CN101238148A - Method of producing antibodies with modified fucosylation level - Google Patents

Abstract

The present invention provides methods for controlling fucosylation levels and improving ADCC activity in antibodies, by inhibiding the expression of FUT8 in antibody-producing cells.

Description

The production method of the antibody of fucosylation level with improvement

This application claims 60/736,982 rights and interests that the U.S. Provisional Application serial number submitted on June 3rd, 2005 is submitted on November 14th, 60/687,625 and 2005, its entire disclosure is taken in herein as reference.

Invention field

The generation of the antibody improved the present invention relates to fucose reduction and Fc functions.

Background of invention

Treatment is recombinated generally to be produced in several mammalian host cell lines with protein, including rat bone marrow tumour NS0 and Chinese hamster ovary (CHO) cell (Anderson and Krummen, 2002；Chu andRobinson, 2001).Every kind of cell line has its Pros and Cons in terms of the characteristic of the protein produced by productivity and the cell.The selection of commodity production cell line usually balance to high productivity the need for being presented to the ability of product quality characteristic required by fixed output quota thing.It is monoclonal antibody with recombinant protein to usually require that the important treatment of a class of high titre process.Some monoclonal antibodies need effector functions, are mediated by Fc areas, to trigger their biological function.With Rituximab (Rituxan^TM, Genentech and Biogen-Idec) exemplified by, it is a kind of chimeric mAb (Carton et al., 2002 for being combined and being caused B cell to cut down with cell surface CD20；Idusogie et al., 2000).Other antibody, such as Bevacizumab (Avastin^TM, Genentech), a kind of humanization anti-vegf (VEGF) antibody, their activity does not need Fc effector functions.

Asn of the monoclonal antibody produced in mammalian host cell in every heavy chain (each complete antibody molecule has two)²⁹⁷Place includes the glycosylation site of a N- connection.Glycan on antibody is typically complicated double feeler structure, with N-acetyl-glucosamine (decile GlcNAc) seldom or without decile, and has high-caliber core fucosylation (Saba et al., 2002).Glycan end is comprising seldom or not comprising terminal sialic acid, and the galactolipin comprising variable.The summary of influence about glycosylation to antibody function refers to such as Wright Morrison, Trend Biotechnol.15：26-31(1997).Considerable work shows, the change of the sugar composition of antibody glycan structures can change Fc effector functions (Kumpel et al., 1994；Kumpel et al., 1995；Schuster et al., 2005；Shields etal., 2002；Umana et al., 1999).Important carbohydrate structure contributive to antibody activity is considered as fucosyl residues (the Shields et al., 2002 that Fc areas N- connections oligosaccharides penetralia N-acetyl-glucosamine (GlacNAc) residue is attached to by the keys of α 1,6；Shinkawa et al., J.Biol.Chem.278 (5)：3466-3473(2003)).Fc γ R, which are combined, requires there is the oligosaccharides (Wright ＆ Morrison, 1997) for being covalently attached to and being guarded in Fc areas at Asn297.The notable rise of cytotoxicity (ADCC) activity of non-fucosylated structures and external antibody dependent cellular is connected to (Shields et al., 2002 recently；Shinkawa et al., 2003).Several laboratories including us successfully using RNA interference (RNAi) or knockout technology to transform Chinese hamster ovary celI to reduce FUT8 mRNA transcript levels or to knock out gene expression (Mori et al., 2004 completely；Yamane-Ohnuki et al., 2004).Mori et al. was reported in 2004, and by the way that cell is transformed into constitutive expression for the siRNA of FUT8 genes and application LCA selections, the antibody producing cells system for the stabilization set up is transformed into the cell line for producing the antibody that ADCC is improved.Mori demonstrates the generation for including the antibody until 70% non-fucosylated glycan.NiwaR.et al., Cancer Res.64 (6)：2127-2133 (2004) has been reported in the mouse model of human PBMC is implanted with, and the anti-CD 20 antibodies with relatively low fucose content can significantly extend the survival of animal.

In history, at Chinese hamster ovary cell (CHO), about 2-6% non-fucosylated antibody is included in the antibody population generated in one of the most frequently used industrial host.But, YB2/0 (rat bone myeloma) and Lec13 (the agglutinin mutant of CHO systems, it has deficiency GDP- mannoses 4,6- dehydratases, cause α 1, the sugared intermediate of the substrate GDP- fucoses or GDP- of 6- fucosyltransferases lacks (Ripka etal., 1986)) cell line can generate the antibody with the non-fucosylated types of 78-98%.Regrettably, the antibody production from these cells is extremely low, therefore it is unpractical with antibody product that these cell lines prepare treatment for commerciality.FUT8 gene codes α 1,6- fucosyltransferases, it is catalyzed the 6th (the Yanagidani etal., J.Biochem 121 that fucosyl residues are transferred to Asn- connections (N- connections) GlcNac of N- glycan from GDP- fucoses：626-632(1997)).Known α 1-6 fucosyltransferases are unique enzymes for being responsible for adding to fucose into the double feeler carbohydrate of N- connections at Asn297 in IgG antibody CH2 domains.

The antibody that antibody Fc district is attached with the ripe carbohydrate structure for lacking fucose is recorded in U.S. Patent application US 2003/0157108 (Presta, L.).It is related to " de- fucosylated " or " fucose deficiency " antibody, including the example of the publication of anti-CD 20 antibodies includes：US 2003/0157108；WO2000/61739；WO 2001/29246；US 2003/0115614；US 2002/0164328；US2004/0093621, US 2004/0132140, and US 2004/0110704 (3 are all Kyowa HakkoKogyo Co., Ltd's)；US 2004/0110282；US 2004/0109865；WO 2003/085119；WO 2003/084570；WO 2005/035586；WO 2005/035778；WO 2005/053742；US2006/0063254；US 2006/0064781；US 2006/0078990；US 2006/0078991；US6,602,684 and US 2003/0175884 (Glycart Biotechnology)；Yamane-Ohnuki et al., Biotech.Bioeng.87：614(2004)；Mori et al., Biotechnology and Bioengineering88 (7)：901-908(2004)；Li et al., are published in NatureBiology (GlycoFi) on January 22nd, 2006 online；Niwa R.et al., Cancer Res.64 (6)：2127-2133(2004)；Okazaki et al., J.Mol.Biol.336：1239-1249(2004)；Yamane-Ohnuki et al., Biotech.Bioeng.87：614(2004)；Shinkawa et al., J.Biol.Chem.278 (5)：3466-3473(2003).Producing the example for the cell line for taking off fucosylated antibody includes Lec13 Chinese hamster ovary celIs (Ripka the et al., Arch.Biochem.Biophys.249 of the fucosylated defect of protein：533-545(1986)；U.S. Patent application US2003/0157108 A1, Presta, L；With WO 2004/056312 A1, Adams et al., especially embodiment 11), and knock out cell line, Chinese hamster ovary celI (Yamane-Ohnuki et al., Biotech.Bioeng.87 that such as α -1,6- fucosyl transferase genes FUT8 is knocked out：614(2004)).It is relevant that there are the glycosylated antigen binding molecules of improvement referring also to US 2005/0123546 (Umana et al.).

RNA interference (RNAi) is highly conserved, sequence-specific PTGS mechanism, and it uses double-stranded RNA (dsRNA) to trigger the degraded of homologous mRNA as signal.The medium of sequence-specific mRNA degradeds is to cut the 21-23nt interference tiny RNA (siRNA) that longer dsRNA is produced by rnase iii.DsRNA is the potent inducer of I types interferon (IFN) synthesis, is also the activator of the enzyme (the latent ribalgilase RNase L of its product activation) of two class IFN inductions.These nonspecific responses to dsRNA are not shorter than 30bp dsRNA triggerings.Topmost processing product is the duplex of the 21 and 22nt RNA with the symmetrical jags of 2nt 3 ', and it is also most effective medium (Elbashir the et al., Nature 411 of mRNA degradeds：494-498(2001)；Elbashir et al., Methods 26：199-213(2002)).

