CN1761746B - Fusion constructs and being used for produces the purposes of the antibody that Fc receptor binding affinity and effector function improve - Google Patents

Fusion constructs and being used for produces the purposes of the antibody that Fc receptor binding affinity and effector function improve Download PDF

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CN1761746B
CN1761746B CN200480007564.2A CN200480007564A CN1761746B CN 1761746 B CN1761746 B CN 1761746B CN 200480007564 A CN200480007564 A CN 200480007564A CN 1761746 B CN1761746 B CN 1761746B
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polypeptide
antibody
host cell
nucleic acid
cell
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CN1761746A (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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Abstract

The present invention relates to field of glycosylation engineering of proteins.More particularly, the present invention relates to nucleic acid molecule, comprise fusion constructs, it has catalytic activity and the purposes in the glycosylation engineering of host cell thereof to produce the treatment characteristic with strengthening, and comprises the polypeptide of antibody with the effector functions of the Fc receptors bind strengthened and enhancing.

Description

Fusion constructs and being used for produces 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 (glycosylationengineering).More particularly, the present invention relates to the nucleic acid molecule with catalytic activity, comprise fusion constructs, and the purposes in host cell glycosylation engineering, be used for produce treatment characteristic strengthen polypeptide, comprise Fc receptors bind improve with effector function improve antibody.
Background technology
The function of the many necessity in glycoprotein mediation human body, other eukaryote and some prokaryotic organism, comprises catalysis, sends signal, cell-ECM exchanges and molecular recognition and combination.They form most non-cytoplasmic protein matter in eukaryote.(Lis etc., Eur.J.Biochem.218:1-27 (1993)).Developed much glycoprotein and be used for the treatment of object, in nearest Two decades years, the Nature creating glycoprotein secretion of recombinant forms has become the main products in 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., Glycobiology1:115-130 (1991)).
Oligosaccharide ingredient can the remarkably influenced characteristic relevant with therapeutic glycoprotein effect, comprise physical stability, to the resistance of protease attack and immune interaction, pharmacokinetics and particular organisms active.Such characteristic not only depends on the presence or absence of oligosaccharides, also depends on its specific structure.Some general introductions between oligosaccharide structure and glycoprotein function can be formed.Such as, some oligosaccharide structure by and the protein-bonded interaction of particular carbon hydrate and mediated glycoprotein from blood flow and remove fast, and other can cause less desirable immune response by antibodies.(Jenkins etc., NatureBiotechnol.14:975-81 (1996)).
Owing to Protein Glycosylation Overview can be become be most suitable for the ability of people's application form, therefore mammalian cell is the preferred host of manufacture of therapeutic glycoprotein.(Cumming etc., Glycobiology1:115-30 (1991); Jenkins etc., NatureBiotechnol.14:975-81 (1996)).The little glycosylated protein of bacterium, and as the same with vegetable cell in yeast, filamentous fungus, insect with the common host of other kind, create and remove fast from blood flow, less desirable immunity interacts, and in some particular cases, reduce bioactive glycosylation pattern.Two decades years in the past, in mammalian cell, Chinese hamster ovary (CHO) cell uses the most general.Except giving suitable glycosylation pattern, these cells continue the cloned cell line producing inheritance stability, high yield.In simple bio, use serum free medium they can be cultured to high-density, and safe and reproducible biological method can be developed.Other conventional zooblast comprises Baby Hamster Kidney (BHK) cell, NS0-and SP2/0-murine myeloma cell.Recently, tested and produced from transgenic animal.(Jenkins etc., NatureBiotechnol.14:975-81 (1996)).
All antibody is guarded position at CH and is contained sugared structure, and each homotype has distinct N-and connects sugared structural arrangement, and it affects albumen assembling, secretion or functionally active changeably.(Wright, A., and Morrison, S.L., TrendsBiotech.15:26-32 (1997)).The sugared structure that connects the N-connected 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., TrendsBiotech.15:26-32 (1997)).Usually, there is at specific glycosylation site the xenogenesis process connecting core oligosaccharide structure makes monoclonal antibody exist with polyglycosylated form.Equally, the Main Differences having shown antibody glycosylation effect occurs between clone, even observes the nuance that given clone grows under different culture condition.(Lifely, M.R. etc., Glycobiology5 (8): 813-22 (1995)).
Non-conjugated monoclonal antibodies (mAb) can be used as the medicine of Therapeutic cancer, and the following medicine as ratified by food and drug administration is proved, Rituximab (Rituxan tM, IDECPharmaceuticals, SanDiego, CA, and GenentechInc., SanFrancisco, CA), be used for the treatment of CD20 positive B-cells, rudimentary or cryptomere Fei Huojinqi lymphoma, Trastuzumab (Herceptin tM, GenentechInc) be used for the treatment of late-stage breast cancer (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) and be used for the treatment of the acute myeloid of recurrence and Alemtuzumab (CAMPATH tM, MilleniumPharmaceuticals/ScheringAG) 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 depends on their outstanding security features, and (Grillo-Lopez, A.-J., etc., Semin.Oncol.26:66-73 (1999); Goldenberg, M.M., Clin.Ther.21:309-18 (1999)).Although the success of these medicines, at present large interest is existed to the specific antibody obtained than not puting together the usual activity obtained of mAb treatment higher.
Obtain usefulness improve greatly and maintain simple production method and the potential approach avoiding significant undesirable side effect, improve the natural cell-mediated effector function (Umana of mAb by through engineering approaches (engineering) their oligosaccharide ingredient, P. etc., NatureBiotechnol.17:176-180 (1999)).IgG1 type antibody, antibody the most frequently used in cancer immunotherapy is the glycoprotein at the Asn297 place of each CH2 structural domain with conservative N-connection glycosylation site.Two the Composite Double antennary oligosaccharide connecting Asn297 are hidden between CH2 structural domain, formation fully contacts with polypeptide backbone, and they to there is antagonist mediation effector function such as antibody dependent cellular cytotoxicity (ADCC) be required (Lifely, M.R. etc., Glycobiology5:813-822 (1995); Jefferis, R. etc., ImmunolRev.163:59-76 (1998); Wright, A. and Morrison, S.L., TrendsBiotechnol.15:26-32 (1997)).
β (1 is shown before present inventor, 4)-the overexpression of NAG transferase I II (GnTIII) in Chinese hamster ovary (CHO) cell, the formation of glycosyltransferase catalysis bisection (bisected) oligosaccharides, the external ADCC significantly improving the anti-neuroblastoma chimeric mAb (chCE7) that through engineering approaches Chinese hamster ovary celI produces is active.(see, Umana, P. etc., NatureBiotechnol.17:176-180 (1999), International Publication No.WO99/54342, each full content is incorporated herein by reference with its entirety at this).Antibody chCE7 belongs to and has high tumour affinity and specificly do not put together the large class of mAb, but usefulness is too little so that can not Clinical practice (Umana when producing with the standard industry clone lacking GnTIII enzyme, P. etc., NatureBiotechnol.17:176-180 (1999)).This research shows first to be expressed GnTIII by engineered antibody production cell and obtains the very big raising of ADCC activity, it also causes the bisected oligosaccharides that constant region (Fc) is relevant, comprise the increase of bisected, nonfucosylated oligosaccharide ratio, exceed the level found in natural generation antibody.
The result of great majority research shows that Fc-acceptor-dependency mechanism substantially explains cytotoxic antibody to antineoplastic action and to indicate antineoplastic optimum antibody and preferably to minimize and inhibition mating partner Fc γ RIIB combines with activation Fc receptors bind.(Clynes, R.A. etc., NatureMedicine6 (4): 443-446 (2000); Kalergis, A.M., and Ravetch, J.V., J.Exp.Med.195 (12): 1653-1659 (in June, 2002).Such as, to propose particularly Fc γ RIIIa acceptor strongly relevant with Antybody therapy usefulness for result of at least one research.(Cartron, G. etc., Blood99 (3): 754-757 (in February, 2002)).This research shows Fc γ RIIIa homozygous patient and has better reaction to Rituximab than heterozygosis patient.Author infers that preferably reaction is because antibody Binding in vivo Fc γ RIIIa is better, and it causes the ADCC better resisting lymphoma cell active.(Cartron, G. etc., Blood99 (3): 754-757 (in February, 2002)).
Except ADCC, successful anti-cancer monoclonal antibody induces Fc-dependent adjustment target cell to survive usually, propagation, or the direct signal mechanism of death, is caused or block the path of somatomedin by activating cells signal level.(Selenko, N. etc., J.Clin.Immunol.22 (3): 124-130 (2002)).Such as, CD20 is treated with Rituximab +cell demonstrated inducing complement mediation cracking and Mab induction apoptosis and ADCC.(Selenko, N. etc., J.Clin.Immunol.22 (3): 124-130 (2002)).In addition, Rituximab induces lymphoma cell apoptosis, the absorption of the peptide not only killing cell but also promote lymphoma to derive by antigen presentation dendritic cell (DC) and the submission that intersects, induction DC is ripe, and produces specific cells toxic T lymphocyte (CTL).
Invention summary
The huge treatment potential of the antibody improved with effector function recognizing that Fc receptor binding affinity improves, therefore the present invention inventors have developed the method for producing such antibody, and it comprises the glycoforms (profile) in engineered antibody Fc district.
The present invention briefly relates to the method for glycosyl through engineering approaches (glycoengineering) host cell to change the glycoforms of one or more polypeptide of this host cell generation.Method of the present invention may be used for producing and has the glycosylated treatment antibody of modification in Fc district, include reducing fucosylation, wherein has the effector function of raising and/or the Fc receptors bind of raising as the glycosylated result antibody of modification.Glycosyl engineered antibody of the present invention is used in particular for the oncotherapy of patient.In one embodiment, glycosyl through engineering approaches host cell of the present invention expresses the nucleic acid molecule that coding has GnTIII catalytic activity or GalT catalytic activity fusion polypeptide.In preferred embodiment, fusion constructs and coding have the nucleic acid molecule coexpression that the nucleic acid molecule of people ManII catalytic activity polypeptide and/or coding have GnTII catalytic activity polypeptide.Still, in another embodiment, improved by glycosyl through engineering approaches and express the host cell that coding has a nucleic acid molecule of ManII catalytic activity polypeptide and produce glycosyl engineered polypeptide of the present invention.
Therefore, one aspect of the present invention relates to the nucleic acid of the separation containing encode fusion peptide sequence, wherein said fusion polypeptide has β (1,4) the active Golgi localization domain (localizationdomain) also containing Golgi resident polypeptide (residentpolypeptide) of-NAG transferase I II (β (Isosorbide-5-Nitrae)-N-acetylglucosaminyltransferaseIII) (" GnTIII ").In one embodiment, fusion polypeptide contains the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II.In further embodiment, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of the localization domain of-NAG transferase I (" GnTI "), β (1, the 2)-localization domain of NAG transferase I I (" GnTII "), the localization domain of mannosidase I and α 1-6 core fucosyltransferase.In preferred embodiment, the nucleotide sequence of separation contains the nucleotide sequence shown in Figure 24 or Figure 25.In another preferred embodiment, the nucleic acid sequence encoding of separation has the polypeptide of aminoacid sequence shown in Figure 24 or Figure 25.Still, in another preferred embodiment, the nucleic acid sequence encoding of separation has the polypeptide of the aminoacid sequence identical with aminoacid sequence at least 80% shown in Figure 24 or Figure 25.
On the other hand, the present invention relates to the nucleic acid of the separation containing encode fusion peptide sequence, wherein said fusion polypeptide has the active Golgi localization domain also containing Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-galactosyltransferase (" GalT ").In one embodiment, fusogenic peptide contains the catalyst structure domain of β (Isosorbide-5-Nitrae)-galactosyltransferase.In another embodiment, fusogenic peptide contains the catalyst structure domain of β (Isosorbide-5-Nitrae) galactosyltransferase.In further embodiment, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of the localization domain of-NAG transferase I (" GnTI "), β (1, the 2)-localization domain of NAG transferase I I (" GnTII "), the localization domain of mannosidase I and α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to the expression vector containing separative nucleic acid, this nucleic acid contains the sequence of encode fusion polypeptide, wherein said fusion polypeptide has the active Golgi localization domain also containing Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II.In one embodiment, expression vector codes contains β (1,4) fusion polypeptide of-NAG transferase I II catalyst structure domain and Golgi localization domain, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of the localization domain of-NAG transferase I, β (1, the 2)-localization domain of NAG transferase I I, the localization domain of mannosidase I and α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to the expression vector containing separative nucleic acid, this nucleic acid contains the sequence of encode fusion polypeptide, and wherein said fusion polypeptide has β (Isosorbide-5-Nitrae)-galactosyltransferasactivity activity and contains the Golgi localization domain of Golgi resident polypeptide.In one embodiment, expression vector codes contains β (1,4) fusion polypeptide of the Golgi localization domain of-galactosyltransferase catalyst structure domain and Golgi resident polypeptide, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of the localization domain of-NAG transferase I, β (1, the 2)-localization domain of NAG transferase I I, the localization domain of mannosidase I and α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to the host cell containing 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 one coding and there is β (1,4)-NAG transferase I II (nucleic acid of the fusion polypeptide that " GnTIII " is active, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment containing Fc district and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.In one embodiment, the fusion polypeptide with GnTIII activity contains β (1,4)-the catalyst structure domain of NAG transferase I II and the Golgi localization domain of heterologous Golgi resident polypeptide, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain 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 one coding and there is β (1,4) nucleic acid of the fusion polypeptide that-galactosyltransferase (" GalT ") is active, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, the described polypeptide that wherein said host cell produces is selected from whole antibody molecule, antibody fragment containing Fc district and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.In one embodiment, the fusion polypeptide with GalT activity contains β (1,4) catalyst structure domain of-galactosyltransferase and the Golgi localization domain of heterologous Golgi resident polypeptide, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain of α 1-6 core fucosyltransferase.
Preferably, Golgi localization domain is from mannosidase II or β (1,2)-NAG transferase I or galactosyltransferase.
On the other hand, the present invention relates to the fusion polypeptide with the active Golgi localization domain also containing heterologous Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II.In one embodiment, fusion polypeptide of the present invention contains the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II.In another embodiment, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain of α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to the fusion rotein with the Golgi localization domain of β (Isosorbide-5-Nitrae)-galactosyltransferasactivity activity also containing heterologous Golgi resident polypeptide.In one embodiment, fusion polypeptide of the present invention contains the catalyst structure domain of β (Isosorbide-5-Nitrae)-galactosyltransferase.In another embodiment, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain of α 1-6 core fucosyltransferase.
Preferably, Golgi localization domain is from mannosidase II or β (1,2)-NAG transferase I (" GnTI ") or galactosyltransferase (" GalT ").
On the other hand, the present invention relates to production and there is β (1,4) method of the fusion polypeptide of-NAG transferase I II activity, cultivates host cell of the present invention in the medium under being included in the condition of the expression of nucleic acid of permission encode fusion polypeptide and collect fusion polypeptide from generation culture.In one embodiment, fusion polypeptide contains the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II.Preferably, fusion polypeptide contains the Golgi localization domain of heterologous Golgi resident polypeptide, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain of α 1-6 core fucosyltransferase.
On the other hand, the present invention relates to production and there is β (1,4) method of the fusion rotein of-galactosyltransferasactivity activity, cultivates host cell of the present invention in the medium under being included in the condition of the expression of nucleic acid of permission encoding fusion protein and collect fusion polypeptide from generation culture.In one embodiment, fusion polypeptide contains the catalyst structure domain of β (Isosorbide-5-Nitrae)-galactosyltransferase.Preferably, fusion polypeptide contains the Golgi localization domain of heterologous Golgi resident polypeptide, Golgi localization domain is selected from localization domain, the β (1 of mannosidase II, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain of α 1-6 core fucosyltransferase.
Preferably, Golgi localization domain is from mannosidase II or β (1,2)-NAG transferase I or galactosyltransferase (" GalT ").
Again on the one hand, the present invention relates to the method for the glycoforms (profile) changing the polypeptide that host cell produces, comprise and nucleic acid of the present invention at least one or expression vector are introduced in host cell.Preferably, polypeptide be IgG or its contain the fragment in polypeptide Fc district.In particularly preferred embodiment, polypeptide be IgG1 or its contain the fragment in polypeptide Fc district.Or polypeptide is fusion rotein, fusion rotein comprises the region being equivalent to human IgG Fc district.
On the other hand, the present invention relates to the method for producing polypeptide in host cell, comprise: (a) is allowing to cultivate host cell under the condition producing polypeptide, this host cell through engineering approaches is expressed at least one coding and is had β (1, 4)-NAG transferase I II activity or β (1, 4) nucleic acid of the fusion polypeptide that-galactosyltransferase (" GalT ") is active, the polypeptide produced is selected from whole antibody molecule, antibody fragment containing Fc district and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, the expression amount of wherein said fusion rotein is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, (b) described polypeptide is separated.Preferably, fusion polypeptide contains β (1, 4)-NAG transferase I be II's or β (1, 4) catalyst structure domain of-galactosyltransferase (" GalT ") and comprise the Golgi localization domain of heterologous Golgi resident polypeptide further, Golgi localization domain is selected from the localization domain of mannosidase II, β (1, 2) localization domain of-NAG transferase I, the localization domain of mannosidase I, β (1, 2) localization domain of-NAG transferase I I and the localization domain of α 1-6 core fucosyltransferase.In preferred embodiment, the polypeptide produced as the result host cell modified has the effector function of raising and/or the Fc receptors bind of raising.In particularly preferred embodiment, the effector function improved be the raising that combines of the raising that combines of the Cytotoxic raising of Fc mediation and the raising of NK Cell binding and scavenger cell and monocyte and polymorphonuclear cell combine raising, the raising of apoptosis of direct signal induction, the raising of dendritic cell maturation and/or T cell sensitization (priming) raising, the Fc receptors bind of raising is the raising combined as Fc γ RIIIA with Fc activated receptor.Preferably, present that the polypeptide that effector function improves and/or Fc receptors bind improves is antibody, antibody fragment or fusion rotein have and carry the non-fucosylated oligosaccharide in a high proportion of Fc district, fusion rotein comprises the region being equivalent to immunoglobulin fc region.
On the other hand, the present invention relates to pharmaceutical composition, containing antibody of the present invention, antibody fragment containing Fc district or fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, and relates to this pharmaceutical composition in treatment tumour as the purposes in cancer or Other diseases.In one embodiment, treatment is that B cell exhausts (Bcelldepletion), by this pharmaceutical composition for the treatment of significant quantity is delivered medicine to the patient of needs as people.
Still more on the one hand, the invention provides host cell, comprise the expression vector containing encode fusion polypeptide-nucleic acid molecule, wherein said fusion polypeptide has the active Golgi localization domain also containing Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII); With the expression vector of the nucleic acid molecule containing coded polypeptide, it is active that wherein said polypeptide has mannosidase II (ManII).In preferred embodiment, fusion polypeptide contains catalyst structure domain and the Golgi localization domain of GnTIII, and Golgi localization domain is selected from the localization domain of the localization domain of ManII, the localization domain of GnTI, the localization domain of GnTII, the localization domain of ManI and α 1-6 core fucosyltransferase.In one embodiment, host cell comprises the expression vector that coding has the polypeptide of GnTII activity further.Encode fusion polypeptide, there is the polypeptide of ManII activity, there is the nucleic acid molecule of the polypeptide of GnTII activity, can be each in the expression vector separated or in same expression vector.
In addition on the one hand, the present invention relates to host cell, comprise the expression vector containing encode fusion polypeptide-nucleic acid molecule, wherein said fusion polypeptide has β (Isosorbide-5-Nitrae)-galactotransferase activity (GalT) and contains the Golgi localization domain of Golgi resident polypeptide; With the expression vector of the nucleic acid molecule containing coded polypeptide, it is active that wherein said polypeptide has mannosidase II (ManII).In preferred embodiment, fusion polypeptide comprises catalyst structure domain and the Golgi localization domain of GnTIII, Golgi localization domain is selected from the localization domain of the localization domain of ManII, the localization domain of GnTI, the localization domain of GnTII, the localization domain of ManI and α 1,6 core fucosyltransferase.In one embodiment, host cell comprises the expression vector that coding has the polypeptide of GnTII activity further.Encode fusion polypeptide, there is the polypeptide of ManII activity, there is the nucleic acid molecule of the polypeptide of GnTII activity, can be each in the expression vector separated or in same expression vector.
Again in one side, the present invention relates to and relate to host cell, this host cell engineering is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, expression amount is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, 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 region being equivalent to immunoglobulin fc region.
In other embodiments, the invention provides host cell, this host cell through engineering approaches expresses that at least one coding has the nucleic acid of the fusion polypeptide of GnTIII activity, at least one coding has the nucleic acid of the polypeptide of ManII activity, with at least one coding, there is the nucleic acid of the polypeptide of GnTII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of described host cell generation, 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 region being equivalent to immunoglobulin fc region.
Again on the one hand, the invention provides host cell, this host cell engineering is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GalT activity and at least one coding have the polypeptide of ManII activity, expression amount is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, 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 region being equivalent to immunoglobulin fc region.
In addition on the one hand, the invention provides host cell, this host cell through engineering approaches expresses that at least one coding has the nucleic acid of the fusion polypeptide of GalT activity, at least one coding has the nucleic acid of the polypeptide of ManII activity, with at least one coding, there is the nucleic acid of the polypeptide of GnTII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of described host cell generation, 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 region being equivalent to immunoglobulin fc region.
Again on the one hand, the present invention relates to the method for producing polypeptide in host cell, host cell is cultivated under being included in the condition allowing polypeptide to produce, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, the expression amount of wherein said fusion polypeptide is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, with be separated described polypeptide.
On the other hand, the present invention relates to the method for producing polypeptide in host cell, host cell is cultivated under being included in the condition allowing polypeptide to produce, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GalT activity and at least one coding have the polypeptide of ManII activity, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, the expression amount of wherein said fusion polypeptide is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, with be separated described polypeptide.
In addition on the one hand, the production method of the polypeptide that the cytotoxicity of producing Fc mediation in host cell improves, host cell is cultivated under being included in the condition allowing polypeptide to produce, this host cell through engineering approaches expresses the nucleic acid of at least one coding GalT and the nucleic acid of at least one coding ManII, the polypeptide produced is selected from whole antibody molecule, antibody fragment, antibody fragment comprises immunoglobulin fc region, one or two expression level wherein in GalT or ManII be enough to modify oligosaccharides in the described polypeptide Fc district that described host cell produces and wherein as described modification result described in the cytotoxicity that mediates of the polypeptide Fc with raising, with the polypeptide being separated the cytotoxicity raising that described Fc mediates.
On the other hand, the present invention relates to the method for producing polypeptide in host cell, comprise: (a) cultivates host cell under the condition allowing polypeptide to produce, this host cell through engineering approaches expresses the nucleic acid that at least one coding has the polypeptide of alpha-Mannosidase II activity, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, and the wherein said expression amount with the polypeptide of alpha-Mannosidase II activity is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation; (b) the described polypeptide produced by described host cell is separated.
On the other hand, the present invention relates to host cell, this host cell through engineering approaches expresses the nucleic acid that at least one coding has the polypeptide of alpha-Mannosidase II activity under the condition allowing polypeptide to produce, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, and the wherein said expression amount with the polypeptide of alpha-Mannosidase II activity is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation.
Still on the other hand, the present invention relates to the polypeptide produced by such host cell, especially there is as the result of described modification oligosaccharides the antibody of the effector function of raising and/or the Fc receptors bind of raising.
Accompanying drawing is sketched
Fig. 1. the MALDI/TOF-MS from the neutral oligosaccharide mix of unmodified (non-sugar based through engineering approaches) the anti-CD 20 IgG1 antibody of the restructuring produced in BHK composes.With antibody expression vector pETR1502 transfectional cell.Antibody purification from substratum as described in the materials and methods part of embodiment 1 is also prepared and analyzes oligosaccharides.
Fig. 2. carry out the MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of the restructuring produced in the BHK of the engineered nucleic acid of personal encoding wild type (" wt ") GnTIII.With antibody expression vector pETR1502 and GnTIII expression vector pETR1166 cotransfection cell.Antibody purification from substratum as described in the materials and methods part of embodiment 1 is also prepared and analyzes oligosaccharides.
Fig. 3. using by oneself coding has the active and MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of restructuring that is that produce in the BHK of the engineered nucleic acid of the fusion polypeptide (" G1-GnTIII ") of being located by GnTI-Golgi localization domain of GnTIII.With antibody expression vector pETR1502 and GnTIII expression vector pETR1425 cotransfection cell.Antibody purification from substratum as described in the materials and methods part of embodiment 1 is also prepared and analyzes oligosaccharides.
Fig. 4. carry out personal coding and there is the active and MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of restructuring that is that produce in BHK by the engineered nucleic acid of the fusion polypeptide (" M2-GnTIII ") of golgi body alpha-Mannosidase II (ManII)-Golgi localization domain location of GnTIII.With antibody expression vector pETR1502 and GnTIII expression vector pETR1506 cotransfection cell.Antibody purification from substratum as described in embodiment 1 materials and methods part is also prepared and analyzes oligosaccharides.
Fig. 5. the MALDI/TOF-MS from the neutral oligosaccharide mix of unmodified (non-sugar based through engineering approaches) the anti-CD 20 IgG1 antibody of the restructuring produced in HEK293-EBNA cell composes.With antibody expression vector pETR1520 transfectional cell.Antibody purification from substratum as described in the materials and methods part of embodiment 1 is also prepared and analyzes oligosaccharides.
Fig. 6. carry out personal coding and there is the active and MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of restructuring that is that produce in HEK293-EBNA by the engineered nucleic acid of the fusion polypeptide (" M2-GnTIII ") of golgi body alpha-Mannosidase II (ManII)-Golgi localization domain location of GnTIII.With antibody expression vector pETR1520 with GnTIII expression vector pETR1519 cotransfection cell.Antibody purification from substratum as described in the materials and methods part of embodiment 1 is also prepared and analyzes oligosaccharides.
Fig. 7. carry out personal coding and there is the active and MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of the glycosyl through engineering approaches anti-CD 20 IgG1 antibody of restructuring that is that produce in HEK293-EBNA by the engineered nucleic acid of the fusion polypeptide (" M2-GnTIII ") of golgi body alpha-Mannosidase II (ManII)-Golgi localization domain location of GnTIII.With antibody expression vector pETR1520 with GnTIII expression vector pETR1519 cotransfection cell.Antibody purification from substratum as described in the materials and methods part of embodiment 1 is also prepared and analyzes oligosaccharides.A () does not have the PNGaseF-of other ferment treatment to discharge the oligosaccharides characteristic pattern of oligosaccharides.B () discharges the oligosaccharides characteristic pattern of oligosaccharides further with the PNGaseF-of EndoH digestion.
Fig. 8. the schematic diagram of the digestion oligosaccharides of (a) EndoH catalysis.EndoH can digest heterozygosis (hybrid) (halving with heterozygosis) oligosaccharides, but indigestion compound (complex) or compound bisected oligosaccharides.B () be compound and heterozygous oligosaccharides by explanation, structure is distributed to initial from the oligosaccharides peak value in the MALDI/TOF-MS mass spectrum of PNGAseF process with identical m/z ratio by Endo-H process.
Fig. 9. the restructuring to unmodified of " G1-GnTIII "-glycosyl through engineering approaches, anti-CD20 is fitted together to the antibody dependent cellular cytotoxicity (ADCC) of IgG1 antibody.Two antibody all produce in bhk cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Fig. 3 and unmodified antibody those in FIG.Target cell (T) is the lymphoblastoid of SKW6.4 people.Effector cell (E) is the human PBMC of fresh separated.Ratio be 25: 1 E: T be used within 4 hours, hatch ADCC test in, cytotoxicity is measured, relative to maximum release (using stain remover to substitute antibody) and spontaneous release (substratum substitutes antibody) contrast by serum lactic dehydrogenase (LDH) release.Test is described in detail in the materials and methods part of embodiment 1.
Figure 10. the restructuring to unmodified of " M2-GnTIII "-glycosyl through engineering approaches, anti-CD20 is fitted together to the antibody dependent cellular cytotoxicity (ADCC) of IgG1 antibody.Two antibody all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Fig. 6 and unmodified antibody those in Figure 5.Target cell (T) is the lymphoblastoid of SKW6.4 people.Effector cell (E) is the human PBMC of fresh separated.Ratio be 25: 1 E: T be used for 4 hours cultivate ADCC test in, cytotoxicity is measured, relative to maximum release (using stain remover to substitute antibody) and spontaneous release (substratum substitutes antibody) contrast by serum lactic dehydrogenase (LDH) release.Test is described in detail in the materials and methods part of embodiment 1.
Figure 11. the restructuring to " wt-GnTIII " glycosyl through engineering approaches of " M2-GnTIII " glycosyl through engineering approaches, anti-CD20 is fitted together to the antibody dependent cellular cytotoxicity (ADCC) of IgG1 antibody.Two antibody all produce in bhk cell.The generation of M2-GnTIII glycosyl engineered antibody and glycosylation characteristic pattern to be described in Fig. 4 and wt-GnTIII glycosyl engineered antibody those in fig. 2.Target cell (T) is the lymphoblastoid of SKW6.4 people.Effector cell (E) is the human PBMC of fresh separated.Ratio be 25: 1 E: T be used for 4 hours cultivate ADCC test in, cytotoxicity is measured, relative to maximum release (using stain remover to substitute antibody) and spontaneous release (substratum substitutes antibody) contrast by serum lactic dehydrogenase (LDH) release.Test is described in detail in the materials and methods part of embodiment 1.
Figure 12. the restructuring to unmodified of " M2-GnTIII "-glycosyl through engineering approaches, anti-CD20 is fitted together to the combination of the Fc γ RIIIa acceptor on IgG1 antibody and NK cell.Two antibody all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Fig. 6 and unmodified antibody those in Figure 5.Binding tests is carried out as described in the materials and methods part of embodiment 1.Be separated from the known genotype donor not producing Fc γ RIIc acceptor (that is, the homozygous gene variant containing in-frame stop codon in Fc γ RIIc encoding sequence) in the NK cells of human beings of its surface expression Fc γ RIIIa acceptor.The geometric mean fluorescence intensity measured of anti-human IgG antibodies's fragment of FITC mark is used to increase along with the amount of recombinant antibodies and NK Cell binding by FACS.The combination detected in this test is that Fc γ RIIIa is specific, as proved (see Figure 13) by use competition Fc γ RIIIa specific Ab fragments.
