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CN1761746A - Fusion constructs and use of same to produce antibodies with increased Fc receptor binding affinity and effector function - Google Patents

Fusion constructs and use of same to produce antibodies with increased Fc receptor binding affinity and effector function Download PDF

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Publication number
CN1761746A
CN1761746A CNA2004800075642A CN200480007564A CN1761746A CN 1761746 A CN1761746 A CN 1761746A CN A2004800075642 A CNA2004800075642 A CN A2004800075642A CN 200480007564 A CN200480007564 A CN 200480007564A CN 1761746 A CN1761746 A CN 1761746A
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polypeptide
antibody
host cell
cell
nucleic acid
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CNA2004800075642A
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CN1761746B (en
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P·乌马纳
P·布林克
C·费拉拉
T·苏特尔
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Roche Glycart AG
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Roche Glycart AG
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Priority to CN201310218123.8A priority Critical patent/CN103540600B/en
Priority claimed from PCT/IB2004/000844 external-priority patent/WO2004065540A2/en
Publication of CN1761746A publication Critical patent/CN1761746A/en
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Abstract

The present invention relates to the field of glycosylation engineering of proteins. More particularly, the present invention relates to nucleic acid molecules, including fusion constructs, having catalytic activity and the use of same in glycosylation engineering of host cells to generate polypeptides with improved therapeutic properties, including antibodies with increased Fc receptor binding and increased effector function.

Description

Fusion constructs and being used for is produced the purposes of the antibody that Fc receptor binding affinity and effector function improve
Background of invention
Invention field
The present invention relates to the field of protein Glycosylation Engineering (glycosylation engineering).More particularly, the present invention relates to have the nucleic acid molecule of catalytic activity, comprise fusion constructs, and the purposes in host cell glycosylation engineering, be used for producing treatment characteristic enhanced polypeptide, the antibody that improves with effector function that comprises that the Fc receptors bind improves.
Background technology
Many necessary function in glycoprotein mediation human body, other eukaryote and some prokaryotic organism comprise catalysis, send signal, cell-cell communication and molecular recognition and combination.They constitute most non-cytoplasmic protein matter in the eukaryote.(Lis etc., Eur.J.Biochem.218:1-27 (1993)).Developed many glycoprotein and be used for the treatment of purpose, in recent two decades, the secretion glycoprotein that generates naturally of recombinant forms has become main products in the biotechnological industries.Example comprises erythropoietin (EPO), therapeutic monoclonal antibodies (therapeutic mAb), tissue plasminogen activator (tPA), interferon-beta (IFN-β), granulocyte-macrophage colony stimutaing factor (GM-CSF) and human chorionic gonadotropin (hCG).(Cumming etc., Glycobiology 1:115-130 (1991)).
The oligosaccharides composition can remarkably influenced and the relevant characteristic of therapeutic glycoprotein effect, the resistance and immune interaction, pharmacokinetics and the particular organisms activity that comprise physical stability, proteolytic enzyme is attacked.Such characteristic not only depends on the existence of oligosaccharides or does not exist, also depends on the structure that it is specific.Can form some general introductions between oligosaccharide structure and the glycoprotein function.For example, some oligosaccharide structure has been by protein-bonded interaction has mediated glycoprotein and removed fast from blood flow with the particular carbon hydrate, and other can and cause the immune response of not expecting by antibodies.(Jenkins etc., Nature Biotechnol.14:975-81 (1996)).
Owing to Protein Glycosylation Overview can be become be suitable for most the ability of people's application form, so mammalian cell is the preferred host of manufacture of therapeutic glycoprotein.(Cumming etc., Glycobiology1:115-30 (1991); Jenkins etc., Nature Biotechnol.14:975-81 (1996)).Bacterium is glycosylated protein seldom, and the same with the common host of other kind as yeast, filamentous fungus, insect and vegetable cell, produced from blood flow and removed fast, the immunity of not expecting interacts, and in some particular cases, reduced bioactive glycosylation pattern.20 years in the past, in the mammalian cell, Chinese hamster ovary (CHO) cell was to use the most general.Except giving suitable glycosylation pattern, these cells continue to produce the cloned cell line of inheritance stability, high yield.In simple bio, use serum free medium they can be cultured to high-density, and can develop safe and reproducible biological method.Other zooblast commonly used comprises young hamster kidney (BHK) cell, NS0-and SP2/0-murine myeloma cell.Recently, tested and produced from transgenic animal.(Jenkins etc., Nature Biotechnol.14:975-81 (1996)).
All antibody contain sugared structure in the conservative position of CH, and each homotype has distinct N-and connects sugared structural arrangement, and it influences albumen assembling, secretion or functionally active changeably.(Wright, A., and Morrison, S.L., Trends Biotech.15:26-32 (1997)).The sugared structure that connects the N-that connects depends on that processing stage changes considerably and can comprise high mannose, higly branched chain and two feeler composite oligosaccharide.(Wright, A., and Morrison, S.L., Trends Biotech.15:26-32 (1997)).Usually, existing the xenogenesis that connects the core oligosaccharide structure to handle at specific glycosylation site makes monoclonal antibody exist with many glycosylations form.Equally, shown that the main difference of antibody glycosylation occurs between the clone, even observed the nuance that the clone of giving is grown under different culture condition.(Glycobiology 5 (8) for Lifely, M.R. etc.: 813-22 (1995)).
Do not put together monoclonal antibody (mAb) and can prove Rituximab (Rituxan as following medicine as the medicine of treatment cancer by food and drug administration's approval TM, IDECPharmaceuticals, San Diego, CA and Genentech Inc., San Francisco CA), is used for the treatment of the positive B-cell of CD20, rudimentary or cryptomere Fei Huojinqi lymphoma, Trastuzumab (Herceptin TM, Genentech Inc) be used for the treatment of breast cancer in late period (Grillo-Lopez, A.-J. etc., Semin.Oncol.26:66-73 (1999); Goldenberg, M.M., Clin.Ther.21:309-18 (1999)), Gemtuzumab (Mylotarg TM, Celltech/Wyeth-Ayerst) be used for the treatment of the acute myelomatosis of recurrence, and Alemtuzumab (CAMPATH TM, MilleniumPharmaceuticals/Schering AG) and be used for the treatment of B cell lymphocytic leukemia.The effectiveness that the success of these products not only depends on them also depend on their outstanding security features (Grillo-Lopez, A.-J. is etc., Semin.Oncol.26:66-73 (1999); Goldenberg, M.M., Clin.Ther.21:309-18 (1999)).Although there is not big interest to obtaining in the success of these medicines than puting together the common higher specific antibody of activity that obtains of mAb treatment at present.
Obtain usefulness raising greatly and keep a simple production method and a potential approach avoiding significant undesirable side effect, be to become to assign to improve the natural cell-mediated effector function (Umana of mAb by their oligosaccharides of through engineering approaches (engineering), P. etc., NatureBiotechnol.17:176-180 (1999)).IgG1 type antibody, the most frequently used antibody in the cancer immunotherapy is to have the glycoprotein that conservative N-connects glycosylation site at the Asn297 place of each CH2 structural domain.Two Composite Double feeler oligosaccharides that connect Asn297 are hidden between the CH2 structural domain, fully contacting of formation and polypeptide main chain, and they to have antagonist mediation effector function such as antibody dependent cellular cytotoxicity (ADCC) be essential (Lifely, M.R. etc., Glycobiology 5:813-822 (1995); Jefferis, R. etc., ImmunolRev.163:59-76 (1998); Wright, A. and Morrison, S.L., TrendsBiotechnol.15:26-32 (1997)).
Shown β (1 before the present inventor, 4)-overexpression of N-acetylglucosaminyltrVnsferase III (GnTIII) in Chinese hamster ovary (CHO) cell, the formation that (bisected) oligosaccharides is halved in glycosyltransferase catalysis has significantly improved the external ADCC activity of the anti-neuroblastoma chimeric mAb (chCE7) that the through engineering approaches Chinese hamster ovary celI produces.(referring to, Umana, P. etc., Nature Biotechnol.17:176-180 (1999), international open No.WO 99/54342, the full content of each is incorporated herein by reference with its integral body at this).Antibody chCE7 belongs to and has high tumour affinity and the big class of the specific mAb of not puting together, but usefulness is too little so that can not clinically use (Umana when producing with the standard industry clone that lacks the GnTIII enzyme, P. etc., Nature Biotechnol.17:176-180 (1999)).This research shows that first expressing GnTIII by engineered antibody production cell obtains the active very big raising of ADCC, it also causes the relevant bisection oligosaccharides of constant region (Fc), comprise the increase of halving non-fucosylation oligosaccharide ratio, surpass the level of finding in the natural generation antibody.
The results of great majority research show that Fc-acceptor-dependency mechanism explained that basically cytotoxic antibody is to antineoplastic action and shown antineoplastic optimum antibody preferably with activation Fc receptors bind and minimize the combination with inhibition mating partner Fc γ RIIB.(Nature Medicine 6 (4) for Clynes, R.A. etc.: 443-446 (2000); Kalergis, A.M., and Ravetch, J.V., J.Exp.Med.195 (12): 1653-1659 (in June, 2002).For example, the result of at least one research proposes that particularly Fc γ RIIIa acceptor is relevant strongly with Antybody therapy usefulness.(Blood 99 (3) for Cartron, G. etc.: 754-757 (in February, 2002)).This studies show that the Fc γ RIIIa patient of isozygotying has better reaction to Rituximab than heterozygosis patient.The author infers that more excellent reaction is that it causes better resisting the ADCC activity of lymphoma cell because better in conjunction with Fc γ RIIIa in the antibody body.(Cartron, G. etc., Blood99 (3): 754-757 (in February, 2002)).
Except ADCC, the anticancer monoclonal antibody of success is induced the survival of the dependent adjusting of Fc-target cell, propagation usually, or dead direct signal mechanism, by the path of initiation of activating cells signal level or blocking-up somatomedin.(Selenko, N. etc., J.Clin.Immunol.22 (3): 124-130 (2002)).For example, treat CD20 with Rituximab +Cell has demonstrated cracking and Mab inductive apoptosis and the ADCC that induces complement-mediated.(Selenko, N. etc., J.Clin.Immunol.22 (3): 124-130 (2002)).In addition, Rituximab induces the lymphoma cell apoptosis, and not only cell killing but also promote the absorption of lymphoma deutero-peptide and the submission that intersects by antigen presentation dendritic cell (DC) is induced the DC maturation, and produced specific cells toxic T lymphocyte (CTL).
The invention summary
Recognize huge treatment potential that the Fc receptor binding affinity improves and the antibody that effector function improves, so inventor of the present invention developed the method for producing such antibody, it comprises the glycosylation form (profile) in engineered antibody Fc district.
The method that the present invention briefly relates to glycosyl through engineering approaches (glycoengineering) host cell changes the glycosylation form of one or more polypeptide that this host cell produces.Method of the present invention can be used for production and have the glycosylated treatment antibody of modification in the Fc district, comprises and has reduced fucosylation, wherein has the effector function of raising and/or the Fc receptors bind of raising as modifying glycosylated antibody as a result.Glycosyl engineered antibody of the present invention is used in particular for patient's oncotherapy.In one embodiment, glycosyl through engineering approaches host cell of the present invention is expressed the nucleic acid molecule that coding has GnTIII catalytic activity or GalT catalytic activity fusion polypeptide.In the preferred embodiment, fusion constructs and coding have the nucleic acid molecule of people ManII catalytic activity polypeptide and/or the nucleic acid molecule coexpression that coding has GnTII catalytic activity polypeptide.Still in another embodiment, improve the host cell of the nucleic acid molecule that expressing encodes has ManII catalytic activity polypeptide by the glycosyl through engineering approaches and produce glycosyl through engineering approaches polypeptide of the present invention.
Therefore, one aspect of the present invention relates to the isolating nucleic acid that contains coding fusion polypeptide sequence, wherein said fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (β (1,4)-N-acetylglucosaminyltransferase III) (" GnTIII ") is active and contain the golgi body locating structure territory (localization domain) that golgi body stops polypeptide (resident polypeptide).In one embodiment, fusion polypeptide contains the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III.Further in the embodiment, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of locating structure territory, β (1, the 2)-locating structure territory of N-acetylglucosaminyltrVnsferase II (" GnTII "), the locating structure territory of mannosidase I and the α 1-6 core fucosyltransferase of N-acetylglucosaminyltrVnsferase I (" GnTI ").In the preferred embodiment, isolated nucleic acid sequences contains Figure 24 or nucleotide sequence shown in Figure 25.In another preferred embodiment, the isolated nucleic acid sequences coding has the polypeptide of Figure 24 or aminoacid sequence shown in Figure 25.Still in another preferred embodiment, the isolated nucleic acid sequences coding has the polypeptide of the aminoacid sequence identical with Figure 24 or aminoacid sequence shown in Figure 25 at least 80%.
On the other hand, the present invention relates to contain the isolating nucleic acid of coding fusion polypeptide sequence, wherein said fusion polypeptide has β (1,4)-galactosyltransferase (" GalT ") activity and contains the golgi body locating structure territory that golgi body stops polypeptide.In one embodiment, fusogenic peptide contains the catalyst structure domain of β (1,4)-galactosyltransferase.In another embodiment, fusogenic peptide contains the catalyst structure domain of β (1,4) galactosyltransferase.Further in the embodiment, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of locating structure territory, β (1, the 2)-locating structure territory of N-acetylglucosaminyltrVnsferase II (" GnTII "), the locating structure territory of mannosidase I and the α 1-6 core fucosyltransferase of N-acetylglucosaminyltrVnsferase I (" GnTI ").
On the other hand, the present invention relates to contain separative expression of nucleic acids carrier, this nucleic acid contains the sequence of the fusion polypeptide of encoding, and wherein said fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III activity and contains the golgi body locating structure territory that golgi body stops polypeptide.In one embodiment, expression vector codes contains β (1,4)-fusion polypeptide in N-acetylglucosaminyltrVnsferase III catalyst structure domain and golgi body locating structure territory, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of locating structure territory, β (1, the 2)-locating structure territory of N-acetylglucosaminyltrVnsferase II, the locating structure territory of mannosidase I and the α 1-6 core fucosyltransferase of N-acetylglucosaminyltrVnsferase I.
On the other hand, the present invention relates to contain separative expression of nucleic acids carrier, this nucleic acid contains the sequence of the fusion polypeptide of encoding, and wherein said fusion polypeptide has β (1,4)-galactosyltransferasactivity activity and contains the golgi body locating structure territory that golgi body stops polypeptide.In one embodiment, expression vector codes contains β (1,4)-galactosyltransferase catalyst structure domain and golgi body stop the fusion polypeptide in the golgi body locating structure territory of polypeptide, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of locating structure territory, β (1, the 2)-locating structure territory of N-acetylglucosaminyltrVnsferase II, the locating structure territory of mannosidase I and the α 1-6 core fucosyltransferase of N-acetylglucosaminyltrVnsferase I.
On the other hand, the present invention relates to contain the host cell of above-mentioned expression vector.
On the other hand, the present invention relates to host cell, this host cell through engineering approaches is expressed at least a coding have β (1,4)-the N-acetylglucosaminyltrVnsferase III (nucleic acid of " GnTIII " active fusion polypeptide, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, contains the antibody fragment and the fusion rotein in Fc district, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.In one embodiment, have the active fusion polypeptide of GnTIII and contain β (1,4)-catalyst structure domain of N-acetylglucosaminyltrVnsferase III and the golgi body locating structure territory of allos golgi body stop polypeptide, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to host cell, this host cell through engineering approaches is expressed at least a coding have β (1,4)-nucleic acid of the active fusion polypeptide of galactosyltransferase (" GalT "), expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, contains the antibody fragment and the fusion rotein in Fc district, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.In one embodiment, have the active fusion polypeptide of GalT and contain β (1,4)-catalyst structure domain of galactosyltransferase and the golgi body locating structure territory of allos golgi body stop polypeptide, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.
Preferably, golgi body locating structure territory is from mannosidase II or β (1,2)-N-acetylglucosaminyltrVnsferase I or galactosyltransferase.
On the other hand, (1,4)-N-acetylglucosaminyltrVnsferase III that the present invention relates to have β is active and contain the fusion polypeptide that the allos golgi body stops the golgi body locating structure territory of polypeptide.In one embodiment, fusion polypeptide of the present invention contains the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III.In another embodiment, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.
On the other hand, (1, the 4)-galactosyltransferasactivity activity that the present invention relates to have β and contain the fusion rotein that the allos golgi body stops the golgi body locating structure territory of polypeptide.In one embodiment, fusion polypeptide of the present invention contains the catalyst structure domain of β (1,4)-galactosyltransferase.In another embodiment, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.
Preferably, golgi body locating structure territory is from mannosidase II or β (1,2)-N-acetylglucosaminyltrVnsferase I (" GnTI ") or galactosyltransferase (" GalT ").
On the other hand, the present invention relates to produce and have β (1,4)-and the method for the active fusion polypeptide of N-acetylglucosaminyltrVnsferase III, be included under the condition of the expression of nucleic acid that allows the coding fusion polypeptide and in substratum, cultivate host cell of the present invention and from generate culture, collect fusion polypeptide.In one embodiment, fusion polypeptide contains the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III.Preferably, fusion polypeptide contains the golgi body locating structure territory that the allos golgi body stops polypeptide, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to produce and have β (1,4)-and the method for the fusion rotein of galactosyltransferasactivity activity, be included under the condition of the expression of nucleic acid that allows encoding fusion protein and in substratum, cultivate host cell of the present invention and from generate culture, collect fusion polypeptide.In one embodiment, fusion polypeptide contains the catalyst structure domain of β (1,4)-galactosyltransferase.Preferably, fusion polypeptide contains the golgi body locating structure territory that the allos golgi body stops polypeptide, golgi body locating structure territory is selected from locating structure territory, the β (1 of mannosidase II, 2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.
Preferably, golgi body locating structure territory is from mannosidase II or β (1,2)-N-acetylglucosaminyltrVnsferase I or galactosyltransferase (" GalT ").
Again on the one hand, the present invention relates to change the method for the glycosylation form (profile) of the polypeptide that host cell produces, comprise at least a nucleic acid of the present invention or expression vector are introduced in the host cell.Preferably, polypeptide is that IgG or its contain the fragment in polypeptide Fc district.In the particularly preferred embodiment, polypeptide is that IgG1 or its contain the fragment in polypeptide Fc district.Perhaps, polypeptide is a fusion rotein, and fusion rotein comprises the zone that is equivalent to human IgG Fc district.
On the other hand, the present invention relates in host cell, produce the method for polypeptide, comprise: (a) under the condition that allows the generation polypeptide, cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-nucleic acid of the active fusion polypeptide of galactosyltransferase (" GalT "), the polypeptide that produces is selected from whole antibody molecule, the antibody fragment and the fusion rotein that contain the Fc district, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and wherein said Expression of Fusion Protein amount is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; (b) separate described polypeptide.Preferably, fusion polypeptide contains β (1,4)-the N-acetylglucosaminyltrVnsferase be III's or β (1,4)-catalyst structure domain of galactosyltransferase (" GalT ") and comprise that further the allos golgi body stops the golgi body locating structure territory of polypeptide, golgi body locating structure territory is selected from the locating structure territory of mannosidase II, β (1,2)-the locating structure territory of N-acetylglucosaminyltrVnsferase I, the locating structure territory of mannosidase I, the locating structure territory of the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and α 1-6 core fucosyltransferase.In the preferred embodiment, the polypeptide that produces as the host cell of modifying as a result has the effector function of raising and/or the Fc receptors bind of raising.In the particularly preferred embodiment, the effector function that improves is Cytotoxic raising and the raising of NK cell bonded and the raising of scavenger cell bonded and the raising of monocyte bonded and the raising of polymorphonuclear cell bonded, the raising of direct signal inductive apoptosis, the raising of dendritic cell maturation and/or the raising of T cell sensitization (priming) of Fc mediation, and the Fc receptors bind of raising is to improve with Fc activated receptor such as Fc γ RIIIA bonded.Preferably, presenting the polypeptide that effector function improves and/or the Fc receptors bind improves is the non-fucosylation oligosaccharides of antibody, antibody fragment or fusion rotein and the Fc district with raising ratio, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
On the other hand, the present invention relates to pharmaceutical composition, contain antibody of the present invention, contain the antibody fragment or the fusion rotein in Fc district, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region and relates to the purposes of this pharmaceutical composition in treatment tumour such as cancer or other disease.In one embodiment, treatment is B cell depleting (B cell depletion), delivers medicine to patient such as the people who needs by this pharmaceutical composition that will treat significant quantity.
Still more on the one hand, the invention provides host cell, comprise the expression vector that contains coding fusion polypeptide nucleic acid molecule, wherein said fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) activity and contains the golgi body locating structure territory that golgi body stops polypeptide; With the expression vector that contains the nucleic acid encoding molecule, wherein said polypeptide has mannosidase II (ManII) activity.In the preferred embodiment, fusion polypeptide contains catalyst structure domain and the golgi body locating structure territory of GnTIII, and golgi body locating structure territory is selected from the locating structure territory of ManII, the locating structure territory of GnTI, the locating structure territory of GnTII, the locating structure territory of ManI and the locating structure territory of α 1-6 core fucosyltransferase.In one embodiment, host cell comprises that further coding has the active polypeptide expression carrier of GnTII.The coding fusion polypeptide, have the active polypeptide of ManII, have the nucleic acid molecule of the active polypeptide of GnTII, can each in the expression vector that separates or in same expression vector.
In addition on the one hand, the present invention relates to host cell, comprise the expression vector that contains coding fusion polypeptide nucleic acid molecule, wherein said fusion polypeptide has β (1,4)-galactotransferase activity (GalT) and contains the golgi body locating structure territory that golgi body stops polypeptide; With the expression vector that contains the nucleic acid encoding molecule, wherein said polypeptide has mannosidase II (ManII) activity.In the preferred embodiment, fusion polypeptide comprises catalyst structure domain and the golgi body locating structure territory of GnTIII, golgi body locating structure territory is selected from the locating structure territory of ManII, the locating structure territory of GnTI, the locating structure territory of GnTII, the locating structure territory of ManI and the locating structure territory of α 1,6 core fucosyltransferase.In one embodiment, host cell comprises that further coding has the active polypeptide expression carrier of GnTII.The coding fusion polypeptide, have the active polypeptide of ManII, have the nucleic acid molecule of the active polypeptide of GnTII, can each in the expression vector that separates or in same expression vector.
Again in the one side, the present invention relates to relate to host cell, this host cell engineering is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GnTIII and the nucleic acid that at least a coding has the active polypeptide of ManII, expression amount is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
In the other embodiments, the invention provides host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid that the nucleic acid of the active fusion polypeptide of GnTIII, at least a coding have the active polypeptide of ManII, has the nucleic acid of the active polypeptide of GnTII with at least a coding, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
Again on the one hand, the invention provides host cell, this host cell engineering is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GalT and the nucleic acid that at least a coding has the active polypeptide of ManII, expression amount is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
In addition on the one hand, the invention provides host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid that the nucleic acid of the active fusion polypeptide of GalT, at least a coding have the active polypeptide of ManII, has the nucleic acid of the active polypeptide of GnTII with at least a coding, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
Again on the one hand, the present invention relates in host cell, produce the method for polypeptide, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GnTIII and the nucleic acid that at least a coding has the active polypeptide of ManII, the polypeptide that produces is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and the expression amount of wherein said fusion polypeptide is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; With separate described polypeptide.
On the other hand, the present invention relates in host cell, produce the method for polypeptide, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GalT and the nucleic acid that at least a coding has the active polypeptide of ManII, the polypeptide that produces is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and the expression amount of wherein said fusion polypeptide is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; With separate described polypeptide.
In addition on the one hand, the production method of the polypeptide that the cytotoxicity of production Fc mediation improves in host cell, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell through engineering approaches is expressed the nucleic acid of at least a coding GalT and the nucleic acid of at least a coding ManII, the polypeptide that produces is selected from whole antibody molecule, antibody fragment, antibody fragment comprises immunoglobulin fc region, and wherein the expression level of one or two among GalT or the ManII is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces and wherein has the cytotoxicity that the Fc of raising mediates as the described polypeptide of the result of described modification; Polypeptide with the cytotoxicity raising that separates described Fc mediation.
On the other hand, the present invention relates in host cell, produce the method for polypeptide, comprise: (a) under the condition that allows polypeptide to produce, cultivate host cell, this host cell through engineering approaches is expressed the nucleic acid that at least a coding has the active polypeptide of alpha-Mannosidase II, the polypeptide that produces is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and wherein said have the active polypeptide expression amount of alpha-Mannosidase II and be enough to modify oligosaccharides in the described polypeptide Fc district that described host cell produces; (b) separate the described polypeptide that produces by described host cell.
On the other hand, the present invention relates to host cell, this host cell through engineering approaches is expressed the nucleic acid that at least a coding has the active polypeptide of alpha-Mannosidase II under the condition that allows polypeptide to produce, the polypeptide that produces is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and wherein said have the active polypeptide expression amount of alpha-Mannosidase II and be enough to modify oligosaccharides in the described polypeptide Fc district that described host cell produces.
Still on the other hand, the present invention relates to the polypeptide that produces by such host cell, especially have the antibody of the Fc receptors bind of the effector function of raising and/or raising as the result of described modification oligosaccharides.
The accompanying drawing summary
Fig. 1. from the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of unmodified (non-glycosyl through engineering approaches) the anti-CD 20 IgG1 antibody of the reorganization that produces among the BHK.With antibody expression vector pETR1502 transfectional cell.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 1.
Fig. 2. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of the reorganization that produces among the BHK of the engineered nucleic acidization of the encoding wild type of using by oneself (" wt ") GnTIII.With antibody expression vector pETR1502 and GnTIII expression vector pETR1166 cotransfection cell.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 1.
Fig. 3. the MALDI/TOF-MS of neutral oligosaccharides mixture that comes personal coding to have the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of the reorganization that produces among the BHK of the active and engineered nucleic acidization by the localized fusion polypeptide in GnTI-golgi body locating structure territory (" G1-GnTIII ") of GnTIII composes.With antibody expression vector pETR1502 and GnTIII expression vector pETR1425 cotransfection cell.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 1.
Fig. 4. the MALDI/TOF-MS of neutral oligosaccharides mixture that comes personal coding to have the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of the reorganization that produces among the BHK of the active and engineered nucleic acidization by golgi body alpha-Mannosidase II (ManII)-localized fusion polypeptide in golgi body locating structure territory (" M2-GnTIII ") of GnTIII composes.With antibody expression vector pETR1502 and GnTIII expression vector pETR1506 cotransfection cell.Antibody purification from substratum described in embodiment 1 material and method part and preparation and analysis oligosaccharides.
Fig. 5. from the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of unmodified (non-glycosyl through engineering approaches) the anti-CD 20 IgG1 antibody of the reorganization that produces in the HEK293-EBNA cell.With antibody expression vector pETR1520 transfectional cell.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 1.
Fig. 6. the MALDI/TOF-MS of neutral oligosaccharides mixture that comes personal coding to have the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of the reorganization that produces among the HEK293-EBNA of the active and engineered nucleic acidization by golgi body alpha-Mannosidase II (ManII)-localized fusion polypeptide in golgi body locating structure territory (" M2-GnTIII ") of GnTIII composes.With antibody expression vector pETR1520 with GnTIII expression vector pETR1519 cotransfection cell.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 1.
Fig. 7. the MALDI/TOF-MS of neutral oligosaccharides mixture that comes personal coding to have the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of the reorganization that produces among the HEK293-EBNA of the active and engineered nucleic acidization by golgi body alpha-Mannosidase II (ManII)-localized fusion polypeptide in golgi body locating structure territory (" M2-GnTIII ") of GnTIII composes.With antibody expression vector pETR1520 with GnTIII expression vector pETR1519 cotransfection cell.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 1.(a) PNGaseF-that does not have other enzyme to handle discharges the oligosaccharides characteristic pattern of oligosaccharides.(b) PNGaseF-that further digests with EndoH discharges the oligosaccharides characteristic pattern of oligosaccharides.
Fig. 8. (a) synoptic diagram of the catalytic digestion oligosaccharides of EndoH.EndoH can digest heterozygosis (hybrid) (halving with heterozygosis) oligosaccharides, but indigestion compound (complex) or compound bisection oligosaccharides.(b) pass through the compound and heterozygous oligosaccharides of explanation, Endo-H handles structure is distributed to the oligosaccharides peak value that has identical m/z ratio in the initial MALDI/TOF-MS mass spectrum from the PNGAseF processing.
Fig. 9. " G1-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the antibody dependent cellular cytotoxicity (ADCC) of the chimeric IgG1 antibody of anti-CD20.Two antibody all produce in bhk cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Fig. 3 and those of unmodified antibody in Fig. 1.Target cell (T) is SKW6.4 people's a lymphoblastoid.Effector cell (E) is the human PBMC of fresh separated.Ratio is that 25: 1 E: T is used for hatching in 4 hours the ADCC test, discharges by serum lactic dehydrogenase (LDH) and measures cytotoxicity, discharges (using stain remover to substitute antibody) and spontaneous release (substratum substitutes antibody) contrasts with respect to maximum.Describe test in detail in the material of embodiment 1 and the method part.
Figure 10. " M2-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the antibody dependent cellular cytotoxicity (ADCC) of the chimeric IgG1 antibody of anti-CD20.Two antibody all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Fig. 6 and those of unmodified antibody in Fig. 5.Target cell (T) is SKW6.4 people's a lymphoblastoid.Effector cell (E) is the human PBMC of fresh separated.Ratio is that 25: 1 E: T is used for 4 hours cultivation ADCC test, discharges by serum lactic dehydrogenase (LDH) and measures cytotoxicity, discharges (using the alternative antibody of stain remover) and spontaneous release (substratum substitutes antibody) contrast with respect to maximum.Describe test in detail in the material of embodiment 1 and the method part.
Figure 11. " M2-GnTIII " glycosyl through engineering approaches to the reorganization of " wt-GnTIII " glycosyl through engineering approaches, the antibody dependent cellular cytotoxicity (ADCC) of the chimeric IgG1 antibody of anti-CD20.Two antibody all produce in bhk cell.The generation of M2-GnTIII glycosyl engineered antibody and glycosylation characteristic pattern be described among Fig. 4 and those of wt-GnTIII glycosyl engineered antibody in Fig. 2.Target cell (T) is SKW6.4 people's a lymphoblastoid.Effector cell (E) is the human PBMC of fresh separated.Ratio is that 25: 1 E: T is used for 4 hours cultivation ADCC test, discharges by serum lactic dehydrogenase (LDH) and measures cytotoxicity, discharges (using the alternative antibody of stain remover) and spontaneous release (substratum substitutes antibody) contrast with respect to maximum.Describe test in detail in the material of embodiment 1 and the method part.
Figure 12. " M2-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the combination of the Fc γ RIIIa acceptor on chimeric IgG1 antibody of anti-CD20 and the NK cell.Two antibody all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Fig. 6 and those of unmodified antibody in Fig. 5.As the material of embodiment 1 and the carrying out combination test described in the method part.NK cells of human beings at its surface expression Fc γ RIIIa acceptor is separated from the known genotype donor that does not produce Fc γ RIIc acceptor (that is the homozygous gene variant that, contains in-frame stop codon in the Fc γ RIIc encoding sequence).Use geometric mean fluorescence intensity that anti-human IgG antibody's fragment of FITC mark measures along with recombinant antibodies and NK cell bonded amount increase by FACS.The combination that detects in this test is that Fc γ RIIIa is specific, as proving (referring to Figure 13) by use competition Fc γ RIIIa specific antibody fragment.
Figure 13. increasing in the presence of the anti-Fc γ of the competition RIII antibody fragment of concentration, " M2-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the combination of the Fc γ RIIIa acceptor on chimeric IgG1 antibody of anti-CD20 and the NK cell.Two recombinant antibodies all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Fig. 6 and those of unmodified antibody in Fig. 5.Carrying out described in the material of embodiment 1 and method part be in conjunction with test, but hatch the NK cell of purifying altogether with the anti-Fc γ of the competition 3G8-Fab2 RIII antibody fragment of recombinant antibodies (final concentration is 3 μ g/ml usually) and increase and change concentration (referring to chart).NK cells of human beings at its surface expression Fc γ RIIIa acceptor is separated from the known genotype donor that does not produce Fc γ RIIc acceptor (that is the homozygous gene variant that, contains in-frame stop codon in the Fc γ RIIc encoding sequence).Use geometric mean fluorescence intensity that anti-human IgG antibody's fragment of FITC mark measures along with recombinant antibodies and NK cell bonded amount increase by FACS.
Figure 14. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the ED-B+ isoform of identification fibronectin and reorganization IgG1 " L19 " antibody that in the HEK293-EBNA cell, produces.(a) the unmodified antibody that in HEK293-EBNA cell, produces with antibody expression vector pETR1546 transfection.(b) the M2-GnTIII glycosyl engineered antibody that in HEK293-EBNA cell, produces with antibody expression vector pETR1546 and GnTIII expression vector pETR1519 cotransfection.By a-protein affinity chromatograph size exclusion chromatographic step two kinds of antibody of purifying from substratum subsequently, go up in Superdex200 matrix (Amersham) damping fluid is changed salt solution (PBS) to phosphate buffered.Material and preparation described in the method and analysis oligosaccharides as embodiment 1.
Figure 15. " M2-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the combination of the Fc γ RIIb acceptor on anti-ED-B+ fibronectin IgG1 antibody and the Raji human lymphoma cell.Two antibody all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Figure 14 b and those of unmodified antibody in Figure 14 a.As the material of embodiment 1 and the carrying out combination test described in the method part.Use geometric mean fluorescence intensity that anti-human IgG antibody's fragment of FITC mark measures along with recombinant antibodies and Raji B cell lymphoma cell bonded amount increase by FACS.
Figure 16. " M2-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the combination of the Fc γ RIIIa acceptor on the chimeric IgG1 antibody of anti-CD20 and the different donor NK cells.Two antibody all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Fig. 6 and those of unmodified antibody in Fig. 5.As the material of embodiment 1 and the carrying out combination test described in the method part.NK cells of human beings at its surface expression Fc γ RIIIa acceptor is separated from the known genotype donor that does not produce Fc γ RIIc acceptor (that is the homozygous gene variant that, contains in-frame stop codon in the Fc γ RIIc encoding sequence).The genotype of two donors is isozygotying of Fc γ RIIIa acceptor 158V-" high-affinity " variant.The genotype of two other donor is the 158V/F heterozygosis of Fc γ RIIIa acceptor 158V-" high-affinity " variant and 158F-" low affinity " variant.Use geometric mean fluorescence intensity that anti-human IgG antibody's fragment of FITC mark measures along with recombinant antibodies and NK cell bonded amount increase by FACS.The combination that detects in this test is that Fc γ RIIIa is specific, as proving (referring to Figure 13) by use competition Fc γ RIIIa specific antibody fragment.
Figure 17. the facs analysis that the CD4 of brachymemma (tCD4) expresses, (a) BHK-1502-28 (wild-type) and (b) chimeric anti-CD 20 IgG1 antibody produced cell that clone BHK-1502-28-11 (M2-GnTIII glycosyl through engineering approaches) is stable be.By the IRES sequence in the pETR1537 GnTIII expression vector expression of tCD4 is operationally expressed the indirect labelling that is connected and therefore expresses as GnTIII with M2-GnTIII.Average and geometric mean fluorescence intensity separately is, glycosyl through engineering approaches clone 27.6 and 19.9, and wild-type cell is 4.7 and 4.1.
Figure 18. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the chimeric IgG1 antibody of the anti-CD20 of reorganization of the M2-GnTIII-glycosyl through engineering approaches that produces from BHK-1502-28-11 clone.Clone, antibody purification and oligosaccharides preparation and analysis are described in the material and method part of embodiment 1.
Figure 19. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the chimeric IgG1 antibody of the anti-CD20 of reorganization of the M2-GnTIII-glycosyl through engineering approaches that produces from BHK-1502-28-11 clone discharges oligosaccharides and further digests with EndoH by PNGaseF.Clone, antibody purification and oligosaccharides preparation and analysis are described in the material and method part of embodiment 1.
Figure 20 .M2-GnTIII " the glycosyl through engineering approaches to the reorganization of unmodified, the combination of the Fc γ RIIIa acceptor on chimeric IgG1 antibody of anti-CD20 and the NK cell, antibody is produced by stable cell lines.The generation of glycosyl engineered antibody and glycosylation characteristic pattern are described in Figure 18 and 19.As the material of embodiment 1 and the carrying out combination test described in the method part.NK cells of human beings at its surface expression Fc γ RIIIa acceptor is separated from the known genotype donor that does not produce Fc γ RIIc acceptor (that is the homozygous gene variant that, contains in-frame stop codon in the Fc γ RIIc encoding sequence).Use geometric mean fluorescence intensity that anti-human IgG antibody's fragment of FITC mark measures along with recombinant antibodies and NK cell bonded amount increase by FACS.The combination that detects in this test is that Fc γ RIIIa is specific, as proving (referring to Figure 13) by use competition Fc γ RIIIa specific antibody fragment.
Figure 21. " M2-GnTIII " glycosyl through engineering approaches to the reorganization of unmodified, the complement-mediated cracking (CML) of the chimeric IgG1 antibody of anti-CD20.Two antibody all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation feature description in Fig. 6 and those of unmodified antibody in Fig. 5.Target cell (T) is SKW6.4 people's a lymphoblastoid.People's complement is used for test.Discharge by LDH and to measure cracking.Test is described in detail in the material and method part of embodiment 1.
Figure 22. come self-identifying human epidermal growth factor (EGFR) and the MALDI/TOF-MS of the neutral oligosaccharides mixture of the chimeric IgG1 of reorganization " C225 " antibody that in the HEK293-EBNA cell, produces spectrum.(a) the unmodified antibody that produces in the HEK293-EBNA cell with antibody expression vector pURSI28 transfection.(b) the M2-GnTIII glycosyl engineered antibody that produces in the HEK293-EBNA cell with antibody expression vector pETRURSI28 and GnTIII expression vector pETR1519 cotransfection.By a-protein affinity chromatograph size exclusion chromatographic step two antibody of purifying from substratum subsequently, go up in Superdex200 matrix (Amersham) damping fluid is changed salt solution (PBS) to phosphate buffered.Material and preparation described in the method and analysis oligosaccharides as embodiment 1.
Figure 23. " M2-GnTIII "-glycosyl through engineering approaches to the reorganization of unmodified, the antibody dependent cellular cytotoxicity (ADCC) of the chimeric IgG1 of anti-EGFR " C225 " antibody.Two antibody all produce in the HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern be described among Figure 22 b and those of unmodified antibody in Figure 22 a.Target cell (T) is A431 people's squamous cancer cell (ECACC No.85090402).Effector cell (E) is the human PBMC of fresh separated.Ratio is that 25: 1 E: T is used for 4 hours cultivation ADCC test, discharges by serum lactic dehydrogenase (LDH) and measures cytotoxicity, discharges (the use stain remover substitutes antibody) and spontaneous release (substratum substitutes antibody) contrast with respect to maximum.Describe test in detail in the material of embodiment 1 and the method part.
Figure 24. be respectively the nucleotide sequence and the aminoacid sequence of mannosidase II-GnTIII fusion polypeptide of the present invention.
Figure 25. be respectively the nucleotide sequence and the aminoacid sequence of GnT-I-GnT-III fusion polypeptide of the present invention.
Figure 26. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the chimeric IgG1 antibody of the anti-CD20 of unmodified recombinant C 2B8 (" Cwt ") that produces in the HEK293-EBNA cell of the antibody expression vector pETR1520 transfection of using by oneself.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 5.