Paying close attention to the patent and patent publications of CD20 antibody includes United States Patent (USP) 5,776,456,5,736,137,5,843,439,6,399,061 and 6,682,734, and U.S. Patent application US 2002/0197255A1, US2003/0021781A1, US2003/0082172A1, US2003/0095963A1, US2003/0147885A1 (Anderson etc.)；United States Patent (USP) 6,455,043B1 and WO00/09160 (Grillo-Lopez, A.)；WO00/27428 (Grillo-Lopez and White)；WO00/27433 (Grillo-Lopez and Leonard)；WO00/44788 (Braslawsky etc.)；WO01/10462 (Rastetter, W.)；WO01/10461 (Rastetter and White)；WO01/10460 (White and Grillo-Lopez)；US2001/0018041A1, US2003/0180292A1, WO01/34194 (Hanna and Hariharan)；U. S. application US2002/0006404 and WO02/04021 (Hanna and Hariharan)；U. S. application US2002/0012665A1 and WO01/74388 (Hanna, N.)；U. S. application US2002/0058029A1 (Hanna, N.)；U. S. application US2003/0103971A1 (Hariharan and Hanna)；U. S. application US2002/0009444A1 and WO01/80884 (Grillo-Lopez, A.)；WO01/97858 (White, C.)；U. S. application US2002/0128488A1 and WO02/34790 (Reff, M.)；WO02/060955 (Braslawsky etc.)；WO02/096948 (Braslawsky etc.)；WO02/079255 (Reff and Davies)；United States Patent (USP) 6,171,586B1 and WO98/56418 (Lam etc.)；WO98/58964 (Raju, S.)；WO99/22764 (Raju, S.)；WO99/51642, United States Patent (USP) 6,194,551B1, United States Patent (USP) 6,242,195B1, United States Patent (USP) 6,528,624B1 and United States Patent (USP) 6,538,124 (Idusogie etc.)；WO00/42072 (Presta, L.)；WO00/67796 (Curd etc.)；WO01/03734 (Grillo-Lopez etc.)；U. S. application US2002/0004587A1 and WO01/77342 (Miller and Presta)；U. S. application US2002/0197256 (Grewal, I.)；U. S. application US2003/0157108A1 (Presta, L.)；United States Patent (USP) 6,565,827B1,6,090,365B1,6,287,537B1,6,015,542,5,843,398 and 5,595,721 (Kaminski etc.)；United States Patent (USP) 5,500,362,5,677,180,5,721,108,6,120,767,6,652,852B1 (Robinson etc.)；United States Patent (USP) 6,410,391B1 (Raubitschek etc.)；United States Patent (USP) 6,224,866B1 and WO00/20864 (Barbera-Guillem, E.)；WO01/13945 (Barbera-Guillem, E.)；WO00/67795(Goldenberg)；U. S. application US2003/0133930A1 and WO00/74718 (Goldenberg and Hansen)；WO00/76542 (Golay etc.)；WO01/72333 (Wolin and Rosenblatt)；United States Patent (USP) 6,368,596B1 (Ghetie etc.)；United States Patent (USP) 6,306,393 and U. S. application US2002/0041847A1 (Goldenberg, D.)；U. S. application US2003/0026801A1 (Weiner and Hartmann)；WO02/102312 (Engleman, E.)；U.S. Patent application US2003/0068664 (Albitar etc.)；WO03/002607 (Leung, S.)；WO03/049694, US2002/0009427A1 and US2003/0185796A1 (Wolin etc.)；WO03/061694 (Sing and Siegall)；US2003/0219818A1 (Bohen etc.)；US2003/0219433A1 and WO03/068821 (Hansen etc.)；US2002/0136719A1 (Shenoy etc.)；WO2004/032828 (Wahl etc.)；WO2004/035607 (Teeling etc.)；US2004/0093621 (Shitara etc.).Referring also to United States Patent (USP) 5,849,898 and European application 330,191 (Seed etc.)；United States Patent (USP) 4,861,579 and EP332,865A2 (Meyer and Weiss)；WO95/03770 (Bhat etc.), US2001/0056066 (Bugelski etc.)；WO2004/035607 (Teeling etc.)；WO2004/056312 (Lowman etc.)；US2004/0093621 (Shitara etc.)；With WO2004/103404 (Watkins etc.).The publication of concern CD20 antibody includes：Teeling, J.et al., " Characterisation of new human CD20 monoclonal antibodieswith potent cytolytic activity against non-Hodgkin ' s lymphomas " Blood, Jun2004；10.1182.

In the FUT8 as described in Yamane-Ohnuki 2004 and Kyowa Hakko patents knocks out cell line, the generation of antibody needs to be transfected into the gene for encoding expectation antibody into the knockout cell line set up.A kind of effective method is needed to produce antibody in cell line is expected, while the fucose content of recombined engineering antibody is controlled, and without undergoing the complicated processes of the establishment FUT8 gene knockouts in selected cell line every time.Needed and with obvious other advantages in being detailed below present invention accomplishes this.

Summary of the invention

It is to change the amino acid sequence in Fc areas to an approach of Fc γ RIII binding affinity to improve antibody (refering to Shields et al., 2002).Humanized anti-cd 20 antibodies variant shown in table 3 is mixed with enhancing Fc γ RIII with reference to the amino acid replacement with ADCC in Fc.The invention provides the method for producing the antibody with relatively low fucose content, the additive effect to Fc γ RIII affinity and ADCC is shown during the amino acid change joint that enhancing Fc γ RIII are combined in Yu Fc areas.

The invention provides produce the antibody comprising IgG Fc in mammalian host cell to reduce the method for antibody fucose content simultaneously, including second of nucleic acid of the nucleic acid of at least one encoding antibody and at least two siRNA in coding targeting SEQ IDNO.1 FUT8 gene order different codings area is introduced into host cell simultaneously, wherein siRNA suppresses FUT8 expression and reduces the fucosylation level of antibody.

Low (knockdown) fucosylated method is struck simultaneously present invention also offers the more effective antibody producing cells system that produces, and the antibody of the generation ADCC compared with the antibody synthesized in mammalian cell with normal fucosylation level improves to some extent.Such method can be taken to build the cell line of the high antibody producing power of presentation and controlled fucosylation level.Such cell line can be used for the scale expanding production of antibody, as in the commodity production for the treatment of antibody.Therefore, there is provided the method for producing the IgG antibody with improved ADCC, including second of nucleic acid of the nucleic acid of at least one encoding antibody and at least two siRNA in coding targeting SEQ ID NO.1 FUT8 gene order different codings area introduced into host cell simultaneously, wherein antibody and siRNA expressed in cell with produce the antibody produced by the cell without siRNA compared to fucosylated reduce the elevated antibody of ADCC activity.

In an embodiment of the method for producing the IgG antibody that ADCC improves to some extent, the antibody is included in Fc areas improves antibody to Fc γ RIII combination and/or ADCC amino acid change at least one.The antibody can include S298A, E333A, K334A Fc amino acid replacements.

Present invention also offers the method for the composition for preparing the humanization CD20 binding antibodies for lacking fucose.

In an embodiment of the inventive method, light (L) chain of the nucleic acid encoding antibody of encoding antibody and again (H) both chains.In one embodiment, H the and L chains and siRNA of antibody are encoded on same expression vector.In an alternate embodiment, H and L chains are encoded on separated expression vector, in addition, each the expression vector of coding H and L chains also includes at least two siRNA of coding nucleic acid.

In an embodiment of all preceding methods, two siRNA are expressed under the control of different promoters.When Pol III promoters are used to drive siRNA transcriptions in expression vector, a siRNA can be expressed under the control of H1 promoters, and second siRNA is expressed under different Pol III promoters U6 control.

In a specific embodiment, first and second siRNA target SEQ ID NO.1 respectively FUT8 gene orders 733-751 and 1056-1074 nucleotides.

In any embodiment of above method, preferred antibody fucosylation level reduction at least 90%, more preferably at least 95%, even more desirably at least 99%.

Additionally provide the antibody produced with above method.

In a preferred embodiment of all preceding methods, the antibody is treatment antibody.In one embodiment, the antibody binding CD20.In one embodiment, the antibody binding BR3.In preferred embodiments, the antibody binding people and the CD20 of other primates.In one embodiment, CD20 binding antibodies are humanized antibodies.In preferred embodiments, the humanized antibody is humanization 2H7 antibody, preferably described in table 3 below and 4.In different embodiments, the humanized antibody includes one of these following paired VL and VH areas：SEQ ID NO.2 L chain variable region sequences and SEQ ID NO.8 H chain variable region sequences；SEQ ID NO.25 L chain variable region sequences and SEQ ID NO.22 H chain variable region sequences；Or SEQ ID NO.25 L chain variable region sequences and SEQ ID NO.33 H chain variable region sequences.In specific embodiments, humanization 2H7 antibody includes following sequence of a pair of L and H chains：SEQ ID NO.13 and SEQ ID NO.14；SEQ ID NO.26 and SEQ ID NO.27；And SEQ ID NO.26 and SEQ ID NO.34.