Figure 13. under the competition anti-Fc γ RIII antibody fragment increasing concentration exists, the restructuring to unmodified of " M2-GnTIII "-glycosyl through engineering approaches, anti-CD20 is fitted together to the combination of the Fc γ RIIIa acceptor on IgG1 antibody and NK cell.Two recombinant antibodies all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Fig. 6 and unmodified antibody those in Figure 5.Binding tests is carried out as described in the materials and methods part of embodiment 1, but with recombinant antibodies (usual final concentration is 3 μ g/ml) with to increase and the competition 3G8-Fab2 anti-Fc γ RIII antibody fragment changing concentration (see chart) hatches the NK cell of purifying altogether.Be separated from the known genotype donor not producing Fc γ RIIc acceptor (that is, the homozygous gene variant containing in-frame stop codon in Fc γ RIIc encoding sequence) in the NK cells of human beings of its surface expression Fc γ RIIIa acceptor.The geometric mean fluorescence intensity measured of anti-human IgG antibodies's fragment of FITC mark is used to increase along with the amount of recombinant antibodies and NK Cell binding by FACS.
Figure 14. identify that the ED-B+ isoform of fibronectin the MALDI/TOF-MS of the neutral oligosaccharide mix of restructuring IgG1 " L19 " antibody produced in HEK293-EBNA cell compose.A unmodified antibody that () produces in the HEK293-EBNA cell with antibody expression vector pETR1546 transfection.B M2-GnTIII glycosyl engineered antibody that () produces in the HEK293-EBNA cell with antibody expression vector pETR1546 and GnTIII expression vector pETR1519 cotransfection.By a-protein affinity chromatograph size exclusion chromatographic step purifying two kinds of antibody from substratum subsequently, damping fluid is exchanged the salt solution (PBS) to phosphate buffered by Superdex200 matrix (Amersham).Preparation as described in the materials and methods of embodiment 1 and analysis oligosaccharides.
Figure 15. the restructuring to unmodified of " M2-GnTIII "-glycosyl through engineering approaches, the combination of the Fc γ RIIb acceptor on anti-ED-B+ fibronectin IgG1 antibody and Raji human lymphoma cell.Two antibody all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Figure 14 b and those of unmodified antibody in Figure 14 a.Binding tests is carried out as described in the materials and methods part of embodiment 1.The amount using the geometric mean fluorescence intensity measured of anti-human IgG antibodies's fragment of FITC mark to combine along with recombinant antibodies and RajiB cell lymphoma cell by FACS and increasing.
Figure 16. the restructuring to unmodified of " M2-GnTIII "-glycosyl through engineering approaches, anti-CD20 is fitted together to the combination of the Fc γ RIIIa acceptor on IgG1 antibody and different donor NK cell.Two antibody all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Fig. 6 and unmodified antibody those in Figure 5.Binding tests is carried out as described in the materials and methods part of embodiment 1.Be separated from the known genotype donor not producing Fc γ RIIc acceptor (that is, the homozygous gene variant containing in-frame stop codon in Fc γ RIIc encoding sequence) in the NK cells of human beings of its surface expression Fc γ RIIIa acceptor.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.The geometric mean fluorescence intensity measured of anti-human IgG antibodies's fragment of FITC mark is used to increase along with the amount of recombinant antibodies and NK Cell binding by FACS.The combination detected in this test is that Fc γ RIIIa is specific, as proved (see Figure 13) by use competition Fc γ RIIIa specific Ab fragments.
Figure 17. the facs analysis that the CD4 (tCD4) of brachymemma expresses, (a) BHK-1502-28 (wild-type) and (b) clone the stable inosculating antibody-CD20IgG1 antibody produced cell system of BHK-1502-28-11 (M2-GnTIII glycosyl through engineering approaches).By the IRES sequence in pETR1537GnTIII expression vector, the expression of tCD4 is operationally expressed the indirect labelling being connected and being also therefore used as GnTIII and expressing with M2-GnTIII.Respective average and geometric mean fluorescence intensity is, glycosyl engineered cell lines 27.6 and 19.9, wild-type cell system 4.7 and 4.1.
Figure 18. the anti-CD20 of restructuring from the M2-GnTIII-glycosyl through engineering approaches of BHK-1502-28-11 clone generation is fitted together to the MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of IgG1 antibody.The preparation of clone, antibody purification and oligosaccharides and analysis are described in the materials and methods part of embodiment 1.
Figure 19. the anti-CD20 of restructuring from the M2-GnTIII-glycosyl through engineering approaches of BHK-1502-28-11 clone generation is fitted together to the MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of IgG1 antibody, discharges oligosaccharides and digest with EndoH further by PNGaseF.The preparation of clone, antibody purification and oligosaccharides and analysis are described in the materials and methods part of embodiment 1.
Figure 20 .M2-GnTIII " restructuring to unmodified of glycosyl through engineering approaches, anti-CD20 is fitted together to the combination of the Fc γ RIIIa acceptor on IgG1 antibody and NK cell, antibody is produced by stable cell lines.Generation and the glycosylation characteristic pattern of glycosyl engineered antibody are described in Figure 18 and 19.Binding tests is carried out as described in the materials and methods part of embodiment 1.Be separated from the known genotype donor not producing Fc γ RIIc acceptor (that is, the homozygous gene variant containing in-frame stop codon in Fc γ RIIc encoding sequence) in the NK cells of human beings of its surface expression Fc γ RIIIa acceptor.The geometric mean fluorescence intensity measured of anti-human IgG antibodies's fragment of FITC mark is used to increase along with the amount of recombinant antibodies and NK Cell binding by FACS.The combination detected in this test is that Fc γ RIIIa is specific, as proved (see Figure 13) by use competition Fc γ RIIIa specific Ab fragments.
Figure 21. the restructuring to unmodified of " M2-GnTIII " glycosyl through engineering approaches, anti-CD20 is fitted together to the complement mediated lysis (CML) of IgG1 antibody.Two antibody all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation feature description in Fig. 6 and unmodified antibody those in Figure 5.Target cell (T) is the lymphoblastoid of SKW6.4 people.People's complement is used for test.Discharged by LDH and measure cracking.Test is described in detail in the materials and methods part of embodiment 1.
Figure 22. carry out self-identifying human epidermal growth factor (EGFR) and the restructuring produced in HEK293-EBNA cell be fitted together to the neutral oligosaccharide mix of IgG1 " C225 " antibody MALDI/TOF-MS spectrum.The unmodified antibody produced in (a) HEK293-EBNA cell with antibody expression vector pURSI28 transfection.B () is with the M2-GnTIII glycosyl engineered antibody produced in the HEK293-EBNA cell of antibody expression vector pETRURSI28 and GnTIII expression vector pETR1519 cotransfection.By a-protein affinity chromatograph size exclusion chromatographic step purifying two antibody from substratum subsequently, damping fluid is exchanged the salt solution (PBS) to phosphate buffered by Superdex200 matrix (Amersham).Preparation as described in the materials and methods of embodiment 1 and analysis oligosaccharides.
Figure 23. the restructuring to unmodified of " M2-GnTIII "-glycosyl through engineering approaches, anti-EGFR is fitted together to the antibody dependent cellular cytotoxicity (ADCC) of IgG1 " C225 " antibody.Two antibody all produce in HEK293-EBNA cell.The generation of glycosyl engineered antibody and glycosylation characteristic pattern to be described in Figure 22 b and those of unmodified antibody in Figure 22 a.Target cell (T) is A431 people's squamous cancer cell (ECACCNo.85090402).Effector cell (E) is the human PBMC of fresh separated.Ratio be 25: 1 E: T be used for 4 hours cultivate ADCC test in, cytotoxicity is measured, relative to maximum release (using stain remover to substitute antibody) and spontaneous release (substratum substitutes antibody) contrast by serum lactic dehydrogenase (LDH) release.Test is described in detail in the materials and methods part of embodiment 1.
Figure 24. be respectively nucleotide sequence and the aminoacid sequence of mannosidase II-GnTIII fusion polypeptide of the present invention.
Figure 25. be respectively nucleotide sequence and the aminoacid sequence of GnT-I-GnT-III fusion polypeptide of the present invention.
Figure 26. the anti-CD20 of unmodified recombinant C 2B8 coming to produce in the HEK293-EBNA cell of personal antibody expression vector pETR1520 transfection is fitted together to the MALDI/TOF-MS spectrum of the neutral oligosaccharide mix of IgG1 antibody (" Cwt ").Antibody purification from substratum as described in the materials and methods part of embodiment 5 is also prepared and analyzes oligosaccharides.
Figure 27. carry out personal antibody expression vector pETR1520 and merge the MALDI/TOF-MS spectrum that the anti-CD20 of restructuring glycosyl through engineering approaches C2B8 produced in the HEK293-EBNA cell of GnTIII polypeptide expression vector (pETR1519) cotransfection is fitted together to the neutral oligosaccharide mix of IgG1 antibody (" Cbrt ").Antibody purification from substratum as described in the materials and methods part of embodiment 5 is also prepared and analyzes oligosaccharides.(A) PNGaseF-of other ferment treatment is not had to discharge the oligosaccharides characteristic pattern of oligosaccharides.(B) the oligosaccharides characteristic pattern of oligosaccharides is discharged further with the PNGaseF-of EndoH digestion.
Figure 28. carry out personal antibody expression vector pETR1520, merge the MALDI/TOF-MS spectrum that the anti-CD20 of restructuring glycosyl through engineering approaches C2B8 produced in the HEK293-EBNA cell of GnTIII polypeptide expression vector (pETR1519) and mannosidase II polypeptide expression vector (pCLF9) cotransfection is fitted together to the neutral oligosaccharide mix of IgG1 antibody (" Cm ").Antibody purification from substratum as described in the materials and methods part of embodiment 5 is also prepared and analyzes oligosaccharides.(A) PNGaseF-of other ferment treatment is not had to discharge the oligosaccharides characteristic pattern of oligosaccharides.(B) the oligosaccharides characteristic pattern of oligosaccharides is discharged further with the PNGaseF-of EndoH digestion.
Figure 29. by the antibody dependent cellular cytotoxicity (ADCC) that the inosculating antibody CD20 that carrys out glycosyl through engineering approaches at the nucleic acid of HEK293-EBNA cells coding ManII-GnTIII fusion polypeptide is antibody-mediated, wherein GnTIII catalyst structure domain is located by ManII Golgi localization domain, the nucleic acid of the ManII-GnTIII that wherein encodes himself (antibody Cbrt) upper express or and the nucleic acid (Cm) of the ManII that encodes in antibody produced cell together with coexpression.Cwt is that the anti-CD20 of recombinant C 2B8 of the unmodified produced in the HEK293-EBNA cell with antibody expression vector pETR1520 transfection is fitted together to IgG1 antibody (" Cwt ").Test is described in detail in the materials and methods part of embodiment 1.
Figure 30. by carrying out the Fc γ RIIIa receptors bind of the chimeric anti-CD20 antibodies of glycosyl through engineering approaches at the nucleic acid of HEK293-EBNA cells coding ManII-GnTIII fusion polypeptide, wherein GnTIII catalyst structure domain is located by ManII Golgi localization domain, wherein ManII-GnTIII coding nucleic acid be himself (antibody Cbrt) upper express or and the nucleic acid (Cm) of the ManII that encodes in antibody produced cell together with coexpression.Cwt is that the anti-CD20 of recombinant C 2B8 of the unmodified produced in the HEK293-EBNA cell with antibody expression vector pETR1520 transfection is fitted together to IgG1 antibody (" Cwt ").Test is described in detail in the materials and methods part of embodiment 1.Be separated from the known genotype donor not producing Fc γ RIIc acceptor (that is, the homozygous gene variant containing in-frame stop codon in Fc γ RIIc encoding sequence) in the NK cells of human beings of its surface expression Fc γ RIIIa acceptor.The geometric mean fluorescence intensity measured of anti-human IgG antibodies's fragment of FITC mark is used to increase along with the amount of recombinant antibodies and NK Cell binding by FACS.The combination detected in this test is that Fc γ RIIIa is specific, as proved (see Figure 13) by use competition Fc γ RIIIa specific Ab fragments.
Figure 31. by carrying out the complement-mediated cytotoxicity of the chimeric anti-CD20 antibodies of glycosyl through engineering approaches at the nucleic acid of HEK293-EBNA cells coding ManII-GnTIII fusion polypeptide, wherein GnTIII catalyst structure domain is located by ManII Golgi localization domain, wherein ManII-GnTIII coding nucleic acid be himself (antibody Cbrt) upper express or and the nucleic acid (Cm) of the ManII that encodes in antibody produced cell together with coexpression.Cwt is that the anti-CD20 of recombinant C 2B8 of the unmodified produced in the HEK293-EBNA cell with antibody expression vector pETR1520 transfection is fitted together to IgG1 antibody (" Cwt ").
Figure 32 (A-C). expression vector pCLF9 (A), pETR1842 (B) and pETR1843 (C).
Figure 33 (A and B). 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 produced under alpha-Mannosidase II exists and find with the relative percentage of antibody Fc portion dependency structure.
Figure 35 (A and B). the oligosaccharides characteristic pattern of the anti-CD-20 monoclonal antibody produced under fusion rotein ManII-GalT exists and find with the relative percentage of antibody Fc portion dependency structure.PNGaseF (A) and the postdigestive oligosaccharides characteristic pattern of EndoH (B).
Figure 36. the antibody produced under alpha-Mannosidase II (ManII) exists and Fc γ RIIIA receptors bind, have the affinity higher than wild-type antibodies.
Figure 37. the antibody dependent cellular cytotoxicity that glycosyl through engineering approaches inosculating antibody CD-20 mediates.
Detailed Description Of The Invention
Term and this area are usually used the same as used herein, unless in addition as given a definition.
As used in this, term antibody is defined as and comprises whole antibody molecule, comprises mono-clonal, polyclone and polyspecific (such as, dual specific) antibody, and there is antibody fragment and the fusion rotein in Fc district, fusion rotein comprises the region being equivalent to immunoglobulin fc region.Also comprise humanization and chimeric antibody.
As used in this, term Fc area definition is the C-terminal region of IgG heavy chain.Although the boundary in IgG heavy chain Fc district can change slightly, human IgG heavy chain Fc district is normally defined one section to C-terminal from the Cys226 amino-acid residue of position.
As used in this, the region that term is equivalent to immunoglobulin fc region is defined as the natural generation allele variant comprising immunoglobulin fc region and the variant with change, change create alternative, add or disappearance but there is no reduce immunoglobulin-mediated effector function (as antibody dependent cellular cytotoxicity) ability.Such as, one or more amino acid can be lacked from the N-terminal of immunoglobulin fc region or C-terminal and there is no and lose biological function.Such variant can be selected to make the impact of activity according to general rule known in the art minimum.(see, such as, Bowie, J.U. etc., Science247:1306-10 (1990).
As used in this, the fusion polypeptide of " have β (Isosorbide-5-Nitrae)-NAG transferase I II active " refers to and catalysis 2-Acetamido-2-deoxy-D-glucose (GlcNAc) residue in β-1-4 key can be added into fusion polypeptide on mannoside that β that N-connects three mannose group cores of oligosaccharides connects.This comprises and presenting and β (1,4) fusion polypeptide that-NAG transferase I II enzymic activity is similar but required not identical, this enzyme is also referred to as β-1-4 mannosyl-glycoprotein 4-β-NAG transferring enzyme (EC2.4.1.144), according to biochemical and the molecular biosciences Intemational Nomenclature council (NC-IUBMB), as measured in particular organisms test, having and there is no dose-dependently.When dose-dependently exists, do not need to be equal to β (1,4)-NAG transferase I II, but with β (1,4)-NAG transferase I II compares substantially similar (namely with given active dose dependency, relative to β (1,4)-NAG transferase I II, candidate polypeptide will present higher activity or active unlike it low more than 25 times, preferably active unlike it low more than 10 times, most preferably unlike its activity of active low more than 3 times).
As used in this, " there is β (Isosorbide-5-Nitrae)-galactosyltransferasactivity activity " with the fusion polypeptide of " there is GalT activity " and refer to and catalysis the galactose residue of UDP semi-lactosi can be added into the fusion polypeptide that N is connected the non reducing end GlcNAc found in oligosaccharides.This comprises and presents enzymic activity and β (1,4) fusion polypeptide that-galactosyltransferase is similar but required not identical, this enzyme is also referred to as UDP-Gal:GlcNAc β-1,4-galactosyltransferase (E.C.2.4.1.38), according to biochemical and the molecular biosciences Intemational Nomenclature council (NC-IUBMB), as measured in particular organisms test, having and there is no dose-dependently.When dose-dependently exists, do not need to be equal to β (1,4)-galactosyltransferase, but with β (1,4)-galactosyltransferase is compared substantially similar (namely with given active dose dependency, relative to β (1,4)-NAG transferase I II, candidate polypeptide will present higher activity or active unlike it low more than 25 times, preferably active unlike it low more than 10 times, most preferably unlike its activity of active low more than 3 times).
Have with the present invention with reference to the nucleotide sequence at least such as nucleic acid of the nucleotide sequence of 95% " identical " or polypeptide, the nucleotide sequence being defined as polynucleotide is equal to canonical sequence, except polynucleotide sequence can comprise with reference to every 100 Nucleotide of nucleotide sequence up to five point mutation.In other words, in order to obtain the polynucleotide with the nucleotide sequence identical with reference nucleotide sequence at least 95%, can delete by another nucleotide substitution up to the Nucleotide of 5% in canonical sequence, or can insert in canonical sequence up to multiple Nucleotide of total nucleotide in canonical sequence 5%.Search sequence can be shown in the whole sequence in Figure 24 or Figure 25.
As practical situation, the whether any specific nucleic acid molecule of known computer program conventional determining or polypeptide and nucleotide sequence of the present invention or peptide sequence can be used to be at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.Measure the preferred method of the best overall coupling between search sequence (sequence of the present invention) and object sequence, also referred to as global sequence's comparison, the FASTDB computer program of the algorithm based on Brutlag etc. can be used to measure, Brutlag etc., Comp.App.Biosci.6:237-245 (1990).In sequence alignment, search sequence and object sequence are all DNA sequence dnas.RNA sequence can be compared by U being converted to T.The result of described global sequence comparison is % homogeny.Calculating preferred parameter used in the FASTDB comparison of the DNA sequencing of % homogeny is: matrix=single entry, k-tuple=4, mispairing punishment=1, connect punishment=30, Randomization Group Length=0, close value=1, gap penalty=5, breach size punishment 0.05, the length of window size=500 or object nucleotide sequence, is as the criterion with shorter one.
If because 5 ' or 3 ' lacks, instead of due to inside disappearance, object sequence is shorter than search sequence, must carry out manual synchronizing to this result.This is because FASTDB program does not have 5 ' and 3 ' brachymemma of calculating object sequence when calculating % homogeny.For 5 ' or 3, the object sequence of end brachymemma, relative to search sequence, is not had the base quantity of the search sequence of pairing/comparison, as the per-cent of the total base of search sequence, corrects % homogeny by calculating object sequence 5 ' and 3 '.Measure Nucleotide by the result of FASTDB sequence alignment and whether mate/comparison.Then from the % homogeny of the FASTDB program computation by above-mentioned use special parameter, deduct this per-cent, obtain final % homogeny value.The value of this correction is the value for the object of the invention.Only calculate as shown by FASTDB comparison, not have and base outside the object sequence 5 ' of search sequence coupling/comparison and 3 ' base is object in order to manually adjust % homogeny value.
Such as, the search sequence of the object sequence of 90 bases and 100 bases is compared measure % homogeny.There occurs disappearance at 5 ' end of object sequence, therefore, FASTDB comparison does not show the coupling/comparison of 5 ' end head, 10 bases.10 unpaired bases represent 10% (5 ' and 3 ' end does not mate base sum in base quantity/search sequence) of sequence, therefore from the % homogeny value of FASTDB program computation, deduct 10%.If remaining 90 bases are mated completely, final % homogeny is 90%.In another embodiment, the object sequence of 90 bases is compared with the search sequence of 100 bases.Current disappearance is inner disappearance, makes object sequence not have not mate with search sequence/the base of comparison 5 ' and 3 '.In this case, the % homogeny calculated by FASTDB does not have manual synchronizing.Again, 5 ' or 3 ' of manual synchronizing object sequence does not have and the base of search sequence coupling/comparison.Do not carry out other manual synchronizings in order to the object of the invention.
There is the polypeptide inquiring about the aminoacid sequence at least such as aminoacid sequence of 95% " identical " with the present invention, the aminoacid sequence being defined as subject polypeptide is equal to search sequence, except subject polypeptide sequence may to comprise in inquiry aminoacid sequence every 100 amino acid up to the change of five amino acid.In other words, in order to obtain the polypeptide with the aminoacid sequence identical with inquiry aminoacid sequence at least 95%, can insert up to the amino-acid residue of 5%, delete in object sequence, or using 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, in the single one or more adjacent group be dispersed in the residue of canonical sequence or in canonical sequence.
As practical situation, known computer program can be used to carry out the whether any specific polypeptide of conventional determining and be at least 80% with reference to polypeptide, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.Measure the preferred method of the best overall coupling between search sequence (sequence of the present invention) and object sequence, also referred to as global sequence's comparison, the FASTDB computer program of the algorithm based on Brutlag etc. can be used to measure, Brutlag etc., Comp.App.Biosci.6:237-245 (1990).In sequence alignment, search sequence and object sequence are all nucleotide sequences and are all aminoacid sequences.The result of described global sequence comparison is % homogeny.Preferred parameter used in FASTDB amino acid alignment is: matrix=PAM0, k-tuple=2, mispairing punishment=1, connects punishment=20, Randomization Group Length=0, close value=1, window size=sequence length, gap penalty=5, breach size punishment=0.05, the length of window size=500 or object nucleotide sequence, is as the criterion with shorter one.
If because N-or C-end is deleted, instead of owing to deleting inside, object sequence is shorter than search sequence, must carry out manual synchronizing to this result.This is because FASTDB program does not have N-and C-of calculating object sequence to hold brachymemma when calculating overall % homogeny.N-and C-is held to the object sequence of brachymemma, relative to search sequence, do not have, with the residue quantity of corresponding object sequences match/comparison, as the per-cent of the total base of search sequence, to correct % homogeny by the search sequence of calculating object sequence N-and C-end.Measure residue by the result of FASTDB sequence alignment and whether mate/comparison.Then from the % homogeny of the FASTDB program computation by above-mentioned use special parameter, deduct this per-cent, obtain final % homogeny value.This final percent identity is the homogeny for the object of the invention.Only consider not have and search sequence match/residue that the object sequence N-of comparison and C-holds is object in order to manually adjust % homogeny value.That is, just N-and C-farthest of object sequence hold outside inquiry resi-dues.
Such as, the search sequence of the object sequence of 90 amino-acid residues and 100 residues is compared measure % homogeny.There occurs deletion at the N-end of object sequence, therefore, FASTDB comparison does not show the pairing/comparison of 10 bases in N-termination.10 unpaired bases represent 10% (N-and C-end do not match base quantity/search sequence in base sum) of sequence, therefore from the % homogeny value of FASTDB program computation, deduct 10%.If remaining 90 residues match completely, final % homogeny is 90%.In another embodiment, the object sequence of 90 residues is compared with the search sequence of 100 residues.Current deletion is inner deletion, makes object sequence not have the base of/comparison unpaired with search sequence at N-or C-end.In this case, the % homogeny calculated by FASTDB does not have manual synchronizing.Again, the resi-dues outside N-and the C-end of manual synchronizing object sequence, as shown in FASTDB comparison, it is not and search sequence pairing/comparison.Do not carry out other manual synchronizings in order to the object of the invention.
As used in this, with the nucleic acid of nucleotide sequence of the present invention " hybridize under stringent condition ", refer to and containing 50% methane amide, 5 × SSC (750mMNaCl, 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 DEG C be incubated overnight, then use the 0.1 × SSC of about 65 DEG C to wash filter membrane and the polynucleotide of hybridizing.
As used in this, term Golgi localization domain refers to the aminoacid sequence of Golgi resident polypeptide, and it is responsible for being fixed in position in Golgi complex.Usually, localization domain comprises enzyme N-terminal " tail ".
As used in this, term effector function (effectorfunction) refers to those biological activitys that can cause antibody Fc district (native sequences Fc district or amino acid sequence variation Fc district).The example of antibody mediated effect subfunction comprises, but be not restricted to, the downward etc. of Fc receptor binding affinity, antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagolysis (ADCP), cytokine secretion, the antigen absorption mediated by the immunocomplex of antigen presenting cell, cell surface receptor.
As used in this, term engineering, through engineering approaches, through engineering approaches and glycosylation engineering think any operation of the glycosylation pattern comprising natural generation polypeptide or its fragment.Glycosylation engineering comprises the metabolic engineering of the glycosylation process of cell, comprises the genetic manipulation of oligosaccharide synthesis pathways to obtain the glycosylation of the change of cells glycoprotein.In addition, glycosylation engineering comprise sudden change and cellular environment on glycosylated impact.
As used in this, term host cell contains the cell system of any kind, and it through engineering approaches can produce target protein matter, protein fragments or the peptide of modifying glycosylated form, comprises antibody, antibody fragment and fusion rotein.Usually, carried out operating the GnTIII expressing optimum level to host cell.Host cell comprises cultured cells, such as, Mammals cultured cells is as Chinese hamster ovary celI, bhk cell, NS0 cell, SP2/0 cell, YO myeloma cell, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, yeast cell, insect cell and vegetable cell, only to list minority, but also comprise the cell in transgenic animal, the plant of transgenic plant or cultivation or animal tissues.
As used in this, the cytotoxicity that the cytotoxicity that term Fc mediates is comprised antibody dependent cellular cytotoxicity and mediated by the soluble Fc-fusion protein containing people Fc district.This is the immune mechanism being caused " antibody target cell " cracking by " people's immune effector cell ", wherein:
" people's immune effector cell " is leukocyte population, its surface display Fc acceptor its combine by the Fc district of these acceptors and antibody or Fc fusion rotein and realize effector function.Such colony comprises, but is not restricted to peripheral blood lymphocytes (PBMC) and/or natural killer (NK) cell.
" antibody target cell " is the cell combined by antibody or Fc fusion rotein.Antibody or Fc fusion rotein are held by the N-of albumen Fc district part and target cell combines.
As used in this, under the cytotoxicity that the Fc that term improves mediates is defined as antibody given in target cell surrounding media or Fc fusion rotein concentration, the increase of " antibody target cell " quantity of the cytotoxic mechanism cracking mediated by Fc defined above within preset time, and/or the cytotoxic mechanism mediated by Fc within preset time obtains the reduction of antibody or Fc fusion rotein concentration in the target cell surrounding media that cracking needs to determined number " antibody target cell ".The Cytotoxic raising of Fc mediation is with respect to same antibody or the fusion protein mediated cytotoxicity of Fc, these antibody or Fc fusion rotein are produced by the host cell of identical type, use identical standard production well known by persons skilled in the art, purifying, preparation and store method, but be not express the host cell generation of glycosyltransferase GnTIII by said methods engineering.
The antibody with the antibody dependent cellular cytotoxicity (ADCC) of raising is meant to the antibody with the ADCC of raising as recorded by any appropriate method known to persons of ordinary skill in the art.A kind of generally acknowledged external ADCC test is as follows:
1) test uses target cell, and this target cell known expresses the target antigen identified by the antigen binding domain of antibody;
2) test uses human peripheral blood mononuclear cell (PBMC) the action effect cell be separated from the healthy donors blood of Stochastic choice;
3) test according to following scheme:
I) standard density centrifugal method is used to be separated PBMC and with 5 × 10 6individual cell/ml is suspended in RPMI cell culture medium;
Ii) cultivate target cell by standard tissue culture methods, from exponential phase of growth collect viability higher than 90% cell, with the washing of RPMI cell culture medium, with 100 microcuries 51cr marks, and washes twice with cell culture medium, and with 10 5the density of individual cell/ml is resuspended in cell culture medium;
Iii) the above-mentioned final target cell suspension liquid of 100 microlitre is transferred in each hole of 96 hole microtiter plates;
Iv) in the medium by antibody from 4000ng/ml serial dilution to 0.04ng/ml, and the antibody-solutions that 50 microlitres obtain is added in the target cell in 96 hole microtiter plates, the various antibody concentration containing above-mentioned whole concentration range are tested in triplicate;
V) maximum release (MR) is contrasted, other 3 holes containing labels targets cell on flat board, accept 50 microlitre 2% (V/V) nonionic detergent (Nonidet, Sigma, St.Louis) aqueous solution, substitutes antibody-solutions (above-mentioned i-th v point);
Vi) for spontaneous release (SR) contrast, other 3 holes containing labels targets cell on flat board, accept 50 microlitre RPMI cell culture mediums and substitute antibody-solutions (above-mentioned i-th v point)
Vii) then by 96 hole microtiter plates at 50 × g centrifugal 1 minute, and 4 DEG C of incubations 1 hour;
Viii) 50 microlitre PBMC suspension (above-mentioned i-th point) added in each hole produce effector: target cell 25: 1 ratio, and flat board is positioned over 5%CO 2in the incubator of atmosphere in 37 DEG C 4 hours;
Ix) cell-free supernatants in each hole is collected and the radioactivity (ER) using gamma counter quantitative experiment to discharge;
X) the specific cleavage percentage ratio of each antibody concentration is calculated according to formula (ER-MR)/(MR-SR) × 100, wherein ER is the quantitative mean radio (see above-mentioned i-th x point) for that antibody concentration, MR is the quantitative mean radio (see above-mentioned v point) for MR contrast, and SR is the quantitative mean radio (see above-mentioned i-th x point) for SR contrast (see above-mentioned vi point);
4) " ADCC of raising " is defined as the raising of the largest percentage of the specific cleavage observed in the antibody concentration range of above-mentioned test, and/or obtains the minimizing of the antibody concentration needed for specific cleavage largest percentage half observed in the antibody concentration range of above-mentioned test.The raising of ADCC relative to above-mentioned test determination, mediated by same antibody, produced by identical host cell types, the production that uses identical standard well known by persons skilled in the art, purifying, preparation and storage method, but the ADCC that the host cell not carrying out overexpression glycosyltransferase GnTIII by through engineering approaches produces.
