof can be performed as described above
and in the examples using methotrexate.
[0284] Further preferred embodiments are described in the
examples.
[0285] In a preferred embodiment of the invention the host cell of
the invention with introduced nucleic acids of the invention are
preferably used after at least one round of single cell cloning and
selection of those cell clones with suitable expression and
secretion of said protein/antibody composition. Preferably said
single cell cloning and selection of those cell clones with
suitable expression and secretion of said protein/antibody
composition occurs after at least one round of methotrexate
amplification described elsewhere herein. In a further preferred
embodiment, said cell clones are further amplified by at least one
additional round of amplification with methotrexate, preferable an
increased concentration of methotrexate, preferably at least about
the double concentration of methotrexate, and even more preferred
followed by a further round of single cell cloning and selection of
those cell clones with suitable expression and secretion of said
protein/antibody composition. With these preferred embodiments cell
clones with particularly high expression yields can be
selected.
[0286] In accordance with the present invention the term culturing
said host cell under conditions which permits the production of
said antibody molecule composition or a respective formulation for
proteins in general means that the host cell of the invention
comprising at least one nucleic acid encoding a protein/antibody
molecule, preferably the preferred embodiments of said nucleic acid
of the invention described elsewhere herein, is cultured under
culture conditions which allow the expression of the
protein/antibody molecule in form of a protein/antibody molecule
composition, preferably secretion into the medium, preferably with
high yields and/or high activity and/or high homogeneity as
described elsewhere herein. Those skilled in the art are able to
select the most suitable culture conditions by using suitable media
and culture conditions such as but not limited to suitable time,
temperature, pH, gasing, feed, medium, medium supplements, vessel
or reactor sizes and principles known to those skilled in the art.
Those skilled in the art are able to select and optimise the most
suitable conditions. Preferred embodiments are described in
examples but are not limited to those.
[0287] Culturing of the cells of the present invention can be
carried out by any of general culturing methods for animal cells
capable of efficiently producing the desired antibody molecule
composition, for example, batch culture, repeated batch culture,
fed-batch culture and perfusion culture. Preferably, fed-batch
culture or perfusion culture is employed in order to raise the
productivity of the desired polypeptides.
[0288] In a further preferred embodiment of the invention said
culturing is performed under serum-free conditions and even further
preferred with protein free media or animal component free
media.
[0289] Adaptation of host cells of the present invention to a
serum-free medium in accordance with the present invention is
surprisingly fast and robust. Adaptation can be carried out, for
example, by adapting cells subcultured in a serum-containing medium
directly to a commercially available serum-free medium, or by
continuous adaptation whereby the direct adaptation to serum-free
medium is preferred and advantageous. During the process of
adaptation to a serum-free medium, the viability of cells lowers
temporarily, which sometimes causes extinction of cells. Therefore,
it is preferred to inoculate cells into a medium for the adaptation
to a serum-free medium at a cell density of 1.times.10<5> to
5.times.10<5> cells/ml, preferably 2.times.10<5>
cells/ml, in order to restore the viability of cells or to keep it
high. After 4 to 7 days of culturing, the cells whose density
reached 5.times.10<5> to 10.times.10<5> cells/ml are
selected as the cells adapted to a serum-free medium. Adaptation to
serum-free medium can be also performed by successive dilution of
the medium supplemented with FCS by a serum-free medium composition
(continuous adaptation). Those skilled in the art are able to
select and optimise the most suitable conditions. Preferred
embodiments are described in examples but are not limited to
those.
[0290] After the cells of the present invention are adapted to a
serum-free medium, a cloned cell line can be prepared by using the
limiting dilution method with a 96-well plate, the colony forming
method, or the like, and cells or cell line are selected due to
properties of those cells which are advantageous when compared to
their parent cell or cell line such as but not limited to shorter
doubling times, faster growth, possibility to grow under higher
densities, can produce more, higher cloning efficiencies, higher
transfection efficiencies for DNA, higher expression rates of
antibody molecule compositions, higher activities for an antibody
molecule composition expressed therein, higher homogeneities of an
antibody molecule composition expressed herein, and/or higher
robustness to scaling up. Methods for selecting the cell with
advantageous properties are known to those skilled in the art or
described herein.
[0291] In accordance with the present invention the term isolating
said antibody molecule composition or a corresponding formulation
for proteins in general means that the protein/antibody molecule
composition expressed by said host cell comprising at least one of
said nucleic acids encoding the protein/antibody molecule or
fraction thereof described elsewhere herein is gained by using the
culture media after culturing or further enriching or purifying the
protein/antibody molecule composition or parts of said
protein/antibody molecule composition by methods known to those
skilled in art. Said protein/antibody molecule composition in sense
of the invention also means parts of said protein/antibody molecule
composition enriched for certain protein/antibody molecules
described elsewhere herein.
[0292] In one preferred embodiment the protein/antibody molecule
composition is isolated by separating the media after culturing
from the cells and/or cell debris for example by centrifugation
techniques.
[0293] In a further preferred embodiment of the invention a
Protein/antibody molecule composition of the invention is isolated
or further enriched by ultrafiltration, precipitation methods or
other concentration methods known to those skilled in the art.
[0294] In a further preferred embodiment of the invention a
protein/antibody molecule composition of the invention is isolated
by purification of the protein/antibody molecule composition by
chromatographic methods such as but not limited to affinity
chromatography using according affinity materials such as but not
limited to Protein A, Protein G, anti-antibody isotype antibodies,
lectin chromatography, antibodies against a certain tag introduced
into antibody molecule such as HIS-tag or myc-tag, or antigen, or
by ion exchange chromatography known to those skilled in the
art.
[0295] Further methods of purifying or enriching proteins or
certain glycoforms of proteins are known to those skilled in the
art and can be selected, adopted, optimised and used alone or in
combination with afore described methods by those skilled in the
art to isolate or further purify, fractionate or enrich the protein
molecule composition or fractions thereof of the invention.
[0296] In a preferred embodiment of the invention an antibody
molecule composition of the invention of mainly IgG is isolated by
Protein A chromatography with or without prior
ultracentrifugation.
[0297] In another preferred embodiment of the invention an antibody
molecule composition of the invention of mainly IgM is isolated by
anti-IgM antibody chromatography with or without prior
ultracentrifugation.
[0298] In another preferred embodiment of the invention an antibody
molecule composition of the invention enriched in certain
glycoforms of the antibody molecule is isolated by lectin affinity
chromatography with or without prior ultracentrifugation. Further
methods of purifying or enriching proteins or certain glycoforms of
proteins are known to those skilled in the art and can be selected,
adopted, optimised and used alone or in combination with afore
described methods by those skilled in the art to isolate or further
purify, fractionate or enrich the antibody molecule composition or
fractions thereof of the invention.
[0299] In a preferred embodiment the antibody molecule composition
is isolated using Protein A columns. In another preferred
embodiment the antibody molecule composition is isolated using an
anti-IgM column.
[0300] In accordance with the present invention the term "increased
activity" means that the activity of a protein and/or antibody
molecule composition of the invention expressed in a host cell of
human myeloid leukaemia origin is higher than the activity of at
least one protein/antibody molecule composition from the same
protein/antibody molecule when expressed in at least one of the
cell lines CHO, or CHOdhfr-, or BHK, or NS0, or SP2/0, or PerC.6 or
mouse hybridoma. In the preferred embodiment of the invention it
means that the activity of a protein/antibody molecule composition
of the invention expressed in a host cell of human myeloid
leukaemia origin is higher than the activity of a protein/antibody
molecule composition from the same protein/antibody molecule when
expressed in CHOdhfr- [ATCC No. CRL-9096]. For antibodies, said
increased activity is an increased Fc-mediated cellular
cytotoxicity or an increased binding activity.
[0301] In the meaning of the invention, "activity" is also a
function or set of functions performed by a protein molecule in a
biological context. In the meaning of the invention, the term
"increased activity", equivalents of the contents and grammatical
equivalents thereof are to understood as an improved or optimal
activity with regard to the selected application, whereby the
activity could be either approximated to a limiting value, for
example being minimized or maximized, or set to a medium value
representing a higher or lower activity compared to the
corresponding protein molecule produced by prior art. Improved or
increased activity in sense of the invention also means a favorable
activity in sense of its biological and/or pharmaceutical meaning
as improved serum half-life, pharmacokinetics, stability,
biological activity, binding, antigenicity and/or immunogenicity.
For example, the biological activity of a protein molecule
composition could be increased to an extend by decreasing adverse
biological effects, e.g. by the reduced stimulation of adverse
immune effects or a decreased immunogenicity.
[0302] Activity of the protein molecule composition according to
the invention can be determined in a suitable bioassay which is
able to determine the activity of the protein. Those skilled in the
art are able to identify suitable bioassays or to build up suitable
bioassays. According to the present invention such bioassays
include for example biological in vitro assays, including cellular
or molecular or mixed assays, such as proliferation assays,
apoptosis assays, cell adhesion assays, signaling assays, migration
assays, cell cytotoxicity assays, phagocytosis assays, lysis
assays, and binding assays. Such bioassays also include in vivo
assays using animal models or humans, such as biodistribution,
pharmacokinetic, pharmacodynamic test, serum-half life tests, tests
for bioavailability, efficacy test, localization tests, treatment
and prophylaxe tests of diseases including clinical studies. Such
bioassays also include chemical, physical, physiochemical,
biophysical and biochemical tests, such as stability towards
temperature, shear stress, pressure, pH, conjugation and others.
Such bioassays also include tests for the immunogenicity and/or
antigenicity in order to improve the properties of the protein
molecule composition in respect to its clinical use. Those skilled
in the art are able to determine the activity or a combination of
described activities of protein molecule compositions.
[0303] In a preferred embodiment the higher activity of the protein
molecule composition is characterized by a higher activity in at
least one in vitro model and/or a higher activity in at least one
in vivo model and/or a higher stability and/or a longer serum
half-life and/or a longer bioavailability and/or an improved
immunogenicity and/or an improved antigenicity determined by at
least one bioassay. The improvement in the overall activity which
is also called herein higher activity can lead for examples to
improvements like lower dosages, longer time intervals for
administration, less side effects and no or lower toxicity of the
product when used in humans or according organisms resulting in
largely improved pharmaceuticals.
[0304] In a preferred embodiment of the invention the activity of a
protein molecule composition of the invention expressed in a host
cell of human myeloid leukaemia origin is higher than the activity
of at least one protein molecule composition from the same protein
molecule produced by prior art.
[0305] Said increased Fc-mediated cellular cytotoxicity is an
increased antibody dependent cellular cytotoxicity (ADCC activity),
complement dependent cytotoxicity (CDC activity), and/or
cytotoxicity caused by phagocytosis (phagocytosis activity). The
increased Fc-mediated cellular cytotoxicity, including ADCC
activity, CDC activity or phagocytosis activity can be determined
by various methods known to those skilled in the art and some are
described in detail in examples without limiting it to those
methods whereby the methods described in the examples are preferred
embodiments of the invention.
[0306] Said increased binding activity is an increased binding to
the epitope of the antibody molecule, such as the epitope, the
antigen, another polypeptide comprising the epitope, or a cell
comprising the epitope of the antibody molecule, or an increased
binding to at least one Fc receptor or another effector ligand,
such as Fc-gammaRI, Fc-gammaRII, Fe-gammaRIII, and subclasses
therefrom such as Fc-gammaRIIa, Fc-gammaRIIIa, Fc-gammaRIIIb, or
C1q component of complement, or FcRn or a molecule or cell
comprising any of those Fc receptors or effector ligands. The
increased binding activity can be a higher affinity, a higher
avidity, and/or a higher number of binding sites or combinations
thereof. The increased binding activity can result in various
effects and activities such as but not limited to forms of receptor
mediated activity as described in the Background of the art. The
increased binding affinity, avidity and receptor mediated activity
can be determined by at least one of the various methods known to
those skilled in the art such as but not limited to Biacore
measurement, Scatchard analysis, ELISA or RIA based measurements,
flow cytometry measurements, test for determining the apoptosis
induction in suitable target cells, tests for determination of the
proliferation of suitable target cells, test for antagonistic,
agonistic and/or receptor blockade of an antibody molecule
composition such as but not limited to inhibition of cell-cell
mediated binding, trigger of cell internal molecular events. Those
skilled in the art are able to select and/or adopt and/or modify a
suitable method or combination of methods for testing the binding
affinity, avidity, number of binding sites and/or receptor mediated
activity.
[0307] The methods of the invention can be used to test the ability
of an antibody to be able to obtain an increased Fc-mediated
cellular cytotoxicity, preferably ADCC activity, CDC activity
and/or cytotoxicity caused by phagocytosis, and/or an increased
binding activity to the epitope of the antibody molecule or
preferably to at least one Fc receptor or another effector ligand,
in general and/or in particular with the host cells of the
invention and its preferred embodiments described elsewhere
herein.
[0308] In a preferred embodiment of the invention the activity of a
protein/antibody molecule composition of the invention expressed in
a host cell of human myeloid leukaemia origin is higher than the
activity of at least one antibody molecule composition from the
same antibody molecule from at least one of the cell lines CHO, or
CHOdhfr-, or BHK, or NS0, or SP2/0, or PerC.6 or mouse hybridoma,
preferably CHOdhfr- [ATCC No. CRL-9096], when expressed
therein.
[0309] In a preferred embodiment of the invention the activity of a
protein/antibody molecule composition of the invention expressed in
a host cell of human myeloid leukaemia origin is at least 50%
higher than the activity of the antibody molecule composition from
the same antibody molecule expressed in the cell line CHOdhfr-
[ATCC No. CRL-9096], more preferably at least 2 times, more
preferably at least 3 times, more preferably at least 4 times, more
preferably at least 5 times, more preferably at least 7 times, more
preferably at least 10 times, more preferably at least 15 times,
more preferably at least 23 times, more preferably at least 30
times, more preferably at least 50 times, more preferably at least
75 times, more preferably at least 100 times, more preferably at
least 150 times, more preferably at least 150 times, more
preferably at least 230 times, more preferably at least 300 times,
more preferably at least 500 times, more preferably at least 750
times, and most preferably more than 1000 times.
[0310] Thereby not each bioassays has to show a higher activity but
depending on the use and the features of a particular protein
molecule composition some favourable biological effects can
compensate for others which are less favourable and still resulting
in a overall higher activity of the protein molecule composition in
sense of the invention. For example, a certain protein molecule
composition can result in a much higher activity by binding to its
receptors to cells thereby triggering secondary effect, such as
induction of proliferation, but show a slightly decreased
serum-half life. In combination the higher activity triggering the
receptor more then compensates for the shorter bioavailability in
the overall bioactivity. In another example, a shorter half-life
and a higher activity towards the receptor triggering are both
advantageous. In yet another example the activity in vivo is not
improved but the stability in vitro improves the production and
storage of the protein molecule composition. In yet another
example, a long half-life but a lower activity is needed.
[0311] In a preferred embodiment of the invention the increased
activity of an antibody molecule composition is an increased ADCC
activity. In another preferred embodiment the increased activity of
an antibody molecule composition is an increased CDC. In another
preferred embodiment the increased activity of an antibody molecule
composition is an increased cytotoxicity caused by phagocytosis. In
another preferred embodiment the increased activity of an antibody
molecule composition is an increased binding activity to the
epitope. In another preferred embodiment the increased activity of
an antibody molecule composition is an increased binding activity
to at least one Fc receptor, preferably Fc.gamma.RIIIA. In a
further preferred embodiment the increased activity of an antibody
molecule composition is an increased Fc-mediated cellular
cytotoxicity and an increased binding activity to the epitope. In a
further preferred embodiment the increased activity of an antibody
molecule composition is an increased ADCC activity and an increased
binding activity to the epitope. In a further preferred embodiment
the increased activity of an antibody molecule composition is an
increased ADCC activity, an increased CDC activity, an increased
binding activity to the epitope and an increased binding activity
to at least one Fc receptor. In a further preferred embodiment the
increased activity of an antibody molecule composition is an
increased ADCC activity, increased cytotoxicity caused by
phagocytosis, an increased binding activity to the epitope and an
increased binding activity to at least one Fc receptor. In the most
preferred embodiment the increased activity of an antibody molecule
composition is an increased ADCC, increased cytotoxicity caused by
phagocytosis, an increased CDC activity and an increased binding
activity to the epitope and an increased binding activity to at
least one Fc receptor.
[0312] In accordance with the present invention the term "improved
homogeneity" means that an protein/antibody molecule composition of
the invention expressed in a host cell of human myeloid leukaemia
origin of the invention comprises fewer different glycoforms, or
more of a favourable glycoform or of favourable glycoforms, or less
of at least one glycoform of an antibody molecule (preferably of
those glycoforms which represent at least 1% of the total antibody
molecule composition on its own) than at least one antibody
molecule composition of the same antibody molecule isolated from at
least one of the cell lines CHO, or CHOdhfr-, or BHK, or NS0, or
SP2/0, or PerC.6 or mouse hybridoma when expressed therein. In a
preferred embodiment of the invention it means that an antibody
molecule composition of the invention expressed in a host cell of
human myeloid leukaemia origin of the invention comprises fewer
different glycoforms, or more of a favourable glycoform or of
favourable glycoforms, or less of at least one glycoform of an
antibody molecule than an antibody molecule composition of the same
antibody molecule isolated from the cell line CHOdhfr- [ATCC No.