Other embodiments of Humanized anti-cd 20 antibodies have hA20 (also known as IMMU-106, or 90Y-hLL2；US 2003/0219433, Immunomedics)；With AME-133 (US 2005/0025764；Applied Molecular Evolution/Eli Lilly).In a different embodiment, the CD20 binding antibodies are human antibodies, preferably HUMAX-CD20^TM(GenMab).In another different embodiment, the CD20 binding antibodies are chimeric antibodies, and preferred embodiment is rituximab (Genentech, Inc.) and chimeric cA20 antibody (referring to US 2003/0219433, Immunomedics).

In another embodiment, the antibody produced with the inventive method is the antibody with reference to BR3.

Invention additionally provides the nucleic acid of the sequence comprising SEQ ID NO.42 and SEQ ID NO.43, SEQ ID NO.42 and SEQ ID NO.43 sequential coding two siRNAs complementary with the Liang Ge different codings area of FUT8 genes.

There is provided comprising humanization CD20 binding antibodies and the composition of carrier with Fc areas, wherein at least 95% antibody does not have fucose in composition.

In a preferred embodiment, the host cell is Chinese hamster ovary (CHO) cell or derivatives thereof.

On the other hand it is the host cell of the nucleic acid comprising at least one encoding antibody and at least two siRNA in coding targeting SEQ ID NO.1 FUT8 gene order different codings area second of nucleic acid, antibody described in host cell expression therein and the siRNA.

Additionally provide the purposes for treating disease with foregoing fucose deficiency antibody compositions.

Brief description

Fig. 1：It is attached with (N- connections) GlcNac of the Asn- connections of the N- glycan of fucosyl residues.

Fig. 2：RNAi technology mediates the schematic diagram of the suppression to gene expression.

Fig. 3：For the diagram for the pSilencer3.1-H1 Puro plasmids for producing FUT8 specific siRNAs.Referring to embodiment 1.

Fig. 4：RNAi probe sequences.Five kinds of sequences are devised according to the rule announced in document.Bolded sequence is mutually complementary and represents produced RNA hairpin portion.The RNAi1-5 that probe 1-5 corresponds in Fig. 5 B.Referring to embodiment 1.

Fig. 5 A and 5B：Fig. 5 A show total length and flag-FUT8 fusion constructs and the schematic diagram of probe region.Fig. 5 B show the Western blotting of the part CHO FUT8 albumen with the anti-flag antibody tests flag marks of M2.Referring to embodiment 1.

Fig. 6：The fucose content (being expressed as % non-fucosylated) of the 2H7 antibody of cell is transiently transfected from RNAi 2 and RNAi 4, as described in Example 2.

Fig. 7 A-E：Binding activity of the 2H7 antibody containing less fucose to different Fc γ acceptors：Fc γ RI (Fig. 7 A)；Fc γ RIIA (Fig. 7 B)；Fc γ RIIB (Fig. 7 C)；Fc γ RIII F158 (Fig. 7 D)；With Fc γ RIII V158 (Fig. 7 E), as described in Example 2.

Fig. 8：Northern engram analysis.FUT8 mRNA are about 3.5kb, and size is similar to rat FUT8.Swimming lane 2 and swimming lane 3 show the FUT8 fewer than the control of swimming lane 1.Referring to embodiment 2.

Fig. 9 A and 9B：Summarize the flow chart of the fucosylated less clone of exploitation.Fig. 9 A：Standard cell lines system develops code.Fig. 9 B：New cell line exploitation code comprising RNAi units in expression plasmid.

Figure 10 A, 10B and 10C：Plasmid construct.Figure 10 A：Control plasmid groups of the antibody HC and LC on separated plasmid；Figure 10 B：Test plasmids of the HC and LC on the separated plasmid comprising one or two RNAi transcriptional units；Figure 10 C：Test plasmids of the HC and LC on the same plasmid comprising one or two RNAi transcriptional units.Abbreviation：HC, heavy chain；LC, light chain；CMV：Cytomegalovirus promoter and enhancer sequence；PUR-DHFR：Puromycin and dihyrofolate reductase fusion.Referring to embodiment 4.

Figure 11 A and 11B：The antibody expression for the clone that stable transfection is obtained, as described in Example 4.For each plasmid transfection, 72 MTX resistance clones of picking are simultaneously screened with ELISA to antibody expression.Figure 11 A：The expression titre of CMV.PD.v511.RNAi4 plasmid transfections.Figure 11 B：The expression titre of CMV.PD.v511.RNAi2.4 plasmid transfections.

Figure 12：The Taqman analyses of FUT8 mRNA level in-sites.From the clone purification total serum IgE derived from CMV.PD.v511RNAi4 and CMV.PD.v511.RNAi2.4 plasmid transfections.FUT8 mRNA level in-sites are measured with the Taqman primer and probes to FUT8 gene specifics.Referring to embodiment 4.

Figure 13：Equal inoculum density determination method.By two control clones from CMV.PD.v511 plasmid transfections, two from CMV.PD.v511.RNAi4 plasmid transfections and with minimum non-fucosylated clone and four are from CMV.PD.v511.RNAi2.4 plasmid transfections and have minimum non-fucosylated clone with 5 × 10⁴The density of individual cells/well, which is inoculated in 96 orifice plates, supplies antibody producing.Antibody titer is determined with ELISA.

Referring to embodiment 4.

Figure 14：The non-fucosylation level of the humanization 2H7.v511 antibody produced with the clone of RNAi4 or RNAi2.4 plasmid transfections.It is included in about 5% non-fucosylated 2H7.v511 (v511 in figure) as control in determination method.Referring to embodiment 4.

Figure 15 A and 15B：The Fc γ RIII binding affinities of the fucosylated variant of humanization 2H7.v511 antibody.Figure 15 A compare binding affinity of the antibody to the F158 low-affinity isotypes of Fc γ RIII acceptors；Figure 15 B compare the binding affinity to V158 high-affinity receptor isotypes.Control is that have about 5% non-fucosylated h2H7.v511.Referring to embodiment 4.

Figure 16 A and 16B：ADCC activity determination method.The ADCC activity of two humanization 2H7 variants, i.e. v16 and v511 and their non-fucosylated (NF) variant is compared with the determination method based on cell using Wil2-S cells.2H7.v16 and .v511 antibody compositions have about 5% it is non-fucosylated.V16-NF and v511-NF variants have about 65-70%'s non-fucosylated.Figure 16 A show the ADCC activity that VF158 donor NK cells are used in determination method, and Figure 16 B show the activity using VV158 donorcellses.

Figure 17 shows encoding full leng CHO FUT8 DNA sequence dna (SEQ ID NO.1).

The detailed description of preferred embodiment

" CD20 " antigen is non-glycosylated cross-film phosphoprotein found on the B cell surface more than 90% from peripheral blood or lymphoid organ, molecular weight about 35kD.CD20 is expressed before early stage in (pre) B cell growth course, and is retained to plasma cell differentiation；It can not find on human stem cell, lymphoid progenitors or normal plasma cells.CD20 is present on both normal B cells and malignant B cell.The other titles of CD20 in the literature include " bone-marrow-derived lymphocyte limitation differentiation antigen " and " Bp35 ".CD20 antigens are recorded in such as Clark and Ledbetter, Adv.Can.Res.52：81-149 (1989) and Valentine et al., J.Biol.Chem.264 (19)：11282-11287(1989).

Term " antibody " is used with broadest herein, monoclonal antibody (including full length monoclonal antibodies), multi-specificity antibody (such as bispecific antibody) and antibody fragment are clearly covered, as long as they show desired biological activity or function.

The biological activity of the humanization CD20 binding antibodies of the present invention will at least include the combination of antibody and h CD20, more preferably and people and other primate CD20 (including macaque, rhesus macaque, chimpanzee, baboon) combination.Antibody can be to be not higher than 1 × 10^-8K_dValue, is preferably no greater than about 1 × 10^-9K_dValue combination CD20, and can kill in vivo or cut down B cell, preferably at least up to 20% compared with the suitable negative control of this unused antibody-like processing.B cell abatement can be the result of ADCC, CDC, apoptosis or other mechanism one or more of which.In some embodiments of disease treatment herein, it may be desirable to which specific effect device function or mechanism surpass others, and those biological functions, such as ADCC are preferably realized with humanization 2H7 some variants.

" Fv " is that the minimum antibody fragment with binding site is recognized comprising intact antigen.The fragment is made up of the dimer of close, Non-covalent binding a heavy chain variable domain and a light-chain variable domain.Six hypervariable loops (heavy chain and each 3 rings of light chain) are given out from the foldable structure of the two domains, facilitates the amino acid residue with reference to antigen and assigns antibody with antigen-binding specificity.However, even single variable domain (or only including half of Fv of three CDR to antigen-specific) also has the ability for recognizing and combining antigen, simply affinity is less than entire binding site.