As used in this, term anti-CD 20 antibodies is defined as the antibody of the daltonian non-glycosylated phosphoprotein of specific recognition cell surface 35,000, and the restricted differentiation antigen Bp35 of this phosphorprotein usual called after human B lymphocyte, is commonly referred to as CD20.
The present invention is based on following discovery: engineered antibody produces cell to express new fusion polypeptide to form the antibody that Fc receptor binding affinity improves and effector function improves, this new polypeptide has β (1,4)-NAG transferase I II (GnTIII) or β (1,4)-galactosyltransferase (" GalT ") is active, and comprises the Golgi localization domain of Golgi resident polypeptide.Or the antibody that effector function improves and/or Fc receptors bind improves can produce cell to obtain by engineered antibody, this antibody produced cell has the expression that coding has the nucleic acid molecule increase of the polypeptide of alpha-Mannosidase II catalytic activity.In preferred embodiment, there is the fusion constructs of GnTIII or GalT activity and the nucleic acid molecule coexpression of coding ManII or GnTII.
Therefore, in one embodiment, the present invention relates to the isolating nucleic acid comprising encode fusion peptide sequence, wherein fusion polypeptide has the active Golgi localization domain with comprising Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII).In preferred embodiment, fusion polypeptide comprises the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II, and Golgi localization domain is the localization domain of mannosidase II.In further embodiment, Golgi localization domain is the localization domain of GalT.
Preferably, the nucleic acid of separation has the nucleotide sequence shown in Figure 24 and SEQIDNO:14.In another preferred embodiment, fusion polypeptide comprises β (1,4) catalyst structure domain of-NAG transferase I II, and Golgi localization domain is the catalyst structure domain of β (1,2)-NAG transferase I (GnTI).Preferably, nucleic acid has the nucleotide sequence shown in Figure 25 and SEQIDNO:12.Or, the Golgi localization domain of another Golgi resident polypeptide can be used.In another preferred embodiment, Golgi localization domain is selected from β (1, the 2)-localization domain of NAG transferase I I, the localization domain of mannosidase I, and the localization domain of α 1-6 core fucosyltransferase.
In another preferred embodiment, the present invention relates to the nucleic acid of separation, there is containing coding the sequence of the polypeptide of aminoacid sequence shown in Figure 24 and SEQIDNO:15 or Figure 25 and SEQIDNO:13.The present invention also comprises the nucleic acid of separation, and containing the sequence of hybridizing with hybridization probe under strict conditions, its nucleotide sequence is made up of the nucleotide sequence shown in Figure 24 and SEQIDNO:14 or Figure 25 and SEQIDNO:12.The invention further relates to the nucleic acid of separation, comprise with nucleotide sequence at least 80% shown in Figure 24 with SEQIDNO:14 or Figure 25 with SEQIDNO:12,85%, 90%, 95%, 96%, 97%, 98% or 99% identical sequence.In another embodiment, the present invention relates to the nucleic acid of separation, comprising coding have with aminoacid sequence at least 80% shown in Figure 24 with SEQIDNO:15 or Figure 25 with SEQIDNO:13,85%, 90%, 95%, 96%, 97%, the sequence of the polypeptide of 98% or 99% identical aminoacid sequence.The present invention also comprises the nucleic acid of separation, comprising the sequence that coding has the polypeptide of aminoacid sequence shown in Figure 24 and SEQIDNO:15 or Figure 25 and SEQIDNO:13 that conserved amino acid substitutes.
In another embodiment, the present invention relates to expression vector, comprise the present invention be separated nucleic acid, described above those.
In further embodiment, the present invention relates to and there is β (1,4) fusion polypeptide of-NAG transferase I II activity or the active Golgi localization domain also containing heterologous Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-galactosyltransferase (" GalT ").In preferred embodiment, fusion polypeptide of the present invention comprises the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II.In particularly preferred embodiment, fusion polypeptide comprises the Golgi localization domain of mannosidase II or β (1,2)-NAG transferase I (GnTI) further.In interchangeable embodiment, Golgi localization domain is selected from the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I, and the localization domain of α 1-6 core fucosyltransferase.Can host cell of the present invention be cultivated in the medium and collect described fusion polypeptide from the culture generated under the condition of expression of nucleic acid allowing the described fusion polypeptide of coding and carry out obtained fusion polypeptide of the present invention.
The invention further relates to the method for the glycoforms (profile) modifying the polypeptide that host cell produces, comprise and nucleic acid of the present invention or carrier are introduced in described host cell.Preferably, the polypeptide of modification be 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, the polypeptide of modification is fusion rotein, and this fusion rotein comprises the region being equivalent to human IgG Fc district.
The invention further relates to the host cell containing 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 one coding and is had β (1,4)-NAG transferase I II activity or β (1,4) nucleic acid of the fusion polypeptide that-galactosyltransferase (" GalT ") is active, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of described host cell generation, wherein said polypeptide is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.In preferred embodiment, the polypeptide that described host cell produces is IgG or its fragment.More preferably, the polypeptide that described host cell produces is IgG1 or its fragment.Or the polypeptide that described host cell produces is fusion rotein, and this fusion rotein comprises the region in the Fc district being equivalent to human IgG such as IgG1.
The modified polypeptide produced as the result host cell of the present invention modified presents the Fc receptor binding affinity of raising and/or the effector function of raising.Preferably, the Fc receptor binding affinity of raising is and the raising of Fc γ activated receptor as Fc γ RIIIa receptors bind.The effector function improved is following one or more raising preferably: 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 mediated by the immunocomplex of antigen presenting cell, the Cytotoxic raising of Fc mediation, with the raising of NK Cell binding, with the raising that scavenger cell combines, with the raising that polymorphonuclear cell (PMN) combines, with the raising that monocyte combines, the raising that target binding antibody is crosslinked, the enhancing of apoptosis-induced direct signal, the raising of dendritic cell maturation and the raising of T cell sensitization.
In particularly preferred embodiment, host cell of the present invention is Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, and the polypeptide that described host cell produces is that anti-CD20 antibodies is as IDEC-C2B8.In another preferred embodiment, host cell is inosculating antibody Human epidermal growth factor receptor monoclonal antibody C225.
Except the nucleic acid containing encode fusion polypeptide of the present invention, host cell of the present invention (transected) nucleic acid further containing at least one brachymemma, the antibody fragment in this nucleic acid encoding antibody molecule, reservation function Fc district or fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region.In preferred embodiment, the nucleic acid encoding anti-CD20 antibodies of at least one brachymemma, chimeric anti-human neuroblastoma monoclonal antibody chCE7, chimeric anti-human kidney cell carcinoma monoclonal antibody chG250, chimeric anti-human colon, lung and breast cancer monoclonal antibody ING-1, Humanized anti-human 17-1A antigen monoclonal antibody 3622W94, Humanized anti-human colorectal tumours antibody A 33, the anti-human melanoma antigens of facedown GD3 Sphingolipids,sialo R24, chimeric anti-human squamous cell carcinoma monoclonal antibody SF-25, anti-human EGFR antibody, anti-human EGFRvIII antibody, anti-human PSMA antibody, anti-human psca antibody, anti-human CD22 antibody, anti-human 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 McAb, anti-LeY antibody, anti-stick protein antibodies, anti-MUC18 antibody, anti-human CD33 antibody, anti-human CD38 antibody, antibodies against human CD 40, anti-human CD45 antibody, antihuman CD 52 antibody, anti-human CD138 antibody, anti-human HLA-DR variant antibodies, anti-human epcam antibody, anti-human CEA antibody, anti-human MUC1 antibody, anti-human MUC1 nucleoprotein antibody, the MUC1 antibody of anti-human Aberrant glycosylation, the antibody of the people fibronectin variant of antagonism containing ED-B structural domain or anti-human HER2/neu antibody.
The invention still further relates to the method for producing polypeptide in host cell, comprise (a) and cultivate host cell under the condition allowing polypeptide to produce, host cell through engineering approaches is expressed at least one coding and is had β (1, 4)-NAG transferase I II activity or β (1, 4) nucleic acid of the fusion polypeptide that-galactosyltransferase (" GalT ") is active, the polypeptide produced is selected from whole antibody molecule, antibody fragment containing Fc district and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, the expression amount of the fusion polypeptide that the wherein said GnTIII of having is active or GalT is active is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, (b) described polypeptide is separated.In preferred embodiment, fusion polypeptide comprises the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II.In particularly preferred embodiment, fusion polypeptide comprises the Golgi localization domain of Golgi resident polypeptide further.Preferably, Golgi localization domain is the Golgi localization domain of mannosidase II or β (1,2)-NAG transferase I (GnTI).Or Golgi localization domain is selected from the localization domain of mannosidase I, the localization domain of β (1,2)-NAG transferase I I and the localization domain 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 improved 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 mediated by the immunocomplex of antigen presenting cell, with the raising of NK Cell binding, with the raising that scavenger cell combines, with the raising that monocyte combines, with the raising that polymorphonuclear cell combines, the raising that target binding antibody is crosslinked, the enhancing of apoptosis-induced direct signal, the raising of dendritic cell maturation and the raising of T cell sensitization.The Fc receptor binding affinity improved preferably and Fc activated receptor as the raising of Fc γ RIIIa receptors bind.
In another embodiment, the present invention relates to the polypeptide produced by the inventive method, it has bisection (bisected) oligosaccharides of increase ratio in the Fc district of described polypeptide.Still, in another embodiment, to be there is by the polypeptide that the inventive method is produced as the result of described modification the oligosaccharides of non-fucosylation in the Fc district of increase ratio.The oligosaccharides of non-fucosylation can be heterozygous or compound.In particularly preferred embodiment, the polypeptide produced by host cell of the present invention and method has the oligosaccharides of bisection, non-fucosylation in the Fc district of increase ratio.The oligosaccharides of bisection, non-fucosylation can be heterozygosis or compound.Especially, the inventive method may be used for producing polypeptide, at least 15% of oligosaccharides wherein in 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%, it is non-fucosylation.The inventive method can also be used to produce polypeptide, oligosaccharides at least 15% wherein in 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%, binary.Still further, the inventive method can be used for producing polypeptide, oligosaccharides at least 15% wherein in 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%, binary, non-fucosylation.The inventive method can also for the production of polypeptide, and the oligosaccharides at least 15% wherein in polypeptide Fc district, more preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 35% are binary hybrid nonfucosylated.
In another embodiment, the present invention relates to the antibody produced by the inventive method, this antibody engineering is with the Fc receptor binding affinity of the effector function and/or raising with raising.Preferably, the effector function improved 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 mediated by the immunocomplex of antigen presenting cell, with the raising of NK Cell binding, with the raising that scavenger cell combines, with the raising that monocyte combines, with the raising that polymorphonuclear cell combines, the raising that target binding antibody is crosslinked, the enhancing of apoptosis-induced direct signal, the raising of dendritic cell maturation and the raising of T cell sensitization.In preferred embodiment, the Fc receptor binding affinity of raising is the raising combined with Fc activated receptor, is more preferably the raising with Fc γ RIIIa receptors bind.The invention further relates to the antibody fragment containing Fc district and fusion rotein, fusion rotein comprises the region being 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 retaining Fc district and fusion rotein and pharmacology, fusion rotein comprises the region being equivalent to immunoglobulin fc region.
The invention further relates to the purposes of this pharmaceutical composition in Therapeutic cancer method.Especially, the present invention relates to the method for Therapeutic 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 containing encode fusion polypeptide, wherein fusion polypeptide has β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII is active) and contains the localization domain of Golgi resident polypeptide; With the expression vector of nucleic acid molecule comprising coded polypeptide, wherein to have alpha-Mannosidase II (ManII) active for polypeptide.In preferred embodiment, the nucleic acid molecule of encode fusion polypeptide and coding have the nucleic acid molecule of the polypeptide of Man II activity on same expression vector or on the expression vector separated.In another preferred embodiment, fusion polypeptide contains the catalyst structure domain of GnTIII.In other preferred embodiment, Golgi localization domain is ManII, β (1,2)-NAG transferase I, β (1,2) localization domain of-NAG transferase I I, mannosidase I or α 1,6-N core fucosyltransferase.In 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, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma.
The present invention further provides host cell, comprise the expression vector of the nucleic acid molecule containing encode fusion polypeptide, fusion polypeptide has β (1,4)-NAG transferase I II (GnTIII active) localization domain containing Golgi resident polypeptide, the expression vector of the nucleic acid molecule containing coded polypeptide, wherein polypeptide has alpha-Mannosidase II (ManII) activity, with the expression vector of the nucleic acid molecule containing coded polypeptide, wherein polypeptide has β (1,2)-NAG transferase I I (GnTII) activity.In preferred embodiment, the nucleic acid molecule of encode fusion polypeptide, coding have the nucleic acid molecule of the polypeptide of ManII activity and coding has the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector or on the expression vector separated.Further preferably the nucleic acid molecule of encode fusion polypeptide is on an expression vector, and the nucleic acid molecule that coding has ManII active polypeptide has the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector with coding.Further preferably, coding has the nucleic acid molecule of the polypeptide of ManII activity on an expression vector, and the nucleic acid molecule of encode fusion polypeptide and coding have the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector.In another embodiment, GnTII is on an expression vector, and the nucleic acid molecule of encode fusion polypeptide and coding have the nucleic acid molecule of the polypeptide of ManII activity on same expression vector.
In addition on the one hand, the present invention relates to host cell, comprise the expression vector of the nucleic acid molecule containing encode fusion polypeptide, wherein fusion polypeptide has β (Isosorbide-5-Nitrae)-galactosyltransferase (GalT) activity and comprises the Golgi localization domain of Golgi resident polypeptide; With the expression vector of the nucleic acid molecule containing coded polypeptide, wherein polypeptide has mannosidase II (ManII) activity.In preferred embodiment, the nucleic acid molecule of encode fusion polypeptide, coding have the nucleic acid molecule of the polypeptide of ManII activity on same expression vector or on the expression vector separated.Preferably, fusion polypeptide contains the catalyst structure domain of GalT.In other embodiments, Golgi localization domain is ManII, β (1,2) localization domain of-NAG transferase I, β (1,2)-NAG transferase I I, mannosidase I or α 1-6 core fucosyltransferase.In 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, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma.
In addition on the one hand, the present invention relates to host cell, comprise the expression vector of the nucleic acid molecule containing encode fusion polypeptide, wherein fusion polypeptide has β (Isosorbide-5-Nitrae)-galactosyltransferase (GalT) activity and comprises the Golgi localization domain of Golgi resident polypeptide; With the expression vector of the nucleic acid molecule containing coded polypeptide, wherein polypeptide has mannosidase II (ManII) activity; And the expression vector of nucleic acid molecule containing coded polypeptide, wherein to have β (1,2)-NAG transferase I I (GnTII) active for polypeptide.Preferably, each nucleic acid molecule is on same expression vector.In embodiment separately, each nucleic acid molecule is on the carrier separated.The nucleic acid molecule that the present invention further provides encode fusion polypeptide is on an expression vector, and the nucleic acid molecule that coding has the polypeptide of ManII activity has the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector with coding.Present invention also offers the nucleic acid molecule of coding ManII on an expression vector, and the nucleic acid molecule of encode fusion polypeptide and coding has the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector.Present invention also offers coding and there is the nucleic acid molecule of the polypeptide of GnTII activity on an expression vector, and the nucleic acid molecule of encode fusion polypeptide and coding have the nucleic acid molecule of the polypeptide of ManII activity on same expression vector.In preferred embodiment, fusion polypeptide contains the catalyst structure domain of GalT.In further preferred embodiment, Golgi localization domain is ManII, β (1,2)-NAG transferase I, β (1,2) localization domain of-NAG transferase I I, mannosidase I or α 1,6-N core fucosyltransferase.
Invention further provides 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 fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, the polypeptide that wherein host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.
Present invention also offers host cell, this host cell through engineering approaches expresses that at least one coding has the nucleic acid of the fusion polypeptide of GnTIII activity, at least one coding has the nucleic acid of the polypeptide of ManII activity and at least one coding has the nucleic acid of the polypeptide of GnTII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, the polypeptide that wherein host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.
Invention additionally provides 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 fusion polypeptide of GalT activity and at least one coding have the polypeptide of ManII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, the polypeptide that wherein host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.
In embodiment separately, present invention also offers host cell, this host cell through engineering approaches expresses that at least one coding has the nucleic acid of the fusion polypeptide of GalT activity, at least one coding has the nucleic acid of the polypeptide of ManII activity and at least one coding has the nucleic acid of the polypeptide of GnTII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, the polypeptide that wherein host cell produces is selected from whole antibody molecule, antibody fragment and fusion rotein, and fusion rotein comprises the region being equivalent to immunoglobulin fc region.Preferably, presented the Fc receptor binding affinity of raising by the polypeptide that host cell produces as the result of modifying.In other preferred embodiment, the polypeptide as the result host cell generation of modifying presents the effector function of raising.Preferably, the effector function of raising is following one or more: the raising of the raising that the Cytotoxic raising of Fc mediation and the raising of NK Cell binding and scavenger cell combine and the raising that polymorphonuclear cell combines and the raising that monocyte combines, the enhancing of apoptosis-induced direct signal, the raising of dendritic cell maturation and/or T cell sensitization.
In other embodiments, the present invention relates to the method for producing polypeptide in host cell, host cell is cultivated under being included in the condition allowing polypeptide to produce, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, the amount that wherein fusion polypeptide is expressed is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, and isolated polypeptide.Preferably, further host cell through engineering approaches is expressed the nucleic acid that at least one coding has the polypeptide of GnTII activity.In other preferred embodiment, fusion polypeptide comprises the catalyst structure domain of GnTIII.In further preferred embodiment, fusion polypeptide comprises the Golgi localization domain of heterologous Golgi resident polypeptide.Preferably, Golgi localization domain is mannosidase II, β (1,2) localization domain of-NAG transferase I, mannosidase I, β (1,2)-NAG transferase I I 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 producing polypeptide in host cell, host cell is cultivated under being included in the condition allowing polypeptide to produce, to be through engineering approaches have to express at least one coding the nucleic acid that the nucleic acid of the fusion polypeptide of GalT activity and at least one coding have the polypeptide of ManII activity to this host cell, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, and wherein the expression amount of fusion polypeptide is enough to the oligosaccharides in the polypeptide Fc district of modification host cell generation.In other embodiments, further host cell through engineering approaches is expressed the nucleic acid that at least one coding has the polypeptide of GnTII activity.Preferably, fusion polypeptide comprises the catalyst structure domain of GalT.Further preferably fusion polypeptide comprises the Golgi localization domain of heterologous Golgi resident polypeptide further.Preferably, Golgi localization domain is mannosidase II, β (1,2) localization domain of-NAG transferase I, mannosidase I, β (1,2)-NAG transferase I I or α 1-6 core fucosyltransferase.Preferably, as the result of above-mentioned modification, polypeptide has the effector function of raising.Especially, in preferred embodiment, the polypeptide that host cell produces has the bisection of ratio increase, the oligosaccharides of non-fucosylation in Fc district.Preferably, the oligosaccharides of bisection, non-fucosylation is heterozygosis.More preferably, the oligosaccharides of bisection, non-fucosylation is compound.In preferred embodiment, the oligosaccharides in polypeptide Fc district is bisection, non-fucosylation at least about 10% to 95%.Oligosaccharides about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% particularly preferably in glycosylated polypeptides Fc district of the present invention is bisection, non-fucosylation.
In other preferred embodiment, the invention provides the antibody with the effector function of raising according to the inventive method through engineering approaches.
In other embodiments, invention further provides the pharmaceutical composition of the antibody containing with good grounds the inventive method through engineering approaches.Preferably, pharmaceutical composition of the present invention comprises acceptable carrier on pharmacology.
Invention further provides the method for the treatment of malignant tumour, comprise the patient pharmaceutical composition of the present invention for the treatment of significant quantity being delivered medicine to needs.
The invention further relates to the method for the Cytotoxic polypeptide producing the Fc mediation with raising in host cell, host cell is cultivated under being included in the condition allowing polypeptide to produce, this host cell is that through engineering approaches is to the nucleic acid of the nucleic acid and at least one coding ManII of expressing at least one coding GalT, the polypeptide produced is selected from whole antibody molecule, comprise the antibody fragment of immunoglobulin fc region, one or two expression amount wherein in GalT or ManII is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, and wherein there is as the result polypeptide modified the cytotoxicity of the Fc mediation of raising, with the Cytotoxic polypeptide being separated the Fc mediation with raising.In other preferred embodiment, the expression level of GalT creates the Cytotoxic antibody molecule of the Fc mediation with raising or comprises the antibody fragment of immunoglobulin fc region.
The invention further relates to the method for the Cytotoxic polypeptide producing the Fc mediation with raising in host cell, host cell is cultivated under being included in the condition allowing polypeptide to produce, this host cell is that through engineering approaches is to the nucleic acid of the nucleic acid and at least one coding ManII of expressing at least one coding GalT, the polypeptide produced is selected from whole antibody molecule, comprise the antibody fragment of immunoglobulin fc region, one or two expression level wherein in GalT or ManII is enough to the oligosaccharides modified in the polypeptide Fc district of host cell generation, and wherein there is as the result polypeptide modified the cytotoxicity of the Fc mediation of raising, with the Cytotoxic polypeptide being separated the Fc mediation with raising.In preferred embodiment, above-mentioned host cell comprises the nucleic acid of at least one coding GnTIII further, wherein the expression amount of GnTIII is enough to the oligosaccharides in the polypeptide Fc district of modification host cell generation, and wherein has the cytotoxicity of the Fc mediation of raising as the result polypeptide modified.Preferably, the one or more expression level in GalT, ManII or GnTIII is enough to form the bisected oligosaccharides in polypeptide Fc district.More preferably, the bisected oligosaccharides in Fc district to the ratio of oligosaccharides total in Fc district at least about 25,35,45,55,60,65,70,75,80,85,90 or 95%.Preferably, the bisected oligosaccharides in Fc district to the ratio of oligosaccharides total in Fc district at least about 45%.In preferred embodiment, bisected oligosaccharides is compound or heterozygosis.Preferably, host cell is mammalian cell, yeast cell, insect cell or vegetable cell.More preferably, host cell is vegetable cell.
On the other hand, the present invention relates to the method for producing polypeptide in host cell:
A. allowing to cultivate host cell under the condition producing polypeptide, this host cell is that through engineering approaches is to express the nucleic acid molecule that at least one coding has alpha-Mannosidase II activity, the polypeptide produced is selected from whole antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region, and the wherein said expression amount with the polypeptide of alpha-Mannosidase II activity is enough to the oligosaccharides modified in the Fc district of the described polypeptide that described host cell produces; With
B. the described polypeptide that described host cell produces is separated.
The result that the invention still further relates to as described modification oligosaccharides has the polypeptide of the effector function of raising and/or the Fc receptors bind of raising, especially antibody, and their purposes in the therapeutic composition of disease therapy especially tumour.
Need qualification and the generation of the coding nucleic acid of the albumen of modification of glycosylation patterns
The invention provides the method for production and the purposes of host system, be used for producing the antibody of glycoforms, the antibody fragment containing Fc district and fusion rotein, a region of fusion rotein is equivalent to the Fc district of immunoglobulin (Ig), there is the Fc receptor binding affinity of raising, preferred Fc activated receptor, and/or there is the effector function of raising, comprise antibody dependent cellular cytotoxicity.The qualification of target epi-position and its glycosylation pattern with potential therapeutic value need the generation of the antibody modified, and the separation of nucleic acid sequence encoding is separately all within the scope of the invention.
Various methods known in the art can be used to carry out the antibody of production object target epi-position.The fragment that such antibody is included, but are not limited to polyclone, mono-clonal, chimeric, humanization, total man, strand, Fab fragment and produced by ScFv, Fab, VH, IgG expression library.Such antibody can be used as diagnostic reagent or therapeutical agent.As therapeutical agent, particularly preferably neutralizing antibody, namely those combine with part, substrate and connector molecule the antibody competed mutually.
For antibody producing, carry out immune various host animal by injection object target protein, include, but are not limited to rabbit, mouse, rat etc.By repeatedly subcutaneous (sc) or intraperitoneal (ip) injects related antigen and adjuvant produces polyclonal antibody in animal.According to host species, various adjuvant can be used to strengthen immunne response, include, but are not limited to, freund's adjuvant (completely and incomplete), mineral rubber are if aluminium hydroxide, tensio-active agent are as lysolecithin, poly alcohol, polyanion, peptide, saponin(e, oily emulsion, and keyhole limpet hemocyanin, dinitrophenol and people's adjuvant of coming in handy are as BCG (bacille Calmette-Guerin vaccine) and corynebacterium parvum (Corynebacteriumparvum).By will as 100g or 5g protein or conjugate (separately for rabbit and mouse) and 3 volume Freund's complete adjuvants, and at multiple position by solution intradermal injection by animal immune to resist antigen, immunoconjugates or derivative.After one month, strengthen animal by the peptide of 1/5 to 1/10 original vol in multiple position subcutaneous injection freund's adjuvant and conjugate, after 7 to 14 days, by animal unhairing and the antigen titration of serum analysis.Strengthen animal until titration is steady.Preferably, by same antigen but and different protein and/or the conjugate puted together by different linking agents strengthen animal.Conjugate can also be obtained in recombinant cell culture thing is as protein fusion.
Use the monoclonal antibody being prepared target by any technology of continuous cell line generation antibody molecule.These comprise, but are not restricted to, hybridoma technology, be described in Kohler and Milstein at first, Nature256:495-97 (1975), human B-lymphocyte hybridoma technology (Kosbor etc., ImmunologyToday4:72 (1983); Cote etc., Proc.Natl.Acad.Sci.U.S.A.80:2026-30 (1983), and EBV-hybridoma technology (Cole etc., MonoclonalAntibodiesandCancerTherapy77-96 (AlanR.Liss, Inc., 1985)); In addition, development can be used produce technology (Morrison etc., the Proc.Natl.Acad.Sci.U.S.A.81:6851-55 (1984) of " chimeric antibody "; Neuberger etc., Nature312:604-08 (1984); Takeda etc., Nature314:452-54 (1985), by splicing the gene from the gene of the specific amouse antibody molecule of suitable antigen and the human antibody molecules from suitable bioactive.These technology can be used for producing chimeric antibody, also comprise other mammiferous antibody molecules.Or, can use to describe and be used for the technology (United States Patent (USP) 4,946,778) of manufacture order chain antibody and produce the single-chain antibody with desired specificity.The invention further relates to the humanized antibody according to the inventive method glycosyl through engineering approaches.The method of producing humanized antibodies is disclosed in such as, and the U.S. Patent No. 6,180,320 of Queen etc., is incorporated herein by reference with its entirety at this.
Antibody fragment containing object target protein specific binding site can be produced by known technology.Such as, such fragment comprises, but is not restricted to, the F (ab ') produced by pepsin digested antibody molecule 2fragment and by reduction F (ab ') 2the Fab fragment of the disulfide bridge bond generation of fragment.Or ((Huse etc., Science246:1275-81 (1989)) identify the Monoclonal Fab fragments with object target protein desired specificity quickly and easily can to build Fab expression library.
Once identify glycosylation pattern there is the antibody or antibody fragment that need to modify, use technical evaluation well known in the art and be separated nucleic acid sequence encoding.
A. for generation of the production of clone of protein of glycosylation pattern with change
The invention provides the host cell expression system of the protein for the production of the glycosylation pattern with change.Especially, the invention provides the host cell systems for the production of the protein with the glycoforms improving therapeutic value.Therefore, on the one hand, the invention provides host cell expression system, it expresses through selection or through engineering approaches such as to have β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII) activity and the fusion polypeptide comprising the localization domain of heterologous Golgi resident polypeptide.Especially, such host cell expression system through engineering approaches can contain the recombinant nucleic acid molecules of the such fusion polypeptide of coding, is operationally connected with the promoter systems of composing type or adjustment.
In one particular, the invention provides host cell, this host cell through engineering approaches expresses the nucleic acid that at least one coding has the fusion polypeptide of the active Golgi localization domain also containing heterologous Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII).On the one hand, host cell through engineering approaches is comprised the nucleic acid molecule that at least one coding has the gene of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII).
Usually, the culturing cell system of any type can be used as the background of through engineering approaches host cell system of the present invention.In preferred embodiment, Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, other mammalian cells, yeast cell, insect cell or vegetable cell are used as the background cell line producing engineered host cell of the present invention.(see Ma, J.K.-C., etc., NatureGenetics4:794-805 (in October, 2003) and the reference quoted thereof) (its full content is hereby incorporated by).
The present invention relates to any engineered host cell comprising and express the fusion polypeptide with the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII) as defined in this.
Can under constitutive promoter or adjustable expression system control, express one and several coding and there is β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII is active) and the nucleic acid of the fusion polypeptide of Golgi localization domain containing xenogenesis Golgi resident polypeptide.Suitable adjustable expression system comprises, but be not restricted to, the promoter systems of tsiklomitsin is adjustable expression system, 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.If host cell systems comprises several different coding have β (1,4) nucleic acid of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII), wherein some can be expressed under constitutive promoter controls, and other express under adjustable expression system controls.Think that maximum expression level is that cell growth rate that stable fusion polypeptide is expressed does not have the highest of remarkable side effect may level, use routine test to measure.The method usually known by this area measures expression level, comprise western blot analysis, use to the antibody of the polypeptid specificity with GnTIII activity or to the peptide-labeled specific antibody of peptide fusion with GnTIII activity, Northern engram analysis, use and to coding, there is the nucleic acid probe of the gene specific of the polypeptide of GnTIII activity or to coding and the probe of peptide-labeled nucleic acid specificity of peptide fusion with GnTIII activity, or it is active to measure GnTIII.Or, the lectin combined with the biosynthetic products of GnTIII can be used, such as, E 4-PHA lectin.Or, can using function test, it measures the Fc receptors bind of the antibody-mediated raising produced by cell and the effector function of raising, and this is cell engineered and have the nucleic acid of coding GnTIII active polypeptide.In further alternative, nucleic acid can be operably connected with reporter gene; The expression level of the fusion polypeptide with the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII) is measured by the signal that measurement report gene expression dose is relevant.Reporter gene can be transcribed together with single mRNA molecule with the nucleic acid of encode fusion polypeptide; Respective encoding sequence is connected with by cap independence translational enhancer (CITE) by internal ribosome entry site (IRES).Reporter gene can have β (1 with at least one coding, 4) nucleic acid of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII) is translated together, makes to form single peptide chain.The nucleic acid of code book invention fusion polypeptide operationally can be connected with reporter gene under the control of single promotor, make the nucleic acid of encode fusion polypeptide become RNA molecule with reporter gene transcription, its alternative splicing becomes two messenger RNA(mRNA) separated (mRNA) molecules; Of obtaining in mRNA translate into reporter protein, another translates into fusion polypeptide.