Figure 27. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the chimeric IgG1 antibody of the anti-CD20 of reorganization glycosyl through engineering approaches C2B8 (" Cbrt ") that produces in the HEK293-EBNA cell of use by oneself antibody expression vector pETR1520 and fusion GnTIII expression of polypeptides carrier (pETR1519) cotransfection.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 5.(A) PNGaseF-that does not have other enzyme to handle discharges the oligosaccharides characteristic pattern of oligosaccharides.(B) PNGaseF-that further digests with EndoH discharges the oligosaccharides characteristic pattern of oligosaccharides.
Figure 28. the MALDI/TOF-MS spectrum of the neutral oligosaccharides mixture of the chimeric IgG1 antibody of the anti-CD20 of reorganization glycosyl through engineering approaches C2B8 (" Cm ") that produces in the HEK293-EBNA cell of the antibody expression vector pETR1520 that uses by oneself, fusion GnTIII expression of polypeptides carrier (pETR1519) and mannosidase II expression of polypeptides carrier (pCLF9) cotransfection.Material and the antibody purification from substratum described in the method part and preparation and analysis oligosaccharides as embodiment 5.(A) PNGaseF-that does not have other enzyme to handle discharges the oligosaccharides characteristic pattern of oligosaccharides.(B) PNGaseF-that further digests with EndoH discharges the oligosaccharides characteristic pattern of oligosaccharides.
Figure 29. come the antibody-mediated antibody dependent cellular cytotoxicity (ADCC) of inosculating antibody CD20 of glycosyl through engineering approaches by the nucleic acid of in the HEK293-EBNA cell, expressing coding ManII-GnTIII fusion polypeptide, wherein the GnTIII catalyst structure domain is by ManII golgi body locating structure territory location, and the nucleic acid of the ManII-GnTIII that wherein encodes is expressed or and the nucleic acid (Cm) of coding ManII coexpression together in antibody produced cell himself (antibody Cbrt) going up.Cwt is the chimeric IgG1 antibody of the anti-CD20 of recombinant C 2B8 (" Cwt ") of the unmodified that produces in the HEK293-EBNA cell with antibody expression vector pETR1520 transfection.Test is described in detail in the material and method part of embodiment 1.
Figure 30. the Fc γ RIIIa receptors bind of coming the chimeric anti-CD20 antibodies of glycosyl through engineering approaches by the nucleic acid of in the HEK293-EBNA cell, expressing coding ManII-GnTIII fusion polypeptide, wherein the GnTIII catalyst structure domain is localized by ManII golgi body locating structure territory, and wherein the ManII-GnTIII coding nucleic acid is himself (antibody Cbrt) go up to express or and the nucleic acid (Cm) of coding ManII coexpression together in antibody produced cell.Cwt is the chimeric IgG1 antibody of the anti-CD20 of recombinant C 2B8 (" Cwt ") of the unmodified that produces in the HEK293-EBNA cell with antibody expression vector pETR1520 transfection.Test is described in detail in the material and method part of embodiment 1.NK cells of human beings at its surface expression Fc γ RIIIa acceptor is separated from the known genotype donor that does not produce Fc γ RIIc acceptor (that is the homozygous gene variant that, contains in-frame stop codon in the Fc γ RIIc encoding sequence).Use geometric mean fluorescence intensity that anti-human IgG antibody's fragment of FITC mark measures along with recombinant antibodies and NK cell bonded amount increase by FACS.The combination that detects in this test is that Fc γ RIIIa is specific, as proving (referring to Figure 13) by use competition Fc γ RIIIa specific antibody fragment.
Figure 31. the complement-mediated cytotoxicity of coming the chimeric anti-CD20 antibodies of glycosyl through engineering approaches by the nucleic acid of in the HEK293-EBNA cell, expressing coding ManII-GnTIII fusion polypeptide, wherein the GnTIII catalyst structure domain is localized by ManII golgi body locating structure territory, and wherein the ManII-GnTIII coding nucleic acid is himself (antibody Cbrt) go up to express or and the nucleic acid (Cm) of coding ManII coexpression together in antibody produced cell.Cwt is the chimeric IgG1 antibody of the anti-CD20 of recombinant C 2B8 (" Cwt ") of the unmodified that produces in the HEK293-EBNA cell with antibody expression vector pETR1520 transfection.
Figure 32 (A-C). expression vector pCLF9 (A), pETR1842 (B) and pETR1843 (C).
Figure 33 (A and B). be used for the expression vector of fusion rotein ManII-GalT (A) and GalT (B).
Figure 34. the oligosaccharides characteristic pattern of the anti-CD-20 monoclonal antibody that in the presence of alpha-Mannosidase II, produces and that found and relative percentage antibody Fc part correlation structure.
Figure 35 (A and B). the oligosaccharides characteristic pattern of the anti-CD-20 monoclonal antibody that in the presence of fusion rotein ManII-GalT, produces and that found and relative percentage antibody Fc part correlation structure.PNGaseF (A) and the postdigestive oligosaccharides characteristic pattern of EndoH (B).
Figure 36. the antibody and the Fc γ RIIIA receptors bind that produce in the presence of alpha-Mannosidase II (ManII) have the affinity higher than wild-type antibody.
Figure 37. the antibody dependent cellular cytotoxicity of glycosyl through engineering approaches inosculating antibody CD-20 mediation.
Detailed Description Of The Invention
Unless used usually the same in term and this area as used herein is in addition as giving a definition.
As used in this, term antibody is defined as and comprises whole antibody molecule, comprises mono-clonal, polyclone and polyspecific (for example, dual specific) antibody, and antibody fragment and fusion rotein with Fc district, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.Also comprise humanization and chimeric antibody.
As used in this, term Fc area definition is the C-terminal zone of IgG heavy chain.Although the boundary in IgG heavy chain Fc district can slight modification, human IgG heavy chain Fc district is normally defined from position Cys226 amino-acid residue and begins to one section of C-terminal.
As used in this, the zone definitions that term is equivalent to immunoglobulin fc region is to comprise the natural generation allele variant of immunoglobulin fc region and the variant with change, changes to have produced to substitute, add or disappearance but do not reduce the ability of immunoglobulin-mediated effector function (as antibody dependent cellular cytotoxicity) basically.For example, can lack one or more amino acid and not lose biological function basically from the N-terminal or the C-terminal of immunoglobulin fc region.It is minimum to select such variant to make to active influence according to general rule known in the art.(referring to, for example, Bowie, J.U. etc., Science 247:1306-10 (1990).
As used in this, the fusion polypeptide that " has β (1,4)-N-acetylglucosaminyltrVnsferase III activity " refers to and can catalysis N-acetylglucosamine (GlcNAc) residue in β-1-4 key be added into fusion polypeptide on the mannoside that β that N-connects three mannose group cores of oligosaccharides connects.This comprises and presenting and β (1,4)-the similar but essential identical fusion polypeptide of N-acetylglucosaminyltrVnsferase III enzymic activity, this enzyme is also referred to as β-1-4 mannosyl-glycoprotein 4-β-N-acetylglucosaminyltrVnsferase (EC2.4.1.144), according to the biochemical and international NK (NC-IUBMB) of molecular biosciences, as measuring in the particular organisms test, there is and do not have dose-dependently.In the situation that dose-dependently exists, do not need to be equal to β (1,4)-N-acetylglucosaminyltrVnsferase III, but and β (1,4)-N-acetylglucosaminyltrVnsferase III compares similar with giving active dose dependency basically (that is, with respect to β (1,4)-N-acetylglucosaminyltrVnsferase III, candidate's polypeptide will present higher activity or active low more than 25 times unlike it, and will be preferably active low more than 10 times unlike it, most preferably unlike the low activity more than 3 times of its activity).
As used in this, " have β (1,4)-galactosyltransferasactivity activity " with the fusion polypeptide of " having the GalT activity " and refer to and can catalysis the galactose residue of UDP semi-lactosi be added into the fusion polypeptide that N is connected the non reducing end GlcNAc that finds in the oligosaccharides.This comprises and presents enzymic activity and β (1,4)-the similar but essential identical fusion polypeptide of galactosyltransferase, this enzyme is also referred to as UDP-Gal:GlcNAc β-1,4-galactosyltransferase (E.C.2.4.1.38), according to the biochemical and international NK (NC-IUBMB) of molecular biosciences, as measuring in the particular organisms test, there is and do not have dose-dependently.In the situation that dose-dependently exists, do not need to be equal to β (1,4)-galactosyltransferase, but and β (1,4)-galactosyltransferase is compared similar with giving active dose dependency basically (that is, with respect to β (1,4)-N-acetylglucosaminyltrVnsferase III, candidate's polypeptide will present higher activity or active low more than 25 times unlike it, and will be preferably active low more than 10 times unlike it, most preferably unlike the low activity more than 3 times of its activity).
Have with the present invention with reference to the nucleotide sequence at least for example nucleic acid or the polypeptide of the nucleotide sequence of 95% " identical ", the nucleotide sequence that is defined as polynucleotide is equal to canonical sequence, except polynucleotide sequence can comprise with reference to per 100 Nucleotide of nucleotide sequence up to five point mutation.In other words, in order to obtain to have and polynucleotide with reference to the identical nucleotide sequence of nucleotide sequence at least 95%, Nucleotide up to 5% in the canonical sequence can be deleted by another nucleotide substitution, or can insert in the canonical sequence up to a plurality of Nucleotide of total nucleotide in the canonical sequence 5%.Search sequence can be shown in the whole sequence among Figure 24 or Figure 25.
As practical situation, any specific nucleic acid molecule or polypeptide and nucleotide sequence of the present invention or peptide sequence are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical can to use known computer program conventional determining.Measure the preferred method of the best overall coupling between search sequence (sequence of the present invention) and the object sequence, be also referred to as global sequence's comparison, can use based on the FASTDB computer program of the algorithm of Brutlag etc. and measure, Brutlag etc., Comp.App.Biosci.6:237-245 (1990).In the sequence alignment, search sequence and object sequence all are dna sequence dnas.Can compare the RNA sequence by U being converted to T.The result of described global sequence comparison is the % homogeny.Calculate that used preferred parameter is in the FASTDB comparison of dna sequencing of % homogeny: matrix=single entry, k-tuple=4, mispairing punishment=1, connect punishment=30, randomization group length=0, close value=1, breach punishment=5, breach size punishment 0.05, the length of window size=500 or object nucleotide sequence is as the criterion with the shorter one.
If because 5 ' or 3 ' disappearance, rather than because inner disappearance, object sequence is shorter than search sequence, must carry out manual synchronizing to this result.This is 5 ' and the 3 ' brachymemma that does not have the calculating object sequence because of FASTDB program when calculating the % homogeny.Object sequence for 5 ' or 3 ' end brachymemma with respect to search sequence, does not have the base quantity of the search sequence of pairing/comparison by calculating object sequence 5 ' and 3 ', as the per-cent of the total base of search sequence, proofread and correct the % homogeny.Whether the result by the FASTDB sequence alignment measures Nucleotide mates/compares.From the % homogeny that calculates by the FASTDB program of above-mentioned use special parameter, deduct this per-cent then, obtain final % homogeny value.This gauged value is the value that is used for the object of the invention.Only calculate as show by the FASTDB comparison, do not have and the object sequence 5 ' of search sequence coupling/comparison and the base outside the 3 ' base are for the artificial purpose of adjustment % homogeny value.
For example, the search sequence of the object sequence of 90 bases and 100 bases is compared measure the % homogeny.Disappearance has taken place in 5 ' end in object sequence, and therefore, the FASTDB comparison does not show the coupling/comparison of 5 ' terminal 10 bases.10 unpaired bases are represented 10% (5 ' and 3 ' end does not mate base sum in base quantity/search sequence) of sequence, therefore deduct 10% from the % homogeny value that the FASTDB program is calculated.If remaining 90 bases are mated fully, final % homogeny is 90%.Among another embodiment, the search sequence of the object sequence of 90 bases and 100 bases is compared.Current disappearance is inner disappearance, makes object sequence not have the base that do not match/compare with search sequence 5 ' and 3 '.In this case, the % homogeny that calculates by FASTDB does not have manual synchronizing.Once more, a manual synchronizing 5 ' or 3 ' not having and the base of search sequence coupling/comparison of object sequence.Do not carry out other manual synchronizings for the object of the invention.
Have with the present invention and inquire about at least for example polypeptide of the aminoacid sequence of 95% " identical " of aminoacid sequence, the object peptide sequence is defined as the object amino acid sequence of polypeptide and is equal to search sequence, except may comprise that per 100 amino acid are up to the change of five amino acid in the inquiry aminoacid sequence.In other words, in order to obtain to have the polypeptide of the aminoacid sequence identical with inquiry aminoacid sequence at least 95%, the amino-acid residue up to 5% in the object sequence can insert, delete, or uses another amino acid replacement.These changes of canonical sequence can occur in reference between the aminoterminal of aminoacid sequence or carboxyl terminal or those terminal positions Anywhere, one or more in group in the single residue that is dispersed in canonical sequence or in canonical sequence.
As practical situation, can use the known computer program come conventional determining whether any specific polypeptide be at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical with reference to polypeptide.Measure the preferred method of the best overall coupling between search sequence (sequence of the present invention) and the object sequence, be also referred to as global sequence's comparison, can use based on the FASTDB computer program of the algorithm of Brutlag etc. and measure, Brutlag etc., Comp.App.Biosci.6:237-245 (1990).In the sequence alignment, search sequence and object sequence all are nucleotide sequences and all are aminoacid sequences.The result of described global sequence comparison is the % homogeny.Used preferred parameter is in the comparison of FASTDB amino acid: matrix=PAM 0, k-tuple=2, mispairing punishment=1 connects punishment=20, randomization group length=0, close value=1, window size=sequence length, breach punishment=5, breach size punishment=0.05, the length of window size=500 or object nucleotide sequence is as the criterion with the shorter one.
If because the terminal deletion of N-or C-, rather than because inner deletion, object sequence is shorter than search sequence, must carry out manual synchronizing to this result.This be because when calculating overall % homogeny the FASTDB program do not have the calculating object sequence of N-and C-hold brachymemma.For the object sequence of N-and C-end brachymemma, with respect to search sequence, the search sequence by calculating object sequence N-and C-end not have the residue quantity with corresponding object sequences match/comparison, as the per-cent of the total base of search sequence, proofreaies and correct the % homogeny.Whether the result by the FASTDB sequence alignment measures residue mates/compares.From the % homogeny that calculates by the FASTDB program of above-mentioned use special parameter, deduct this per-cent then, obtain final % homogeny value.This final percentage homogeny is the homogeny that is used for the object of the invention.Only the residue of object sequence N-that consideration does not have and search sequence matches/compares and C-end is the purpose for the artificial % of adjustment homogeny value.That is, just the N-farthest of object sequence and C-hold outer inquiry residue position.
For example, the search sequence of the object sequence of 90 amino-acid residues and 100 residues is compared measure the % homogeny.At the N-of object sequence end deletion has taken place, therefore, the FASTDB comparison does not show the pairing/comparison of 10 bases in N-termination.10 unpaired bases are represented 10% (N-and C-end do not match base quantity/search sequence in base sum) of sequence, therefore deduct 10% from the % homogeny value that the FASTDB program is calculated.If remaining 90 residues match fully, final % homogeny is 90%.Among another embodiment, the search sequence of the object sequence of 90 residues and 100 residues is compared.Current deletion is inner deletion, make object sequence at N-or the C-end not have and search sequence is unpaired/base compared.In this case, the % homogeny that calculates by FASTDB does not have manual synchronizing.Once more, a manual synchronizing N-of object sequence and the outer residue position of C-end, as showing in the FASTDB comparison, it is not and search sequence pairing/comparison.Do not carry out other manual synchronizings for the object of the invention.
As used in this, nucleic acid with nucleotide sequence of the present invention " hybridize under stringent condition ", refer to and containing 50% methane amide, 5 * SSC (750mM NaCl, the 75mM Trisodium Citrate), in the solution of the salmon sperm dna of the shearing of 50mM sodium phosphate (pH7.6), 5 * Denhardt solution, 10% dextran sulfate and 20 μ g/ml sex change 42 ℃ be incubated overnight, then wash filter membrane and the polynucleotide of hybridizing with 0.1 about 65 ℃ * SSC.
As used in this, term golgi body locating structure territory refers to golgi body and stops amino acid sequence of polypeptide, and it is responsible for being fixed in position in the Golgi complex.Usually, the locating structure territory comprises enzyme N-terminal " tail ".
As used in this, term effector function (effector function) refers to those biological activitys that can cause antibody Fc district (native sequences Fc district or aminoacid sequence variant Fc district).The example of antibody mediated effect subfunction comprises, but be not restricted to Fc receptor binding affinity, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagolysis (ADCP), cytokine secretion, the antigen absorption of passing through the immunocomplex mediation of antigen presenting cell, the downward modulation of cell surface receptor etc.
As used in this, term engineering, through engineering approaches, through engineering approaches and glycosylation engineering think any operation that comprises natural generation polypeptide or its segmental glycosylation pattern.The glycosylation engineering comprises the metabolic engineering of the glycosylation process of cell, comprises that the genetic manipulation of oligosaccharides route of synthesis obtains to express in the cell glycosylation of the change of glycoprotein.In addition, glycosylation engineering comprises that sudden change and cellular environment are to glycosylated influence.
As used in this, the term host cell is contained the cell system of any kind of, and it can through engineering approaches produce target protein matter, protein fragments or the peptide of modifying glycosylated form, comprises antibody, antibody fragment and fusion rotein.Usually, host cell has been carried out operating the GnTIII that expresses optimum level.Host cell comprises cultured cells, for example, Mammals cultured cells such as Chinese hamster ovary celI, bhk cell, NS0 cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, yeast cell, insect cell and vegetable cell, only enumerate minority, but also comprised the plant of transgenic animal, transgenic plant or cultivation or the cell in the animal tissues.
As used in this, the cytotoxicity of term Fc mediation comprises antibody dependent cellular cytotoxicity and by containing the fusion protein mediated cytotoxicity of solubility Fc-in people Fc district.This is to cause " antibody target cell " cracked immune mechanism by " people's immune effector cell ", wherein:
" people's immune effector cell " is leukocyte population, its Fc district by these acceptors and antibody or Fc fusion rotein of its surface display Fc acceptor in conjunction with and realize effector function.Such colony comprises, but is not restricted to peripheral blood lymphocytes (PBMC) and/or natural killer (NK) cell.
" the antibody target cell " is by antibody or Fc fusion rotein bonded cell.Antibody or Fc fusion rotein are by the N-end and the target cell combination of albumen Fc district part.
As used in this, the cytotoxicity of the Fc mediation that term improves is defined as around target cell under the antibody or Fc fusion rotein concentration given in the substratum, the increase of cytotoxic mechanism cracked " antibody target cell " quantity of Fc mediation by above definition in preset time, and/or the cytotoxic mechanism by the Fc mediation obtains the reduction that antibody in the substratum or Fc fusion rotein concentration are given around the target cell that determined number " antibody target cell " needs in cracking in preset time.The Cytotoxic raising of Fc mediation is with respect to passing through same antibody or the fusion protein mediated cytotoxicity of Fc, these antibody or Fc fusion rotein are produced by the host cell of same type, use identical standard production well known by persons skilled in the art, purifying, preparation and store method, but be not to produce by the host cell that said methods engineeringization is expressed glycosyltransferase GnTIII.
The antibody meaning with antibody dependent cellular cytotoxicity (ADCC) of raising is the antibody as the ADCC with raising that records by the known any appropriate method of those of ordinary skills.A kind of generally acknowledged external ADCC test is as follows:
1) target cell is used in test, and known this target cell is expressed the target antigen by the antigen binding domain identification of antibody;
2) isolating human peripheral blood mononuclear cell (PBMC) action effect cell from the healthy donors blood of selecting is at random used in test;
3) test according to following scheme:
I) use the standard density centrifugal method to separate PBMC and with 5 * 10 6Individual cell/ml is suspended in the RPMI cell culture medium;
Ii) cultivate target cell, collect viability from exponential phase of growth and be higher than 90% cell by the normal structure cultural method, with the washing of RPMI cell culture medium, with 100 microcuries 51The Cr mark is used the cell culture medium washed twice, and with 10 5The density of individual cell/ml is resuspended in the cell culture medium;
Iii) the above-mentioned final target cell suspension liquid of 100 microlitres is transferred in each hole of 96 hole microtiter plates;
Iv) in substratum with antibody from the 4000ng/ml serial dilution to 0.04ng/ml, and the resulting antibody-solutions of 50 microlitres added in the target cell in the 96 hole microtiter plates, the various antibody concentration that will contain above-mentioned whole concentration ranges are tested in triplicate;
V) discharge (MR) contrast for maximum, contain other 3 holes of labels targets cell on the flat board, (St.Louis) aqueous solution substitutes antibody-solutions (above-mentioned iv point) for Nonidet, Sigma to accept 50 microlitre 2% (V/V) nonionic detergent;
Vi), contain other 3 holes of labels targets cell on the flat board, accept 50 microlitre RPMI cell culture mediums and substitute antibody-solutions (above-mentioned iv point) for spontaneous release (SR) contrast
Vii) then with 96 hole microtiter plates centrifugal 1 minute at 50 * g, and 4 ℃ of incubations 1 hour;
Viii) 50 microlitre PBMC suspension (above-mentioned i point) are added and produce effector in each hole: 25: 1 the ratio of target cell, and flat board is positioned over 5%CO 2In the incubator of atmosphere in 37 ℃ 4 hours;
Ix) radioactivity (ER) of collecting the acellular supernatant liquor in each hole and using the gamma counter quantitative experiment to discharge;
X) calculate the specific cleavage percentage ratio of each antibody concentration according to formula (ER-MR)/(MR-SR) * 100, wherein ER is the quantitative average radioactivity (referring to above-mentioned ix point) for that antibody concentration, MR is that the quantitative average radioactivity (referring to above-mentioned v point) and the SR that contrast for MR are the quantitative average radioactivity (referring to above-mentioned ix point) that contrasts (referring to above-mentioned vi point) for SR;
4) " ADCC of raising " is defined as the raising of the largest percentage of observed specific cleavage in the antibody concentration scope of above-mentioned test, and/or obtains the minimizing of observed half required antibody concentration of specific cleavage largest percentage in the antibody concentration scope of above-mentioned test.The raising of ADCC is with respect to above-mentioned test determination, by same antibody mediation, that produce by the same type host cell, as to use identical standard well known by persons skilled in the art production, purifying, preparation and storage method, but the ADCC that does not come the host cell of overexpression glycosyltransferase GnTIII to produce by through engineering approaches.
As used in this, the term anti-CD 20 antibodies is defined as the antibody of specific recognition cell surface 35,000 daltonian non-glycosylated phosphorproteins, and the restricted differentiation antigen Bp35 of the common called after human B lymphocyte of this phosphorprotein is commonly referred to as CD20.
The present invention is based on following discovery: engineered antibody produces cell and expresses the antibody that new fusion polypeptide forms raising of Fc receptor binding affinity and effector function raising, this new polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) or β (1,4)-galactosyltransferase (" GalT ") activity, and comprise that golgi body stops the golgi body locating structure territory of polypeptide.Perhaps, antibody that effector function improves and/or that the Fc receptors bind improves can produce cell by engineered antibody and obtain, and this antibody produced cell has coding and has the expression that the nucleic acid molecule of the polypeptide of alpha-Mannosidase II catalytic activity increases.In the preferred embodiment, has the nucleic acid molecule coexpression of the active fusion constructs of GnTIII or GalT and coding ManII or GnTII.
Therefore, in one embodiment, the present invention relates to comprise the isolating nucleic acid of coding fusion polypeptide sequence, wherein fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) activity and comprises that golgi body stops the golgi body locating structure territory of polypeptide.In the preferred embodiment, fusion polypeptide comprises the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III, and golgi body locating structure territory is the locating structure territory of mannosidase II.Further in the embodiment, golgi body locating structure territory is the locating structure territory of GalT.
Preferably, isolating nucleic acid has the nucleotide sequence shown in Figure 24 and the SEQ ID NO:14.In another preferred embodiment, fusion polypeptide comprises the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III, and golgi body locating structure territory is the catalyst structure domain of β (1,2)-N-acetylglucosaminyltrVnsferase I (GnTI).Preferably, nucleic acid has the nucleotide sequence shown in Figure 25 and the SEQ ID NO:12.Perhaps, can use another golgi body to stop the golgi body locating structure territory of polypeptide.In another preferred embodiment, golgi body locating structure territory is selected from the locating structure territory of β (1,2)-locating structure territory of N-acetylglucosaminyltrVnsferase II, the locating structure territory of mannosidase I and α 1-6 core fucosyltransferase.
In another preferred embodiment, the present invention relates to isolating nucleic acid, contain coding and have the sequence of the polypeptide of aminoacid sequence shown in Figure 24 and SEQ ID NO:15 or Figure 25 and the SEQ ID NO:13.The present invention also comprises isolating nucleic acid, contains the sequence of hybridizing with hybridization probe under stringent condition, and its nucleotide sequence is made up of the nucleotide sequence shown in Figure 24 and SEQ ID NO:14 or Figure 25 and the SEQ IDNO:12.The invention further relates to isolating nucleic acid, comprise and Figure 24 and SEQ ID NO:14 or Figure 25 and nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97% shown in the SEQ ID NO:12,98% or 99% identical sequence.In another embodiment, the present invention relates to isolating nucleic acid, comprising that coding has the sequence with the polypeptide of Figure 24 and SEQ ID NO:15 or Figure 25 and aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97% shown in the SEQ ID NO:13,98% or 99% identical aminoacid sequence.The present invention also comprises isolating nucleic acid, comprises that coding has the sequence of the polypeptide of aminoacid sequence shown in conserved amino acid alternate Figure 24 and SEQ ID NO:15 or Figure 25 and the SEQ ID NO:13.
In another embodiment, the present invention relates to expression vector, comprise the isolating nucleic acid of the present invention, as above-mentioned those.
Further in the embodiment, the present invention relates to have β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-galactosyltransferase (" GalT ") are active and contain the fusion polypeptide that the allos golgi body stops the golgi body locating structure territory of polypeptide.In the preferred embodiment, fusion polypeptide of the present invention comprises the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III.In the particularly preferred embodiment, fusion polypeptide further comprises the golgi body locating structure territory of mannosidase II or β (1,2)-N-acetylglucosaminyltrVnsferase I (GnTI).In the interchangeable embodiment, golgi body locating structure territory is selected from the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.Can under the condition of the expression of nucleic acid that allows the described fusion polypeptide of coding, in substratum, cultivate host cell of the present invention and from the culture that generates, collect described fusion polypeptide and make fusion polypeptide of the present invention.
The invention further relates to the method for the glycosylation form (profile) of the polypeptide of modifying the host cell generation, comprise nucleic acid of the present invention or carrier are introduced in the described host cell.Preferably, modified polypeptides is that IgG or its contain the fragment in Fc district.More preferably, polypeptide is IgG1, or it contains the fragment in Fc district.In another preferred embodiment, modified polypeptides is a fusion rotein, and this fusion rotein comprises the zone that is equivalent to human IgG Fc district.
The invention further relates to the host cell that contains nucleic acid of the present invention and expression vector.In one embodiment, the present invention relates to host cell, this host cell through engineering approaches is expressed at least a coding and is had β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-nucleic acid of the active fusion polypeptide of galactosyltransferase (" GalT "), expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that described host cell produces, wherein said polypeptide is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.In the preferred embodiment, the polypeptide that described host cell produces is IgG or its fragment.More preferably, the polypeptide of described host cell generation is IgG1 or its fragment.Perhaps, the polypeptide that described host cell produces is a fusion rotein, and this fusion rotein comprises and is equivalent to for example zone in the Fc district of IgG1 of human IgG.
Present the Fc receptor binding affinity of raising and/or the effector function of raising as the modified polypeptide of modifying that host cell of the present invention produced as a result.Preferably, the Fc receptor binding affinity of raising is the raising with Fc γ activated receptor such as Fc γ RIIIa receptors bind.The preferably following one or more raising of the effector function that improves: the raising of antibody dependent cellular cytotoxicity, the raising of antibody dependent cellular phagolysis (ADCP), the raising of cytokine secretion, the raising of the antigen uptake of the immunocomplex mediation by antigen presenting cell, the Cytotoxic raising of Fc mediation, improve with NK cell bonded, improve with the scavenger cell bonded, and polymorphonuclear cell (PMN) bonded improves, improve with the monocyte bonded, the raising that the target binding antibody is crosslinked, the enhancing of apoptosis-induced direct signal, the raising of the raising of dendritic cell maturation and T cell sensitization.
In the particularly preferred embodiment, host cell of the present invention is that the polypeptide that Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma and described host cell produce is anti-CD20 antibodies such as IDEC-C2B8.In another preferred embodiment, host cell is inosculating antibody people EGFR monoclonal antibody C225.
Except containing the nucleic acid of coding fusion polypeptide of the present invention, host cell of the present invention further contains at least a brachymemma (transected) nucleic acid, the antibody fragment or the fusion rotein in this nucleic acid encoding antibody molecule, reservation function Fc district, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.In the preferred embodiment, the nucleic acid encoding anti-CD20 antibodies of at least a brachymemma, inosculating antibody people neuroblastoma monoclonal antibody chCE7, inosculating antibody people kidney cell carcinoma monoclonal antibody chG250, inosculating antibody people colon, lung and breast cancer monoclonal antibody ING-1, Humanized anti-human 17-1A antigen monoclonal antibody 3622W94, Humanized anti-human colorectum tumour antibody A33, the anti-human melanoma antibody of facedown GD3 Sphingolipids,sialo R24, inosculating antibody people squamous cell carcinoma monoclonal antibody SF-25, anti-people EGFR antibody, anti-people EGFRvIII antibody, anti-people PSMA antibody, anti-people's psca antibody, anti-people CD22 antibody, anti-people CD30 antibody, anti-TAG72 antibody, the antibody of anti-high molecular melanoma associated antigen (HMWMAA), anti-GD3 ganglioside antibody, anti-GD2 ganglioside antibody, anti-GM2 ganglioside antibody, anti human nerve joint glycosides fat antibody, anti-EGFRvIII antibody, anti-alpha 2 integrin antibodies, anti-CD80 antibody, anti-LeY antibody, anti-stick protein antibodies, anti-MUC18 antibody, anti-people CD33 antibody, anti-people CD38 antibody, antibodies against human CD 40, anti-people CD45 antibody, antihuman CD 52 antibody, anti-people CD138 antibody, anti-people HLA-DR variant antibody, anti-people EpCAM antibody, anti-people CEA antibody, anti-people MUC1 antibody, anti-people MUC1 nucleoprotein antibody, the unusual glycosylated MUC1 antibody of anti-people, antagonism contains the antibody or the anti-people HER2/neu antibody of people's fibronectin variant of ED-B structural domain.
The invention still further relates to the method for in host cell, producing polypeptide, comprise that (a) cultivates host cell under the condition that allows polypeptide to produce, the host cell through engineering approaches is expressed at least a coding and is had β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-nucleic acid of the active fusion polypeptide of galactosyltransferase (" GalT "), the polypeptide that produces is selected from whole antibody molecule, the antibody fragment and the fusion rotein that contain the Fc district, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and the expression amount of the wherein said GnTIII of having activity or the active fusion polypeptide of GalT is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; (b) separate described polypeptide.In the preferred embodiment, fusion polypeptide comprises the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III.In the particularly preferred embodiment, fusion polypeptide comprises that further golgi body stops the golgi body locating structure territory of polypeptide.Preferably, golgi body locating structure territory is the golgi body locating structure territory of mannosidase II or β (1,2)-N-acetylglucosaminyltrVnsferase I (GnTI).Perhaps, golgi body locating structure territory is selected from the locating structure territory of mannosidase I, the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II and the locating structure territory of α 1-6 core fucosyltransferase.The polypeptide that the inventive method produces has the Fc receptor binding affinity of raising and/or the effector function of raising.Preferably, the effector function of raising is following one or more: the Cytotoxic raising (comprising the raising of antibody dependent cellular cytotoxicity) of Fc mediation, the raising of antibody dependent cellular phagolysis (ADCP), the raising of cytokine secretion, the raising of the antigen uptake of the immunocomplex mediation by antigen presenting cell, improve with NK cell bonded, improve with the scavenger cell bonded, improve with the monocyte bonded, improve with the polymorphonuclear cell bonded, the raising that the target binding antibody is crosslinked, the enhancing of apoptosis-induced direct signal, the raising of the raising of dendritic cell maturation and T cell sensitization.The Fc receptor binding affinity that improves preferably with the raising of Fc activated receptor such as Fc γ RIIIa receptors bind.
In another embodiment, the present invention relates to the polypeptide by the inventive method production, it has bisection (bisected) oligosaccharides of increase ratio in the Fc district of described polypeptide.Still in another embodiment, the polypeptide of producing by the inventive method as the result of described modification has the oligosaccharides of non-fucosylation in the Fc district of increase ratio.The oligosaccharides of non-fucosylation can be a heterozygous or compound.In the particularly preferred embodiment, the polypeptide of producing by host cell of the present invention and method has the oligosaccharides of bisection in the Fc district of increase ratio, non-fucosylation.Halve, the oligosaccharides of non-fucosylation can be heterozygosis or compound.Especially, the inventive method can be used to produce polypeptide, wherein at least 15% of the oligosaccharides in the polypeptide Fc district, 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 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, be non-fucosylation.The inventive method can also be used to producing polypeptide, the oligosaccharides at least 15% in the polypeptide Fc district wherein, 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 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, be binary.Still further, the inventive method can be used for producing polypeptide, the oligosaccharides at least 15% in the polypeptide Fc district wherein, 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 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, be binary, non-fucosylation.The inventive method can also be used to produce polypeptide, wherein the oligosaccharides at least 15% in the polypeptide Fc district, more preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 35% be binary heterozygosis non-fucosylation.
In another embodiment, the present invention relates to the antibody produced by the inventive method, this antibody engineeringization is with the effector function with raising and/or the Fc receptor binding affinity of raising.Preferably, the effector function of raising is following one or more: the Cytotoxic raising (comprising the raising of antibody dependent cellular cytotoxicity) of Fc mediation, the raising of antibody dependent cellular phagolysis (ADCP), the raising of cytokine secretion, the raising of the antigen uptake of the immunocomplex mediation by antigen presenting cell, improve with NK cell bonded, improve with the scavenger cell bonded, improve with the monocyte bonded, improve with the polymorphonuclear cell bonded, the raising that the target binding antibody is crosslinked, the enhancing of apoptosis-induced direct signal, the raising of the raising of dendritic cell maturation and T cell sensitization.In the embodiment preferred, the Fc receptor binding affinity of raising is to improve with Fc activated receptor bonded, is more preferably the raising with Fc γ RIIIa receptors bind.The invention further relates to the antibody fragment and the fusion rotein that contain the Fc district, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.Such antibody fragment and fusion rotein present the Fc receptor binding affinity of raising and/or the effector function of raising.
The invention further relates to pharmaceutical composition, comprise acceptable carrier on antibody of the present invention, the antibody fragment that keeps the Fc district and fusion rotein and the pharmacology, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
The invention further relates to the purposes of this pharmaceutical composition in treatment cancer method.Especially, the present invention relates to treat method for cancer, comprise the pharmaceutical composition of the present invention of drug treatment significant quantity.
The invention still further relates to host cell, comprise the expression vector of the nucleic acid molecule that contains the fusion polypeptide of encoding, wherein fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII activity) and contains the locating structure territory that golgi body stops polypeptide; With the expression vector that comprises the nucleic acid encoding molecule, wherein polypeptide has alpha-Mannosidase II (ManII) activity.In the preferred embodiment, the nucleic acid molecule and the coding of coding fusion polypeptide have the nucleic acid molecule of the active polypeptide of mannosidase II on the same expression vector or on the expression vector that separates.In another preferred embodiment, fusion polypeptide contains the catalyst structure domain of GnTIII.In the other preferred embodiment, golgi body locating structure territory is ManII, β (1,2)-and N-acetylglucosaminyltrVnsferase I, β (1,2)-N-acetylglucosaminyltrVnsferase II, mannosidase I or α 1, the locating structure territory of 6-N core fucosyltransferase.In the further preferred embodiment, host cell is selected from mammalian cell, yeast cell, insect cell and vegetable cell.Preferably, host cell is Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma.
The present invention further provides host cell, the expression vector that comprises the nucleic acid molecule that contains the fusion polypeptide of encoding, fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII activity) and contain the locating structure territory that golgi body stops polypeptide, the expression vector that contains the nucleic acid encoding molecule, wherein polypeptide has alpha-Mannosidase II (ManII) activity, with the expression vector that contains the nucleic acid encoding molecule, wherein polypeptide has β (1,2)-N-acetylglucosaminyltrVnsferase II (GnTII) activity.In the preferred embodiment, the nucleic acid molecule of coding fusion polypeptide, coding have the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that coding has the active polypeptide of GnTII on the same expression vector or on the expression vector that separates.The nucleic acid molecule of the fusion polypeptide of further preferably encoding is on an expression vector, and nucleic acid molecule and the coding with ManII active polypeptide of encoding has the nucleic acid molecule of the active polypeptide of GnTII on same expression vector.Further preferably, the nucleic acid molecule that coding has the active polypeptide of ManII is on an expression vector, and the nucleic acid molecule of coding fusion polypeptide and coding have the nucleic acid molecule of the active polypeptide of GnTII on same expression vector.In another embodiment, GnTII is on an expression vector, and the nucleic acid molecule of coding fusion polypeptide and nucleic acid molecule that coding has the active polypeptide of ManII are on same expression vector.
In addition on the one hand, the present invention relates to host cell, the expression vector that comprises the nucleic acid molecule that contains the fusion polypeptide of encoding, wherein fusion polypeptide has β (1,4)-galactosyltransferase (GalT) activity and comprises that golgi body stops the golgi body locating structure territory of polypeptide; With the expression vector that contains the nucleic acid encoding molecule, wherein polypeptide has mannosidase II (ManII) activity.In the preferred embodiment, nucleic acid molecule, the coding of coding fusion polypeptide have the nucleic acid molecule of the active polypeptide of ManII on the same expression vector or on the expression vector that is separating.Preferably, fusion polypeptide contains the catalyst structure domain of GalT.In the other embodiments, golgi body locating structure territory is the locating structure territory of ManII, β (1,2)-N-acetylglucosaminyltrVnsferase I, β (1,2)-N-acetylglucosaminyltrVnsferase II, mannosidase I or α 1-6 core fucosyltransferase.In the preferred embodiment, host cell is selected from mammalian cell, yeast cell, insect cell or vegetable cell.Preferably, host cell is Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma.