CRL-9096] when expressed therein.
[0313] One heterogeneity particularly problematic for production
and use in human is the sialylation. In a preferred embodiment the
protein/antibody molecule composition of the invention has an
improved homogeneity by comprising no glycoform with the sialic
acid N-glycolylneuraminic acid (NeuGc), whereby in this case no
glycoform means no glycoform of more than 1% of all carbohydrate
chains obtainable from the purified antibody molecule composition
and more preferable no carbohydrate chain detectable at all as
being detectable by the methods known to those skilled in the art.
Since NeuGc is known to be able to be immunogenic in humans this is
a large advantage of the host cells of the invention over other
production systems such as CHO, NS0, SP2/0.
[0314] In a preferred embodiment the protein/antibody molecule
composition of the invention has an improved homogeneity in respect
to sialylation by comprising less than 5% of glycoforms, more
preferably less than 3%, even more preferably less than 1%, and
most preferably no glycoforms of the protein/antibody molecule
composition with sialic acid detectable as described in examples.
In a further preferred embodiment a protein/antibody molecule
composition with improved homogeneity in respect to sialylation is
achieved by using a host cell of human myeloid leukaemia origin of
the invention which has a defect in the sugar nucleotide precursor
pathway and therefore is deficient for or has reduced CMP-sialic
acid which results in no or a largely reduced sialylation of the
carbohydrate sugar chains of the proteins/antibody molecules when
the cells are grown in a serum-free medium. In an even further
preferred embodiment such protein/antibody molecule composition
with improved homogeneity in respect to sialylation can be achieved
by using NM-F9 [DSM ACC2606] or NM-D4 [DSM ACC2605] as a host cell
of the invention grown in a serum-free medium as and described in
more detail in examples.
[0315] In another further preferred embodiment a protein/antibody
molecule composition with improved homogeneity in respect to
sialylation is achieved by using a host cell of human myeloid
leukaemia origin of the invention which has a defect in the sugar
nucleotide transporter of GMP-sialic acid or in at least one
sialyltransferase which results in no or a reduced sialylation of
the carbohydrate sugar chains of the proteins/antibody molecules
when the cells are grown in a serum-free medium. Examples are
NM-F9, NM-D4 and GT-2X.
[0316] In another preferred embodiment of the invention the
protein/antibody molecule composition with improved homogeneity in
respect to sialylation is achieved by using a host cell of human
myeloid leukaemia origin of the invention which has an increased
sialylation degree. Said sialylation degree means that the amount
of the sialic acid N-acetylneuraminic (NeuNc or NeuNAc) on the
protein/antibody molecules in an antibody molecule composition is
at least 5%, more preferably at least 15%, more preferably at least
20%, more preferably at least 25%, more preferably at least 30%,
more preferably at least 35%, more preferably at least 40%, more
preferably at least 50%, more preferably at least 60%, more
preferably at least 70%, more preferably at least 80%, more
preferably at least 90%, more preferably at least 100%, more
preferably at least 3 times, more preferably at least 5 times, more
preferably at least 10 times, more preferably at least 25 times,
more preferably at least 50 times, and most preferably more than
100 times, higher than the amount of sialic acid N-acetylneuraminic
(NeuNc or NeuNAc) of the total carbohydrate units or the particular
carbohydrate chain at a particular Glycosyaltion site of the
protein/antibody molecule when comparing with the same amount of
protein/antibody molecules of a protein/antibody molecule
composition of the same protein/antibody molecule isolated from at
least one of the cell lines CHO, or CHOdhfr-, or BHK, or NS0, or
SP2/0, or PerC.6 or mouse hybridoma, preferably CHOdhfr- [ATCC No.
CRL-9096] when expressed therein. The sialylation degree can be
detected by methods known to those skilled in the art, such as but
not limited to immuno blot analysis or ELISA using lectins which
binding depends on the sialylation of the carbohydrate structure,
such as SNA, MAL, MAL I or PNA, by chemical detection methods such
as the thiobarbituric acid method, by HPLC or mass spectrometry or
combination thereof. Those skilled in the art can select the most
suitable method and adopt and optimise it to the purpose and more
details are described in the examples. Preferred is an immunoblot
analysis using SNA.
[0317] In another further preferred embodiment a protein/antibody
molecule composition with improved homogeneity in respect to
sialylation is achieved by using a host cell of human myeloid
leukaemia origin of the invention, preferably K562, NM-F9 [DSM
ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X and cells or cell lines derived
therefrom, and most preferably K562, NM-E-2F9, NM-C-2F5, NM-H9D8,
or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X which has an increased
sialylation degree, and results in a protein/antibody molecule
composition which comprises alpha 2-6 linked sialic acid, for
detectable for example by SNA binding, in a more preferred version
the protein/antibody molecule composition comprises alpha 2-6 and
alpha 2-3 linked sialic acids.
[0318] In another preferred embodiment a protein/antibody molecule
composition with improved homogeneity in respect to sialylation is
achieved by using a host cell of human myeloid leukaemia origin
which comprise at least one sialyltransferase able to attach NeuNAc
in alpha 2-3 and at least one sialyltransferase able to attach
NeuNAc in alpha 2-6 linkage to sugar groups, such as but not
limited to K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605],
NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X
and cells or cell lines derived therefrom, thereby resulting in a
more preferred composition of glycoforms of the protein/antibody
molecule in the protein/antibody molecule composition.
[0319] In an even further (preferred) embodiment of the invention
said antibody molecule composition of the invention with improved
homogeneity in respect to sialylation comprises 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 (Cgamma2
domain) of the Fc region.
[0320] In an even further preferred embodiment of the former
antibody molecule composition with improved homogeneity in respect
to sialylation is expressed in a host cell of human myeloid
leukaemia origin which has an increased sialylation degree and/or
comprise at least one sialyltransferase able to attach sialic acid
in alpha 2-3 and at least one sialyltransferase able to attach
sialic acid in alpha 2-6 linkage to sugar groups, such as K562,
NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X
and cells or cell lines derived therefrom.
[0321] In an even further preferred embodiment the former antibody
molecule has at least one N-glycosylation site (Asn-X-Ser/Thr,
whereby X can be any amino acid except Pro) and/or at least one
O-glycosylation site in a sequence of the Fab region.
[0322] In a further preferred embodiment the antibody molecule
composition of the invention with improved homogeneity in respect
to sialylation comprising 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 (Cgamma2 domain) of the Fc part has an extended serum-half
life and/or bioavailability when measured in at least one mammal
such as mice, rats, or preferably in humans, than the antibody
molecule composition of the same antibody molecule isolated from at
least one of the cell lines CHO, or CHOdhfr-, or BHK, or NS0, or
SP2/0, NM-F9, NM-D4 or PerC.6 or mouse hybridoma, preferably the
cell line CHOdhfr- [ATCC No. CRL-9096] when expressed therein.
[0323] The bioavailability of antibodies can be optimized using the
present invention. Expression of antibody molecules in particular
in cells having a high or even a very high sialylation (groups 1
and 3 discussed above) can lead to an antibody composition with a
prolonged bioavailability, while expression of antibody molecules
in the cells of the group 2 may lead to an antibody composition
with a comparably shortened bioavailability. The bioavailability
can be tested as known by those skilled in the art and as described
in the examples using animals or preferably humans. Animals include
mice, rats, guinea pigs, dogs, or monkeys but are not restricted to
those species. Due to the human nature those animals which have a
glycosylation and most important sialylation closest to the human
are preferred, most preferred are humans.
[0324] In an even more preferred embodiment the antibody molecule
composition with improved homogeneity in respect to sialylation is
expressed in a host cell of human myeloid leukaemia origin of the
invention which has an increased sialylation degree and/or comprise
at least one sialyltransferase able to attach sialic acid in alpha
2-3 and at least one sialyltransferase able to attach sialic acid
in alpha 2-6 linkage to sugar groups, such as K562, NM-F9 [DSM
ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, and cells or cell lines derived therefrom, and comprise
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 and has an
extended serum-half life and/or bioavailability when measured in at
least one mammal such as mice, rats, or preferably in humans, than
the antibody molecule composition of the same antibody molecule
isolated from at least one of the cell lines CHO, or CHOdhfr-, or
BHK, or NS0, or SP2/0, or PerC.6 or mouse hybridoma, preferably the
cell line CHOdhfr- [ATCC No. CRL-9096] when expressed therein. In a
further preferred embodiment the antibody molecule expressed is
Erbitux (Cetuximab).
[0325] In accordance with the present invention the term "increased
yield" means that the average or the maximum yield of a
protein/antibody molecule composition of the invention produced in
a host cell of human myeloid leukaemia origin is higher than the
respective average or maximum yield of at least one
protein/antibody molecule composition from the same
protein/antibody molecule when expressed in at least one of the
cell lines CHO, or CHOdhfr-, or BHK, or NS0, or SP2/0, or PerC.6 or
mouse hybridoma. In the preferred embodiment of the invention it
means that the average or maximum yield of a protein/antibody
molecule composition of the invention expressed in a host cell of
human myeloid leukaemia origin is higher than the respective
average or maximum yield of a protein/antibody molecule composition
from the same protein/antibody molecule when expressed in CHOdhfr-
[ATCC No. CRL-9096] using the murine dhfr gene and methotrexate for
amplification in CHOdhfr-. The average and maximum yield is
measured in SPR, which reflects the productivity of a cell, cell
mixture or a cell line, can be determined by those skilled in the
art and is described in its preferred embodiment in the
examples.
[0326] In a further preferred embodiment of the invention at least
the average or the maximum yield of a protein/antibody molecule
composition of the invention expressed in a host cell of human
myeloid leukaemia origin, preferably K562, NM-F9 [DSM ACC2606],
NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived therefrom, is
at least 10% higher than the according of the protein/antibody
molecule composition from the same protein/antibody molecule
expressed in the cell line CHOdhfr- [ATCC No. CRL-9096] using the
murine dhfr gene and methotrexate for amplification in CHOdhfr-,
more preferably at least 15%, more preferably at least 20%, more
preferably at least 25%, more preferably at least 30%, more
preferably at least 35%, more preferably at least 45%, more
preferably at least 50%, more preferably at least 55%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, more preferably at least 90%, more
preferably at least 100%, more preferably at least 3 times, more
preferably at least 4 times, and most preferably more than 5
times.
[0327] The invention further provides a nucleic acid comprising
[0328] (a) a sequence encoding a protein, preferably an antibody
molecule or at least one part thereof as described elsewhere
herein, and [0329] (b) at least one sequence encoding a sequence
from the group of sequence #1 to sequence #9.
[0330] In a preferred embodiment of the invention the nucleic acid
of the invention comprises [0331] (a) a sequence encoding a
protein, preferably an antibody molecule or at least one part
thereof as described elsewhere herein, and [0332] (b) a sequence
encoding sequence #1.
[0333] In a further preferred embodiment of the invention the above
described nucleic acid of the invention further comprises a
sequence encoding a selection marker, preferably a sequence
encoding for a polypeptide which induces an antibiotic resistance
of a host cells in which said nucleic acid is introduced, such as
but not limited to neomycin or puromycin.
[0334] In a further preferred embodiment of the invention the above
described nucleic acid of the invention further comprises at least
one sequence of at least one genetic element described elsewhere
herein.
[0335] The invention further provides a host cell of human myeloid
leukaemia origin or any human myeloid or myeloid precursor cell or
cell line which can be obtained from a leukaemia patient, or any
myeloid or myeloid precursor cell or cell line which can be
obtained from a human donor or a mixture of cells or cell lines
comprising at least one cell of human myeloid leukaemia origin, or
cell, cells, or cell line which was obtained by fusing at least one
cell, cells, or a cell line of human myeloid leukaemia origin or
any human myeloid or myeloid precursor cell or cell line which can
be obtained from a leukaemia patient, or any myeloid or myeloid
precursor cell or cell line which can be obtained from a human
donor, with another cell of human or animal origin, such as but not
limited to B cells, CHO cells, comprising at least one nucleic acid
encoding a protein/antibody molecule or parts thereof which was
introduced into said cells.
[0336] The invention provides a host cell of the invention
described above comprising at least one nucleic acid encoding a
protein/antibody molecule or at least one part thereof.
[0337] In a preferred embodiment the invention provides the host
cell K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9,
NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or
cell line derived from anyone of said host cells, preferably those
which grows under serum-free conditions and from which a
protein/antibody molecule composition is isolated under serum-free
conditions, and even more preferred those into which the nucleic
acid encoding the antibody molecule was introduced under serum-free
conditions, comprising at least one nucleic acid encoding a
protein/antibody molecule or parts thereof, preferably a nucleic
acid comprising a sequence encoding a protein/antibody molecule or
at least one part thereof as described elsewhere herein.
[0338] The invention provides a host cell of the invention
described above comprising at least one nucleic acid encoding at
least one polypeptide of the group of sequence #1 to sequence #9,
preferably sequence #1.
[0339] The invention provides a host cell of the invention
described above comprising at least one nucleic acid encoding an
antibody molecule or parts thereof and at least one nucleic acid
encoding at least one polypeptide of the group of sequence #1 to
sequence #9, preferably sequence #1.
[0340] In a preferred embodiment the invention provides the host
cell K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9,
NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or
cell line derived from anyone of said host cells, preferably those
which grows under serum-free conditions and from which a
protein/antibody molecule composition is isolated under serum-free
conditions, and even more preferred those into which the nucleic
acid encoding the protein/antibody molecule was introduced under
serum-free conditions, comprising at least one nucleic acid
encoding a protein/antibody molecule or parts thereof, preferably a
nucleic acid comprising a sequence encoding a protein/antibody
molecule or at least one part thereof as described elsewhere
herein, and at least one sequence encoding a sequence from the
group of sequence #1 to sequence #9, preferably sequence #1.
[0341] In a further preferred embodiment the invention provides the
above described host cells wherein the antibody molecule encoded is
an antibody of WO2004/065423.
[0342] In an even further preferred embodiment the invention
provides the above described host cells wherein the antibody
molecule encoded is the antibody PankoMab [Cancer Immunol
Immunother. 2006 November; 55(11):1337-47. Epub 2006 February
PankoMab: a potent new generation anti-tumour MUC1 antibody.
Danielczyk et al], preferably a chimaeric form of PankoMab with all
human constant domains, and more preferably a humanized
PankoMab.
[0343] The invention further provides a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity and fully human glycosylation produced by any
of the methods of the invention as described somewhere herein.
[0344] In a preferred embodiment the invention provides a
protein/antibody molecule composition produced by any of the
methods of the invention has an increased activity and/or increased
yield and/or improved homogeneity and fully human glycosylation
produced by any of the methods of the invention when compared to a
protein/antibody molecule composition of the same protein/antibody
molecule isolated from at least one of the cell lines CHO, or
CHOdhfr-, or BHK, or NS0, or SP2/0, or PerC.6 or mouse hybridoma,
preferably CHOdhfr- [ATCC No. CRL-9096], when expressed
therein.
[0345] The protein molecule or part thereof expressed can be any
protein or protein part or protein fragment. The antibody molecule
or part thereof expressed can be any antibody or antibody part or
antibody fragment.
[0346] Protein molecule compositions of the present invention can
be used for prophylactic and/or therapeutic treatment of diseases,
such as leukemia, neutropenia, cytopenia, cancer, bone marrow
transplantation, diseases of hematopoietic systems, infertility and
autoimmune diseases. The spectrum of therapeutic applications known
to people of the field of art, of protein molecule compositions is
very wide. For example, G-CSF is an important therapeutic to treat
neutropenia, a life-threatening decrease in neutrophils as
consequence of a chemotherapy of leukemic cancer patients. GM-CSF
is specifically used for treatment of AML patients at relative high
age after chemotherapy to achieve a fast recovery from neutropenia.
GM-CSF is additionally approved as therapeutic for several
applications in bone marrow transplantations and for mobilization
of peripheral blood stem cells. In addition, there are several
clinical applications of GM-CSF that are currently under
investigation, such as for treatment of HIV and cancer. Certain
diseases of the hematopoietic system are treated with EPO, and
IFN-beta is currently an important therapeutic for treatment of
multiple sclerosis, an autoimmune disease. Another example is FSH
which is widely used for treatment of male and female infertility.
hCG is also applied for the treatment of infertility, but focusing
on the anovulation in women. hGH has clinically-proven benefits,
such as bodyfat reduction and muscle tissue increase.
[0347] Protein molecule compositions of the present invention can
also be used for the manufacture of a medicament for prophylactic
and/or therapeutic treatments of diseases selected from the group
comprising leukemia, neutropenia, cytopenia, cancer, bone marrow
transplantation, diseases of hematopoietic systems, infertility and
autoimmune diseases.