If have expressed several different coding there is β (1,4) nucleic acid of the active Golgi localization domain fusion polypeptide also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII), they can arrange they are transcribed with one and several mRNA molecule like this.If they are transcribed as single mRNA molecule, be connected respective encoding sequence by internal ribosome entry site (IRES) with by cap independence translational enhancer (CITE).They can be become RNA molecule from single promoter transcription, its alternative splicing becomes several messenger RNA(mRNA) (mRNA) molecule separated, then its each fusion polypeptide translating into each own coding.
In another embodiment, the invention provides host expression system, for the production for the treatment of antibody, there is the Fc receptor binding affinity of raising, especially combine with Fc activated receptor, and the effector function improved, comprise antibody dependent cellular cytotoxicity.Usually, the nucleic acid that coding needs the antibody of mutagenic glycoforms is expressed in host cell expression system through engineering approaches and/or selection, express together with the nucleic acid that at least one coding has the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII).In one embodiment, carry out transfection host cell system with the encode nucleic acid of such fusion polypeptide of at least one.Usually, the cell of transfection is selected to identify the clone expressing fusion polypeptide of the present invention with separating stable.
The culturing cell system of any type can be used as the background of through engineering approaches host cell system of the present invention.In preferred embodiment, Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, other mammalian cells, yeast cell, insect cell or vegetable cell can be used.Usually, such clone through engineering approaches comprises the nucleic acid of at least one transfection further, and the whole antibody molecule of this nucleic acid encoding, antibody fragment containing immunoglobulin fc region or fusion rotein, fusion rotein comprises the region being equivalent to immunoglobulin fc region.Usually such antibody producing cells system be derived from 20 to 120pg/ (cell. sky) high specific output produces and the clone of secretory antibody.In interchangeable embodiment, the hybridoma cell line of expressing object specific antibodies is used as the background cell line of production through engineering approaches cell of the present invention.
In one embodiment, enter in antibody expression vector by the nucleic acid clone of encoding antibody, antibody fragment or Fc fusion polypeptide, then transfection is entered in host cell, and selects and screen the high and stable cell clone of specific antibody output.Then with the clone that glycoprotein-modification glycosyltransferases expression carrier transfection is selected like this, this expression vector contains the following nucleic acid of coding, such as, a () has β (1, 4) fusion polypeptide that-NAG transferase I II (GnTIII) is active, or (b) has β (1, 4) fusion polypeptide that-galactosyltransferase (GalT) is active, or (c) has the active polypeptide of golgi body alpha-Mannosidase II (ManII), or (d) has the fusion polypeptide of GnTIII activity and has the polypeptide of ManII activity further, or (e) has the fusion polypeptide of GalT activity and has the polypeptide of ManII activity further.Then select and screening and cloning, this be cloned in cause antibodies specific high yield level on stably express antibody-encoding genes, glycosyltransferase gene is modified with stably express glycoprotein on the expression level causing Fc district glycosylation pattern to be modified, described modification comprises the increase of non-fucosylated oligosaccharide part, oligosaccharides is bisection or non-binary, it can be compound or heterozygous further, it is relevant with the Fc receptors bind increased, 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 Fc dependent cellular cytotoxicity.Below describe and select and screening method.
In another embodiment, put upside down the order that the transfection of glycosyltransferase expression system carrier is modified in above-mentioned two transfections and antibody expression vector transfection and glycoprotein, namely first modify glycosyltransferases expression carrier transfection host cell with glycoprotein, then use antibody expression vector transfection.In such method, can by the clone of the following either method screening further described from the glycosyltransferase gene of first enough stably express level of transfection, or with the replicon of this clone of antibody expression vector transient transfection, then with the following screening method further described, to 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 Fc acceptor comprising the binding affinity of Fc γ RIII acceptor and improving the receptor-mediated effector function of Fc, comprise Fc dependent cellular cytotoxicity.
In further embodiment, transfection antibody-encoding genes and glycosyltransferase gene together in single transfection procedure, in single expression vector or in the carrier separated.
Usually, at least one nucleic acid encoding in host cell systems has β (1,4)-NAG transferase I II (GnTIII) activity or β (1,4) fusion polypeptide of the Golgi localization domain of-galactosyltransferasactivity activity also containing xenogenesis Golgi resident polypeptide, or its coding has the polypeptide of golgi body alpha-Mannosidase II activity.
The nucleic acid of expression one or several code book invention fusion polypeptide of can getting off in the control of constitutive promoter or adjustable expression system.Suitable adjustable expression system comprises, but be not restricted to, the expression system of tsiklomitsin is adjustable expression system, the derivable expression system of moulting hormone, lac-switch is adjustable expression system, the derivable expression system of glucocorticosteroid, temperature inducible promoter system and metallothionein(MT) metal inducement.If host cell systems comprises several different coding have β (1,4) nucleic acid of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII), wherein some can be expressed under constitutive promoter controls, and other express under adjustable expression system controls.Think that maximum expression level is that cell growth rate that stable fusion polypeptide is expressed does not have the highest of remarkable side effect may level, use routine test to measure.The method usually known by this area measures expression level, comprise western blot analysis, such as use to the antibody of the polypeptid specificity with GnTIII activity or to the peptide-labeled specific antibody of peptide fusion with GnTIII activity, Northern engram analysis, such as use and to coding, there is the nucleic acid probe of the gene specific of the polypeptide of GnTIII activity or to coding and the peptide-labeled nucleic acid specificity probe of peptide fusion with GnTIII activity, or it is active to measure GnTIII.Or, the lectin combined with the biosynthetic products of GnTIII can be used, such as, E 4-PHA lectin.Or, can using function test, it measures the Fc receptors bind of the antibody-mediated raising produced by cell and the effector function of raising, and this is cell engineered and have the nucleic acid of coding GnTIII active polypeptide.In further alternative, nucleic acid can be operably connected with reporter gene; The signal of being correlated with by measurement report gene expression dose measures the expression level of fusion polypeptide of the present invention.Reporter gene can be transcribed together with single mRNA molecule with the nucleic acid of coding described glycoprotein-modification glycosyltransferase; Respective encoding sequence is connected with by cap independence translational enhancer (CITE) by internal ribosome entry site (IRES).Reporter gene can have β (1 with at least one coding, 4) nucleic acid of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII) is translated together, makes to form single peptide chain.The nucleic acid of encode fusion polypeptide operationally can be connected with reporter gene under the control of single promotor, make the nucleic acid of code book invention fusion polypeptide become RNA molecule with reporter gene transcription, its alternative splicing becomes two messenger RNA(mRNA) separated (mRNA) molecules; Of obtaining in mRNA translate into reporter protein, another translates into fusion polypeptide.
If have expressed several different coding there is β (1,4) nucleic acid of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII), they can arrange like this and make them be transcribed into one and several mRNA molecule.If they are transcribed as single mRNA molecule, be connected respective encoding sequence by internal ribosome entry site (IRES) with by cap independence translational enhancer (CITE).They can become RNA molecule from single promoter transcription, and its alternative splicing becomes several messenger RNA(mRNA) (mRNA) molecule separated, then its each fusion polypeptide translating into each own coding.
I. expression system
This area can be used to carry out construction of expression vector according to the known method of technician, this expression vector contains the encoding sequence of target protein matter and has the active and encoding sequence of the fusion polypeptide of Golgi localization domain containing xenogenesis Golgi resident polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII) and suitable to transcribe/translate control signal.These methods comprise recombinant DNA technology in vi, synthetic technology and In vivo recombination/gene recombination.See, such as be described in Maniatis etc., MolecularCloningALaboratoryManual, ColdSpringHarborLaboratory, N.Y. (1989) and Ausubel etc., CurrentProtocolsinMolecularBiology, GreenePublishingAssociates and WileyInterscience, the technology in N.Y (1989).
Various host-expression systems can be utilized to the encoding sequence of the encoding sequence and fusion polypeptide of expressing the object of the invention protein.Preferably, mammalian cell is used as host cell systems, carrys out transfection with the recombinant plasmid dna containing the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide or cosmid DNA expression vectors.More particularly, Chinese hamster ovary celI, bhk cell, NSO cell, SP2/0 cell, YO myeloma cell, P3 × 63 murine myeloma cell, PER cell, PER.C6 cell or hybridoma, other mammalian cell, yeast cell, insect cell or vegetable cell are used as host cell systems.Some examples of expression system and system of selection are described in following reference, in this as reference: Borth etc., Biotechnol.Bioen.71 (4): 266-73 (2000-2001), Werner etc., Arzneimittelforschung/DrugRes.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, other eukaryotic host cell system can be used, comprise yeast cell, with the recombinant yeast expression vector transfection containing the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide of the present invention; Insect cell system, infects with the recombinant virus expression vector (such as, baculovirus) containing the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide of the present invention; Vegetable cell system, with recombinant virus expression vector (such as, cauliflower mosaic virus, CaMV; Tobacco mosaic virus (TMV), TMV) to infect or with recombinant plasmid expression vector (such as, 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 (such as, adenovirus, vaccinia virus) infect, expression system comprises through engineering approaches to (such as mouse cell lines) stable amplification (CHO/dhfr) or the unstable clone increased in double minute chromosome of the sequence of coding target protein matter DNA containing multiple copied and the encoding sequence of fusion polypeptide of the present invention.
For method of the present invention, usual stably express is more preferred than transient expression, because stably express obtains more how reproducible result usually be also easier to scale operation, that is, in production-scale clone, produces glycosyl engineered antibody of the present invention.Except using the expression vector containing virus origin of replication, host cell can also be transformed with the respective coding nucleic acid controlled by suitable expression controlling elements (such as, promotor, enhanser, sequence, transcription terminator, site of polyadenylation etc.) and selectable markers.After introducing foreign DNA, make through engineering approaches cell grow 1-2 days in enrichment medium, be then converted to Selective agar medium.Selectable markers in recombinant plasmid gives the resistance for selecting and selects cell, and this cytotostatic ground plasmid integration is entered their karyomit(e) and growth forms locus (foci), then clones and be extended to clone.
Multiple choices system can be used, include, but are not limited to, herpes simplex virus thymidine kinase ((Wigler etc., Cell11:223 (1977)), xanthoglobulin-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc.Natl.Acad.Sci.USA48:2026 (1962)), with adenine phosphoribosyl transferase (Lowy etc., Cell22:817 (1980)) gene, it may be used for tk separately -, hgprt -or aprt -in cell.Further, metabolic antagonist resistance can with following basis: the dhfr that elects, and it gives methotrexate resistance (Wigler etc., Natl.Acad.Sci.USA77:3567 (1989); O ' Hare etc., Proc.Natl.AcadSci.USA78:1527 (1981)); Gpt, it gives mycophenolic acid (Mulligan & Berg, Proc.Natl.Acad.Sci.USA78:2072 (1981)); Neo, it gives aminoglycoside G-418 resistance (Colberre-Garapin etc., J.Mol.Biol.150:1 (1981)), and hygro, it gives hygromycin resistance (Santerre etc., Gene30:147 (1984).Recently, describe other Select gene, i.e. trpB, it makes cell utilize indole in place of tlyptophan; HisD, it makes cell utilize histidinol alternate sets propylhomoserin (Hartman & Mulligan, Proc.Natl.Acad.Sci.USA85:8047 (1988)); Glutamin synthase system; With ODC (ornithine decarboxylase), it gives the resistance (McConlogue:CurrentCommunicationinMolecularBiology, ColdSpringHarborLaboratory edit (1987)) of ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO.
Ii. the transfectant of glycosylation pattern protein or the qualification of transformant with modification is expressed
Identify containing encoding sequence and the host cell of expressing biologically active gene product by least four ordinary methods; A () DNA-DNA or DNA-RNA is hybridized; The existence of (b) " mark " gene function or disappearance; C () evaluates transcriptional level, measured by the expression of mRNA transcript respective in host cell; (d) detect gene product, measured by immunity test or its biological activity.
In first method, use containing and the probe of nucleotide sequence of respective encoding sequence or its part or derivatives thereof homology detected the existence of inserting the encoding sequence of target protein matter in expression vector and the encoding sequence of fusion polypeptide of the present invention by DNA-DNA or DNA-RNA hybridization.
In second method, the presence or absence formation etc. of inclusion body (such as, in thymidine kinase activity, antibiotics resistance, methotrexate resistance, transformation phenotype, baculovirus) based on specific " mark " gene function is identified and selects recombinant expression vector/host system.Such as, if in the marker gene sequence of the encoding sequence of target protein matter and fusion polypeptide encoding sequence insertion vector of the present invention, identify the recon containing respective encoding sequence by the disappearance of marker gene function.Or, marker gene can with encoding sequence series connection place, be used for control coding sequence express identical or different promotor control under.Mark response induction or the expression selected indicate the expression of the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide.
In 3rd method, evaluated the transcriptional activity of the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide of the present invention by cross experiment.Such as, the probe of use and the encoding sequence of target protein matter and the encoding sequence of fusion polypeptide of the present invention or its specific part homology divides analysis of variance RNA by Northern trace.Or, the total nucleic acid of host cell can be extracted and the hybridization of test and such probe.
In 4th method, can immunological evaluation target protein protein product expression and there is β (1,4) expression of the encoding sequence of the fusion polypeptide of the active Golgi localization domain also containing xenogenesis Golgi resident polypeptide of-NAG transferase I II (GnTIII), such as by western blot, immunity test is as Radioimmunoprecipitation, enzyme linked immune assay etc.But the final test of success of expression system comprises the detection of biologically active gene product.
B. there is the protein of the glycosylation pattern of change and the generation of protein fragments and purposes
The effector function i. with raising comprises generation and the purposes of the antibody of antibody dependent cellular cytotoxicity
In preferred embodiment, the invention provides antibody and antibody fragment that the Fc receptors bind with raising and/or effector function comprise the glycoforms of antibody dependent cellular cytotoxicity.
The clinical trial that non-conjugated monoclonal antibodies (mAb) is used for the treatment of certain cancers has created inspirer result recently.Dillman, CancerBiother. & Radiopharm.12:223-25 (1997); Deo etc., ImmunologyToday18:127 (1997).Be fitted together to, do not put together IgG1 and ratified for rudimentary or follicular B-cell non Hodgkin lymphom.Dillman, CancerBiother. & Radiopharm.12:223-25 (1997), and another does not put together mAb, the humanization IgG1 of target solid breast tumors, in III clinical trial phase, demonstrates distant view result.Deo etc., ImmunologyToday18:127 (1997).The antigen of these two mAb their respective tumour cell camber express and by effector cell in vitro or internal antibody mediate effective tumor destruction.On the contrary, many other is not puted together mAb and can not be caused the effector function being enough to be effective to clinical application with trickle tumour-specific.Frost etc., Cancer80:317-33 (1997); Surfus etc., J.Immunother.19:184-91 (1996).For some in these more weak mAb, test adjunct cytokine therapy recently.Add cytokine and can stimulate antibody dependent cellular cytotoxicity (ADCC) by the activity and quantity improving 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, and are initiated due to the combination of leukocyte receptors and antibody constant region (Fc).Deo etc., ImmunologyToday18:127 (1997).
Improve and do not put together the difference of ADCC activity of IgG1 but the method for complementation is the Fc fragment of engineered antibody.The lower hinge area that protein engineering research has shown Fc γ R and IgGCH2 structural domain interacts.Lund etc., J.Immunol.157:4963-69 (1996).But Fc γ R combines also needs existence and CH2 district to guard the covalently bound oligosaccharides in Asn297 place.Lund etc., J.Immunol.157:4963-69 (1996); Wright and Morrison, TrendsBiotech.15:26-31 (1997), propose 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 improving interaction affinity.
Carry two N-in IgG molecule Qi Fc district and connect oligosaccharides, in each heavy chain one.The same with any glycoprotein, antibody produces with the colony of glycoforms, and it is shared identical polypeptide backbone but has the different oligosaccharides be connected with glycosylation site.The oligosaccharides be typically found in serum IgG Fc district is Composite Double feeler type (bi-antennarytype) ((Wormald etc., Biochemistry36:130-38 (1997)), with terminal sialic acid and the bisection NAG (GlcNAc) of low levels, and the terminal galactosylation of variable pitch and core fucosylation.Some researchs show that the minimum sugared structure of Fc γ R combination needs is arranged in oligosaccharides core.Lund etc., J.Immunol.157:4963-69 (1996).
Treat the mouse of mAb or the clone of hamster origin usually required oligosaccharide determinant is connected to Fc site for producing in industry and academia not put together.But the IgG of these expression of cell lines lacks the bisection GlcNAc found in low amounts serum IgG.Lifely etc., Glycobiology318:813-22 (1995).On the contrary, the humanization IgG1 (CAMPATH-1H) observing rat bone myeloma generation recently carries bisection GlcNAc in its some glycoforms.Lifely etc., Glycobiology318:813-22 (1995).The antibody in rat cell source reaches active with the external ADCC of the similar maximum of CAMPATH-1H antibody produced in standard cell lines system, but in low-down antibody concentration.
CAMPATH antigen exists on lymphoma cell with high level usually, and this chimeric mAb has high ADCC activity under bisection GlcNAc lacks.Lifely etc., Glycobiology318:813-22 (1995).N-connects in glycosylation pathway differ, adds bisection GlcNAc by enzyme β (Isosorbide-5-Nitrae)-NAG transferase I II (GnTIII).Schachter，Biochem.CellBiol.64：163-81(1986)。
Research before employs single antibody and produces Chinese hamster ovary celI system, before this clone, through engineering approaches expresses the cloned GnT III gene enzyme (Umana of different levels with outside regulative mode, P. etc., NatureBiotechnol.17:176-180 (1999)).The method first time establishes the strict correlation between GnTIII expression with modified antibodies ADCC activity.
Further the present invention has the antibody of the Fc receptor binding affinity of raising and the effector function of raising, include, but are not limited to, by the anti-human neuroblastoma monoclonal antibody (chCE7) that the inventive method produces, the chimeric anti-human renal cell's carcinoma monoclonal antibody (chG250) produced by the inventive method, the Humanized anti-HER 2 monoclonal antibody produced by the inventive method (such as, Trastuzumab (HERCEPTIN), by the chimeric anti-human colon that the inventive method produces, lung and breast cancer monoclonal antibody (ING-1), the Humanized anti-human 17-1A antigen monoclonal antibody (3622W94) produced by the inventive method, the colorectal tumour antibody of the Humanized anti-human produced by the inventive method (A33), the anti-human melanoma antigens (R24) of the facedown GD3 Sphingolipids,sialo produced by the inventive method, with the chimeric anti-human squamous cell carcinoma monoclonal antibody (SF-25) produced by the inventive method, monoclonal antibody resisting human small cell lung carcinoma (the BEC2 produced by the inventive method, ImcloneSystems, MerckKgaA), by the anti-human Fei Huojinqi lymphomas monoclonal antibody (Bexxar (tositumomab that the inventive method produces, CoulterPharmaceuticals), Oncolym (Techniclone, AlphaTherapeutic)), the anti-human squamous cell head obtained by the inventive method and neck carcinoma monoclonal antibody (C225, ImCloneSystems), the anti-human rectum obtained by the inventive method and colorectal carcinoma monoclonal antibody (Panorex (edrecolomab), Centocor, GlaxoWellcome), human ovary carcinoma resisting monoclonal antibody (the Theragyn produced by the inventive method, Antisoma), by the anti-human acute myelogenous leukemia carcinoma monoclonal antibody (SmartM195 that the inventive method produces, ProteinDesignLabs, Kanebo), by the anti-human glioblastoma monoclonal antibody (Cotara that the inventive method produces, Techniclone, CambridgeAntibodyTechnology), by the anti-human B cell Fei Huojinqi lymphomas monoclonal antibody (IDEC-Y2B8 that the inventive method produces, IDECPharmaceuticals), monoclonal antibody (the CEA-Cide of the anti-human noumenal tumour produced by the inventive method, Immunomedics), by the anti-human colorectum carcinoma monoclonal antibody (iodine 131-MN-14 that the inventive method produces, Immunomedics), by the anti-human ovary that the inventive method produces, kidney, mammary gland and prostate cancer monoclonal antibody (MDX-210, Medarex, Novartis), the anti-human colorectum produced by the inventive method and carcinoma of the pancreas monoclonal antibody (TTMA, Pharmacie & Upjohn), the anti-human TAG-72 produced by the inventive method expresses carcinoma monoclonal antibody (MDX-220, Medarex), the anti-human EGFr-produced by the inventive method expresses carcinoma monoclonal antibody (MDX-447), the Anti-X activity (Genentech) produced by the inventive method, by the anti-human mammary gland that the inventive method produces, lung, prostate gland and carcinoma of the pancreas and malignant melanoma monoclonal antibody (BrevaRex, AltaRex), by the anti-human acute myelogenous leukemia monoclonal antibody (MonoclonalAntibodyConjugate that the inventive method produces, Immunex).In addition, the present invention relates to antibody fragment and fusion rotein, fusion rotein contains the region being equivalent to immunoglobulin fc region.
Ii. promote generation and the purposes of the fusion rotein of Fc mediating cytotoxicity, fusion rotein contains the region being equivalent to immunoglobulin fc region
As mentioned above, the present invention relates to the raising treatment Fc receptor binding affinity of antibody and/or the method for effector function.This is realized by the glycosylation pattern in the Fc district of the such antibody of through engineering approaches, produce especially by engineered antibody generation cell and such as there is the polypeptide that GnTIII is active or GalT is active or ManII is active, the oligosaccharides that this is peptide modified is connected with such antibody Fc district.This strategy can be used for improving by being not only the cytotoxicity of the Fc mediation by treating the undesirable cell of antibody-mediated antagonism with region any numerator mediated being equivalent to immunoglobulin fc region, because the change introduced by glycosylation engineering only have impact on Fc district, therefore with the Fc acceptor interaction on the effector cell surface that relates in ADCC mechanism.The molecule containing Fc of method disclosed by the invention can be used, include, but are not limited to, a soluble fusion protein (Chamov and Ashkenazi that () You He Fc district N-holds the target protein structural domain merged to obtain, TrendsBiotech.14:52 (1996)), (b) by the fusion rotein (Stabila being positioned the protoplast membrane grappling of holding the II type membrane spaning domain of the plasmalemma merged to obtain with Fc fragment N-, P.F., NatureBiotech.16:1357 (1998)).
When soluble fusion protein (a), target structural domain guide fusion rotein and undesirably cell such as cancer cells combine, that is, with treat mode like antibody class.Therefore, these numerator mediated effector functions of raising disclosed by the invention comprise Fc mediation cellular cytoxicity activity method application with use to treatment antibody method identical.
When film grappling fusion rotein (b), in body, less desirable cell must express the gene of encoding fusion protein.This can be obtained by gene therapy method, namely with the extremely undesirably cell of transfectional cell in the plasmid instructing fusion rotein encoding gene to express or virus vector body, or by the Transplanted cells of genetically engineered expressed fusion protein in its surface being entered in body.Latter cell is implanted into (encapsulated cell therapy) in body usually in polymer capsule, and wherein they are not subject to the destruction of Fc mediating cytotoxicity mechanism.But capsule apparatus cell that is broken and that escape becomes undesirable, can be eliminated by Fc mediating cytotoxicity.Stabila etc., NatureBiotech.16:1357 (1998).In this case, method disclosed by the invention can be used, by will other expression casette of the suitable or maximum expression level of fusion polypeptide of the present invention be guided to be incorporated in gene therapy vector, or through engineering approaches cell of expressing the fusion polypeptide of the present invention of suitable or maximum expression amount to be implanted.
According to the treatment use of antibody, antibody fragment and fusion polypeptide that the inventive method produces
Antibody of the present invention (i.e. antibody, antibody fragment and fusion rotein, it contains the region being equivalent to immunoglobulin fc region) may be used solely to target and kills the tumour cell in body.Antibody can also make in conjunction with suitable therapeutical agent for treatment human cancer.Such as, antibody can use together as chemotherapy, radiotherapy in conjunction with conventional treatments or put together with medicine or toxin and lymphokine or tumor-inhibitory growth factor or be connected and therapeutical agent is sent to cancer position.
By the technology of such therapeutical agent and antibody conjugate be known [see, such as Arnon etc., " MonoclonalAntibodiesforImmunotargetingofDrugsinCancerThe rapy ", MonoclonalAntibodiesandCancerTherapy, Reisfeld etc. (editor), pp.243-56 (AlanR.Liss, Inc.1985); Hellstrom etc., " AntibodiesForDrugDelivery ", ControlledDrugDelivery (the 2nd edition), Robinson etc. (editor), pp.623-53 (MarcelDekker, Inc.1987); Thorpe, " AntibodyCarriersOfCytotoxicAgentsInCancerTherapy:AReview ", MonoclonalAntibodies ' 84:BiologicalAndClinicalApplications, Pinchera etc. (editor), pp.475-506 (1985); With Thorpe etc., " ThePreparationAndCytotoxicPropertiesOfAntibody-ToxinConj ugates ", Immunol.Rev., 62:119-58 (1982)].
Or, Glyco-engineered antibodies can combine with high-energy radiation such as isotropic substance such as <131>I, when it is positioned tumor locus, cause the killing of several cell dia [see, such as, Order, " Analysis, Results, andFutureProspectiveofTheTherapeuticUseofRadiolabeledAnt ibodyinCancerTherapy ", MonoclonalAntibodiesForCancerDetectionAndTherapy, Baldwin etc. (editor), pp.303-16 (AcademicPress1985)].Still according to another embodiment, antibody of the present invention can be puted together with second antibody and forms antibody heteroconjugate thing to treat tumour cell, if Segal is in U.S. Patent No. 4, and 676, describe in 980.
Still for other treatment use of antibody of the present invention comprise such as puted together by recombinant DNA technology or be connected to can by the enzyme of Prodrug converting one-tenth cytotoxic drug and use antibody-enzyme conjugate in conjunction with prodrug by prodrug tumor locus change into cytotoxic agent [see, such as, Senter etc., " Anti-TumorEffectsofAntibody-alkalinePhosphatase ", Proc.Natl.Acad.Sci.USA85:4842-46 (1988); " EnhancementoftheinvitroandinvivoAntitumorActivitesofPhos phorylatedMitocycinCandEtoposideDerivativesbyMonoclonal. Antibody-AlkalinePhosphataseConjugates ", CancerResearch49:5789-5792 (1989); And Senter, " ActivationofProdrugsbyAntibody-EnzymeConjugates:ANewAppr oachtoCancerTherapy, " FASEBJ.4:188-193 (1990)].
Still another treatment use of antibody of the present invention, relates under complement or partial antibody-medicine or Antibody-toxin conjugates existence, for removing tumour cell from the marrow of cancer patients.According to the method, by can clean autologous bone marrow in vitro by antibody treatment and marrow is inculcated back patient [see, such as, Ramsay etc., " BoneMarrowPurgingUsingMonoclonalAntibodies ", J.Clin.Immunol., 8 (2): 81-88 (1988)].
In addition, the present invention contains the chimeric antibody in the territory, antigen-specific binding region of required monoclonal antibody, recombinant immunotoxin and other recombinant precursor and may be used for treatment.Such as, monochain immunotoxin of the present invention can be used for interior therapeutic human cancer.
Similarly, and the fusion rotein of antigen binding domain containing antibody at least of the present invention that at least functionally active part of another kind of albumen such as lymphokine or oncostatin with anti-tumor activity combines, interior therapeutic human cancer can be used for.In addition, recombinant technology known in the art can be used for building bi-specific antibody, and wherein a binding specificity of antibody 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 off tumor cells, the antigen of this tumor cells expression and monoclonal antibody of the present invention or function equivalent specific binding.The method comprises and contacting with described tumour cell by glycosyl engineered antibody of the present invention or containing its immunoconjugates (such as immunotoxin).These tumour cells may form human cancer.
In addition, the invention provides the method for interior therapeutic cancer (such as human cancer).The method comprises the composition for the treatment of significant quantity is delivered medicine to patient, and composition comprises at least one glycosyl engineered antibody of the present invention or containing its immunoconjugates (such as, immunotoxin).
Again on the one hand, the present invention relates to and exhaust the modification method of (depletion) treatment by the Immunological diseases of all or part of generation of pathogenic autoantibody based on B cell, comprise the patient immunoreactivity antibody for the treatment of significant quantity being delivered medicine to needs, improvement comprises the antibody with raising ADCC obtained according to the inventive method of drug treatment significant quantity.In preferred embodiment, antibody is anti-CD20 antibodies.Autoimmune disorders or imbalance, include, but are not limited to, immune-mediated thrombocytopenia, as acute idiopathic thrombocytopenia purpura and chronic idiopathic thrombocytopenia purpura, dermatomyositis, tarantism, lupus nephritis, rheumatic fever, polyglandular syndromes, Henoch-Schonlein purpura, latter stage streptoccal nephritis, erythema nodosum, Takayasu ' s arteritis, addison's disease, erythema multiforme, polyarteritis nodosa, stiff spondylitis, Goodpasture's syndrome, thromboangiitis ubiteran, primary biliary cirrhosis, Hashimoto ' s thyroiditis, thyrotoxicosis, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pamphigusvulgaris, wegner's granulomatosis, film ephrosis, amyotrophic lateral sclerosis, myelophthisis, polymyalgia, pernicious anemia, quick Progressive symmetric erythrokeratodermia glomerulonephritis and fibrous tissue form pulmonary alveolitis, Inflammatory response is as dermatitis, comprise psoriasis and dermatitis (such as, atopic dermatitis), whole body scleroderma and sclerosis, the reaction (such as, Crohn disease and ulcerative colitis) relevant to enteritis, respiratory distress syndrome (comprises adult respiratory distress syndrome, ARDS), dermatitis, meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, such as eczema and asthma and other illness, comprise T cell and infiltrate and chronic inflammatory reaction, atherosclerosis, white corpuscle adhesion is not enough, rheumatoid arthritis, Systemic Lupus Eryhamatosus (SLE), diabetes (such as, type i diabetes or insulin-dependent diabetes), multiple sclerosis, Reynaud ' s syndromes, autoimmune thyroiditis, allergic encephalitis, Sjorgen ' s syndromes, the juvenile diabetes started, with the usual immunne response relevant with retardance allergy with the acute of T-cell mediated by cytokine found in pulmonary tuberculosis, sarcoidosis, polymyositis, granulomatosis and nodular vasculitis, pernicious amenorrhoea (addison's disease), relate to the disease of leukocyte infiltration, central nervous system (CNS) inflammation, multiple organ injury's syndromes, hemolytic anemia (including, but are not limited to cryoglobulinemia or Ku Musishi positive anemia), myasthenia gravis, the disease of antigen-antibody complexes mediation, anti-angiogenic bead sarolemma is sick, anti-phospholipid syndrome, allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome, pemphigoid bleb, it bleb, autoimmunization polyendocrinopathy, Reiter ' s disease, tetanic people's syndromes, Behcet disease, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, immunity thrombocytopenia purpura (ITP) or Autoimmune thrombocytopenia etc.In this respect of the present invention, the normal B cells in antibody consumption blood of the present invention is with the time period extended.