In addition on the one hand, the present invention relates to host cell, the expression vector that comprises the nucleic acid molecule that contains the fusion polypeptide of encoding, wherein fusion polypeptide has β (1,4)-galactosyltransferase (GalT) activity and comprises that golgi body stops the golgi body locating structure territory of polypeptide; With the expression vector that contains the nucleic acid encoding molecule, wherein polypeptide has mannosidase II (ManII) activity; And the expression vector that contains the nucleic acid encoding molecule, wherein polypeptide has β (1,2)-N-acetylglucosaminyltrVnsferase II (GnTII) activity.Preferably, each nucleic acid molecule is on same expression vector.In the embodiment separately, each nucleic acid molecule is on the carrier that separates.The nucleic acid molecule that the present invention further provides the coding fusion polypeptide is on an expression vector, and nucleic acid molecule and the coding with the active polypeptide of ManII of encoding has the nucleic acid molecule of the active polypeptide of GnTII on same expression vector.The present invention also provides the nucleic acid molecule of coding ManII on an expression vector, and the nucleic acid molecule of coding fusion polypeptide and coding have the nucleic acid molecule of the active polypeptide of GnTII on same expression vector.The present invention also provides nucleic acid molecule that coding has an active polypeptide of GnTII on an expression vector, and the nucleic acid molecule of coding fusion polypeptide and coding have the nucleic acid molecule of the active polypeptide of ManII on same expression vector.In the preferred embodiment, fusion polypeptide contains the catalyst structure domain of GalT.In the further preferred embodiment, golgi body locating structure territory is ManII, β (1,2)-and N-acetylglucosaminyltrVnsferase I, β (1,2)-N-acetylglucosaminyltrVnsferase II, mannosidase I or α 1, the locating structure territory of 6-N core fucosyltransferase.
The present invention further provides host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GnTIII and the nucleic acid that at least a coding has the active polypeptide of ManII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, wherein the polypeptide of host cell generation is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
The present invention also provides host cell, this host cell through engineering approaches expresses that at least a coding has the nucleic acid of the active fusion polypeptide of GnTIII, at least a coding has the nucleic acid of the active polypeptide of ManII and the nucleic acid that at least a coding has the active polypeptide of GnTII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, wherein the polypeptide of host cell generation is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
The present invention provides host cell in addition, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GalT and the nucleic acid that at least a coding has the active polypeptide of ManII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, wherein the polypeptide of host cell generation is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
In the embodiment separately, the present invention also provides host cell, this host cell through engineering approaches expresses that at least a coding has the nucleic acid of the active fusion polypeptide of GalT, at least a coding has the nucleic acid of the active polypeptide of ManII and the nucleic acid that at least a coding has the active polypeptide of GnTII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, wherein the polypeptide of host cell generation is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.Preferably, the polypeptide that produces by host cell as the result who modifies presents the Fc receptor binding affinity of raising.In the other preferred embodiment, present the effector function of raising as the polypeptide of the generation of host cell as a result of modifying.Preferably, the effector function of raising is following one or more: the Cytotoxic raising of Fc mediation and NK cell bonded improve and the scavenger cell bonded improves and the polymorphonuclear cell bonded improves and the monocyte bonded improves, enhancing, the raising of dendritic cell maturation and/or the raising of T cell sensitization of apoptosis-induced direct signal.
In the other embodiments, the present invention relates in host cell, produce the method for polypeptide, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GnTIII and the nucleic acid that at least a coding has the active polypeptide of ManII, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and wherein the amount expressed of fusion polypeptide is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces; And isolated polypeptide.Preferably, further the host cell through engineering approaches is expressed the nucleic acid that at least a coding has the active polypeptide of GnTII.In the other preferred embodiment, fusion polypeptide comprises the catalyst structure domain of GnTIII.In the further preferred embodiment, fusion polypeptide comprises that the allos golgi body stops the golgi body locating structure territory of polypeptide.Preferably, golgi body locating structure territory is the locating structure territory of mannosidase II, β (1,2)-N-acetylglucosaminyltrVnsferase I, mannosidase I, β (1,2)-N-acetylglucosaminyltrVnsferase II or α 1-6 core fucosyltransferase.Preferably, as the result of above-mentioned modification, polypeptide has the effector function of raising.
The invention further relates to the method for in host cell, producing polypeptide, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell is that through engineering approaches is expressed at least a coding and had the nucleic acid of the active fusion polypeptide of GalT and the nucleic acid that at least a coding has the active polypeptide of ManII, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and wherein the expression amount of fusion polypeptide is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces.In the other embodiments, further the host cell through engineering approaches is expressed the nucleic acid that at least a coding has the active polypeptide of GnTII.Preferably, fusion polypeptide comprises the catalyst structure domain of GalT.Further preferably fusion polypeptide comprises that further the allos golgi body stops the golgi body locating structure territory of polypeptide.Preferably, golgi body locating structure territory is the locating structure territory of mannosidase II, β (1,2)-N-acetylglucosaminyltrVnsferase I, mannosidase I, β (1,2)-N-acetylglucosaminyltrVnsferase II or α 1-6 core fucosyltransferase.Preferably, as the result of above-mentioned modification, polypeptide has the effector function of raising.Especially, in the preferred embodiment, the polypeptide that host cell produces has the bisection of ratio increase, the oligosaccharides of non-fucosylation in the Fc district.Preferably, the oligosaccharides of bisection, non-fucosylation is a heterozygosis.More preferably, the oligosaccharides of bisection, non-fucosylation is a compound.In the preferred embodiment, the oligosaccharides in the polypeptide Fc district at least about 10% to 95% be halve, non-fucosylation.Oligosaccharides in the preferred especially glycosylated polypeptides Fc of the present invention district is about the 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, the 95%th, bisection, non-fucosylation.
In the other preferred embodiment, the invention provides antibody according to the effector function with raising of the inventive method through engineering approaches.
In the other embodiments, the present invention further provides the pharmaceutical composition of the antibody that contains with good grounds the inventive method through engineering approaches.Preferably, pharmaceutical composition of the present invention comprises acceptable carrier on the pharmacology.
The present invention further provides the method for treatment malignant tumour, comprised that the pharmaceutical composition of the present invention with the treatment significant quantity delivers medicine to the patient of needs.
The invention further relates to the method for the Cytotoxic polypeptide of in host cell, producing Fc mediation with raising, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell is that through engineering approaches is expressed the nucleic acid of at least a coding GalT and the nucleic acid of at least a coding ManII, the polypeptide that is produced is selected from whole antibody molecule, the antibody fragment that comprises immunoglobulin fc region, wherein the expression amount of one or two among GalT or the ManII is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, and wherein has the cytotoxicity that the Fc of raising mediates as the polypeptide of modifying as a result; With the Cytotoxic polypeptide that separates Fc mediation with raising.In the other preferred embodiment, the expression level of GalT has produced the Cytotoxic antibody molecule of the Fc mediation with raising or has comprised the antibody fragment of immunoglobulin fc region.
The invention further relates to the method for the Cytotoxic polypeptide of in host cell, producing Fc mediation with raising, be included under the condition that allows polypeptide to produce and cultivate host cell, this host cell is that through engineering approaches is expressed the nucleic acid of at least a coding GalT and the nucleic acid of at least a coding ManII, the polypeptide that is produced is selected from whole antibody molecule, the antibody fragment that comprises immunoglobulin fc region, wherein the expression level of one or two among GalT or the ManII is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, and wherein has the cytotoxicity that the Fc of raising mediates as the polypeptide of modifying as a result; With the Cytotoxic polypeptide that separates Fc mediation with raising.In the preferred embodiment, above-mentioned host cell further comprises the nucleic acid of at least a coding GnTIII, wherein the expression amount of GnTIII is enough to modify the oligosaccharides in the polypeptide Fc district that host cell produces, and wherein has the cytotoxicity that the Fc of raising mediates as the polypeptide of modifying as a result.Preferably, the one or more expression level among GalT, ManII or the GnTIII is enough to form the bisection oligosaccharides in the polypeptide Fc district.More preferably, the bisection oligosaccharides in the Fc district to the ratio of total oligosaccharides in the Fc district at least about 25,35,45,55,60,65,70,75,80,85,90 or 95%.Preferably, the bisection oligosaccharides in the Fc district to the ratio of total oligosaccharides in the Fc district at least about 45%.In the preferred embodiment, the bisection oligosaccharides is compound or heterozygosis.Preferably, host cell is mammalian cell, yeast cell, insect cell or vegetable cell.More preferably, host cell is a vegetable cell.
On the other hand, the present invention relates in host cell, produce the method for polypeptide:
A. under the condition that allows the generation polypeptide, cultivate host cell, this host cell is that through engineering approaches is expressed at least a coding and had the active nucleic acid molecule of alpha-Mannosidase II, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, the oligosaccharides in the wherein said Fc district that has the active polypeptide expression amount of alpha-Mannosidase II and be enough to modify a described polypeptide that described host cell produces; With
B. separate the described polypeptide that described host cell produces.
The invention still further relates to polypeptide, especially antibody that result as described modification oligosaccharides has the Fc receptors bind of the effector function of raising and/or raising, and their purposes in treatment disease especially tumor treatment composition.
Need the evaluation and the generation of the proteic coding nucleic acid of modification of glycosylation patterns
The invention provides the method for production and the purposes of host system, be used for the antibody of production glycosylation form, the antibody fragment that contains the Fc district and fusion rotein, a zone of fusion rotein is equivalent to the Fc district of immunoglobulin (Ig), Fc receptor binding affinity with raising, preferred Fc activated receptor, and/or have the effector function of raising, comprise antibody dependent cellular cytotoxicity.The evaluation of target epi-position and have the production of antibodies that its glycosylation pattern of potential therapeutic value need be modified, and separately the separation of nucleic acid sequence encoding all within the scope of the invention.
Can use various methods known in the art to come the antibody of production purpose target epi-position.The fragment that such antibody includes, but are not limited to polyclone, mono-clonal, chimeric, humanization, total man, strand, Fab fragment and produces by ScFv, Fab, VH, IgG expression library.Such antibody can be used as diagnostic reagent or therapeutical agent.As therapeutical agent, preferred especially neutralizing antibody, promptly those and part, substrate and connector molecule are in conjunction with the antibody of competing mutually.
For antibody producing, come immune various host animals by injection purpose target protein, include, but are not limited to rabbit, mouse, rat etc.By repeatedly subcutaneous (sc) or intraperitoneal (ip) injection related antigen and adjuvant produce polyclonal antibody in animal.According to host species, can use various adjuvants to come enhancing immunity to reply, include, but are not limited to, freund's adjuvant (fully with incomplete), mineral rubber such as aluminium hydroxide, tensio-active agent such as lysolecithin, poly alcohol, polyanion, peptide, saponin(e, oily emulsion, keyhole limpet hemocyanin, dinitrophenol and the people's adjuvant that comes in handy such as BCG (bacille Calmette-Guerin vaccine) and corynebacterium parvum (Corynebacteriumparvum).By will be as 100g or 5g protein or conjugate (separately for rabbit and mouse) and 3 volume Freund's complete adjuvants, and at a plurality of positions with the solution intradermal injection with animal immune with antagonism antigen, immunoconjugates or derivative.After one month, strengthen animal by the peptide and the conjugate of 1/5 to 1/10 original vol in a plurality of position subcutaneous injection freund's adjuvants, after 7 to 14 days, with the antigen titration of animal unhairing and serum analysis.It is steady until titration to strengthen animal.Preferably, with same antigen but strengthen animal with different protein and/or by the conjugate that different linking agents is puted together.Can also in reconstitution cell culture such as albumen fusion, make conjugate.
Use prepares the monoclonal antibody of target by any technology of continuous cell line generation antibody molecule.These comprise, but are not restricted to, and hybridoma technology is described in Kohler and Milstein at first, Nature 256:495-97 (1975), human B cell hybridoma technology (Kosbor etc., Immunology Today 4:72 (1983); Cote etc., Proc.Natl.Acad.Sci.U.S.A.80:2026-30 (1983) and EBV-hybridoma technology (Cole etc., Monoclonal Antibodies and Cancer Therapy 77-96 (AlanR.Liss, Inc., 1985)); In addition, can use development to produce technology (Morrison etc., the Proc.Natl.Acad.Sci.U.S.A.81:6851-55 (1984) of " chimeric antibody "; Neuberger etc., Nature 312:604-08 (1984); Takeda etc., Nature 314:452-54 (1985), by splicing from the gene of the specific mouse antibodies molecule of suitable antigen with from the gene of suitable bioactive human antibody molecules.These technology can be used for producing chimeric antibody, also comprise other mammiferous antibody molecules.Perhaps, can use to describe and be used for the technology (United States Patent (USP) 4,946,778) of manufacture order chain antibody and produce and have desirable specific single-chain antibody.The invention further relates to according to the inventive method humanized antibody of glycosyl through engineering approaches.The method of producing the humanized antibodies for example is disclosed in, and the U.S. Patent No. 6,180,320 of Queen etc. is incorporated herein by reference with its integral body at this.
The antibody fragment that can contain purpose target protein specific binding site by known technology production.For example, such fragment comprises, but is not restricted to, the F (ab ') that produces by the pepsin digested antibody molecule 2The fragment and the F (ab ') that passes through to reduce 2The Fab fragment that segmental disulfide bridge bond produces.Perhaps, ((Huse etc., Science 246:1275-81 (1989)) identifies quickly and easily and has the desirable specific mono-clonal Fab fragment of purpose target protein can to make up the Fab expression library.
In case identify antibody or antibody fragment that glycosylation pattern has needs modification, use technical evaluation well known in the art and separate nucleic acid sequence encoding.
A. be used to produce the production of the proteinic clone of glycosylation pattern with change
The invention provides the proteinic host cell expression system that is used to produce glycosylation pattern with change.Especially, the invention provides the proteinic host cell systems that is used to produce glycosylation form with raising therapeutic value.Therefore, on the one hand, the invention provides the host cell expression system, its be through select or through engineering approaches express for example have β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) is active and comprise that the allos golgi body stops the fusion polypeptide in the locating structure territory of polypeptide.Especially, such host cell expression system can through engineering approaches contains the recombinant nucleic acid molecules of the such fusion polypeptide of coding, and operationally the promoter systems with composing type or adjusting is connected.
In one particular, the invention provides host cell, this host cell through engineering approaches is expressed at least a coding to have a β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) active and contain the nucleic acid of fusion polypeptide that the allos golgi body stops the golgi body locating structure territory of polypeptide.On the one hand, it is active and contain the nucleic acid molecule of gene of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide that the host cell through engineering approaches is comprised that at least a coding has a β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII).
Usually, the culturing cell of any kind system can be used as the background of through engineering approaches host cell of the present invention system.In the preferred embodiment, Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, other mammalian cells, yeast cell, insect cell or vegetable cell are as the background clone that produces through engineering approaches host cell of the present invention.(referring to Ma, J.K.-C., etc., Nature Genetics 4:794-805 (in October, 2003) and the reference of quoting thereof) (its full content is hereby incorporated by).
The present invention relates to comprise as defined in this and to express that to have β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) active and contain any through engineering approaches host cell of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide.
Can be under constitutive promoter or the control of adjustable expression system, expressing one and several codings has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII activity) and contains the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide.Suitable adjustable expression system comprises, but be not restricted to the promoter systems of the adjustable expression system of tsiklomitsin, the derivable expression system of moulting hormone, lac-switch expression system, the derivable expression system of glucocorticosteroid, thermoinducible promoter systems and metallothionein(MT) metal inducement.Has β (1 if comprise several different codings in the host cell systems, 4)-N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide, wherein some can be expressed under constitutive promoter control, and other are expressed under adjustable expression system control.Think that maximum expression level is to stablize cell growth rate that fusion polypeptide expresses not have the highest of remarkable side effect may level, uses routine test to measure.Measure expression level by the common known method in this area, comprise the Western engram analysis, use to antibody with the active polypeptid specificity of GnTIII or to have the peptide-labeled specific antibody that the active polypeptide of GnTIII merges, the Northern engram analysis, use coding is had the active polypeptide of GnTIII gene specific nucleic acid probe or to coding with have the probe of the peptide-labeled nucleic acid specificity that the active polypeptide of GnTIII merges, or measure the GnTIII activity.Perhaps, can use the biosynthetic products bonded lectin with GnTIII, for example, E 4-PHA lectin.Perhaps, can the functions of use test, it has measured the Fc receptors bind of the antibody-mediated raising that produces by cell and the effector function of raising, this cell engineeringization and have the nucleic acid of coding GnTIII active polypeptide.Further in the alternative, nucleic acid can be operably connected with reporter gene; It is active and contain the expression level of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide to measure (1, the 4)-N-acetylglucosaminyltrVnsferase III (GnTIII) that has β by the relevant signal of measurement report gene expression dose.Reporter gene can be transcribed as single mRNA molecule together with the nucleic acid of coding fusion polypeptide; By internal ribosome entry site (IRES) and the encoding sequence that is connected by cap independence translational enhancer (CITE) separately.Reporter gene can have β (1 with at least one coding, 4)-and N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide and translate together, make to form single peptide chain.The nucleic acid of code book invention fusion polypeptide can operationally be connected with reporter gene under the control of single promotor, makes the nucleic acid and the reporter gene of coding fusion polypeptide be transcribed into the RNA molecule, and its alternative splicing becomes two messenger RNA(mRNA)s that separate (mRNA) molecule; Translate into reporter protein for one among the resultant mRNA, another translates into fusion polypeptide.
Has β (1 if expressed several different codings, 4)-and N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the nucleic acid that the xenogenesis golgi body stops the golgi body locating structure territory fusion polypeptide of polypeptide, and they can arrange to make them to transcribe with several mRNA molecules with one like this.If they are transcribed as single mRNA molecule, by internal ribosome entry site (IRES) and the encoding sequence that is connected by cap independence translational enhancer (CITE) separately.They can be become the RNA molecule from single promoter transcription, its alternative splicing becomes several messenger RNA(mRNA) that separates (mRNA) molecules, then its each translate into the fusion polypeptide of each own coding.
In another embodiment, the invention provides host expression system, be used for production for treating antibody, have the Fc receptor binding affinity of raising, especially, comprise antibody dependent cellular cytotoxicity with combination of Fc activated receptor and the effector function that improves.Usually, the host cell expression system is through engineering approaches and/or select to express the nucleic acid that coding needs the antibody of mutagenic glycosylation form, to have a β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) active and contain the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide and express together with at least a coding.In one embodiment, come the transfection host cell system with the nucleic acid of the such fusion polypeptide of at least a coding.Usually, select cells transfected to identify the clone who expresses fusion polypeptide of the present invention with separating stable.
The culturing cell system of any kind can be used as the background of through engineering approaches host cell of the present invention system.In the preferred embodiment, can use Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, other mammalian cells, yeast cell, insect cell or vegetable cell.Usually, such clone through engineering approaches further comprises the nucleic acid of at least a transfection, the whole antibody molecule of this nucleic acid encoding, the antibody fragment that contains immunoglobulin fc region or fusion rotein, and fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.Usually such antibody producing cells system be derived from 20 to 120pg/ (cell. day) high specific output produces and the clone of secretory antibody.In the interchangeable embodiment, express the background clone of the hybridoma cell line of purpose specific antibodies as production through engineering approaches cell of the present invention.
In one embodiment, the nucleic acid clone of encoding antibody, antibody fragment or Fc fusion polypeptide is advanced in the antibody expression vector, transfection is advanced in the host cell then, and selects and the high and stable cell clone of screening specific antibody output.Then with glycoprotein-selected like this clone of modification glycosyltransferase expression vector transfection, this expression vector contains the following nucleic acid of coding, for example, (a) has β (1,4)-the active fusion polypeptide of N-acetylglucosaminyltrVnsferase III (GnTIII), or (b) has a β (1,4)-the active fusion polypeptide of galactosyltransferase (GalT), or (c) has an active polypeptide of golgi body alpha-Mannosidase II (ManII), or (d) have an active fusion polypeptide of GnTIII and further have the active polypeptide of ManII, or (e) has the active fusion polypeptide of GalT and further have the active polypeptide of ManII.Select then and screening and cloning, this is cloned in stably express antibody coding gene on the level that causes the antibodies specific high yield, modify the glycosylation transferase gene with stably express glycoprotein on the expression level that causes Fc district glycosylation pattern to be modified, described modification comprises the increase of non-fucosylation oligosaccharides part, oligosaccharides is bisection or non-binary, it further can be compound or heterozygous, it is relevant with the Fc receptors bind that increases, the particularly raising of Fc-Fc γ RIII binding affinity, with the raising of the receptor-mediated effector function of Fc, include but not limited to the Fc dependent cellular cytotoxicity.Below describe and select and screening method.
In another embodiment, putting upside down above-mentioned two transfections is the order that antibody expression vector transfection and glycoprotein are modified the carrier transfection of glycosylation transferase expression system, promptly at first modify glycosyltransferase expression vector transfection host cell, use the antibody expression vector transfection then with glycoprotein.In such method, can be by the clone of the following arbitrary method screening that further describes from the glycosyltransferase gene of the enough stably express levels of first transfection, perhaps be somebody's turn to do clone's replicon with the antibody expression vector transient transfection, the screening method that further describes below using then, so that identify the clone of the glycosyltransferase gene of stably express level, this expression level causes the modification of Fc district glycosylation pattern and causes improving the Fc acceptor comprising the binding affinity of Fc γ RIII acceptor and improving the receptor-mediated effector function of Fc, comprises the Fc dependent cellular cytotoxicity.
Further in the embodiment, transfection antibody coding gene and glycosyltransferase gene together in single transfection step are in single expression vector or in the carrier that is separating.
Usually, at least a nucleic acid encoding in the host cell systems has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) activity or β (1,4)-galactosyltransferasactivity activity and contain the fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide, perhaps, its coding has the active polypeptide of golgi body alpha-Mannosidase II.
Can under the control of constitutive promoter or adjustable expression system, express the nucleic acid of one or several code book invention fusion polypeptide.Suitable adjustable expression system comprises, but be not restricted to the expression system of the adjustable expression system of tsiklomitsin, the derivable expression system of moulting hormone, the adjustable expression system of lac-switch, the derivable expression system of glucocorticosteroid, temperature inducible promoter system and metallothionein(MT) metal inducement.Has β (1 if comprise several different codings in the host cell systems, 4)-N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide, wherein some can be expressed under constitutive promoter control, and other are expressed under adjustable expression system control.Think that maximum expression level is to stablize cell growth rate that fusion polypeptide expresses not have the highest of remarkable side effect may level, uses routine test to measure.Measure expression level by the common known method in this area, comprise the Western engram analysis, for example use to antibody with the active polypeptid specificity of GnTIII or to have the peptide-labeled specific antibody that the active polypeptide of GnTIII merges, the Northern engram analysis, for example use to coding have the active polypeptide of GnTIII gene specific nucleic acid probe or to coding with have the peptide-labeled nucleic acid specificity probe that the active polypeptide of GnTIII merges, or measure the GnTIII activity.Perhaps, can use the biosynthetic products bonded lectin with GnTIII, for example, E 4-PHA lectin.Perhaps, can the functions of use test, it has measured the Fc receptors bind of the antibody-mediated raising that produces by cell and the effector function of raising, this cell engineeringization and have the nucleic acid of coding GnTIII active polypeptide.Further in the alternative, nucleic acid can be operably connected with reporter gene; Measure the expression level of fusion polypeptide of the present invention by the relevant signal of measurement report gene expression dose.Reporter gene can be transcribed as single mRNA molecule together with the nucleic acid of coding described glycoprotein-modification glycosyltransferase; By internal ribosome entry site (IRES) and the encoding sequence that is connected by cap independence translational enhancer (CITE) separately.Reporter gene can have β (1 with at least one coding, 4)-and N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide and translate together, make to form single peptide chain.The nucleic acid of coding fusion polypeptide can operationally be connected with reporter gene under the control of single promotor, makes the nucleic acid of code book invention fusion polypeptide and reporter gene be transcribed into the RNA molecule, and its alternative splicing becomes two messenger RNA(mRNA)s that separate (mRNA) molecule; Translate into reporter protein for one among the resultant mRNA, another translates into fusion polypeptide.
Has β (1 if expressed several different codings, 4)-and N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the nucleic acid of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide, and they can be arranged like this and make them be transcribed into one and several mRNA molecules.If they are transcribed as single mRNA molecule, by internal ribosome entry site (IRES) and the encoding sequence that is connected by cap independence translational enhancer (CITE) separately.They can become the RNA molecule from single promoter transcription, and its alternative splicing becomes several messenger RNA(mRNA) that separates (mRNA) molecules, then its each translate into the fusion polypeptide of each own coding.
I. expression system
Can use this area to come construction of expression vector according to technician's known method, this expression vector contains the encoding sequence of target protein matter and has a β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) active and contain the xenogenesis golgi body and stop the encoding sequence of fusion polypeptide in golgi body locating structure territory of polypeptide and suitable transcribing/translate control signal.These methods comprise reorganization/gene recombination in extracorporeal recombinant DNA technology, synthetic technology and the body.Referring to, for example be described in Maniatis etc., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989) and Ausubel etc., Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, the technology among the N.Y (1989).
Can utilize various host-expression systems to express the encoding sequence of proteinic encoding sequence of the object of the invention and fusion polypeptide.Preferably, mammalian cell is as host cell systems, comes transfection with the recombinant plasmid dna or the cosmid DNA expression vector of the encoding sequence of encoding sequence that contains target protein matter and fusion polypeptide.More particularly, Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, other mammalian cell, yeast cell, insect cell or vegetable cell are as host cell systems.Some case descriptions of expression system and system of selection are in following reference, at this as a reference: Borth etc., Biotechnol.Bioen.71 (4): 266-73 (2000-2001), Werner etc., Arzneimittelforschung/Drug Res.48 (8): 870-80 (1998), Andersen and Krummen, Curr.Op.Biotechnol.13:117-123 (2002), Chadd and Chamow, Curr.Op.Biotechnol.12:188-194 (2001), and Giddings, Curr.Op.Biotechnol.12:450-454 (2001).In another embodiment, can use other eukaryotic host cell system, comprise yeast cell, with the recombinant yeast expression vector transfection of the encoding sequence that contains the encoding sequence of target protein matter and fusion polypeptide of the present invention; Insect cell system infects with the recombinant virus expression vector (for example, baculovirus) of the encoding sequence that contains the encoding sequence of target protein matter and fusion polypeptide of the present invention; The vegetable cell system is with recombinant virus expression vector (for example, cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV) TMV) infects or with recombinant plasmid expression vector (for example, Ti-plasmids) transfection, expression vector contains the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide of the present invention; Or zooblast system, with recombinant virus expression vector (for example, adenovirus, vaccinia virus) infect, expression system comprises that through engineering approaches contains (for example mouse cell lines) the stable amplification (CHO/dhfr) in double minute chromosome or the unstable expanded cells system of the encoding sequence of the sequence of coding target protein matter DNA of multiple copied and fusion polypeptide of the present invention.
For method of the present invention, stably express is more preferred than transient expression usually, also is easier to scale operation because stably express obtains how reproducible result usually,, produces glycosyl engineered antibody of the present invention in production-scale clone that is.The expression vector that contains the virus replication starting point except use, host cell can also with by suitable expression controlling elements (for example, promotor, enhanser, sequence, transcription terminator, polyadenylation site etc.) but the conversion of the coding nucleic acid of controlling separately and selective marker.After introducing foreign DNA, the through engineering approaches cell was grown 1-2 days in enrichment medium, be converted to the selection substratum then.But the selective marker in the recombinant plasmid has given the resistance that is used for selecting and has selected cell, and this cytotostatic ground forms locus (foci) with karyomit(e) and the growth that plasmid integration advances them, then clones and be extended to clone.
Can use the multiple choices system, include, but are not limited to, herpes simplex virus thymidine kinase ((Wigler etc., Cell 11:223 (1977)), xanthoglobulin-guanine phosphoribosyltransferase (Szybalska ﹠amp; Szybalski, Proc.Natl.Acad.Sci.USA 48:2026 (1962)), and adenine phosphoribosyl transferase (Lowy etc., Cell 22:817 (1980)) gene, it can be used for tk separately -, hgprt -Or aprt -In the cell.Also have, the metabolic antagonist resistance can be with following basis: the dhfr that elects, and it has given methotrexate resistance (Wigler etc., Natl.Acad.Sci.USA77:3567 (1989); O ' Hare etc., Proc.Natl.Acad.Sci.USA 78:1527 (1981)); Gpt, it gives mycophenolic acid resistance (Mulligan ﹠amp; Berg, Proc.Natl.Acad.Sci.USA 78:2072 (1981)); Neo, it has given aminoglycoside G-418 resistance (Colberre-Garapin etc., J.Mol.Biol.150:1 (1981)), and hygro, and it has given hygromycin resistance (Santerre etc., Gene 30:147 (1984).Recently, described other and selected gene, i.e. trpB, it makes cell utilize indoles to replace tryptophane; HisD, it makes cell utilize histidinol alternate sets propylhomoserin (Hartman ﹠amp; Mulligan, Proc.Natl.Acad.Sci.USA85:8047 (1988)); The glutamine synthase system; And ODC (ornithine decarboxylase), it has given the resistance (McConlogue:Current Communication in Molecular Biology, Cold Spring Harbor Laboratory edits (1987)) of ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO.
Ii. express and have the proteinic transfectant of glycosylation pattern of modification or the evaluation of transformant
Identify the host cell that contains encoding sequence and express the biologically active gene product by at least four ordinary methods; (a) DNA-DNA or DNA-RNA hybridization; (b) existence or the disappearance of " mark " gene function; (c) estimate transcriptional level, by in the host cell separately the expression of mRNA transcript measure; (d) detect gene product, measure by immunity test or its biological activity.
In first method, use and to contain and the probe of encoding sequence or its part or derivatives thereof homologous nucleotide sequence is hybridized the encoding sequence that detects the encoding sequence that inserts the target protein matter in the expression vector and fusion polypeptide of the present invention by DNA-DNA or DNA-RNA separately existence.
In second method, based on the existence of specific " mark " gene function or do not exist (for example, thymidine kinase activity, antibiotics resistance, methotrexate resistance, transform the formation of inclusion body in phenotype, the baculovirus etc.) to identify and select recombinant expression vector/host system.For example, if the encoding sequence of target protein matter and fusion polypeptide encoding sequence of the present invention insert in the marker gene sequence of carrier, identify by the disappearance of marker gene function and contain the recon of encoding sequence separately.Perhaps, marker gene can be placed with encoding sequence series connection, under the control that is used for controlling the identical or different promotor that encoding sequence expresses.Mark response is induced or the expression selected has shown the expression of the encoding sequence of the encoding sequence of target protein matter and fusion polypeptide.
In the 3rd method, estimate the transcriptional activity of the encoding sequence of the encoding sequence of target protein matter and fusion polypeptide of the present invention by cross experiment.For example, use and the encoding sequence of the encoding sequence of target protein matter and fusion polypeptide of the present invention or its specific part homologous probe to separate and analyze RNA by the Northern trace.Perhaps, can extract total nucleic acid and the test and the hybridization of probe like this of host cell.
In the 4th method, the expression of protein product that can the immunological evaluation target protein and have β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII) is active and contain the expression of encoding sequence of fusion polypeptide that the xenogenesis golgi body stops the golgi body locating structure territory of polypeptide, for example by Western trace, immunity test such as radioimmunoassay precipitation, enzyme linked immune assay etc.Yet the final test of the success of expression system comprises the detection of biologically active gene product.
B. have the protein of glycosylation pattern of change and the generation and the purposes of protein fragments
I. the effector function that has raising comprises the production of antibodies and the purposes of antibody dependent cellular cytotoxicity
In the preferred embodiment, the invention provides the antibody and the antibody fragment of the glycosylation form that Fc receptors bind with raising and/or effector function comprise antibody dependent cellular cytotoxicity.
Do not put together the clinical trial that monoclonal antibody (mAb) is used for the treatment of certain cancers and produced inspirer result recently.Dillman, Cancer Biother.﹠amp; Radiopharm.12:223-25 (1997); Deo etc., Immunology Today 18:127 (1997).Chimeric, do not put together that IgG1 has ratified to be used for rudimentary or folliculus B-cell non-Hodgkin lymphomas.Dillman, Cancer Biother.﹠amp; Radiopharm.12:223-25 (1997), and another does not put together mAb, the humanization IgG1 of target entity breast tumor has demonstrated the distant view result in the III clinical trial phase.Deo etc., Immunology Today18:127 (1997).The antigen of these two mAb is expressed and is mediated effective tumor destruction by the effector cell at external or internal antibody at their tumour cell camber separately.On the contrary, many other do not puted together mAb and had the effector function that trickle tumour-specific can not cause is enough to be effective to clinical application.Frost etc., Cancer 80:317-33 (1997); Surfus etc., J.Immunother.19:184-91 (1996).For among these more weak mAb some, tested the helper factor in treatment recently.Add cytokine and can stimulate antibody dependent cellular cytotoxicity (ADCC) by activity and the quantity that improves circulating lymphocyte.Frost etc., Cancer80:317-33 (1997); Surfus etc., J.Immunother.19:184-91 (1996).ADCC, the molten born of the same parents of antibody target cell attack, owing to the combination of leukocyte receptors and antibody constant region (Fc) is initiated.Deo etc., Immunology Today 18:127 (1997).
The active difference of ADCC of IgG1 is not puted together in raising but the complementary method is the Fc fragment of engineered antibody.Protein engineering research has shown that low hinge area of Fc γ R and IgG CH2 structural domain interacts.Lund etc., J.Immunol.157:4963-69 (1996).Yet Fc γ R is in conjunction with also needing to exist the covalently bound oligosaccharides with conservative Asn297 place, CH2 district.Lund etc., J.Immunol.157:4963-69 (1996); Wright and Morrison, TrendsBiotech.15:26-31 (1997) proposes oligosaccharides and polypeptide all directly provides interaction sites or need oligosaccharides to keep active CH2 polypeptide conformation.Therefore, the modification of oligosaccharide structure can develop into the method that improves the interaction affinity.
The IgG molecule carries two N-and connects oligosaccharides in its Fc district, in each heavy chain one.The same with any glycoprotein, antibody produces with the colony of glycosylation form, its shared phase homopolypeptide main chain but have the different oligosaccharides that are connected with glycosylation site.The oligosaccharides that is typically found in the serum IgG Fc district is Composite Double feeler type (bi-antennary type) ((Wormald etc., Biochemistry 36:130-38 (1997)), have the terminal sialic acid of low levels and the terminal galactose baseization and the core fucosylation of bisection N-n acetylglucosamine n (GlcNAc) and variable pitch.Some studies show that Fc γ R is arranged in the oligosaccharides core in conjunction with the minimum sugared structure of needs.Lund etc., J.Immunol.157:4963-69 (1996).
Being used for industry and academia produces and does not put together the clone that the mouse for the treatment of mAb or hamster originate and usually required oligosaccharide determinant is connected to the Fc site.Yet the IgG of these expression of cell lines lacks the bisection GlcNAc that finds in low amount serum IgG.Lifely etc., Glycobiology 318:813-22 (1995).On the contrary, the humanization IgG1 (CAMPATH-1H) that observes rat bone myeloma generation recently carries bisection GlcNAc in its some glycosylation forms.Lifely etc., Glycobiology 318:813-22 (1995).The antibody in rat cell source reach with standard cell lines system in the external ADCC activity of the similar maximum of CAMPATH-1H antibody that produces, but in low-down antibody concentration.
CAMPATH antigen exists on the lymphoma cell with high level usually, and this chimeric mAb has high ADCC activity under bisection GlcNAc disappearance.Lifely etc., Glycobiology318:813-22 (1995).N-connects in the glycosylation pathway differ, adds bisection GlcNAc by enzyme β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII).Schachter，Biochem.CellBiol.64：163-81(1986)。
Research has before used single antibody to produce Chinese hamster ovary celI system, before this clone through engineering approaches express the clone GnTIII gene enzyme (Umana, P. etc., Nature Biotechnol.17:176-180 (1999)) of different levels with outside regulative mode.This method has been set up the strict dependency between GnTIII expression and the modified antibodies ADCC activity for the first time.
Further the present invention has the antibody of the effector function of the Fc receptor binding affinity of raising and raising, include, but are not limited to, anti-people's neuroblastoma monoclonal antibody (chCE7) by the inventive method generation, inosculating antibody human kidney cells carcinoma monoclonal antibody (chG250) by the inventive method generation, the Humanized anti-HER 2 monoclonal antibody that produces by the inventive method (for example, Trastuzumab (HERCEPTIN), inosculating antibody people colon by the inventive method generation, lung and breast cancer monoclonal antibody (ING-1), Humanized anti-human 17-1A antigen monoclonal antibody (3622W94) by the inventive method generation, the colorectal tumour antibody of Humanized anti-human (A33) by the inventive method generation, the anti-human melanoma antibody (R24) of the facedown GD3 Sphingolipids,sialo that produce by the inventive method, with the inosculating antibody people squamous cell carcinoma monoclonal antibody (SF-25) that produces by the inventive method, monoclonal antibody resisting human small cell lung carcinoma (BEC2 by the inventive method generation, Imclone Systems, Merck KgaA), anti-people Fei Huojinqi lymphomas monoclonal antibody (Bexxar (tositumomab by the inventive method generation, Coulter Pharmaceuticals), Oncolym (Techniclone, Alpha Therapeutic)), the anti-people's squamous cell head and the neck carcinoma monoclonal antibody (C225 that make by the inventive method, ImClone Systems), the anti-people's rectum and the colorectal carcinoma monoclonal antibody (Panorex (edrecolomab) that make by the inventive method, Centocor, Glaxo Wellcome), human ovary carcinoma resisting monoclonal antibody (Theragyn by the inventive method generation, Antisoma), anti-people's acute myelogenous leukemia carcinoma monoclonal antibody (SmartM195 by the inventive method generation, Protein Design Labs, Kanebo), anti-people's glioblastoma monoclonal antibody (Cotara by the inventive method generation, Techniclone, Cambridge Antibody Technology), anti-human B cell Fei Huojinqi lymphomas monoclonal antibody (IDEC-Y2B8 by the inventive method generation, IDECPharmaceuticals), monoclonal antibody (the CEA-Cide of the anti-people's noumenal tumour that produces by the inventive method, Immunomedics), anti-human colorectal cancer monoclonal antibody (iodine 131-MN-14 by the inventive method generation, Immunomedics), anti-people's ovary by the inventive method generation, kidney, mammary gland and prostate cancer monoclonal antibody (MDX-210, Medarex, Novartis), anti-people's colorectum and carcinoma of the pancreas monoclonal antibody (TTMA, Pharmacie ﹠amp by the inventive method generation; Upjohn), the anti-people TAG-72 that produces by the inventive method expresses carcinoma monoclonal antibody (MDX-220, Medarex), the anti-people EGFr-that produces by the inventive method expresses carcinoma monoclonal antibody (MDX-447), anti-VEGF monoclonal antibody (Genentech) by the inventive method generation, anti-people's mammary gland by the inventive method generation, lung, prostate gland and carcinoma of the pancreas and malignant melanoma monoclonal antibody (BrevaRex, AltaRex), the anti-people's acute myelogenous leukemia monoclonal antibody that produces by the inventive method (MonoclonalAntibody Conjugate, Immunex).In addition, the present invention relates to antibody fragment and fusion rotein, fusion rotein contains the zone that is equivalent to immunoglobulin fc region.
Ii. promote the generation and the purposes of the toxic fusion rotein of Fc mediated cell, fusion rotein contains the zone that is equivalent to immunoglobulin fc region
As mentioned above, the present invention relates to improve the Fc receptor binding affinity of treatment antibody and/or the method for effector function.This glycosylation pattern by the Fc district of the such antibody of through engineering approaches is realized, especially producing cell by engineered antibody produces and for example has GnTIII activity or GalT activity or the active polypeptide of ManII, the oligosaccharides that this is peptide modified and such antibody Fc district is connected.This strategy can be used for improving by having any numerator mediated cytotoxicity of being not only by the Fc mediation for the treatment of the antibody-mediated undesirable cell of antagonism in the zone that is equivalent to immunoglobulin fc region, therefore and the lip-deep Fc acceptor interaction of effector cell that relates in the ADCC mechanism because the change of introducing by the glycosylation engineering has only influenced the Fc district.Can use the molecule that contains Fc of method disclosed by the invention, include, but are not limited to, (a) soluble fusion protein (Chamov and the Ashkenazi that makes by target protein structural domain with the fusion of Fc district N-end, Trends Biotech.14:52 (1996)), (b) fusion rotein (Stabila of the protoplast membrane grappling that makes by the II type membrane spaning domain of the plasmalemma that is positioned to merge with Fc fragment N-end, P.F., Nature Biotech.16:1357 (1998)).