Term " monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity as used herein, that is each antibody of composition colony is identical in terms of primary amino acid sequences and/or combines same epitope, issuable during except production monoclonal antibody to become external, such variant is general to be existed with indivisible.Such monoclonal antibody is typically include the antibody for including the peptide sequence for combining target, and wherein target Binding peptide sequence is by including selecting the process including single target Binding peptide sequence to obtain in many peptide sequences of comforming.For example, selection course can be comform polyclonal such as hybridoma clone, phage clone or recombinant DNA clone set in select Unique clones.It should be understood that, selected target binding sequence can further change, such as in order to improve affinity to target, by target binding sequence humanization, improve its yield in cell culture, reduce its immunogenicity in vivo, create multi-specificity antibody, and the antibody comprising the target binding sequence after change is also the monoclonal antibody of the present invention.From the typical polyclonal antibody preparations difference included for the different antibodies of different determinants (epitope), every kind of monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.Specificity at them is outer, and the advantage of monoclonal antibody preparations is that they are generally not affected by the pollution of other immunoglobulins.Modifier " monoclonal " show antibody basically homogeneity antibody population obtain feature, should not be construed as require antibody is produced by any ad hoc approach.For example, the monoclonal antibody used according to the present invention can be generated by multiple technologies, including such as hybridoma (such as Kohler et al., Nature 256：495(1975)；Harlow et al., Antibodies：A Laboratory Manual, ColdSpring Harbor Laboratory Press, 2nd ed.1988；Hammerling et al., in：Monoclonal Antibodies and T-Cell Hybridomas, 563-681, Elsevier, N.Y, 1981), recombinant DNA method is (see, for example, United States Patent (USP) No.4,816,567), display technique of bacteriophage is (see, for example, Clackson et al., Nature 352：624-628(1991)；Marks et al., J.Mol.Biol.222：581-597(1991)；Sidhu et al., J.Mol.Biol.338 (2)：299-310(2004)；Lee et al., J.Mol.Biol.340 (5)：1073-1093(2004)；Fellouse, Proc.Nat.Acad.Sci.USA101 (34)：12467-12472(2004)；Lee et al., J.Immunol.Methods 284 (1-2)：119-132 (2004)) and for generating the technology of people or human-like antibodies (see, for example, WO 1998/24893 in the animal of the gene with part or whole human immunoglobulin gene's seat or encoding human immunoglobulin's sequence；WO 1996/34096；WO 1996/33735；WO 1991/10741；Jakobovits et al., Proc.Natl.Acad.Sci.USA 90：2551(1993)；Jakobovits et al., Nature 362：255-258(1993)；Bruggemann et al., Year in Immuno.7：33(1993)；United States Patent (USP) No.5,545,806；5,569,825；5,591,669 (belonging to GenPharm)；5,545,807；WO 1997/17852；United States Patent (USP) No.5,545,807；5,545,806；5,569,825；5,625,126；5,633,425；5,661,016；Markset al., Bio/Technology 10：779-783(1992)；Lonberg et al., Nature 368：856-859(1994)；Morrison, Nature 368：812-813(1994)；Fishwild et al., NatureBiotechnology 14：845-851(1996)；Neuberger, Nature Biotechnology 14：826(1996)；Lonberg and Huszar, Intern.Rev.Immunol.13：65-93(1995)).

" functional fragment " of the CD20 binding antibodies of the present invention refers to those and retains the fragment that the biological activity including abatement B cell is shown with they derivative intact full length molecule with substantially the same affinity combination CD20 and according to external or in vivoassay method (such as those are as described herein) measurement.

Term " variable " refers to the extensive truth of some of variable domain section sequence difference between antibody.V structure domain mediate antigen combines and limits specificity of the specific antibodies to its specific antigen.However, variability is not uniformly distributed in 110 amino acid of variable domain leap.In fact, V areas are by 15-30 amino acid, the referred to as section not made a variation relatively of framework region (FR) and each length for distinguishing framework is 9-12 amino acid, is referred to as the shorter region composition of the extreme variation of " hypervariable region ".Each self-contained four FR of variable domain of native heavy and light chain, they take beta-pleated sheet conformation mostly, are connected by three hypervariable regions for forming loop connecting and formed in some cases a beta-pleated sheet structure part.Hypervariable region in every chain the keeping together closely by FR, and facilitate the formation of the antigen binding site of antibody together with the hypervariable region of another chain (referring to Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD (1991)).Constant domain does not participate in the combination of antibody and antigen directly, but shows the participation of antibody in a variety of effector functions, the cytotoxicity (ADCC) of such as antibody dependent cellular.

Term " hypervariable region " refers to the amino acid residue for being responsible for antigen binding in antibody as used herein.Hypervariable region generally comprises amino acid residue (such as V from " complementary determining region " or " CDR "_LIn residue 24-34 (L1), 50-56 (L2) and 89-97 (L3) nearby and V_HIn residue 31-35B (H1), 50-65 (H2) and 95-102 (H3) near；Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residue (such as V from " hypervariable loop "_LIn residue 26-32 (L1), 50-52 (L2) and 91-96 (L3) and V_HIn residue 26-32 (H1), 52A-55 (H2) and 96-101 (H3)；Chothia and Lesk, J.Mol.Biol.196：901-917(1987)).

When referenced herein, " consensus sequence " or shared V structure domain sequence refer to the artificial sequence compared derived from known human immunoglobulin(HIg) variable region sequences amino acid sequence.Compared based on these, prepare the recombinant nucleic acid sequence of coding V structure domain amino acid sequence, the V structure domain amino acid sequence is the consensus sequence of derived from human κ chains and people's H chain subclass IIIV domains.Shared V sequences do not have any of antibody binding specificity or affinity.

A part for heavy chain and/or light chain is identical or homologous with derived from particular species or the corresponding sequence belonged in the antibody of specific antibodies classification or subclass in " chimeric " antibody (immunoglobulin), and the remainder of chain is identical or homologous with derived from another species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass, and the fragment of this antibody-like, as long as they show desired biological activity (United States Patent (USP) No.4,816,567；Morrison et al., Proc.Natl.Acad.Sci.USA 81：6851-6855(1984)).Humanized antibody used herein is a subset of chimeric antibody.

" humanization " form of inhuman (such as mouse) antibody refers to the chimeric antibody that bottom line includes the sequence derived from non-human immunoglobulin.Largely, some hypervariable region residues immunoglobulin that some hypervariable region residues with non-human species' (donor antibody) such as mouse, rat, rabbit or the non-human primate for expecting specificity, affinity and ability are replaced that humanized antibody refers in human immunoglobulin(HIg) (receptor antibody).In some cases, Fv framework regions (FR) residue of human immunoglobulin(HIg) is replaced with corresponding non-human residues.In addition, humanized antibody can be included in the residue not found in receptor antibody or donor antibody.It is to further improve the performance of antibody such as binding affinity to carry out these modifications.In general, humanized antibody will include at least one, usually two substantially whole following variable domains, wherein entirely or substantially upper whole hypervariable loop corresponds to the hypervariable loop of non-human immunoglobulin, and entirely or substantially upper whole FR is the FR of human immunoglobulin sequence, although FR can include the amino acid replacement of one or more raising binding affinities.The number of the amino acid replacement of these in FR is generally no more than at 6 in heavy chain, is no more than in light chain at 3.Humanized antibody optionally will also include at least part constant region for immunoglobulin (Fc), the typically constant region of human immunoglobulin(HIg).More details are referring to Jones et al., Nature 321：522-525(1986)；Riechmann et al., Nature 332：323-329(1988)；Presta, Curr.Op.Struct.Biol.2：593-596(1992).

Antibody " effector functions " refers to those and is attributable to antibody Fc district (native sequences Fc areas or amino acid sequence variation Fc areas) and the biological activity changed with antibody isotype.The example of antibody mediated effect device function includes：C1q is combined and complement-dependent cytotoxicity；Fc acceptors are combined；The cytotoxicity (ADCC) of antibody dependent cellular mediation；Phagocytosis；Cell surface receptor (such as B-cell receptor) is lowered；With B cell activation.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " refer to the secreting type Ig being wherein attached to present on some cytotoxic cells (such as natural killer (NK) cell, neutrophil cell and macrophage) on Fc acceptors (FcR) and enable these cytotoxic effect cells to specifically bind the target cell for carrying antigen, and the cytotoxic form of target cell is then killed with cytotoxin.Antibody " arms " (arm) cytotoxic cell, and such lethal effect definitely requires.Mediation ADCC main cell, NK cells, an expression Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetchand Kinet, Annu.Rev.Immunol.9：The page tables 3 of 457-92 (1991) the 464th summarize the FcR expression on hematopoietic cell.For the ADCC activity of purpose of appraisals molecule, external ADCC determination methods, such as United States Patent (USP) No.5 can be carried out, 500,362 or 5,821,337 or Presta United States Patent (USP)s No.6 is described in 737,056.Effector cell available for such determination method includes PMNC (PBMC) and natural killer (NK) cell.Or can purpose of appraisals molecule in vivo ADCC activity, such as in animal model, such as Clynes et al., PNAS (USA) 95：Disclosed in 652-656 (1998).If antibody is CD20 binding antibodies, ADCC activity can be tested in the transgenic mice (hCD20+/hCD16+Tg mouse) that expression h CD20 adds CD16 as described below.