According to enforcement of the present invention, patient can be people, horse, pig, ox, mouse, dog, cat and bird patient.Other warm-blooded animal is also included in the present invention.
Present invention also offers the method for Therapeutic cancer patient.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 usually characterized by the cell mass with antigen related neoplasms on cell surface.The method comprise by the cancer amount of killing with cytotoxic agent combine cancer target antibody administration in patient.Usually, under the condition allowing the target on the antibodies cell surface combined, form the combination of cancer target antibody and cytotoxic agent.By combining target, cancer target antibody directly or indirectly causes or causes killing in conjunction with cell, therefore treats patient.
Additionally provide the method suppressing mammalian tumor cell hyperplasia, it comprises and mammalian tumor cell contact with the glycosyl engineered antibody of the present invention of enough concentration or the immunoconjugates containing it, makes the hyperplasia of suppression mammalian tumor cell.
Invention further provides the method suppressing growth of human tumor cells, treatment patient tumors and treatment patient Accretive Type disease.These methods comprise the present composition of significant quantity are delivered medicine to patient.
Therefore very clearly present invention comprises the treatment pharmaceutical composition of human cancer, mixture and method.Such as, the present invention includes the pharmaceutical composition being used for the treatment of human cancer, composition comprises acceptable carrier on the antibody of the present invention of medicine effective quantity and pharmacology.
Composition can contain antibody or antibody fragment, unmodified and therapeutical agent (such as, medicine, toxin, enzyme or second antibody) put together or in recombinant form (such as, fragment, the bi-specific antibody of chimeric antibody, chimeric antibody).Composition can comprise other antibody or the conjugate (such as, mixtures of antibodies) of Therapeutic cancer in addition.
Conventional administration mode can be used to come administration antibody of the present invention, antibody conjugates and immunotoxin composition, include, but are not limited to intravenously, intraperitoneal, oral, lymph interior or directly administration is entered in 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, polymeric microcapsule or microvesicle, liposome and injectable maybe can inject.Preferred form depends on administering mode and treatment use.
Composition of the present invention also preferably includes conventional pharmaceutical known in the art can accept carrier and adjuvant if human serum albumin, ion-exchanger, aluminum oxide, Yelkin TTS, buffer substance are if phosphoric acid salt, glycine, Sorbic Acid, potassium sorbate and salt or ionogen are as Protamine sulfates.
The most effective administering mode of the present composition and dosage regimen depend on the severity of disease and process, patient health and to the response for the treatment of and the judgement for the treatment of physician.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, polymeric microcapsule or microvesicle, liposome and injectable maybe can inject.Preferred form depends on administering mode and treatment use.
The most effective administering mode of molecule of the present invention and dosage regimen depend on the position of tumour to be treated, the severity of cancer and process, patient health and to the response for the treatment of and the judgement for the treatment of physician.Therefore, should the composition dosage of titration single patient.
Dosage mutual relationship based on all size of surface-area mg/kg and the animal of species and people is described in Freireich, the CancerChemother. such as E.J., Rep.50 (4): 219-244 (1966).The adjustment can carrying out dosage regimen carrys out optimizing growth of tumour cell and suppresses and kill response, such as, can with day basis by dosage separately and administration or according to circumstances part reduce dosage (such as, daily several dosage of separating or according to the minimizing of particular treatment case part).
Should be understood that the present composition dosage obtaining treatment needs can reduce further along with schedule optimizing.
According to enforcement of the present invention, pharmaceutical carrier can be lipid carrier.Lipid carrier can be phosphatide.In addition, lipid carrier can be lipid acid.Further, lipid carrier can be stain remover.As used in this, stain remover changes any material that surface tension of liquid normally reduces surface tension of liquid.
In one embodiment of the invention, stain remover is nonionic detergent.The example of nonionic detergent, includes, but are not limited to, and polysorbate 80 (also referred to as tween 80 or (polyoxyethylene sorbitan monooleate), Brij and Triton (such as TritonWR-1339 and TritonA-20).
Or stain remover can be zwitterionic detergent.The example of zwitterionic detergent, includes, but are not limited to, alkyl trimethyl ammonium bromide.
In addition, according to the present invention, lipid carrier can be 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 given preparation and embodiment can make those skilled in the art more clearly understand and implement the present invention.But the present invention is not restricted to the scope of exemplary embodiment, it is only 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 before and accompanying drawing, those become apparent those skilled in the art for various change of the present invention.The scope falling into claims is determined in such change.
Embodiment
Embodiment 1
Materials and methods
1. the structure of antibody expression vector
anti-CD20 antibodies expression vector pETR1502
C2B8 anti-CD20 antibodies expression vector pETR1502 by the expression cassette that four independently, are separated form (one for C2B8 light chain of antibody, one for C2B8 heavy chain of antibody, one for neomycin resistance gene and one for mouse dhfr gene).The synthesis of all genes under the control of myeloproliferative sarcoma virus (MPSV) promotor and containing the polyadenylation signal being derived from rabbit beta globin gene has polyadenylation signal.
In single stage method, use PCR to assemble the variable heavy chain (VH) of coding anti-CD20 antibodies C2B8 and the CDNA (Kobayashi of variable light (VL) from the single stranded oligonucleotide of a series of overlap, N. etc., Biotechniques23:500-503 (1997)).The original series (international publication number: WO94/11026) of coding C2B8VL and VH fragment is obtained from disclosed international patent application.VL and the VHcDNA fragment subclone of assembling is entered in pBluescriptIIKS (+) to produce pBlue-C2B8VH and pBlue-C2B8VL plasmid, and checks order.
From the variable chains of corresponding pBlue-C2B8VH and pBlue-C2B8VL plasmid amplification C2B8, be used in primer that 5 ' end introduces AscI restriction site and introduces convenient restriction sites with constant region junction variable (BsiWI is used for light chain and NheI for heavy chain).From human lymphocyte cDNA library (Quickclone, Clontech) increase IgGI constant region, is used in the primer (BsiWI and BamHI is used for constant light and NheI and BamHI is used for constant heavy) that 5 ' and 3 ' end introduces convenient restriction sites.
After confirming correct DNA sequence dna, the light chain of C2B8 antibody and heavy chain are combined with MPSV promotor and polyadenylation signal separately.In the first step, build two different expression vectors: one for C2B8 light chain (pETR1315), another is for C2B8 heavy chain (pETR1316).In second step, by neomycin resistance expression cassette (be derived from Tn5 transposon neomycin resistance gene and under being positioned over the control of minimum MPSV promotor) introduce in carrier pETR1315 and form plasmid pETR1481.Plasmid pETR1328 is obtained in dhfr expression casette insertion vector pETR1316 under MPSV promotor being controlled.In final step, two expression module (C2B8 light chain+neo and C2B8 heavy chain+dhfr) are incorporated in a carrier and form plasmid pETR1502.
anti-CD20 antibodies expression vector pETR1520
PETR1520 is combined with C2B8 anti-CD20 antibodies expression vector and replication orgin, replication orgin from EpsteinBarr virus (oriP), for episomal vector in produce EpsteinBarr Virus Nuclear Antigen (EBNA) cell in copying and keeping.In order to build C2B8 expression vector pETR1520, the C2B8 from pETR1416 being expressed module and inserts in the carrier pETR1507 containing oriP with HinDIII fragment.Carrier pETR1416 with pETR1502 is similar, except the neomycin resistance gene of this plasmid is under the control of whole MPSV promotor, instead of 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 each free antibody L19, the encode fragment of people's antibody of identifying the ED-B domain of fibronectin substitutes.By the DNA fragmentation of Overlap extension PCR method composite coding variable region, use the synthetic oligonucleotide (Pini, A. etc., J.Biol.Chem.273 (34): 21769-76 (1998)) based on L19 antibody variable sequences.
anti-egfr antibodies (C225) expression vector pURS128
This carrier is identical with carrier pETR1520, except the variable heavy chain of coding C2B8 anti-CD20 antibodies and light chain by antibody C225, identify that the respective encode fragment of the chimeric antibody of Human epidermal growth factor receptor substitutes.By the DNA fragmentation of Overlap extension PCR method composite coding variable region, use based on the synthetic oligonucleotide of C225 antibody variable sequences that (sequence is found in disclosed patent application, international publication number WO96/40210, Figure 16 and 17 of this patent application corresponds to heavy chain and light chain separately).
The structure of 2.GnTIII fusion expression vector
pETR1166. for the carrier of GnTIII constitutive expression
In order to build GnTIII expression vector pETR1166, from rat kidney cDNA library (Clontech) by pcr amplification rat GnTIII.In order to detect GnTIII easily by western blotting subsequently, c-myc-Epitope tag is held by C to add the upstream (aminoacid sequence: PEQKLISEEDL) of and then gene end codon.After confirming the correct sequence of GnTIII, gene is inserted in MPSV promotor and controls and the polyadenylation signal adding synthesis rabbit beta globin gene.Final GnTIII expression vector is also containing the puromycin-resistant box separated for selecting, and puromycin resistance gene is also under MPSV promotor controls and the polyadenylation signal of the rabbit beta globin gene of synthesis.
102 amino acid replacements of PETR1425: people GnTI, 76 ammonia of GnTIII base acid
The structure of this heterozygosis glycosyltransferase gene is carried out by ensuing over-lap PCR reaction.In a reaction, use the main region of primer GAB-179 and GAB-180 amplification people GnTI.In this PCR reacts, also introduce Kozak consensus sequence and AscI restriction site at 5 ' end.The PCR fragment obtained has the overlap of 23bp from position 229 with GnTIII.In second PCR reaction, the GnTIII district of position 229 to 380 of increasing with primer GAB-177 and GAB-178, creates the PCR fragment having unique BstXI site in 3 ' end band, has the overlap of 22bp at 5 ' end and GnTI main region.The template (primer GAB-179 and GAB-178) of reacting by two PCR fragment purifying and as the 3rd PCR.The fragment that purifying obtains also digests with AscI, and connection carrier pETR1001 (with AscI and SmaI cutting) obtains plasmid pETR1404.After confirmation insertion sequence, MPSV promoter sequence is added in the pETR1404 of AscI (partial digested)/PmeI fragment, produces plasmid pETR1423.From the SphI/BstXI fragment of pETR1166, with the expression vector of original rat GnTIII gene, then substitute by the corresponding fragment of pETR1423 and obtain plasmid pETR1425, containing the GnTI-GnTIII fusion under the control of MPSV promotor and the puromycin-resistant box for selecting.
Primer sequence:
GAB-177：GCGTGTGCCTGTGACCCCCGCGCCCCTGCTCCAGCCACTGTCCCC
GAB-178：GAAGGTTTCTCCAGCATCCTGGTACC
GAB-179：CTGAGGCGCGCCGCCACCATGCTGAAGAAGCAGTCTGCAGGGC
GAB-180：
GGGGACAGTGGCTGGAGCAGGGGCGCGGGGGTCACAGGCACACGCGGC
100 amino acid replacements of pETR1506: people's mannosidase II GnTIII's 76 n terminal amino acids
Carry out the structure of pETR1506, the structure of similar pETR1425.Use carrier pBlueman as the main region of template amplification people Mannosidase II gene with primer GAB-252 and GAB-253.In this PCR process, introduce FseI site and Kozak consensus sequence at 5 ' end.The PCR fragment obtained starts and GnTIII gene overlap 23bp in position 229.In second PCR reaction, with primer GAB-254 and GAB-255 amplification part GnTIII gene (position 229-460).This PCR creates and holds containing having 43bp overlap with mannosidase II and containing the fragment in unique StuI site at 3 ' end 5 '.Purifying two fragments also use the template in the PCR of primer GAB-252 and GAB-255 as the 3rd.Obtained fragment is inserted pIC19H and obtains carrier pETR1484.After confirming the correct sequence of this insertion, build complete fusion gene by the StuI/BamHI fragment of the FseI/StuI fragment and pETR1166 that connect pETR1484 in carrier pETR12177 (FseI/BamHI).The plasmid (pETR1500) obtained is containing the heterozygosis manII-GnTIII gene (SEQIDNO:14) under the control of MPSV promotor.In order to select plasmid in mammalian cell, inserting puromycin-resistant box with the StuI fragment of pETR1166, producing 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 sick from EpsteinBarr the combination of the replication orgin oriP of poison
Use primer GAB-261 and GAB-262, from the 2kb fragment of plasmid pCEP4 (Invitrogen) amplification containing oriP.In this PCR reaction process, introduce SspI and EcoRI site at two ends of fragment.In order to check order, by oriP fragment insertion vector pIC19H.After verified correct sequence, by oriP (to fill 5 ' overlapping ends with BsmBI digestion and use Klenow polysaccharase) in SspI fragment insertion vector pETR1001.The plasmid called after pETR1507 obtained.SphI/NruImanII-GnTIII expression cassette from pETR1510 is inserted in the pETR1507 digested by identical restriction endonuclease and obtains plasmid pETR1519.
Primer sequence:
GAB-261：GCTAAATATTGAATTCCCTTTATGTGTAACTCTTGGCTGAAGC
GAB-262：TAGCAATATTGAATTCGCAGGAAAAGGACAAGCAGCGAAAATT
CACGC
the cd4 cell surface mark of pETR1537: heterozygosis manII-GnTIII fusion gene and brachymemma the combination of note gene
Modify the cd4 cell surface markers gene that pETR1506 expression vector expresses brachymemma in addition.Speak briefly, heterozygosis manII-GnTIII track fusion box being changed into bicistronic expression cassettes from monocistron, being inserted the downstream (containing the people CD4 leader sequence for secreting of then cross-film and ectodomain) of the terminator that manII-GnTIII merges by the cDNA of the human CD 4 protein matter of brachymemma of poliovirus IRES sequence then being encoded.
3. with GnTIII fusion expression vector and antibody expression vector transfection mammalian cell
the transfection of bhk cell
Before electroporation 24 hours by the cell (vitality 90-95%) that exponentially grows with 0.9 × 10 6the concentration of individual cell/ml is inoculated in the T75 culturing bottle of suitable quantity.As substratum, use the Invitrus (CellCultureTechnologies, Switzerland) of supplementary 10% foetal calf serum (FCS).By cell counting before electroporation.8 × 10 are collected by centrifugal (5 minutes, 200 × g) 6individual cell also abandons supernatant liquor.Cell resuspension to be transferred to containing (0.4cm interval) in the aseptic electroporation cuvette of 8 μ g circular plasmids DNA and incubated at room 5 minutes in 800 μ lInvitrus substratum.Use GenePulserII (BioRad) by following condition by cell electroporation: 400V, 960 μ F, two subpulse 30 seconds intervals.After electroporation, cell is transferred to immediately in the T25 culturing bottle containing 5ml growth medium (Invitrus/20% (V/V) FCS/1.25% (V/V) DMSO), and at 5%CO 237 DEG C of cultivations in atmosphere incubator.For the production of unmodified (non-glycosylation) antibody, only use antibody expression vector transfectional cell.For the production of glycosylated antibodies, with two plasmid co-transfection cells, one for antibody expression, another for merge GnTIII polypeptide (SEQIDNO:15) express, respective ratio is 3: 1.
the transfection of HEK293-EBNA cell
The HEK293-EBNA cell that transfection exponentially grows is carried out by calcium phosphate precipitation method.Use the DMEM substratum of supplementary 10%FCS make in T culturing bottle cell as adhesivity monolayer culture thing growth, when they be 50 to 80% be paved with time carry out transfection.For the transfection of T75 culturing bottle, 800 ten thousand cells were inoculated in 14ml in first 24 hours in transfection and supplement in the DMEM substratum of FCS (final 10%V/V) and 250 μ g/ml Liu Suanyan NEOMYCIN SULPHATEs, and cell is positioned over 5%CO 2in atmosphere incubator, 37 DEG C are spent the night.For each T75 culturing bottle to be transfected, by mixing the CaCl of the total plasmid vector DNA of 47 μ g, 235 μ lM 2solution to add water to final volume be that 469 μ l come obtained DNA, CaCl 2with the solution of water.50mMHEPES, 280mMNaCl, 1.5mMNa of 469 μ lpH7.05 is added in this solution 2hPO 4solution, mixed for 10 seconds immediately and at room temperature placed for 20 seconds.Supplement the DMEM diluted suspension of 2%FCS with 12ml, and add alternative existing substratum in T75.Cell is at 37 DEG C of 5%CO 2under hatch 17 to 20 hours, then use the DMEM substitutive medium of 12ml10%FCS.For the production of unmodified (non-glycosylation) antibody, only use antibody expression vector transfectional cell.For the production of glycosylated antibodies, with two plasmid co-transfection cells, one for antibody expression, another is for merging GnTIII expression of polypeptides, and respective ratio is 4: 1.Transform after 5 days, collect supernatant liquor, at 1200rpm centrifugal 5 minutes, second time is centrifugal subsequently, 4000rpm10 minute, and 4 DEG C of preservations.
express the generation of the stable mammal cell line of restructuring anti-CD20 antibodies
With the pETR1502C2B8 antibody expression vector containing neomycin resistance gene expression cassette by electroporation transfection bhk cell (BHK21-13c).First neomycin-resistant clones is selected to obtain a set of clone with the pETR1502 carrier DNA of chromosomal integration.Then ELISA experiment sieving is used to be used for the clone of recombinant antibodies production.Speak briefly, electroporation is after 24 hours, counts viable cell and the fluorocyte carrying out contrasting electroporation by counting parallel pEYFP-expression vector measures transfection efficiency.By cell dilution in the Invitrus Selective agar medium containing 10%FCS and 1mg/ml Liu Suanyan NEOMYCIN SULPHATE.The viable transfectional cell (1 × 10 of usual 8 96 hole plating different concns 3, 5 × 10 2with 1 × 10 2individual cell per well) and hatch until can clone be identified at 37 DEG C.Once clonal growth is to almost converging, produced by the antibody of elisa assay supernatant liquor.First ELISA positive colony is expanded to 24 holes subsequently on 6 hole flat boards, then to T25 culturing bottle.Grow 5 days in T25 culturing bottle after, use the final antibody titers of ELISA test determination.Use this electroporation and system of selection, isolate bhk cell clone (BHK-1502-28) of expressing C2B8 anti-CD20 antibodies, this cell clone produces 13 μ g/ml antibody under above-mentioned culture condition.
express the product of the stable mammal cell line of restructuring anti-CD20 antibodies and GnTIII fusion raw
The clone BHK-1502-28 of electroporation transfection constitutive expression anti-CD-20 monoclonal antibody gene and neomycin resistance gene is passed through with pETR1537 expression vector.PETR1537 is the carrier for constitutive expression ManII-GntIII gene and clipped form people CD4, and the latter is IRES dependent expression.Carrier is also containing puromycin resistance gene expression cassette.First puromycin-resistant clone is selected to obtain a set of clone with the pETR1537 carrier DNA of chromosomal integration.Then screening and cloning is used for the surface expression of the CD4 (tCD4) of brachymemma, and it is as the mark of bicistronic mRNA ManII-GnTIII+tCD4 gene expression unit expression level.Use the generation of the recombinant antibodies of the selected clone of ELISA verification experimental verification.
Speak briefly, with XmnI by the linearizing of pETR1537 carrier DNA.Contrast transfection is carried out with EYFP expression vector is parallel.The cell of whole intracellular expression EYFP is counted to measure transfection efficiency after 24 hours.58% of whole cell expresses EYFP.Cell viability is 91%.Transfection one day after, the cell serial dilution of pETR1537 and pEYFP transfection is 1: 100,1: 500,1: 1000 and 1: 5000 extent of dilution and the final volume be inoculated on 96 hole flat boards is (Invitrus in the Selective agar medium of 0.2ml, 10%FCS, 20 μ g/ml tetracyclines, 1mg/ml Liu Suanyan NEOMYCIN SULPHATE).Two Zhou Houke see clone.They are expanded and screens brachymemma CD4 (tCD4) and express and antibody expression.
In order to the screening of tCD4 expression level, about 500,000 cell FACS buffer solution also hatches 20 minutes with the anti-human CD4 (BectonDickinson, Switzerland) of 5 μ lFITC on ice.After twice washing, Cell resuspension is also used facs analysis (Figure 17 A-B) in 0.5mlFACS damping fluid.Isolate the clone (BHK-1502-28-11) with good tCD4 expression level.Grow 5 days in T25 culturing bottle after, produce the anti-CD20 antibodies that whole titre is about 17 μ g/ml, as passed through measured by ELISA.
4. the production of unmodified and glycosylated antibodies and purifying
the collection of substratum
With antibody expression vector transfection or add in the situation of GnTIII fusion expression vector cotransfection bhk cell with antibody expression vector, cultivate transfectional cell 96 h before harvest culture supernatants after transfection.According to the productivity of expection, several electroporation (10-15) is carried out to same vehicle.
With antibody expression vector transfection or add in the situation of GnTIII fusion expression vector cotransfection HEK293-EBNA cell with expression vector, within after transfection about 16 hours, replace substratum with fresh culture, then cultivate transfectional cell 120 h before harvest substratum afterwards further.
For stable BHK-1502-28-11 clone, 500,000 cell/ml inoculum culture, and collect supernatant liquor in cultivation after 4 days, cell density is 1.7 × 10 6have vitality cell/ml, cell viability is 69%.
antibody purification
Use two continuous print chromatographic step monoclonal antibody purification from culture supernatants.First step comprises protein A chromatography, uses pH gradient elution, has effectively been separated ox and human IgG.Subsequently for sample buffer is exchanged for phosphate buffered saline (PBS) (PBS) by cation-exchange chromatography step.
5. Oligosaccharide Analysis
By PNGaseF digestion from abzyme solution release oligosaccharides, antibody to be fixed on pvdf membrane or in the solution.
The digestion solution containing release oligosaccharides obtained directly obtains uses EndoH glycosidase digestions for MALDI/TOF-MS analysis or taking a step forward of preparation MALDI/TOF-MS analytic sample.
the Oligosaccharide release method of pvdf membrane sessile antibody
With PVDF (ImmobilonP, Millipore, Bedford, Massachusetts) the Kong Zhongyong 100 μ l methyl alcohol of 96 hole flat boards that obtains of film is moistening, and vacuum is 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 membrane three times.Then 50 μ lRCM damping fluid (8M urea, 360mMTris, 3.2mMEDTA, pH8.6) washing holes are used.30-40 μ g antibody is loaded in the hole containing 10 μ lRCM damping fluids.By using the liquid in vacuum pumping hole to make it by film, using 50 μ lRCM buffer solution films twice subsequently, hatching 1 hour to carry out the reduction of disulfide bridge bond at 37 DEG C by the 0.1M dithiothreitol (DTT) that adds in 50 μ lRCM.
After reduction, use vacuum from hole, remove dithiothreitol (DTT) solution.With 300 μ l water, hole is washed three times, then in room temperature dark, hatch 30 minutes carry out cysteine residues carboxymethylation by the 0.1M acetic acid iodine that adds in 50 μ lRCM.
After carboxymethylation, use vacuum pumping hole, use 300 μ l water washing three times subsequently.Then by 1% aqueous solution of 100 μ l polyvinylpyrrolidones 360 is carried out closed pvdf membrane for 1 hour to prevent the absorption of endoglycosidase in incubated at room.Then remove encapsulant by light vacuum, then use 300 μ l water washing three times.
By adding 2.5mU peptide-N-glycosylase F (restructuring N-glycanase, GLYKO, Novato, and 0.1mU sialidase (GLYKO CA), Novato, CA) discharge N connection oligosaccharides and remove any possible charged monosaccharide residue, enzyme is at the 20mMNaHCO of the pH7.0 of 25 μ l final volume 3in, 37 DEG C of digestion 3 hours.
the Oligosaccharide release method of antibody in solution
By 2.5mUPNGaseF (Glyko, the U.S.A.) mixing in the 2mMTris of the antibody of 40-50 μ g and 25 microliter final volume pH7.0, mixture is hatched 3 hours at 37 DEG C.
the PNGaseF of endoglycosidase digestion discharges oligosaccharides and heterozygosis bisected oligosaccharides structure is distributed to the purposes of MALDI/TOF-MS neutral oligosaccharides peak value
Endoglycosidase H (EC3.2.1.96) is used to digest the oligosaccharides of PNGaseF release subsequently.In order to EndoH digestion, 15mUEndoH (Roche, Switzerland) is added the final volume that PNGaseF Digestive system (antibody in aforesaid method solution) obtains 30 microlitres, at 37 DEG C, mixture is hatched 3 hours.EndoH cracking N-connects the NAG residue of the chitobiose core of oligosaccharides.Enzyme only can digest MOS and most of hybrid type glycans, and complex type oligosaccharides is not hydrolyzed.
the sample preparation of MALDI/TOF-MS
After acetic acid is added into final concentration 150mM, enzymic digestion liquid containing release oligosaccharides is hatched 3 hours further in room temperature, subsequently through loading micro--biology-rotary chromatography post (BioRad, Switzerland) 0.6ml Zeo-karb (the AG50W-X8 resin in, hydrogen form, 100-200 mesh, BioRad, Switzerland) come decationize and protein.The sample obtained by 1 microlitre uses to stainless steel target plate, with 1 μ lsDHB matrix mixing on flat board.By obtained sDHB matrix in 1: 1 (V/V) ethanol/10mM sodium chloride aqueous solution of 2mg2,5-resorcylic acid and 0.1mg5-methoxysalicylic acid being dissolved in 1ml.Sample air is dry, use 0.2 μ l ethanol, finally make sample recrystallize under air.
MALDI/TOF-MS
Being used for obtaining mass spectrographic MALDI-TOF mass spectrograph is VoyagerElite (PerspectiveBiosystems).Operating equipment in linear configurations, postpones with 20kV acceleration and 80ns.The external calibration of oligosaccharides standard is used to distribute for the mass spectrum of ion.Summation obtains final mass spectrum from the mass spectrum of 200 Laser emission.
prepared by 6.PBMC
Use Histopaque-1077 (SigmaDiagnosticsInc., St.Louis, MO63178USA) and substantially obtain peripheral blood lymphocytes (PBMC) according to the guidance system of manufacturers.Tout court, take the venous samples can of volunteer with heparinized syringe, volunteer requires to run 1 minute, to improve the per-cent of the natural killer cell (NK) in blood with all strength.With the PBS not containing Ca or Mg by blood thinning to 1: 0.75-1.3 is also laid on Histopaque-1077.Room temperature (RT) 400 × g free of discontinuities gradient centrifugation 30 minutes.Collect the mesophase spherule containing PBMC and wash (the every 50ml cells from two gradients) with PBS and pass through within centrifugal 10 minutes, to collect at RT300 × g.With PBS by after pellet resuspended, PBMC is counted and passes through within centrifugal 10 minutes, to wash second time at RT200 × g.Then Cell resuspension is used in suitable substratum program subsequently.
7.NK cellular segregation
Be separated NK cells of human beings from PBMC, use negative selection methods, use not in conjunction with the magnetic bead (MACS system, from MiltenyiBiotecGmbH, 51429BergischGladbach, GER) of CD16 and CD56 positive cell.In ice-cold MACS damping fluid, (PBS containing 2%FCS and 2mMEDTA) by PBMC washing once, hatches 10 minutes at 4 DEG C with 1: the 1FCS of the every ml of 20Mio cell and MACS buffer solution mixture resuspension.Then cell precipitation is also used the MACS damping fluid resuspension containing 10%FCS, every 1,000 ten thousand cell 80 μ l.Then every 1,000 ten thousand cells add 20 μ l haptens-antibody-solutions.With repetition vortex pipe, cell is hatched 10 minutes at 4 DEG C.After washing twice with the MACS of at least 10 × mark volume, by Cell resuspension in containing in the MACS damping fluid of 10%, every 80 μ l10 1,000,000 cells, and every 1,000 ten thousand cells add 20 μ l antihapten-microballons.With repetition vortex pipe, pipe is hatched 15 minutes at 4 DEG C.With MACS damping fluid by cell washing once after, by Cell resuspension, up to 10,000 ten thousand cells in 500 μ lMACS damping fluids, and be loaded on the LSMACS post that is positioned in MINI-MACS magnet, balance with 3mlMACS damping fluid.With 3 × 3mlMACS buffer solution pillar.Collect the cell flow through in fraction and be also used as NK cell subsequently.Expressing by CD56 the purity measured is 88-95%.