In the situation of soluble fusion protein (a), target structural domain guiding fusion rotein and do not expect cell such as the cancer cells combination, that is, and with treat mode like the antibody class.Therefore, these numerator mediated effector functions of raising disclosed by the invention comprise that the application of method of cellular cytoxicity activity of Fc mediation is identical to the method for the treatment of antibody with utilization.
In the situation of film grappling fusion rotein (b), the cell of not expecting in the body must be expressed the gene of encoding fusion protein.This can obtain by gene therapy method, does not promptly extremely expect cell with transfectional cell in plasmid that instructs the fusion rotein encoding gene to express or the virus vector body, or by the Transplanted cells of genetically engineered expressed fusion protein is in its surface gone in the body.Latter cell is implanted into (encapsulated cell therapy) in the body usually in polymer capsule, wherein they are not subjected to the destruction of Fc mediated cell toxicity mechanism.Yet capsule apparatus cell broken and that escape becomes undesirable, can eliminate by Fc mediated cell toxicity.Stabila etc., NatureBiotech.16:1357 (1998).In this case, can use method disclosed by the invention, by will guiding fusion polypeptide of the present invention other expression casette suitable or maximum expression level to incorporate in the gene therapy vector, or through engineering approaches waits to implant the cell of the fusion polypeptide of the present invention of expressing suitable or maximum expression amount.
The treatment of antibody, antibody fragment and the fusion polypeptide that produces according to the inventive method is used
Antibody of the present invention (be antibody, antibody fragment and fusion rotein, it contains the zone that is equivalent to immunoglobulin fc region) may be used solely to target and kills intravital tumour cell.Antibody can also make in conjunction with suitable therapeutical agent and be used for treating human cancer.For example, antibody can be in conjunction with conventional treatments such as chemotherapy, radiotherapy is used together or put together or be connected with medicine or toxin and lymphokine or tumor suppression somatomedin therapeutical agent is sent to the cancer position.
The technology that such therapeutical agent and antibody are puted together be known [referring to, Arnon etc. for example, " Monoclonal Antibodies for Immunotargeting of Drugs inCancer Therapy ", Monoclonal Antibodies and Cancer Therapy, Reisfeld etc. (editor), pp.243-56 (Alan R.Liss, Inc.1985); Hellstrom etc., " Antibodies For Drug Delivery ", Controlled DrugDelivery (the 2nd edition), Robinson etc. (editor), pp.623-53 (MarcelDekker, Inc.1987); Thorpe, " Antibody Carriers Of CytotoxicAgents In Cancer Therapy:A Review ", Monoclonal Antibodies ' 84:Biological And Clinical Applications, Pinchera etc. (editor), pp.475-506 (1985); With Thorpe etc., " The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates ", Immunol.Rev., 62:119-58 (1982)].
Perhaps, Glyco-engineered antibodies can with high-energy radiation for example isotropic substance as＜131 the I combination, when it is positioned tumor locus, cause the killing of several cell dias [referring to, for example, Order, " Analysis; Results; and Future Prospective of The TherapeuticUse of Radiolabeled Antibody in Cancer Therapy ", MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin etc. (editor), pp.303-16 (Academic Press 1985)].Still according to another embodiment, antibody of the present invention can be puted together with second antibody and forms antibody xenogenesis conjugate and treat tumour cell,, describes in 980 in U.S. Patent No. 4,676 as Segal.
Still for other treatment of antibody of the present invention use comprise for example put together by recombinant DNA technology or be connected to prodrug can be changed into the enzyme of cytotoxic drug and use antibody-enzyme conjugate in conjunction with prodrug with prodrug tumor locus change into cytotoxic agent [referring to, for example, Senter etc., " Anti-Tumor Effects of Antibody-alkalinePhosphatase ", Proc.Natl.Acad.Sci.USA 85:4842-46 (1988); " Enhancement of the in vitro and in vivo Antitumor Activitesof Phosphorylated Mitocycin C and Etoposide Derivatives byMonoclonal.Antibody-Alkaline Phosphatase Conjugates ", CancerResearch 49:5789-5792 (1989); And Senter, " Activation ofProdrugs by Antibody-Enzyme Conjugates:A New Approach toCancer Therapy, " FASEB is (1990) J.4:188-193].
Still another treatment of antibody of the present invention is used, and relates in the presence of complement or partial antibody-medicine or antibody-toxin conjugate, is used for removing tumour cell from cancer patients's marrow.According to this method, by with antibody treatment can external cleaning autologous bone marrow and with marrow inculcate back the patient [referring to, for example, Ramsay etc., " Bone Marrow Purging UsingMonoclonal Antibodies ", J.Clin.Immunol., 8 (2): 81-88 (1988)].
In addition, the present invention's chimeric antibody, recombinant immunotoxin and other recombinant precursor of containing the specific antigens calmodulin binding domain CaM of required monoclonal antibody can be used for the treatment of.For example, monochain immunotoxin of the present invention can be used for the interior therapeutic human cancer.
Similarly and the another kind of albumen with anti-tumor activity for example the part of the functionally active at least bonded of lymphokine or oncostatin contain the fusion rotein of the antigen binding domain of antibody of the present invention at least, can be used for the interior therapeutic human cancer.In addition, recombinant technology known in the art can be used for making up bi-specific antibody, and wherein antibody binding specificity relates to tumor associated antigen, and another binding specificity of antibody molecule except described tumor associated antigen.
The invention provides the method for optionally killing tumour cell, this tumor cells expression and monoclonal antibody of the present invention or function equivalent specificity bonded antigen.This method comprises glycosyl engineered antibody of the present invention or the immunoconjugates (for example immunotoxin) that contains it is contacted with described tumour cell.These tumour cells may form human cancer.
In addition, the invention provides the method for interior therapeutic cancer (for example human cancer).This method comprises that the composition with the treatment significant quantity delivers medicine to the patient, and composition comprises at least a glycosyl engineered antibody of the present invention or contains its immunoconjugates (for example, immunotoxin).
Again on the one hand, the present invention relates to based on the modification method of B cell depleting (depletion) treatment by the Immunological diseases of all or part of generation of autoantibody of causing a disease, comprise will the treatment significant quantity immunoreactivity antibody deliver medicine to the patient of needs, improvement comprises the antibody with raising ADCC that makes according to the inventive method of drug treatment significant quantity.In the preferred embodiment, antibody is anti-CD20 antibodies.Autoimmune disorders or imbalance include, but are not limited to, immune-mediated thrombocytopenia, as acute spy's property sent out thrombopenia purpura and chronic idiopathic thrombopenia purpura, dermatomyositis, tarantism, lupus nephritis, rheumatic fever, polyadenous syndromes, the Henoch-Schonlein purpura, latter stage streptoccal nephritis, erythema nodosum, Takayasu ' s arteritis, addison's disease, erythema multiforme, polyarteritis nodosa, stiff spondylitis, Goodpasture, thromboangiitis ubiteran, primary bile liver cirrhosis, Hashimoto ' s thyroiditis, thyrotoxicosis, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris, wegner's granulomatosis, film ephrosis, amyotrophic lateral sclerosis, myelophthisis, polymyalgia, pernicious anemia, carrying out property glomerulonephritis and fibrous tissue form pulmonary alveolitis fast, Inflammatory response such as dermatitis comprise psoriasis and dermatitis (for example, atopic dermatitis); Whole body scleroderma and sclerosis; The reaction relevant (for example, Crohn disease and ulcerative colitis) with enteritis; Respiratory distress syndrome (comprises adult respiratory distress syndrome; ARDS); Dermatitis; Meningitis; Encephalitis; Uveitis; Colitis; Glomerulonephritis; Allergic conditions, for example eczema and asthma and other illness comprise T cellular infiltration and chronic inflammatory reaction; Atherosclerosis; The white corpuscle adhesion is not enough; Rheumatoid arthritis; Whole body lupus erythematosus (SLE); Diabetes (for example, type i diabetes or insulin-dependent diabetes); The multiple sclerosis disease; Reynaud ' s syndromes, the autoimmunization thyroiditis; Allergic encephalitis; Sjorgen ' s syndromes; The diabetes of juvenile beginning; With usually in pulmonary tuberculosis, sarcoidosis, polymyositis, granulomatosis and nodular vasculitis, find by cytokine and the acute of the T-cell mediated immunne response relevant with the retardance allergy; Pernicious amenorrhoea (addison's disease); Relate to the disease that white corpuscle oozes out, central nervous system (CNS) inflammation; Multiple organ injury's syndromes; Hemolytic anemia (including, but are not limited to the positive anaemia of cryoglobulinemia or Ku Musishi); Myasthenia gravis; The disease of antigen-antibody complexes mediation; Anti-angiogenic bead sarolemma disease; Anti-phosphatide syndromes; Allergic neuritis; Graves' disease; The Lambert-Eaton myasthenic syndrome; The pemphigoid bleb; It bleb; The autoimmunization polyendocrinopathy; Reiter ' s disease; Tetanic people's syndromes; The Behcet disease; Giant cell arteritis; Immunocomplex ephritis; IgA nephropathy; The IgM polyneuropathy; Immunity thrombopenia purpura (ITP) or autoimmunization thrombocytopenia etc.In this respect of the present invention, the time period of the normal B cell in the antibody consumption blood of the present invention to prolong.
According to enforcement of the present invention, the patient can be people, horse, pig, ox, mouse, dog, cat and bird patient.Other warm-blooded animal also is included among the present invention.
The present invention also provides treatment cancer patients's method.The patient can be people, dog, cat, mouse, rat, rabbit, horse, goat, sheep, ox, chicken.Cancer can be accredited as mammary gland, bladder, retinoblastoma, papillarycystadenocarcinomaofovary, Wilm ' s tumour or small cell lung cancer, is characterized by the cell mass with antigen related neoplasms on the cell surface usually.This method comprise with the cancer amount of killing with cytotoxic agent bonded cancer target antibody administration in the patient.Usually, under the condition that allows the target on the bonded antibodies cell surface, form the combination of cancer target antibody and cytotoxic agent.By combining target, cancer target antibody directly or indirectly causes or causes in conjunction with the killing of cell, and therefore treats the patient.
Also provide the inhibition mammalian tumor cell outgrowth method, it comprises the glycosyl engineered antibody of the present invention of mammalian tumor cell and enough concentration or the immunoconjugates that contains it contact, makes the hyperplasia of inhibition mammalian tumor cell.
The present invention further provides the method that suppresses growth of human tumor cells, treatment patient tumors and treatment patient hyperplasia type disease.These methods comprise that the present composition with significant quantity delivers medicine to the patient.
Therefore very clear pharmaceutical composition, mixture and the method that present invention includes the treatment human cancer.For example, the present invention includes the pharmaceutical composition that is used for the treatment of human cancer, composition comprises acceptable carrier on the antibody of the present invention of medicine effective quantity and the pharmacology.
Composition can contain antibody or antibody fragment, and is unmodified and that therapeutical agent (for example, medicine, toxin, enzyme or second antibody) is puted together or with recombinant forms (for example, the fragment of chimeric antibody, chimeric antibody, bi-specific antibody).Composition can comprise other antibody or the conjugate (for example, mixtures of antibodies) for the treatment of cancer in addition.
Can use conventional administering mode to come administration antibody of the present invention, antibody conjugates and immunotoxin composition, include, but are not limited in intravenously, intraperitoneal, oral, the lymph or directly administration advance in the tumour.Preferred intravenous administration.
The present composition can be multiple formulation, includes, but are not limited to the solution that liquor or suspension, tablet, pill, pulvis, suppository, polymerization microcapsule or microvesicle, liposome and injectable maybe can inject.Preferred form depends on administering mode and treatment application.
Composition of the present invention also preferably includes conventional medicine known in the art can accept carrier and adjuvant such as human serum albumin, ion-exchanger, aluminum oxide, Yelkin TTS, buffer substance such as phosphoric acid salt, glycine, Sorbic Acid, potassium sorbate and salt or ionogen such as Protamine sulfates.
Severity that the most effective administering mode of the present composition and dosage regimen depend on disease and process, patient healthy and to the response of treatment and treatment doctor's judgement.Therefore, the composition dosage of titration single patient.However, the significant quantity of the present composition is about 1 to about 2000mg/kg.
Molecule described here can be multiple formulation, includes, but are not limited to the solution that liquor or suspension, tablet, pill, pulvis, suppository, polymerization microcapsule or microvesicle, liposome and injectable maybe can inject.Preferred form depends on administering mode and treatment application.
The position that the most effective administering mode of molecule of the present invention and dosage regimen depend on tumour to be treated, the severity of cancer and process, patient healthy and to the response of treatment and treatment doctor's judgement.Therefore, composition dosage that should the titration single patient.
Be described in Freireich, Cancer Chemother. such as E.J., Rep.50 (4): 219-244 (1966) based on all size of surface-area mg/kg and the animal of species and people's dosage mutual relationship.The adjustment that can carry out dosage regimen comes the inhibition of optimizing growth of tumour cell and kills response, for example, can with day the basis with dosage separately and administration or according to circumstances part reduce dosage (for example, several dosage that separate of administration every day or according to the minimizing of particular treatment case part).
Should be understood that obtaining to treat the present composition dosage that needs can further reduce along with the schedule optimizing.
According to enforcement of the present invention, pharmaceutical carrier can be a lipid carrier.Lipid carrier can be a phosphatide.In addition, lipid carrier can be a lipid acid.Also have, lipid carrier can be a stain remover.As used in this, stain remover is to change any material that surface tension of liquid normally reduces surface tension of liquid.
In one embodiment of the invention, stain remover is the nonionic stain remover.The example of nonionic detergent includes, but are not limited to, and polysorbate 80 (is also referred to as tween 80 or (polyoxyethylene sorbitan monooleate), Brij and Triton (for example Triton WR-1339 and TritonA-20).
Perhaps, stain remover can be the ion stain remover.The example of ion stain remover includes, but are not limited to, alkyl trimethyl ammonium bromide.
In addition, according to the present invention, lipid carrier can be a liposome.As used in this application, " liposome " is any film in conjunction with carrier, and it contains any molecule of the present invention or its composition.
Following examples will be explained in more detail the present invention.Following preparation of giving and embodiment can make those skilled in the art more be expressly understood and implement the present invention.Yet the present invention is not restricted to the scope of exemplary embodiment, and it only is the explanation of the single aspect of the present invention, and the method for function equivalence is also within the scope of the invention.In fact, except described here, according to description and accompanying drawing before, those become apparent those skilled in the art for various changes of the present invention.Such change determines to fall into the scope of claims.
Embodiment
Embodiment 1
Material and method
1. the structure of antibody expression vector
Anti-CD20 antibodies expression vector pETR1502
C2B8 anti-CD20 antibodies expression vector pETR1502 forms (one is used for the C2B8 light chain of antibody, and one is used for the C2B8 heavy chain of antibody, and one is used for neomycin resistance gene and one and is used for mouse dhfr gene) by four expression cassettes that independently, separate.All genes are under the control of myeloproliferative sarcoma virus (MPSV) promotor and contain the synthetic total polyadenylation signal of the polyadenylation signal that is derived from the rabbit beta globin gene.
In single stage method, use PCR from the variable heavy chain (VH) of a series of eclipsed single stranded oligonucleotide assembling coding anti-CD20 antibodies C2B8 and the CDNA (Kobayashi, N. etc., Biotechniques 23:500-503 (1997)) of variable light chain (VL).From disclosed international patent application, obtain coding C2B8 VL and the segmental original series (international publication number: WO94/11026) of VH.The VL and the VH cDNA fragment subclone of assembling are advanced to produce pBlue-C2B8VH and pBlue-C2B8VL plasmid among the pBluescriptIIKS (+), and order-checking.
From the variable chains of corresponding pBlue-C2B8VH and pBlue-C2B8VL plasmid amplification C2B8, use 5 ' and hold the primer (BsiWI is used for light chain and NheI is used for heavy chain) of introducing the AscI restriction site and introducing suitable restriction site in variable and constant region junction.(Quickclone, Clontech) amplification IgGI constant region is used the primer (BsiWI and BamHI are used for constant light chain and NheI and BamHI and are used for constant heavy chain) of introducing suitable restriction site at 5 ' and 3 ' end from human lymphocyte cDNA library.
After confirming correct dna sequence dna, with the light chain of C2B8 antibody and heavy chain separately with MPSV promotor and polyadenylation signal combination.In the first step, make up two different expression vectors: one is used for C2B8 light chain (pETR1315), and another is used for C2B8 heavy chain (pETR1316).In second step, neomycin resistance expression cassette (be derived from the neomycin resistance gene of Tn5 transposon and be positioned under the control of minimum MPSV promotor) introduced forming plasmid pETR1481 among the carrier pETR1315.Dhfr expression casette under the control of MPSV promotor is inserted acquisition plasmid pETR1328 among the carrier pETR1316.In the final step, (C2B8 light chain+neo and C2B8 heavy chain+dhfr) are incorporated in and form plasmid pETR1502 in the carrier to express modules with two.
Anti-CD20 antibodies expression vector pETR1520
PETR1520 has made up C2B8 anti-CD20 antibodies expression vector and replication orgin, and replication orgin is from Epstein Barr virus (oriP), is used for episome carrier duplicating and keep in the cell that produces EpsteinBarr virus nuclear antigen (EBNA).In order to make up C2B8 expression vector pETR1520, will express module from the C2B8 of pETR1416 and contain among the carrier pETR1507 of oriP with the insertion of HinDIII fragment.Carrier pETR1416 is similar with pETR1502, except the neomycin resistance gene of this plasmid under the control of whole MPSV promotors, rather than minimum MPSV promotor.
Anti-fibronectin antibody expression vector pETR1546
This carrier is identical with carrier pETR1520, except the variable heavy chain of coding C2B8 anti-CD20 antibodies and light chain the encode fragment of people's antibody each free antibody L19, the identification the ED-B domain of fibronectin substitute.By the dna fragmentation of overlapping extension PCR method composite coding variable region, use synthetic oligonucleotide (Pini, A. etc., J.Biol.Chem.273 (34): 21769-76 (1998)) based on L19 antibody variable region sequence.
Anti-egfr antibodies (C225) expression vector pURSI28
This carrier is identical with carrier pETR1520, except the variable heavy chain and the light chain of coding C2B8 anti-CD20 antibodies substituted by the encode fragment separately of chimeric antibody antibody C225, the identification Human epidermal growth factor receptor.Dna fragmentation by overlapping extension PCR method composite coding variable region, (sequence is found in the disclosed patent application based on the synthetic oligonucleotide of C225 antibody variable region sequence in use, international publication number WO96/40210, Figure 16 of this patent application and 17 are separately corresponding to heavy chain and light chain).
2.GnTIII the structure of fusion expression vector
PETR1166. the carrier that is used for the GnTIII constitutive expression
In order to make up GnTIII expression vector pETR1166, pass through pcr amplification rat GnTIII from rat kidney cDNA library (Clontech).In order to detect GnTIII easily by western blotting subsequently, hold c-myc-epi-position mark to add the and then upstream (aminoacid sequence: PEQKLISEEDL) of gene terminator codon C.After confirming the correct sequence of GnTIII, be inserted under the control of MPSV promotor gene and the polyadenylation signal of the synthetic rabbit beta globin gene of adding.Final GnTIII expression vector also contains the tetracycline resistance box that separates that is useful on selection, and puromycin resistance gene is also under the control of MPSV promotor and the polyadenylation signal of synthetic rabbit beta globin gene.
PETR1425: 102 amino acid replacements of people GnTI 76 ammonia of GnTIII Base acid
Structure by ensuing overlapping this heterozygosis glycosyltransferase gene of PCR reaction carrying out.In the reaction, use the main region of primer GAB-179 and GAB-180 amplification people GnTI.In this PCR reaction, Kozak consensus sequence and AscI restriction site have also been introduced at 5 ' end.229 beginnings and GnTIII have that 23bp's is overlapping to resulting PCR fragment from the position.In second PCR reaction, the GnTIII district with primer GAB-177 and GAB-178 amplification position 229 to 380 has produced the PCR fragment that has unique BstXI site at 3 ' end, holds and the GnTI main region has that 22bp's is overlapping 5 '.Two PCR fragment purifications also are used as the template (primer GAB-179 and GAB-178) of the 3rd PCR reaction.The resulting fragment of purifying and with AscI digestion, and connection carrier pETR1001 (cutting with AscI and SmaI) obtains plasmid pETR1404.After confirming insertion sequence, the MPSV promoter sequence is added as producing plasmid pETR1423 among AscI (part digestion)/segmental pETR1404 of PmeI.SphI/BstXI fragment from pETR1166, have original rat GnTIII expression carrier, substitute with the corresponding fragment of pETR1423 then and obtain plasmid pETR1425, contain the tetracycline resistance box that the GnTI-GnTIII under the control of MPSV promotor merges and is used to select.
Primer sequence:
GAB-177：GCGTGTGCCTGTGACCCCCGCGCCCCTGCTCCAGCCACTGTCCCC
GAB-178：GAAGGTTTCTCCAGCATCCTGGTACC
GAB-179：CTGAGGCGCGCCGCCACCATGCTGAAGAAGCAGTCTGCAGGGC
GAB-180：
GGGGACAGTGGCTGGAGCAGGGGCGCGGGGGTCACAGGCACACGCGGC
PETR1506: 100 amino acid replacements of people's mannosidase II GnTIII 76 n terminal amino acids
Carry out the structure of pETR1506, the structure of similar pETR1425.Use the main region of carrier pBlueman with primer GAB-252 and GAB-253 as template amplification people mannosidase II gene.In this PCR process, introduce FseI site and Kozak consensus sequence at 5 ' end.Resulting PCR fragment is 229 beginning and GnTIII gene overlap 23bp in the position.In second PCR reaction, with primer GAB-254 and GAB-255 amplification part GnTIII gene (position 229-460).This PCR has produced to contain with mannosidase II at 5 ' end has 43bp overlapping and contain the fragment in unique StuI site at 3 ' end.Two fragments of purifying are also as the template among the PCR of the 3rd use primer GAB-252 and GAB-255.Resultant fragment is inserted pIC19H obtain carrier pETR1484.After confirming the correct sequence of this insertion, make up complete fusion gene by the FseI/StuI fragment of connection pETR1484 and the StuI/BamHI fragment of pETR1166 in carrier pETR12177 (FseI/BamHI).Resulting plasmid (pETR1500) contains the heterozygosis manII-GnTIII gene (SEQ ID NO:14) under the control of MPSV promotor.In order in mammalian cell, to select plasmid, insert tetracycline resistance box with the StuI fragment of pETR1166, produce plasmid pETR1506.
Primer sequence:
GAB-252：
GCTAGGCCGGCCGCCACCATGAAGTTAAGCCGCCAGTTCACCGTGTTCGG
GAB-253：
GGGGACAGTGGCTGGAGCAGGGGTGAGCCAGCACCTTGGCTGAAATTGCTTTGTG
AACTTTTCGG
GAB-254：
TCCGAAAAGTTCACAAAGCAATTTCAGCCAAGGTGCTGGCTCACCCCTGCT
CCAGCCACTGTCCCC
GAB-255：ATGCCGCATAGGCCTCCGAGCAGGACCCC
PETR1519: heterozygosis manII-GnTIII fusion gene and from Epstein Barr disease The combination of the replication orgin oriP of poison
Use primer GAB-261 and GAB-262, contain the 2kb fragment of oriP from plasmid pCEP4 (Invitrogen) amplification.In this PCR reaction process, SspI and EcoRI site have been introduced at segmental two ends.In order to check order, the oriP fragment is inserted among the carrier pIC19H.After the verified correct sequence, oriP is inserted (with BsmBI digestion and the flat 5 ' overlapping ends of use Klenow polysaccharase benefit) among the carrier pETR1001 with the SspI fragment.Resulting plasmid called after pETR1507.To insert from the SphI/NruI manII-GnTIII expression cassette of pETR1510 among the pETR1507 with the enzymic digestion of same restrictions endonuclease and obtain plasmid pETR1519.
Primer sequence:
GAB-261：GCTAAATATTGAATTCCCTTTATGTGTAACTCTTGGCTGAAGC
GAB-262：TAGCAATATTGAATTCGCAGGAAAAGGACAAGCAGCGAAAATT
CACGC
PETR1537: the cd4 cell surface mark of heterozygosis manII-GnTIII fusion gene and brachymemma The combination of note gene
Modify the cd4 cell surface markers gene that the pETR1506 expression vector is expressed brachymemma in addition.Speak briefly, heterozygosis manII-GnTIII fusion gene expression cassette is changed into the bicistronic mRNA expression cassette from monocistron, and the cDNA of the human CD 4 protein matter by brachymemma that poliovirus IRES sequence is then encoded inserts the downstream (contain then stride film and ectodomain be used for excretory people CD4 leader sequence) of the terminator that manII-GnTIII merges.
3. with GnTIII fusion expression vector and antibody expression vector transfection mammalian cell
The transfection of bhk cell
Will be before electroporation 24 hours by the cell (vitality 90-95%) of index law growth with 0.9 * 10 6The concentration of individual cell/ml is inoculated in the T75 culturing bottle of suitable quantity.As substratum, and the Invitrus of additional 10% foetal calf serum (FCS) of use (Cell CultureTechnologies, Switzerland).Before electroporation with cell counting.(5 minutes, 200 * g) collected 8 * 10 by centrifugal 6Individual cell also abandons supernatant liquor.Be resuspended to cell in the 800 μ l Invitrus substratum and be transferred in the aseptic electroporation cuvette that contains 8 μ g circular plasmids DNA (0.4cm at interval) and incubated at room 5 minutes.Use GenePulser II (BioRad) with following condition with cell electroporation: 400V, 960 μ F, 30 seconds intervals of two subpulses.Behind the electroporation, cell is transferred in the T25 culturing bottle that contains 5ml growth medium (Invitrus/20% (V/V) FCS/1.25% (V/V) DMSO) immediately, and at 5%CO 237 ℃ of cultivations in the atmosphere incubator.For the production of unmodified (not glycosylation) antibody, only use the antibody expression vector transfectional cell.For the production of glycosylated antibodies, with two plasmid co-transfection cells, one is used for antibody expression, and another is used to merge GnTIII polypeptide (SEQ IDNO:15) expresses, and ratio separately is 3: 1.
The transfection of HEK293-EBNA cell
Come the HEK293-EBNA cell of transfection with the calcium phosphate precipitation method by the index law growth.The DMEM substratum that use to replenish 10%FCS makes cell as the growth of adhesivity monolayer culture thing in the T culturing bottle, when they are 50 to 80% to carry out transfection when being paved with.For the transfection of T75 culturing bottle, preceding 24 hours of transfection 800 ten thousand cell inoculations are replenished in the DMEM substratum of FCS (final 10%V/V) and 250 μ g/ml Xin Meisus in 14ml, and cell is positioned in the 5%CO2 atmosphere incubator 37 ℃ spends the night.Treat the T75 culturing bottle of transfection for each, by mixing the CaCl of the total plasmid vector DNA of 47 μ g, 235 μ l 1M 2Solution and to add entry to final volume be that 469 μ l make DNA, CaCl 2Solution with water.50mM HEPES, the 280mM NaCl, the 1.5mM Na that in this solution, add 469 μ l pH7.05 2HPO 4Solution mixed for 10 seconds immediately and also at room temperature placed for 20 seconds.Replenish the DMEM diluted suspension of 2%FCS with 12ml, and add alternative existing substratum among the T75.Cell is at 37 ℃ of 5%CO 2Under hatched 17 to 20 hours, the DMEM with 12ml 10%FCS substitutes substratum then.For the production of unmodified (not glycosylation) antibody, only use the antibody expression vector transfectional cell.For the production of glycosylated antibodies, with two plasmid co-transfection cells, one is used for antibody expression, and another is used to merge the GnTIII expression of polypeptides, and ratio separately is 4: 1.Transform after 5 days, collect supernatant liquor, at 1200rpm centrifugal 5 minutes, for the second time centrifugal subsequently, 4000rpm 10 minutes, and 4 ℃ of preservations.
The generation of the stable mammal cell line of express recombinant anti-CD20 antibodies
Pass through electroporation transfection bhk cell (BHK21-13c) with the pETR1502C2B8 antibody expression vector that contains the neomycin resistance gene expression cassette.At first select neomycin resistance to clone and obtain the clone that a tackling has the pETR1502 carrier DNA of chromosomal integration.Use the ELISA experiment sieving to be used for the clone that recombinant antibodies is produced then.Speak briefly, electroporation is after 24 hours, counts viable cell and measures transfection efficiency by counting the parallel fluorocyte that contrasts electroporation with the pEYFP-expression vector.Cell dilution is selected in the substratum in the Invitrus that contains 10%FCS and 1mg/ml Xin Meisu.The viable transfectional cell (1 * 10 of common 8 96 hole plating different concns 3, 5 * 10 2With 1 * 10 2The every hole of individual cell) and at 37 ℃ hatch until identifying the clone.In case clonal growth is to almost converging, by the antibody generation of elisa assay supernatant liquor.The ELISA positive colony is at first expanded to 24 holes subsequently on the 6 hole flat boards, then to the T25 culturing bottle.Growth was used the final antibody titers of ELISA test determination after 5 days in the T25 culturing bottle.Use this electroporation and system of selection, isolate the bhk cell clone (BHK-1502-28) who expresses the C2B8 anti-CD20 antibodies, this cell clone produces 13 μ g/ml antibody under above-mentioned culture condition.
The product of the stable mammal cell line that express recombinant anti-CD20 antibodies and GnTIII merge Give birth to
With the clone BHK-1502-28 of pETR1537 expression vector by electroporation transfection constitutive expression anti-CD-20 monoclonal antibody gene and neomycin resistance gene.PETR1537 is the carrier that is used for constitutive expression ManII-GntIII gene and clipped form people CD4, and the latter is that the IRES dependency is expressed.Carrier also contains the puromycin resistance gene expression cassette.At first select the tetracycline resistance clone to obtain the clone that a tackling has the pETR1537 carrier DNA of chromosomal integration.Screening and cloning is used for the surface expression of the CD4 (tCD4) of brachymemma then, and it is as the mark of bicistronic mRNA ManII-GnTIII+tCD4 genetic expression unit expression level.Use the generation of the selected clone's of ELISA verification experimental verification recombinant antibodies.
Speak briefly, with XmnI with the linearizing of pETR1537 carrier DNA.Contrast transfection with the EYFP expression vector is parallel.The cell of the whole intracellular expression EYFP of counting is measured transfection efficiency after 24 hours.All 58% of cell is to express EYFP's.Cell viability is 91%.Transfection one day after, be 1: 100,1: 500,1: 1000 and 1: 5000 extent of dilution with pETR1537 and the serial dilution of pEYFP cells transfected and be inoculated in (Invitrus in the selection substratum that final volume on the 96 hole flat boards is 0.2ml, 10%FCS, 20 μ g/ml tetracyclines, the 1mg/ml Xin Meisu).Two Zhou Houke see the clone.Their expansions and screening brachymemma CD4 (tCD4) are expressed and antibody expression.
For the screening of tCD4 expression level, (Becton Dickinson Switzerland) is hatched 20 minutes to about 500,000 cells on ice with the washing of FACS damping fluid and with the anti-people CD4 of 5 μ l FITC.After twice washing, cell is resuspended to 0.5ml FACS damping fluid and uses facs analysis (Figure 17 A-B).Isolate clone (BHK-1502-28-11) with good tCD4 expression level.Growth produced the anti-CD20 antibodies that whole titre is about 17 μ g/ml after 5 days in the T25 culturing bottle, as measured by ELISA.
4. the production of unmodified and glycosylated antibodies and purifying
The collection of substratum
With the antibody expression vector transfection or add with antibody expression vector in the situation of GnTIII fusion expression vector cotransfection bhk cell, collect culture supernatants after 96 hours cultivating transfectional cell after the transfection.According to the productivity of expection, same vehicle is carried out several electroporation (10-15).
With the antibody expression vector transfection or add with expression vector in the situation of GnTIII fusion expression vector cotransfection HEK293-EBNA cell, replace substratum with fresh culture in about 16 hours after the transfection, collect afterwards substratum after 120 hours further cultivating transfectional cell then.
For stable BHK-1502-28-11 clone, with 500,000 cells/ml inoculation culture thing, and after cultivating 4 days, collect supernatant liquor, cell density is 1.7 * 10 6Vitality cell/ml is arranged, and cell viability is 69%.
Antibody purification
Use two successive chromatographic step monoclonal antibody purification from culture supernatants.First step comprises the a-protein chromatography, uses the pH gradient elution, has separated ox and human IgG effectively.For the cation-exchange chromatography step sample buffer is exchanged for phosphate buffered saline (PBS) (PBS) subsequently.
5. oligosaccharides analysis
Separate the release oligosaccharides by PNGaseF digestion from abzyme, antibody is fixed on the pvdf membrane or in solution.
Resulting contain the digestion solution that discharges oligosaccharides directly make be used for that MALDI/TOF-MS analyzes or before preparation MALDI/TOF-MS analytic sample further with the digestion of EndoH Glycosylase.
The oligosaccharides method for releasing of pvdf membrane sessile antibody
With PVDF (Immobilon P, Millipore, Bedford, Massachusetts) the Kong Zhongyong 100 μ l methyl alcohol of the 96 hole flat boards that make of film are moistening, and vacuum used to Multiscreen vacuum manifold (Millipore, Bedford, Massachusetts) draw liquid that comes up makes it pass through pvdf membrane.With 300 μ l water washing pvdf membranes three times.Use 50 μ l RCM damping fluid (8M urea, 360mM Tris, 3.2mM EDTA, pH8.6) washing holes then.30-40 μ g antibody is loaded in the hole of containing 10 μ l RCM damping fluids.Make it pass through film by the liquid in the utilization vacuum pumping hole, wash film twice with 50 μ l RCM damping fluids subsequently, by adding the 0.1M dithiothreitol (DTT) among the 50 μ l RCM and hatching the reduction of carrying out disulfide bridge bond in 1 hour at 37 ℃.
After the reduction, the utilization vacuum is removed dithiothreitol (DTT) solution from the hole.With hole washing three times, carried out the cysteine residues carboxymethylation in 30 minutes by adding the 0.1M acetic acid iodine among the 50 μ l RCM and in the room temperature dark, hatching with 300 μ l water then.
After the carboxymethylation, use the vacuum pumping hole, use 300 μ l water washings three times subsequently.Sealed pvdf membrane in 1 hour to prevent the absorption of endoglycosidase by 1% aqueous solution in incubated at room then with 100 μ l polyvinylpyrrolidones 360.Remove encapsulant by slight vacuum then, then with 300 μ l water washings three times.
By adding 2.5mU peptide-N-glycosylase F (reorganization N-glycanase, GLYKO, Novato, CA) and 0.1mU sialidase (GLYKO, Novato CA) discharges N connection oligosaccharides and removes any possible charged monosaccharide residue, and enzyme is at the 20mMNaHCO of the pH7.0 of 25 μ l final volume 3In, 37 ℃ of digestion 3 hours.
The oligosaccharides method for releasing of antibody in the solution
(Glyko U.S.A.) mixes, and mixture was hatched 3 hours at 37 ℃ with the 2.5mU PNGaseF among the 2mM Tris of the antibody of 40-50 μ g and 25 microlitre final volume pH7.0.
The PNGaseF of endoglycosidase digestion discharges oligosaccharides the heterozygosis oligosaccharide structure of halving is distributed Give the purposes of MALDI/TOF-MS neutral oligosaccharides peak value
The oligosaccharides of using endoglycosidase H (EC 3.2.1.96) digestion PNGaseF to discharge subsequently.For EndoH digestion, (Roche Switzerland) adds the final volume that PNGaseF Digestive system (antibody in the aforesaid method solution) obtains 30 microlitres, at 37 ℃ mixture is hatched 3 hours with 15mU EndoH.EndoH cracking N-connects the N-n acetylglucosamine n residue of the chitobiose core of oligosaccharides.Enzyme only can digest few seminose and most of heterozygous glycan, and compound oligosaccharides does not obtain hydrolysis.
The specimen preparation of MALDI/TOF-MS
After acetic acid is added into final concentration 150mM, to contain the enzymic digestion liquid that discharges oligosaccharides further hatched 3 hours in room temperature, pass the little-biology-rotation chromatographic column (BioRad that packs into subsequently, Switzerland) 0.6ml Zeo-karb (the AG50W-X8 resin in, hydrogen form, the 100-200 mesh, BioRad Switzerland) comes decationize and protein.The resulting sample of 1 microlitre is used to stainless steel target flat board, on flat board, mix with 1 μ lsDHB matrix.By with 2mg 2,5-resorcylic acid and 0.1mg 5-methoxyl group Whitfield's ointment are dissolved in 1: 1 (V/V) ethanol/10mM sodium chloride aqueous solution of 1ml and make sDHB matrix.With the sample air drying, use 0.2 μ l ethanol, finally make sample recrystallize under air.
MALDI/TOF-MS
Being used for obtaining mass spectral MALDI-TOF mass spectrograph is Voyager Elite (Perspective Biosystems).Operating equipment in linear configurations postpones with 20kV acceleration and 80ns.Use the external calibration of oligosaccharides standard to be used for the distribution of ionic mass spectrum.Summation obtains final mass spectrum from the mass spectrum of 200 Laser emission.
6.PBMC preparation
Use Histopaque-1077 (Sigma Diagnostics Inc., St.Louis, MO63178 USA) also to get peripheral blood lymphocytes (PBMC) according to the guidance system of manufacturers basically.Tout court, take volunteer's venous samples can with the heparinization syringe, the volunteer requires to run 1 minute with all strength, so that improve the per-cent of the natural killer cell (NK) in the blood.With the PBS that does not contain Ca or Mg with blood thinning to 1: 0.75-1.3 and be laid on the Histopaque-1077.Room temperature (RT) 400 * g free of discontinuities gradient centrifugation 30 minutes.Collection contains the intermediate phase of PBMC and also passes through to collect in centrifugal 10 minutes at RT 300 * g with PBS washing (from every 50ml cell of two gradients).With PBS with pellet resuspended after, with PBMC counting and by washed the second time in centrifugal 10 minutes at RT 200 * g.Then cell is resuspended to the program that is used in the suitable medium subsequently.
7.NK cellular segregation
Separate NK cells of human beings from PBMC, use negative system of selection, and the magnetic bead of use debond CD16 and CD56 positive cell (the MACS system, from Miltenyi Biotec GmbH, 51429 Bergisch Gladbach, GER).(PBS that contains 2%FCS and 2mM EDTA) with PBMC washing once hatched 10 minutes with 1 of the every ml of 20Mio cell: 1FCS and MACS buffer solution mixture resuspension and at 4 ℃ in ice-cold MACS damping fluid.Then cell precipitation is also used the MACS damping fluid resuspension that contains 10%FCS, per 1,000 ten thousand cells, 80 μ l.Per then 1,000 ten thousand cells add 20 μ l haptens-antibody-solutions.With repeating the vortex pipe cell was hatched 10 minutes at 4 ℃.After the MACS washed twice with at least 10 * mark volume, cell is resuspended to contains in 10% the MACS damping fluid, per 80 μ l, 1,000 ten thousand cells, and per 1,000 ten thousand cells add 20 μ l antihapten-microballons.With repeating the vortex pipe pipe was hatched 15 minutes at 4 ℃.With the MACS damping fluid with cell washing once after, with the cell resuspension,, and be loaded on the LSMACS post that is positioned in the MINI-MACS magnet, with 3ml MACS damping fluid balance up to 10,000 ten thousand cells in the 500 μ lMACS damping fluids.With 3 * 3mlMACS damping fluid washing pillar.Collection is flow through the cell in the fraction and is used as the NK cell subsequently.Expressing the purity of measuring by CD56 is 88-95%.