[0348] In a preferred embodiment of the invention the antibody
molecule expressed is an antibody molecule recognizing psoriasis,
rheumatoid arthritis, Crohn's disease, ulcerative colitis,
autoimmune diseases, SLE, Multiple Sclerosis, autoimmune
haematological disorders, asthma, allergy, graft-versus-host
disease, allograft rejection, glaucoma surgery, myocardial
infarction, viruses as RSV, HIV, Hep B, or CMV, cancer, sarcoma,
CLL, AML, or NHL.
[0349] In an even preferred embodiment of the invention the
antibody molecule expressed is an antibody molecule recognizing the
cancer, tumor or metastasis, at least one cancer cell or tumor
cell, in at least one human, preferably selected from the group of
cancerous diseases or tumor diseases of the ear-nose-throat region,
of the lungs, mediastinum, gastrointestinal tract, urogenital
system, gynecological system, breast, endocrine system, skin, bone
and soft-tissue sarcomas, mesotheliomas, melanomas, neoplasms of
the central nervous system, cancerous diseases or tumor diseases
during infancy, lymphomas, leukemias, paraneoplastic syndromes,
metastases with unknown primary tumor (CUP syndrome), peritoneal
carcinomatoses, immunosuppression-related malignancies and/or tumor
metastases.
[0350] In a preferred embodiment of the invention the expressed
antibody or part thereof is an anti MUC1 antibody.
[0351] In a further preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is the antibody Rituximab, Herceptin, Erbitux,
Campath 1H or antibodies derived therefrom.
[0352] In an even more preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/050707, and even more
preferred of WO2004/065423, and even more preferred PankoMab
[Cancer Immunol Immunother. 2006 November; 55(11):1337-47. Epub
2006 February PankoMab: a potent new generation anti-tumour MUC1
antibody. Danielczyk et al], and even more preferred a chimaeric
version thereof comprising all human constant domains, and even
more preferred a humanized antibody thereof.
[0353] In an even more preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is any whole antibody of the invention, preferably
Rituximab, Herceptin, Erbitux, more preferably WO2004/065423, most
preferably PankoMab, whereby the antibody molecule composition
isolated from any host cell of the invention, preferably K562,
NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5,
NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line
derived from anyone of said host cells, comprises no detectable
NeuGc. The same applies to proteins in general.
[0354] In an even more preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is any whole antibody molecule or an antibody
molecule comprising the Cgamma2 domain of the invention, preferably
Rituximab, Herceptin, Erbitux, more preferably WO2004/065423, most
preferably PankoMab, whereby the antibody molecule composition
isolated from a host cell of the invention, preferably K562, NM-F9
[DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6012, GT-2X or a cell or cell line derived
from anyone of said host cells, comprises at least one glycoform
with alpha 2-6 sialic acid. The same applies to proteins in
general.
[0355] In an even more preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is any whole antibody molecule or an antibody
molecule comprising the Cgamma2 domain of the invention, preferably
Rituximab, Herceptin, Erbitux, Campath 1H, more preferably
WO2004/065423, most preferably PankoMab, whereby the antibody
molecule composition isolated from host cell NM-E-2F9, NM-C-2F5,
NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line
derived from anyone of said host cells, comprises alpha 2-6 sialic
acid, which is detectable by immune blot analysis with the lectin
SNA as described in examples. The same applies to proteins in
general.
[0356] In another preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is any antibody molecule, preferably Rituximab,
Herceptin, Erbitux, Campath 1H, more preferably WO2004/065423, most
preferably PankoMab, and the antibody molecule composition isolated
from the host cell NM-F9 or NM-D4 of the invention after
cultivation in serum-free and more preferably protein-free medium
has an improved homogeneity with no detectable sialic acids. The
same applies to proteins in general.
[0357] In a further preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is the antibody Rituximab, Herceptin, Campath 1H, or
antibodies derived therefrom, and the antibody molecule composition
isolated from the host cell of the invention has an increased ADCC
activity of at least 4 fold higher than the activity of the
antibody molecule composition from the same antibody molecule
expressed in the cell line CHOdhfr- [ATCC No. CRL-9096].
[0358] In an even more preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab, and the antibody molecule composition isolated from the
host cell of the invention has an increased ADCC activity of at
least 4 fold higher when produced in at least one of the cells
K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5,
NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line
derived from anyone of said host cells than the activity of the
antibody molecule composition from the same antibody molecule
expressed in the cell line CHOdhfr- [ATCC No. CRL-9096].
[0359] In another preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab, and the antibody molecule composition isolated from the
host cell of the invention K562, NM-F9 [DSM ACC2606], NM-D4 [DSM
ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived from anyone of
said host cells has an increased binding activity to its epitope of
at least 50% higher, preferably 2 fold higher than the activity of
the antibody molecule composition from the same antibody molecule
expressed in the cell line CHOdhfr- [ATCC No. CRL-9096].
[0360] In another preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab, and the antibody molecule composition isolated from the
host cell NM-F9 or NM-D4 of the invention after cultivation in
serum-free and more preferably protein-free medium has an improved
homogeneity with no detectable sialic acids. The same applies to
proteins in general.
[0361] In another preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab, and the antibody molecule composition isolated from the
host cell K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9,
NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or
cell line derived from anyone of said host cells has an improved
homogeneity with at least 10% more sialic acids than the antibody
molecule composition from the same antibody molecule expressed in
the cell line CHOdhfr- [ATCC No. CRL-9096]. The same applies to
proteins in general.
[0362] In another preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab, and the antibody molecule composition isolated from the
host cell of the invention has an improved homogeneity with a
higher degree of no detectable sialic acids when expressed in NM-F9
or NM-D4 when compared to an antibody molecule composition from the
same antibody molecule expressed in the cell line CHOdhfr- [ATCC
No. CRL-9096]. The same applies to proteins in general.
[0363] In another preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab, and the antibody molecule composition isolated from the
host cell of the invention has an improved homogeneity as described
above, and an increased ADCC activity of at least 4 fold higher
than the activity of the antibody molecule composition from the
same antibody molecule expressed in the cell line CHOdhfr- [ATCC
No. CRL-9096].
[0364] In the most preferred embodiment of the invention the
antibody molecule encoded by the nucleic acid or nucleic acids of
the invention is an antibody of WO2004/065423, more preferably
PankoMab.
[0365] The invention further provides a host cell for producing a
protein/antibody molecule composition having increased activity
and/or increased yield and/or improved homogeneity and fully human
glycosylation in sense of the invention as described elsewhere
herein, wherein the host cell is any cell, cells, or cell line of
human myeloid leukaemia origin or any human myeloid or myeloid
precursor cell or cell line which can be obtained from a leukaemia
patient, or any myeloid or myeloid precursor cell or cell line
which can be obtained from a human donor or a mixture of cells or
cell lines comprising at least one cell of human myeloid leukaemia
origin, or a cell or cell line derived therefrom as described
elsewhere herein, or a mixture of cells or cell lines comprising at
least one of those aforementioned cells.
[0366] The invention also provides a host cell for producing a
protein/antibody molecule composition having increased activity
and/or increased yield and/or improved homogeneity and fully human
glycosylation in sense of the invention as described elsewhere
herein, wherein the host cell is any cell, cells, or cell line
which was obtained by fusing at least one cell, cells, or a cell
line of human myeloid leukaemia origin or any human myeloid or
myeloid precursor cell or cell line which can be obtained from a
leukaemia patient, or any myeloid or myeloid precursor cell or cell
line which can be obtained from a human donor, with another cell of
human or animal origin, such as but not limited to B cells, CHO
cells.
[0367] In a preferred embodiment said host cell the invention
provides for producing a protein/antibody molecule composition
having increased activity and/or increased yield and/or improved
homogeneity in sense of the invention as described elsewhere
herein, is the cell or cell line KG1, MUTZ-3, K562, NM-F9 [DSM
ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line derived
therefrom, or a mixture of cells or cell lines comprising at least
one of those aforementioned cells.
[0368] In a further preferred embodiment said host cell the
invention provides for producing a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity in sense of the invention as described
elsewhere herein, is the cell or cell line K562, NM-F9 [DSM
ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line derived
therefrom.
[0369] In a further preferred embodiment said host cell the
invention provides for producing a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity in sense of the invention as described
elsewhere herein, is the cell or cell line NM-F9 [DSM ACC2606],
NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived therefrom or a
cell or cell line derived therefrom as described elsewhere
herein.
[0370] In an even further preferred embodiment said host cell the
invention provides for producing a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity in sense of the invention as described
elsewhere herein, is a cell or a cell line derived from KG1,
MUTZ-3, K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9,
NM-C-2F5, NM-H9D8, or NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or
cell line derived therefrom as described elsewhere herein, which
grows under serum-free conditions, and preferably those in which
the nucleic acid encoding the protein/antibody molecule can be
introduced in these cells and an antibody molecule composition is
isolated under serum-free conditions.
[0371] In an even further preferred embodiment said host cell the
invention provides for producing a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity in sense of the invention as described
elsewhere herein, is a cell or a cell line derived from K562, NM-F9
[DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line derived
therefrom as described elsewhere herein, which grows under
serum-free conditions.
[0372] In an even further preferred embodiment said host cell the
invention provides for producing a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity in sense of the invention as described
elsewhere herein, is a cell or a cell line derived from K562, NM-F9
[DSM ACC2606], NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or
NM-H9D8-E6, NM-H9D8-E6Q12, GT-2X or a cell or cell line derived
therefrom as described elsewhere herein, which grows under
serum-free conditions and in which the nucleic acid encoding the
protein/antibody molecule can be introduced in these cells and a
protein/antibody molecule composition is isolated under serum-free
conditions.
[0373] In the most preferred embodiment said host cell the
invention provides for producing a protein/antibody molecule
composition having increased activity and/or increased yield and/or
improved homogeneity in sense of the invention as described
elsewhere herein, is the cell or cell line NM-F9 [DSM ACC2606],
NM-D4 [DSM ACC2605], NM-E-2F9, NM-C-2F5, NM-H9D8, or NM-H9D8-E6,
NM-H9D8-E6Q12, GT-2X or a cell or cell line derived therefrom as
described elsewhere herein, which grows under serum-free
conditions, and preferably those in which the nucleic acid encoding
the protein/antibody molecule can be introduced in these cells and
the protein/antibody molecule composition is isolated under
serum-free conditions.
[0374] The invention further provides a protein/protein composition
isolated by any of the methods of the invention described elsewhere
herein.
[0375] The invention further provides a protein molecule
composition isolated by any of the methods of the invention
described elsewhere herein which has an increased activity and/or
increased yield and/or improved homogeneity and fully human
glycosylation in sense of the invention and described elsewhere
herein.
[0376] In a further preferred embodiment of the invention the
protein molecule or part thereof has a size of at least 10 kDa,
preferably a size of at least 15 kDa, more preferably a size of at
least 20 kDa, more preferably a size of at least 25 kDa, more
preferably a size of at least 30 kDa, more preferably a size of at
least 35 kDa, more preferably a size of at least 40 kDa, more
preferably a size of at least 45 kDa, more preferably a size of at
least 50 kDa, more preferably a size of at least 55 kDa, more
preferably a size of at least 60 kDa, more preferably a size of at
least 65 kDa, more preferably a size of at least 70 kDa, more
preferably a size of at least 75 kDa, more preferably a size of at
least 80 kDa, more preferably a size of at least 85 kDa, more
preferably a size of at least 90 kDa, more preferably a size of at
least 95 kDa, more preferably a size of at least 100 kDa, more
preferably a size of at least 105 kDa, more preferably a size of at
least 110 kDa, more preferably a size of at least 115 kDa, more
preferably a size of at least 120 kDa, more preferably a size of at
least 125 kDa, more preferably a size of at least 130 kDa, more
preferably a size of at least 135 kDa, more preferably a size of at
least 140 kDa, more preferably a size of at least 145 kDa, more
preferably a size of at least 150 kDa, more preferably a size of at
least 155 kDa, more preferably a size of at least 160 kDa, more
preferably a size of at least 165 kDa, more preferably a size of at
least 170 kDa, more preferably a size of at least 175 kDa, more
preferably a size of at least 180 kDa, more preferably a size of at
least 185 kDa, more preferably a size of at least 190 kDa, more
preferably a size of at least 195 kDa, most preferably a size of at
least 200 kDa.
[0377] In a preferred embodiment of the invention said protein
molecule composition originates from any of the protein molecule of
the group of cytokines and their receptors, for instance the tumor
necrosis factors TNF-alpha and TNF-beta; renin; human growth
hormone and bovine growth hormone; growth hormone releasing factor;
parathyroid hormone; thyroid stimulating hormone; lipoproteins;
alpha-1-antitrypsin; insulin A-chain and B-chain; gonadotrophins,
e.g. follicle stimulating hormone (FSH), luteinizing hormone (LH),
thyrotrophin, and human chorionic gonadotrophin (hCG); calcitonin;
glucagon; clotting factors such as factor VIIIC, factor IX, factor
VII, tissue factor and von Willebrands factor; anti-clotting
factors such as protein C; atrial natriuretic factor; lung
surfactant; plasminogen activators, such as urokinase, human urine
and tissue-type plasminogen activator; bombesin; thrombin;
hemopoietic growth factor; enkephalinase; human macrophage
inflammatory protein; a serum albumin such as 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 such as bone-derived neurotrophic factor,
neurotrophin-3, -4, -5, -6 and nerve growth factor-beta;
platelet-derived growth factor; fibroblast growth factors;
epidermal growth factor; transforming growth factor such as
TGF-alpha and TGF-beta; insulin-like growth factor-I and -II;
insulin-like growth factor binding proteins; CD proteins such as
CD-3, CD-4, CD-8 and CD-19; erythropoietin (EPO); osteoinductive
factors; immunotoxins; a bone morphogenetic protein; an interferon
such as interferon-alpha, -beta, and -gamma; colony stimulating
factors (CSF's), e.g. M-CSF, GM-CSF and G-CSF; interleukins (IL's),
e.g. IL-1 to IL-12; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; antibodies and
immunoadhesins; Glycophorin A; MUC1.
[0378] In a more preferred embodiment of the invention said protein
molecule composition originates from any of the protein molecule of
the group of Glycophorin A, EPO, G-CSF, GM-CSF, FSH, hCG, LH,
interferons, interleukins, antibodies and/or fragments thereof.
[0379] Also provided is a glycol protein or protein composition
obtainable by the production methods according to the present
invention. Said protein preferably has the glycosylation
characteristics as defined in claim 27.
[0380] Preferably, said protein composition is an antibody molecule
composition. The invention further provides an antibody molecule
composition isolated by any of the methods of the invention
described elsewhere herein which has an increased activity and/or
increased yield and/or improved homogeneity in sense of the
invention and described elsewhere herein.
[0381] Examples of such antibodies include antibodies against
ganglioside GD3, human interleukin-5 receptor alpha-chain, HER2, CC
chemokine receptor 4, CD20, CD22, neuroblastoma, MUC1, epidermal
growth factor receptor (EGFR).
[0382] In a preferred embodiment of the invention said antibody
molecule composition originates from any of the antibody molecule
of the group of Muromomab, Daclizumab, Basiliximab, Abciximab,
Rituximab, Herceptin, Gemtuzumab, Alemtuzumab, Ibritumomab,
Cetuximab (Erbitux), Bevacizumab, Tositumomab, Pavlizumab,
Infliximab, Eculizumab, Epratuzumab, Omalizumab, Efalizumab,
Adalimumab, Campath-1H, C2B8, Panorex, BrevaRex, Simulect, Antova,
OKT3, Zenapax, ReoPro, Synagis, Ostavir, Protovir, OvaRex,
Vitaxin.
[0383] In a more preferred embodiment of the invention said
antibody molecule composition originates from any of the antibody
molecule of the group of Rituximab, Herceptin, anti-CC chemokine
receptor 4 antibody KM2160, Campath-1H, C2B8, Erbitux,
anti-neuroblastoma antibody chCE7. In an even more preferred
embodiment of the invention said antibody molecule composition
originates from the antibody molecule of the group of
WO2004/065423, more preferably PankoMab, more preferably its
chimaeric and even more preferably its humanized form.
[0384] Also provided is a protein or protein composition obtainable
by the production method of the present invention, wherein the
protein is an antibody which binds to the MUC1 epitope, comprising
the amino acid sequence DTR.
[0385] MUC-1 is an established tumor marker expressed on a variety
of epithelial tumours and is a potential tumor target. MUC-1 is a
large, highly 0-glycosylated transmembrane glycoprotein. The
extracellular portion consists of a variable number of 20 to 120
tandem repeats (TR), each of which consists of 20 amino acids with
five potential O-glycosylation sites. MUC 1 is not only expressed
on epithelial tissues but also on haematopoietic cells. Several
antibodies are known which bind the DTR motif of MUC 1 which are
also suitable proteins/antibodies in the context of the present
invention (for an overview see Karsten et al, 1998). Karsten also
described a novel carbohydrate induced conformational epitope on
MUC 1 (TA MUC) of the structure . . . PDT*RP . . . (SEQ ID NO: 22)
where T* is O glycosylated. The glycans present at this site are
themselves tumour-specific carbohydrate structures.