8.ADCC tests
Obtained PBMC or NK being used as effector cell as mentioned above.Be 25: 1 and 10: 1 for the respective cytological effect thing of PBMC and NK to the ratio of target.The effector cell of obtained suitable concn in AIM-V substratum, to add 50 μ l in each hole of 96 hole circle base plates.The target cell of C2B8 antibody is grown on SKW6.4 or the NamalwaB lymphocyte containing in the DMEM of 10%FCS.In PBS, wash target cell, count and be resuspended to AIM-V, every ml0.3 1,000,000, to add 30000 cells in each micropore of 100 μ l.By antibody dilution in AIM-V, add in 50 μ l pre-bed board target cell and RT combining target 10 minutes.Then effector cell is added and by flat board containing 5%CO 2moistening air in 37 DEG C hatch 4 hours.Citotoxicity detection kit (RocheDiagnostics, Rotkreuz, Switzerland) is used to test killing of target cell by measuring serum lactic dehydrogenase (LDH) release destroying cell.After flat board hatches 4 hours, centrifugal at 800 × g, the 100 μ l supernatant liquors in each hole are transferred on 96 new hole clear flat bottom flat boards.100 μ l color substrate buffer in test kit are added in each hole.The Vmax value at least 10 minutes of color reaction is measured, use SOFTmaxPRO software (MolecularDevices, Sunnyvale, CA94089, USA) at ELISA reader 490nm.From only there is no to measure spontaneous LDH the hole of antibody discharge containing target cell and effector cell.Maximum release is measured from the hole only containing target cell and 1%TritonX-100.The per-cent killed of following calculating specific antibodies mediation: (x-SR)/(MR-SR) * 100, average Vmax, the MR of wherein x to be average Vmax, the SR of specific antibodies concentration be spontaneous release are the average Vmax of maximum release.
Fc γ RIIIA on 9.NK cell combines
At the NK cell centrifugation 5 minute of 200 × g by fresh separated, with 0.09% (wt/vol) lactic acid solution (140mMNaCl, 5mMKCl, pH3.9) in room temperature by 3 × 10 5cell/ml hatches the pre-treatment of 5 minutes to remove the relevant IgG of NK cell.(DeHaasM.，J.Immunol.156：2948(1996))。
With PBS, 0.1%BSA, 0.01% sodiumazide by cell washing twice, and by concentration adjustment to PBS, 0.15BSA, 0.01% sodiumazide 2 × 10 6individual cell/ml.With 0,0.1,0.3,1,3,10 μ g/ml antibody variants by 5 × 10 5cell hatches 30 minutes at 4 DEG C.Then by cell washing twice, and the F (ab ') by puting together with the fluorescein isothiocyanate of 1: 200 2goat anti human IgG (JacksonImmunoReasearch, WestGrove, PA) and anti-human CD56-PE is with 5 μ l/5 × 10 5cell (BDPharmingen, sanDiego, CA) hatches 30 minutes to detect antibodies at 4 DEG C.(ShieldsR. etc., J.Biol.Chem.277 (30): 26733-26740 (2002)).
In the upper fluorescence intensity relating to binding antibody variant detecting CD56+ cell of FACSCalibur (BDBioscience, SanJose, CA).
The combination of Fc γ RIIb on 10.Raji lymphocyte
In PBS, RajiB cell human lymphocyte is washed 20 minutes (concentration is 0.3Mio cell/ml) at 37 DEG C.Then by Cell resuspension in 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 to be added in Raji cell and to hatch 30 minutes (whole cell concn is 200 ten thousand cells/ml) at 4 DEG C.After twice washing, the F (ab ') that the fluorescein isothiocyanate of 1: 200 is puted together 2goat anti human IgG (JacksonImmunoReasearch, WestGrove, PA) to add in cell and hatches 30 minutes at 4 DEG C.After washing once, by Cell resuspension in 0.5mlPBS, 0.1%BSA, 0.01%NaN 3in, and (BDBioscience, SanJose, CA) measures the fluorescence intensity relating to binding antibody variant of viable cell on FACSCalibur.
11. CDC tests
Target cell is counted, with PBS washing, is resuspended in AIM-V (Invitrogen), every ml1 1,000,000 cell.By 50 μ l cell seedings in each hole of 96 hole flat bottom plate.50 μ l also add in cell by obtained antibody diluent in AIM-V.Make antibody at room temperature and Cell binding 10 minutes.Complement (Quidel) is fresh to thaw, and dilutes 3 times and added in hand-hole by 50 μ l with AIM-V.Obtained rabbit complement (CedarlaneLaboratories) as described in manufacturers, dilutes 3 times with AIM-V and is added in hand-hole by 50 μ l.In contrast, before adding test, Complement source is heated 30 minutes at 56 DEG C.
Test panel is hatched 2 hours at 37 DEG C.Killing of cell is measured by measuring LDH release.Tout court, by flat board at 300 × g centrifugal 3 minutes.Every hole 50 μ l supernatant liquor to be transferred in 96 new hole flat boards and to add the test reagent of 50 μ l from cytotoxic reagent box (Roche).The corresponding Vmax of LDH concentration in supernatant liquor is measured with the kinetic measurement of ELISA reader.Maximum release is measured by incubated cell under 1%TritonX-100 existence.
Results and discussions
By with expressing the carrier of antibody gene and expressing the anti-CD20 that coding produces glycosyl engineered forms with the culture of the carrier cotransfection mammalian cell of each peptide species of GnTIII activity and be fitted together to IgG1 antibody (C2B8 chimeric antibody, also referred to as rituximab).The same antibody of unmodified form (non-sugar based through engineering approaches) is produced by the mammalian cell of the carrier transfection only expressed with antibody gene.The cell of transfection at least keeps three days, by the recombinant antibodies that a-protein affinity chromatography is purifying secreted from substratum in culture.The genetic expression that coding has a polypeptide of GnTIII activity produces have no significant effect relative to not producing the cell by cell vitality of such antibody, Growth of Cells and antibody.
Then the glycosylation pattern of antibody purification is analyzed.These antibody carry the N-be only connected with the Asn297 residue in human IgG1 Fc district and connect oligosaccharides.Analyzed by MALDI/TOF-MS subsequently from abzyme solution removing oligosaccharides by PNGaseF digestion.Use this technology, the part of different oligosaccharide species in total original Fc oligosaccharides colony can be detected, structure can also be distributed to different peak values (Umana, P. etc., NatureBiotechnol.17:176-180 (1999)) in mass spectrum.
Fig. 1 shows the neutral oligosaccharides MALDI/TOF-MS characteristic pattern that the anti-CD20 of the recombinant C 2B8 produced in bhk cell is fitted together to IgG1 antibody.With these cells of antibody expression vector pETR1502 transfection.Fig. 2 to 4 shows the individual features figure of the same antibody produced with the bhk cell that coding has the engineered nucleic acid of the polypeptide of GnTIII activity.Characteristic pattern in Fig. 2 is formed from the wild-type GnTIII nucleic acid that carrier pETR1166 expresses by using coding.Characteristic pattern in Fig. 3 is formed by the nucleic acid using coding to be included in the fusion polypeptide of the GnTI localization domain that N-holds and GnTIIIC-holds catalyst structure domain to merge.This fusion gene is expressed from carrier pETR1425.Characteristic pattern in Fig. 4 is formed by the nucleic acid using nucleic acid encoding to be included in the fusion polypeptide of the localization domain of golgi body α mannosidase II (ManII) that N holds and GnTIII catalyst structure domain merges.This fusion gene is expressed from carrier pETR1506.
Unmodified antibody has typical oligosaccharides form (Fig. 1), m/z ratio be 1485,1648 with 1810 peak value respectively with 0,1 with two feelers of 2 galactose residues, core fucosylation, composite oligosaccharide is consistent.Similar characteristic formp (Lifely is found in the Fc district oligosaccharides of the non-through engineering approaches IgG1 antibody produced as CHO and murine myeloma cell at other standards Mammals industrial cell lines, M.R. etc., Glycobiology5:813-822 (1995)).By the expression of wild-type GnTIII the celliferous through engineering approaches of antagonist mainly formed bisection, core fucosylation, Composite Double antennary oligosaccharide (Fig. 2), are bisection counterparts of the non-bisection be found in unmodified antibody, fucosylated oligosaccharide peak value at the peak value of m/z ratio 1689,1851 and 2014.By expressing the nucleic acid of coding GnTI-GnTIII fusion polypeptide, the celliferous through engineering approaches of antagonist also mainly forms bisection Composite Double antennary oligosaccharide (noting the peak value of m/z1689 and 1851 in Fig. 3), and wherein GnTIII catalyst structure domain is located by GnTI Golgi localization domain.But, relative to wild-type GnTIII, use GnTI-GnTIII to merge to cause halve, non-fucosylation and bisection, hybrid structure raising (m/z1664 between comparison diagram 2 and Fig. 3,1810, the peak value of 1826 and 1973 is relative to overall per-cent).For the oligosaccharides material that GnTI-modifies, halve, non-fucosylation and halve, the synthesis of hybrid structure by recombinate GnTIII catalyst structure domain and following between competition formed, (i) endogenous core α 1,6-fucosyltransferase, (ii) golgi body alpha-Mannosidase II (ManII) and (iii) GnTII, due to once modify oligosaccharides with the bisection GlcNAc that added by GnTIII catalyzed reaction, these three other enzymes just no longer act on modification bisected oligosaccharides.Because GnTII effect N-connects the ManII downstream in oligosaccharides biosynthetic pathway, the ManII blocking effect therefore by adding bisection GlcNAc has also effectively blocked GnTII.The peak value of m/z1664 and 1826 is non-fucosylations, and the peak value of m/z1810 and 1973 is fucosylations.The EndoH glycosidase digestions of heterozygosis and composite oligosaccharide (Fig. 8 A) can be offered an explanation, be used for confirming that the raising of these peak values is raisings (vide infra) of bisection because Fc connects, non-fucosylation and bisection, hybrid oligosaccharides ratio.
Contrast is formed with at the active coding nucleic acid of this other GnTIII used, the through engineering approaches forming cell by the expression antagonist of coding ManII-GnTIII fusion polypeptide (SEQIDNO:14) nucleic acid mainly forms bisections, non-fucosylation and bisection, hybrid structure (m/z1664 in attention Fig. 4,1810, the peak value of 1826 and 1973), and wherein GnTIII catalyst structure domain is located by ManII Golgi localization domain.Therefore, merge (SEQIDNO:12 and 13) relative to wild-type GnTIII and GnTI-GnTIII, ManII-GnTIII to merge in the synthesis of the bisection connected at Fc, non-fucosylation and bisection, hybrid oligosaccharides more effectively (peak value of comparison diagram 2, m/z1664 and 1810 between 3 and 4 is relative to overall per-cent).The bisection formed by the expression of nucleic acid that encoding wild type GnTIII, GnTI-GnTIII merge and ManII-GnTIII merges, non-fucosylation Fc oligosaccharide ratio are 4,13 and 33% separately.Bisected oligosaccharides is not detected in unmodified (non-through engineering approaches) antibody.
The raising that ManII-GnTIII fusion constructs is expressed in antibody produced cell causes the further raising of bisection, non-fucosylated oligosaccharide ratio.This is by being proven at the ManII-GnTIII construct from carrier (pETR1519) of transfection HEK293-EBNA cells with the OriP for free replicon.This expression system known causes high-caliber expression, also for the expression from carrier pETR1520 antibody gene.In this system, the purifying of high level expression, the oligosaccharides characteristic formp of unmodified (non-sugar based through engineering approaches) antibody are shown in Fig. 5, its again show there is 0,1 and 2 galactose residue non-bisection, fucosylation peak value typical oligosaccharides characteristic formp (such as, comparison diagram 1 and 5, that be presented at the unmodified antibody of expressing in bhk cell or that higher level is expressed in HEK293-EBNA cell similar oligosaccharides characteristic formp).Carry out engineered antibody with the nucleic acid that the ManII-GnTIII that the higher level within the system of encoding is expressed merges and produce the generation that cell causes antibody, wherein most of Fc oligosaccharides be halve, non-fucosylation (see Fig. 6, wherein halve, the m/z1664 of non-fucosylation hybrid oligosaccharides form together with 1826 peak values exceed 90% of total oligosaccharides).
As mentioned above, endoglycosidase H (EndoH) is used for confirming the distribution at the different oligosaccharides peaks that bisected, nonfucosylated structure and bisection, hybrid structure are observed in MALDI characteristic pattern.PNGaseF-and PNGaseF+EndoH-digestion is derived from the MALDI/TOF-MS neutral oligosaccharides characteristic pattern that anti-CD20 is fitted together to the glycan of IgG1 antibody and is shown in Fig. 7, produces antibody by the HEK293 cell of GnTIII (M2) overexpression glycosyl through engineering approaches.The peak value of m/z1664 can distribute to two different glycan, and namely the compound of the binary or non-fucosylation of the heterozygosis of non-fucosylation is non-binary.It is due to identical monose composition (Fig. 8 B) that different structures shows identical m/z ratio.
The glycan digesting PNGaseF release with endoglycosidase H creates new structure, and main peak value is converted to 1460 (Fig. 7 B) from m/z1664.Difference corresponds to the quality of GlcNAc residue.As mentioned above, EndoH can not divide complex type oligosaccharides.Therefore, after endoglycosidase H digests, the main peak value of m/z1664 can distribute to the second-class somatotype of non-fucosylation heterozygosis.
Compound or heterozygosis bisection glycan can be distributed in other peak.After EndoH digestion, the peak of m/z1810 disappears, and therefore structure can distribute to the second-class somatotype of heterozygosis of fucosylation.From a GlcNAc residue of m/z1810 peak value and Fucose (from core α-1,6 fucosylation, reduction end GlcNAc residue) minimizing of residue creates the structure of m/z1460.The peak being digested (elimination of GlcNAc residue) m/z1502 by EndoH is disappeared and has occurred the peak of m/z1298, demonstrates 1502 peaks and can distribute to the second-class somatotype of non-fucosylation heterozygosis.After EndoH digestion, the disappearance at m/z1647 peak demonstrates the heterozygosis bisection structure that this peak is fucosylation.The removal of a GlcNAc and Fucose creates the structure of m/z1298.The peak of m/z1826, the second-class somatotype of heterozygosis of non-fucosylation, is digested by EndoH.This generates the structure of m/z1622.After EndoH digestion, high mannose type (1257m/z) can be distributed in the peak of m/z1053, is digested by EndoH.As expected, the peak of m/z1689 (compound bisection) is not subject to the impact of EndoH digestion.In synthesis, from the data that table 1 obtains, we infer that the oligosaccharide structure of 88% is with bisection GlcNAc, and wherein 60% is the heterozygosis bisection structure of non-fucosylation, and 22% is that fucosylation heterozygosis is binary and 6% be the compound bisected oligosaccharides structure of fucosylation.
Table 1. oligosaccharides distributes
m/z Possible structure Relative % before EndoH The m/z expected after EndoH The m/z observed after EndoH Relative % after 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 is 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 is 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 is binary (49%)
1689 The compound of fucosylation is binary 3 1689 1689 5 The compound of fucosylation is binary (3%)
1810 The compound of the binary or fucosylation of the heterozygosis of fucosylation 15 1460 or 1810 1460 1810 60 2 (2%) of the heterozygosis binary (13%) of fucosylation and the compound of fucosylation
1826 The heterozygosis of non-fucosylation is binary 4 1622 1622 7 The heterozygosis of non-fucosylation is binary (4%)
1851 Fucosylation binary 3 1851 1851 2 The compound of fucosylation is binary (3%)
1972 The heterozygosis of fucosylation is binary 3 1622 1622 7 The heterozygosis of fucosylation is binary (3%)
Mass balance (with molar fraction %):
A) peak of m/z1502 and 1647: 7+7%=14% (expection).The digestion of EndoH to two peaks creates m/z1298 (obtaining 13% after EndoH)
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
With bisection GlcNAc:88%
The heterozygosis of non-fucosylation is binary: 60%
The heterozygosis of fucosylation is binary: 22%
The compound of fucosylation is binary: 6%
Above-mentioned data (Fig. 1 to 6) show GnTIII and express and be used for the level of the certain position structural domain two of GnTIII catalyst structure domain target golgi body, have impact on restructuring GnTIII catalyst structure domain and endogenous core α 1,6-fucosyltransferase, between (ManII) and GnTII enzyme to the competition of the oligosaccharide substrates that GnTI modifies.In this competition, the comparatively high expression level of GnTIII is conducive to this, cause halving, hybrid oligosaccharides and bisection, the high level of non-fucosylated oligosaccharide and adjoint bisection composite oligosaccharide and bisection, fucosylated oligosaccharide content reduce.This is for also noticing before wild-type GnTIII (Umana, P. etc., NatureBiotechnol.17:176-180 (1999)).But, although form the bisected oligosaccharides of similar aggregate level, relative to wild-type GnTIII, the oligosaccharide substrates that GnTI is modified and endogenous core α 1,6-fucosyltransferase, ManII and GnTII enzyme, by GnTI or locate GnTIII catalyst structure domain by ManII localization domain and will cause more effective competition.
Compare with wild-type GnTIII, GnTI-GnTIII merges for halving, hybrid oligosaccharides and bisection, the synthesis of non-fucosylated oligosaccharide more efficient power, can explaining along distributing to the golgi body in the other direction by transmitting relative to the glycoprotein material of GnTIII at GnTI early.((Rabouille, C. etc., J.CellSci.108:1617-27 (1995)) is measured by quantitative immunological electron microscopy before the meticulous golgi body distribution of GnTI and ManII.Two enzymes are divided into cloth along golgi body, be mainly positioned at inside and the blister cavities (cisternae) through golgi body heap (stack), and relative to through blister cavities, the content in inner blister cavities is higher.Also do not measure the accurate quantification spatial distribution of core α 1,6-fucosyltransferase, GnTII and wild-type GnTIII.But, above-mentionedly be not interpreted as what is halved for synthesis, hybrid oligosaccharides and bisection, non-fucosylated oligosaccharide, ManII-GnTIII fusion ratio GnTI-GnTIII merges more effective, because GnTI and ManII has identical spatial distribution along Golgi compartments (subcompartment).
The more efficient power that ManII-GnTIII merges indicates in inner-and the existence of functional sugar glycosylation reaction compartment (subcompartment) through the relative organization in-golgi body blister cavities physics compartment (subcompartment).Believe so-called " inside-golgi body glycosylase ", GnTI, GnTII and ManII are present in golgi body as high molecular complex body.But if localization domain makes these enzymes form a part for these complex bodys, this merges for GnTI-GnTIII and ManII-GnTIII fusion is identical.Any significance degree that the expression that restructuring GnTI-GnTIII merges does not cause endogenous wild type GnTI enzyme replacement paired Fc-oligosaccharides to synthesize, due to the modification causing most oligosaccharides to obtain GnTI and GnTIII two reaction at these all GnTIII constructs used.
Our data show, due to ManII localization domain, between the catalyst structure domain that endogenous GnTI and restructuring ManII-GnTIII merges, there occurs the pairing of accurate function.The systematism pairing of the enzyme of catalysis following reaction in biosynthetic pathway, with preference, the product that first is reacted is transferred to the mode of second catalytic site spread away from enzyme relative to this product, be known occur in another biosynthetic pathway as glycolysis-and polyketone biosynthesizing.Reported that GnTI and ManII formed " kin oligopolymer ", relocated when the endoplasmic reticulum (Nilsson, T. etc., EMBOJ.13 (3) .562-74 (1994)) in these enzymes of major general.Find a pair charged amino acid residue in each main region of these two enzymes identifies it is critical for this kin.Contrary in GnTI in the electric charge of residue and ManII.We have identified the Similar residues that this part N-connects the main region equivalent position of other golgi body glycosylase related in oligosaccharides biosynthetic pathway, i.e. core α 1,6-fucosyltransferase (identical with the electric charge of GnTI, instead of as be complementary electric charge in ManII situation), ManI and GnTII.We also identify these residues is conservative between species.Although proposed these residues for be integrated into enzyme formed polymer composite in or even for the dispensable (Opat of Golgi localization, A.S. etc., J.Biol.Chem.275 (16): 11836-45 (2000)), possible they relate to the perfect match of catalyst structure domain in oligosaccharides biosynthetic process.Such pairing needs not be irreversible, but can be mediated by instantaneous, the dynamic (dynamical) interaction between enzyme.Other pairing determinant may be there is in the other places in main region or catalysis region.But, will lose from the right any contribution of the specific GnTI-ManII of ManII catalyst structure domain in the restructuring with GnTIII catalyst structure domain is merged.
Fig. 9 to 11 demonstrates the raising of antibody dependent cellular cytotoxicity (ADCC) that the nucleic acid overexpression that has the polypeptide of GnTIII activity by encoding in antibody produced cell causes, and polypeptide is positioned golgi body by different localization domain.There is the active and expression being positioned the recombinant polypeptide of golgi body by GnTI Golgi localization domain the is caused ADCC of GnTIII improve and be shown in Fig. 9.The oligosaccharides characteristic formp of the control antibodies in testing for Fig. 9 ADCC is shown in Fig. 1.The oligosaccharides characteristic formp of the glycosyl engineered antibody in testing for Fig. 9 ADCC is shown in Fig. 3.There is GnTIII activity and improved by the ADCC that the expression that Glycosylase ManII Golgi localization domain is positioned the recombinant polypeptide of golgi body causes and be shown in Figure 10.The oligosaccharides characteristic formp of the control antibodies in testing for Figure 10 ADCC is shown in Fig. 5.The oligosaccharides characteristic formp of the glycosyl engineered antibody in testing for Figure 10 ADCC is shown in Fig. 6.
Figure 11 shows has that GnTIII is active and the expression being positioned the recombinant polypeptide of golgi body by ManII Golgi localization domain causes ADCC activity to improve, relative to the wild-type GnTIII polypeptide using self GnTIII gorky localization domain.Wild-type GnTIII expresses and oligosaccharides characteristic pattern for the glycosyl engineered antibody in Figure 11 ADCC test is shown in Fig. 2.There is GnTIII activity, be positioned by ManII Golgi localization domain that the fusion polypeptide of golgi body is expressed and be shown in Fig. 4 for the oligosaccharides characteristic pattern of glycosyl through engineering approaches during Figure 11 ADCC tests.These data also show relative to compound, fucosylation, the antibody of non-bisected oligosaccharides, the ADCC that the antibody comprising bisection hybrid oligosaccharides and bisected, nonfucosylated oligosaccharides with bisected oligosaccharides has raising is active.It should be noted that all bisected oligosaccharides of the higher antibody of activity in testing for Figure 10 ADCC are the hybrid oligosaccharides of bisection, non-fucosylation.As described above, use that to have GnTIII active and cause more effectively synthesizing the bisected oligosaccharides of non-fucosylation by the fusion polypeptide that ManII Golgi localization domain is positioned golgi body, and Figure 11 shows the antibody relative to these oligosaccharides with lower level, the antibody with these bisected, nonfucosylated oligosaccharides of improving the standard more has activity in ADCC.ADCC activity improve and Fc Related Oligosaccharides colony in this bisection, non-fucosylated oligosaccharide part increase be associated, when this part is higher than 15-20%, great raising can be found out.
Known natural killer (NK) cell is the important amboceptor of ADCC.These cells carry on its surface and activate Fc γ receptor II IA, also referred to as CD16a.On the Fc district of target cell binding antibody and NK cell, the combination of Fc γ RIIIA acceptor is required for the induction that is crosslinked and ADCC subsequently of these acceptors on NK cell.Therefore, evaluate antibody that said method produces and the combination of Fc acceptor is important, especially people's immune effector cell illustrates the acceptor in their natural form.The affinity activating Fc acceptor Fc γ RIIIA with people that Figure 12 demonstrates that glycosyl engineered antibody that the expression of nucleic acid that has the fusion polypeptide of GnTIII activity by coding in antibody produced cell produces has a raising combines.As above for ADCC test, these antibody have the bisection improving content, non-fucosylated oligosaccharide, and oligosaccharides is produced by the expression of fusion polypeptide in antibody produced cell with GnTIII activity.NK cell used in this test carrys out the genotype donor (Metes, D. etc., J.Immunol.Methods258 (1-2): 85-95 (2001)) of its NK cell comfortable not being expressed Fc γ RIIc acceptor.Therefore, the Fc acceptor on these cell surfaces is only had to be activate Fc γ RIIIA acceptor.Figure 13 shows binding tests and measures the specific binding affinity with this receptor.This is by showing with the competition of Fc γ RIII-specific inhibition antibody fragment (3G8Fab2-fragment).
The Fc-FcR improved interacts on the strong evidence of anti-tumour antibody treatment result impact from effect and the dependency between isozygotying compared with high-affinity Fc γ RIIIA recipient genotypes, be found in the Lymphoma ((Cartron accepting rituximab, G. etc., Blood99 (3): 754-8 (2002)).This finds the single parameter relevant with the molecules in response ratio of raising with the target responsivity greatly improved.The effect improved due to the Fc γ RIIIA-Fc acceptor interaction improved is derived from the function being comprised the realization of natural killer (NK) cell, scavenger cell, monocyte and dendritic cell by various immunocyte.NK cell, scavenger cell and monocyte activate crosslinked tumor cell lysis (believing it is FcR dependency kill mechanism main in the body widely) (Maloney that can result through ADCC of Fc γ RIIIA acceptor, D.G. etc., Semin.Oncol.29 (1Suppl.2): 2-9 (2002), AmigorenaS., 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, and Heard M.E., C.CritRevOncolHematol.40 (1): 25-35 (2001)), and cause at the contiguous release cells factor of tumour cell (Carson, W.E. etc., Eur.J.Immunol.31:3016-3025 (2001)).These cytokines cause the direct cytotoxic effect on tumour cell subsequently, with cause blood vessel formation against function, it by depriving oxygen and the growth of nutrition Tumor suppression, and causes the tumour antigen submission of raising, as a part for the immunne response to 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 (such as, from in the body of antibodies by dying tumour cell that ADCC attacks at first) dendritic cell maturation that improves can be caused, antigen uptake and the submission to T cell, and the crossed sensitization of cytotoxic T cell, the latter is very effective potential mechanism (AmigorenaS. to activation antineoplastic immune, J.Exp.Med.195 (1): F1-3 (2002), Kalergis, and Ravetch A.M., J.V.J.Exp.Med.195 (12): 1653-1659 (2002), Selenko, N. etc., J.Clin.Immunol.22 (3): 124-130 (2002)).By the crosslinked raising that also results in target cell and directly kill of the antibodies target cell of Fc acceptor on immune effector cell, such as apoptosis (the Reff of induction by the crosslinked of antibody-mediated target antigen molecule, and Heard M.E., C.CritRevOncolHematol.40 (1): 25-35 (2001), Cragg, M.S. etc., Blood101 (3): 1045-1052 (2003).In all these immune effector cells, only have NK cell only to have in its surface and activate Fc γ R.In other cell type, activate Fc γ RIII and exist together with inhibition Fc γ RIIb, the induction of anti-tumour effect subfunction is balanced by the forward exceeding the activation suppressing signal and causes.
The Fc receptors bind that Figure 15 shows raising compare with inhibition Fc γ RIIb be activated receptor optionally.As explained above, the effector function that this selectivity realizes for the immunocyte beyond NK cell is important.In addition, what the combination that obtains by using method glycosyl through engineering approaches Fc antibody regions described herein improved the standard that the accepts unmodified antibody observed than those opinion of natures isozygotys compared with much higher (Figure 16) of high-affinity Fc γ RIIIA genotype patient/donor, and this combination improves the effect relevant (Cartron, G. etc. Blood99 (3): 754-8 (2002)) improved with anticancrin.
Activate the binding domains of Fc γ RIIIB acceptor and the almost identical of Fc γ RIIIA.Therefore, above-mentioned data also demonstrate glycosyl engineered antibody described herein can result through the effector cell showing Fc γ RIIIB, as polymorphic nucleus (PMN) cell, the raising of the effector function of medium, comprise release and the phagolysis ((Reff of toxic product, and Heard M.E., C.CritRevOncolHematol.40 (1): 25-35 (2001), Daeron, FM.Annu.Rev.Immunol.15:203-34 (1997), Ravetch, J.V. and BollandS.Annu.Rev.Immunol.19:275-90 (2001)).
Figure 18 shows the oligosaccharides characteristic formp of anti-CD20 antibodies that bhk cell produces, and bhk cell is grown in suspension and through engineering approaches is carried out composing type high level expression recombinant antibodies and had the fusion polypeptide of GnTIII activity.For the antibody from fusion GnTIII through engineering approaches cell, this oligosaccharides characteristic formp shows the increase (also see table 2) of bisected, nonfucosylated oligosaccharides and bisection hybrid oligosaccharides level.These structures (see Fig. 1) are not found in the non-sugar based engineered antibody produced by non-sugar based through engineering approaches bhk cell.Express the through engineering approaches cell that GnTIII merges and present normal growth in suspension and good antibody production.
The relative percentage of the oligosaccharides of the glycosyl through engineering approaches monoclonal antibody produced by stable BHK-1502-28-11 clone is listed in table 2.
Table 2: the relative percentage at the peak obtained 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 is binary: 17% (6+7+10+12)
Heterozygosis is binary: 71.6% (4+5+8+9+11)
The structure that Oligosaccharide Analysis shows 88.6% carries bisection GlcNAc residue, 50.8% be fucosylation be non-fucosylation with 37.8%.The most of peaks demonstrating acquisition with the oligosaccharides of endoglycosidase H digestion PNGaseF release are second-class somatotypes of heterozygosis (Figure 19).Figure 20 shows the raising of activation Fc acceptor Fc γ RIIIA binding affinity on glycosyl engineered antibody and NK cells of human beings that BHK-1502-28-11 clone produces.The clone growing also composing type stably express antibody gene and fusion GnTIII polypeptide in suspension is desirable to scale operation treatment antibody.Use standard cell lines engineering method, can by introducing merging GnTIII gene the clone containing antibody gene, or by antibody gene being introduced the clone (" the production clone of pre-glycosyl through engineering approaches ") containing merging GnTIII gene, or by introducing antibody gene and GnTIII fusion gene realizes glycosyl through engineering approaches simultaneously.
Also have detected the complement mediated lysis (CML) of the anti-CD20 antibodies produced in through engineering approaches cell, this project cell is used for high level expression coding and has GnTIII activity and the nucleic acid being positioned the fusion polypeptide of golgi body by ManII localization domain, and CML is the different effect subfunction not relying on Fc acceptor on immune effector cell.Most oligosaccharides of this glycosyl engineered antibody are the hybrid nonfucosylated types that halves.Compare with unmodified antibody, observe the CML activity (Figure 21) that this anti-CD20 antibodies reduces.For some application, band is improved ADCC and is desirable with reducing the antibody of CML, such as, be reduced by the side effect of CML mediation, as the vasculitis in tumor locus blood vessel.Anti-CD20 antibodies treatment is observed to other remarkable side effect (vanderKolkL.E. etc., BrJHaematol.115 (4): 807-11 (2001)) of CML mediation.But above-mentioned oligosaccharides characteristic formp also show that engineered antibody produces that cell expresses GnTIII fusion polypeptide with intermediate expression level and the composite oligosaccharide that causes the hybrid nonfucosylated oligosaccharides of bisection (higher than 15%) of medium level in the Fc oligosaccharides colony of glycosyl engineered antibody instead of have a signal portion is possible.Such composite oligosaccharide, with normal, do not fall low-level CML and is correlated with.Therefore data sheet understands that can produce band is like this improved the antibody of ADCC, and the CML of its closely similar level that can keep comparing with non-engineered antibody is active.