8.ADCC test
Make PBMC or NK as mentioned above as the effector cell.Is 25: 1 and 10: 1 for PBMC and NK cytological effect thing separately to the ratio of target.In the AIM-V substratum, make the effector cell of suitable concn, so that in each hole of 96 hole circle base plates, add 50 μ l.The target cell of C2B8 antibody is SKW6.4 or the Namalwa bone-marrow-derived lymphocyte that grows among the DMEM that contains 10%FCS.Wash target cell in PBS, count and be resuspended to AIM-V, every ml 0.3 hundred ten thousand is so that add 30000 cells in each micropore of 100 μ l.Antibody dilution in AIM-V, is added the target cell of pre-bed board and RT combining target 10 minutes in 50 μ l.Add the effector cell then and flat board is being contained 5%CO 2Moistening atmosphere in 37 ℃ hatched 4 hours.(Roche Diagnostics, Rotkreuz Switzerland) test killing of target cell by measuring serum lactic dehydrogenase (LDH) release that destroys cell to use the cytotoxicity detection kit.After flat board is hatched 4 hours, centrifugal at 800 * g, the 100 μ l supernatant liquors in each hole are transferred on the transparent flat flat board in 96 new holes.100 μ l color substrate buffer solutions in the test kit are added in each hole.Measure the Vmax value at least 10 minutes of color reaction at ELISA reader 490nm, and use SOFTmaxPRO software (MolecularDevices, Sunnyvale, CA94089, USA).From only containing target cell and effector cell but do not have to measure the hole of antibody spontaneous LDH and discharge.From the hole of only containing target cell and 1%TritonX-100, measure maximum release.The per-cent of killing of following calculating specific antibodies mediation: (x-SR)/(MR-SR) * 100, and wherein x is the average Vmax of specific antibodies concentration, and SR is the average Vmax of spontaneous release, and MR is the maximum average Vmax that discharges.
9.NK the Fc γ RIIIA combination on the cell
At 200 * g with the NK cell centrifugation of fresh separated 5 minutes, with 0.09% (wt/vol) lactic acid solution (140mM NaCl, 5mM KCl, pH3.9) in room temperature with 3 * 10 5The relevant IgG of NK cell is removed in the pre-treatment that cell/ml was hatched 5 minutes.(De Haas M.，J.Immunol.156：2948(1996))。
With PBS, 0.1%BSA, 0.01% sodiumazide with cell washing twice, and with concentration adjustment to PBS, 0.15 BSA, 0.01% sodiumazide 2 * 10 6Individual cell/ml.With 0,0.1,0.3,1,3,10 μ g/ml antibody variants with 5 * 10 5Cell was hatched 30 minutes at 4 ℃.Then with cell washing twice, and the F (ab ') by puting together with 1: 200 fluorescein isothiocyanate 2The anti-human IgG of goat (Jackson ImmunoReasearch, West Grove, PA) and anti-people CD56-PE with 5 μ l/5 * 10 5Cell (CA) hatch at 4 ℃ and detected antibodies in 30 minutes by BD Pharmingen, san Diego.(Shields R. etc., J.Biol.Chem.277 (30): 26733-26740 (2002)).
(BD Bioscience, San Jose CA) go up the fluorescence intensity that relates to the binding antibody variant that detects the CD56+ cell at FACS Calibur.
10.Raji the combination of Fc γ RIIb on the lymphocyte
(concentration was 0.3Mio cell/ml) in 20 minutes 37 ℃ of washings with RajiB cell human lymphocyte in PBS.Then cell is resuspended to PBS, 0.1%BSA, 0.01%NaN 3In, 2.22 100 ten thousand cells/ml add 180 μ l in each FACS pipe.The L19-unmodified of ten times of antibody diluents (0,0.1,0.3,1,3,10,30 μ g/ml) and L19 glycosyl through engineering approaches monoclonal antibody added in the Raji cell and hatch 30 minutes at 4 ℃ (whole cell concn is 200 ten thousand cells/ml).After twice washing, the F (ab ') that 1: 200 fluorescein isothiocyanate is puted together 2The anti-human IgG of goat (PA) hatched 30 minutes in the adding cell and at 4 ℃ by Jackson ImmunoReasearch, West Grove.After the washing once, cell is resuspended to 0.5mlPBS, 0.1%BSA, 0.01%NaN 3In, and (BD Bioscience, San Jose CA) measure the fluorescence intensity that relates to the binding antibody variant of viable cell on FACS Calibur.
11. CDC test
Target cell is counted, washs, is resuspended among the AIM-V (Invitrogen) every ml 100 ten thousand cells with PBS.With 50 μ l cell seedings in each hole of the flat flat board in 96 holes.In AIM-V, make antibody diluent and 50 μ l are added in the cell.Make antibody at room temperature and cell in conjunction with 10 minutes.Fresh the thawing of human serum complement (Quidel) adds in the hand-hole with 3 times of AIM-V dilutions and with 50 μ l.As the described rabbit complement (CedarlaneLaboratories) that makes of manufacturers, add in the hand-hole with 3 times of AIM-V dilutions and with 50 μ l.In contrast, before adding test, the complement source was heated 30 minutes at 56 ℃.
Test panel was hatched 2 hours at 37 ℃.Discharge and measure killing of cell by measuring LDH.Tout court, with flat board centrifugal 3 minutes at 300 * g.Be transferred to every hole 50 μ l supernatant liquors in the 96 new hole flat boards and add the test reagent of 50 μ l from cytotoxic reagent box (Roche).Measure the corresponding Vmax of LDH concentration in the supernatant liquor with the kinetic measurement of ELISA reader.Measure maximum release by incubated cell in the presence of 1%Triton X-100.
Result and discussion
Produce the chimeric IgG1 antibody of anti-CD20 (the C2B8 chimeric antibody is also referred to as rituximab) of glycosyl engineered forms by the culture that carrier and expression coding with the expressing antibodies gene have the carrier cotransfection mammalian cell of active each peptide species of GnTIII.Produce the same antibody of unmodified form (non-glycosyl through engineering approaches) by the carrier mammalian cells transfected of only expressing with antibody gene.Cells transfected kept in culture three days at least, by a-protein affinity chromatography purifying secreted recombinant antibodies from substratum.The genetic expression that coding has the active polypeptide of GnTIII has no significant effect with respect to the cell pair cell vitality that does not produce such antibody, cell growth and antibody generation.
Analyze the glycosylation pattern of antibody purification then.These antibody carry the N-that only is connected with the Asn297 residue in human IgG1 Fc district and connect oligosaccharides.Separate from abzyme by PNGaseF digestion and to remove oligosaccharides and analyze by MALDI/TOF-MS subsequently.Use this technology, can detect the part of different oligosaccharides kinds in the total original Fc oligosaccharides colony, can also be in mass spectrum structure be distributed to different peak value (Umana, P. etc., Nature Biotechnol.17:176-180 (1999)).
Fig. 1 has shown the neutral oligosaccharides MALDI/TOF-MS characteristic pattern of the chimeric IgG1 antibody of the anti-CD20 of the recombinant C 2B8 that produces in the bhk cell.With these cells of antibody expression vector pETR1502 transfection.Fig. 2 to 4 has shown the individual features figure of the same antibody that the bhk cell of the engineered nucleic acidization that has the active polypeptide of GnTIII with encoding produces.Characteristic pattern among Fig. 2 is formed by the wild-type GnTIII nucleic acid that uses coding to express from carrier pETR1166.Characteristic pattern among Fig. 3 is formed by the nucleic acid of fusion polypeptide that uses coding to be included in the GnTI locating structure territory of N-end and the fusion of GnTIII C-end catalyst structure domain.This fusion gene is expressed from carrier pETR1425.Characteristic pattern among Fig. 4 is formed by the nucleic acid of fusion polypeptide in locating structure territory that uses nucleic acid encoding to be included in the golgi body α mannosidase II (ManII) of N end and the fusion of GnTIII catalyst structure domain.This fusion gene is expressed from carrier pETR1506.
Unmodified antibody has typical oligosaccharides form (Fig. 1), the m/z ratio be 1485,1648 with 1810 peak value respectively with have 0,1 with 2 galactose residues two feelers, the core fucosylation, composite oligosaccharide is consistent.In the Fc district oligosaccharides of other standard Mammals industry clones such as the CHO and the non-through engineering approaches IgG1 antibody of murine myeloma cell generation, find similar characteristic formp (Lifely, M.R. etc., Glycobiology 5:813-822 (1995)).By the expression of wild-type GnTIII the celliferous through engineering approaches of antagonist mainly form bisection, core fucosylation, Composite Double feeler oligosaccharides (Fig. 2), the peak value in m/z ratio 1689,1851 and 2014 is to be found in the non-bisection in the unmodified antibody, the bisection counterpart of fucosylation oligosaccharides peak value.The celliferous through engineering approaches of antagonist also mainly forms bisection Composite Double feeler oligosaccharides (noting the peak value of m/z1689 among Fig. 3 and 1851) by the nucleic acid of expressing coding GnTI-GnTIII fusion polypeptide, and wherein the GnTIII catalyst structure domain is to locate by GnTI golgi body locating structure territory.Yet, with respect to wild-type GnTIII, use GnTI-GnTIII to merge to cause halve, the raising of non-fucosylation and bisection, hybrid structure (m/z1664 between comparison diagram 2 and Fig. 3,1810,1826 and 1973 peak value are with respect to overall per-cent).Oligosaccharides material for the GnTI-modification, halve, non-fucosylation and bisection, hybrid structure synthetic by reorganization GnTIII catalyst structure domain and following between competition form, (i) endogenous core α 1, the 6-fucosyltransferase, (ii) golgi body alpha-Mannosidase II (ManII) and (iii) GnTII, because in case modify oligosaccharides with the bisection GlcNAc that adds by the GnTIII catalyzed reaction, these three other enzymes just no longer act on modifies the bisection oligosaccharides.Because GnTII effect N-connects the ManII downstream in the oligosaccharides biosynthetic pathway, therefore also effectively blocked GnTII by the ManII blocking effect that adds bisection GlcNAc.M/z1664 and 1826 peak value are non-fucosylations, and the peak value of m/z1810 and 1973 is fucosylations.Can offer an explanation the EndoH Glycosylase digestion of heterozygosis and composite oligosaccharide (Fig. 8 A), be used for confirming that the raising of these peak values is owing to bisection, non-fucosylation and the bisection that Fc connects, the raising (vide infra) of heterozygosis oligosaccharide ratio.
Contrast with forming at the active coding nucleic acid of this other used GnTIII, the through engineering approaches that forms cell by coding ManII-GnTIII fusion polypeptide (SEQ ID NO:14) expression of nucleic acids antagonist mainly forms bisections, non-fucosylation and bisection, hybrid structure (m/z1664,1810 among attention Fig. 4,1826 and 1973 peak value), and wherein the GnTIII catalyst structure domain is localized by ManII golgi body locating structure territory.Therefore, merge (SEQ ID NO:12 and 13) with respect to wild-type GnTIII and GnTI-GnTIII, ManII-GnTIII merges in bisection, non-fucosylation and the bisection that Fc connects, heterozygosis oligosaccharides synthetic more effective (m/z1664 between the comparison diagram 2,3 and 4 and 1810 peak value are with respect to overall per-cent).Respectively do for oneself 4,13 and 33% by bisection, non-fucosylation Fc oligosaccharide ratio that the expression of nucleic acid that encoding wild type GnTIII, GnTI-GnTIII merge and ManII-GnTIII merges forms.In unmodified (non-through engineering approaches) antibody, do not detect the bisection oligosaccharides.
The raising that the ManII-GnTIII fusion constructs is expressed in antibody produced cell causes halving, the further raising of non-fucosylation oligosaccharide ratio.This obtains proof by express the ManII-GnTIII construct from carrier (pETR1519) that has the OriP that is used for free replicon in transfection HEK293-EBNA cell.Known this expression system causes high-caliber expression, also is used for the expression from carrier pETR1520 antibody gene.The oligosaccharides characteristic formp of the purifying of high level expression, unmodified (non-glycosyl through engineering approaches) antibody is shown among Fig. 5 in this system, its typical oligosaccharides characteristic formp that has shown non-bisection with 0,1 and 2 galactose residue, fucosylation peak value once more (for example, comparison diagram 1 and 5 is presented at the similar oligosaccharides characteristic formp of expressing in bhk cell unmodified antibody or that higher level is expressed in the HEK293-EBNA cell).Come engineered antibody to produce cell with the nucleic acid that is coded in the ManII-GnTIII fusion that higher level is expressed in this system and cause production of antibodies, wherein most of Fc oligosaccharides be halve, non-fucosylation (referring to Fig. 6, wherein halve, the m/z1664 of non-fucosylation heterozygosis oligosaccharides and 1826 peak values constitute together surpass total oligosaccharides 90%).
As mentioned above, endoglycosidase H (EndoH) is used for confirming the distribution of halving non-fucosylation structure and bisection, hybrid structure observed different oligosaccharides peak in the MALDI characteristic pattern.The MALDI/TOF-MS neutral oligosaccharides characteristic pattern that PNGaseF-and PNGaseF+EndoH-digestion are derived from the glycan of the chimeric IgG1 antibody of anti-CD20 is shown among Fig. 7, by the HEK293 cell generation antibody of GnTIII (M2) overexpression glycosyl through engineering approaches.The peak value of m/z1664 can be distributed to two different glycan, and promptly binary the or non-fucosylation of the heterozygosis of non-fucosylation is compound non-binary.Different structures shows that identical m/z ratio is because identical monose is formed (Fig. 8 B).
The glycan that discharges with endoglycosidase H digestion PNGaseF has produced new structure, and main peak value is converted to 1460 (Fig. 7 B) from m/z1664.Difference is corresponding to the quality of GlcNAc residue.As mentioned above, EndoH can not divide compound oligosaccharides.Therefore, after endoglycosidase H digestion, the main peak value of m/z1664 can be distributed to the second-class somatotype of non-fucosylation heterozygosis.
Other peak can be distributed to compound or heterozygosis bisection glycan.After the EndoH digestion, the peak of m/z1810 has disappeared, so structure can be distributed to the second-class somatotype of heterozygosis of fucosylation.From GlcNAc residue of m/z1810 peak value and Fucose (from core α-1,6 fucosylation, reduction end GlcNAc residue) minimizing of residue produced the structure of m/z1460.Peak by EndoH digestion (elimination of GlcNAc residue) m/z1502 has disappeared and the peak of m/z1298 occurred, has proved that 1502 peaks can distribute to the second-class somatotype of non-fucosylation heterozygosis.The disappearance at m/z1647 peak, EndoH digestion back has proved that this peak is the heterozygosis bisection structure of fucosylation.The removal of a GlcNAc and a Fucose has produced the structure of m/z1298.The peak of m/z1826, the second-class somatotype of the heterozygosis of non-fucosylation digests by EndoH.This has produced the structure of m/z1622.After the EndoH digestion, high mannose type (1257m/z) can be distributed in the peak of m/z1053, digests by EndoH.As expected, the peak of m/z1689 (compound bisection) is not subjected to the influence of EndoH digestion.In synthetic, data from table 1 acquisition, we infer that 88% oligosaccharide structure has bisection GlcNAc, and wherein 60% is the heterozygosis bisection structure of non-fucosylation, the 22%th, and the fucosylation heterozygosis is binary and 6% be the compound bisection oligosaccharide structure of fucosylation.
Table 1. oligosaccharides distributes
m/z Possible structure Relative % before the EndoH The m/z that expects behind the EndoH Observed m/z behind the EndoH Relative % behind the EndoH Distribute
1256 High mannose 9 1053 1053 11 High mannose (9%)
1502 The compound of the binary or non-fucosylation of the heterozygosis of non-fucosylation 7 1298 or 1502 1298 - 13 The heterozygosis of non-fucosylation binary (7%)
1647 The compound of the binary or fucosylation of the heterozygosis of fucosylation 7 1298 or 1647 1298 - 13 The heterozygosis of fucosylation binary (7%)
1664 The compound of the binary or non-fucosylation of the heterozygosis of non-fucosylation 49 1460 or 1664 1460 - 60 The heterozygosis of non-fucosylation binary (49%)
1689 Fucosylation compound binary 3 1689 1689 5 Compound binary (3%) of fucosylation
1810 The compound of the binary or fucosylation of the heterozygosis of fucosylation 15 1460 or 1810 1460 1810 60 2 The compound (2%) of heterozygosis of fucosylation binary (13%) and fucosylation
1826 The heterozygosis of non-fucosylation is binary 4 1622 1622 7 The heterozygosis of non-fucosylation binary (4%)
1851 Fucosylation binary 3 1851 1851 2 Compound binary (3%) of fucosylation
1972 The heterozygosis of fucosylation is binary 3 1622 1622 7 The heterozygosis of fucosylation binary (3%)
Mass balance (with molar fraction %):
A) m/z1502 and 1647 peak: 7+7%=14% (expection).EndoH has produced m/z1298 (obtaining 13% behind EndoH) to the digestion at two peaks
B) m/z1664 and 1810 peaks: 49+13%=62% (expection).EndoH produces m/z1460 (obtaining 60%)
C) m/z1826 and 1972 peaks: 4+3%=7% (expection).EndoH produces m/z1622 (7%)
The relative percentage that little junction structure is total
Have bisection GlcNAc:88%
The heterozygosis of non-fucosylation is binary: 60%
The heterozygosis of fucosylation is binary: 22%
Fucosylation compound binary: 6%
Above-mentioned data (Fig. 1 to 6) have shown that GnTIII expresses and be used for level with two of the certain position structural domains of GnTIII catalyst structure domain target golgi body, reorganization GnTIII catalyst structure domain and endogenous core α 1 have been influenced, the competition of the oligosaccharides substrate of between 6-fucosyltransferase, (ManII) and the GnTII enzyme GnTI being modified.In this competition, GnTIII helps this than high expression level, causes halving, the content of heterozygosis oligosaccharides and bisection, the high level of non-fucosylation oligosaccharides and the bisection composite oligosaccharide of following and bisection, fucosylation oligosaccharides reduces.This is for also having noticed (Umana, P. etc., Nature Biotechnol.17:176-180 (1999)) before the wild-type GnTIII.Yet, although form the bisection oligosaccharides of similar aggregate level, with respect to wild-type GnTIII, oligosaccharides substrate and endogenous core α 1 for the GnTI modification, 6-fucosyltransferase, ManII and GnTII enzyme are by GnTI or locate the GnTIII catalyst structure domain by ManII locating structure territory and will cause more effective competition.
GnTIII compares with wild-type, the GnTI-GnTIII fusion can be explained by distributing with respect to the golgi body in the suitable extremely opposite direction of the glycoprotein material transmission of GnTIII at GnTI of morning for bisection, heterozygosis oligosaccharides and bisection, the higher effectiveness of non-fucosylation oligosaccharides synthetic.Before distributing, the meticulous golgi body of GnTI and ManII measures ((Rabouille, C. etc., J.Cell Sci.108:1617-27 (1995)) by quantitative immune electron microscopy.Two enzymes are divided into cloth along golgi body, mainly are positioned at inner and pass the blister cavities (cisternae) of golgi body heap (stack), and with respect to passing blister cavities, the content in the inner blister cavities is higher.Also do not measure core α 1, the accurate quantification spatial distribution of 6-fucosyltransferase, GnTII and wild-type GnTIII.Yet, above-mentioned be not interpreted as what for synthetic halve, heterozygosis oligosaccharides and bisection, non-fucosylation oligosaccharides, ManII-GnTIII fusion ratio GnTI-GnTIII merges more effective, because GnTI has identical spatial distribution with ManII along golgi body compartment (subcompartment).
The higher effectiveness that ManII-GnTIII merges shown inner-and pass-existence of the function glycosylation compartment (subcompartment) of relative organizationization in the golgi body blister cavities physics compartment (subcompartment).Believe so-called " inside-golgi body glycosylase ", GnTI, GnTII and ManII are present in the golgi body as the high molecular complex body.Yet if the locating structure territory makes these enzymes form the part of these complex bodys, this merges for GnTI-GnTIII, and fusion is identical with ManII-GnTIII.The expression that reorganization GnTI-GnTIII merges does not cause any significance degree of endogenous wild-type GnTI enzyme replacement paired Fc-oligosaccharides synthetic, owing to cause most oligosaccharides to obtain the modification of GnTI and two reactions of GnTIII at these all used GnTIII constructs.
Our data show, because ManII locating structure territory between the catalyst structure domain of endogenous GnTI and reorganization ManII-GnTIII fusion accurate function pairing has taken place.Catalysis is with the systematism pairing of the enzyme of afterreaction in the biosynthetic pathway, with preference the product of first reaction is transferred to respect to the mode of this product away from second catalytic site of enzyme diffusion, be known in another biosynthetic pathway, take place as glycolysis-and polyketone biosynthesizing.Reported that GnTI and ManII formed " kin oligopolymer ", one to these enzymes of major general relocates when endoplasmic reticulum (Nilsson, T. etc., EMBO be (3) .562-74 (1994) J.13).Find that a pair of charged amino-acid residue in each main region of this two enzymes is critical for this kin identification.Opposite among the GnTI among the electric charge of residue and the ManII.We have identified that this part N-connects the similar residue of equivalent site in the main region of other golgi body glycosylase that relates in the oligosaccharides biosynthetic pathway, be core α 1,6-fucosyltransferase (identical, rather than as be complementary electric charge in the ManII situation), ManI and GnTII with the electric charge of GnTI.We also identify these residues is conservative between species.Although proposed these residues for be integrated in the polymer composite that enzyme forms or even for the dispensable (Opat in golgi body location, A.S. etc., J.Biol.Chem.275 (16): 11836-45 (2000)), possible they relate to the accurate pairing of catalyst structure domain in the oligosaccharides biosynthetic process.Such pairing needs not be irreversible, but can mediate by instantaneous, the dynamic (dynamical) interaction between the enzyme.May there be other pairing determinant in other places in main region or catalysis region.Yet, in the reorganization that has the GnTIII catalyst structure domain is merged, will lose right any contribution from the specific GnTI-ManII of ManII catalyst structure domain.
Fig. 9 to 11 has proved the raising that is had the antibody dependent cellular cytotoxicity (ADCC) that the nucleic acid overexpression of the active polypeptide of GnTIII causes by coding in the antibody produced cell, and polypeptide is positioned golgi body by different locating structures territory.It is active and be positioned ADCC that the expression of the recombinant polypeptide of golgi body causes by GnTI golgi body locating structure territory and improve and be shown among Fig. 9 to have GnTIII.The oligosaccharides characteristic formp that is used for the control antibodies of Fig. 9 ADCC test is shown among Fig. 1.The oligosaccharides characteristic formp that is used for the glycosyl engineered antibody of Fig. 9 ADCC test is shown among Fig. 3.It is active and be positioned ADCC that the expression of the recombinant polypeptide of golgi body causes by Glycosylase ManII golgi body locating structure territory and improve and be shown among Figure 10 to have GnTIII.The oligosaccharides characteristic formp that is used for the control antibodies of Figure 10 ADCC test is shown among Fig. 5.The oligosaccharides characteristic formp that is used for the glycosyl engineered antibody of Figure 10 ADCC test is shown among Fig. 6.
Figure 11 has shown that to have GnTIII active and cause by the expression that ManII golgi body locating structure territory is positioned the recombinant polypeptide of golgi body that ADCC is active to be improved, with respect to the wild-type GnTIII polypeptide that uses self GnTIII gorky locating structure territory.Oligosaccharides characteristic pattern wild-type GnTIII expression and that be used for the glycosyl engineered antibody of Figure 11 ADCC test is shown among Fig. 2.The fusion polypeptide oligosaccharides characteristic pattern that express and that be used for the glycosyl through engineering approaches of Figure 11 ADCC test that have the GnTIII activity, is positioned golgi body by ManII golgi body locating structure territory is shown among Fig. 4.These data have also shown with respect to having antibody compound, fucosylation, non-bisection oligosaccharides, have the bisection oligosaccharides and comprise that the bisection heterozygosis oligosaccharides and the antibody of the non-fucosylation oligosaccharides of halving have the ADCC activity of raising.All bisection oligosaccharides that it should be noted that the higher antibody of activity that is used for Figure 10 ADCC test are heterozygosis oligosaccharides of bisection, non-fucosylation.As described above, it is active and cause the bisection oligosaccharides of more effective synthetic non-fucosylation by the fusion polypeptide that ManII golgi body locating structure territory is positioned golgi body that use has GnTIII, and Figure 11 has shown the antibody with respect to these oligosaccharides that have lower level, and the antibody that has these non-fucosylation oligosaccharides of halving of improving the standard more has activity in ADCC.The increase of this bisection in the relevant oligosaccharides with Fc of the active raising of the ADCC colony, non-fucosylation oligosaccharides part is associated, and when this part is higher than 15-20%, improves greatly as can be seen.
Known natural killer (NK) cell is the important amboceptor of ADCC.These cells carry on its surface and activate Fc γ receptor II IA, are also referred to as CD16a.On the Fc district of target cell binding antibody and the NK cell combination of Fc γ RIIIA acceptor for the crosslinked of these acceptors on the NK cell and subsequently inducing of ADCC be essential.Therefore, it is important estimating the antibody that said method produces and the combination of Fc acceptor, and especially people's immune effector cell has been showed the acceptor in their natural form.Figure 12 proved have by coding in antibody produced cell that glycosyl engineered antibody that the expression of nucleic acid of the active fusion polypeptide of GnTIII produces has a raising activate the affinity combination of Fc acceptor Fc γ RIIIA with the people.Aforesaid these antibody have bisection, the non-fucosylation oligosaccharides that improves content for the ADCC test, and oligosaccharides is to produce by having the expression of the active fusion polypeptide of GnTIII in antibody produced cell.Used NK cell expresses on comfortable its NK cell the genotype donor (Metes, D. etc., J.Immunol.Methods 258 (1-2): 85-95 (2001)) of Fc γ RIIc acceptor in this test.Therefore, having only the Fc acceptor on these cell surfaces is to activate Fc γ RIIIA acceptor.Figure 13 has shown in conjunction with experimental measurement the specificity binding affinity with this receptor.This shows by the competition with Fc γ RIII-specific inhibition antibody fragment (3G8 Fab2-fragment).
The strong evidence of the Fc-FcR interaction partners anti-tumour antibody treatment result influence that improves is from rendeing a service and isozygotying than the dependency between the high-affinity Fc γ RIIIA acceptor gene type, be found in lymphoma the patient ((Cartron that accepts rituximab, G. etc., Blood 99 (3): 754-8 (2002)).This is to find and the target response rate that improves greatly and the relevant single parameter of molecules in response ratio of raising.Because being derived from by various immunocytes, the effectiveness that the Fc γ RIIIA-Fc acceptor interaction that improves improves comprises the function that natural killer (NK) cell, scavenger cell, monocyte and dendritic cell are realized.The NK cell, activate crosslinked tumour cell cracking (believing it is main FcR dependency kill mechanism in the body widely) (Maloney that can cause of Fc γ RIIIA acceptor on scavenger cell and the monocyte by ADCC, D.G. etc., Semin.Oncol.29 (1 Suppl.2): 2-9 (2002), Amigorena S., J.Exp.Med.195 (1): F1-3 (2002)), also cause antibody dependent cellular phagolysis (Hazenbos, W.L. etc., J.Immunol.161 (6): 3026-32 (1998), Reff, M.E. and Heard, C.Crit Rev Oncol Hematol.40 (1): 25-35 (2001)), and cause in the contiguous cytokine (Carson, W.E. etc., Eur.J.Immunol.31:3016-3025 (2001)) that discharges of tumour cell.These cytokines cause the direct cytotoxic effect on the tumour cell subsequently, with cause blood vessel formation against function, it is by depriving tumour antigen submission that oxygen and nutrition suppress tumor growth and cause improving, as the part to the immunne response of antitumor cell of activated T cell mediation.Dendritic cell are conclusive for the antigen presentation of T cell, Fc γ RIIIA in its surface crosslinked (for example, from in the body of antibodies by the initial dying tumour cell of attacking of ADCC) can cause the dendritic cell maturation that improves, antigen uptake and to the submission of T cell, and the crossed sensitization of cytotoxic T cell, the latter is very effective potential mechanism (Amigorena S. to activating antineoplastic immune, J.Exp.Med.195 (1): F1-3 (2002), Kalergis, A.M. and Ravetch, J.V.J.Exp.Med.195 (12): 1653-1659 (2002), Selenko, N. etc., J.Clin.Immunol.22 (3): 124-130 (2002)).The crosslinked raising that has also caused target cell directly to be killed of antibodies target cell by Fc acceptor on the immune effector cell, for example pass through the crosslinked of antibody-mediated target antigen molecule and inductive apoptosis (Reff, M.E. and Heard, C.Crit Rev Oncol Hematol.40 (1): 25-35 (2001), Cragg, M.S. etc., Blood 101 (3): 1045-1052 (2003).In all these immune effector cells, have only the NK cell only to have the Fc of activation γ R in its surface.In other cell type, activate Fc γ RIII and inhibition Fc γ RIIb and exist together, inducing by surpassing the activated forward balance that suppresses signal of anti-tumour effect subfunction causes.
Figure 15 shown the Fc receptors bind that improves and inhibition Fc γ RIIb compare be activated receptor optionally.As explained above, this selectivity is important for the effector function of the realization of the immunocyte beyond the NK cell.In addition, naturally observed accept the isozygotying of standard unmodified antibody by what use that method glycosyl through engineering approaches Fc antibody regions described herein obtains in conjunction with improving than much higher (Figure 16) of high-affinity Fc γ RIIIA genotype patient/donor than those, and this combination improves the effectiveness relevant (Cartron, Blood99 such as G. (3): 754-8 (2002)) that improves with anticancrin.
The binding domains of activation Fc γ RIIIB acceptor and Fc γ RIIIA are much at one.Therefore, above-mentioned data have shown that also glycosyl engineered antibody described herein can cause by showing the effector cell of Fc γ RIIIB, as polymorphic nucleus (PMN) cell, the raising of the effector function of medium, the release and the phagolysis ((Reff that comprise toxic product, M.E. and Heard, C.CritRev Oncol Hematol.40 (1): 25-35 (2001), Daeron, FM.Annu.Rev.Immunol.15:203-34 (1997), Ravetch, J.V. and Bolland S.Annu.Rev.Immunol.19:275-90 (2001)).
Figure 18 has shown the oligosaccharides characteristic formp of the anti-CD20 antibodies that bhk cell produces, and bhk cell grows in the suspension and through engineering approaches is come composing type high level expression recombinant antibodies and had the active fusion polypeptide of GnTIII.For the antibody from fusion GnTIII through engineering approaches cell, this oligosaccharides characteristic formp has shown the increase (also referring to table 2) of halving non-fucosylation oligosaccharides and bisection heterozygosis oligosaccharides level.In the non-glycosyl engineered antibody that produces by non-glycosyl through engineering approaches bhk cell, do not find these structures (referring to Fig. 1).Express through engineering approaches cell that GnTIII merges and presented normal growth in suspension and good antibody production.
The relative percentage of the oligosaccharides of the glycosyl through engineering approaches monoclonal antibody that produces by stable BHK-1502-28-11 clone is listed in the table 2.
Table 2: the relative percentage at the peak that obtains by MALDI/TOF-MS
Peak (m/z) Relative percentage
1 1257 2.5％
2 1486 2.8％
3 1647 6％
4 1664 22.30％
5 1680 2.5％
6 1689 4.8％
7 1705 3％
8 1810 27.8％
9 1826 10％
10 1851 7.5％
11 1972 9％
12 2012 1.75％
Total is binary: 88.6% (4+5+6+7+8+9+10+11+12)
Total non-fucosylation binary: 37.8% (4+5+7+9)
Total fucosylation binary: 50.8% (6+8+10+11+12)
Compound binary: 17% (6+7+10+12)
Heterozygosis is binary: 71.6% (4+5+8+9+11)
The oligosaccharides analysis has shown that 88.6% structure carries bisection GlcNAc residue, the 50.8%th, fucosylation with 37.8% be non-fucosylation.The oligosaccharides that discharges with endoglycosidase H digestion PNGaseF has proved that the most of peaks that obtain are second-class somatotypes of heterozygosis (Figure 19).Figure 20 has shown the glycosyl engineered antibody of BHK-1502-28-11 clone generation and the raising of the activation Fc acceptor Fc γ RIIIA binding affinity on the NK cells of human beings.The clone of growth and composing type stably express antibody gene and fusion GnTIII polypeptide is ideal to scale operation treatment antibody in suspension.Use the standard cell lines engineering method, can introduce the clone that contains antibody gene by merging the GnTIII gene, or contain the clone (" the production clone of pre-glycosyl through engineering approaches ") that merges the GnTIII gene, or by introducing antibody gene simultaneously and the GnTIII fusion gene is realized the glycosyl through engineering approaches by antibody gene is introduced.
Also detected the complement-mediated cracking (CML) of the anti-CD20 antibodies that produces in the through engineering approaches cell, this project cell is used for high level expression coding, and to have a GnTIII active and be positioned the nucleic acid of the fusion polypeptide of golgi body by ManII locating structure territory, and CML is the different effect subfunction that does not rely on Fc acceptor on the immune effector cell.Most oligosaccharides of this glycosyl engineered antibody are the non-fucosylation types of bisection heterozygosis.Compare with unmodified antibody, observe the CML activity (Figure 21) that this anti-CD20 antibodies reduces.Use for some, having raising ADCC and having the antibody that reduces CML is ideal, for example reduces the side effect by the CML mediation, as the vasculitis in the tumor locus blood vessel.Other remarkable side effect (van der Kolk L.E. etc., Br J Haematol.115 (4): 807-11 (2001)) of CML mediation has been observed in treatment for anti-CD20 antibodies.Yet above-mentioned oligosaccharides characteristic formp has shown also that engineered antibody produces that cell is expressed the GnTIII fusion polypeptide with medium expression level and the composite oligosaccharide that causes the non-fucosylation oligosaccharides of bisection heterozygosis (being higher than 15%) of medium level in the Fc oligosaccharides colony of glycosyl engineered antibody rather than have a signal portion is possible.Such composite oligosaccharide and normal, the CML that does not reduce level is relevant.Therefore data sheet is understood to produce like this and is had the antibody that improves ADCC, and it can keep and the compare CML activity of closely similar level of non-engineered antibody.
Another chimeric IgG1 antibody C225 by method glycosylation identification Human epidermal growth factor receptor described herein (EGFR) is also referred to as cetuximab.Figure 22 has shown the oligosaccharides feature of the same antibody of unmodified anti-egfr antibodies C225 and glycosyl engineered forms.To have a GnTIII active and be positioned by ManII locating structure territory to produce in the cell of nucleic acid of fusion polypeptide of golgi body expressing coding for the latter.Figure 23 has shown the ADCC of the anti-egfr antibodies increase that is formed by this glycosyl through engineering approaches.That produce by method described herein and ADCC improves and the glycosyl engineered antibody that improves with the binding affinity that activates the Fc acceptor, Antybody therapy for cancer and autoimmune disorders is the molecule that distant view is arranged, treat them and cause the effectiveness that improves owing to be used for these, with respect to the corresponding unmodified of these antibody (non-glycosyl through engineering approaches) form.In addition and the antibody of unmodified compare, may reduce therapeutic dose for the antibody of glycosyl through engineering approaches, these favourable influence the economy of antibody producing.
Embodiment 2
Treatment chronic graft versus host disease patient's immune-mediated thrombopenia
Autoimmunity thrombopenia in the chronic graft versus host disease has been represented the example of the B cell dysregulation that causes clinical disease.In order to treat chronic graft versus host disease patient's immune-mediated thrombopenia, with prepared in accordance with the present invention and have the anti-CD20 chimeric mAb that improves ADCC and deliver medicine to the patient, as Ratanatharathorn, V. etc., (its integral body being incorporated herein by reference) described in Ann.Intern.Med.133 (4): the 275-79 (2000) at this.Particularly, infusion antibody weekly, 375mg/m 2, deliver medicine to 4 weeks of patient.Antybody therapy has produced the content that B cell in the peripheral blood significantly reduces and reduced platelet-associated antibody.
Embodiment 3
Treat the incomplete and hemolytic anemia of serious, immune-mediated simple property red blood cell development
Immune-mediated, acquired simple property red blood cell development not complete (PRCA) is the disease of seldom seeing, and is relevant with the autoimmunity phenomenon usually.For treat the patient immune-mediated, acquired simple property red blood cell development is incomplete, that to make according to the present invention and have the anti-CD20 chimeric mAb that improves ADCC and deliver medicine to the patient, as Zecca, M. etc., (its integral body being incorporated herein by reference) described in the Blood 12:3995-97 (1997) at this.Particularly, give the antibody of two dosage of patient of PRCA and autoimmune hemolytic anemia, 375mg/m 2, weekly.Behind the Antybody therapy, begin with intravenously immunoglobulin (Ig) replacement therapy.This treatment has produced the remarkable minimizing of B cell and has followed the hemoglobin level of increase to realize significantly improving of reticulocyte count.
Embodiment 4
Material and method
1. make up the GalT fusion expression vector
The carrier that is used for constitutive expression GalT
In order to make up the GalT expression vector, pass through pcr amplification GalT cDNA from cDNA library (Clontech).With C-terminal c-myc-epi-position mark add and then the upstream of gene terminator (aminoacid sequence: PEQKLISEEDL), in order to measure GalT by western blotting easily later on.After confirming the correct sequence of GalT, gene is inserted in the control of MPSV promotor down, and synthetic rabbit beta Globulin polyadenylation signal is added.Final GalT expression vector also contains the tetracycline resistance box that separates that is useful on selection, has also puromycin resistance gene and the synthetic rabbit beta Globulin polyadenylation signal under the control of MPSV promotor.
With the amino acid replacement coding GalT locating structure territory in coding people GnTI locating structure territory Amino acid.
Carry out the structure of this heterozygosis galactosyltransferase gene, for example,, form the plasmid of GnTI-GalT fusion that contains under the control of MPSV promotor and the tetracycline resistance box that is used to select by overlapping PCR reaction.
Amino acid replacement coding GalT location with coding people mannosidase II locating structure territory The amino acid of structural domain.
Carry out the structure of GalT expression vector.Resulting plasmid contains the heterozygosis manII-GalT gene under the control of MPSV promotor.
Combination heterozygosis manII-GalT fusion gene and duplicating from Epstein Barr virus Starting point oriP.
Advance in the above-mentioned heterozygosis ManII-GalT expression vector as the embodiment 1 described dna fragmentation subclone that will have oriP.
The cd4 cell surface markers gene of combination heterozygosis manII-GalT fusion gene and brachymemma
The modification expression vector is used for the other expression of cd4 cell surface markers gene of brachymemma.Speak briefly, convert heterozygosis manII-GalT fusion gene expression cassette to the bicistronic mRNA expression cassette from monocistron, by and then being encoded, the back inserts the downstream (comprise being used for excretory people CD4 leader sequence, film and ectodomain are and then striden in the back) that manII-GalT merges terminator along the poliovirus IRES sequence of anti-human CD 4 protein matter cDNA.