[0386] Hence, it is desirable to use a MUC antibody which can
discriminate between the TA MUC tumour epitope and the
non-glycosylated epitope. One suitable antibody able to
specifically recognize the glycosylated TA MUC epitope is the
PankoMab antibody. His production is described in detail in
Danielczyk et al 2006, herein fully incorporated by reference
(PankoMab: a potent new generation anti-tumor MUC-1 antibody). The
antibody PankoMab or a variant thereof, competitively binding the
same TA-MUC 1 epitope as the parent PankoMab antibody is preferably
used. Such an antibody variant has at least one of the following
characteristics: [0387] it binds an epitope comprising at least the
amino acid sequence PDTRP (SEQ ID NO: 22); [0388] it binds to a
short MUC peptide of 30 amino acids comprising 1,5 TRs when it is
glycosylated with Gal-NAcalpha at the PDTRP sequence (SEQ ID NO:
22) but not if the same peptide is not glycosylated; [0389] it
shows an additive length effect of more than 25, preferably 28
(most preferably a ratio of 29.5); [0390] it depicts a low or even
no binding to cells of the haematopoietic system (regarding the
detection method, please see Danielczyk et al 2006, herein
incorporated by reference); [0391] it has a high affinity towards
tumour cells ranging from approximately at least
K.sub.ass=0.2-1.times.10.sup.9M.sup.-1 as determined by Scatchard
plot analyses.
[0392] Suitable examples of respective variants are given in the
examples. The antibody can be of murine, origin, chimeric or
humanised.
[0393] The antibody binding the TA-MUC 1 epitope, preferably the
PankoMab antibody or the antibodies Panko 1 and Panko 2 as
described herein, has at least one of the following glycosylation
characteristics: [0394] (i) 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; [0395] (ii) 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; [0396] (iii) it comprises a detectable amount of
bisecGlcNAc; [0397] (iv) it has no or less than 2% hybrid or high
mannose structures.
[0398] A respective glycosylation pattern results in the following
surprising and beneficial activity pattern: [0399] (i) a CDC
activity which is more than 15% higher than the activity of the
same antibody expressed in CHO cells; [0400] (ii) a serum half life
which is elongated by factor 2 (more than 1,5) compared to an
antibody which carries no detectable sialylation; [0401] (iii) 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].
[0402] A respective antibody can be obtained by producing it in
cell lines according to the present invention which provide a high
sialylation and galactosylation degree, but preferably a lower
fucosylation. Suitable examples are NM-H9D8 and NM-H9D8-E6.
Example 1
[0403] Analyses of fut8 Expression in Cells
[0404] RNA was extracted from NM-F9 [DSM ACC2606], NM-D4 [DSM
ACC2605], NM-H9, K562, NM-H9D8 [DSM ACC2806], GT-2x [DSM ACC2858]
and HepG2 (control) cells according to standard procedures
(RNeasy-Mini-Kit, Qiagen). The mRNA was isolated using magnetic
beads technology according to manufacturers instruction
(HDynabeads.RTM. Oligo(dT)25H, Invitrogen) For the first-strandP
.sup.PcDNA synthesis, 50 .mu.l bead suspension of each sample and
Omniscript Reverse Transcriptase (Qiagen) were used according to
manufacturers instructions. For the subsequentP .sup.PRT-PCR
reaction 5 .mu.l of the cDNA productP .sup.Pand specific fut8
primers were used resulting in a 212 bp fragment. As a control
actin specific primers were used resulting in 240 bp fragment. The
resulting PCR-product(s)P .sup.Pwere analysed on a 1.5% gel.
[0405] NM-F9, NM-D4, NM-H9, K562, NM-H9D8 and GT-2x do express the
mRNA for fut8.
[0406] FIG. 1 shows as an example the fut8 mRNA expression of
NM-F9, NM-D4, and NM-H9D8.
Example 2
[0407] Glycoengineering of K562 Cells
[0408] Glycoengineering of K562 cells and generation of NM-D4 and
NM-F9 cell line are described in EP1654353.
[0409] NM-H9 cells, and a cell or cell line derived therefrom with
high sialylation potential were generated as follows. Random
mutagenesis was performed by treating K562 cells with the
alkylating agent ethyl methanesulfonate. Per sample K562 cells were
washed in PBS and seeded at 10P.sup.6P cells per ml cell culture
medium supplemented with EMS (0.1 mg/ml, ethyl methanesulfonate,
Sigma-Aldrich) overnight at 37.degree. C. and 5% COB.sub.2B. Cells
were washed and provided with fresh medium. Every second day cell
vitality was determined by trypan blue staining, and cells were
analysed by immunocytochemical staining.
[0410] Subsequently, cells exposing the novel phenotype of high TF
expression were selected by means of a TF-specific antibody. K562
cells were washed in B-PBS (0.5% BSA in PBS), incubated with 50
.mu.l of supernatant of hybridoma cultures of the monoclonal
antibody A78-G/A7 or PankoMab and 950 .mu.l of B-PBS at 4.degree.
C. for 30 min. After washing the procedure was repeated with 50
.mu.l of rat-anti-mouse-IgM-antibody or rat-anti-mouse-IgG-antibody
conjugated with MicroBeads (Miltenyi Biotec, Koln, Germany). After
washing the magnetically labelled TF-positive K562 cells were
separated by two successive columns provided by Miltenyi Biotec
(Koln, Germany) as described in the manufacturers manual. Following
nine days of cultivation, the isolation procedure was repeated in
total three times. FACS analysis (flow cytometry) started with
antibody staining: About 3.times.10.sup.5 cells were incubated at
4.degree. C. for 1.5 h with primary monoclonal antibody (hybridoma
culture supernatants of A78-G/A7 (IgM), PankoMab (IgG1), all
diluted 1:2 in cell culture medium) followed by the secondary
Cy3-conjugated goat anti-mouse IgM or IgG antibody 1:200 diluted in
PBS, at 4.degree. C. for 30 min and were washed again. Resuspended
cells (200 .mu.l PBS) were investigated by flow cytometry (flow
cytometer: Coulter Epics, Beckman Coulter, Krefeld, Ger).
Quantitative analyses were carried out using the Expo32 software
(Becton Coulter) with following parameter for antibody labelled
cells: forward scatter (FS): 26 V, gain 1, sideward scatter (SS):
807 V, gain 5, FL2: 740 V, gain 1, and following parameter for
lectin labeled cells: FS: 26 V, gain 1, SS: 807 V, gain 5, FL1:740
V, gain 1).
[0411] After three rounds of isolation a K562 cell population of
93% TF-positive cells was received. However, the percentage of
TF-positive K562 cells decreased over time reaching a bottom level
of about 20% TF-positive cells during a period of 14 days following
the isolation procedure. For stable expression of the TF-positive
phenotype, K562 cells were isolated for a forth time and finally,
the isolated TF-positive K562 cells were cloned thereafter by
limited dilution in 96-well plates (1 cell/100 .mu.l). Among thirty
K562 cell clones that were obtained, seventeen cell clones
expressed low amounts of the TF antigen or no TF antigen. These
cell clones were analysed for SNA binding in flow cytometry and for
proliferation rates (analysis of doubling time see below). Cell
clones showing high SNA binding and high proliferation rate were
selected. Stable NM-H9 clone was selected for further clone
development by single cell cloning and to optimise growth under
serum-free conditions. As the most preferred cell clone NM-H9D8
[DSM ACC2806] was selected and deposited under DSM ACC2806 at the
"DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH"
in Braunschweig (Germany), by Glycotope GmbH, Robert-Rossle-Str.
10, 13125 Berlin (Germany) at the Sep. 15, 2006.
Example 3
[0412] Cultivation of K562, NM-F9, NM-D4, NM-H9, NM-E-2F9,
NM-C-2F5, NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, and GT-2x Cell Lines
and CHOdhfr- Cells and Generation of Serum Free Cell Lines
[0413] K562, NM-F9, NM-D4, NM-H9, NM-E-2F9, NM-C-2F5, NM-H9D8 [DSM
ACC2806], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856], or
GT-2x [DSM ACC2858] were cultured in RPMI 1640 supplemented with
10% FCS and 2 mM glutamine or serum free in X-Vivo 20 medium and
grown at 37.degree. C. in a humidified atmosphere of 8%.
[0414] CHOdhfr- cells (ATCC No. CRL-9096) were cultured in DMEM
supplemented with 10% FCS, 2 mM glutamine, and 2% HT supplement or
serum free in CHO-S-SFM II medium and grown at 37.degree. C. in a
humidified atmosphere of 8%.
[0415] K562, NM-F9, NM-D4, NM-H9, NM-E-2F9, NM-C-2F5, NM-H9D8 [DSM
ACC2806], NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856], or
GT-2x [DSM ACC2858] and cell or cell line derived from said host
cells are easily adapted to serum free conditions by a complete
change of cell culture medium. Cells and cell lines according to
the present invention are inoculated into a serum-free medium as
X-Vivo 20 at a density of 1.times.10<5> to
5.times.10<5> cells/ml, preferably 2.times.10<5>
cells/ml, and cultured by an ordinary culturing method for animal
cells. After 4 to 7 days of culturing, the cells whose density
reached 5.times.10<5> to 10.times.10<5> cells/ml are
selected as the cells adapted to a serum-free medium. Adaptation to
serum-free media can be also performed by successive dilution of
the medium supplemented with FCS by a serum-free medium composition
(continuous adaptation). Productivity of converted FCS production
clones of antibody composition producing host cells of the present
invention to serum free conditions is mostly preserved.
[0416] Adaptation of CHOdhfr- (ATCC No. CRL-9096) to serum free
conditions has to be performed stepwise and takes several weeks
whereby a lost of productivity of at least a half is usual.
Example 4
[0417] Cloning of Vectors to Express the Chimeric Antibodies
PankoMab, Panko1, Panko2, or Cetuximab in Eukaryotic Cells
[0418] Variable sequences of PankoMab were PCR amplified with
specific primers from the murine hybridoma cells producing PankoMab
[HCancer Immunol Immunother. H 2006 November; 55(11):1337-47. Epub
2006 February PankoMab: a potent new generation anti-tumour MUC1
antibody. Danielczyk et al].
[0419] Variable sequences VH and VL of Panko1 and Panko2 are
described in WO2004/065423, herein incorporated by reference.
TABLE-US-00001 Variable Heavy chain of Panko1: (SEQ ID NO: 11)
EVKLVESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE
IRSKANNHATYYAESVKGRFTISRDVSKSSVYLQMNNLRAEDTGIYYCTR
GGYGFDYWGQGTTLTVS. Variable Light chain of Panko1: (SEQ ID NO: 12)
DIVLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPK
LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP LTFGDGTKLELKR.
Variable Heavy chain of Panko2: (SEQ ID NO: 13)
EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAE
IRLKSNNYTTHYAESVKGRFTISRDDSKSSVSLQMNNLRVEDTGIYYCTR
HYYFDYWGQGTTLTVS. Variable Light chain of Panko2: (SEQ ID NO: 14)
DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYFFWYLQKPGLSPQ
LLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELP
PTFGGGTKLEIKR.
[0420] Variable amino acid sequences of Cetuximab VH and VL were
obtained from
http://redpoll.pharmacy.ualberta.ca/drugbank/cgi-bin/getCard.cgi?CAR-
D=BTD00071.txt and reverse translated into cDNA coding sequences by
using VectorNTI.
TABLE-US-00002 Variable Heavy chain of Cetuximab: (SEQ ID NO: 15)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT
YYDYEFAYWGQGTLVTVS. Variable Light chain of Cetuximab: (SEQ ID NO:
16) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY
ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA GTKLELKR.
[0421] The cDNA sequence was extended by NcoI/NheI in the case for
the VL and NcoI/SalI in the case of the VH and the cDNA was
generated. The NcoI/XhoI-cut variable heavy chain fragments VH was
cloned into the NcoI/SalI-cut BS-Leader vector as described in
WO2004/065423. The BS-Leader vector includes a cloning cassette to
introduce the T cell receptor signal peptide sequence at the 5' end
and a splice donor sequence at the 3' end of the variable domains.
The variable light chain VL of the corresponding antibody was
amplified with a specific primer at the 3' end encoding
additionally the splice donor site and was cloned via NcoI/NheI
into the likewise digested BS-Leader vector. Thereafter, each
HindIII/BamHI fragment from the BS-Leader vector was cloned into
the corresponding eukaryotic expression vector. These vectors
(pEFpuroCgamma1VB.sub.HB, pEFdhfrCkappaVB.sub.LB,
pEFdhfrB.sub.mutBCkappaVB.sub.LB) comprise EF-1alpha-promoter and
HCMV enhancer, SV40 origin, polyadenylation signal, puromycin
resistance gene in the vector for the heavy chain and the murine
dihydrofolase gene (dhfr) for CHO cell expression or SEQ ID 1
(Sequence 1#) for K562, NM-F9 [DSM ACC2606], NM-D4 [DSM ACC2605],
NM-H9, NM-E-2F9, NM-C-2F5, NM-H9D8 [DSM ACC2806], NM-H9D8-E6 [DSM
ACC2807], NM-H9D8-E6Q12 [DSM ACC2856], or GT-2x [DSM ACC2858]
expression for selection and gene amplification in the vector for
the light chain, as well as the genomic sequences of the human
constant gamma1 region for the heavy chain or the human constant
kappa region for the light chain (primers for amplification from
genomic human DNA and vector map see WO2004/065423).
Example 5
[0422] Transfection of Eukaryotic Cells to Express the Chimeric
Antibodies PankoMab, Panko1, Panko2, or Cetuximab and Gene
Amplification Procedure to Generate High Producing Cell Clones in
the Presence of Serum
[0423] To express the chimeric antibodies in NM-F9 [DSM ACC2606] or
CHOdhfr- [ATCC No. CRL-9096] cells were co-transfected with a
mixture of above described vectors for the heavy and light chains
(1:1 to 1:3) by lipofection using DMRIE-C or electroporation for
the suspension cells as NM-F9 and lipofectamin or electroporation
for the adherent cell line CHOdhfr-. Two days post-transfection,
growth medium was changed to selection medium (NM-F9 in RPMI
1640+10% FCS+2 mM L-glutamine+0.75 .mu.g/ml puromycin+50 nM
methotrexate; CHOdhfr- in DMEM+10% dialysed FCS+2 mM L-glutamine+5
.mu.g/ml puromycin+50 mM methotrexate) for 1 week. First
amplification was performed by increasing methotrexate
concentration to 100 nM for additional 2 weeks. Part of amplified
cell population was single cell cloned in medium without addition
of puromycin and methotrexate, rest of the cells were subjected to
a new round of gene amplification by increasing the methotrexate
concentration. In this manner four to six rounds of gene
amplification (100, 200, 500, 1000, 2000, 3000 nM methotrexate)
were performed.
[0424] Additionally, best producing clones identified by clone
screening and analysis were amplified further similarly.
[0425] Following single cell cloning in 96-well plates using
limited dilution (0.5 cells per well), plates were cultivated for 2
to 3 weeks, microscopically analysed for growing cell clones, and
cloning efficiency in % (number of wells with growing cell
clones.times.100/theoretical number of seeded cells) was
determined. Growing clones were screened for productivity using
different procedures for adherent CHOdhfr- cells or for NM-F9
suspension cells.
[0426] CHOdhfr-:
[0427] Cells of growing clones were washed with PBS and harvested
by Accutase treatment. Half of resuspended cells were seeded in a
96-well test plate, the other half was seeded in a 24-well plate
for further cultivation. Test plate was cultivated for 20 to 24 h.
Supernatant of each well was analysed for antibody titre as
described in Determination of specific productivity rate (SPR) and
doubling time (g) (see below). Relative cell density was measured
using the MTT assay. In detail, cell were incubated with MTT
solution for 2 h, solution was discarded and cell lysed by a 0.04 M
HCl solution in 2-propanol. After 2 hours plate was moderately
mixed and measured using a microtiter plate photometer with 570 nm
filter in dual mode versus 630 nm reference filter.
[0428] NM-F9:
[0429] 96-well plates were centrifuged and supernatant was
discarded. Cells were resuspended in 200 .mu.l fresh medium. Half
of resuspended cells were seeded in a 96-well test plate and
diluted with 100 .mu.l medium, the other half remains in the
cloning plates for further cultivation. After 2 days of
cultivation, test plate was centrifuged and 20 .mu.l of supernatant
were analysed for antibody titre as described in Determination of
specific productivity rate (SPR) and doubling time (g) (see below).