" human effector cell " refers to expression one or more FcR and exercises the leucocyte of effector functions.Preferably, the cell at least expresses Fc γ RIII and exercises ADCC effector functions.Mediating the example of ADCC human leukocytes includes PMNC (PBMC), natural killer (NK) cell, monocyte, cytotoxic T cell and neutrophil cell, preferably PBMC and NK cells.Effector cell can separate from its natural origin, such as blood.

The acceptor in " Fc acceptors " or " FcR " description binding antibody Fc areas.It is preferred that FcR be native sequences people FcR.Furthermore it is preferred that FcR be FcR (γ acceptors) with reference to IgG antibody, including Fc γ RI, the acceptor of Fc γ RII and Fc γ RIII subclass include the allelic variant and alternative splice forms of these acceptors.Fc γ RII acceptors include Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" suppression acceptor "), and they have similar amino acid sequence, and difference is main in its cytoplasmic domains.Activated receptor Fc γ RIIA are in its cytoplasmic domains comprising immunity receptor the activation motifs (ITAM) based on tyrosine.Suppress acceptor Fc γ RIIB and suppression motif (ITIM) of the immunity receptor based on tyrosine is included in its cytoplasmic domains (referring to summary Da ё ron, Annu.Rev.Immunol.15：203-234(1997)).FcR summary is referring to Ravetch and Kinet, Annu.Rev.Immunol.9：457-492(1991)；Capel et al., Immunomethods 4：25-34(1994)；De Haas et al., J.Lab.Clin.Med.126：330-41(1995).Term " FcR " covers other FcR herein, including those futures will be identified.The term also include neonatal receptor, FcRn, it is responsible for Maternal immunoglobulin G being transferred to fetus (Guyer et al., J.Immunol.117：587 (1976) and Kim et al., J.Immunol.24：249 (1994)) and adjust the dynamic equilibrium of immunoglobulin.

WO 00/42072 (Presta) describes the antibody variants that the combination to FcR is improved or reduced.The content of the patent publications is clearly taken in herein as reference.Referring also to Shields et al., J.Biol.Chem.9 (2)：6591-6604(2001).

On the binding affinity to FcRn, in one embodiment, the EC50 of antibody or apparent Kd (in pH 6.0) ＜=100nM, more preferably ＜=10nM.As for Fc γ RIII (F158；That is low-affinity isotype) binding affinity that improves, in one embodiment, EC50 or apparent Kd ＜=10nM, and for Fc γ RIII (V158；High-affinity), EC50 or apparent Kd ＜=3nM.It is known (see, for example, Ghetie 1997, Hinton 2004) and described below to measure to the method for FcRn combination.The Binding in vivo and serum half-life of people's FcRn high-affinities Binding peptide and people FcRn can be determined, such as in expression people FcRn transgenic mice or the human cell line through transfection, or in the primate that application of Fc variant polypeptides.In certain embodiments, the humanization 2H7 antibody of the present invention also includes the amino acid change in IgG Fc and shows the binding affinity improved to people FcRn, higher than the antibody with wild type IgG Fc at least 60 times, at least 70 times, at least 80 times, more preferably at least 100 times, preferably at least 125 times, even more desirably at least 150 times to about 170 times.

" complement-dependent cytotoxicity " or " CDC " refers to dissolving when there is complement to target cell.The activation of classic complement approach is antibody (suitable subclass) starting combined by the component of complement system first (C1q) with reference to its associated antigen.In order to assess complement activation, CDC determination methods can be carried out, such as such as Gazzano-Santoro et al., J.Immunol.Methods 202：Described in 163 (1996).

The polypeptide variants of the C1q binding abilities of Fc region amino acid sequences and raising or reduction with change are recorded in United States Patent (USP) No.6,194,551 B1 and WO 99/51642.The content of those patent publications is clearly taken in herein as reference.Referring also to Idusogie et al., J.Immunol.164：4178-4184(2000).

Through present specification and claims, unless otherwise indicated, the residue numbering mode of heavy chain immunoglobulin constant domain is such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, the numbering of EU indexes in MD (1991), clearly income is used as reference herein." the EU indexes in such as Kabat " refers to the residue numbering mode of human IgG1's EU antibody.The residue in V areas is numbered according to Kabat numberings, unless expressly stated sequentially or other numbering systems.

The example of CD20 antibody includes：" C2B8 ", is referred to as " rituximab " (" RITUXAN /MABTHERA  ") (United States Patent (USP) No.5,736,137) now；The 2B8 mouse antibody of yttrium [90] mark, is referred to as " Y2B8 " or " Ibritumomab Tiuxetan " (ZEVALIN ), can be from BiogenIdec, Inc. purchases (such as United States Patent (USP) No.5,736,137；2B8 was preserved in ATCC, numbering HB11388 on June 22nd, 1993)；Mouse IgG2a " B1 ", also referred to as " Tositumomab ", is optionally used¹³¹I marks to produce "¹³¹I-B1 " or " iodine 131 tositumomab " antibody (BEXXAR^TM), can be from Corixa purchases (referring also to United States Patent (USP) No.5,595,721)；Mouse monoclonal antibody " 1F5 " (such as Press et al., Blood 69 (2)：584-591 (1987)) and its variant, include 1F5 (such as WO 2003/002607, Leung, S. of " framework repairing " or humanization；ATCC preserved material HB-96450)；Mouse 2H7 and chimeric 2H7 antibody (such as United States Patent (USP) No.5,677,180)；Humanization 2H7 (such as WO 2004/056312 (Lowmanet al.) and listed hereinafter)；HUMAX-CD20^TMCD20 molecules (Genmab, Denmark in the high-affinity antibody of complete people, targeting B cell cell membrane；See, for example, Glennie and van de Winkel, DrugDidcovery Today 8：503-510 (2003) and Cragg et al., Blood 101：1045-1052(2003))；Listed human monoclonal antibodies in WO 04/035607 and WO 2005/103081 (Teeling et al., GenMab/Medarex)；Described Fc areas are combined with the antibody of the sugar chain of complicated N- glucosides connection in US 2004/0093621 (Shitara et al.)；With reference to CD20 monoclonal antibody and antigen-binding fragment (such as WO 2005/000901, Tedde et al.), such as HB20-3, HB20-4, HB20-25 and MB20-11；With reference to CD20 single chain protein matter (such as US 2005/0186216 (Ledbetter andHayden-Ledbetter)；US 2005/0202534(Hayden-Ledbetter and Ledbetter)；US2005/0202028(Hayden-Ledbetter and Ledbetter)；US 2005/0202023(Hayden-Ledbetter and Ledbetter)-Trubion Pharm Inc.)；Listed AME-133 in the CD20 binding molecules of such as AME series antibodies, such as WO 2004/103404 and US 2005/0025764 (Watkins et al., Applied Molecular Evolution, Inc.)^TMThe listed CD20 antibody with Fc mutation in antibody and such as WO2005/070963 (Allan et al., Applied Molecular Evolution, Inc.)；Described CD20 binding molecules in such as WO 2005/016969 and US 2005/0069545 (Carr et al.)；Such as in WO 2005/014618 (Chang et al.) listed bispecific antibody；Humanization LL2 monoclonal antibodies, such as US 2005/0106108 (Leung and Hansen；Described in Immunomedics)；For CD20 chimeric or humanization B-Ly1 antibody, such as WO2005/044859 and (the Umana et al. of US 2005/0123546；GlycArt BiotechnologyAG) in it is described；A20 antibody or its variant, such as chimeric or humanization A20 antibody (being cA20, hA20 respectively) and IMMUN-106 (such as US 2003/0219433, Immunomedics)；And monoclonal antibody L27, G28-2,93-1B3, B-C1 or NU-B2, (such as Valentine et al., in can be obtained from international leukocyte differential count seminar (International Leukocyte Typing Workshop)：Leukocyte Typing III, McMichael volumes, p.440, Oxford University Press (1987)).CD20 antibody preferred herein is humanization, the CD20 antibody of chimeric or people, more preferably rituximab, a kind of humanization 2H7, chimeric or humanization A20 antibody (Immunomedics), HUMAX-CD20^TMH CD20 antibody (Genmab) and the immunoglobulin/protein (Trubion Pharm Inc.) for combining CD20.