3. with GalT fusion expression vector and antibody expression vector transfection mammalian cell
The transfection bhk cell
Cultivation as described in example 1 above, collection and transfection subsequently are by the cell (vitality 90-95%) of exponential growth.In order to produce the glycosyl engineered antibody, with two plasmid co-transfection cells, one is used for antibody expression and another and is used to merge the GalT expression of polypeptides, and ratio is 3: 1 separately.
Transfection HEK293-EBNA cell
As the HEK293-EBNA cell of embodiment 1 described transfection by exponential growth.In order to produce the glycosyl engineered antibody, with two plasmid co-transfection cells, one is used for antibody expression and another and is used to merge the GalT expression of polypeptides, and ratio is 4: 1 separately.After the transfection 5 days, collect supernatant liquor, at 1200rpm centrifugal 5 minutes, then 4000rpm centrifugal 10 minutes for the second time and remain in 4 ℃.
The generation of the stable mammal cell line that express recombinant anti-CD20 antibodies and GalT merge
By the clone BHK-1502-28 of electroporation with expression vector transfection constitutive expression anti-CD-20 monoclonal antibody gene and neomycin resistance gene.Carrier allows the constitutive expression of the people CD4 of ManII-GalT gene and clipped form, and the latter is that the IRES dependency is expressed.Carrier also contains the puromycin resistance gene expression cassette.At first select the tetracycline resistance clone to obtain the clone of a cover karyomit(e) integrative vector DNA.Brachymemma CD4 (tCD4) surface expression of screening and cloning then, it is as the mark of bicistronic mRNA ManII-GalT+tCD4 genetic expression unit expression level.Use ELISA to test the generation of checking selected clone's recombinant antibodies.
As described in example 1 above carry out transfection and the screening of tCD4 expression level subsequently.
4. the production and the purifying of unmodified antibody and glycosyl engineered antibody
With the antibody expression vector transfection or add with antibody expression vector in the situation of GalT fusion expression vector cotransfection bhk cell, after after the transfection cells transfected being cultivated 96h hour, collect culture supernatants.According to the productivity of expection, same vehicle is carried out several electroporation (10-15).
With the antibody expression vector transfection or add with antibody expression vector in the situation of GalT fusion expression vector cotransfection HEK293-EBNA cell, transfection is replaced substratum with fresh culture after about 16 hours, further cultivates transfectional cell then and collects the substratum that the back adds after 120 hours.
For stable BHK-1502-28-11 clone, collect supernatant liquor with 500,000 cells/ml inoculation culture thing and after cultivating 4 days.
Antibody purification
Use two continuous chromatography steps monoclonal antibody purification from culture supernatants, as described in example 1 above a-protein chromatography and cation-exchange chromatography.
5. oligosaccharides analysis
Separate the release oligosaccharides by PNGaseF digestion from abzyme, antibody is fixed on the pvdf membrane or in solution.
The resulting Digestive system that discharges oligosaccharides that contains is used for the MALDI/TOF-MS analysis or further used the EndoH Glycosylase to digest before preparation MALDI/TOF-MS analytic sample direct the preparation.
As described in example 1 above, carry out the oligosaccharides method for releasing of antibody in the oligosaccharides method for releasing of pvdf membrane sessile antibody and the solution.
The PNGaseF of endoglycosidase H digestion discharges oligosaccharides with heterozygosis glycosylation oligosaccharide structure branch The purposes at dispensing MALDI/TOF-MS neutral oligosaccharides peak
The oligosaccharides of using endoglycosidase H (EC 3.2.1.96) digestion PNGase to discharge subsequently as described in example 1 above.
MALDI/TOF-MS
Preparation as described in example 1 above contains the enzymolysis, digestion liquid sample that discharges oligosaccharides and carries out the MALDI/TOF mass spectrum subsequently.
Cell preparation with separate
Basically use Histopaque-1077 (Sigma DiagnosticsInc., St.Louis, MO63178 USA) preparation peripheral blood lymphocytes (PBMC) according to the guidance and the method described in the embodiment 1 of manufacturers.
Use negative system of selection as described in example 1 above from PBMC, to isolate NK cells of human beings.
8.ADCC test
The PBMC of aforesaid preparation action effect cell or NK and the toxic ability of their mediated cells of test in antibody dependent cellular cytotoxicity (ADCC) test as described in example 1 above.
9.NK Fc γ RIIIA combination on the cell and the Fc γ RIIb combination on the Raji lymphocyte
Fc γ RIIIA combination on the NK cell of mensuration fresh separated as described in example 1 above and the Fc γ RIIb combination on the Raji lymphocyte.
10. CDC test
Use antibody diluent to carry out the CDC test according to the method described in the embodiment 1.
Result and discussion
In order to prove with respect to the cell that does not produce this polypeptide, coding has the influence of genetic expression pair cell vitality, cell growth or the antibody production of the active polypeptide of GalT, tests.
The glycosylation pattern that passes through MALDI/TOF-MS analysis antibody purification as described in example 1 above.Use this technology, can measure the ratio of the different oligosaccharides kinds in the total original Fc oligosaccharides colony, structure can also be distributed to peak different in the mass spectrum (Umana, P. etc., NatureBiotechnol.17:176-180 (1999)).
Measure the characteristic formp of unmodified antibody oligosaccharides.Particularly, whether mensuration mainly forms galactosylation, the core fucosylation, Composite Double feeler oligosaccharides by the through engineering approaches of the antibody produced cell of wild-type GalT expression.Whether the through engineering approaches of also measuring the antibody produced cell of the expression of nucleic acid by the GnTI-GalT fusion polypeptide (wherein the GalT catalyst structure domain is localized by GnTI golgi body locating structure territory) of encoding mainly forms the Composite Double feeler oligosaccharides of galactosylation with respect to wild-type GalT.If fucosylation structure galactosylation, non-and galactosylation, hybrid structure is synthetic, these are formed by GalT and other glycosyltransferase or Glycosylase competition.In a single day expection has modified oligosaccharides by the semi-lactosi that the catalytic reaction of GalT adds, and α 1, and 6-core fucosyltransferase, golgi body alpha-Mannosidase II (ManII) and GnTII no longer work and modify the oligosaccharides of galactosylation.
The EndoH Glycosylase that use can be offered an explanation heterozygosis and composite oligosaccharide digests the ratio of the galactosylation of estimating Fc and being connected, non-fucosylation oligosaccharides and galactosylation heterozygosis oligosaccharides.
Test the through engineering approaches of antibody produced cell of measuring the expression of nucleic acid by coding ManII-GalT fusion polypeptide (wherein the GalT catalyst structure domain is localized by ManII golgi body locating structure territory) and whether mainly form galactosylation, non-fucosylation and galactosylation, heterozygosis.Particularly, measure with respect to wild-type GalT and GnTI-GalT and merge, whether ManII-GalT merges in the galactosylation, non-fucosylation oligosaccharides and the galactosylation that synthesize the Fc connection, heterozygosis oligosaccharides more effective.
As mentioned above, use endoglycosidase H (EndoH) to confirm that non-fucosylation structure of galactosylation and galactosylation hybrid structure distribute to observed different oligosaccharides peak in the MALDI characteristic pattern.In having the oligosaccharides galactose residue of galactose residue, measured the per-cent of those non-fucosylation hybrid structure, fucosylation heterozygosis and fucosylation composite oligosaccharide structure.
The oligosaccharides substrate of having measured the GalT expression level and being used for the certain position structural domain of GalT catalyst structure domain target golgi body is modified GnTI is in reorganization GalT catalyst structure domain and endogenous core α 1, the influence of competing between 6-fucosyltransferase, ManII and the GnTII enzyme.Measured the level of the antibody dependent cellular cytotoxicity (ADCC) that antibody produced cell amplifying nucleic acid overexpression causes, this nucleic acid encoding has the active polypeptide by the localized GalT in different locating structures territory.
Also measured the glycosyl engineered antibody that produces by the expression of antibody produced cell amplifying nucleic acid and whether had activating the binding affinity of Fc acceptor Fc γ RIIIA or be used to suppress Fc γ RIIb with the people of raising, this nucleic acid encoding has the active fusion polypeptide of GalT.
The GalT construct will be competed endogenous core α 1, the activity of 6-fucosyltransferase, and ADCC is also improved in the Fc district of glycosyl engineered antibody therefore.
Measured the oligosaccharides characteristic formp of the anti-CD20 antibodies of bhk cell generation, bhk cell grows in the suspension and through engineering approaches is come composing type high level expression recombinant antibodies and had the active fusion polypeptide of GalT.Also measured oligosaccharides relative percentage by the glycosylation monoclonal antibody of stablizing the generation of BHK-1502-28-11 clone.
Embodiment 5
Material and method
1.ManII and the structure of GnTII expression vector
In order to make up the ManII expression vector, people's mannosidase II (SEQ ID NO:17) cDNA subclone is entered the downstream of MPSV promotor in the expression vector plasmid and the upstream of synthetic rabbit beta Globulin polyadenylation signal.For GnTII expresses, used at the upstream subclone of the downstream of people CMV promotor/enhanser and Trobest polyadenylation signal the expression vector plasmid of people GnTIIcDNA.
Combination expression vector and from the replication origin oriP of Epstein Barr virus.
Advance to obtain ManII expression vector pCLF9 in the above-mentioned ManII expression vector as the embodiment 1 described dna fragmentation subclone that will have oriP.Advance to obtain GnTII expression vector pGnTII in the above-mentioned GnTII expression vector as the embodiment 1 described dna fragmentation subclone that will have oriP.
2. transfection HEK293-EBNA cell
Transfection as described in example 1 above is by the HEK293-EBNA cell of exponential growth.In order to produce unmodified antibody " Cwt ", with antibody expression vector (pETR1520) transfectional cell.In order to produce glycosyl engineered antibody " Cbrt ", with two plasmid co-transfection cells, one is used for antibody expression (pETR1520) and another is used to merge GnTIII expression of polypeptides (pETR1519), and ratio is 4: 1.In order to produce glycosyl engineered antibody " Cm ", with three plasmid co-transfection cells, one is used for antibody expression (pETR1520), and one is used to merge GnTIII expression of polypeptides (pETR1519), with one be used for mannosidase II and express (pCLF9), ratio is 3: 1: 1.In order to produce glycosyl engineered antibody " Cmg ", with four plasmid co-transfection cells, one is used for antibody expression (pETR1520), one is used to merge GnTIII expression of polypeptides (pETR1519), one is used for mannosidase II and expresses (pCLF9), with one be used for GnTII and express (pGnTII), ratio is 4: 0.33: 0.33: 0.33.
3. the production and the purifying of unmodified antibody and glycosyl engineered antibody
The substratum of above-mentioned transfectional cell is replaced in transfection with fresh culture after about 16 hours, collect afterwards substratum after 120 hours further cultivating transfectional cell then.The supernatant liquor of collecting centrifugal 5 minutes at 1200rpm is then 4000rpm centrifugal 10 minutes for the second time and be stored in 4 ℃.
Antibody purification
Use two continuous chromatography steps monoclonal antibody purification from culture supernatants, as described in example 1 above a-protein chromatography and cation-exchange chromatography.For antibody cwt7, cbrt5 and cm1, after cation-exchange step, use Superdex200 post (AmershamPharmacia) to carry out other size exclusion chromatography step and add phosphate buffered saline (PBS), collect the monomeric igg peak.
4. oligosaccharides analysis
The abzyme that passes through in the PNGaseF digestion solution is as described in example 1 above separated the oligosaccharides of release.
The PNGaseF of endoglycosidase H digestion discharges oligosaccharides with the heterozygosis oligosaccharide structure branch of halving The purposes at dispensing MALDI/TOF-MS neutral oligosaccharides peak
The oligosaccharides of using endoglycosidase H (EC 3.2.1.96) digestion PNGaseF to discharge subsequently as described in example 1 above.
MALDI/TOF-MS
Preparation as described in example 1 above contains the enzymolysis, digestion liquid sample that discharges oligosaccharides and moves on the MALDI/TOF mass spectrograph subsequently.
Cell preparation with separate
Basically use Histopaque-1077 (Sigma Diagnostics Inc., St.Louis, MO63178 USA) preparation peripheral blood lymphocytes (PBMC) according to the guidance and the method described in the embodiment 1 of manufacturers.
6.NK cellular segregation
Use negative system of selection as described in example 1 above from PBMC, to isolate NK cells of human beings.
7.ADCC test
Aforesaid PBMC or NK and the toxic ability of their mediated cells of test in antibody dependent cellular cytotoxicity (ADCC) test as described in example 1 above that makes the action effect cell.
8.NK the Fc γ RIIIA combination on the cell
Fc γ RIIIA combination on the NK cell that is determined at fresh separated as described in example 1 above and the combination of Fc γ RIIb.
9. CDC test
Use antibody diluent to carry out the CDC test according to the method described in the embodiment 1, use following change for preparation people complement source.Tout court, from healthy volunteer's blood, make normal human serum (NHS).Made blood coagulation 1 hour, centrifugal 20 minutes then at 1200g.With acellular supernatant liquor serum five equilibrium and be stored in-80 ℃.Whole test volume with 20% uses NHS.
Result and discussion
Cotransfection cultivation by mammalian cell makes chimeric IgG1 antibody (the C2B8 chimeric antibody of anti-CD20, be also referred to as rituximab) the glycosyl engineered forms, mammalian cell is with the carrier of expressing antibodies gene and express the carrier cotransfection that coding has the gene of GnTIII and the active polypeptide of mannosidase II.Cultivate the other glycosyl engineered antibody form that also makes by mammiferous cotransfection, mammalian cell is with the carrier of expressing antibodies gene and express the carrier cotransfection of the gene with GnTIII activity, mannosidase II activity and the active polypeptide of GnTII of encoding.In order to produce glycosyl engineered antibody " Cbrt ", with two plasmid co-transfection cells, one is used for antibody expression (pETR1520) and another is used to merge GnTIII expression of polypeptides (pETR1519).In order to produce glycosyl engineered antibody " Cm ", with three plasmid co-transfection cells, one is used for antibody expression (pETR1520), and one is used to merge GnTIII expression of polypeptides (pETR1519) and one and is used for mannosidase II and expresses (pCLF9).In order to produce glycosyl engineered antibody " Cmg ", with four plasmid co-transfection cells, one is used for antibody expression (pETR1520), and one is used to merge GnTIII expression of polypeptides (pETR1519), and one is used for mannosidase II and expresses (pCLF9) and one and be used for GnTII expression (pGnTII).Only come unmodified (the non-glycosyl through engineering approaches) form " Cwt " of production same antibody with the carrier transfection mammalian cell of expressing antibodies gene.Transfectional cell was kept 5 days purifying secreted recombinant antibodies from substratum in culture.With respect to the cell that does not produce such glycosyltransferase or Glycosylase polypeptide, genetic expression pair cell vitality, cell growth and antibody producing that coding has GnTIII and the active polypeptide of ManII have no significant effect.
Analyze the glycosylation pattern of antibody purification then.These antibody have the N-that only is connected in human IgG1 Fc district Asn297 residue and connect oligosaccharides.Separate from abzyme by PNGaseF digestion and to remove oligosaccharides and analyze by MALDI/TOF-MS subsequently.Use this technology, can measure the ratio of the different oligosaccharides kinds in the total original Fc oligosaccharides colony, structure can also be distributed to peak different in the mass spectrum (Umana, P. etc., Nature Biotechnol.17:176-180 (1999)).
Figure 26 has shown the neutral oligosaccharides MALDI/TOF-MS characteristic pattern of the chimeric IgG1 antibody of the anti-CD20 of unmodified recombinant C 2B8 Cwt.Described in embodiment 1, Fig. 5 before for unmodified antibody, Cwt has typical oligosaccharides characteristic pattern, m/ z 1485,1648 and 1810 peak, respectively and to have a composite oligosaccharide of two feelers, core fucosylation of 0,1 and 2 galactose residue consistent.The antibody produced cell through engineering approaches of the nucleic acid by expressing coding ManII-GnTIII fusion polypeptide (wherein by location, ManII golgi body locating structure territory GnTIII catalyst structure domain) causes producing antibody (Cbrt), and wherein most of Fc oligosaccharides is (referring to Figure 27) of bisection, non-fucosylation heterozygosis.As described in example 1 above, endoglycosidase H (EndoH) the non-fucosylation structure that is used for confirm halving is distributed to viewed different oligosaccharides peak in the MALDI characteristic pattern with the bisection hybrid structure.
Cause producing antibody (Cm) by the nucleic acid of coexpression coding ManII-GnTIII fusion polypeptide (wherein by location, ManII golgi body locating structure territory GnTIII catalyst structure domain) and the antibody produced cell through engineering approaches of the nucleic acid of coding ManII, wherein most of Fc oligosaccharides is bisection, non-fucosylation compound (referring to Figure 28).The antibody produced cell through engineering approaches of the nucleic acid of nucleic acid, the nucleic acid of coding ManII and the GnTII that encodes by coexpression coding ManII-GnTIII fusion polypeptide (wherein by location, ManII golgi body locating structure territory GnTIII catalyst structure domain) causes producing antibody (Cmg), wherein most of Fc oligosaccharides be halve, non-fucosylation compound, the oligosaccharides part that halve, the compound Fc of non-fucosylation connects even be higher than antibody Cm's.
Figure 29 has shown the data that proved the antibody dependent cellular cytotoxicity (ADCC) that improves, the ADCC of this raising is expressed in antibody produced cell by the nucleic acid of coding ManII-GnTIII fusion polypeptide (wherein by location, ManII locating structure territory GnTIII catalyst structure domain) to form, wherein the ManII-GnTIII coding nucleic acid be own (antibody Cbrt) expression or with the nucleic acid of the ManII (Cm) that encodes coexpression together in antibody produced cell.Therefore, the content that improves heterozygosis in the glycosyl engineered antibody Fc district or the compound non-fucosylation oligosaccharides of bisection will cause the active raising of ADCC.
Known natural killer (NK) cell is the important media of ADCC.These cells carry on its surface and activate Fc γ receptor II IA, are also referred to as CD16a.On the Fc district of target cell binding antibody and the NK cell combination of Fc γ RIIIA acceptor for the crosslinked of these acceptors on the NK cell and subsequently inducing of ADCC be essential.Therefore estimate antibody that said method produces and Fc acceptor especially wherein people's immunoeffectors molecular display their combination of natural form acceptor be important.Figure 30 proved have by coding in antibody produced cell that glycosyl engineered antibody that the expression of nucleic acids of the active fusion polypeptide of GnTIII produces has a raising activate the affinity combination of Fc acceptor Fc γ RIIIA with the people, this nucleic acid oneself (antibody Cbrt) is expressed or and the nucleic acid of the ManII (Cm) that encodes coexpression together in antibody produced cell.Aforesaid these antibody have the bisection of improving the standard, non-fucosylation oligosaccharides for the ADCC test, and oligosaccharides forms by having the expression of the active fusion polypeptide of GnTIII in antibody produced cell.
Therefore, the level that improves heterozygosis in the glycosyl engineered antibody Fc district or the compound non-fucosylation oligosaccharides of bisection will cause the active raising of ADCC.Used NK cell expresses on comfortable its NK cell the genotype donor (Metes, D. etc., J.Immunol.Methods 258 (1-2): 85-95 (2001)) of Fc γ RIIc acceptor in this test.Therefore having only the Fc acceptor on these cell surfaces is to activate Fc γ RIIIA acceptor.
The binding domains of activation Fc γ RIIIB acceptor and Fc γ RIIIA are much at one.Therefore, above-mentioned data have shown that also glycosyl engineered antibody described herein can improve by effector cell such as the cell-mediated effector function of polymorphic nucleus (PMN) of showing Fc γ RIIIB, the release and the phagolysis ((Reff that comprise toxic product, M.E. and Heard, C.Crit RevOncol Hematol.40 (1): 25-35 (2001), Daeron, FM.Annu.Rev.Immunol.15:203-34 (1997), Ravetch, J.V. and Bolland S.Annu.Rev.Immunol.19:275-90 (2001)).
Also detected the complement-mediated cracking (CML) of the anti-CD20 antibodies Cbrt that produces in the through engineering approaches cell, this project cell is used to express coding, and to have a GnTIII active and be positioned the nucleic acid of the fusion polypeptide of golgi body by ManII locating structure territory, and CML is the different effect subfunction that does not rely on Fc acceptor on the immune effector cell.Most oligosaccharides of this glycosyl engineered antibody are the non-fucosylation types of bisection heterozygosis.Cwt compares with unmodified antibody, observes the CML activity (Figure 31) that Cbrt antibody reduces.Use for some, having raising ADCC and having the antibody that reduces CML is ideal, for example reduces the side effect by the CML mediation, as the vasculitis in the tumor locus blood vessel.Other remarkable side effect (van der Kolk L.E. etc., Br J Haematol.115 (4): 807-11 (2001)) of CML mediation has been observed in treatment for anti-CD20 antibodies.Yet, can produce the glycosyl engineered antibody, this antibody has the active and Fc γ RIII combination of ADCC of raising with respect to unmodified antibody, but does not significantly reduce the CML activity, as in the situation of antibody Cm (Figure 31).It is ideal that such antibody needs high ADCC and high complement activation and the active application of CML for the elimination of maximum target cell wherein.Above-mentioned oligosaccharides characteristic formp shown can be by GnTIII fusion polypeptide and coding ManII (antibody Cm) nucleic acid together the nucleic acid of coexpression or GnTIII fusion polypeptide and coding ManII and coding GnTII (antibody Cmg) nucleic acid together coexpression cell engineeringization is produced antibody, wherein the oligosaccharides that most of Fc connects in the antibody is the bisection of non-heterozygous, non-fucosylation are compound.The glycosyl engineered antibody has the ADCC activity of the raising relevant with the level of the non-fucosylation oligosaccharides raising of halving and the Fc γ RIII binding affinity that improves, and because composite oligosaccharide has partly improved their CML activity with respect to the increase partly of heterozygosis oligosaccharides.
This and embodiment have before described the expression of fusion polypeptide coding nucleic acid, wherein fusion polypeptide is positioned Golgi complex and has the catalyst structure domain of competing with the endogenous fucosyltransferase, the oligosaccharides acceptor of modifying by the catalytic reaction of GnTI before being used for.The recombinant glycoprotein that produces by this project host cell has the non-fucosylation oligosaccharides of improving the standard.The nucleic acid that this embodiment has proved coding ManII and/or GnTII nucleic acid and the above-mentioned coding fusion polypeptide coexpression in this host cell together causes the increase of biosynthesizing flux towards composite oligosaccharide, rather than heterozygosis oligosaccharides, and the non-fucosylation composite oligosaccharide that therefore causes synthetic glycoprotein to have improving the standard, with respect to the glycoprotein that produces in the cell that does not have coexpression coding ManII and/or GnTII nucleic acid.
Embodiment 6
The overexpression of alpha-Mannosidase
Molecular cloning
People's alpha-Mannosidase II
Coding people alpha-Mannosidase II (" hManII ") gene (SEQ ID NO:17) (E.C.3.2.1.114), under the control of MPSV promotor, the clone advances to contain in the expression vector of OriP element.Resulting expression vector pCLF9 is shown among Figure 32 A.The expression vector of coding light chain of anti-CD-20 monoclonal antibody and heavy chain respectively do for oneself pETR1842 and pETR1843 (Figure 32 B and 32C).
Fusion rotein ManII-GalT
Construction of fusion protein as described below (SEQ ID NO:20) is made up of the catalyst structure domain of hManII CTS and people β (1,4)-galactosyltransferase (M22921, amino acid/11 26-397).By PCR from pETR1484 (CF33, GAB252) amplification hManII CTS zone.Use CF31 and CF35 catalyst structure domain (amino acid/11 26-397) from pBlueGalT amplification GalT.The catalyst structure domain of hManII CTS and GalT is merged the fusion rotein (pCLF24) that obtains by the control of MPSV promotor.Obtain whole GalT gene from pBlueGalT.Gene sequencing (SEQ ID NO:16) with coding GalT:
MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSR
LPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDS
SPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDL
ELVAKQNPNVKMGGRYAPRDCVSPHKVAMPFRNRQEHLKYWLYYLHP
VLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDV
DLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLT
INGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKK
NEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS
With 5 ' of CF32/CF38 amplification GalT, and at gene front adding FseI restriction site.Find that by order-checking sequence correctly and by FseI/DraIII digestion exchanges (pCLF26) in pCLF24.OriP added produce pCLF25 and pCLF27 (Figure 33 A and 33B) among pCLF24 and the pCLF26 respectively.
Alpha-Mannosidase and the ManII-GalT expression in the HEK293-EBNA cell
With calcium phosphate method transfection HEK293-EBNA cell.Tout court, for transfection T150, preceding 24 hours of transfection with 1,500 ten thousand cell inoculations in 28ml DMEM, 10%FCS, 250 μ g/ml Xin Meisus and, 5%CO at 37 ℃ 2Overnight incubation.
Treat the T150 culturing bottle of transfection for each, by mixing the CaCl of the total plasmid vector DNA of 94 μ g, 469 μ l 1M 2Solution also adds entry and makes DNA, CaCl to final volume 938 μ l 2Solution with water.The 1.5mM Na that in this solution, adds 938 μ l 50mM HEPES, 280mM NaCl, pH7.05 2HPO 4Solution, and mixed immediately 10 seconds and left standstill 20 seconds in room temperature.Replenish the DMEM diluted suspension of 2%FCS with 24ml, and add alternative existing substratum among the T150.With cell at 37 ℃, 5%CO 2Hatched about 17 to 20 hours, and substitute substratum with the DMEM of 30ml 10%FCS.
In order to test the influence of alpha-Mannosidase II, with ratio separately 2: 2: 1 pETR1842, pETR1843 and pCLF9 transfectional cell to core fucosyltransferase competition.For fused protein ManII-GalT, with ratio separately 2: 2: 1 pETR1842, pETR1843 and pCLF25 transfectional cell.After the transfection 5 days, collect supernatant liquor, at 1200rpm centrifugal 5 minutes, then, filter and be stored in 4 ℃ 4000rpm centrifugal 10 minutes for the second time.
The purifying of anti-CD-20 monoclonal antibody
By two step chromatographies monoclonal antibody purification from the 30ml supernatant liquor, comprise first a-protein chromatography, the Niu Kangti from be present in serum isolates monoclonal antibody, and then cation-exchange chromatography exchanges to PBS with sample buffer.
Oligosaccharides is analyzed
PNGaseF digestion
(reorganization, Roche Switzerland) is hatched monoclonal antibody sample (50 μ g) with the N-Glycosylase F of 0.1mU/ μ l.37 ℃ digested 3 hours in the Tris of 2mM pH7.0 damping fluid.Then with the neutral oligosaccharides that discharges incubated at room 3 hours in 150mM acetic acid.Then with load in a subtle way-biology-rotation chromatographic column (BioRad, Hercules, CA) the 0.6ml Zeo-karb in (the AG50W-X8 resin, hydrogen form, the 100-200 order, BioRad, Hercules is CA) with the sample desalination.
EndoH digestion
Before acetic acid was handled, (EC3.2.1.96, ROCHE, the Switzerland) oligosaccharides that discharges of digestion PNGaseF, endoglycosidase H were the enzymes of the N-n acetylglucosamine n residue of the division N chitobiose core that connects oligosaccharides with endoglycosidase H.Enzyme will decompose few seminose, and majority is the heterozygous glycan, and compound oligosaccharides does not obtain hydrolysis.
With the digestion of the 0.2mU/ μ l endoglycosidase H among 2mM pH7.0 Tris oligosaccharides.Digestion was carried out 3 hours at 37 ℃.Oligosaccharides incubated at room 3 hours in 150mM acetic acid is subsequently with loading in a subtle way-biology-rotation chromatographic column (BioRad, Suitzerland) 0.6ml Zeo-karb (the AG50W-X8 resin in, hydrogen form, the 100-200 order, BioRad is Switzerland) with the sample desalination.
Matrix and specimen preparation
By with 2 of 2mg, make 2,5-resorcylic acid matrix (sDHB) in the 5-methoxyl group Whitfield's ointment of 5-resorcylic acid+0.1mg the is dissolved in 1ml ethanol/10mM sodium chloride aqueous solution of 1: 1 (v/v).1 μ l sample is used to the stainless steel target, and and 1 μ l sDHB mixing.Sample air is dry and use 0.2 μ l ethanol.
MALDI/TOF analyzes
Being used for obtaining mass spectral MALDI-TOF mass spectrograph is to have equipped the VoyagerElite (Perspective Biosystems) that time-delay is extracted.Operate this device in the reverberator mode.Positively charged ion is postponed after-acceleration to 20kV at 75ns.Five mass spectrums (200 emissions) merging of 40 emissions is obtained final mass spectrum.Use the external perimysium reference of oligosaccharides standard to be used for the distribution of ionic mass spectrum.
The oligosaccharides characteristic formp
The oligosaccharides characteristic formp of the anti-CD20 antibodies that produces in the presence of ManII is shown among Figure 34.The oligosaccharides of discovery and antibody Fc part correlation is a composite structure, and wherein 48% lacks the core Fucose.Alpha-Mannosidase II and the competition of core fucosyltransferase produce 48% non-fucosylation oligosaccharide structure.Under alpha-Mannosidase II disappearance, the oligosaccharides of antibody Fc part only comprises the structure (wild-type antibody) of fucosylation.
The oligosaccharides characteristic formp of the anti-CD20 antibodies that produces in the presence of the ManII-GalT fusion rotein is shown among Figure 35 A-B.For alpha-Mannosidase II, also have under ManII-GalT fusion rotein situation, the amount of non-fucosylation oligosaccharide structure has increased.The high per-cent of non-fucosylation structure is consistent with high mannose structures (m/ z 1256,1419 and 1581).For this 67% non-fucosylation sugar, found other 30% the non-fucosylation structure of heterozygosis (m/z1622).Therefore, the sample that produces in the presence of the ManII-GalT fusion rotein has shown almost 100% non-fucosylation structure.
The biological activity of the antibody that in the presence of alpha-Mannosidase II or ManII-GalT fusion rotein, produces
In order to measure the effectiveness of alpha-Mannosidase II and ManII-GalT enzyme effect in the competition of core fucosyltransferase, carried out the associated biomolecule test.Tested sample external antibody dependent cellular cytotoxicity (ADCC) and and through engineering approaches Chinese hamster ovary celI system (CHO-1708-37) surface on the combination of CD16 acceptor of expression.
IgG combination on CHO-1708-37
CHO-1708-37 clone is expressed the γ chain of Fc γ RIIIA acceptor (CD16) and Fc γ RI acceptor in its surface.Use the 3G8-FITC monoclonal antibody to test the expression (Figure 36) of Fc γ RIIIA acceptor (CD16) by facs analysis.In the PBS of 0.1%BSA, hatch 1Mio/ml CHO-1708-37 cell, use the different antibodies variant of different concns (10,3,1,0.3,0.1 μ g/ml), triplicate.Cell is washed at 4 ℃ of PBS of hatching 30 minutes and using subsequently 0.1%BSA.By the F (ab ') that puts together with 1: 200 fluorescein isothiocyanate 2The anti-human IgG of goat (PA) hatch at 4 ℃ and detected antibodies in 30 minutes by Jackson Immuno Reasearch, West Grove.(BDBioscience, San Jose CA) measure the fluorescence intensity that relates to the binding antibody variant on the FACS of gate viable cell Calibur.
Following antibody variants is included in conjunction with in the test experiment:
Cwt8 (wild-type 1)
ManII (alpha-Mannosidase II)
The antibody that produces in the presence of alpha-Mannosidase II (Man II) and the binding affinity of Fc γ RIIIA acceptor are than the height of wild-type antibody.
External antibody dependent cellular cytotoxicity (ADCC)
Use Histopaque-1077 (Sigma Diagnostics Inc., St.Louis, MO63178 USA) also to get peripheral blood lymphocytes (PBMC) according to the guidance system of manufacturers basically.Tout court, take venous samples can with the heparinization syringe from the volunteer, the volunteer is required to run 1 minute with all strength, so that improve the per-cent of the natural killer cell (NK) in the blood., and be laid on the Histopaque-1077 blood thinning 1: 0.75-1.3 with the PBS that does not contain Ca or Mg.In room temperature (RT) in the unremitting gradient centrifugation of 400g 30 minutes.Collection contains the intermediate phase of PBMC and collected in centrifugal 10 minutes with PBS washing (from every 50ml cell of two gradients) and by RT300g.With PBS with pellet resuspended after, washed in centrifugal 10 minutes for the second time with PBMC counting and by RT 200g.Then cell is resuspended to the program that is used in the suitable medium subsequently.
Washing Raji target cell in PBS, counting also is resuspended to DMEM, 10%FCS, 1%Glutamax with every ml 1Mio.Add 1 in these: 100calcein is also hatched cell 30 minutes at 37 ℃.Washed cell in PBS then, counting and every ml 0.3Mio are resuspended among the AIM-V.This cell suspending liquid of 100 μ l is added in each hole of round bottom 96 hole flat boards.In AIM-V, dilute antibody, and 50 μ l are added in the pre-incubated target cell and at RT and target in conjunction with 10 minutes.As above make the PBMC of action effect cell.Effector is 25: 1 to the ratio of target cell.In AIM-V, make the effector cell and 50 μ l are added in each hole with every ml 15Mio.Flat board is being contained 5%CO 2Moistening atmosphere in 37 ℃ hatched 4 hours.With cell washed cell and add 200 μ l borate solutions in PBS.Measure kill (Figure 37) that measures target cell with the calcein that in substratum, discharges after the borate solution cracking.
From only containing target cell and effector cell but do not have and measure spontaneous release the hole of antibody.From the hole of only containing target cell and 1%Triton X-100, measure maximum release.The per-cent of killing of following calculating specific antibodies mediation: ((x-SR)/(MR-SR) * 100, wherein x is the average Vmax in specific antibodies concentration, and SR is the average Vmax of spontaneous release, and MR is the maximum average Vmax that discharges.
Embodiment 7
The purposes of the monoclonal antibody against EGFR of glycosyl through engineering approaches in the treatment psoriasis
Can treat psoriasis people patient with the glycosyl through engineering approaches monoclonal antibody against EGFR of prepared in accordance with the method for the present invention.Especially, the patient to accept capacity value weekly be 400mg/m 2The infusion of glycosyl engineered antibody.With week be basic administration 250mg/m 2Maintenance dosage until obtaining complete reaction.
Embodiment 8
The purposes of glycosyl through engineering approaches anti-ErbB monoclonal antibody in treatment metastatic prostate cancer, transitivity breast cancer, metastatic colorectal cancer and IIIb phase or V phase nonsmall-cell lung cancer
RhuMAb2C4 is total length, the Humanized monoclonal antibodies (producing at Chinese hamster ovary celI) of facedown ErbB2.RhuMab2C4 has blocked the related of ErbB2 and other ErbB family member, has therefore suppressed the extracellular signal by the ErbB approach.RhuMAb2C4 has not only suppressed ErbB2 overexpression growth of tumor, but also has blocked the growth of tumor that needs ErbB ligand dependent signal.
The RhuMAb2C4 of the glycosyl engineered forms that makes by the inventive method can be used for the treatment of carcinoma of prostate patient, transitivity patients with mastocarcinoma, metastatic colorectal cancer patient and IIIb phase or the IV phase nonsmall-cell lung cancer patient that hormone is difficult to treatment (male sex hormone independence) as single medicament.Particularly, with glycosyl through engineering approaches RhuMAb2C4 weekly or per three all intravenouslys (IV) administration 2 or 4mg/kg, stop until progression of disease.Replenish antibody (20mL is full of concentration 20mg/mL or higher concentration) with the multiple doses liquid preparation.
Be clear that and implement the present invention with the special different modes of describing among specification sheets and the embodiment before.According to above-mentioned instruction various modifications of the present invention and change is possible, therefore within the scope of the appended claims.
Whole openly being hereby incorporated by at these all publications of quoting (comprising patent, patent application, periodical literature, laboratory manual, books or other document).