By another chimeric IgG1 antibody C225 of method glycosylation identification Human epidermal growth factor receptor (EGFR) described herein, also referred to as cetuximab.Figure 22 shows the oligosaccharides feature of the same antibody of unmodified anti-egfr antibodies C225 and glycosyl engineered forms.The latter is had GnTIII activity at expression coding and is positioned by ManII localization domain to produce in the cell of the nucleic acid of the fusion polypeptide of golgi body.Figure 23 shows the ADCC of the anti-egfr antibodies increase formed by this glycosyl through engineering approaches.By method described herein produce and ADCC improves and the glycosyl engineered antibody that improves with the binding affinity activating Fc acceptor, Antybody therapy for cancer and autoimmune disorders is the molecule having distant view, owing to treating the effect that they cause improving for these, relative to the corresponding unmodified of these antibody (non-sugar based through engineering approaches) form.In addition, compare with the antibody of unmodified, the antibody for glycosyl through engineering approaches may reduce therapeutic dose, these favourable influence economy of antibody producing.
Embodiment 2
The immune-mediated thrombopenia for the treatment of chronic graft versus host Disease
Autoimmunity thrombopenia in chronic graft versus host disease represents the example of the B cell dysregulation causing clinical disease.In order to treat the immune-mediated thrombopenia of chronic graft versus host Disease, by prepared in accordance with the present invention and have and improve the anti-CD20 chimeric mAb of ADCC and deliver medicine to patient, as Ratanatharathorn, V. etc., (its entirety being incorporated herein by reference at this) described in Ann.Intern.Med.133 (4): 275-79 (2000).Particularly, infusion antibody weekly, 375mg/m 2, deliver medicine to patient 4 weeks.Antybody therapy creates B cell in peripheral blood significantly to be reduced and reduces the content of platelet-associated antibody.
Embodiment 3
Treat the complete and hemolytic anemia of serious, immune-mediated pure red blood cell development
Immune-mediated, acquired pure red blood cell development complete (PRCA) is the disease of seldom seeing, usually relevant to autoimmune phenomena.In order to treat patient immune-mediated, acquired pure red blood cell development is incomplete, by obtain according to the present invention and have and improve the anti-CD20 chimeric mAb of ADCC and deliver medicine to patient, as Zecca, M. etc., (its entirety being incorporated herein by reference at this) described in Blood12:3995-97 (1997).Particularly, the antibody of patient's two dosage of PRCA and autoimmune hemolytic anemia is given, 375mg/m 2, weekly.After Antybody therapy, start to use Intravenous immunoglobuin replacement therapy.This treatment creates the remarkable minimizing of B cell and achieves significantly improving of reticulocyte count with the hemoglobin level increased.
Embodiment 4
Materials and methods
1. build GalT fusion expression vector
for the carrier of constitutive expression GalT
In order to build GalT expression vector, from cDNA library (Clontech) by pcr amplification GalTcDNA.C-terminal c-myc-Epitope tag is added the upstream (aminoacid sequence: PEQKLISEEDL) of and then gene terminator, in order to measure GalT conveniently by western blotting later.After confirming the correct sequence of GalT, under gene being inserted in the control of MPSV promotor, and the rabbit beta Globulin polyadenylation signal of synthesis is added.Final GalT expression vector also containing the puromycin-resistant box separated for selecting, with the rabbit beta Globulin polyadenylation signal of the puromycin resistance gene also under MPSV promotor controls and synthesis.
with the amino acid replacement of encoding human GnTI localization domain coding GalT localization domain amino acid.
Carrying out the structure of this heterozygosis galactosyltransferase gene, such as, reacted by over-lap PCR, forming the plasmid of puromycin-resistant box merged containing the GnTI-GalT under controlling in MPSV promotor and for selecting.
with the amino acid replacement coding GalT location of encoding human mannosidase II localization domain the amino acid of structural domain.
Carry out the structure of GalT expression vector.The plasmid obtained contains the heterozygosis manII-GalT gene under MPSV promotor controls.
combination heterozygosis manII-GalT fusion gene and copying from EpsteinBarr virus starting point oriP.
As described in Example 1 the DNA fragmentation subclone with oriP is entered in above-mentioned heterozygosis ManII-GalT expression vector.
the cd4 cell surface markers gene of combination heterozygosis manII-GalT fusion gene and brachymemma
Modify expression vector and be used for the other expression of the cd4 cell surface markers gene of brachymemma.Speak briefly, heterozygosis manII-GalT track fusion box is converted to bicistronic expression cassettes from monocistron, by by below and then coding insert along the poliovirus IRES sequence of anti-human CD 4 protein matter cDNA the downstream (comprise the people CD4 leader sequence for secreting, after and then cross-film and ectodomain) that manII-GalT merges terminator.
3. with GalT fusion expression vector and antibody expression vector transfection mammalian cell
transfection bhk cell
Cultivation as described in example 1 above, to collect and the cell (vitality 90-95%) that exponentially grows of transfection subsequently.In order to produce glycosyl engineered antibody, with two plasmid co-transfection cells, one for antibody expression and another for merging GalT expression of polypeptides, respective ratio is 3: 1.
transfection HEK293-EBNA cell
The HEK293-EBNA cell that transfection as described in Example 1 exponentially grows.In order to produce glycosyl engineered antibody, with two plasmid co-transfection cells, one for antibody expression and another for merging GalT expression of polypeptides, respective ratio is 4: 1.Transfection is after 5 days, collects supernatant liquor, at 1200rpm centrifugal 5 minutes, is then held in 4 DEG C centrifugal 10 minutes of 4000rpm second time.
express the generation of the stable mammal cell line of restructuring anti-CD20 antibodies and GalT fusion
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 IRES dependent expression.Carrier is also containing puromycin resistance gene expression cassette.First puromycin-resistant clone is selected to obtain the clone of a set of chromosomal integration vector DNA.Then brachymemma CD4 (tCD4) surface expression of screening and cloning, it is as the mark of bicistronic mRNA ManII-GalT+tCD4 gene expression unit expression level.ELISA test is used to check the generation of selected clone's recombinant antibodies.
The screening carrying out transfection and tCD4 expression level subsequently as described in example 1 above.
4. the production of unmodified antibody and glycosyl engineered antibody and purifying
With antibody expression vector transfection or add in the situation of GalT fusion expression vector cotransfection bhk cell with antibody expression vector, after transfection by the cell cultures 96h h before harvest culture supernatants of transfection.According to the productivity of expection, several electroporation (10-15) is carried out to same vehicle.
With antibody expression vector transfection or add in the situation of GalT fusion expression vector cotransfection HEK293-EBNA cell with antibody expression vector, transfection replaced substratum with fresh culture after about 16 hours, the substratum added after then cultivating transfectional cell 120 h before harvest further.
For stable BHK-1502-28-11 clone, with 500,000 cell/ml inoculum culture also collects supernatant liquor in cultivation after 4 days.
antibody purification
Use two continuous chromatography steps monoclonal antibody purification from culture supernatants, protein A chromatography as described in example 1 above and cation-exchange chromatography.
5. Oligosaccharide Analysis
By PNGaseF digestion from abzyme solution release oligosaccharides, antibody to be fixed on pvdf membrane or in the solution.
The obtained Digestive system containing release oligosaccharides is directly analyzed for the preparation of MALDI/TOF-MS or uses EndoH glycosidase digestions taking a step forward of preparation MALDI/TOF-MS analytic sample.
As described in example 1 above, the Oligosaccharide release method of antibody in the Oligosaccharide release method of pvdf membrane sessile antibody and solution is carried out.
the PNGaseF that endoglycosidase H digests discharges oligosaccharides and is divided by heterozygosis glycosylation oligosaccharide structure the purposes at dispensing MALDI/TOF-MS neutral oligosaccharides peak
The endoglycosidase H (EC3.2.1.96) that uses subsequently as described in example 1 above digests the oligosaccharides of PNGase release.
MALDI/TOF-MS
Preparation as described in example 1 above contains the enzymolysis, digestion liquid sample of release oligosaccharides and carries out MALDI/TOF mass spectrum subsequently.
6. cell is prepared and is separated
Substantially Histopaque-1077 (SigmaDiagnosticsInc., St.Louis, MO63178USA) is used to prepare peripheral blood lymphocytes (PBMC) according to the method described in the guidance of manufacturers and embodiment 1.
Negative selection methods is as described in example 1 above used to isolate NK cells of human beings from PBMC.
8.ADCC tests
Prepare as above action effect cell PBMC or NK and as described in example 1 above in antibody dependent cellular cytotoxicity (ADCC) test, test the ability of their mediating cytotoxicity.
Fc γ RIIIA combination on 9.NK cell and the Fc γ RIIb on Raji lymphocyte combine
Fc γ RIIIA combination on the NK cell of mensuration fresh separated as described in example 1 above and the Fc γ RIIb on Raji lymphocyte combine.
10. CDC test
Antibody diluent is used to carry out CDC test according to the method described in embodiment 1.
Results and discussions
In order to prove the cell relative to not producing this polypeptide, coding has the genetic expression of the polypeptide of GalT activity to the impact of cell viability, Growth of Cells or antibody production, tests.
The glycosylation pattern being analyzed antibody purification by MALDI/TOF-MS as described in example 1 above.Use this technology, the ratio of the different oligosaccharide species in total original Fc oligosaccharides colony can be measured, structure can also be distributed to peaks (umana, P. etc., NatureBiotechnol.17:176-180 (1999)) different in mass spectrum.
Measure the characteristic formp of unmodified antibody oligosaccharides.Particularly, the through engineering approaches measuring the antibody produced cell of being expressed by wild-type GalT whether mainly formed galactosylation, core fucosylation, Composite Double antennary oligosaccharide.Whether the through engineering approaches also measuring the antibody produced cell of the expression of nucleic acid by coding GnTI-GalT fusion polypeptide (wherein GalT catalyst structure domain is located by GnTI Golgi localization domain) mainly forms the Composite Double antennary oligosaccharide of galactosylation relative to wild-type GalT.If galactosylation, non-fucosylated structures and galactosylation, hybrid structure is synthesis, these are competed by GalT and other glycosyltransferase or Glycosylase and are formed.Expection is once oligosaccharides modified by the semi-lactosi added by the reaction of GalT catalysis, and α 1,6-core fucosyltransferase, golgi body alpha-Mannosidase II (ManII) and GnTII no longer work the oligosaccharides modifying galactosylation.
Use the EndoH glycosidase digestions that can offer an explanation heterozygosis and composite oligosaccharide to the ratio of the estimate Fc galactosylation be connected, non-fucosylated oligosaccharide and galactosylation hybrid oligosaccharides.
Whether the through engineering approaches of carrying out testing the antibody produced cell of the expression of nucleic acid measured by coding ManII-GalT fusion polypeptide (wherein GalT catalyst structure domain is located by ManII Golgi localization domain) mainly forms galactosylation, non-fucosylation and galactosylation, heterozygosis.Particularly, measure and merge relative to wild-type GalT and GnTI-GalT, whether ManII-GalT merges in the galactosylation of synthesis Fc connection, non-fucosylated oligosaccharide and galactosylation, hybrid oligosaccharides more effective.
As mentioned above, endoglycosidase H (EndoH) is used to confirm that the non-fucosylated structures of galactosylation and galactosylation hybrid structure distribute to the different oligosaccharides peaks observed in MALDI characteristic pattern.With in the oligosaccharides galactose residue of galactose residue, determine the per-cent of those non-fucosylation hybrid structure, fucosylation heterozygosis and fucosylation complex oligosaccharide structure.
The impact determining GalT expression level and compete between restructuring GalT catalyst structure domain and endogenous core α 1,6-fucosyltransferase, ManII and GnTII enzyme for oligosaccharide substrates that the certain position structural domain of GalT catalyst structure domain target golgi body is modified GnTI.Determine the level of the antibody dependent cellular cytotoxicity (ADCC) that antibody produced cell amplifying nucleic acid overexpression causes, this nucleic acid encoding has the polypeptide of the GalT activity of being located by different localization domain.
Also measured were by antibody produced cell amplifying nucleic acid express produce glycosyl engineered antibody whether have raising activate the binding affinity of Fc acceptor Fc γ RIIIA with people or for suppressing Fc γ RIIb, this nucleic acid encoding has the fusion polypeptide of GalT activity.
GalT construct will compete the activity of endogenous core α 1,6-fucosyltransferase, and ADCC is also improved in the Fc district of glycosyl engineered antibody therefore.
Determine the oligosaccharides characteristic formp of anti-CD20 antibodies that bhk cell produces, bhk cell is grown in suspension and through engineering approaches is carried out composing type high level expression recombinant antibodies and had the fusion polypeptide of GalT activity.Also measured were the oligosaccharides relative percentage of the glycosylation monoclonal antibody produced by stable BHK-1502-28-11 clone.
Embodiment 5
Materials and methods
The structure of 1.ManII and GnTII expression vector
In order to build ManII expression vector, people's mannosidase II (SEQIDNO:17) cDNA subclone is entered the upstream of the downstream of MPSV promotor in expression vector plasmid and the rabbit beta Globulin polyadenylation signal of synthesis.In order to GnTII expresses, employ at the downstream of people's CMV promoter/enhanser and the upstream subclone of the Trobest polyadenylation signal expression vector plasmid of people GnTIIcDNA.
combinational expression carrier and the replication origin oriP from EpsteinBarr virus.
As described in Example 1 the DNA fragmentation subclone with oriP is entered in above-mentioned ManII expression vector to obtain ManII expression vector pCLF9.As described in Example 1 the DNA fragmentation subclone with oriP is entered in above-mentioned GnTII expression vector to obtain GnTII expression vector pGnTII.
2. transfection HEK293-EBNA cell
The HEK293-EBNA cell that transfection as described in example 1 above exponentially grows.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 for antibody expression (pETR1520) with another is for merging GnTIII expression of polypeptides (pETR1519), ratio is 4: 1.In order to produce glycosyl engineered antibody " Cm ", with three plasmid co-transfection cells, one for antibody expression (pETR1520), one for merging GnTIII expression of polypeptides (pETR1519), express (pCLF9) with one for mannosidase II, ratio is 3: 1: 1.In order to produce glycosyl engineered antibody " Cmg ", with four plasmid co-transfection cells, one for antibody expression (pETR1520), one for merging GnTIII expression of polypeptides (pETR1519), (pCLF9) is expressed for one for mannosidase II, express (pGnTII) with one for GnTII, ratio is 4: 0.33: 0.33: 0.33.
3. the production of unmodified antibody and glycosyl engineered antibody and purifying
The substratum of above-mentioned transfectional cell is replaced in transfection with fresh culture after about 16 hours, then cultivating transfectional cell 120 h before harvest substratum afterwards further.The supernatant liquor collected at 1200rpm centrifugal 5 minutes, is then stored in 4 DEG C centrifugal 10 minutes of 4000rpm second time.
antibody purification
Use two continuous chromatography steps monoclonal antibody purification from culture supernatants, protein A chromatography as described in example 1 above and cation-exchange chromatography.For antibody cwt7, cbrt5 and cm1, after cation-exchange step, use Superdex200 post (AmershamPharmacia) carry out other size exclusion chromatography step and add phosphate buffered saline (PBS), collect monomeric igg peak.
4. Oligosaccharide Analysis
The oligosaccharides by abzyme solution release in PNGaseF digestion solution as described in example 1 above.
the PNGaseF that endoglycosidase H digests discharges oligosaccharides and heterozygosis bisected oligosaccharides structure is divided the purposes at dispensing MALDI/TOF-MS neutral oligosaccharides peak
The endoglycosidase H (EC3.2.1.96) that uses subsequently as described in example 1 above digests the oligosaccharides of PNGaseF release.
MALDI/TOF-MS
Preparation as described in example 1 above contains the enzymolysis, digestion liquid sample of release oligosaccharides and runs on MALDI/TOF mass spectrograph subsequently.
5. cell is prepared and is separated
Substantially Histopaque-1077 (SigmaCDiagnosticsInc., St.Louis, MO63178USA) is used to prepare peripheral blood lymphocytes (PBMC) according to the method described in the guidance of manufacturers and embodiment 1.
6.NK cellular segregation
Negative selection methods is as described in example 1 above used to isolate NK cells of human beings from PBMC.
7.ADCC tests
PBMC or NK of action effect cell as above obtained the ability of testing their mediating cytotoxicity in antibody dependent cellular cytotoxicity (ADCC) test as described in example 1 above.
Fc γ RIIIA on 8.NK cell combines
The Fc γ RIIIA on the NK cell of fresh separated of being determined at as described in example 1 above combines and the combination of Fc γ RIIb.
9. CDC test
Use antibody diluent to carry out CDC test according to the method described in embodiment 1, following change is used for preparation people Complement source.Tout court, obtained normal human serum (NHS) from the blood of healthy volunteer.Make blood coagulation 1 hour, then at 1200g centrifugal 20 minutes.Acellular supernatant liquor serum decile is stored in-80 DEG C.NHS is used with the whole test volume of 20%.
Results and discussions
Cultivate obtained anti-CD20 by the cotransfection of mammalian cell and be fitted together to IgG1 antibody (C2B8 chimeric antibody, also referred to as rituximab) glycosyl engineered forms, mammalian cell is expressed the carrier of antibody gene and is expressed coding and have the carrier cotransfection of the gene of the polypeptide of GnTIII and Man II activity.Cultivate also obtained other glycosyl engineered antibody form by mammiferous cotransfection, mammalian cell is expressed the carrier of antibody gene and is expressed the carrier cotransfection that coding has the gene of the polypeptide of GnTIII activity, Man II activity and GnTII activity.In order to produce glycosyl engineered antibody " Cbrt ", with two plasmid co-transfection cells, one for antibody expression (pETR1520) with another is for merging GnTIII expression of polypeptides (pETR1519).In order to produce glycosyl engineered antibody " Cm ", with three plasmid co-transfection cells, for antibody expression (pETR1520), express (pCLF9) for merging GnTIII expression of polypeptides (pETR1519) and one for mannosidase II for one for one.In order to produce glycosyl engineered antibody " Cmg ", with four plasmid co-transfection cells, one for antibody expression (pETR1520), for merging GnTIII expression of polypeptides (pETR1519), express (pGnTII) for mannosidase II expression (pCLF9) and one for GnTII for one for one.Unmodified (non-sugar based through engineering approaches) form " Cwt " of production same antibody is only carried out with the carrier transfection mammalian cell of expressing antibody gene.Transfectional cell is kept 5 days in culture, recombinant antibodies purifying secreted from substratum.Relative to the cell not producing such glycosyltransferase or Glycosylase polypeptide, the genetic expression that coding has the polypeptide of GnTIII and ManII activity has no significant effect cell viability, Growth of Cells and antibody producing.
Then the glycosylation pattern of antibody purification is analyzed.These antibody connect oligosaccharides with the N-being only connected to human IgG1 Fc district Asn297 residue.Also analyzed by MALDI/TOF-MS subsequently from abzyme solution removing oligosaccharides by PNGaseF digestion.Use this technology, the ratio of the different oligosaccharide species in total original Fc oligosaccharides colony can be measured, structure can also be distributed to peaks (Umana, P. etc., NatureBiotechnol.17:176-180 (1999)) different in mass spectrum.
Figure 26 shows the neutral oligosaccharides MALDI/TOF-MS characteristic pattern that the anti-CD20 of unmodified recombinant C 2B8 is fitted together to IgG1 antibody Cwt.As described in embodiment 1, Fig. 5 before for unmodified antibody, Cwt has typical oligosaccharides characteristic pattern, m/z1485,1648 and 1810 peak, respectively and have two feelers of 0,1 and 2 galactose residue, core fucosylation composite oligosaccharide consistent.Cause producing antibody (Cbrt) by the antibody produced cell through engineering approaches of the nucleic acid of expressing coding ManII-GnTIII fusion polypeptide (wherein by ManII Golgi localization domain location GnTIII catalyst structure domain), wherein most of Fc oligosaccharides is bisection, non-fucosylation heterozygosis (see Figure 27).As described in example 1 above, endoglycosidase H (EndoH) is used for confirming that bisected, nonfucosylated structure and bisection hybrid structure distribute to viewed different oligosaccharides peak in MALDI characteristic pattern.
Cause producing antibody (Cm) by the antibody produced cell through engineering approaches of the nucleic acid of coexpression coding ManII-GnTIII fusion polypeptide (wherein by ManII Golgi localization domain location GnTIII catalyst structure domain) and the nucleic acid of coding ManII, wherein most of Fc oligosaccharides is bisection, non-fucosylation compound (see Figure 28).Cause producing antibody (Cmg) by the antibody produced cell through engineering approaches of the nucleic acid of the nucleic acid of coexpression coding ManII-GnTIII fusion polypeptide (wherein locating GnTIII catalyst structure domain by ManII Golgi localization domain), the nucleic acid of coding ManII and coding GnTII, wherein most of Fc oligosaccharides is bisection, non-fucosylation compound, and the oligosaccharide portions that bisection, non-fucosylation compound Fc connect is even higher than antibody Cm's.
Figure 29 shows the data of the antibody dependent cellular cytotoxicity (ADCC) demonstrating raising, the ADCC of this raising is expressed in antibody produced cell by the nucleic acid of coding ManII-GnTIII fusion polypeptide (wherein locating GnTIII catalyst structure domain by ManII localization domain) to be formed, wherein ManII-GnTIII coding nucleic acid be oneself (antibody Cbrt) expression or together with the nucleic acid of the ManII (Cm) that encodes is in antibody produced cell coexpression.Therefore, the content improving heterozygosis or compound bisected, nonfucosylated oligosaccharides in glycosyl engineered antibody Fc district will cause the raising of ADCC activity.
Known natural killer (NK) cell is the important medium of ADCC.These cells carry on its surface and activate Fc γ receptor II IA, also referred to as CD16a.On the Fc district of target cell binding antibody and NK cell, the combination of Fc γ RIIIA acceptor is required for the induction that is crosslinked and ADCC subsequently of these acceptors on NK cell.Therefore the combination evaluating antibody that said method produces and Fc acceptor especially wherein their natural form acceptor of people's immunoeffectors molecular display is important.The affinity activating Fc acceptor Fc γ RIIIA with people that Figure 30 demonstrates that glycosyl engineered antibody that the expression that has the nucleic acid of the fusion polypeptide of GnTIII activity by coding in antibody produced cell produces has a raising combines, this nucleic acid oneself (antibody Cbrt) express or and the nucleic acid of the ManII (Cm) that encodes in antibody produced cell together with coexpression.As above for ADCC test, these antibody have the bisection of improving the standard, non-fucosylated oligosaccharide, and oligosaccharides is formed by the expression of fusion polypeptide in antibody produced cell with GnTIII activity.
Therefore, the level improving heterozygosis or compound bisected, nonfucosylated oligosaccharides in glycosyl engineered antibody Fc district will cause the raising of ADCC activity.NK cell used in this test carrys out the genotype donor (Metes, D. etc., J.Immunol.Methods258 (1-2): 85-95 (2001)) of its NK cell comfortable not being expressed Fc γ RIIc acceptor.Therefore the Fc acceptor on these cell surfaces is only had to be activate Fc γ RIIIA acceptor.
Activate the binding domains of Fc γ RIIIB acceptor and the almost identical of Fc γ RIIIA.Therefore, above-mentioned data also demonstrate glycosyl engineered antibody described herein can by showing the effector function raising that the effector cell of Fc γ RIIIB is as cell-mediated in polymorphic nucleus (PMN), comprise release and the phagolysis ((Reff of toxic product, and Heard M.E., C.CritRevOncolHematol.40 (1): 25-35 (2001), Daeron, FM.Annu.Rev.Immunol.15:203-34 (1997), Ravetch, J.V. and BollandS.Annu.Rev.Immunol.19:275-90 (2001)).
Also have detected the complement mediated lysis (CML) of the anti-CD20 antibodies Cbrt produced in through engineering approaches cell, this project cell has GnTIII activity and the nucleic acid being positioned the fusion polypeptide of golgi body by ManII localization domain for expressing coding, and CML is the different effect subfunction not relying on Fc acceptor on immune effector cell.Most oligosaccharides of this glycosyl engineered antibody are the hybrid nonfucosylated types that halves.Compare with unmodified antibody Cwt, observe the CML activity (Figure 31) that Cbrt antibody reduces.For some application, band is improved ADCC and is desirable with reducing the antibody of CML, such as, be reduced by the side effect of CML mediation, as the vasculitis in tumor locus blood vessel.Anti-CD20 antibodies treatment is observed to other remarkable side effect (vanderKolkL.E. etc., BrJHaematol.115 (4): 807-11 (2001)) of CML mediation.But can produce glycosyl engineered antibody, the ADCC that this antibody is improved relative to unmodified antibody band active and Fc γ RIII combines, but remarkable reduction CML activity, as when antibody Cm (Figure 31).Such antibody needs the application of high ADCC and high complement activation and CML activity to be desirable for the elimination of wherein maximum target cell.Above-mentioned oligosaccharides characteristic formp show can by GnTIII fusion polypeptide together with the nucleic acid of coding ManII (antibody Cm) together with the nucleic acid of coexpression or GnTIII fusion polypeptide and coding ManII and the nucleic acid of coding GnTII (antibody Cmg) coexpression produce antibody by cell engineered, wherein in antibody, the oligosaccharides of most of Fc connection is that the bisection of non-heterozygous, non-fucosylation are compound.Glycosyl engineered antibody has the Fc γ RIII binding affinity that the ADCC of the raising relevant with the level that bisected, nonfucosylated oligosaccharides improves is active and improve, and the CML that improve them relative to the increase of hybrid oligosaccharides part due to composite oligosaccharide part is active.
This and the expression that embodiment has described fusion polypeptide coding nucleic acid before, wherein fusion polypeptide is positioned Golgi complex and has the catalyst structure domain with the competition of endogenous fucosyltransferase, for the oligosaccharide acceptor that the reaction before by GnTI catalysis is modified.The recombinant glycoprotein produced by this project host cell has the non-fucosylated oligosaccharide of improving the standard.This example demonstrates the coexpression of coding ManII and/or GnTII nucleic acid together with the nucleic acid of above-mentioned encode fusion polypeptide in this host cell causes biosynthesizing flux towards the increase of composite oligosaccharide, instead of hybrid oligosaccharides, and therefore cause the glycoprotein synthesized to have the non-fucosylation composite oligosaccharide of improving the standard, relative to the glycoprotein not having to produce in the cell of coexpression coding ManII and/or GnTII nucleic acid.
Embodiment 6
The overexpression of alpha-Mannosidase
Molecular cloning
human α-mannosidase II
The gene (SEQIDNO:17) of encoding human alpha-Mannosidase II (" hManII ") (E.C.3.2.1.114), under MPSV promotor controls, clones in the expression vector containing OriP element.The expression vector pCLF9 obtained is shown in Figure 32 A.The coding light chain of anti-CD-20 monoclonal antibody and the expression vector of heavy chain are pETR1842 and pETR1843 (Figure 32 B and 32C) separately.
fusion rotein ManII-GalT
Construction of fusion protein (SEQIDNO:20) as described below, is made up of the catalyst structure domain of hManIICTS and people β (Isosorbide-5-Nitrae)-galactosyltransferase (M22921, amino acid/11 26-397).To be increased hManIICTS region from pETR1484 (CF33, GAB252) by PCR.Use CF31 and CF35 from the catalyst structure domain (amino acid/11 26-397) of pBlueGalT amplification GalT.The catalyst structure domain of hManIICTS and GalT is merged and obtains the fusion rotein (pCLF24) controlled by MPSV promotor.Whole GalT gene is obtained from pBlueGalT.Gene sequencing (SEQIDNO:16) by coding GalT:
MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSR
LPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDS
SPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDL
ELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP
VLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDV
DLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLT
INGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKK
NEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYRLYTQITVDIGTPS
Increase with CF32/CF38 5 ' of GalT, and before gene, add FseI restriction site.Find that sequence correctly and by FseI/DraIII digests by order-checking and exchange (pCLF26) in pCLF24.OriP is added in pCLF24 and pCLF26 and produce pCLF25 and pCLF27 (Figure 33 A and 33B) respectively.
Alpha-Mannosidase and the expression of ManII-GalT in HEK293-EBNA cell
With calcium phosphate procedure transfection HEK293-EBNA cell.Tout court, in order to transfection T150, within first 24 hours, 1,500 ten thousand cells are inoculated in 28mlDMEM, 10%FCS in transfection, 250 μ g/ml Liu Suanyan NEOMYCIN SULPHATEs at 37 DEG C, 5%CO 2overnight incubation.
For each T150 culturing bottle to be transfected, by mixing the CaCl of the total plasmid vector DNA of 94 μ g, 469 μ l1M 2solution also adds water and obtains DNA, CaCl to final volume 938 μ l 2with the solution of water.The 1.5mMNa of 938 μ l50mMHEPES, 280mMNaCl, pH7.05 is added in this solution 2hPO 4solution, and mix 10 seconds immediately and leave standstill 20 seconds in room temperature.Supplement the DMEM diluted suspension of 2%FCS with 24ml, and add alternative existing substratum in T150.By cell at 37 DEG C, 5%CO 2hatch about 17 to 20 hours, and with the DMEM substitutive medium of 30ml10%FCS.
In order to test the impact that alpha-Mannosidase II competes core fucosyltransferase, be pETR1842, pETR1843 and pCLF9 transfectional cell of 2: 2: 1 by respective ratio.For fused protein ManII-GalT, be pETR1842, pETR1843 and pCLF25 transfectional cell of 2: 2: 1 by respective ratio.Transfection is after 5 days, collects supernatant liquor, at 1200rpm centrifugal 5 minutes, then centrifugal 10 minutes of 4000rpm second time, filters and is stored in 4 DEG C.