Term " BR3 ", " BR3 polypeptides " or " BR3 acceptors " are covered " native sequences BR3 polypeptides " as used herein.People BR3 sequences (SEQ ID NO：44)

1   MRRGPRSLRG RDAPAPTPCV PAECFDLLVR HCVACGLLRT PRPKPAGASS PAPRTALQPQ

61  ESVGAGAGEA ALPLPGLLFG APALLGLALV LALVLVGLVS WRRRQRRLRG ASSAEAPDGD

121 KDAPEPLDKV IILSPGISDA TAPAWPPPGE DPGTTPPGHS VPVPATELGS TELVTTKTAG

181 PEQQ

As used herein, " B cell abatement " refers to after medicine or Antybody therapy, compared with the level before treatment, the reduction of b cell level in animal or human body.Well-known determination method can be used to measure for b cell level, such as by obtaining whole blood count, by the facs analysis that is dyed to known B cell mark and by the method described in such as EXPERIMENTAL EXAMPLESThe.B cell abatement can be part or complete.In one embodiment, the abatement of expression CD20 B cell is at least 25%.In the patient for receiving B cell abatement medicine, the duration of B cell abatement is usually the time recovered circulation time and B cell of the medicine in patient's body.

" separation " antibody refers to the antibody that a kind of identified and from its natural surroundings composition is separated and/or reclaimed.The contaminant component of its natural surroundings refers to the material of the diagnosis that will disturb the antibody or therapeutical uses, it may include the solute of enzyme, hormone and other oroteins property or non-proteinaceous.In preferred embodiments, by antibody purification to the measure of (1) according to Lowry methods, antibody weight is more than 95%, most preferably weight is more than 99%, (2) the N- ends by using spinning cup sequenator at least 15 residues of acquisition or the degree of internal amino acid sequence are enough, or (3) are according to the SDS-PAGE under reproducibility or non-reducing conditions and use Coomassie blue or preferred Silver stain, reach homogeneity.Since at least one composition of antibody natural surroundings is not in, then the antibody of separation includes the antibody iM situ in recombinant cell.However, the antibody of separation will generally be prepared by least one purification step.

" separation " nucleic acid molecules refer to the nucleic acid molecules that at least one contaminative nucleic acid molecules being generally associated in identified and natural origin with antibody nucleic acids are separated.The nucleic acid molecules of separation are different from finding in nature at the form of it or background.Therefore the nucleic acid molecules of separation have any different with being present in nucleic acid molecules when in n cell.However, the nucleic acid molecules of separation include being often expressed as the nucleic acid molecules included in the cell of the antibody, such as when the chromosome mapping when the nucleic acid molecules in the cell is different from its chromosome mapping in n cell.

Statement " control sequence " refers to DNA sequence dna necessary to the coded sequence expressed and be operatively connected in specific host organism.For example, the control sequence suitable for prokaryotes includes promoter, optional operator sequence and ribosome bind site.Known eukaryotic utilizes promoter, polyadenylation signal and enhancer.

If one section of nucleic acid is in functional interrelationship with another section of nucleotide sequence, it is " being operatively connected ".If for example, presequence (presequence) or secreting the DNA of leading (secretory leader) and being expressed as participating in the preceding protein (preprotein) of polypeptide secretion, the DNA of it and the polypeptide is operatively connected；If promoter or enhancer influence the transcription of coded sequence, it is operatively connected with the sequence；Or, if the position of ribosome bind site promotes translation, it is operatively connected with coded sequence.In general, " being operatively connected " means that connected DNA sequence dna is adjacent, and mean adjacent in the case of secretion is leading and be in read state.However, enhancer need not be adjacent.Connection can be realized by the coupled reaction at convenient restriction site.If without such site, according to oligonucleotides adapter or joint of the conventional practice using synthesis.

" carrier " includes shuttle vector and expression vector.Typically, plasmid construction thing will also include replication orgin (such as ColE1 replication orgins) and selection marker (such as ampicillin or tetracyclin resistance), be respectively used to duplication and selection of the plasmid in bacterium." expression vector " refers to the carrier that control sequence or controlling element necessary to antibody includes the antibody fragment of the present invention are expressed included in bacterium or eukaryotic.Following discloses suitable carrier.

The cell of generation humanization CD20 binding antibodies such as humanization 2H7 antibody of the invention is by bacterium and eukaryotic host cell including wherein having had been introduced into the nucleic acid for encoding the antibody.Following discloses suitable host cell.

Term " label " refers to the detectable compounds or composition being directly or indirectly coupled with antibody as used herein.Label itself can be by itself just detectable (such as radioisotopic tracer or fluorescent marker), or in the case of enzyme marker, the chemical modification of detectable substrate compounds or composition can be catalyzed.

The method and composition of the present invention

RNAi is disturbed

Long double-stranded RNA (dsRNA；Usual ＞ 200nt) it can be used for expression of the silencing of target genes in a variety of organisms and cell type (such as worm, drosophila and plant).After pickup, long dsRNA enters the cellular pathways that commonly referred to as RNA disturbs (RNAi) approach.First, dsRNA is processed to 20-25 nucleotides (nt) siRNA (siRNA) (initial step) by being referred to as Dicer RNase III sample enzymes.Then, siRNA is assembled into the compound containing endoribonuclease of referred to as RNA induction silencing complex (RISC), untwists in the process.SiRNA chains then guide RISC at complementary RNA molecule, and they cut there associates RNA (effect step) with destruction.RNA cutting generations are associated in the near middle of siRNA chains institute calmodulin binding domain CaM, cause specific gene silencing.But, because strong antiviral response is presented in most of mammalian cells, it is characterized in that introduce be longer than after 30bp dsRNA it is non-specific suppress protein synthesis and RNA degradeds, so researcher with 21-23bp siRNA transfectional cells to induce RNAi in such systems without causing antiviral response.In the method for the invention, RNAi approach is induced using the specific dsRNA of at least one targeting specific gene transcription product (being FUT8 in this case).DsRNA is delivered into cell by any suitable dsRNA delivery systems.Suitable negative control by be any transcription product not in target biology body dsRNA (dsRNA for for example targetting luciferase).

In the method for the invention, RNAi approach is induced using the specific dsRNA of at least one targeting specific gene transcription product (being FUT8 in this case).In the culture cell of mammal, generally RNAi is induced by being introduced directly into siRNA or from DNA construct being expressed as hairpin structure in the cell.

The method for producing siRNA

There are 5 kinds of commonly known methods to produce the siRNA studied for gene silencing：(i) chemical synthesis；(ii) in-vitro transcription；(iii) long dsRNA is digested with the enzyme (such as Dicer, RNase III) of RNase III families；(iv) expressed in cell from siRNA expression plasmids or viral vector, and (vi) is expressed in cell from siRNA expression cassettes derived from PCR.First three methods are related to prepares siRNA in vitro, is then introduced directly into mammalian cell by fat transfection, electroporation or other technologies.Latter two method expresses the siRNA carrier and expression cassette based on DNA by introducing in the cell.All these methods, in addition to by digesting long dsRNA generations siRNA groups, are required for carefully designing siRNA making the silence of target gene maximize while minimizing the influence to (off-target) gene that misses the target.Chemical synthesis is preferred and most widely used siRNA preparation methods, the mammalian cell for transiently transfecting culture, and RNAi effects are then monitored by measured downstream method.SiRNA is easier transfection than plasmid.

Exemplary SiRNA expression vector has the pSilencer from Ambion companies (Austin, Texas)^TMSiRNA expression vector, it uses U6 (Kunkel and Pederson, 1988；Miyashiand Taira, 2002) or H1 polymerase III promoters siRNA is expressed in mammalian cell.For example, pSilencer3.0-H1 (plasmid composition is as shown in Figure 3) is using H1 RNA promoters as characteristic (H1 RNA are RNase P parts).Multiple choices mark such as hygromycin, neomycin, puromycin can be included in these carriers.PSilencer2.0-U6 and 3.0-H1 SiRNA expression vectors are linearized with BamH I and Hind III, the jag for being easy to directed cloning is left.In order to cause silence, the small DNA Insert Fragments for encoding the short hairpin RNA for targetting target gene are cloned into carrier, positioned at Pol III promoters downstream.Once being transfected into mammalian cell, the carrier containing Insert Fragment just expresses short hairpin RNA, and it is processed into rapidly siRNA by cell system.