Relative cell density was measured using the WST-1 assay by
addition of 10 .mu.l WST-1 solution (Roche) in each well. After 1
to 3 hours incubation measurement was performed using a microtiter
plate photometer with 450 nm filter in dual mode versus 630 nm
reference filter.
[0430] Cell clones with a high ratio of antibody titre to cell
density were selected and cultivated and analysed further.
[0431] The conditions for K562, NM-D4 and NM-H9 were the same.
[0432] Determination of specific productivity rate (SPR) and
doubling time (g)
[0433] For each clone, 2.times.10P.sup.4P cells were seeded per
well of a 24-well tissue culture plate in 500 .mu.l growth media.
The cells were allowed to grow for 3 days, conditioned media
harvested for analysis, and the cells were removed if necessary by
Accutase and counted. Specific antibody titres were quantitatively
determined from media samples by ELISA. Assay plates were coated
with a human kappa chain specific antibody (BD). Bound recombinant
antibody was detected with anti-human IgG (H+L) horseradish
peroxidase (HRP) conjugate (Jackson Immunoresearch Laboratories).
For the quantification, purified recombinant chimeric antibody was
used as a standard.
[0434] The SPR measured in picograms of specific protein per cell
per day (pcd) is a function of both growth rate and productivity,
and was calculated by following equations:
SPR = total protein mass integral cell area ( ICA ) ##EQU00001##
ICA = ( final cell number - initial cell number ) .times. days in
culture log B eB ( final cell number / initial cell number )
##EQU00001.2##
[0435] Doubling time was calculated by following equation:
g=log 2.times.(hours in culture)/log (final cell number/initial
cell number)
[0436] Determination of Average Yield and Maximum Yield for the
Cell Lines
[0437] Following single cell cloning in 96-well plates using
limited dilution (0.5 cells per well) as described above, from 200
theoretically plated single clones, the SPR is determined for
growing cell clones and the average and deviation is determined, as
well as the maximum yield for the different cell lines and
different conditions. Table 1 compares the data of chimeric
PankoMab-producing cell clones of CHOdhfr- and NM-F9 developed
under serum-containing conditions.
Example 6
[0438] Generation of Serum-Free High Yield Cell Clones Producing
Antibody Molecule Composition
[0439] Transfection of NM-H9D8, NM-H9D8-E6, NM-H9D8-E6Q12, or GT-2x
adapted to serum-free medium X-Vivo 20 was performed under
serum-free conditions using DMRIE-C or electroporation
(Nucleofector, Amaxa). Two days post-transfection, growth medium
was changed to selection medium (X-Vivo 20+0.75 .mu.g/ml
puromycin+50 nM methotrexate) for 1 week. First amplification was
performed by increasing methotrexate concentration to 100 nM for
additional 2 weeks. Part of amplified cell population was single
cell cloned in X-Vivo without addition of puromycin and
methotrexate, rest of the cells were subjected to a new round of
gene amplification by increasing the methotrexate concentration. In
this manner four to six rounds of gene amplification (100, 200,
500, 1000, 2000, 3000 nM methotrexate) were performed.
Additionally, best producing clones identified by clone screening
and analysis were amplified further similarly. Following single
cell cloning in 96-well plates using limited dilution (0.5 cells
per well), plates were cultivated for 2 to 3 weeks, microscopically
analysed for growing cell clones, and cloning efficiency in %
(number of wells with growing cell clones.times.100/theoretical
number of seeded cells) was determined. Growing clones were
screened for productivity using the same procedure as for NM-F9
suspension cells in the presence of serum (see above).
[0440] Determination of Average Yield and Maximum Yield for the
Cell Clones
[0441] Following single cell cloning in 96-well plates using
limited dilution (0.5 cells per well) as described above, from 200
theoretically plated single clones, the SPR is determined for
growing cell clones and the average and deviation is determined, as
well as the maximum yield for different conditions. Table 2 shows
data of chimeric PankoMab-producing cell clones of NM-H9D8 [DSM
ACC2806] developed completely under serum-free conditions.
[0442] Specific production rates (SPR) following amplification in
cell lines according to the invention are higher than in CHOdhfr-,
and highest in chimeric PankoMab-producing NM-H9D8 [DSM ACC2806]
cells developed under serum-free conditions.
[0443] The production rate of the majority of clones is highly
stable over at least 6 weeks without selection pressure. Best clone
was stable with a SPR of 30 pcd over 6 weeks with a doubling rate
of 24 h.
[0444] Date reflects productivity of clones in small-lab scale with
further potential in productivity increase by process development,
media optimisation and fermentation.
[0445] Higher productivity and yield of production clones developed
under serum-free conditions are advantageous and conditions are the
same for all other antibodies and for all cell lines as K562,
NM-F9, NM-D4, NM-H9, NM-E-2F9, NM-C-2F5, NM-H9D8-E6 [DSM ACC2807],
NM-H9D8-E6Q12 [DSM ACC2856], or GT-2x [DSM ACC2858] adapted to
serum-free conditions.
Example 7
[0446] Isolation of an Antibody Molecule Composition
[0447] For production and isolation of an antibody molecule
composition according to the invention, the stably transfected
cells secreting the chimeric antibodies PankoMab, Panko1, Panko2,
or Cetuximab were cultivated in serum free medium until a cell
density of about 1 to 2.times.10P.sup.6P cells/ml was reached.
Following removal of the cells from the cell culture supernatant by
centrifugation (400.times.g, 15 min), the chimeric antibody was
purified using a protein A column (HiTrap r-protein A FF, Amersham
Pharmacia Biotech). The purified antibody fraction eluted by sudden
pH change was re-buffered in PBS and concentrated using Centriprep
centrifuge tubes (cut off 50 kDa, Millipore).
Example 8
[0448] Determination of Fc-Mediated Cellular Cytotoxicity of
Antibody Molecule Compositions According to the Invention
[0449] PBMC Isolation from Blood Donors as Effector Cells
[0450] PBMC were isolated from blood of healthy donors by density
centrifugation with Ficoll-Hypaque (Biochrom). Cells were washed 3
times with RPMI 1640 supplemented with 5% FCS and cryopreserved in
separate batches of 5.times.10.sup.7 cells. PBMC were thawed and
used directly or kept overnight in RPMI 1640 supplemented with 10%
FCS (RPMI/FCS) before use in flow cytometry or as effector cells in
the cytotoxic assays.
[0451] Detection of Antibody-Dependent Cellular Cytotoxicity in an
In Vitro Model
[0452] The antibody-dependent cellular cytotoxicity (ADCC) of the
recombinant antibodies according to the invention was investigated
in an europium release assay. Target cells (ZR-75-1,
5.times.10.sup.6) were incubated for 6 minutes on ice in 800 .mu.l
of europium buffer (50 mM HEPES, pH 7.4, 93 mM NaCl, 5 mM KCl, 2 mM
MgCl.sub.2, 10 mM diethylenetriaminepentaacetic acid, 2 mM
europium(III)acetate), electroporated (710 V, 1 pulse, 30 .mu.s) in
a Multiporator (eppendorf), and subsequently incubated on ice for
another 6 min. Thereafter, the cells were washed 5 times in RPMI
1640/5% FCS and seeded in a 96-well round-bottom plate (Nunc;
1.times.10.sup.4 cells in 100 .mu.l per well). Following addition
of 20 .mu.l of recombinant antibodies at varying concentration (0.5
to 25 .mu.g/ml final concentration in 200 .mu.l incubation volume)
or the corresponding controls (medium, isotype control human IgG),
human peripheral blood cells (80 .mu.l per well) were added as
effector cells, using an effector/target cell ratio of 50:1. 80
.mu.l RPMI/FCS with no effector cells was added to determine
spontaneous release. Maximum release was determined after complete
lysis of the target cells with ethanol. Following incubation in an
incubator at 37.degree. C. for 4 hours, the plate was centrifuged
at 500.times.g for 5 minutes, and 25 .mu.l of each sample was
pipetted in 200 .mu.l per well of enhancement solution
(Perkin-Elmer Wallac). Following incubation for 15 min at room
temperature, the fluorescence was determined (VictorP.sup.2P
Fluorometer, Perkin-Elmer Wallac). The specific cytotoxicity is
obtained from the equation (experimental lysis-spontaneous
lysis)/(maximum lysis-spontaneous lysis)*100.
[0453] Alternatively, target cells (LS174T or ZR-75-1,
3.times.10.sup.6 cells) were incubated for 6 minutes on ice in 100
.mu.l of europium buffer (50 mM HEPES, pH 7.4, 93 mM NaCl, 5 mM
KCl, 2 mM MgCl.sub.2, 10 mM diethylenetriaminepentaacetic acid, 2
mM europium(III)acetate), electroporated in a Nucleofector II
(Amaxa) with program A-011, and subsequently incubated on ice for
another 6 min. Thereafter, the cells were washed 4 times in RPMI
1640/5% FCS and seeded in a 96-well round-bottom plate (Nunc; 5 to
10.times.10.sup.3 cells in 100 .mu.l per well). Following addition
of 20 .mu.l of recombinant antibodies at varying concentration
(0.001 to 100 ng/ml final concentration in 200 .mu.l incubation
volume for Cetuximab; 0.16-5 .mu.g/ml for chimeric PankoMab,
Panko1, or Panko2) or the corresponding controls (medium, isotype
control human IgG), human peripheral blood cells (80 .mu.l per
well) were added as effector cells, using an effector/target cell
ratio of 100:1 to 50:1. 80 .mu.l RPMI/FCS with no effector cells
was added to determine spontaneous release. Maximum release was
determined after complete lysis of the target cells with 1% Triton
X-100. Following incubation in an incubator at 37.degree. C. for 4
hours or over night, the plate was centrifuged at 500.times.g for 5
minutes, and 25 .mu.l of each sample was pipetted in 200 .mu.l per
well of enhancement solution (Perkin-Elmer Wallac). Following
incubation for 10 min at room temperature, the fluorescence was
determined (Victor.sup.2 Fluorometer, Perkin-Elmer Wallac). The
specific cytotoxicity is obtained from the equation (experimental
lysis-spontaneous lysis)/(maximum lysis-spontaneous lysis)*100.
[0454] The ADCC activity of the Cetuximab isolated from fut8.sup.-
cell line NM-H9D8-E6 is significantly higher than the ADCC activity
of the Cetuximab isolated from the CHOdhfr- cells or SP2/0 cells.
As shown in FIG. 2, Cetuximab isolated from NM-H9D8-E6 induces the
same specific lysis of LS174T cells at about 30 times lower
antibody concentrations as Cetuximab isolated from CHOdhfr- or from
SP2/0 cells (Erbitux, obtained from Merck). This indicates a 30
times higher ADCC activity compared with the commercially available
product. This result is obtained by using the production method of
the present invention, resulting in an optimised glycosylation
pattern.
[0455] Also the ADCC activity of the chimeric PankoMab isolated
from NM-F9 [DSM ACC2606], K562, NM-H9, NM-D4 [DSM ACC2605],
NM-H9D8-E6 [DSM ACC2807], NM-H9D8-E6Q12 [DSM ACC2856], GT-2x [DSM
ACC2858], or NM-H9D8 [DSM ACC2806] is about 5 times higher than the
ADCC activity of the chimeric PankoMab isolated from the CHOdhfr-
cells. About a fifth of the chimaeric PankoMab concentration leads
to the same ADCC activity as the chimeric PankoMab expressed in the
CHOdhfr- cells. FIG. 3 shows the respective results using the NM-F9
cell line for comparison purposes.
[0456] The ADCC activity of the chimeric Panko1 isolated from
NM-H9D8-E6 or NM-H9D8-E6Q12 is about 8 times higher than the ADCC
activity of the chimeric Panko1 isolated from CHOdhfr- cells. About
an eighth of the chimeric Panko1 concentration leads to the same
ADCC activity as the chimeric Panko1 expressed in the CHOdhfr-
cells. The respective results are shown in FIG. 4.
[0457] The ADCC activity of the chimeric Panko2 isolated from
NM-H9D8 or GT-2x is about 6 times higher than the ADCC activity of
the chimeric Panko2 isolated from CHOdhfr- cells. About a sixth of
the chimeric Panko2 concentration leads to the same ADCC activity
as the chimeric Panko2 expressed in the CHOdhfr- cells. The
respective results are shown FIG. 5.
[0458] The ADCC activity of the chimeric Panko2 isolated from
NM-H9D8-E6 is lower than the ADCC activity of the chimeric Panko2
isolated from NM-H9D8 cells. The respective results are shown FIG.
6.
[0459] Detection of Complement-Dependent Cellular Cytotoxicity in
an In Vitro Model
[0460] The complement-dependent cellular cytotoxicity (CDC) of
antibodies was investigated in an europium release assay.
Eu.sup.3+-loaded target cells (ZR-75-1, 3.times.10.sup.6) were
prepared as described above.
[0461] Thereafter, the cells were seeded in a 96-well round-bottom
plate (Nunc; 5.times.10.sup.3 cells in 100 .mu.l per well).
Following addition of 20 .mu.l of antibodies at varying
concentration (0.5 to 50 .mu.g/ml final concentration in 200 .mu.l
incubation volume) or the corresponding controls (medium, isotype
control human IgG), cells were incubated half an hour at room
temperature. Thereafter, 10 .mu.l per well baby rabbit complement
(Cedarline, 1:5 to 1:10 diluted) were added. 10 .mu.l RPMI/FCS with
no complement was added to determine spontaneous release. Maximum
release was determined after complete lysis of the target cells
with ethanol or 1% TritonX-100. Following incubation in an
incubator at 37.degree. C. for 3 to 3.5 hours, the plate was
centrifuged at 500.times.g for 5 minutes, and 25 .mu.l of each
sample was pipetted in 200 .mu.l per well of enhancement solution
(Perkin-Elmer Wallac). Following incubation for 10 min at room
temperature, the fluorescence was determined (VictorP.sup.2P
Fluorometer, Perkin-Elmer Wallac). The specific cytotoxicity is
obtained from the equation (experimental lysis-spontaneous
lysis)/(maximum lysis-spontaneous lysis)*100.
[0462] CDC activity of chimeric PankoMab isolated from NM-F9 cells
is 8 times higher than CDC activity of chimeric PankoMab isolated
form CHOdhfr- cells. Same specific lysis is induced at 8 times
lower concentration of chimeric PankoMab isolated from NM-F9
compared to chimeric PankoMab isolated from CHOdhfr- cells. The
respective results are shown FIG. 7.
[0463] Conjugate Formation Assay (CFA) to Analyse Phagocytosis
Activity of Antibody Molecule Compositions
[0464] Tumor Cell Staining with PKH26:
[0465] 1.times.10P.sup.7P to 2.times.10P.sup.7P tumor cells were
washed twice in PBS, resuspended in 1 ml diluent C, 1 ml PKH26 was
added at concentration 12.times.10P.sup.-6P M for ZR-74-1,
ZR-74-1TF, MCF-7 and MT-3. After 3 min incubation at RT, staining
was stopped by adding 2 ml FCS for 1 min at RT. Medium (4 ml, RPMI
supplemented with 1% L-glutamine, 10% FCS) was added, cells were
spun down and washed 4 times with medium. Tumor cells were
incubated o.n. at 37.degree. C., 5% COB.sub.2B to allow the release
of excess PKH-26.
[0466] Optimization of PKH-26 staining was performed with half of
the cell numbers and volumes.
[0467] CFA:
[0468] PKH-26 labeled tumor cells were harvested with trypsin/EDTA,
washed once with PBS and resuspended at 0.8.times.10P.sup.6P
cells/ml in MAK cell medium (Invitrogen) in the presence or absence
of recombinant antibody molecule compositions. Tumor cells (250
.mu.l, 0.2.times.10P.sup.6P cells) were seeded in non-adherent
polypropylene tubes.
[0469] MAK cells were prepared according to Boyer et al., Exp.
Hematol. 27, 751-761 (1999), washed and resuspended at
1.6.times.10P.sup.6P cells/ml. 250 .mu.l MAK cells
(0.2.times.10P.sup.6P cells) were added to PKH-26 labeled tumor
cells (E:T ratio 2:1). Individual samples were incubated in
duplicates at 4.degree. C. and 37.degree. C./5% COB.sub.2B for 3 h
or o.n. (18-20 h).
[0470] After incubation at 3 h or o.n. cells were washed with PBS
and stained with CD11c-FITC (1:18.5)+7-AAD (1:500) in PBS/10% FCS.
Cells were washed with PBS, resuspended in 400 .mu.l PBS.
Acquisition was done on 10.000 cells in an Epics XL (Beckman
Coulter).
[0471] Percentage of colocalized MAK cells was determined as
percentage of PKH-26 positive cells in the cell population gated
for all viable CD11c-FITC positive cells.