Sequence table
<110>GlycArt Biotechnology AG
Umana，Pablo
Bruenker，Peter
Ferrara，Claudia
Suter，Tobias
＜120〉fusion constructs and be used for producing the Fc receptor binding affinity
The purposes of the antibody that improves with effector function
<130>1975.018PC04
<150>US 60/441,307
<151>2003-01-22
<150>US 60/491,254
<151>2003-07-31
<150>US 60/495,142
<151>2003-08-15
<160>20
<170>PatentIn version 3.2
<210>1
<211>11
<212>PRT
＜213〉unknown
<220>
＜223〉c-myc epi-position tag
<400>1
Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210>2
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-177 PCR primer
<400>2
gcgtgtgcct gtgacccccg cgcccctgct ccagccactg tcccc 45
<210>3
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-178 PCR primer
<400>3
gaaggtttct ccagcatcct ggtacc 26
<210>4
<211>43
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-179 PCR primer
<400>4
ctgaggcgcg ccgccaccat gctgaagaag cagtctgcag ggc 43
<210>5
<211>48
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-180 PCR primer
<400>5
ggggacagtg gctggagcag gggcgcgggg gtcacaggca cacgcggc 48
<210>6
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-252 PCR primer
<400>6
gctaggccgg ccgccaccat gaagttaagc cgccagttca ccgtgttcgg 50
<210>7
<211>65
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-253 PCR primer
<400>7
ggggacagtg gctggagcag gggtgagcca gcaccttggc tgaaattgct ttgtgaactt 60
ttcgg 65
<210>8
<211>66
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-254 PCR primer
<400>8
tccgaaaagt tcacaaagca atttcagcca aggtgctggc tcacccctgc tccagccact 60
gtcccc 66
<210>9
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-255 PCR primer
<400>9
atgccgcata ggcctccgag caggacccc 29
<210>10
<211>43
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-261 PCR primer
<400>10
gctaaatatt gaattccctt tatgtgtaac tcttggctga agc 43
<210>11
<211>48
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAB-262 PCR primer
<400>11
tagcaatatt gaattcgcag gaaaaggaca agcagcgaaa attcacgc 48
<210>12
<211>1715
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of GnTI-GnTIII
<400>12
atgaagttaa gccgccagtt caccgtgttc ggcagtgcga tcttctgtgt ggtgattttc 60
tcgctctacc tgatgctgga ccggggtcac ttagactacc ccaggaaccc gcgccgcgag 120
ggctccttcc ctcagggcca gctctcaatg ttgcaagaaa aaatagacca tttggagcgt 180
ttgctagctg agaataatga gatcatctca aatattagag actcagtcat caatttgagt 240
gagtctgtgg aggatggtcc gaaaagttca caaagcaatt tcagccaagg tgctggctca 300
cccctgctcc agccactgtc ccctagcaag gccaccgaag aactgcaccg ggtggacttc 360
gtgttgccgg aggacaccac agagtatttt gtgcgcacca aagctggcgg tgtgtgcttc 420
aaaccaggta ccaggatgct ggagaaacct tctccagggc ggacagagga gaagaccaag 480
gtggctgagg ggtcctcggt ccggggtcct gctcggaggc ctatgcggca tgtgttgagt 540
gcacgggagc gcctgggagg ccggggcact aggcgcaagt gggttgagtg tgtgtgcctg 600
ccaggctggc acgggcccag ctgcggggtg cccactgtgg tccagtattc caacctgccc 660
accaaggagc gcctggtacc cagggaggtg ccgaggcggg ttatcaacgc catcaacatc 720
aaccatgagt tcgacctgct ggatgtgcgc ttccatgagc tgggcgatgt tgtggacgcc 780
tttgtggtct gcgaatccaa tttcaccgcc tacggggagc ctcggccgct caagttccga 840
gagatgctga ccaatggcac cttcgagtac atccgccaca aggtgctcta cgtcttcctg 900
gaccacttcc cacctggtgg ccgtcaggac ggctggattg cagacgacta cctgcgtacc 960
ttcctcaccc aggatggtgt ctcccgcctg cgcaacctgc gacctgatga cgtctttatc 1020
atcgacgacg cggacgagat ccctgcgcgt gatggtgtgc tgttcctcaa gctctacgat 1080
ggctggacag agcccttcgc cttccatatg cgcaagtccc tgtatggttt cttttggaag 1140
caaccaggca cacggaggtg gtgtcaggct gcaccattga catgctgcag gctgtgtatg 1200
ggctggacgg catccgcctg cgccgccgtc agtactacac catgcccaac tttcgacagt 1260
atgagaaccg caccggccac atcctagtgc agtggtctct cggcagcccc ctgcacttcg 1320
cgggctggca ctgctcctgg tgcttcacac ccgagggcat ctacttcaaa ctcgtgtcgg 1380
cccagaatgg tgacttcccc cgctggggtg actacgagga caagagggac ctcaattaca 1440
tccgaagctt gattcgcact gggggatggt tcgacggcac gcagcaggag taccctcctg 1500
cagaccccag tgaacacatg tatgctccta agtacctgct caagaactat gaccagttcc 1560
gctacttgct cgaaaatccc taccgggagc ccaagagcac tgtagagggt gggcgccgga 1620
accagggctc agacggaagg tcatctgctg tcaggggcaa gttggataca acggagggcc 1680
cggaacagaa actgatctct gaagaggacc tgtag 1715
<210>13
<211>571
<212>PRT
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of GnTI-GnTIII
<400>13
Met Lys Leu Ser Arg Gln Phe Thr Val Phe Gly Ser Ala Ile Phe Cys
1 5 10 15
Val Val Ile Phe Ser Leu Tyr Leu Met Leu Asp Arg Gly His Leu Asp
20 25 30
Tyr Pro Arg Asn Pro Arg Arg Glu Gly Ser Phe Pro Gln Gly Gln Leu
35 40 45
Ser Met Leu Gln Glu Lys Ile Asp His Leu Glu Arg Leu Leu Ala Glu
50 55 60
Asn Asn Glu Ile Ile Ser Asn Ile Arg Asp Ser Val Ile Asn Leu Ser
65 70 75 80
Glu Ser Val Glu Asp Gly Pro Lys Ser Ser Gln Ser Asn Phe Ser Gln
85 90 95
Gly Ala Gly Ser Pro Leu Leu Gln Pro Leu Ser Pro Ser Lys Ala Thr
100 105 110
Glu Glu Leu His Arg Val Asp Phe Val Leu Pro Glu Asp Thr Thr Glu
115 120 125
Tyr Phe Val Arg Thr Lys Ala Gly Gly Val Cys Phe Lys Pro Gly Thr
130 135 140
Arg Met Leu Glu Lys Pro Ser Pro Gly Arg Thr Glu Glu Lys Thr Lys
145 150 155 160
Val Ala Glu Gly Ser Ser Val Arg Gly Pro Ala Arg Arg Pro Met Arg
165 170 175
His Val Leu Ser Ala Arg Glu Arg Leu Gly Gly Arg Gly Thr Arg Arg
180 185 190
Lys Trp Val Glu Cys Val Cys Leu Pro Gly Trp His Gly Pro Ser Cys
195 200 205
Gly Val Pro Thr Val Val Gln Tyr Ser Asn Leu Pro Thr Lys Glu Arg
210 215 220
Leu Val Pro Arg Glu Val Pro Arg Arg Val Ile Asn Ala Ile Asn Ile
225 230 235 240
Asn His Glu Phe Asp Leu Leu Asp Val Arg Phe His Glu Leu Gly Asp
245 250 255
Val Val Asp Ala Phe Val Val Cys Glu Ser Asn Phe Thr Ala Tyr Gly
260 265 270
Glu Pro Arg Pro Leu Lys Phe Arg Glu Met Leu Thr Asn Gly Thr Phe
275 280 285
Glu Tyr Ile Arg His Lys Val Leu Tyr Val Phe Leu Asp His Phe Pro
290 295 300
Pro Gly Gly Arg Gln Asp Gly Trp Ile Ala Asp Asp Tyr Leu Arg Thr
305 310 315 320
Phe Leu Thr Gln Asp Gly Val Ser Arg Leu Arg Asn Leu Arg Pro Asp
325 330 335
Asp Val Phe Ile Ile Asp Asp Ala Asp Glu Ile Pro Ala Arg Asp Gly
340 345 350
Val Leu Phe Leu Lys Leu Tyr Asp Gly Trp Thr Glu Pro Phe Ala Phe
355 360 365
His Met Arg Lys Ser Leu Tyr Gly Phe Phe Trp Lys Gln Pro Gly Thr
370 375 380
Leu Glu Val Val Ser Gly Cys Thr Ile Asp Met Leu Gln Ala Val Tyr
385 390 395 400
Gly Leu Asp Gly Ile Arg Leu Arg Arg Arg Gln Tyr Tyr Thr Met Pro
405 410 415
Asn Phe Arg Gln Tyr Glu Asn Arg Thr Gly His Ile Leu Val Gln Trp
420 425 430
Ser Leu Gly Ser Pro Leu His Phe Ala Gly Trp His Cys Ser Trp Cys
435 440 445
Phe Thr Pro Glu Gly Ile Tyr Phe Lys Leu Val Ser Ala Gln Asn Gly
450 455 460
Asp Phe Pro Arg Trp Gly Asp Tyr Glu Asp Lys Arg Asp Leu Asn Tyr
465 470 475 480
Ile Arg Ser Leu Ile Arg Thr Gly Gly Trp Phe Asp Gly Thr Gln Gln
485 490 495
Glu Tyr Pro Pro Ala Asp Pro Ser Glu His Met Tyr Ala Pro Lys Tyr
500 505 510
Leu Leu Lys Asn Tyr Asp Gln Phe Arg Tyr Leu Leu Glu Asn Pro Tyr
515 520 525
Arg Glu Pro Lys Ser Thr Val Glu Gly Gly Arg Arg Asn Gln Gly Ser
530 535 540
Asp Gly Arg Ser Ser Ala Val Arg Gly Lys Leu Asp Thr Thr Glu Gly
545 550 555 560
Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
565 570
<210>14
<211>1722
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of ManII-GnTIII
<400>14
atgctgaaga agcagtctgc agggcttgtg ctgtggggcg ctatcctctt tgtggcctgg 60
aatgccctgc tgctcctctt cttctggacg cgcccagcac ctggcaggcc accctcagtc 120
agcgctctcg atggcgaccc cgccagcctc acccgggaag tgattcgcct ggcccaagac 180
gccgaggtgg agctggagcg gcagcgtggg ctgctgcagc agatcgggga tgccctgtcg 240
agccagcggg ggagggtgcc caccgcggcc cctcccgccc agccgcgtgt gcctgtgacc 300
cccgcgcccc tgctccagcc actgtcccct agcaaggcca ccgaagaact gcaccgggtg 360
gacttcgtgt tgccggagga caccacagag tattttgtgc gcaccaaagc tggcggtgtg 420
tgcttcaaac caggtaccag gatgctggag aaaccttctc cagggcggac agaggagaag 480
accaaggtgg ctgaggggtc ctcggtccgg ggtcctgctc ggaggcctat gcggcatgtg 540
ttgagtgcac gggagcgcct gggaggccgg ggcactaggc gcaagtgggt tgagtgtgtg 600
tgcctgccag gctggcacgg gcccagctgc ggggtgccca ctgtggtcca gtattccaac 660
ctgcccacca aggagcgcct ggtacccagg gaggtgccga ggcgggttat caacgccatc 720
aacatcaacc atgagttcga cctgctggat gtgcgcttcc atgagctggg cgatgttgtg 780
gacgcctttg tggtctgcga atccaatttc accgcctacg gggagcctcg gccgctcaag 840
ttccgagaga tgctgaccaa tggcaccttc gagtacatcc gccacaaggt gctctacgtc 900
ttcctggacc acttcccacc tggtggccgt caggacggct ggattgcaga cgactacctg 960
cgtaccttcc tcacccagga tggtgtctcc cgcctgcgca acctgcgacc tgatgacgtc 1020
tttatcatcg acgacgcgga cgagatccct gcgcgtgatg gtgtgctgtt cctcaagctc 1080
tacgatggct ggacagagcc cttcgccttc catatgcgca agtccctgta tggtttcttt 1140
tggaagcaac caggcacact ggaggtggtg tcaggctgca ccattgacat gctgcaggct 1200
gtgtatgggc tggacggcat ccgcctgcgc cgccgtcagt actacaccat gcccaacttt 1260
cgacagtatg agaaccgcac cggccacatc ctagtgcagt ggtctctcgg cagccccctg 1320
cacttcgcgg gctggcactg ctcctggtgc ttcacacccg agggcatcta cttcaaactc 1380
gtgtcggccc agaatggtga cttcccccgc tggggtgact acgaggacaa gagggacctc 1440
aattacatcc gaagcttgat tcgcactggg ggatggttcg acggcacgca gcaggagtac 1500
cctcctgcag accccagtga acacatgtat gctcctaagt acctgctcaa gaactatgac 1560
cagttccgct acttgctcga aaatccctac cgggagccca agagcactgt agagggtggg 1620
cgccggaacc agggctcaga cggaaggtca tctgctgtca ggggcaagtt ggatacaacg 1680
gagggcccgg aacagaaact gatctctgaa gaggacctgt ag 1722
<210>15
<211>573
<212>PRT
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of ManII-GnTIII fusion
<400>15
Met Leu Lys Lys Gln Ser Ala Gly Leu Val Leu Trp Gly Ala Ile Leu
1 5 10 15
Phe Val Ala Trp Asn Ala Leu Leu Leu Leu Phe Phe Trp Thr Arg Pro
20 25 30
Ala Pro Gly Arg Pro Pro Ser Val Ser Ala Leu Asp Gly Asp Pro Ala
35 40 45
Ser Leu Thr Arg Glu Val Ile Arg Leu Ala Gln Asp Ala Glu Val Glu
50 55 60
Leu Glu Arg Gln Arg Gly Leu Leu Gln Gln Ile Gly Asp Ala Leu Ser
65 70 75 80
Ser Gln Arg Gly Arg Val Pro Thr Ala Ala Pro Pro Ala Gln Pro Arg
85 90 95
Val Pro Val Thr Pro Ala Pro Leu Leu Gln Pro Leu Ser Pro Ser Lys
100 105 110
Ala Thr Glu Glu Leu His Arg Val Asp Phe Val Leu Pro Glu Asp Thr
115 120 125
Thr Glu Tyr Phe Val Arg Thr Lys Ala Gly Gly Val Cys Phe Lys Pro
130 135 140
Gly Thr Arg Met Leu Glu Lys Pro Ser Pro Gly Arg Thr Glu Glu Lys
145 150 155 160
Thr Lys Val Ala Glu Gly Ser Ser Val Arg Gly Pro Ala Arg Arg Pro
165 170 175
Met Arg His Val Leu Ser Ala Arg Glu Arg Leu Gly Gly Arg Gly Thr
180 185 190
Arg Arg Lys Trp Val Glu Cys Val Cys Leu Pro Gly Trp His Gly Pro
195 200 205
Ser Cys Gly Val Pro Thr Val Val Gln Tyr Ser Asn Leu Pro Thr Lys
210 215 220
Glu Arg Leu Val Pro Arg Glu Val Pro Arg Arg Val Ile Asn Ala Ile
225 230 235 240
Asn Ile Asn His Glu Phe Asp Leu Leu Asp Val Arg Phe His Glu Leu
245 250 255
Gly Asp Val Val Asp Ala Phe Val Val Cys Glu Ser Asn Phe Thr Ala
260 265 270
Tyr Gly Glu Pro Arg Pro Leu Lys Phe Arg Glu Met Leu Thr Asn Gly
275 280 285
Thr Phe Glu Tyr Ile Arg His Lys Val Leu Tyr Val Phe Leu Asp His
290 295 300
Phe Pro Pro Gly Gly Arg Gln Asp Gly Trp Ile Ala Asp Asp Tyr Leu
305 310 315 320
Arg Thr Phe Leu Thr Gln Asp Gly Val Ser Arg Leu Arg Asn Leu Arg
325 330 335
Pro Asp Asp Val Phe Ile Ile Asp Asp Ala Asp Glu Ile Pro Ala Arg
340 345 350
Asp Gly Val Leu Phe Leu Lys Leu Tyr Asp Gly Trp Thr Glu Pro Phe
355 360 365
Ala Phe His Met Arg Lys Ser Leu Tyr Gly Phe Phe Trp Lys Gln Pro
370 375 380
Gly Thr Leu Glu Val Val Ser Gly Cys Thr Ile Asp Met Leu Gln Ala
385 390 395 400
Val Tyr Gly Leu Asp Gly Ile Arg Leu Arg Arg Arg Gln Tyr Tyr Thr
405 410 415
Met Pro Asn Phe Arg Gln Tyr Glu Asn Arg Thr Gly His Ile Leu Val
420 425 430
Gln Trp Ser Leu Gly Ser Pro Leu His Phe Ala Gly Trp His Cys Ser
435 440 445
Trp Cys Phe Thr Pro Glu Gly Ile Tyr Phe Lys Leu Val Ser Ala Gln
450 455 460
Asn Gly Asp Phe Pro Arg Trp Gly Asp Tyr Glu Asp Lys Arg Asp Leu
465 470 475 480
Asn Tyr Ile Arg Ser Leu Ile Arg Thr Gly Gly Trp Phe Asp Gly Thr
485 490 495
Gln Gln Glu Tyr Pro Pro Ala Asp Pro Ser Glu His Met Tyr Ala Pro
500 505 510
Lys Tyr Leu Leu Lys Asn Tyr Asp Gln Phe Arg Tyr Leu Leu Glu Asn
515 520 525
Pro Tyr Arg Glu Pro Lys Ser Thr Val Glu Gly Gly Arg Arg Asn Gln
530 535 540
Gly Ser Asp Gly Arg Ser Ser Ala Val Arg Gly Lys Leu Asp Thr Thr
545 550 555 560
Glu Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
565 570
<210>16
<211>398
<212>PRT
＜213〉unknown
<220>
＜223〉from the GalT aminoacid sequence of pBlueGalT
<400>16
Met Arg Leu Arg Glu Pro Leu Leu Ser Gly Ser Ala Ala Met Pro Gly
1 5 10 15
Ala Ser Leu Gln Arg Ala Cys Arg Leu Leu Val Ala Val Cys Ala Leu
20 25 30
His Leu Gly Val Thr Leu Val Tyr Tyr Leu Ala Gly Arg Asp Leu Ser
35 40 45
Arg Leu Pro Gln Leu Val Gly Val Ser Thr Pro Leu Gln Gly Gly Ser
50 55 60
Asn Ser Ala Ala Ala Ile Gly Gln Ser Ser Gly Glu Leu Arg Thr Gly
65 70 75 80
Gly Ala Arg Pro Pro Pro Pro Leu Gly Ala Ser Ser Gln Pro Arg Pro
85 90 95
Gly Gly Asp Ser Ser Pro Val Val Asp Ser Gly Pro Gly Pro Ala Ser
100 105 110
Asn Leu Thr Ser Val Pro Val Pro His Thr Thr Ala Leu Ser Leu Pro
115 120 125
Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met Leu Ile Glu
130 135 140
Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala Lys Gln Asn Pro Asn
145 150 155 160
Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp Cys Val Ser Pro His
165 170 175
Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu His Leu Lys
180 185 190
Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln Arg Gln Gln Leu Asp
195 200 205
Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp Thr Ile Phe Asn Arg
210 215 220
Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala Leu Lys Asp Tyr Asp
225 230 235 240
Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro Met Asn Asp
245 250 255
His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg His Ile Ser Val Ala
260 265 270
Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val Gln Tyr Phe Gly Gly
275 280 285
Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr Ile Asn Gly Phe Pro
290 295 300
Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile Phe Asn Arg
305 310 315 320
Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro Asn Ala Val Val Gly
325 330 335
Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn Glu Pro Asn
340 345 350
Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr Met Leu Ser
355 360 365
Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu Asp Val Gln Arg Tyr
370 375 380
Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly Thr Pro Ser
385 390 395
<210>17
<211>3435
<212>DNA
＜213〉people (Homo sapiens)
<400>17
atgaagttaa gccgccagtt caccgtgttc ggcagtgcga tcttctgtgt ggtgattttc 60
tcgctctacc tgatgctgga ccggggtcac ttagactacc ccaggaaccc gcgccgcgag 120
ggctccttcc ctcagggcca gctctcaatg ttgcaagaaa aaatagacca tttggagcgt 180
ttgctagctg agaataatga gatcatctca aatattagag actcagtcat caatttgagt 240
gagtctgtgg aggatggtcc gaaaagttca caaagcaatt tcagccaagg tgctggctca 300
catcttctgc cctcacaatt atccctctGa gttgacactg cagactgtct gtttgcttca 360
caaagtggaa gtcacaattc agatgtgcag atgttggatg tttacagtct aatttctttt 420
gacaatccag atggtggagt ttggaagcaa ggatttgaca ttacttatga atctaatgaa 480
tgggacactg aaccccttca agtctttgtg gtgcctcatt cccataacga cccaggttgg 540
ttgaagactt tcaatgacta ctttagagac aagactcagt atatttttaa taacatggtc 600
ctaaagctga aagaagactc acggaggaag tttatttggt ctgagatctc ttacctttca 660
aagtggtggg atattataga tattcagaag aaggatgctg ttaaaagttt aatagaaaat 720
ggtcagGttg aaattgtgac aggtggctgg gttatgcctg atgaagctac tccacattat 780
tttgccttaa ttgatcaact aattgaagga catcagtggc tggaaaataa tataggagtg 840
aaacctcggt ccggctgggc tattgatccc tttggacact caccaacaat ggcttatctt 900
ctaaaccgtg ctggactttc tcacatgctt atccagagag ttcattatgc agttaaaaaa 960
cactttgcac tgcataaaac attggagttt ttttggagac agaattggga tctgggatct 1020
gtcacagata ttttatgcca catgatgccc ttctacagct atgacatccc tcacacttgt 1080
ggacctgatc ctaaaatatg ctgccagttt gattttaaac gtcttcctgg aggcagattt 1140
ggttgtccct ggggagtccc cccagaaaca atacatcctg gaaatgtcca aagcagggct 1200
cggatgctac tagatcagta ccgaaagaag tcaaagcttt ttcgtaccaa agttctcctg 1260
gctccactag gagatgattt ccgctactgt gaatacacgg aatgggattt acagtttaag 1320
aattatcagc agctttttga ttatatgaat tctcagtcca agtttaaagt taagatacag 1380
tttggaactt tatcagattt ttttgatgcg ctggataaag cagatgaaac tcagagagac 1440
aagggccagt cgatgttccc tgttttaagt ggagattttt tcacttatgc cgatcgagat 1500
gatcattact ggagtggcta ttttacatcc agaccctttt acaaacgaat ggacagaatc 1560
atggaatctc atttaagggc tgctgaaatt ctttactatt tcgccctgag acaagctcac 1620
aaatacaaga taaataaatt tctctcatca tcactttaca cggcactgac agaagccaga 1680
aggaatttgg gactgtttca acatcatgat gctatcacag gaactgcaaa agactgggtg 1740
gttgtggatt atggtaccag actttttcat tcgttaatgg ttttggagaa gataattgga 1800
aattctgcat ttcttcttat tttgaaggac aaactcacat acgactctta ctctcctgat 1860
accttcctgg agatggattt gaaacaaaaa tcacaagatt ctctgccaca aaaaaatata 1920
ataaggctga gtgcggagcc aaggtacctt gtggtctata atcctttaga acaagaccga 1980
atctcgttgg tctcagtcta tgtgagttcc ccgacagtgc aagtgttctc tgcttcagga 2040
aaacctgtgg aagttcaagt cagcgcagtt tgggatacag caaatactat ttcagaaaca 2100
gcctatgaga tctcttttcg agcacatata ccgccattgg gactgaaagt gtataagatt 2160
ttggaatcag caagttcaaa ttcacattta gctgattatg tcttgtataa gaataaagta 2220
gaagatagcg gaattttcac cataaagaat atgataaata ctgaagaagg tataacacta 2280
gagaactcct ttgttttact tcggtttgat caaactggac ttatgaagca aatgatgact 2340
aaagaagatg gtaaacacGa tgaagtaaat gtgcaatttt catggtatgg aaGcaGaatt 2400
aaaagagaca aaagtggtgc ctacctcttc ttaGctgatg gtaatgccaa gccttatgtt 2460
tacacaacac cgccctttgt cagagtgaca catggaagga tttattcgga agtgaGttgc 2520
ttttttgacc atgttactca tagagtccga ctataccaca tacagggaat agaaggacag 2580
tctgtggaag tttccaatat tgtggacatc cgaaaagtat ataaccgtga gattgcaatg 2640
aaaatttctt ctgatataaa aagcGaaaat agattttata ctgacctaaa tgggtaccag 2700
attcaaccta gaatgacact gagcaaattg cctcttcaag caaatgtcta tcccatgacc 2760
acaatggcct atatccagga tgccaaacat cgtttgacac tgctctctgc tcagtcttta 2820
ggggtttcga gtttgaatag tggtcagatt gaagttatca tggatcgaag actcatgcaa 2880
gatgataatc gtggccttga gcaaggtatc caggataaca agattacagc taatctattt 2940
cgaatactac tagaaaaaag aagtgctgtt aatacggaag aagaaaagaa gtcggtcagt 3000
tatccttctc tccttagcca cataacttct tctctcatga atcatccagt cattccaatg 3060
gcaaataagt tcttctcacc tacccttgag ctgcaaggtg aattctctcc attacagtca 3120
tctttgcctt gtgacattca tctggttaat ttgagaacaa tacagtcaaa ggtgggcaat 3180
gggcactcca atgaggcagc cttgatcctc cacagaaaag ggtttgattg tcggttctct 3240
agcaaaggca cagggctgtt ttgttctact actcagggaa agatattggt acagaaactt 3300
ttaaacaagt ttattgtcga aagtctcaca ccttcatcac tatccttgat gcattcacct 3360
cccggcactc agaatataag tgagatcaac ttgagtccaa tggaaatcag cacattccga 3420
atccagttga ggtga 3435
<210>18
<211>1144
<212>PRT
＜213〉people
<400>18
Met Lys Leu Ser Arg Gln Phe Thr Val Phe Gly Ser Ala Ile Phe Cys
1 5 10 15
Val Val Ile Phe Ser Leu Tyr Leu Met Leu Asp Arg Gly His Leu Asp
20 25 30
Tyr Pro Arg Asn Pro Arg Arg Glu Gly Ser Phe Pro Gln Gly Gln Leu
35 40 45
Ser Met Leu Gln Glu Lys Ile Asp His Leu Glu Arg Leu Leu Ala Glu
50 55 60
Asn Asn Glu Ile Ile Ser Asn Ile Arg Asp Ser Val Ile Asn Leu Ser
65 70 75 80
Glu Ser Val Glu Asp Gly Pro Lys Ser Ser Gln Ser Asn Phe Ser Gln
85 90 95
Gly Ala Gly Ser His Leu Leu Pro Ser Gln Leu Ser Leu Ser Val Asp
100 105 110
Thr Ala Asp Cys Leu Phe Ala Ser Gln Ser Gly Ser His Asn Ser Asp
115 120 125
Val Gln Met Leu Asp Val Tyr Ser Leu Ile Ser Phe Asp Asn Pro Asp
130 135 140
Gly Gly Val Trp Lys Gln Gly Phe Asp Ile Thr Tyr Glu Ser Asn Glu
145 150 155 160
Trp Asp Thr Glu Pro Leu Gln Val Phe Val Val Pro His Ser His Asn
165 170 175
Asp Pro Gly Trp Leu Lys Thr Phe Asn Asp Tyr Phe Arg Asp Lys Thr
180 185 190
Gln Tyr Ile Phe Asn Asn Met Val Leu Lys Leu Lys Glu Asp Ser Arg
195 200 205
Arg Lys Phe Ile Trp Ser Glu Ile Ser Tyr Leu Ser Lys Trp Trp Asp
210 215 220
Ile Ile Asp Ile Gln Lys Lys Asp Ala Val Lys Ser Leu Ile Glu Asn
225 230 235 240
Gly Gln Leu Glu Ile Val Thr Gly Gly Trp Val Met Pro Asp Glu Ala
245 250 255
Thr Pro His Tyr Phe Ala Leu Ile Asp Gln Leu Ile Glu Gly His Gln
260 265 270
Trp Leu Glu Asn Asn Ile Gly Val Lys Pro Arg Ser Gly Trp Ala Ile
275 280 285
Asp Pro Phe Gly His Ser Pro Thr Met Ala Tyr Leu Leu Asn Arg Ala
290 295 300
Gly Leu Ser His Met Leu Ile Gln Arg Val His Tyr Ala Val Lys Lys
305 310 315 320
His Phe Ala Leu His Lys Thr Leu Glu Phe Phe Trp Arg Gln Asn Trp
325 330 335
Asp Leu Gly Ser Val Thr Asp Ile Leu Cys His Met Met Pro Phe Tyr
340 345 350
Ser Tyr Asp Ile Pro His Thr Cys Gly Pro Asp Pro Lys Ile Cys Cys
355 360 365
Gln Phe Asp Phe Lys Arg Leu Pro Gly Gly Arg Phe Gly Cys Pro Trp
370 375 380
Gly Val Pro Pro Glu Thr Ile His Pro Gly Asn Val Gln Ser Arg Ala
385 390 395 400
Arg Met Leu Leu Asp Gln Tyr Arg Lys Lys Ser Lys Leu Phe Arg Thr
405 410 415
Lys Val Leu Leu Ala Pro Leu Gly Asp Asp Phe Arg Tyr Cys Glu Tyr
420 425 430
Thr Glu Trp Asp Leu Gln Phe Lys Asn Tyr Gln Gln Leu Phe Asp Tyr
435 440 445
Met Asn Ser Gln Ser Lys Phe Lys Val Lys Ile Gln Phe Gly Thr Leu
450 455 460
Ser Asp Phe Phe Asp Ala Leu Asp Lys Ala Asp Glu Thr Gln Arg Asp
465 470 475 480
Lys Gly Gln Ser Met Phe Pro Val Leu Ser Gly Asp Phe Phe Thr Tyr
485 490 495
Ala Asp Arg Asp Asp His Tyr Trp Ser Gly Tyr Phe Thr Ser Arg Pro
500 505 510
Phe Tyr Lys Arg Met Asp Arg Ile Met Glu Ser His Leu Arg Ala Ala
515 520 525
Glu Ile Leu Tyr Tyr Phe Ala Leu Arg Gln Ala His Lys Tyr Lys Ile
530 535 540
Asn Lys Phe Leu Ser Ser Ser Leu Tyr Thr Ala Leu Thr Glu Ala Arg
545 550 555 560
Arg Asn Leu Gly Leu Phe Gln His His Asp Ala Ile Thr Gly Thr Ala
565 570 575
Lys Asp Trp Val Val Val Asp Tyr Gly Thr Arg Leu Phe His Ser Leu
580 585 590
Met Val Leu Glu Lys Ile Ile Gly Asn Ser Ala Phe Leu Leu Ile Leu
595 600 605
Lys Asp Lys Leu Thr Tyr Asp Ser Tyr Ser Pro Asp Thr Phe Leu Glu
610 615 620
Met Asp Leu Lys Gln Lys Ser Gln Asp Ser Leu Pro Gln Lys Asn Ile
625 630 635 640
Ile Arg Leu Ser Ala Glu Pro Arg Tyr Leu Val Val Tyr Asn Pro Leu
645 650 655
Glu Gln Asp Arg Ile Ser Leu Val Ser Val Tyr Val Ser Ser Pro Thr
660 665 670
Val Gln Val Phe Ser Ala Ser Gly Lys Pro Val Glu Val Gln Val Ser
675 680 685
Ala Val Trp Asp Thr Ala Asn Thr Ile Ser Glu Thr Ala Tyr Glu Ile
690 695 700
Ser Phe Arg Ala His Ile Pro Pro Leu Gly Leu Lys Val Tyr Lys Ile
705 710 715 720
Leu Glu Ser Ala Ser Ser Asn Ser His Leu Ala Asp Tyr Val Leu Tyr
725 730 735
Lys Asn Lys Val Glu Asp Ser Gly Ile Phe Thr Ile Lys Asn Met Ile
740 745 750
Asn Thr Glu Glu Gly Ile Thr Leu Glu Asn Ser Phe Val Leu Leu Arg
755 760 765
Phe Asp Gln Thr Gly Leu Met Lys Gln Met Met Thr Lys Glu Asp Gly
770 775 780
Lys His His Glu Val Asn Val Gln Phe Ser Trp Tyr Gly Thr Thr Ile
785 790 795 800
Lys Arg Asp Lys Ser Gly Ala Tyr Leu Phe Leu Pro Asp Gly Asn Ala
805 810 815
Lys Pro Tyr Val Tyr Thr Thr Pro Pro Phe Val Arg Val Thr His Gly
820 825 830
Arg Ile Tyr Ser Glu Val Thr Cys Phe Phe Asp His Val Thr His Arg
835 840 845
Val Arg Leu Tyr His Ile Gln Gly Ile Glu Gly Gln Ser Val Glu Val
850 855 860
Ser Asn Ile Val Asp Ile Arg Lys Val Tyr Asn Arg Glu Ile Ala Met
865 870 875 880
Lys Ile Ser Ser Asp Ile Lys Ser Gln Asn Arg Phe Tyr Thr Asp Leu
885 890 895
Asn Gly Tyr Gln Ile Gln Pro Arg Met Thr Leu Ser Lys Leu Pro Leu
900 905 910
Gln Ala Asn Val Tyr Pro Met Thr Thr Met Ala Tyr Ile Gln Asp Ala
915 920 925
Lys His Arg Leu Thr Leu Leu Ser Ala Gln Ser Leu Gly Val Ser Ser
930 935 940
Leu Asn Ser Gly Gln Ile Glu Val Ile Met Asp Arg Arg Leu Met Gln
945 950 955 960
Asp Asp Asn Arg Gly Leu Glu Gln Gly Ile Gln Asp Asn Lys Ile Thr
965 970 975
Ala Asn Leu Phe Arg Ile Leu Leu Glu Lys Arg Ser Ala Val Asn Thr
980 985 990
Glu Glu Glu Lys Lys Ser Val Ser Tyr Pro Ser Leu Leu Ser His Ile
995 1000 1005
Thr Ser Ser Leu Met Asn His Pro Val Ile Pro Met Ala Asn Lys
1010 1015 1020
Phe Phe Ser Pro Thr Leu Glu Leu Gln Gly Glu Phe Ser Pro Leu
1025 1030 1035
Gln Ser Ser Leu Pro Cys Asp Ile His Leu Val Asn Leu Arg Thr
1040 1045 1050
Ile Gln Ser Lys Val Gly Asn Gly His Ser Asn Glu Ala Ala Leu
1055 1060 1065
Ile Leu His Arg Lys Gly Phe Asp Cys Arg Phe Ser Ser Lys Gly
1070 1075 1080
Thr Gly Leu Phe Cys Ser Thr Thr Gln Gly Lys Ile Leu Val Gln
1085 1090 1095
Lys Leu Leu Asn Lys Phe Ile Val Glu Ser Leu Thr Pro Ser Ser
1100 1105 1110
Leu Ser Leu Met His Ser Pro Pro Gly Thr Gln Asn Ile Ser Glu
1115 1120 1125
Ile Asn Leu Ser Pro Met Glu Ile Ser Thr Phe Arg Ile Gln Leu
1130 1135 1140
Arg
<210>19
<211>1116
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of ManII-Gal T
<400>19
atgaagttaa gccgccagtt caccgtgttc ggcagtgcga tcttctgtgt ggtgattttc 60
tcgctctacc tgatgctgga ccggggtcac ttagactacc ccaggaaccc gcgccgcgag 120
ggctccttcc ctcagggcca gctctcaatg ttgcaagaaa aaatagacca tttggagcgt 180
ttgctagctg agaataatga gatcatctca aatattagag actcagtcat caatttgagt 240
gagtctgtgg aggatggtcc gaaaagttca caaagcaatt tcagccaagg tgctggctca 300
cccgcctgcc ctgaggagtc cccgctgctt gtgggcccca tgctgattga gtttaacatg 360
cctgtggacc tggagctcgt ggcaaagcag aacccaaatg tgaagatggg cggccgctat 420
gcccccaggg actgcgtctc tcctcacaag gtggccatca tcattccatt ccgcaaccgg 480
caggagcacc tcaagtactg gctatattat ttgcacccag tcctgcagcg ccagcagctg 540
gactatggca tctatgttat caaccaggcg ggagacacta tattcaatcg tgctaagctc 600
ctcaatgttg gctttcaaga agccttgaag gactatgact acacctgctt tgtgtttagt 660
gacgtggacc tcattccaat gaatgaccat aatgcgtaca ggtgtttttc acagccacgg 720
cacatttccg ttgcaatgga taagtttgga ttcagcctac cttatgttca gtattttgga 780
ggtgtctctg ctctaagtaa acaacagttt ctaaccatca atggatttcc taataattat 840
tggggctggg gaggagaaga tgatgacatt tttaacagat tagtttttag aggcatgtct 900
atatctcgcc caaatgctgt ggtcgggagg tgtcgcatga tccgccactc aagagacaaa 960
aaaaatgaac ccaatcctca gaggtttgac cgaattgcac acacaaagga gacaatgctc 1020
tctgatggtt tgaactGact cacctaccag gtgctggatg tacagagata cccattgtat 1080
acccaaatca cagtggacat cgggacaccg agctag 1116
<210>20
<211>371
<212>PRT
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of ManII-Gal T
<400>20
Met Lys Leu Ser Arg Gln Phe Thr Val Phe Gly Ser Ala Ile Phe Cys
1 5 10 15
Val Val Ile Phe Ser Leu Tyr Leu Met Leu Asp Arg Gly His Leu Asp
20 25 30
Tyr Pro Arg Asn Pro Arg Arg Glu Gly Ser Phe Pro Gln Gly Gln Leu
35 40 45
Ser Met Leu Gln Glu Lys Ile Asp His Leu Glu Arg Leu Leu Ala Glu
50 55 60
Asn Asn Glu Ile Ile Ser Asn Ile Arg Asp Ser Val Ile Asn Leu Ser
65 70 75 80
Glu Ser Val Glu Asp Gly Pro Lys Ser Ser Gln Ser Asn Phe Ser Gln
85 90 95
Gly Ala Gly Ser Pro Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly
100 105 110
Pro Met Leu Ile Glu Phe Asn Met Pro Val Asp Leu Glu Leu Val Ala
115 120 125
Lys Gln Asn Pro Asn Val Lys Met Gly Gly Arg Tyr Ala Pro Arg Asp
130 135 140
Cys Val Ser Pro His Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg
145 150 155 160
Gln Glu His Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro Val Leu Gln
165 170 175
Arg Gln Gln Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Asp
180 185 190
Thr Ile Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe Gln Glu Ala
195 200 205
Leu Lys Asp Tyr Asp Tyr Thr Cys Phe Val Phe Ser Asp Val Asp Leu
210 215 220
Ile Pro Met Asn Asp His Asn Ala Tyr Arg Cys Phe Ser Gln Pro Arg
225 230 235 240
His Ile Ser Val Ala Met Asp Lys Phe Gly Phe Ser Leu Pro Tyr Val
245 250 255
Gln Tyr Phe Gly Gly Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Thr
260 265 270
Ile Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp
275 280 285
Asp Ile Phe Asn Arg Leu Val Phe Arg Gly Met Ser Ile Ser Arg Pro
290 295 300
Asn Ala Val Val Gly Arg Cys Arg Met Ile Arg His Ser Arg Asp Lys
305 310 315 320
Lys Asn Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys
325 330 335
Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr Tyr Gln Val Leu
340 345 350
Asp Val Gln Arg Tyr Pro Leu Tyr Thr Gln Ile Thr Val Asp Ile Gly
355 360 365
Thr Pro Ser
370

Claims (286)

1. the isolating nucleic acid that contains coding fusion polypeptide sequence, wherein said fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-galactosyltransferasactivity activity and contain the golgi body locating structure territory that golgi body stops polypeptide.
2. according to the isolating nucleic acid of claim 1, wherein said fusion polypeptide contains the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III or β (1,4)-galactosyltransferase.
3. according to the isolating nucleic acid of claim 2, wherein said golgi body locating structure territory is the locating structure territory of mannosidase II.
4. according to the isolating nucleic acid of claim 3, has the nucleotide sequence shown in Figure 24 and the SEQ ID NO:14.
5. according to the isolating nucleic acid of claim 2, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase I.
6. according to the isolating nucleic acid of claim 5, has the nucleotide sequence shown in Figure 25 and the SEQ ID NO:12.
7. according to the isolating nucleic acid of claim 2, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II.
8. according to the isolating nucleic acid of claim 2, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
9. according to the isolating nucleic acid of claim 2, wherein said golgi body locating structure territory is the locating structure territory of α 1-6 core fucosyltransferase.
10. according to the isolating nucleic acid of claim 3, contain coding and have the sequence of the polypeptide of aminoacid sequence shown in Figure 24 and the SEQ IDNO:15.
11., contain coding and have the sequence of the polypeptide of aminoacid sequence shown in Figure 25 and the SEQ IDNO:13 according to the isolating nucleic acid of claim 5.
12. according to the isolating nucleic acid of claim 3, contain the sequence of hybridizing with hybridization probe under stringent condition, the nucleotide sequence of probe is made of the nucleotide sequence shown in Figure 24 and the SEQ ID NO:14.
13. according to the isolating nucleic acid of claim 5, contain the sequence of hybridizing with hybridization probe under stringent condition, the probe nucleotide sequence is made of the nucleotide sequence shown in Figure 25 and the SEQ ID NO:12.
14., contain the sequence identical with Figure 24 and nucleotide sequence shown in the SEQ ID NO:14 at least 80% according to the isolating nucleic acid of claim 3.
15., contain the sequence identical with Figure 25 and nucleotide sequence shown in the SEQ ID NO:12 at least 80% according to the isolating nucleic acid of claim 5.
16. according to the isolating nucleic acid of claim 3, contain the sequence of coded polypeptide, this polypeptide has the aminoacid sequence identical with Figure 24 and aminoacid sequence shown in the SEQ ID NO:15 at least 80%.
17. according to the isolating nucleic acid of claim 5, contain the sequence of coded polypeptide, this polypeptide has the aminoacid sequence identical with Figure 25 and aminoacid sequence shown in the SEQ ID NO:13 at least 80%.
18. according to the isolating nucleic acid of claim 3, contain the sequence of coded polypeptide, this polypeptide has the aminoacid sequence shown in conserved amino acid alternate Figure 24 and the SEQ ID NO:15.
19. according to the isolating nucleic acid of claim 5, contain the sequence of coded polypeptide, this polypeptide has the aminoacid sequence shown in conserved amino acid alternate Figure 25 and the SEQ ID NO:13.
20. expression vector contains the isolating nucleic acid of with good grounds claim 1-19 in each.
21. fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-galactosyltransferasactivity activity and contains the golgi body locating structure territory that the allos golgi body stops polypeptide.
22., contain the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III or β (1,4)-galactosyltransferase according to the fusion polypeptide of claim 21.
23. according to the fusion polypeptide of claim 21, wherein golgi body locating structure territory is the locating structure territory of mannosidase II.
24. according to the fusion polypeptide of claim 21, wherein golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase I.