SiRNA is delivered into culture cell

For many immortalized cell lines, siRNA transfection is carried out using the reagent based on lipid or amine, such as Ambion siPORT^TMLipid and siPORT^TM Amine Transfection Agents.For delivering into primary cell and suspension cell, carry out electroporation using buffer solution special, gentle to cell and the impulsive condition of optimization and generally result in the survival ability that effectively siRNA is delivered without damaging cell.

The control of siRNA experiments

The negative control dsRNA of luciferase (such as target) for not targetting any endogenous transcription product can be used as control due to introducing the non-specific influences caused by any siRNA to gene expression.(easy-to-assay) positive control easily determined can be used for optimization transfection conditions, it is ensured that siRNA is effectively delivered, and determines that specific measured downstream method is available.Because positive control is used for many different aspects of RNAi experiments, therefore often require over a kind of control.For transfection Optimal Experimental, Silencer^TMGAPDH siRNA are a kind of preferable positive controls.This siRNA effective reticence GAPDH is expressed and its effect can readily be monitored in rna level or by western blot or immunofluorescence by qRT-PCR or other methods in protein level.

The determination method of RNAi effects

There are several determination methods to can be used for measurement RNAi effects.Include the determination method based on cell, enzymatic determination method, array analysis available for the determination method for understanding the biology effect for striking low target gene.SiRNA plays their effect in mRNA level in-site.Confirm and transfect the most simple determination method dependence qRT-PCR of optimization to measure the target transcript levels for negative control siRNA handles cell in gene specific siRNA processing cell for siRNA.Applied Biosystems TaqMan  Gene ExpressionAssays, available for the gene more than 41,000 kinds of people, mouse and rats, it can also be used to this purpose.Ambion siRNA databases are provided and gene specific Silencer^TMThe link for each determination method that siRNA that is pre-designed and confirming is matched.It can also be assessed in protein level and strike low degree.Due to being recovered to native protein in most cases, therefore enzymatic determination method can also be carried out.SiRNA, said target mrna and target protein level can be associated.

The antibody of the present invention is comprising IgG Fc areas and generally combined in vitro and with Fc γ RIIIA in vivo and ADCC is presented.The mammalian host cell for being usually used in producing the antibody of the fragment with IgG Fc areas or its reservation Asn glycosylation site and ADCC effector functions typically produces a group antibody, and wherein 94-98% monoclonal antibody is fucosylated.The siRNA of 2 or more targeting FUT8 genes transfectant cells are produced and expressed by the inventive method will produce antibody expected from a group, they have the fucosylation level reduced compared with the antibody population produced by the host cell with normal, unmodified FUT8 expression, and result is that the antibody population of fucosylated reduction can generally improve Fc γ RIIIA combinations and/or ADCC when there is suitable effector cell.

In one embodiment, the antibody binding CD20, particularly primate of the fucosylated reduction produced by the inventive method CD20.In one embodiment, the CD20 of these antibody bindings people.

In one embodiment, the invention provides the humanization 2H7 antibody with reduced fucose produced by the inventive method.The generation of hu2H7 antibody is recorded in WO 04/056312 in detail, is completely collected herein by reference.In specific embodiments, the variant is 2H7.v16, hu2H7.v511 and hu2H7.v114.

In full length antibody, humanization CD20 binding antibodies of the invention are by the V structure domain comprising connected humanization and the C-structure domain of human immunoglobulin(HIg).In a preferred embodiment, H chains C areas come from human IgG, preferably IgG1 or IgG3.L chain C-structure domain is preferred from people's κ chains.

For the object of the invention, " humanization 2H7 " refers to following complete antibody or antibody fragment, and it includes light chain variable district (V_L) sequence：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP

SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGT

KVEIKR(SEQ ID NO：2)；

With weight chain variable district (V_H) sequence：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSS(SEQ ID NO：8).

If humanization 2H7 antibody is complete antibody, it is preferred that it includes v16 light-chain amino acid sequences：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP

SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC(SEQ ID NO：13)；

And heavy chain amino acid sequence：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPG(SEQ ID NO：14).

A kind of foregoing humanized 2H7 mAb variant is 2H7.v31, and it has and SEQ ID NO above：13 identical L chain-orderings and include following H chain amino acid sequences：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK(SEQ ID NO：15).

Another variant of foregoing humanized 2H7 antibody is such a antibody, its SEQ ID NO.25 comprising 2H7.v114 V_L：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKR(SEQ ID NO.25)；

With SEQ ID NO.22 V_H：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSASYWYFDVWGQGTLVTVSS(SEQ ID NO.22)。

2H7.v114 complete L chain amino acid sequences have following sequence：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC(SEQ ID NO：26)；

2H7.v114 complete H chain amino acid sequences be：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK(SEQ ID NO：27).

Another variant is 2H7.v138, and it includes SEQ ID NO.26 H chain amino acid sequences.

Another variant, 2H7.v477 includes SEQ ID NO.25 V_LWith SEQ ID NO.22 V_H, and with H chain amino acid sequences：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHWHYTQKS

LSLSPGK(SEQ ID NO：31).

Another variant of foregoing humanized 2H7 antibody is such a antibody, its SEQ ID NO.25 comprising 2H7.v511 V_LWith SEQ ID NO.33 V_H(being shown in Table 4)：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSYRYWYFDVWGQGTLVTVSS(SEQ ID NO.33)。

In one embodiment, antibody includes 2H7.v511 light chains (SEQ ID NO.26)：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC；

With 2H7.v511 heavy chains (SEQ ID NO.34)：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPG。

The V areas of all other variant based on v16 are by the amino acid sequence with v16, except the position of amino acid replacement shown in table 3 below.Unless otherwise indicated, 2H7 variants will have and v16 identical L chains.Humanized antibody 2H7.v16 is also referred to as rhuMAb2H7, PRO70769 or Ocrelizumab.

Table 3

2H7 Pattern	Light chain (VL) Change	Heavy chain (VH) Change	Fc Change
				16 supply Reference			-
31	-	-	S298A, E333A, K334A
				73	M32L	N100A
75	M32L	N100A	S298A, E333A, K334A
				96	S92A	D56A, N100A

114	M32L, S92A	D56A, N100A	S298A, E333A, K334A
				115	M32L, S92A	D56A, N100A	S298A, E333A, K334A, E356D, M358L
116	M32L, S92A	D56A, N100A	S298A, K334A, K322A
				138	M32L, S92A	D56A, N100A	S298A, E333A, K334A, K326A
477	M32L, S92A	D56A, N100A	S298A, E333A, K334A, K326A, N434W
				375	-	-	K334L
511	M32L, S92A	D56A, N100Y,  S100aR	S298A, E333A, K334A, K326A
				588	-	-	S298A, E333A, K334A, K326A

Table 4

2H7 Pattern	VL   SEQ ID NO.	VH     SEQ ID NO.	Complete L chains   SEQ ID NO.	Complete H chains   SEQ ID NO.
					16	2	8	13	14
31	2	8	13	15
					73	16	17	18	19
75	16	17	18	20
					96	21	22	23	24
114	25	22	26	27
					115	25	22	26	28
116	25	22	26	29
					138	25	22	26	30
477	25	22	26	31
					375	2	8	13	32
511	25	33	26	34
					588	2	8	35	36

Residue numbering mode is according to Kabat et al., Sequences of Immunological Interest, 5thEd.Public Health Service, National Institutes of Health, Bethesda, Md. (1991), insertion represents that breach is represented in sequence chart with dash with a, b, c, d and e.In the CD20 binding antibodies comprising Fc areas, the C- terminal lysines (residue 447 according to EU numbering systems) in Fc areas can remove, for example, transforming the nucleic acid of encoding antibody polypeptide in antibody purification procedures or by recombined engineering.Therefore, humanization 2H7 antibody compositions of the invention can include the antibody with K447, eliminate all K447 antibody or with the mixture with the antibody without K447 residues.

N- glycosylation sites in IgG are located at the Asn297 in CH2 domains.The humanization 2H7 antibody compositions of the present invention include the composition of any foregoing humanized 2H7 antibody with Fc areas, about 80-100% (preferably approximately 90-99%) antibody includes the ripe core carbohydrate structure for lacking fucose wherein in composition, is attached to the Fc areas of glycoprotein.Such composition confirms to show herein combines the surprising improvement of aspect with Fc γ RIIIA (F158), and Fc γ RIIIA (F158) are effective unlike Fc γ RIIIA (V158) in terms of with human IgG interaction.Fc γ RIIIA (F158) are more more conventional than Fc γ RIIIA (V158) in normal, healthy African American and Caucasian.Referring to Lehrnbeche etal., Blood 94：4220(1999).