Example 9
[0472] Analysis of Antibody Molecule Composition Binding Activity
in ELISA
[0473] To analyse antigen binding of antibody molecule compositions
expressed in different cell lines purified antibody molecule
compositions of chimeric PankoMab and Panko 2 were measured in an
ELISA on synthetic glycosylated MUC1 30-mer peptide with the
sequence APPAHGVTSAPDT [GalNAcalpha]RPAPGSTAPPAHGVISA (SEQ ID NO:
23). The non-glycosylated MUC1 peptide served as a control.
[0474] Using stock solutions (1 mg in 1 ml of bidest. HB.sub.2BO)
stored in portions at -20.degree. C., a dilution of 1 .mu.g/ml in
PBS was produced. 50 .mu.l/well was pipetted in a NUNC Immuno plate
F96 MaxiSorp, and the test plate was incubated at 4.degree. C.
overnight. On the next day, the test plate was washed 3 times with
PBS/0.2% Tween-20. Subsequently, non-specific binding sites were
blocked with 2% BSA in PBS, incubated for at least 1 h at room
temperature, and 50 .mu.l of the recombinant PankoMab was applied
(1-400 ng/ml PBS/1% BSA). After three wash steps with PBS/0.2%
Tween-20, peroxidase-coupled secondary anti-human Fc.gamma.1
antibody was employed to detect the specifically bound antibody. To
detect the bound secondary antibody, a color reaction with TMB
(3,3'',5,5''-tetramethylbenzidine) was performed. After 15 minutes
the reaction was quenched by adding 2.5 N HB.sub.2BSOB.sub.4B.
Measurement was performed using a microtiter plate photometer with
450 nm filter in dual mode versus 630 nm reference filter.
[0475] The binding activity of the chimaeric PankoMab isolated from
NM-F9 is about 50% higher than the binding activity of the
chimaeric PankoMab isolated from the CHOdhfr- cells (FIG. 8).
[0476] The binding activity of the chimeric Panko2 expressed and
isolated from the high sialylating clone NM-H9D8 in ELISA is
comparable to that from GT-2x and higher compared to that from
CHOdhfr- cells (FIG. 9).
Example 10
[0477] Glycan Analysis of Antibody Molecule Compositions Expressed
in NM-F9, NM- and CHOdhfr- Cells
[0478] Glycans of at least 100 .mu.g purified antibody were cleaved
by acid hydrolysis. Sialic acids were labelled specifically by
conjugation with the fluorescence dye DMB. Analysis was performed
by HPLC e.g. on an Asahipak-NH2 column to separate the saccharides.
Identification and calculation of sialic acids was performed by
standard substances of appropriate sialic acids.
[0479] Analysis of chimeric PankoMab antibody molecule composition
expressed in CHOdhfr- or NM-F9 cells revealed in <10%
monosialylation of the CHOdhfr- product and nearly no sialylation
of the NM-F9 or GT-2x product. The latter contained only
non-charged glycans.
[0480] In more detail, glycans of at least 100 .mu.g purified
antibody were cleaved by acid hydrolysis. Sialic acids were
labelled specifically by conjugation with the fluorescence dyes
like DMB. Analysis was performed by HPLC e.g. by reverse phase
chromatography on a RP-18 column. Identification and calculation of
sialic acids was performed by standard substances of appropriate
sialic acids.
[0481] For the determination of the charged glycan structures and
the galactosylation state 100 .mu.g antibodies were digested by
trypsin and the N-Glykans was obtained by PNGaseF treatment. The
purified glycans were labelled with 2-aminobenzamide (2-AB) and
subjected to an anion exchange chromatography HPLC
(Asahi-PAK-column) for the determination of charged N-glycan
structures. For the determination of the galactosylation state the
--N-glycans were treated by neuraminidase for depletion of the
sialic acid content and separated by an Aminophase HPLC
(Luna-NH2-column) and the peaks analysed by mass spectrometry.
[0482] The potentially bisecting GlcNAc carrying glycan containing
peaks were separated in a second step by a reverse phase
chromatography (RP18-column). By this triantennary and
biantennary+bisected structures can be distinguished. The
fucosylation degree of the antibodies was determined by aminophase
HPLC (Luna-NH2-column) of the 2-AB labelled glycans. The peaks were
quantified by integration and underlying glycan structure was
analysed by mass spectrometry. The fucosylated and non-fucosylated
structures were determined and the integrated peak areas were
quantified.
[0483] Different antibody molecule compositions of Cetuximab,
PankoMab and Panko1 expressed in NM-F9, GT-2x, NM-H9D8, NM-H9D8-E6,
or CHOdhfr- cells were analysed and results are summarised in the
tables 3 to 4.
[0484] Lectins which bind preferentially to alpha2-6 (SNA) or
alpha2-3 (MAL-I) linked sialic acids were used to characterize the
antibody sialylation by ELISA or Western blot analysis.
[0485] Western blot analysis was performed to identify the
differently sialylated heavy chain of antibody molecule
compositions expressed in CHOdhfr-, NM-F9, or NM-H9D8 [DSM
ACC2806]. 5 .mu.g of each antibody molecule composition were
separated by SDS-Page in a 10% acrylamide gel under reducing
conditions. Proteins were transferred to nitrocellulose and
visualized by lectins and/or by secondary anti-human IgG
antibodies. FIG. 10 shows the differently sialylated heavy chain of
antibody molecule compositions of chimeric PankoMab expressed in
CHOdhfr-, NM-F9, or NM-H9D8 [DSM ACC2806] either by secondary
anti-human IgG antibodies (FIG. 10A) or SNA (FIG. 10B) which
detects 2-6 sialylation. FIG. 11 shows the differently sialylated
heavy chain of antibody molecule compositions of Cetuximab
expressed in CHOdhfr- or NM-H9D8 visualized by SNA binding.
[0486] ELISA experiments were used to analyse the sialylation of
antibodies isolated from supernatants of NM-F9, NM-H9D8, or
NM-H9D8-E6 cells. Purified antibodies of 2 .mu.g/ml in PBS and 50
.mu.l per well were coated to 96 well microtiter plates (Maxisorp)
at 4.degree. C. over night, blocked (PBS/BSA), washed and incubated
for 1 hour with biotinylated SNA (2 .mu.g/ml, PBS/1% BSA) and
washed again. Wells were than incubated with POD-streptavidin,
washed and developed with TMB. Reaction was stopped by addition of
2.5N H.sub.2SO.sub.4 and the optical density was measured at 450 nm
versus 630 nm as reference. FIG. 12 shows that binding of SNA
occurs to antibodies expressed in NM-H9D8 or NM-H9D8-E6 cells, but
not to antibodies expressed in CHOdhfr- or GT-2x cells. Intensities
of binding are different for the individual antibodies what
indicates a different sialylation degree caused by the fine
structure of the individual antibodies or the availability of the
saccharides under the conditions used.
[0487] ELISA experiments were performed by coating the purified
antibodies expressed in the different cell lines in wells of
microtiter plates (2 .mu.g/ml, 50 .mu.l per well) and detecting by
appropriate biotinylated lectins SNA and MAL I. Dependence of
lectin binding by sialylation was checked by neuraminidase
treatment (0.1 U/ml and incubation at room temperature for 1 hour).
Results for Cetuximab are illustrated in FIGS. 13A and 13B.
[0488] Dot blot analyses were performed to identify differences in
sialylation of antibodies expressed in CHOdhfr-, NM-F9, NM-H9D8,
and NM-H9D8-E6 cells. 3 .mu.g of purified antibodies were spotted
each to nitrocellulose, blocked with PBS/BSA, washed and visualized
by SNA which detects 2,6-sialylation. FIG. 14 shows blots of the
chimeric antibodies Panko1 and Panko2 isolated from CHOdhfr-,
NM-F9, NM-H9D8, or NM-H9D8-E6 cells. 2,6-sialylation is only
detectable on antibodies isolated from NM-H9D8 or NM-H9D8-E6
cells.
Example 11
[0489] Detection of Bioavailability of Antibody Molecule
Compositions Expressed in NM-F9, NM-H9D8 and CHOdhfr- Cells
[0490] A longer bioavailability was measured in nude mice for the
sialylated antibody PankoMab expressed in NM-H9D8 compared to the
non sialylated antibody PankoMab expressed in NM-F9 cells. 5 .mu.g
purified antibody per mouse was i.v. injected for at least 3 mice
per group. Blood samples were collected at different time points
after injection (5 minutes, 2, 5, 8, 24, 48, 72, and 144 hours
after injection), serum was isolated and the samples were stored at
-80.degree. C. until analysis. The antibody titer was determined in
these samples by ELISA. FIG. 15 shows that chimeric PankoMab
isolated from NM-H9D8 cells is longer available than that isolated
from NM-F9 cells which is probably caused by the different
sialylation of the antibodies.
Example 12
[0491] Cloning of Vectors to Express hFSH in Eukaryotic Cells
[0492] Coding sequences of FSH alpha and FSH beta chain were PCR
amplified with specific primers using the BAC clones
RZPDB737B122053D (FSHalpha genomic), IRAUp969E0752D (FSHalpha cDNA)
and RZPDB737H0619D6 (FSHbeta genomic) obtained from the RZPD,
Germany.
TABLE-US-00003 Primers for FSH alpha chain: (SEQ ID NO: 17)/
FSHa-wt-f-KpnI: AAAGGTACCATGGATTACTACAGAAAATATG (SEQ ID NO: 18)
FSHa-b-BamHI: AAAGGATCCTTAAGATTTGTGATAATAAC; Primers for FSH beta
chain: (SEQ ID NO: 19)/ FSHb-wt-f-HindIII:
TTTAAGCTTATGAAGACACTCCAGTTTTTC (SEQ ID NO: 20) FSHb-b-BamHI:
TTTGGATCCTTATTCTTTCATTTCACC
[0493] After amplification the products were cloned via
HindIII/BamHI (FSHgenomicbeta) and KpnI/BamHI (FSHgenomicalpha or
FSHcDNAalpha) into the corresponding eukaryotic expression vector.
A cDNA sequence was produced by Gene synthesis coding for the FSH
beta chain fused to a TCR-leader sequence and cloned into the
eukaryotic expression vector (FSHcDNAbeta).
[0494] Sequence FSHcDNAbeta (TCR-leader sequence underlined):
TABLE-US-00004 (SEQ ID NO: 21)
atggcctgccccggcttcctgtgggccctggtgatcagcacctgcctgga
attctccatggctaacagctgcgagctgaccaacatcaccatcgccatcg
agaaagaggaatgccggttctgcatcagcatcaacaccacctggtgcgcc
ggctactgctacacccgggacctggtgtacaaggaccccgccaggcccaa
gatccagaaaacctgcaccttcaaagaactggtgtacgagaccgtgcggg
tgcccggctgcgcccaccacgccgacagcctgtacacctaccccgtggcc
acccagtgccactgcggcaagtgcgacagcgacagcaccgactgcaccgt
gaggggcctgggccccagctactgcagcttcggcgagatgaaagagtga
[0495] The expression vectors (pEFpuro and pEFdhfrB.sub.mutB)
comprise EF-1alpha-promoter and HCMV enhancer, SV40 origin,
polyadenylation signal, puromycin resistance gene in the vector for
the alpha chain, and the murine dihydrofolase gene (dhfr) for CHO
cell expression or SEQ ID 1 (Sequence 1#) for NM-F9, GT-2x, K562,
NM-H9, NM-D4, or NM-H9D8 expression for selection and gene
amplification in the vector for the beta chain.
Example 13
[0496] Transfection of Eukaryotic Cells to Express Human
Recombinant hFSH and Gene Amplification Procedure to Generate High
Producing Cell Clones Under Serum Free Conditions (GT-2x and
NM-H9D8) or Serum Containing Conditions (CHOdhfr-)
[0497] To express hFSH in GT-2x [DSM ACC2858], NM-H9D8 (DSM
ACC2806), and CHOdhfr- (ATCC No. CRL-9096) cells were
co-transfected with a mixture of above described vectors for alpha
and the beta chains (1:1 to 1:3) by lipofection using DMRIE-C or
electroporation (Nucleofector; Amaxa) for the suspension cells as
GT-2x (FSHcDNAalpha/FSHgenomicbeta) and NM-H9D8
(FSHcDNAalpha/FSHcDNAbeta) and lipofectamin or electroporation for
the adherent cell line CHOdhfr- (FSHgenomicalpha/FSHgenomicbeta).
These are exemplary combinations used in this setup. Each other
combination of a hFSH alpha and a hFSH beta chain expression
plasmid leads to comparable results.
[0498] Two days post-transfection, growth medium was changed to
selection medium (GT-2x and NM-H9D8 in X-Vivo20 medium+0.75
.mu.g/ml puromycin+50 nM methotrexate; CHOdhfr- in DMEM+10%
dialysed FCS+2 mM L-glutamine+5 .mu.g/ml puromycin+50 mM
methotrexate) for 1 week. First amplification was performed by
increasing methotrexate concentration to 100 nM for additional 2
weeks and 200 nM methotrexate for another 2 weeks. Part of
amplified cell population was single cell cloned in medium without
addition of puromycin and methotrexate. Rest of the cells were
subjected to a new round of gene amplification by increasing the
methotrexate concentration. In this manner two to three rounds of
gene amplification (100, 200, 500 nM methotrexate) were performed.
Additionally, best producing clones identified by clone screening
and analysis were amplified further similarly.
[0499] CHOdhfr-:
[0500] Cells of growing clones were washed with PBS and harvested
by Accutase treatment. Half of resuspended cells were seeded in a
96-well test plate, the other half was seeded in a 24-well plate
for further cultivation. Test plate was cultivated for 20 to 24 h.
Supernatant of each well was analysed for antibody titre as
described in Determination of specific productivity rate (SPR) and
doubling time (g) (see below). Relative cell density was measured
using the MTT assay. In detail, cell were incubated with MTT
solution for 2 h, solution was discarded and cell lysed by a 0.04 M
HCl solution in 2-propanol. After 2 hours plate was moderately
mixed and measured using a microtiter plate photometer with 570 nm
filter in dual mode versus 630 nm reference filter.
[0501] GT-2x and NM-H9D8:
[0502] 96-well plates were centrifuged and supernatant was
discarded. Cells were resuspended in 200 .mu.l fresh medium. Half
of resuspended cells were seeded in a 96-well test plate and
diluted with 100 .mu.l medium, the other half remains in the
cloning plates for further cultivation. After 2 days of
cultivation, test plate was centrifuged and 20 .mu.l of supernatant
were analysed for antibody titre as described in Determination of
specific productivity rate (SPR) and doubling time (g) (see below).
Relative cell density was measured using the WST-1 assay by
addition of 10 .mu.l WST-1 solution (Roche) in each well. After 1
to 3 hours incubation measurement was performed using a microtiter
plate photometer with 450 nm filter in dual mode versus 630 nm
reference filter.
[0503] Determination of Specific Productivity Rate (SPR) and
Doubling Time (g)
[0504] For each clone, 2.times.10P.sup.4P cells were seeded per
well of a 24-well tissue culture plate in 500 .mu.l growth media.
The cells were allowed to grow for 3 days, conditioned media
harvested for analysis, and the cells were removed if necessary by
Accutase and counted. Specific antibody titres were quantitatively
determined from media samples by ELISA. Assay plates were coated
with a mouse mAB specific to hFSH beta (ab22473). Bound recombinant
hFSH was incubated with goat polyclonal antibody specific to hCG
alpha (ab20712) and detected with a donkey anti goat IgG H+L-POD
(JacksonImmuno Cat. No 305-035-003). For the quantification,
purified recombinant hFSH was used as a standard.
[0505] The SPR measured in picograms of specific protein per cell
per day (pcd) is a function of both growth rate and productivity,
and was calculated by following equations:
SPR = total protein mass integral cell area ( ICA ) ##EQU00002##
ICA = ( final cell number - initial cell number ) .times. days in
culture log B eB ( final cell number / initial cell number )
##EQU00002.2##
[0506] Doubling time was calculated by following equation:
g=log 2.times.(hours in culture)/log (final cell number/initial
cell number)
Determination of Average Yield and Maximum Yield for the Cell
Lines
[0507] Following single cell cloning in 96-well plates using
limited dilution (0.5 cells per well) as described above, from 200
theoretically plated single clones, the SPR is determined for
growing cell clones and the average and deviation is determined, as
well as the maximum yield for the different cell lines and
different conditions. Table 7 and Table 8 compares the data of hFSH
producing cell clones of CHOdhfr-, GT-2x, and NM-H9D8 developed
under serum-containing conditions (CHOdhfr-) and serum free
conditions (GT-2x and NM-H9D8).
Example 14
[0508] Isolation of hFSH Molecule Composition
[0509] For production and isolation of a hFSH molecule composition
according to the invention, the stably transfected cells secreting
hFSH were cultivated in serum free medium until a cell density of
about 1 to 2.times.10P.sup.6P cells/ml was reached. Following
removal of the cells from the cell culture supernatant by
centrifugation (400.times.g, 15 min), the chimeric antibody was
purified comprising affinity chromatography using a polyclonal
antibody to hCGalpha coupled to an NHS activated column (HiTrap
NHS-activated HP; GE Healthcare). The purified FSH fraction eluted
by sudden pH change was re-buffered in PBS and concentrated using
Centriprep centrifuge tubes (cut off 10 kDa, Millipore) and
analysed by SDS-PAGE (see FIG. 16).