25. according to the fusion polypeptide of claim 21, wherein golgi body locating structure territory is the locating structure territory of mannosidase I.
26. according to the fusion polypeptide of claim 21, wherein golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II.
27. according to the fusion polypeptide of claim 21, wherein golgi body locating structure territory is the locating structure territory of α 1-6 core fucosyltransferase.
28. contain the host cell of the expression vector of claim 20.
Has β (1 29. produce, 4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-method of the fusion polypeptide of galactosyltransferasactivity activity, be included under the condition of the described expression of nucleic acid that allows the described fusion polypeptide of coding and in substratum, cultivate the host cell of claim 28, and from the gained culture, collect described fusion polypeptide.
30. modify the method for the glycosylation form of the polypeptide that host cell produces, comprise that the nucleic acid that claim 1-19 is arbitrary introduces in the described host cell.
31. the method for the glycosylation form of the polypeptide that the modification host cell produces comprises the expression vector of claim 20 is introduced in the described host cell.
32. according to the method for claim 30 or 31, wherein said polypeptide is IgG or its fragment.
33. according to the method for claim 32, wherein said polypeptide is IgG1 or its fragment.
34. according to the method for claim 32, wherein said polypeptide is a fusion rotein, this fusion rotein comprises the fragment that is equivalent to human IgG Fc district.
35. host cell, this host cell through engineering approaches is expressed at least a coding and is had β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-nucleic acid of the fusion polypeptide of galactosyltransferasactivity activity, expression amount is enough to modify the oligosaccharides in the polypeptide Fc fragment that described host cell produces, wherein said polypeptide is selected from whole antibody molecule, antibody fragment and fusion rotein, and this fusion rotein comprises the fragment that is equivalent to immunoglobulin fc region.
36. the host cell of claim 35, the described polypeptide that wherein said host cell produces is IgG or its fragment.
37. the host cell of claim 35, the described polypeptide that wherein said host cell produces is IgG1 or its fragment.
38. the host cell of claim 35, the described polypeptide that wherein said host cell produces is a fusion rotein, and this fusion rotein comprises the fragment that is equivalent to human IgG Fc district.
39. the host cell of claim 35, wherein the described polypeptide that produces as the described host cell of the result of described modification presents the Fc receptor binding affinity of raising.
40. the host cell of claim 35, wherein the described polypeptide that produces as the described host cell of the result of described modification presents the effector function of raising.
41. according to the host cell of claim 35, wherein said fusion polypeptide contains the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III or β (1,4)-galactosyltransferase.
42. according to the host cell of claim 41, wherein said fusion polypeptide comprises that further the allos golgi body stops the golgi body locating structure territory of polypeptide.
43. according to the host cell of claim 42, wherein said golgi body locating structure territory is the locating structure territory of mannosidase II.
44. according to the host cell of claim 42, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase I.
45. according to the host cell of claim 42, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II.
46. according to the host cell of claim 42, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
47. according to the host cell of claim 42, wherein said golgi body locating structure territory is the locating structure territory of α 1-6 core fucosyltransferase.
48. according to the host cell of claim 40, the effector function of wherein said raising is the cytotoxicity of the Fc mediation of raising.
49. according to the host cell of claim 40, the effector function of wherein said raising is to improve with NK cell bonded.
50. according to the host cell of claim 40, the effector function of wherein said raising is to improve with the scavenger cell bonded.
51. according to the host cell of claim 40, the effector function of wherein said raising is to improve with the polymorphonuclear cell bonded.
52. according to the host cell of claim 40, the effector function of wherein said raising is to improve with the monocyte bonded.
53. according to the host cell of claim 40, the effector function of wherein said raising is the raising of apoptosis-induced direct signal.
54. according to the host cell of claim 40, the effector function of wherein said raising is the raising of dendritic cell maturation.
55. according to the host cell of claim 40, the effector function of wherein said raising is the raising of T cell sensitization.
56. according to the host cell of claim 39, wherein said Fc acceptor is a Fc γ activated receptor.
57. according to the host cell of claim 39, wherein said Fc acceptor is a Fc γ RIIIA acceptor.
58. according to the host cell of claim 35, wherein said host cell is Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma.
59. the host cell of claim 35, the described polypeptide that wherein said host cell produces is an anti-CD20 antibodies.
60. the host cell of claim 59, wherein said anti-CD20 antibodies is IDEC-C2B8.
61. the host cell of claim 35, the described polypeptide that wherein said host cell produces is inosculating antibody human kidney cells carcinoma monoclonal antibody chG250.
62. the host cell of claim 35 further comprises (transected) nucleic acid of at least a brachymemma, this nucleic acid encoding antibody molecule and antibody fragment or fusion rotein, and fusion rotein comprises the fragment that is equivalent to immunoglobulin fc region.
63. the host cell of claim 35, wherein said at least a coding has β (1,4)-nucleic acid of the fusion polypeptide of N-acetylglucosaminyltrVnsferase III activity or β (1,4)-galactosyltransferasactivity activity is operationally to be connected with constitutive promoter element.
64. the host cell of claim 62, the nucleic acid encoding anti-CD20 antibodies of wherein said at least one institute brachymemma, inosculating antibody people neuroblastoma monoclonal antibody chCE7, inosculating antibody human kidney cells carcinoma monoclonal antibody chG250, inosculating antibody people colon, lung and breast cancer monoclonal antibody ING-1, Humanized anti-human 17-1A antigen monoclonal antibody 3622W94, Humanized anti-human colorectum tumour antibody A33, anti-human melanoma antibody at GD3 Sphingolipids,sialo R24, inosculating antibody people squamous cell carcinoma monoclonal antibody SF-25, anti-people EGFR antibody, anti-people EGFRvIII antibody, anti-people PSMA antibody, anti-people's psca antibody, anti-people CD22 antibody, anti-people CD30 antibody, anti-people CD33 antibody, anti-people CD38 antibody, antibodies against human CD 40, anti-people CD45 antibody, antihuman CD 52 antibody, anti-people CD138 antibody, anti-people HLA-DR variant antibody, anti-people EpCAM antibody, anti-people CEA antibody, anti-people MUC1 antibody, anti-people MUC1 nucleoprotein antibody, the unusual glycosylation MUC1 antibody of anti-people, antagonism contains the antibody or the anti-people HER2/neu antibody of people's fibronectin variant of ED-B structural domain.
65. in host cell, produce the method for polypeptide, comprising:
A. under the condition that allows the generation polypeptide, cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had β (1,4)-N-acetylglucosaminyltrVnsferase III activity or β (1,4)-nucleic acid of the fusion polypeptide of galactosyltransferasactivity activity, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, this fusion rotein comprises the fragment that is equivalent to immunoglobulin fc region, and the expression amount of wherein said fusion polypeptide is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; With
B. separate described polypeptide.
66. according to the method for claim 65, wherein said fusion polypeptide comprises the catalyst structure domain of β (1,4)-N-acetylglucosaminyltrVnsferase III or β (1,4)-galactosyltransferase.
67. according to the method for claim 65, wherein said fusion polypeptide comprises that further the allos golgi body stops the golgi body locating structure territory of polypeptide.
68. according to the method for claim 67, wherein said golgi body locating structure territory is the locating structure territory of mannosidase II.
69. according to the method for claim 67, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase I.
70. according to the method for claim 67, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
71. according to the method for claim 67, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II.
72. according to the method for claim 67, wherein said golgi body locating structure territory is the locating structure territory of α 1-6 core fucosyltransferase.
73., wherein have the effector function of raising as the described polypeptide of the result of described modification according to the method for claim 65.
74. according to the method for claim 73, the effector function of wherein said raising is the cytotoxicity of the Fc mediation of raising.
75. according to the method for claim 73, the effector function of wherein said raising is to improve with NK cell bonded.
76. according to the method for claim 73, the effector function of wherein said raising is to improve with the scavenger cell bonded.
77. according to the method for claim 73, the effector function of wherein said raising is to improve with the monocyte bonded.
78. according to the method for claim 73, the effector function of wherein said raising is to improve with the polymorphonuclear cell bonded.
79. according to the method for claim 73, the effector function of wherein said raising is the raising of apoptosis-induced direct signal.
80. according to the method for claim 73, the effector function of wherein said raising is the raising of dendritic cell maturation.
81. according to the method for claim 73, the effector function of wherein said raising is the raising of T cell sensitization.
82. according to the method for claim 65, wherein the described polypeptide that produces as the described host cell of the result of described modification presents the Fc receptor binding affinity of raising.
83. 2 method according to Claim 8, wherein said Fc acceptor is the Fc activated receptor.
84. 2 method according to Claim 8, wherein said Fc acceptor is a Fc γ RIIIA acceptor.
85. according to the method for claim 65, the described polypeptide that wherein said host cell produces has the raising ratio in described polypeptide Fc district binary oligosaccharides.
86. according to the method for claim 65, the described polypeptide that wherein said host cell produces has the raising ratio in described polypeptide Fc district non-fucosylation oligosaccharides.
87. 6 method according to Claim 8, wherein said non-fucosylation oligosaccharides is a heterozygosis.
88. 6 method according to Claim 8, wherein said non-fucosylation oligosaccharides is a compound.
89. according to the method for claim 65, the described polypeptide that wherein said host cell produces has the bisection of raising ratio, non-fucosylation oligosaccharides in described polypeptide Fc district.
90. 9 method according to Claim 8, the non-fucosylation oligosaccharides of wherein said bisection is a heterozygosis.
91. 9 method according to Claim 8, the non-fucosylation oligosaccharides of wherein said bisection is a compound.
92. 9 method according to Claim 8, the oligosaccharides at least 20% in the wherein said polypeptide Fc district are the non-fucosylations of halving.
93. 9 method according to Claim 8, the oligosaccharides at least 25% in the wherein said polypeptide Fc district are the non-fucosylations of halving.
94. 9 method according to Claim 8, the oligosaccharides at least 30% in the wherein said polypeptide Fc district are the non-fucosylations of halving.
95. 9 method according to Claim 8, the oligosaccharides at least 35% in the wherein said polypeptide Fc district are the non-fucosylations of halving.
96. the through engineering approaches that produces according to the arbitrary method of claim 65-96 has the antibody that improves effector function.
97. the through engineering approaches that produces according to the arbitrary method of claim 65-96 has the antibody that improves the Fc receptor binding affinity.
98. according to the antibody of claim 97, the effector function of wherein said raising is the cytotoxicity of the Fc mediation of raising.
99. according to the antibody of claim 97, the effector function of wherein said raising is to improve with NK cell bonded.
100. according to the antibody of claim 97, the effector function of wherein said raising is to improve with the scavenger cell bonded.
101. according to the antibody of claim 97, the effector function of wherein said raising is to improve with the monocyte bonded.
102. according to the antibody of claim 97, the effector function of wherein said raising is to improve with the polymorphonuclear cell bonded.
103. according to the antibody of claim 97, the effector function of wherein said raising is the raising of apoptosis-induced direct signal.
104. according to the antibody of claim 97, the effector function of wherein said raising is the raising of dendritic cell maturation.
105. according to the antibody of claim 97, the effector function of wherein said raising is the raising of T cell sensitization.
106. according to the antibody of claim 98, wherein said Fc acceptor is the Fc activated receptor.
107. according to the antibody of claim 98, wherein said Fc acceptor is a Fc γ RIIIa acceptor.
108. contain the antibody fragment that Fc district and through engineering approaches have the raising effector function by what the arbitrary method of claim 65-96 produced.
109. the comprise zone and the through engineering approaches that are equivalent to immunoglobulin fc region that produce by the arbitrary method of claim 65-96 have the fusion rotein that improves effector function.
110. contain the antibody fragment that Fc district and through engineering approaches have raising Fc receptor binding affinity by what the arbitrary method of claim 65-96 produced.
111. the comprise zone and the through engineering approaches that are equivalent to immunoglobulin fc region that produce by the arbitrary method of claim 65-96 have the fusion rotein that improves the Fc receptor binding affinity.
112. comprise claim 97-108 each antibody and pharmacology on can accept the pharmaceutical composition of carrier.
113. comprise the pharmaceutical composition that to accept carrier on the antibody fragment of claim 109 or claim 111 and the pharmacology.
114. comprise the pharmaceutical composition that to accept carrier on the fusion rotein of claim 110 or claim 112 and the pharmacology.
115. the treatment method for cancer comprises that the arbitrary pharmaceutical composition of claim 113-115 with the treatment significant quantity delivers medicine to the patient of needs.
116. treat improving one's methods of disease based on the B cell depleting, comprise the antibody administration of will treat significant quantity in the human experimenter of needs, described improvement comprises the antibody that the arbitrary method of the claim 65-96 of drug treatment significant quantity produces.
117. improving one's methods of claim 117, wherein said antibody is anti-CD-20 monoclonal antibody.
118. improving one's methods of claim 118, wherein said anti-CD20 antibodies is IDEC-C2B8.
119. comprise the isolating nucleic acid of the sequence of the fusion polypeptide of encoding, wherein said fusion polypeptide has β (1,4)-galactosyltransferasactivity activity and comprises that golgi body stops the golgi body locating structure territory of polypeptide.
120. according to the isolating nucleic acid of claim 119, wherein said fusion polypeptide contains the catalyst structure domain of β (1,4)-galactosyltransferase.
121. according to the isolating nucleic acid of claim 2, wherein golgi body locating structure territory is the locating structure territory of mannosidase II.
122. contain the expression vector of the arbitrary isolating nucleic acid of with good grounds claim 119-121.
123. have β (1,4)-galactosyltransferasactivity activity and comprise that the allos golgi body stops the fusion polypeptide in the golgi body locating structure territory of polypeptide.
124., comprise the catalyst structure domain of β (1,4)-galactosyltransferase according to the fusion polypeptide of claim 123.
125. according to the fusion polypeptide of claim 124, wherein golgi body locating structure territory is the locating structure territory of mannosidase II.
126. contain the host cell of the expression vector of claim 122.
Has β (1 127. produce, 4)-method of the fusion polypeptide of galactosyltransferasactivity activity, be included under the condition of the described expression of nucleic acid that allows the described fusion polypeptide of coding and in substratum, cultivate the host cell of claim 126 and from the gained culture, collect described fusion polypeptide.
128. modify the method for the glycosylation form of the polypeptide that host cell produces, comprise that the nucleic acid that claim 119-121 is arbitrary introduces in the described host cell.
129. the method for the glycosylation form of the polypeptide that the modification host cell produces comprises the expression vector of claim 122 is introduced in the described host cell.
130. host cell comprises:
(a) contain the expression vector of the nucleic acid molecule of the fusion polypeptide of encoding, it is active and comprise that golgi body stops the golgi body locating structure territory of polypeptide that wherein said fusion polypeptide has β (1,4)-N-acetylglucosaminyltrVnsferase III (GnTIII); With
(b) contain the expression vector of nucleic acid encoding molecule, wherein said polypeptide has mannosidase II (ManII) activity.
131. the host cell of claim 130, the described nucleic acid molecule of the described fusion polypeptide of wherein encoding has the described nucleic acid molecule of the active polypeptide of mannosidase II on same expression vector with coding is described.
132. the host cell of claim 130, the described nucleic acid molecule of the described fusion polypeptide of wherein encoding has the described nucleic acid molecule of the active polypeptide of mannosidase II on the expression vector that separates with coding is described.
133. the host cell of claim 130, wherein said fusion polypeptide contains the catalyst structure domain of GnTIII.
134. the host cell of claim 130, wherein said golgi body locating structure territory is the locating structure territory of ManII.
135. the host cell of claim 130, wherein said golgi body locating structure territory are the locating structure territories of β (1,2)-N-acetylglucosaminyltrVnsferase I.
136. the host cell of claim 130, wherein said golgi body locating structure territory are the locating structure territories of β (1,2)-N-acetylglucosaminyltrVnsferase II.
137. the host cell of claim 130, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
138. the host cell of claim 130, wherein said golgi body locating structure territory is α 1, the locating structure territory of 6-N core fucosyltransferase.
139. the host cell of claim 130, wherein said host cell is selected from mammalian cell, yeast cell, insect cell or vegetable cell.
140. the host cell of claim 130, wherein said host cell are selected from Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell and hybridoma.
141. the host cell of claim 130 further comprises the expression vector that contains the nucleic acid encoding molecule, wherein said polypeptide has β (1,2)-N-acetylglucosaminyltrVnsferase II (GnTII) activity.
142. the host cell of claim 141, the nucleic acid molecule of wherein said coding fusion polypeptide, described coding have the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that described coding has the active polypeptide of GnTII on same expression vector.
143. the host cell of claim 141, the nucleic acid molecule of wherein said coding fusion polypeptide, described coding have the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that described coding has the active polypeptide of GnTII on the expression vector that separates.
144. the host cell of claim 141, the nucleic acid molecule of wherein said coding fusion polypeptide is on an expression vector, and described coding has the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that described coding has the active polypeptide of GnTII on same expression vector.
145. the host cell of claim 141, the nucleic acid molecule of wherein said coding ManII are on an expression vector, the nucleic acid molecule of described coding fusion polypeptide and described coding have the nucleic acid molecule of the active polypeptide of GnTII on same expression vector.
146. the host cell of claim 141, the nucleic acid molecule of the described GnTII of wherein said coding are on an expression vector, the nucleic acid molecule of described coding fusion polypeptide and described coding have the nucleic acid molecule of the active polypeptide of ManII on same expression vector.
147. host cell comprises:
(a) contain the expression vector of the nucleic acid molecule of the fusion polypeptide of encoding, it is active and comprise that golgi body stops the golgi body locating structure territory of polypeptide that wherein said fusion polypeptide has β (1,4) galactosyltransferase (GalT); With
(b) contain the expression vector of nucleic acid encoding molecule, wherein said polypeptide has mannosidase II (ManII) activity.
148. the host cell of claim 147, the nucleic acid molecule of wherein said coding fusion polypeptide and described coding have the nucleic acid molecule of the active polypeptide of ManII on same expression vector.
149. the host cell of claim 147, the nucleic acid molecule of wherein said coding fusion polypeptide and described coding have the nucleic acid molecule of the active polypeptide of ManII on the expression vector that separates.
150. the host cell of claim 147, wherein said fusion polypeptide contains the catalyst structure domain of GalT.
151. the host cell of claim 147, wherein said golgi body locating structure territory is the locating structure territory of ManII.
152. the host cell of claim 147, wherein said golgi body locating structure territory are the locating structure territories of β (1,2)-N-acetylglucosaminyltrVnsferase I.
153. the host cell of claim 147, wherein said golgi body locating structure territory are the locating structure territories of β (1,2)-N-acetylglucosaminyltrVnsferase II.
154. the host cell of claim 147, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
155. the host cell of claim 147, wherein said golgi body locating structure territory is α 1, the locating structure territory of 6-N core fucosyltransferase.
156. the host cell of claim 147, wherein said host cell is selected from mammalian cell, yeast cell, insect cell or vegetable cell.
157. the host cell of claim 147, wherein said host cell are selected from Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell and hybridoma.
158. the host cell of claim 147 further comprises the expression vector that contains the nucleic acid encoding molecule, wherein said polypeptide has β (1,2)-N-acetylglucosaminyltrVnsferase II (GnTII) activity.
159. the host cell of claim 158, the nucleic acid molecule of wherein said coding fusion polypeptide, described coding have the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that described coding has the active polypeptide of GnTII on same expression vector.
160. the host cell of claim 158, the nucleic acid molecule of wherein said coding fusion polypeptide, described coding have the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that described coding has the active polypeptide of GnTII on the expression vector that separates.
161. the host cell of claim 158, the nucleic acid molecule of wherein said coding fusion polypeptide is on an expression vector, and described coding has the nucleic acid molecule of the active polypeptide of ManII and nucleic acid molecule that described coding has the active polypeptide of GnTII on same expression vector.
162. the host cell of claim 158, the nucleic acid molecule of wherein said coding ManII are on an expression vector, the nucleic acid molecule of described coding fusion polypeptide and described coding have the nucleic acid molecule of the active polypeptide of GnTII on same expression vector.
163. the host cell of claim 158, the nucleic acid molecule of the described GnTII of wherein said coding are on an expression vector, the nucleic acid molecule of described coding fusion polypeptide and described coding have the nucleic acid molecule of the active polypeptide of ManII on same expression vector.
164. the host cell of claim 158, wherein said fusion polypeptide comprises the catalyst structure domain of GalT.
165. the host cell of claim 158, wherein said golgi body locating structure territory is the locating structure territory of ManII.
166. the host cell of claim 158, wherein said golgi body locating structure territory are the locating structure territories of β (1,2)-N-acetylglucosaminyltrVnsferase I.
167. the host cell of claim 158, wherein said golgi body locating structure territory are the locating structure territories of β (1,2)-N-acetylglucosaminyltrVnsferase II.
168. the host cell of claim 158, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
169. the host cell of claim 158, wherein said golgi body locating structure territory is the locating structure territory of α 1,6 core fucosyltransferase.
170. the host cell of claim 158, wherein said host cell is selected from mammalian cell, yeast cell, insect cell or vegetable cell.
171. the host cell of claim 159, wherein said host cell are selected from Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell and hybridoma.
172. host cell, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid of the active polypeptide of GnTIII and the nucleic acid that at least one coding has the active polypeptide of ManII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc fragment that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to the immunoglobulin fc region territory.
173. host cell, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the active polypeptide of GnTIII, nucleic acid that at least one coding has the active polypeptide of ManII and at least one coding have the active polypeptide of GnTII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc fragment that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to the immunoglobulin fc region territory.
174. host cell, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid of the active fusion polypeptide of GalT and the nucleic acid that at least one coding has the active polypeptide of ManII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc zone that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the zone that is equivalent to the immunoglobulin fc region territory.
175. host cell, this host cell through engineering approaches expresses that at least a coding has the nucleic acid of the active fusion polypeptide of GalT, at least a coding has the nucleic acid of the active polypeptide of ManII and the nucleic acid that at least a coding has the active polypeptide of GnTII, expression amount is enough to modify the oligosaccharides in the polypeptide Fc district that described host cell produces, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and this fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
176. according to each host cell of claim 172-175, wherein the described polypeptide that produces as the described host cell of the result of described modification presents the Fc receptor binding affinity of raising.
177. according to the host cell of claim 172-175, wherein the described polypeptide that produces as the described host cell of the result of described modification presents the effector function of raising.
178. according to the host cell of claim 177, the effector function of wherein said raising is the cytotoxicity of the Fc mediation of raising.
179. according to the host cell of claim 177, the effector function of wherein said raising is to improve with NK cell bonded.
180. according to the host cell of claim 177, the effector function of wherein said raising is to improve with the scavenger cell bonded.
181. according to the host cell of claim 177, the effector function of wherein said raising is to improve with the polymorphonuclear cell bonded.
182. according to the host cell of claim 177, the effector function of wherein said raising is to improve with the monocyte bonded.
183. according to the host cell of claim 177, the effector function of wherein said raising is the raising of apoptosis-induced direct signal.
184. according to the host cell of claim 177, the effector function of wherein said raising is the raising of dendritic cell maturation.
185. according to the host cell of claim 177, the effector function of wherein said raising is the raising of T cell sensitization.
186. in host cell, produce the method for polypeptide, comprising:
A. under the condition that allows the generation polypeptide, cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GnTIII and the nucleic acid that at least a coding has the active polypeptide of ManI I, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, this fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and the expression amount of wherein said fusion polypeptide is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; With
B. separate described polypeptide.
187. the method for claim 186, the further through engineering approaches of wherein said host cell is expressed the nucleic acid that at least a coding has the active polypeptide of GnTII.
188. according to the method for claim 186 or 187, wherein said fusion polypeptide comprises the catalyst structure domain of GnTIII.
189. according to the method for claim 188, wherein said fusion polypeptide comprises that further the allos golgi body stops the golgi body locating structure territory of polypeptide.
190. according to the method for claim 189, wherein said golgi body locating structure territory is the locating structure territory of mannosidase II.
191. according to the method for claim 189, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase I.
192. according to the method for claim 189, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
193. according to the method for claim 189, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II.
194. according to the method for claim 189, wherein said golgi body locating structure territory is the locating structure territory of α 1-6 core fucosyltransferase.
195., wherein have the effector function of raising as the described polypeptide of the result of described modification according to the method for claim 186.
196. in host cell, produce the method for polypeptide, comprising:
A. under the condition that allows the generation polypeptide, cultivate host cell, this host cell through engineering approaches is expressed at least a coding and is had the nucleic acid of the active fusion polypeptide of GalT and the nucleic acid that at least a coding has the active polypeptide of ManII, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, this fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and the expression amount of wherein said fusion polypeptide is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; With
B. separate described polypeptide.
197. according to the method for claim 197, the further through engineering approaches of wherein said host cell is expressed the nucleic acid that at least a coding has the active polypeptide of GnTII.
198. according to the method for claim 196 or 197, wherein said fusion polypeptide comprises the catalyst structure domain of GalT.
199. according to the method for claim 198, wherein said fusion polypeptide comprises that further the allos golgi body stops the golgi body locating structure territory of polypeptide.
200. according to the method for claim 199, wherein said golgi body locating structure territory is the locating structure territory of mannosidase II.
201. according to the method for claim 199, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase I.
202. according to the method for claim 199, wherein said golgi body locating structure territory is the locating structure territory of mannosidase I.
203. according to the method for claim 199, wherein said golgi body locating structure territory is the locating structure territory of β (1,2)-N-acetylglucosaminyltrVnsferase II.
204. according to the method for claim 199, wherein said golgi body locating structure territory is the locating structure territory of α 1-6 core fucosyltransferase.
205., wherein have the effector function of raising as the described polypeptide of the result of described modification according to the method for claim 199.
206. according to the method for claim 186 or 196, the described polypeptide that wherein said host cell produces has the raising ratio in described polypeptide Fc district the non-fucosylation oligosaccharides of bisection.
207. according to the method for claim 206, the non-fucosylation oligosaccharides of wherein said bisection is a heterozygosis.
208. according to the method for claim 206, the non-fucosylation oligosaccharides of wherein said bisection is a compound.
209. according to the method for claim 206, the oligosaccharides at least 20% in the wherein said polypeptide Fc district is the non-fucosylation of halving.
210. according to the method for claim 206, the oligosaccharides at least 25% in the wherein said polypeptide Fc district is the non-fucosylation of halving.
211. according to the method for claim 206, the oligosaccharides at least 30% in the wherein said polypeptide Fc district is the non-fucosylation of halving.
212. according to the method for claim 206, the oligosaccharides at least 35% in the wherein said polypeptide Fc district is the non-fucosylation of halving.
213. the through engineering approaches that produces according to the arbitrary method of claim 196-212 has the antibody that improves effector function.
214. comprise the pharmaceutical composition that to accept carrier on the antibody of claim 213 and the pharmacology.
215. the treatment method for cancer comprises that the pharmaceutical composition of will treat the claim 214 of significant quantity delivers medicine to the patient of needs.
216. in host cell, produce the method for the Cytotoxic polypeptide of Fc mediation, comprising with raising:
A. under the condition that allows the generation polypeptide, cultivate host cell, this host cell through engineering approaches is expressed the nucleic acid of at least a coding GalT and the nucleic acid of at least a coding ManII, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment, this antibody fragment comprises immunoglobulin fc region, wherein the expression level of one or two among GalT or the ManII is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces, and has the cytotoxicity that the Fc of raising mediates as the wherein said polypeptide of the result of described modification; With
B. separate described Cytotoxic polypeptide with Fc mediation of raising.
217. the method for claim 216, wherein in the step (a), described host cell comprises the nucleic acid of the whole antibody of at least a coding.
218. the method for claim 216, wherein in the step (a), described host cell comprises the segmental nucleic acid of at least a encoding antibody.
219. the method for claim 216, wherein the expression level of GalT has produced the Cytotoxic antibody molecule of the Fc mediation with raising or has comprised the antibody fragment of immunoglobulin fc region.
220. the method for claim 216, wherein said host cell further comprises the nucleic acid of at least a coding GnTIII, the expression amount of wherein said GnTIII is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces, and has the cytotoxicity that the Fc of raising mediates as the wherein said polypeptide of the result of described modification.
221. claim 216 or 220 arbitrary methods, wherein the one or more expression level among GalT, ManII or the GnTIII is enough to form the bisection oligosaccharides in described polypeptide Fc district.
222. the method for claim 221, wherein in the Fc district bisection oligosaccharides in the Fc district all the ratio of oligosaccharides be at least 45%.
223. the method for claim 221, wherein said bisection oligosaccharides is a compound.
224. the method for claim 221, wherein said bisection oligosaccharides is a heterozygosis.
225. claim 216 or 220 arbitrary methods, wherein said host cell is selected from mammalian cell, yeast cell, insect cell or vegetable cell.
226. the method for claim 225, wherein said host cell is a vegetable cell.
227. arbitrary method of claim 216 or 220, wherein said host cell are selected from Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell and hybridoma.
228. in host cell, produce the method for polypeptide, comprising:
A. under the condition that allows the generation polypeptide, cultivate host cell, this host cell through engineering approaches is expressed the nucleic acid that at least a coding has the active polypeptide of α mannosidase II, the polypeptide that is produced is selected from whole antibody molecule, antibody fragment and fusion rotein, this fusion rotein comprises the zone that is equivalent to immunoglobulin fc region, and the active polypeptide expression amount of the wherein said α of having mannosidase II is enough to modify the oligosaccharides in the described polypeptide Fc district that described host cell produces; With
B. separate the described polypeptide that described host cell produces.
229. according to the method for claim 228, wherein the described polypeptide that produces as the described host cell of the result of described modification has the effector function of raising.
230. according to the method for claim 229, the effector function of wherein said raising is the cytotoxicity of the Fc mediation of raising.
231. according to the method for claim 229, the effector function of wherein said raising is to improve with NK cell bonded.
232. according to the method for claim 229, the effector function of wherein said raising is to improve with the scavenger cell bonded.
233. according to the method for claim 229, the effector function of wherein said raising is to improve with the polymorphonuclear cell bonded.
234. according to the method for claim 229, the effector function of wherein said raising is to improve with the monocyte bonded.
235. according to the method for claim 229, the effector function of wherein said raising is the raising of apoptosis-induced direct signal.
236. according to the method for claim 229, the effector function of wherein said raising is the raising of dendritic cell maturation.
237. according to the method for claim 229, the effector function of wherein said raising is the raising of T cell sensitization.
238. according to the method for claim 228, wherein the described polypeptide that produces as the described host cell of the result of described modification has presented the Fc receptors bind that improves.
239. according to the method for claim 238, wherein said Fc acceptor is a Fc γ activated receptor.
240. according to the method for claim 238, wherein said Fc acceptor is a Fc γ RIIIA acceptor.
241. according to the method for claim 228, wherein said host cell is Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3X63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma.
242. according to the method for claim 228, the described polypeptide that wherein said host cell produces is an anti-CD20 antibodies.
243. according to the method for claim 228, wherein said anti-CD20 antibodies is IDEC-C2B8.
244. according to the method for claim 228, the described polypeptide that wherein said host cell produces is inosculating antibody human kidney cells carcinoma monoclonal antibody chG250.
245. method according to claim 228, wherein said host cell further comprises at least a brachymemma (transected) nucleic acid, (transected) nucleic acid encoding antibody molecule and the antibody fragment or the fusion rotein of this brachymemma, fusion rotein comprises the zone that is equivalent to immunoglobulin fc region.
246. method according to claim 245, wherein said at least a brachymemma (transected) nucleic acid molecule encoding anti-CD20 antibodies, inosculating antibody people neuroblastoma monoclonal antibody chCE7, inosculating antibody human kidney cells carcinoma monoclonal antibody chG250, inosculating antibody people colon, lung and breast cancer monoclonal antibody ING-1, Humanized anti-human 17-1A antigen monoclonal antibody 3622W94, Humanized anti-human colorectum tumour antibody A33, the anti-human melanoma antibody of anti-GD3 Sphingolipids,sialo R24, inosculating antibody people squamous cell carcinoma monoclonal antibody SF-25, anti-people EGFR antibody, anti-people EGFRvIII antibody, anti-people PSMA antibody, anti-people's psca antibody, anti-people CD22 antibody, anti-people CD30 antibody, anti-people CD33 antibody, anti-people CD38 antibody, antibodies against human CD 40, anti-people CD45 antibody, antihuman CD 52 antibody, anti-people CD138 antibody, anti-people HLA-DR variant antibody, anti-people EpCAM antibody, anti-people CEA antibody, anti-people MUC1 antibody, anti-people MUC1 nucleoprotein antibody, the unusual glycosylation MUC1 antibody of anti-people, antagonism contains people's fibronectin variant antibody, anti-people TAG-72 antibody or the anti-people HER2/neu antibody of ED-B structural domain.
247. according to the method for claim 228, the described polypeptide that wherein said host cell produces has the raising ratio in described polypeptide Fc district bisection oligosaccharides.
248. according to the method for claim 228, the described polypeptide that wherein said host cell produces has the raising ratio in described polypeptide Fc district non-fucosylation oligosaccharides.
249. according to the method for claim 248, wherein said non-fucosylation oligosaccharides is a heterozygosis.
250. according to the method for claim 248, wherein said non-fucosylation oligosaccharides is a compound.
251. according to the method for claim 228, the described polypeptide that wherein said host cell produces has the raising ratio in described polypeptide Fc district the non-fucosylation oligosaccharides of bisection.
252. according to the method for claim 251, the non-fucosylation oligosaccharides of wherein said bisection is a heterozygosis.
253. according to the method for claim 251, the non-fucosylation oligosaccharides of wherein said bisection is a compound.
254. according to the method for claim 248, oligosaccharides at least 20% in the wherein said polypeptide Fc district is non-fucosylation.
255. according to the method for claim 248, oligosaccharides at least 25% in the wherein said polypeptide Fc district is non-fucosylation.
256. according to the method for claim 248, oligosaccharides at least 30% in the wherein said polypeptide Fc district is non-fucosylation.
257. according to the method for claim 248, oligosaccharides at least 35% in the wherein said polypeptide Fc district is non-fucosylation.
258. according to the method for claim 248, oligosaccharides at least 40% in the wherein said polypeptide Fc district is non-fucosylation.
259. according to the method for claim 248, at least 45% of oligosaccharides is non-fucosylation in the wherein said polypeptide Fc district.
260. according to the method for claim 248, oligosaccharides at least 48% in the wherein said polypeptide Fc district is non-fucosylation.
261. the through engineering approaches that produces according to claim 228 method has the antibody that improves effector function.
262. the through engineering approaches that produces according to claim 228 method has the antibody that improves the Fc receptor binding affinity.
263. according to the antibody of claim 261, the effector function of wherein said raising is the cytotoxicity of the Fc mediation of raising.
264. according to the antibody of claim 261, the effector function of wherein said raising is to improve with NK cell bonded.
265. according to the antibody of claim 261, the effector function of wherein said raising is to improve with the scavenger cell bonded.
266. according to the antibody of claim 261, the effector function of wherein said raising is to improve with the monocyte bonded.
267. according to the antibody of claim 261, the effector function of wherein said raising is to improve with the polymorphonuclear cell bonded.
268. according to the antibody of claim 261, the effector function of wherein said raising is the raising of apoptosis-induced direct signal.
269. according to the antibody of claim 261, the effector function of wherein said raising is the raising of dendritic cell maturation.
270. according to the antibody of claim 261, the effector function of wherein said raising is the raising of T cell sensitization.
271. according to the antibody of claim 262, wherein said Fc acceptor is the Fc activated receptor.
272. according to the antibody of claim 262, wherein said Fc acceptor is a Fc γ RIIIa acceptor.
Contain the antibody fragment that Fc district and through engineering approaches have the raising effector function 273. claim 228 method produces.
274. the comprise zone and the through engineering approaches that are equivalent to immunoglobulin fc region that claim 228 method produces have the fusion rotein that improves effector function.
Contain the antibody fragment that Fc district and through engineering approaches have raising Fc receptor binding affinity 275. claim 228 method produces.
276. the comprise zone and the through engineering approaches that are equivalent to immunoglobulin fc region that claim 228 method produces have the fusion rotein that improves the Fc receptor binding affinity.
277. comprise the pharmaceutical composition that to accept carrier on the antibody of the arbitrary claim of claim 261-272 and the pharmacology.
278. comprise the pharmaceutical composition that to accept carrier on the antibody fragment of claim 273 or claim 275 and the pharmacology.
279. comprise the pharmaceutical composition that to accept carrier on the fusion rotein of claim 274 or 276 and the pharmacology.
280. the method for treatment tumour comprises that the arbitrary pharmaceutical composition of claim 277-279 with the treatment significant quantity delivers medicine to the patient of needs.
281. treat improving one's methods of disease based on the B cell depleting, comprise the antibody administration that will treat significant quantity in the people experimenter of needs, improve the antibody of the claim 228 method generation that comprises the drug treatment significant quantity.
282. improving one's methods of claim 281, wherein said antibody is anti-CD-20 monoclonal antibody.
283. the method for claim 228, wherein said nucleic acid molecule comprise SEQ ID NO:17.
284. the method for claim 228, wherein said polypeptide with alpha-Mannosidase II comprises SEQ ID NO:18.
285., contain SEQ ID NO:19 according to the isolated nucleic acid molecule of claim 121.
286., contain SEQ ID NO:20 according to the fusion polypeptide of claim 125.
CN200480007564.2A 2003-01-22 2004-01-22 Fusion constructs and being used for produces the purposes of the antibody that Fc receptor binding affinity and effector function improve Expired - Lifetime CN1761746B (en)

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Cited By (7)

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CN102695791A (en) * 2009-09-04 2012-09-26 伦敦卫生及热带医学学院 Protein glycosylation
CN103320388A (en) * 2012-03-20 2013-09-25 无锡药明康德生物技术有限公司 Cell culture method capable of improving antibody expression levels and improving glycosylation levels
CN101679966B (en) * 2007-01-24 2014-03-12 协和发酵麒麟株式会社 Genetically recombinant antibody composition having enhanced effector activity
CN101951946B (en) * 2007-10-01 2014-12-10 百时美施贵宝公司 Human antibodies that bind mesothelin and uses thereof
CN104962579A (en) * 2007-06-15 2015-10-07 麦迪卡格公司 Modifying glycosylation production in plants
CN108882740A (en) * 2015-12-29 2018-11-23 N·V·努特里奇亚 Fermented formulations containing non-digestible oligosaccharides
CN109265552A (en) * 2012-10-30 2019-01-25 埃派斯进有限公司 Anti-CD 40 antibodies and its application method

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679966B (en) * 2007-01-24 2014-03-12 协和发酵麒麟株式会社 Genetically recombinant antibody composition having enhanced effector activity
CN104962579A (en) * 2007-06-15 2015-10-07 麦迪卡格公司 Modifying glycosylation production in plants
CN101951946B (en) * 2007-10-01 2014-12-10 百时美施贵宝公司 Human antibodies that bind mesothelin and uses thereof
CN102695791A (en) * 2009-09-04 2012-09-26 伦敦卫生及热带医学学院 Protein glycosylation
CN103320388A (en) * 2012-03-20 2013-09-25 无锡药明康德生物技术有限公司 Cell culture method capable of improving antibody expression levels and improving glycosylation levels
CN103320388B (en) * 2012-03-20 2015-10-28 无锡药明康德生物技术股份有限公司 Improve the cell culture processes of antibody expression amount and improvement level of glycosylation
CN109265552A (en) * 2012-10-30 2019-01-25 埃派斯进有限公司 Anti-CD 40 antibodies and its application method
CN108882740A (en) * 2015-12-29 2018-11-23 N·V·努特里奇亚 Fermented formulations containing non-digestible oligosaccharides

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