The purifying of anti-CD-20 monoclonal antibody
By two step chromatographies monoclonal antibody purification from 30ml supernatant liquor, comprise first protein A chromatography, isolate monoclonal antibody from the Niu Kangti be present in serum, then cation-exchange chromatography, sample buffer is exchanged to PBS.
Oligosaccharide Analysis
pNGaseF digests
Monoclonal antibody sample (50 μ g) is hatched with the N-Glycosylase F (restructuring, Roche, Switzerland) of 0.1mU/ μ l.In the Tris damping fluid of 2mMpH7.0,37 DEG C are carried out digestion 3 hours.Then by the neutral oligosaccharides of release incubated at room 3 hours in 150mM acetic acid.Then the 0.6ml Zeo-karb (AG50W-X8 resin, hydrogen form, 100-200 order, BioRad, Hercules, CA) in use loading in a subtle way-biology-rotary chromatography post (BioRad, Hercules, CA) is by sample desalination.
endoH digests
Before acetic acid process, digest the oligosaccharides of PNGaseF release with endoglycosidase H (EC3.2.1.96, ROCHE, Switzerland), endoglycosidase H is the enzyme that division N connects the NAG residue of the chitobiose core of oligosaccharides.Enzyme will decompose MOS, and majority is hybrid type glycans, and complex type oligosaccharides is not hydrolyzed.
Oligosaccharides is digested with the 0.2mU/ μ l endoglycosidase H in 2mMpH7.0Tris.Digestion carries out 3 hours at 37 DEG C.Oligosaccharides incubated at room 3 hours in 150mM acetic acid, subsequently with loading in a subtle way-biology-rotary chromatography post (BioRad, Suitzerland) 0.6ml Zeo-karb (the AG50W-X8 resin in, hydrogen form, 100-200 order, BioRad, Switzerland) by sample desalination.
matrix and sample preparation
Obtained DHB matrix (sDHB) in the ethanol/10mM sodium chloride aqueous solution of 1ml1: 1 (v/v) is dissolved in by the 5-methoxysalicylic acid of the DHB+0.1mg by 2mg.1 μ l sample is used in stainless steel target, and mixes with 1 μ lsDHB.Sample air is dry and use 0.2 μ l ethanol.
mALDI/TOF analyzes
Being used for obtaining mass spectrographic MALDI-TOF mass spectrograph is the VoyagerElite (PerspectiveBiosystems) being equipped with time delay extraction.This device is operated in reverberator mode.Positively charged ion is accelerated to 20kV after 75ns postpones.40 five mass spectrums (200 transmittings) launched are merged and obtains final mass spectrum.The external perimysium reference of oligosaccharides standard is used to distribute for the mass spectrum of ion.
oligosaccharides characteristic formp
The oligosaccharides characteristic formp of the anti-CD20 antibodies produced under ManII exists is shown in Figure 34.Find that the oligosaccharides relevant with antibody Fc portion is composite structure, wherein 48% lacks core fucose.Alpha-Mannosidase II and core fucosyltransferase competition, produce 48% non-fucosylated oligosaccharide structure.Under alpha-Mannosidase II lacks, the oligosaccharides of antibody Fc portion only comprises the structure (wild-type antibodies) of fucosylation.
The oligosaccharides characteristic formp of the anti-CD20 antibodies produced under ManII-GalT fusion rotein exists is shown in Figure 35 A-B.For alpha-Mannosidase II, also have in ManII-GalT fusion rotein situation, the amount of non-fucosylated oligosaccharide structure adds.The high per-cent of non-fucosylated structures is consistent with high mannose structures (m/z1256,1419 and 1581).For this 67% non-fucosylation sugar, find the hybrid nonfucosylated structure (m/z1622) of other 30%.Therefore, the sample produced under ManII-GalT fusion rotein exists shows the non-fucosylated structures of almost 100%.
The biological activity of the antibody produced under alpha-Mannosidase II or ManII-GalT fusion rotein exist
In order to measure the effect of alpha-Mannosidase II and the effect in core fucosyltransferase competition of ManII-GalT enzyme, carry out associated biomolecule test.Test external antibody dependent cellular cytotoxicity (ADCC) and and the combination of CD16 acceptor expressed on the surface of through engineering approaches Chinese hamster ovary celI system (CHO-1708-37) of sample.
igG on CHO-1708-37 combines
CHO-1708-37 clone expresses the γ chain of Fc γ RIIIA acceptor (CD16) and Fc γ RI acceptor in its surface.3G8-FITC monoclonal antibody is used to test the expression (Figure 36) of Fc γ RIIIA acceptor (CD16) by facs analysis.In the PBS of 0.1%BSA, hatch 1Mio/mlCHO-1708-37 cell, use the different antibodies variant of different concns (10,3,1,0.3,0.1 μ g/ml), in triplicate.Cell is hatched 30 minutes at 4 DEG C and washed with the PBS of 0.1%BSA subsequently.By the F (ab ') puted together with 1: 200 fluorescein isothiocyanate 2goat anti human IgG (JacksonImmunoReasearch, WestGrove, PA) hatches 30 minutes to detect antibodies at 4 DEG C.On the FACSCalibur of gate viable cell, (BDBioscience, SanJose, CA) measures the fluorescence intensity relating to binding antibody variant.
Following antibody variants is included in binding tests experiment:
Cwt8 (wild-type 1)
ManII (alpha-Mannosidase II)
The lower antibody of generation and the binding affinity of the Fc γ RIIIA acceptor height than wild-type antibodies is there is at alpha-Mannosidase II (ManII).
external antibody dependent cellular cytotoxicity (ADCC)
Use Histopaque-1077 (SigmaDiagnosticsInc., St.Louis, MO63178USA) and substantially obtain peripheral blood lymphocytes (PBMC) according to the guidance system of manufacturers.Tout court, take venous samples can with heparinized syringe from volunteer, volunteer is required to run 1 minute, to improve the per-cent of the natural killer cell (NK) in blood with all strength.With the PBS not containing Ca or Mg by blood thinning 1: 0.75-1.3, and be laid on Histopaque-1077.In room temperature (RT) in the unremitting gradient centrifugation of 400g 30 minutes.Collect the mesophase spherule containing PBMC and wash (the every 50ml cells from two gradients) with PBS and within centrifugal 10 minutes, collected by RT300g.With PBS by after pellet resuspended, PBMC is counted and within centrifugal 10 minutes, washs second time by RT200g.Then Cell resuspension is used in suitable substratum program subsequently.
In PBS, wash Raji target cell, counting is also resuspended to DMEM with every ml1Mio, 10%FCS, 1%Glutamax.In these, add 1: 100calcein and cell is hatched 30 minutes at 37 DEG C.Then washed cell in PBS, counting also every ml0.3Mio is resuspended in AIM-V.100 these cell suspending liquids of μ l are added in each hole of round bottom 96 hole flat board.In AIM-V, dilute antibody, and 50 μ l to be added in pre-incubated target cell and at RT and target in conjunction with 10 minutes.As above the PBMC of obtained action effect cell.The ratio of effector to target cell is 25: 1.In AIM-V, obtain effector cell with every ml15Mio and 50 μ l are added in each hole.By flat board containing 5%CO 2moistening atmosphere in 37 DEG C hatch 4 hours.By cell washed cell add 200 μ l borate solutions in PBS.The calcein discharged in the medium after the cracking of measurement borate solution is to measure killing (Figure 37) of target cell.
From only still measuring spontaneous release without the hole of antibody containing target cell and effector cell.Maximum release is measured from the hole only containing target cell and 1%TritonX-100.The per-cent killed of following calculating specific antibodies mediation: ((x-SR)/(MR-SR) * 100, the average Vmax of wherein x to be average Vmax, the MR being spontaneous release at average Vmax, the SR of specific antibodies concentration be maximum release.
Embodiment 7
The purposes of monoclonal antibody against EGFR in treatment psoriasis of glycosyl through engineering approaches
Psoriasis people patient can be treated with the glycosyl through engineering approaches monoclonal antibody against EGFR of prepared in accordance with the method for the present invention.Especially, patient accepts weekly capacity value is 400mg/m 2the infusion of glycosyl engineered antibody.Administration 250mg/m based on week 2preservation dose until obtain 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 associating of ErbB2 and other ErbB family member, therefore inhibits the extracellular signal by ErbB approach.RhuMAb2C4 not only inhibits the growth of ErbB2 overexpression tumour, but also has blocked the growth of the tumour needing ErbB ligand dependent signal.
The RhuMAb2C4 of the glycosyl engineered forms obtained by the inventive method can be used as single medicament and be used for the treatment of hormone and be difficult to treat the prostate cancer patients of (male sex hormone independence), transitivity patients with mastocarcinoma, metastatic colorectal cancer patient and IIIb phase or IV phase Patients with Non-small-cell Lung.Particularly, by glycosyl through engineering approaches RhuMAb2C4 weekly or intravenously (IV) administration 2 in every three weeks or 4mg/kg, until progression of disease stops.Antibody (20mL is full of concentration 20mg/mL or higher concentration) is supplemented with multiple doses liquid preparation.
Being clear that can with the different modes described special in specification sheets before and embodiment to implement the present invention.Possible according to above-mentioned instruction various modification of the present invention and change, therefore within the scope of the appended claims.
In whole being openly hereby incorporated by of these all publications quoted (comprising patent, patent application, periodical literature, laboratory manual, books or other document).
Figure IYZ000004153108900011
Figure IYZ000004153108900021
Figure IYZ000004153108900031
Figure IYZ000004153108900041
Figure IYZ000004153108900051
Figure IYZ000004153108900061
Figure IYZ000004153108900071
Figure IYZ000004153108900081
Figure IYZ000004153108900091
Figure IYZ000004153108900101
Figure IYZ000004153108900111
Figure IYZ000004153108900121
Figure IYZ000004153108900141
Figure IYZ000004153108900151
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Figure IYZ000004153108900211

Claims (57)

1. the nucleic acid of the separation of the sequence containing encode fusion polypeptide, wherein said fusion polypeptide has β (1,4)-NAG transferase I II is active, and by β (1,4) Golgi localization domain of-NAG transferase I II catalyst structure domain and human Golgi mannosidase II is formed, and wherein said fusion polypeptide is as shown in following aminoacid sequence:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
2. the nucleotide sequence be separated, it comprises the sequence of encode fusion polypeptide, wherein said fusion polypeptide has β (1,4)-NAG transferase I II is active, and by β (1,4) catalyst structure domain of-NAG transferase I II, the Golgi localization domain of human Golgi mannosidase II and C-terminal polypeptide label are formed, and the aminoacid sequence of wherein said catalyst structure domain and Golgi localization domain is by following Sequence composition:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisP heAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
3. the separating nucleotide of claim 2, wherein C-terminal polypeptide label is the c-myc epitope tag shown in SEQIDNO:1, and the aminoacid sequence of wherein said fusion polypeptide is by following Sequence composition: metLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu.
4. expression vector, containing the nucleic acid of the separation any one of with good grounds claims 1 to 3.
5. fusion polypeptide, its encoded by nucleic acid any one of claims 1 to 3.
6. the mammalian host cell of the expression vector containing claim 4.
7. produce and there is β (1,4) method of the fusion polypeptide of-NAG transferase I II activity, be included in the host cell cultivating claim 6 under allowing the condition of the described expression of nucleic acid of the described fusion polypeptide of coding in the medium, and collect described fusion polypeptide from gained culture.
8. modify the method for the glycoforms of the polypeptide that host cell produces, comprise and the nucleic acid any one of claim 1-3 is introduced in described host cell.
9. the method for claim 8, the described polypeptide wherein produced by described host is the fragment in IgG or the Fc district containing IgG.
10. the method for claim 9, the described polypeptide wherein produced by described host is the fragment in IgG1 or the Fc district containing IgG1.
11. the method for claim 8, the described polypeptide wherein produced by described host comprises the fusion rotein with the region of the Fc district equivalence of human IgG.
12. modify the method for the glycoforms of the polypeptide that host cell produces, comprise and the expression vector of claim 4 is introduced in described host cell.
13. method according to claim 12, the described polypeptide wherein produced by described host is the fragment of the IgG in IgG Huo Han Fc district.
14. methods according to claim 13, the described polypeptide wherein produced by described host is the fragment in IgG1 or the Fc district containing IgG1.
15. method according to claim 12, the described polypeptide wherein produced by described host is fusion rotein, and this fusion rotein comprises the fragment being equivalent to human IgG Fc district.
16. Mammalian host cells, the host cells engineered to express a kind of coding has at least beta (1, 4) - N - acetyl glucosamine transferase activity of III fusion peptide nucleic acid, express the amount described enough to modify the host cell to produce peptide Fc area of oligosaccharides, wherein the fusion peptide is composed of the following amino acid sequence:MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
17. mammalian host cells, this host cell through engineering approaches is expressed at least one coding and is had β (1, 4) nucleic acid of the fusion polypeptide of-NAG transferase I II activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of described host cell generation, wherein said polypeptide is selected from complete antibody molecule, antibody fragment, and described fused protein comprises the region with the Fc district equivalence of immunoglobulin (Ig), wherein said fusion polypeptide has β (1, 4)-NAG transferase I II is active, by β (1, 4) catalyst structure domain of-NAG transferase I II, the Golgi localization domain of people's mannosidase and C-terminal polypeptide label are formed, the aminoacid sequence of wherein said catalyst structure domain and described Golgi localization domain is by following Sequence composition: metLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly.
The host cell of 18. claims 17, wherein said C-terminal polypeptide label is the c-myc epitope tag of SEQIDNO:1, and the wherein said aminoacid sequence with the fusion polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II activity is by following Sequence composition:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu。
19. the host cell any one of claim 16-18, the described polypeptide that host cell described in the Fc district containing IgG produces is IgG or its fragment.
The host cell of 20. claims 19, the described polypeptide that wherein said host cell produces is the fragment in IgG1 or the Fc district containing IgG1.
21. the host cell any one of claim 16-18, the described polypeptide that wherein said host cell produces is fusion rotein, and this fusion rotein comprises the fragment being equivalent to human IgG Fc district.
Host cell any one of 22. claim 16-18, wherein as described modification result described in the described polypeptide that produces of host cell present Fc γ RIIIA or the Fc γ RIIIB receptor binding affinity of raising.
Host cell any one of 23. claim 16-18, the described polypeptide that wherein said host cell produces is anti-CD20 antibodies.
The host cell of 24. claims 23, wherein said anti-CD20 antibodies is IDEC-C2B8.
Host cell any one of 25. claim 16-18, the described polypeptide that wherein said host cell produces is chimeric anti-human renal cell's carcinoma monoclonal antibody chG250.
26. the host cell any one of claim 16-18, comprise the nucleic acid of at least one transfection further, this nucleic acid encoding antibody molecule and antibody fragment or fusion rotein, fusion rotein comprises the fragment being equivalent to immunoglobulin fc region.
Host cell any one of 27. claim 16-18, the nucleic acid that wherein said at least one coding has the fusion polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II activity is operationally connected with constitutive promoter element.
The host cell of 28. claims 26, the nucleic acid encoding anti-CD20 antibodies of wherein said at least one transfection, chimeric anti-human neuroblastoma monoclonal antibody chCE7, chimeric anti-human renal cell's carcinoma monoclonal antibody chG250, chimeric anti-human colon, lung and breast cancer monoclonal antibody ING-1, Humanized anti-human 17-1A antigen monoclonal antibody 3622W94, Humanized anti-human colorectal tumours antibody A 33, for the anti-human melanoma antigens of GD3 Sphingolipids,sialo R24, chimeric anti-human squamous cell carcinoma monoclonal antibody SF-25, anti-human EGFR antibody, anti-human EGFRvIII antibody, anti-human PSMA antibody, anti-human psca antibody, anti-human CD22 antibody, anti-human CD30 antibody, anti-human CD33 antibody, anti-human CD38 antibody, antibodies against human CD 40, anti-human CD45 antibody, antihuman CD 52 antibody, anti-human CD138 antibody, anti-human HLA-DR variant antibodies, anti-human epcam antibody, anti-human CEA antibody, anti-human MUC1 antibody, anti-human MUC1 nucleoprotein antibody, anti-human Aberrant glycosylation MUC1 antibody, the antibody of the people fibronectin variant of antagonism containing ED-B structural domain or anti-human HER2/neu antibody.
29. methods of producing polypeptide in mammalian host cell, comprising:
A. allowing to cultivate mammalian host cell under the condition producing polypeptide, this host cell through engineering approaches is expressed at least one coding and is had β (1, 4) nucleic acid of the fusion polypeptide of-NAG transferase I II activity, the polypeptide produced is selected from complete antibody molecule, antibody fragment and fusion rotein, this fusion rotein comprises the fragment being equivalent to immunoglobulin fc region, the expression amount of wherein said fusion polypeptide is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, wherein said have β (1, 4) fusion polypeptide of-NAG transferase I II activity is by β (1, 4) catalyst structure domain of-NAG transferase I II and the Golgi localization domain of people's mannosidase II are formed, with
B. peptide separation stated, wherein a beta (1, 4) - N - acetyl glucosamine transferase III fusion of active peptide is composed of the following amino sequence:MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
30. methods of producing polypeptide in mammalian host cell, comprising:
A. allowing to cultivate mammalian host cell under the condition producing polypeptide, this host cell through engineering approaches is expressed at least one coding and is had β (1, 2) nucleic acid of the fusion polypeptide of-NAG transferase I II activity, the polypeptide produced is selected from complete antibody molecule, antibody fragment and comprise the fusion rotein of fragment in the Fc district being equivalent to immunoglobulin (Ig), the expression amount of wherein said fusion polypeptide is enough to the oligosaccharides modified in the Fc district of the described polypeptide that described host cell produces, and described in there is β (1, 2) fusion polypeptide of-NAG transferase I II activity is by β (1, 2) catalyst structure domain of-N-ethanoyl glucotransferase III, the Golgi localization domain of people's mannosidase III and C-terminal polypeptide label are formed, with
B. be separated described polypeptide, the aminoacid sequence of wherein said catalyst structure domain and described Golgi localization domain is by following Sequence composition:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAsp GlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
The method of 31. claims 30, wherein said C-terminal polypeptide label is the c-myc epitope tag shown in SEQIDNO:1, and the aminoacid sequence with the fusion polypeptide of β (Isosorbide-5-Nitrae)-NAG transferase I II activity is wherein by following Sequence composition:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu。
32. the antibody that produces of the method any one of claim 29-31 for the treatment of significant quantity is for the preparation of the purposes exhausting based on B cell in the medicine of disease therapy.
The purposes of 33. claims 32, wherein said antibody is anti-CD-20 monoclonal antibody.
The purposes of 34. claims 33, wherein said anti-CD20 antibodies is IDEC-C2B8.
35. mammalian host cells, comprising:
The expression vector of (a) nucleic acid molecule containing encode fusion polypeptide, wherein said fusion polypeptide has β (1,4)-NAG transferase I II (GnTIII) is active and be made up of the catalyst structure domain of β (Isosorbide-5-Nitrae)-NAG transferase I II and the Golgi localization domain of people's mannosidase II; With
The expression vector of (b) nucleic acid molecule containing coded polypeptide, it is active that wherein said polypeptide has mannosidase II (ManII), and wherein said fusion polypeptide is by following Sequence composition:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
36. mammalian host cells, comprising:
The expression vector of (a) nucleic acid molecule containing encode fusion polypeptide, wherein said fusion polypeptide has β (1,2)-NAG transferase I II (GnTIII) is active, and be made up of the catalyst structure domain of β (1,2)-NAG transferase I II, the Golgi localization domain of people's mannosidase II and C-terminal polypeptide label; With
The expression vector of (b) nucleic acid molecule containing coded polypeptide, it is active that wherein said polypeptide has mannosidase II (manII),
The aminoacid sequence of wherein said catalyst structure domain and described Golgi localization domain is by following Sequence composition: metLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly
。
37. the host cell of claim 36, wherein said C-terminal polypeptide label is the c-myc epitope tag shown in SEQIDNO:1, and the aminoacid sequence of wherein said fusion polypeptide is by following Sequence composition:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAsnLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu。
Host cell any one of 38. claim 35-37, comprises the expression vector of the nucleic acid molecule containing coded polypeptide further, and it is active that wherein said polypeptide has β (1,2)-NAG transferase I I (GnTII).
The host cell of 39. claims 38, the nucleic acid molecule of wherein said encode fusion polypeptide, described coding have the nucleic acid molecule of the polypeptide of ManII activity and described coding has the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector.
The host cell of 40. claims 38, the nucleic acid molecule of wherein said encode fusion polypeptide, described coding have the nucleic acid molecule of the polypeptide of ManII activity and described coding has the nucleic acid molecule of the polypeptide of GnTII activity on the expression vector separated.
The host cell of 41. claims 38, the nucleic acid molecule of wherein said encode fusion polypeptide is on an expression vector, and described coding has the nucleic acid molecule of the polypeptide of ManII activity and described coding has the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector.
The host cell of 42. claims 38, the nucleic acid molecule of wherein said coding ManII is on an expression vector, and the nucleic acid molecule of described encode fusion polypeptide and described coding have the nucleic acid molecule of the polypeptide of GnTII activity on same expression vector.
The host cell of 43. claims 38, the nucleic acid molecule of the described GnTII of wherein said coding is on an expression vector, and the nucleic acid molecule of described encode fusion polypeptide and described coding have the nucleic acid molecule of the polypeptide of ManII activity on same expression vector.
44. mammalian host cells, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of described host cell generation, the described polypeptide that wherein said host cell produces is selected from complete antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to territory, immunoglobulin fc region, the wherein said fusion polypeptide β (1 with GnT activity, 4) gorky's localization domain of-NAG transferase I II and people's mannosidase II is formed,
Wherein a GnTIII fusion of active polypeptide amino acid sequence consists of the following sequence:MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
45. mammalian host cells, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc district of described host cell generation, the described polypeptide that wherein said host cell produces is selected from complete antibody and divides, antibody fragment and the fusion rotein comprising the region being equivalent to territory, immunoglobulin fc region, and the wherein said fusion polypeptide with GnTIII activity is by β (1, 4) catalyst structure domain of-NAG transferase I II, the Golgi localization domain of human Golgi and C-terminal polypeptide label are formed.
The aminoacid sequence of wherein said catalyst structure domain and described Golgi localization domain is by Sequence composition as follows:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
The host cell of 46. claims 45, wherein said C-terminal polypeptide label is the c-myc epitope tag of SEQIDNO:1, and the wherein said aminoacid sequence with the fusion polypeptide of GnTIII activity is by Sequence composition as follows:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu。
47. mammalian host cells, this host cell through engineering approaches expresses the nucleic acid that at least one coding has the fusion polypeptide of GnTIII activity, at least one coding has the nucleic acid that the nucleic acid of the polypeptide of ManII activity and at least one coding have the polypeptide of GnTII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc fragment of described host cell generation, the described polypeptide that wherein said host cell produces is selected from complete antibody molecule, antibody fragment and fusion rotein, fusion rotein comprises the region being equivalent to territory, immunoglobulin fc region, the wherein said fusion polypeptide with GnTIII activity is by β (1,4) catalyst structure domain of-NAG transferase I II and the Golgi localization domain of people's mannosidase II are formed, wherein, described fusion polypeptide is made up of following aminoacid sequence: MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIl ePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProA rgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAsp HisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSe rValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnP heSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThr GluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheVa lArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysP roSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArg GlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGl yArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyP roSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArg LeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGl uPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheV alValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArg GluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPh eLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrL euArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAsp AspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPh eLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerL euTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThr IleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGl nTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuV alGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCys PheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPhePr oArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleA rgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSer GluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLe uLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnG lnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly.
48. mammalian host cells, this host cell through engineering approaches expresses the nucleic acid that at least one coding has the fusion polypeptide of GnTIII activity, at least one coding has the nucleic acid that the nucleic acid of the polypeptide of ManII activity and at least one coding have the polypeptide of GnTII activity, expression amount is enough to the oligosaccharides modified in the polypeptide Fc fragment of described host cell generation, the described polypeptide that wherein said host cell produces is selected from complete antibody molecule, antibody fragment and the fusion rotein comprising the region being equivalent to immunoglobulin region, the wherein said fusion polypeptide with GnTIII activity is by β (1, 4) catalyst structure domain of-NAG transferase I I, the Golgi localization domain of people's mannosidase II and C-terminal polypeptide label are formed, the aminoacid sequence of wherein said catalyst structure domain and Golgi localization domain is made up of aminoacid sequence as follows:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
The host cell of 49. claims 48, wherein said C-terminal polypeptide label is the c-myc epitope tag shown in SEQIDNO:1, and the aminoacid sequence wherein with the described fusion polypeptide of GnTIII activity is by Sequence composition as follows:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu。
50. methods of producing polypeptide in mammalian host cell, comprising:
A. allowing to cultivate mammalian host cell under the condition producing polypeptide, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, the polypeptide produced is selected from complete antibody molecule, antibody fragment and fusion rotein, this fusion rotein comprises the region being equivalent to immunoglobulin fc region, the expression amount of wherein said fusion polypeptide is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, the wherein said fusion polypeptide with GnTIII activity is by β (1, 4) catalyst structure domain of-NAG transferase I II and the Golgi localization domain of people's mannosidase II are formed, with
B. peptide separation stated, including GnTIII activity described in the fusion peptide sequence of amino acids by as shown in the following sequence:MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
51. methods of producing polypeptide in mammalian host cell, comprising:
A. allowing to cultivate mammalian host cell under the condition producing polypeptide, this host cell through engineering approaches is expressed at least one coding and is had the nucleic acid that the nucleic acid of the fusion polypeptide of GnTIII activity and at least one coding have the polypeptide of ManII activity, the polypeptide produced is selected from complete antibody molecule, antibody fragment and the fusion rotein comprising the region being equivalent to immunoglobulin fc region, the expression amount of wherein said fusion polypeptide is enough to the oligosaccharides modified in the described polypeptide Fc district of described host cell generation, and the fusion polypeptide wherein with GnTIII activity is by β (1, 4) catalyst structure domain of-NAG transferase I II, the Golgi localization domain of people's mannosidase II and C-terminal polypeptide label are formed, with
B. be separated described polypeptide, the aminoacid sequence of wherein said catalyst structure domain and described Golgi localization domain is by Sequence composition as follows:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGly。
The method of 52. claims 51, wherein said C-terminal polypeptide label is the c-myc epitope tag of SEQIDN0:1, and the sequence wherein with the amino of the fusion polypeptide of GnTIII activity is by Sequence composition as follows:
MetLysLeuSerArgGlnPheThrValPheGlySerAlaIlePheCysValValIlePheSerLeuTyrLeuMetLeuAspArgGlyHisLeuAspTyrProArgAsnProArgArgGluGlySerPheProGlnGlyGlnLeuSerMetLeuGlnGluLysIleAspHisLeuGluArgLeuLeuAlaGluAsnAsnGluIleIleSerAsnIleArgAspSerValIleAsnLeuSerGluSerValGluAspGlyProLysSerSerGlnSerAsnPheSerGlnGlyAlaGlySerProLeuLeuGlnProLeuSerProSerLysAlaThrGluGluLeuHisArgValAspPheValLeuProGluAspThrThrGluTyrPheValArgThrLysAlaGlyGlyValCysPheLysProGlyThrArgMetLeuGluLysProSerProGlyArgThrGluGluLysThrLysValAlaGluGlySerSerValArgGlyProAlaArgArgProMetArgHisValLeuSerAlaArgGluArgLeuGlyGlyArgGlyThrArgArgLysTrpValGluCysValCysLeuProGlyTrpHisGlyProSerCysGlyValProThrValValGlnTyrSerAsnLeuProThrLysGluArgLeuValProArgGluValProArgArgValIleAsnAlaIleAsnIleAsnHisGluPheAspLeuLeuAspValArgPheHisGluLeuGlyAspValValAspAlaPheValValCysGluSerAsnPheThrAlaTyrGlyGluProArgProLeuLysPheArgGluMetLeuThrAsnGlyThrPheGluTyrIleArgHisLysValLeuTyrValPheLeuAspHisPheProProGlyGlyArgGlnAspGlyTrpIleAlaAspAspTyrLeuArgThrPheLeuThrGlnAspGlyValSerArgLeuArgAsnLeuArgProAspAspValPheIleIleAspAspAlaAspGluIleProAlaArgAspGlyValLeuPheLeuLysLeuTyrAspGlyTrpThrGluProPheAlaPheHisMetArgLysSerLeuTyrGlyPhePheTrpLysGlnProGlyThrLeuGluValValSerGlyCysThrIleAspMetLeuGlnAlaValTyrGlyLeuAspGlyIleArgLeuArgArgArgGlnTyrTyrThrMetProAsnPheArgGlnTyrGluAsnArgThrGlyHisIleLeuValGlnTrpSerLeuGlySerProLeuHisPheAlaGlyTrpHisCysSerTrpCysPheThrProGluGlyIleTyrPheLysLeuValSerAlaGlnAsnGlyAspPheProArgTrpGlyAspTyrGluAspLysArgAspLeuAsnTyrIleArgSerLeuIleArgThrGlyGlyTrpPheAspGlyThrGlnGlnGluTyrProProAlaAspProSerGluHisMetTyrAlaProLysTyrLeuLeuLysAsnTyrAspGlnPheArgTyrLeuLeuGluAsnProTyrArgGluProLysSerThrValGluGlyGlyArgArgAsnGlnGlySerAspGlyArgSerSerAlaValArgGlyLysLeuAspThrThrGluGlyProGluGlnLysLeuIleSerGluGluAspLeu。
Method any one of 53. claim 50-52, the further through engineering approaches of wherein said host cell expresses the nucleic acid that at least one coding has the polypeptide of GnTII activity.
54. the method any one of claim 50-52, the oligosaccharides at least 20% in wherein said polypeptide Fc district is bisected, nonfucosylated.
55. the method any one of claim 50-52, the oligosaccharides at least 25% in wherein said polypeptide Fc district is bisected, nonfucosylated.
56. the method any one of claim 50-52, the oligosaccharides at least 30% in wherein said polypeptide Fc district is bisected, nonfucosylated.
57. the method any one of claim 50-52, the oligosaccharides at least 35% in wherein said polypeptide Fc district is bisected, nonfucosylated.
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