Example 15
[0510] Determination the Activity of FSH Molecule Compositions
According to the Invention
[0511] The activity of FSH molecules carrying a different
glycosylation pattern can be determined according to the
subsequently described methods. In order to select a suitable cell
line with the screening method according to the present invention,
which provides an optimized glycosylation, the FSH molecule is
expressed in different cell lines, thereby obtaining FSH
compositions from the individual cell lines depicting a diverging
glycosylation pattern with respect to e.g. the sialylation degree,
galactosylation and/or fucosylation degree. The FSH
molecule/composition having the most favourable activity and hence
glycosylation pattern can be determined by at least one of the
following methods:
[0512] Rat Granulosa Cell Assay:
[0513] Granulosa cells were obtained from DES treated
hypophysectomised rats. The method for the preparation of the cells
is described in detail by Jia and Hsueh 1985.
[0514] The obtained cells were cultured in a 24 well scale with
160000 cells/well in McCoy's 5A medium containing 100 nM DES, 0,125
M 1-methyl-3-isobuthylxanthine, 2 mM Glutamine, 1 .mu.M
androstenedione, 100 U/ml penicillin and 100 .mu.g/ml streptomycin,
1 .mu.g/ml bovine insulin and increasing concentrations of rFSH
preparations derived from GT-2x and NM-H9D8 cells (0-1 .mu.g/ml)
for up to 72 hours. The media were collected 72 h after incubation
and stored at -20.degree. C. until quantification of oestradiol by
ELISA (Calbiotech # ES071S).
[0515] 293HEK Assay:
[0516] HEK293 cells stably transfected with the hFSH-receptor
(2.times.10.sup.5 cells/35 mm culture dish) were exposed to
increasing doses of each protein molecule composition of FSH
obtained from GT-2x and NM-H9D8 cells in the range of 0-1 .mu.g/ml
FSH in the presence of 0.125 mM 1-methyl-3-isobuthylxanthine for 24
h at 37.degree. C. for up to 72 hours. After incubation, total
(intra- and extracellular) cAMP concentrations were determined by
the cAMP Direct Biotrak.TM. EIA (GE Healthcare, Cat. no.: RPN225)
according to the manufacturer's instructions.
[0517] GFSHR-17 Cell Assay:
[0518] Cells were cultured with Dulbecco modified Eagle medium Ham
F12 (1:1 v:v; Biochrom KG, Berlin, Germany) containing 5% fetal
calf serum (Biochrom KG, Berlin, Germany). The cells were plated
with 2.times.10.sup.5 cell/24 well plate and incubated with the FSH
protein molecule compositions obtained from GT-2x and NM-H9D8 cells
for 24 h at a range from 0-1 .mu.g/ml for 1-24 hours. The media
were collected after incubation and stored at -20.degree. C. until
quantification of progesterone by using a progesterone ELISA assay
(Biochem Immunosystems, Freiburg, Germany) according to the
manufacturer's instructions.
[0519] Human Granulosa Cell Assay
[0520] Granulosa-lutein cells from follicular aspirates, obtained
from women participating an in vitro fertilization program at the
University of Munster, were enriched as described by Khan et al.,
1990 and seeded into 6-well plates (1-1.5.times.10.sup.5 viable
cells per well). The cells were incubated at 37.degree. C. in HAM's
F12/Dulbecco's modified essential medium (DMEM) (1:1; ICN
Biomedicals, Meckenheim, Germany) supplemented with 10%
heat-inactivated fetal calf serum (FCS; Gibco, Eggenstein,
Germany), penicillin (50 units/nil), streptomycin (50 pg/ml),
gentamycin (100 pg/ml) and amphotericin (0.6 pg/ml) in an
atmosphere of 5% CO.sub.2. The cells were exposed to increasing
doses of each protein molecule composition of FSH obtained from
GT-2x and NM-H9D8 cells in the range of 0-1 .mu.g/ml FSH in the
presence of 0.125 mM 1-methyl-3-isobuthylxanthine for 24 h at
37.degree. C. for up to 72 hours. After incubation, total (intra-
and extracellular) cAMP concentrations were determined by the cAMP
Direct Biotrak.TM. EIA (GE Healthcare, Cat. no.: RPN225) according
to the manufacturer's instructions.
Example 16
[0521] Glycan Analysis of FSH Compositions Expressed in GT-2x and
CHOdhfr- Cells
[0522] Western blot analysis was performed to identify the
differently sialylated FSH molecule compositions expressed in
CHOdhfr-, GT-2x [DSM ACC2858], or NM-H9D8 [DSM ACC2806]. 5 .mu.g of
each antibody molecule composition were separated by SDS-Page in a
10% acrylamide gel under reducing conditions. Proteins were
transferred to nitrocellulose and visualized by SNA (FIG. 17) which
detects 2-6 sialylation.
[0523] A detailed glycan analysis can be performed according to
example 10 for determination of the sialic acid content, charge
distribution, fucose content, galactosylation state and bisecting
GlcNAc content.
[0524] Definitions According to IUPAC-IUBMB Recommendations
(1996):
[0525] Neu5Gc: 5-N-glycolyl-.alpha.-neuraminic acid
[0526] Neu5Ac and NeuNAc: 5-N-acetyl-.alpha.-neuraminic acid
[0527] Gala1,3Gal:
.alpha.-D-galactopyranosyl-(1.fwdarw.*3)-galactopyranosyl-adjacent
saccharide
[0528] 2,3 linked Neuraminic acid:
5-N-acetyl-.alpha.-neuraminyl-(2.fwdarw.3)-adjacent saccharide 2,6
linked neuraminic acid:
5-N-acetyl-.alpha.-neuraminyl-(2.fwdarw.6)-adjacent saccharide
[0529] It is to be understood that this invention is not limited to
the particular methodology, protocols and/or reagents as described
herein as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
TABLE-US-00005 SEQ ID: Sequence #1 (SEQ ID NO: 1)
MVRPLNCIVAVSQDMGIGKNGDLPWPPLRNEWKYFQRMTTTSSVEGKQNL
VIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRL
IEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFP
EIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Sequence #2 (SEQ ID NO: 2)
MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNESRYFQRMTTTSSVEGKQNL
VIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKL
TEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFP
EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Sequence #3 (SEQ ID NO: 3)
MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEARYFQRMTTTSSVEGKQNL
VIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKL
TEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFP
EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Sequence #4 (SEQ ID NO: 4)
MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEGRYFQRMTTTSSVEGKQNL
VIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKL
TEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFP
EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Sequence #5 (SEQ ID NO: 5)
MVGSLNCIVAVSQNMGIGKNGDYPWPPLRNEFRYFQRMTTTSSVEGKQNL
VIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKL
TEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFP
EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Sequence #6 (SEQ ID NO: 6)
MVGSLNCIVAVSQNMGIGKNGDRPWPPLRNEFRYFQRMTTTSSVEGKQNL
VIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKL
TEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFP
EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Sequence #7 (SEQ ID NO: 7)
MVGSLNCIVAVSQNMGIGKNGDFPWPPLRNEFRYFQRMTTTSSVEGKQNL
VIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKL
TEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFP
EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND Sequence #8 (SEQ ID NO: 8)
MVRPLNCIVAVSQNMGIGKNGDRPWPPLRNEFKYFQRMTTTSSVEGKQNL
VIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRL
IEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFP
EIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Sequence #9 (SEQ ID NO: 9)
MVRPLNCIVAVSQNMGIGKNGDYPWPPLRNEFKYFQRMTTTSSVEGKQNL
VIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRL
IEQPELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFP
EIDLGKYKLLPEYPGVLSEVQEEKGIKYKFEVYEKKD Sequence #10 (SEQ ID NO: 10)
MWLGSLLLLGTVACSISA
TABLE-US-00006 TABLE 1 CHOdhfr- NM-F9 Production rate 1.sup.st
round (100 nM MTX) 1.sup.st round (100 nM MTX) (clgG1) Average: 2
+/- 1 pcd; Average: 5 +/- 1,5 pcd; pcd determined Max: 3 pcd Max:
13 pcd after 1.sup.st to 4.sup.th round 2.sup.nd round (200 nM MTX)
2.sup.nd round (200 nM MTX) of gene amplification Average: 5 +/- 1
pcd; Average: 7.5 +/- 4.5 pcd; (methotrexate) and Max: 7 pcd Max:
19 pcd single cell cloning 3.sup.rd round (500 nM MTX) 3.sup.nd
round (500 nM MTX) from theoretically Average: 9 +/- 5 pcd;
Average: 12.5 +/- 4.5 200 seeded clones Max: 17 pcd pcd; Max: 19
pcd (limited dilution) 4.sup.th round (1000 nM MTX) 4.sup.nd round
(1000 nM Average: 13 +/- 5 pcd; MTX) Max: 22 pcd Average: 16.5 +/-
6 pcd; Max: 27 pcd
TABLE-US-00007 TABLE 2 NM-H9D8 Production rate (clgG1) 1.sup.st
round (100 nM MTX) pcd determined after 1.sup.st to 5.sup.th
Average: 7 +/- 4 pcd; Max: 14 pcd round of gene amplification
2.sup.nd round (200 nM MTX) (methotrexate) and single cell Average:
14 +/- 4 pcd; Max: 23 pcd cloning from theoretically 200 3.sup.rd
round (500 nM MTX) seeded clones (limited dilution) Average: 13 +/-
6 pcd; Max: 31 pcd 4.sup.th round (1000 nM MTX) Average: 23 +/- 8
pcd; Max: 34 pcd 5.sup.th round (2000 nM MTX) Average: 23 +/- 9
pcd; Max: 38 pcd
TABLE-US-00008 TABLE 3 pmol pmol pmol pmol Neu5Gc/ Neu5Ac/ Neu5Gc
Neu5Ac .mu.g Protein .mu.g Protein Erbitux (Merck; SP2/0) 0.149
0.000 1.784 0.000 CetuxiMab GT-2x 0.000 0.278 0.000 3.335 CetuxiMab
NM-H9D8 0.000 1.156 0.000 12.003 cPankoMab CHOdhfr- 0.000 0.477
0.000 4.921 cPankoMab GT-2x 0.000 0.248 0.000 2.990 cPankoMab
NM-H9D8 0.000 0.776 0.000 13.856
TABLE-US-00009 TABLE 4 A0 A1 A2 A3 A4 Panko1 CHOdhfr- 75 9 11 4 1
Panko1 NM-H9D8 43 14 32 10 1 Panko1 NM-H9D8-E6 40 16 33 10 1
c-PankoMab CHOdhfr- 93 7 0 0 0 c-PankoMab NM-F9 99 1 0 0 0
c-PankoMab NM-H9D8 62 26 11 1 0
TABLE-US-00010 TABLE 5 G0[%] G1[%] G2[%] G3[%] Panko1 CHOdhfr- 22
45 33 0 Panko1 NM-H9D8 7 8 66 14 Panko1 NM-H9D8-E6 5 18 58 19
cPankoMab CHOdhfr- 28 36 35 1 cPankoMab NM-F9 7 26 65 2 cPankoMab
NM-H9D8 7 32 59 0
TABLE-US-00011 TABLE 6 biantennary + non bisecting fucosylated
fucosylated GlcNAc Panko1 CHOdhfr- 100 0 0 Panko1 NM-H9D8 45 55 5
Panko1 NM-H9D8-E6 21 79 2 biantennary + bisecting non biantennary
GlcNAc fucosylated cPankoMab-CHO 99 0 0 cPankoMab-NM-F9 68 29 6
cPankoMab-NM-H9D8 59 39 1
TABLE-US-00012 TABLE 7 CHOdhfr- GT-2X Production rate 3.sup.rd
round (500 nM MTX) 2.sup.nd round (200 nM MTX) (FSH) pcd Average:
<1 pcd; Max: Average: 2.5 +/- 2.0 pcd; determined after 0.05 pcd
Max: 6 pcd 3.sup.rd round of gene 3.sup.rd round (500 nm MTX)
amplification Average: 2.5 +/- 1.3 pcd; (methotrexate) and Max: 12
pcd single cell cloning from theoretically 200 seeded clones
(limited dilution)
TABLE-US-00013 TABLE 8 NM-H9D8 Production rate (FSH) 2.sup.nd round
(200 nM MTX) pcd determined after 2.sup.nd round Average: 2.2 +/-
1.2 pcd; of gene amplification Max: 4 pcd (methotrexate) and single
cell cloning from theoretically 200 seeded clones (limited
dilution)
TABLE-US-00014 TABLE 9 No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal High Galactose as High
Galactose as High Galactose as defined in claim 2 defined in claim
2 defined in claim 2 and depending and depending and depending
claims claims claims Low Fucose as Low Fucose as Low Fucose as
defined in claim 2 defined in claim 2 defined in claim 2 and
depending and depending and depending claims claims claims
bisecGlcNAc bisecGlcNAc bisecGlcNAc high Activity, in high
Activity, in high Activity, in particular Fc activity particular Fc
activity particular Fc activity higher affinity higher affinity
higher affinity high yield high yield high yield 2-6 NeuNAc low
sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and more
sialic acid as depending claims defined in claim 2 and depending
claims No NeuGc No NeuGc No NeuGc No detectable No detectable No
detectable terminal Galalpha 1-3 terminal Galalpha 1-3 terminal
Galalpha 1-3 Gal Gal Gal High Galactose as High Galactose as High
Galactose as defined in claim 2 defined in claim 2 defined in claim
2 and depending and depending and depending claims claims claims
Low Fucose as Low Fucose as Low Fucose as defined in claim 2
defined in claim 2 defined in claim 2 and depending and depending
and depending claims claims claims bisecGlcNAc bisecGlcNAc
bisecGlcNAc high Activity, in high Activity, in high Activity, in
particular Fc activity particular Fc activity particular Fc
activity high yield high yield high yield 2-6 NeuNAc low sialic
acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and more sialic
acid as depending claims defined in claim 2 and depending claims No
NeuGc No NeuGc No NeuGc No detectable No detectable No detectable
terminal Galalpha 1-3 terminal Galalpha 1-3 terminal Galalpha 1-3
Gal Gal Gal High Galactose as High Galactose as High Galactose as
defined in claim 2 defined in claim 2 defined in claim 2 and
depending and depending and depending claims claims claims Low
Fucose as Low Fucose as Low Fucose as defined in claim 2 defined in
claim 2 defined in claim 2 and depending and depending and
depending claims claims claims bisecGlcNAc bisecGlcNAc bisecGlcNAc
high yield high yield high yield 2-6 NeuNAc low sialic acid as 2-6
NeuNAc 2-3 NeuNAc defined in claim 2 and more sialic acid as
depending claims defined in claim 2 and depending claims No NeuGc
No NeuGc No NeuGc No detectable No detectable No detectable
terminal Galalpha 1-3 terminal Galalpha 1-3 terminal Galalpha 1-3
Gal Gal Gal High Galactose as High Galactose as High Galactose as
defined in claim 2 defined in claim 2 defined in claim 2 and
depending and depending and depending claims claims claims Low
Fucose as Low Fucose as Low Fucose as defined in claim 2 defined in
claim 2 defined in claim 2 and depending and depending and
depending claims claims claims bisecGlcNAc bisecGlcNAc bisecGlcNAc
high Activity, in high Activity, in high Activity, in particular Fc
activity particular Fc activity particular Fc activity 2-6 NeuNAc
low sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and
more sialic acid as depending claims defined in claim 2 and
depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal High Galactose as High
Galactose as High Galactose as defined in claim 2 defined in claim
2 defined in claim 2 and depending and depending and depending
claims claims claims bisecGlcNAc bisecGlcNAc bisecGlcNAc high
Activity, in high Activity, in high Activity, in particular Fc
activity particular Fc activity particular Fc activity high yield
high yield high yield 2-6 NeuNAc low sialic acid as 2-6 NeuNAc 2-3
NeuNAc defined in claim 2 and more sialic acid as depending claims
defined in claim 2 and depending claims No NeuGc No NeuGc No NeuGc
No detectable No detectable No detectable terminal Galalpha 1-3
terminal Galalpha 1-3 terminal Galalpha 1-3 Gal Gal Gal High
Galactose as High Galactose as High Galactose as defined in claim 2
defined in claim 2 defined in claim 2 and depending and depending
and depending claims claims claims bisecGlcNAc bisecGlcNAc
bisecGlcNAc high Activity, in high Activity, in high Activity, in
particular Fc activity particular Fc activity particular Fc
activity 2-6 NeuNAc low sialic acid as 2-6 NeuNAc 2-3 NeuNAc
defined in claim 2 and more sialic acid as depending claims defined
in claim 2 and depending claims No NeuGc No NeuGc No NeuGc No
detectable No detectable No detectable terminal Galalpha 1-3
terminal Galalpha 1-3 terminal Galalpha 1-3 Gal Gal Gal High
Galactose as High Galactose as High Galactose as defined in claim 2
defined in claim 2 defined in claim 2 and depending and depending
and depending claims claims claims bisecGlcNAc bisecGlcNAc
bisecGlcNAc high yield high yield high yield 2-6 NeuNAc low sialic
acid as 6 NeuNAc 2-3 NeuNAc defined in claim 2 and more sialic acid
as depending claims defined in claim 2 and depending claims No
NeuGc No NeuGc No NeuGc No detectable No detectable No detectable
terminal Galalpha 1-3 terminal Galalpha 1-3 terminal Galalpha 1-3
Gal Gal Gal High Galactose as High Galactose as High Galactose as
defined in claim 2 defined in claim 2 defined in claim 2 and
depending and depending and depending claims claims claims high
Activity, in high Activity, in high Activity, in particular Fc
activity particular Fc activity particular Fc activity 2-6 NeuNAc
low sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and
more sialic acid as depending claims defined in claim 2 and
depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal High Galactose as High
Galactose as High Galactose as defined in claim 2 defined in claim
2 defined in claim 2 and depending and depending and depending
claims claims claims high yield high yield high yield 2-6 NeuNAc
low sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and
more sialic acid as depending claims defined in claim 2 and
depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal Low Fucose as Low Fucose as
Low Fucose as defined in claim 2 defined in claim 2 defined in
claim 2 and depending and depending and depending claims claims
claims bisecGlcNAc bisecGlcNAc bisecGlcNAc high Activity, in high
Activity, in high Activity, in particular Fc activity particular Fc
activity particular Fc activity higher affinity higher affinity
higher affinity high yield high yield high yield 2-6 NeuNAc low
sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and more
sialic acid as depending claims defined in claim 2 and depending
claims No NeuGc No NeuGc No NeuGc No detectable No detectable No
detectable terminal Galalpha 1-3 terminal Galalpha 1-3 terminal
Galalpha 1-3 Gal Gal Gal Low Fucose as Low Fucose as Low Fucose as
defined in claim 2 defined in claim 2 defined in claim 2 and
depending and depending and depending claims claims claims
bisecGlcNAc bisecGlcNAc bisecGlcNAc high Activity, in high
Activity, in high Activity, in particular Fc activity particular Fc
activity particular Fc activity high yield high yield high yield
2-6 NeuNAc low sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in
claim 2 and more sialic acid as depending claims defined in claim 2
and depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal Low Fucose as Low Fucose as
Low Fucose as defined in claim 2 defined in claim 2 defined in
claim 2 and depending and depending and depending claims claims
claims bisecGlcNAc bisecGlcNAc bisecGlcNAc high yield high yield
high yield 2-6 NeuNAc low sialic acid as 2-6 NeuNAc 2-3 NeuNAc
defined in claim 2 and more sialic acid as depending claims defined
in claim 2 and depending claims No NeuGc No NeuGc No NeuGc No
detectable No detectable No detectable terminal Galalpha 1-3
terminal Galalpha 1-3 terminal Galalpha 1-3 Gal Gal Gal Low Fucose
as Low Fucose as Low Fucose as defined in claim 2 defined in claim
2 defined in claim 2 and depending and depending and depending
claims claims claims bisecGlcNAc bisecGlcNAc bisecGlcNAc high
Activity, in high Activity, in high Activity, in particular Fc
activity particular Fc activity particular Fc activity 2-6 NeuNAc
low sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and
more sialic acid as depending claims defined in claim 2 and
depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal bisecGlcNAc bisecGlcNAc
bisecGlcNAc high Activity, in high Activity, in high Activity, in
particular Fc activity particular Fc activity particular Fc
activity high yield high yield high yield 2-6 NeuNAc low sialic
acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and
more sialic acid as depending claims defined in claim 2 and
depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal bisecGlcNAc bisecGlcNAc
bisecGlcNAc high Activity, in high Activity, in high Activity, in
particular Fc activity particular Fc activity particular Fc
activity 2-6 NeuNAc low sialic acid as 2-6 NeuNAc 2-3 NeuNAc
defined in claim 2 and more sialic acid as depending claims defined
in claim 2 and depending claims No NeuGc No NeuGc No NeuGc No
detectable No detectable No detectable terminal Galalpha 1-3
terminal Galalpha 1-3 terminal Galalpha 1-3 Gal Gal Gal bisecGlcNAc
bisecGlcNAc bisecGlcNAc high yield high yield high yield 2-6 NeuNAc
low sialic acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and
more sialic acid as depending claims defined in claim 2 and
depending claims No NeuGc No NeuGc No NeuGc No detectable No
detectable No detectable terminal Galalpha 1-3 terminal Galalpha
1-3 terminal Galalpha 1-3 Gal Gal Gal high Activity, in high
Activity, in high Activity, in particular Fc activity particular Fc
activity particular Fc activity 2-6 NeuNAc low sialic acid as 2-6
NeuNAc 2-3 NeuNAc defined in claim 2 and more sialic acid as
depending claims defined in claim 2 and depending claims No NeuGc
No NeuGc No NeuGc No detectable No detectable No detectable
terminal Galalpha 1-3 terminal Galalpha 1-3 terminal Galalpha 1-3
Gal Gal Gal high yield high yield high yield 2-6 NeuNAc low sialic
acid as 2-6 NeuNAc 2-3 NeuNAc defined in claim 2 and more sialic
acid as depending claims defined in claim 2 and depending
claims
TABLE-US-00015 TABLE 10 Panko 1 Fuc neg + BisGlcNAc neg. H9D8 = 54%
H9D8-E6 = 79% Fuc pos + BisGlcNAc pos H9D8 = 5% H9D8-E6 = 2% Fuc
neg + BisGlcNAc pos H9D8 = 0% H9D8-E6 = 0% Panko 2 Fuc neg +
BisGlcNAc neg. H9D8 = ~0% F9 and GT-2X = ~5-6% Fuc pos + BisGlcNAc
pos H9D8 = 36% F9 and GT-2X = 29% Fuc neg + BisGlcNAc pos H9D8 = 0%
F9 and GT-2X = 0%
Sequence CWU 1
1
231187PRTUnknownMTX resistant DHFR variant 1Met Val Arg Pro Leu Asn
Cys Ile Val Ala Val Ser Gln Asp Met Gly1 5 10 15Ile Gly Lys Asn Gly
Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Trp 20 25 30Lys Tyr Phe Gln
Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45Asn Leu Val
Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60Asn Arg
Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu65 70 75
80Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp
85 90 95Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp
Met 100 105 110Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala
Met Asn Gln 115 120 125Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile
Met Gln Glu Phe Glu 130 135 140Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Gly Lys Tyr Lys Leu Leu145 150 155 160Pro Glu Tyr Pro Gly Val
Leu Ser Glu Val Gln Glu Glu Lys Gly Ile 165 170 175Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Lys Asp 180 1852187PRTUnknownMTX resistant DHFR
variant 2Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn
Met Gly1 5 10 15Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg
Asn Glu Ser 20 25 30Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val
Glu Gly Lys Gln 35 40 45Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe
Ser Ile Pro Glu Lys 50 55 60Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu
Val Leu Ser Arg Glu Leu65 70 75 80Lys Glu Pro Pro Gln Gly Ala His
Phe Leu Ser Arg Ser Leu Asp Asp 85 90 95Ala Leu Lys Leu Thr Glu Gln
Pro Glu Leu Ala Asn Lys Val Asp Met 100 105 110Val Trp Ile Val Gly
Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His 115 120 125Pro Gly His
Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu 130 135 140Ser
Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu145 150
155 160Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly
Ile 165 170 175Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp 180
1853187PRTUnknownMTX resistant DHFR variant 3Met Val Gly Ser Leu
Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly1 5 10 15Ile Gly Lys Asn
Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Ala 20 25 30Arg Tyr Phe
Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45Asn Leu
Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60Asn
Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu65 70 75
80Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp
85 90 95Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp
Met 100 105 110Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala
Met Asn His 115 120 125Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile
Met Gln Asp Phe Glu 130 135 140Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Glu Lys Tyr Lys Leu Leu145 150 155 160Pro Glu Tyr Pro Gly Val
Leu Ser Asp Val Gln Glu Glu Lys Gly Ile 165 170 175Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Asn Asp 180 1854187PRTUnknownMTX resistant DHFR
variant 4Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn
Met Gly1 5 10 15Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg
Asn Glu Gly 20 25 30Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val
Glu Gly Lys Gln 35 40 45Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe
Ser Ile Pro Glu Lys 50 55 60Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu
Val Leu Ser Arg Glu Leu65 70 75 80Lys Glu Pro Pro Gln Gly Ala His
Phe Leu Ser Arg Ser Leu Asp Asp 85 90 95Ala Leu Lys Leu Thr Glu Gln
Pro Glu Leu Ala Asn Lys Val Asp Met 100 105 110Val Trp Ile Val Gly
Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His 115 120 125Pro Gly His
Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu 130 135 140Ser
Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu145 150
155 160Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly
Ile 165 170 175Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp 180
1855187PRTUnknownMTX resistant DHFR variant 5Met Val Gly Ser Leu
Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly1 5 10 15Ile Gly Lys Asn
Gly Asp Tyr Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30Arg Tyr Phe
Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45Asn Leu
Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60Asn
Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu65 70 75
80Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp
85 90 95Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp
Met 100 105 110Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala
Met Asn His 115 120 125Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile
Met Gln Asp Phe Glu 130 135 140Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Glu Lys Tyr Lys Leu Leu145 150 155 160Pro Glu Tyr Pro Gly Val
Leu Ser Asp Val Gln Glu Glu Lys Gly Ile 165 170 175Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Asn Asp 180 1856187PRTUnknownMTX resistant DHFR
variant 6Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn
Met Gly1 5 10 15Ile Gly Lys Asn Gly Asp Arg Pro Trp Pro Pro Leu Arg
Asn Glu Phe 20 25 30Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val
Glu Gly Lys Gln 35 40 45Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe
Ser Ile Pro Glu Lys 50 55 60Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu
Val Leu Ser Arg Glu Leu65 70 75 80Lys Glu Pro Pro Gln Gly Ala His
Phe Leu Ser Arg Ser Leu Asp Asp 85 90 95Ala Leu Lys Leu Thr Glu Gln
Pro Glu Leu Ala Asn Lys Val Asp Met 100 105 110Val Trp Ile Val Gly
Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His 115 120 125Pro Gly His
Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu 130 135 140Ser
Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu145 150
155 160Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly
Ile 165 170 175Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp 180
1857187PRTUnknownMTX resistant DHFR variant 7Met Val Gly Ser Leu
Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly1 5 10 15Ile Gly Lys Asn
Gly Asp Phe Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30Arg Tyr Phe
Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45Asn Leu
Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60Asn
Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu65 70 75
80Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp
85 90 95Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp
Met 100 105 110Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala
Met Asn His 115 120 125Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile
Met Gln Asp Phe Glu 130 135 140Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Glu Lys Tyr Lys Leu Leu145 150 155 160Pro Glu Tyr Pro Gly Val
Leu Ser Asp Val Gln Glu Glu Lys Gly Ile 165 170 175Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Asn Asp 180 1858187PRTUnknownMTX resistant DHFR
variant 8Met Val Arg Pro Leu Asn Cys Ile Val Ala Val Ser Gln Asn
Met Gly1 5 10 15Ile Gly Lys Asn Gly Asp Arg Pro Trp Pro Pro Leu Arg
Asn Glu Phe 20 25 30Lys Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val
Glu Gly Lys Gln 35 40 45Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe
Ser Ile Pro Glu Lys 50 55 60Asn Arg Pro Leu Lys Asp Arg Ile Asn Ile
Val Leu Ser Arg Glu Leu65 70 75 80Lys Glu Pro Pro Arg Gly Ala His
Phe Leu Ala Lys Ser Leu Asp Asp 85 90 95Ala Leu Arg Leu Ile Glu Gln
Pro Glu Leu Ala Ser Lys Val Asp Met 100 105 110Val Trp Ile Val Gly
Gly Ser Ser Val Tyr Gln Glu Ala Met Asn Gln 115 120 125Pro Gly His
Leu Arg Leu Phe Val Thr Arg Ile Met Gln Glu Phe Glu 130 135 140Ser
Asp Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu145 150
155 160Pro Glu Tyr Pro Gly Val Leu Ser Glu Val Gln Glu Glu Lys Gly
Ile 165 170 175Lys Tyr Lys Phe Glu Val Tyr Glu Lys Lys Asp 180
1859187PRTUnknownMTX-resistant DHFR variant 9Met Val Arg Pro Leu
Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly1 5 10 15Ile Gly Lys Asn
Gly Asp Tyr Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30Lys Tyr Phe
Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45Asn Leu
Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60Asn
Arg Pro Leu Lys Asp Arg Ile Asn Ile Val Leu Ser Arg Glu Leu65 70 75
80Lys Glu Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser Leu Asp Asp
85 90 95Ala Leu Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys Val Asp
Met 100 105 110Val Trp Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala
Met Asn Gln 115 120 125Pro Gly His Leu Arg Leu Phe Val Thr Arg Ile
Met Gln Glu Phe Glu 130 135 140Ser Asp Thr Phe Phe Pro Glu Ile Asp
Leu Gly Lys Tyr Lys Leu Leu145 150 155 160Pro Glu Tyr Pro Gly Val
Leu Ser Glu Val Gln Glu Glu Lys Gly Ile 165 170 175Lys Tyr Lys Phe
Glu Val Tyr Glu Lys Lys Asp 180 1851018PRTUnknownSecretion peptide
signal 10Met Trp Leu Gly Ser Leu Leu Leu Leu Gly Thr Val Ala Cys
Ser Ile1 5 10 15Ser Ala11117PRTUnknownVariable heavy chain of the
antibody Panko -1 11Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asp Ala 20 25 30Trp Met Asp Trp Val Arg Gln Ser Pro Glu
Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Arg Ser Lys Ala Asn Asn
His Ala Thr Tyr Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Val Ser Lys Ser Ser65 70 75 80Val Tyr Leu Gln Met Asn
Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95Tyr Cys Thr Arg Gly
Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr
Val Ser 11512113PRTUnknownVariable light chain of the antibody
Panko 1 12Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser
Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile
Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu
Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Leu Thr Phe
Gly Asp Gly Thr Lys Leu Glu Leu Lys 100 105
110Arg13116PRTUnknownVariable heavy chain of the antibody Panko 2
13Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Thr Thr His
Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75 80Val Ser Leu Gln Met Asn Asn Leu Arg Val
Glu Asp Thr Gly Ile Tyr 85 90 95Tyr Cys Thr Arg His Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser
11514113PRTUnknownVariable light chain of the antibody Panko 2
14Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly1
5 10 15Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His
Ser 20 25 30Asn Gly Ile Thr Tyr Phe Phe Trp Tyr Leu Gln Lys Pro Gly
Leu Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe
Thr Leu Arg Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Pro Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg15118PRTUnknownVariable
heavy chain of the antibody Cetuximab 15Gln Val Gln Leu Lys Gln Ser
Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys
Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Val
Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp
Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Leu
Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys
Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90
95Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser 11516108PRTUnknownVariable light
chain of the antibody Cetuximab 16Asp Ile Leu Leu Thr Gln Ser Pro
Val Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys
Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30Ile His Trp Tyr Gln Gln
Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Glu
Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser65 70 75 80Glu Asp
Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr
85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100
1051731DNAArtificialPrimer for FSH alpha chain 17aaaggtacca
tggattacta cagaaaatat g 311829DNAartificialPrimer for FSH alpha
chain 18aaaggatcct taagatttgt gataataac 291930DNAArtificialPrimer
for FSH beta chain 19tttaagctta tgaagacact ccagtttttc
302027DNAArtificialPrimer for FSH beta chain 20tttggatcct
tattctttca tttcacc 2721399DNAArtificialcDNA
sequencemisc_feature(1)..(63)TCR leader sequence 21atggcctgcc
ccggcttcct gtgggccctg gtgatcagca cctgcctgga attctccatg 60gctaacagct
gcgagctgac caacatcacc atcgccatcg agaaagagga atgccggttc
120tgcatcagca tcaacaccac ctggtgcgcc ggctactgct acacccggga
cctggtgtac 180aaggaccccg ccaggcccaa gatccagaaa acctgcacct
tcaaagaact ggtgtacgag 240accgtgcggg tgcccggctg cgcccaccac
gccgacagcc tgtacaccta ccccgtggcc 300acccagtgcc actgcggcaa
gtgcgacagc gacagcaccg actgcaccgt gaggggcctg 360ggccccagct
actgcagctt cggcgagatg aaagagtga 399225PRTUnknownEpitope on MUC1
(Thr can be glycosylated) 22Pro Asp Thr Arg Pro1
52330PRTUnknownMUC1 peptide 23Ala Pro Pro Ala His Gly Val Thr Ser
Ala Pro Asp Thr Arg Pro Ala1 5 10 15Pro Gly Ser Thr Ala Pro Pro Ala
His Gly Val Thr Ser Ala 20 25 30
* * * * *
References