Bispecific humanization 2H7 antibody covers such antibody, and wherein the one arm of antibody at least has the antigen binding domain of humanization 2H7 heavy chain of antibody of the present invention and/or L chains, and another arm has the V areas binding specificity for the second antigen.In specific embodiments, second antigen is selected from CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.

In certain embodiments, the humanization 2H7 antibody of the present invention further includes the amino acid change in IgG Fc, and show the binding affinity to people FcRn improved than the antibody with wild type IgG Fc, improve at least 60 times, at least 70 times, at least 80 times, more preferably at least 100 times, preferably at least 125 times, even more desirably at least 150 times to about 170 times.

If FUT8 transcription products or protein level in siRNA transfectional cells have measurable reduction with untransfected and expression FUT8 inhibitions siRNA's compared with the level in like cell, FUT8 expression is suppressed or struck low.The fucose content of FUT8 transcription products or protein and produced antibody in cell can be quantitative by process described below.It is preferred that the suppression level of FUT8 expression causes the fucosylation level of antibody in composition to reduce at least 65%, preferably 75-80%, more preferably 90%, even more preferably 95% or 99%.

Promoter available for driving siRNA expression has Pol type III promoters, such as H1 or U6 promoters.TRNA promoters can also be used.

Host cell will include eukaryotic, such as mammal and plant cell.Preferred host cell is mammalian cell, such as Chinese hamster ovary celI, but also provides other suitable host cells herein.

If antibody display goes out to combine level and ADCC activity has rise than the same sample antibody produced by being struck with normal FUT8 gene functions without RNAi and FUT8 in low host cell, Fc γ RIII are combined and/or ADCC is improved.Measurement Fc γ R are combined and ADCC method is described below.

The production of antibody

Monoclonal antibody

Monoclonal antibody can be used initially by Kohler et al., Nature 256：The hybridoma method that 495 (1975) are recorded is generated, or can be generated by recombinant DNA method (United States Patent (USP) No.4,816,567).

In hybridoma method, immune mouse as described above or other suitable host animals, such as hamster, to trigger generation or can generate the lymphocyte of following antibody, the antibody will specifically bind the protein for being used for being immunized.Or, can immunological lymphocyte in vitro.After immune, lymphocyte is separated, is then merged using suitable fusion agent such as polyethylene glycol with myeloma cell line, to form hybridoma (Goding, Monoclonal Antibodies：Principlrs and Practice, pp.59-103, AcademicPress, 1986).

By thus prepared hybridoma in suitable inoculation of medium and culture, the culture medium preferably comprises the one or more materials for suppressing Parent Myeloma Cell (also referred to as fusion partner) the growth or survival do not merged.For example, if Parent Myeloma Cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the selective medium for hybridoma will typically contain hypoxanthine, aminopterin-induced syndrome and thymidine (HAT culture mediums), and these materials prevent the growth of HGPRT deficient cells.

It is preferred that fusion partner myeloma cell be that those efficiently merge, support selected antibody producing cells stable and high-caliber generation antibody and the myeloma cell sensitive to carrying out the selective medium of selection for the parental cell not merged.It is preferred that myeloma cell line be rat bone marrow tumour system, such as those can be from Sol gram research institute cell distributing center (Salk Institute Cell Distribution Center, SanDiego, California, USA) MOPC-21 the and MPC-11 mouse tumors obtained are derivative and can be from American type culture collection (American Type Culture Collection, Rockville, Maryland, USA) obtain SP-2 and derivative such as X63-Ag8-653.Human myeloma and mouse-people's heteromyeloma cell lines also have record (Kozbor, the J.Immunol.133 for generating human monoclonal antibodies：3001(1984)；Brodeur et aL., Monoclonal Antibody Production Techniquesand Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

Generation of the culture based assays that hybridoma just can be wherein being grown for the monoclonal antibody of antigen.Preferably, by immunoprecipitation or by external binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), the binding specificity of the monoclonal antibody generated by hybridoma is determined.

The binding affinity of monoclonal antibody can be for example, by Munson et al., Anal.Biochem.107：Scatchard described in 220 (1980) analyzes to determine.

Once identifying hybridoma of the generation with the antibody for expecting specificity, affinity and/or activity, the clone can be subcloned by limiting dilution code, and be cultivated (Goding, Monoclonal Antibodies by standard method：Principles and Practice, pp.59-103, AcademicPress, 1986).Culture medium suitable for this purpose includes such as D-MEM or RPMI-1640 culture mediums.In addition, hybridoma can in animal as ascites tumor carry out In vivo culture, for example by by cell intraperitoneal injection into mouse.

Flow can be purified by conventional antibody, such as affinity chromatography (such as using albumin A or Protein G-Sepharose), ion-exchange chromatography, hydroxyapatite, gel electrophoresis, dialysis, will suitably it be separated with culture medium, ascites or serum by the monoclonal antibody of subclone secretion.

Encode the DNA routine protocols separation easy to use and sequencing (such as by using the oligonucleotide probe for the gene that can specifically bind coding mouse heavy chain and light chain) of monoclonal antibody.Hybridoma is such DNA preferred source.Once separation, DNA can be placed in expression vector, then the expression vector is transfected into not in the host cell of generation antibody protein in addition, such as Bacillus coli cells, Simian COS cells, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthesis of monoclonal antibody in recombinant host cell.The summary of recombination expressions of the DNA in bacterium on encoding antibody includes Skerra et al., Curr.Opinion in Immunol.5：256-262 (1993) and Pl ü ckthun, Immunol.Revs.130：151-188(1992).

In another embodiment, can be from use McCafferty et al., Nature 348：Monoclonal antibody or antibody fragment are separated in the phage antibody library of technique construction described in 552-554 (1990).Clackson et al., Nature 352：624-628 (1991) and Marks et al., J.Mol. Biol.222：581-597 (1991) describes the separation of the mouse carried out using phage library and human antibody respectively.Subsequent publications describe human antibody (Marks the et al., Bio/Technology 10 for reorganizing generation high-affinity (nM scopes) by chain：779-783 (1992)), and combination infects and In vivo recombination is used as strategy (Waterhouse et al., the Nuc.Acids Res.21 for building very big phage library：2265-2266(1993)).In this way, these technologies are the feasible alternative methods for separating the conventional monoclonal antibody hybridoma technology of monoclonal antibody.

The DNA of encoding antibody can be modified to generate chimeric or fusion antibody polypeptide, for example, pass through employment heavy chain and constant region of light chain (C_HAnd C_L) sequence replacing homologous murine sequences (United States Patent (USP) No.4,816,567；Morrison et al., Proc.Natl.Acad.Sci.USA 81：6851 (1984)), or by the way that immunoglobulin coding sequence is merged with the coded sequence all or in part of NIg polypeptide (heterologous polypeptide).The constant region of NIg polypeptide sequence replacing antibody can be used, or the variable region of an antigen binding site of antibody is substituted with them, to produce chimeric bivalent antibody, it is comprising having a specific antigen binding site to a kind of antigen and have another specific antigen binding site to not synantigen.

Humanized antibody

This area has described the method for humanizing non-human antibodies.Preferably, humanized antibody has one or more amino acid residues introduced from non-people source.These non-human amino acid residues are often referred to as " inputting " residue, and they are typically derived from " input " variable domain.Humanization can substantially follow Winter and its method for colleague carries out (Jones et al., Nature 321：522-525(1986)；Reichmann et al., Nature 332：323-327(1988)；Verhoeyen et al., Science239：1534-1536 (1988)), substitute corresponding human antibody sequence by using hypervariable region sequence.Therefore, such " humanization " antibody is chimeric antibody (United States Patent (USP) No.4,816,567), wherein substantially less than whole people's variable domain is substituted with the corresponding sequence from non-human species.In practice, humanized antibody is typically such human antibody, and some of some hypervariable region residues are substituted with some possible FR residues with the residue in the similar site in rodent antibodies.

It is extremely important for reduction antigenicity and HAMA (human anti-mouse antibody) response when antibody is intended for human therapeutic use by the selection of people's variable domain for building humanized antibody, including light chain and heavy chains.According to so-called " most suitable " (best-fit) method, the whole library of known people's variable domain sequence is screened with the variable domain sequence of rodent antibodies.Identify with the immediate people's variable domain sequence of rodent and receive people's framework region (FR) therein be used for humanized antibody (Sims et al., J.Immunol.151：2296(1993)；Chothia et al., J.Mol.Biol.196：901(1987)).Another method uses the specific frame area as derived from specific light chain or the consensus sequence of all human antibodies of heavy chain subgroup.Same framework can be used for several different humanized antibody (Carter et al., Proc.Natl.Acad.Sci.USA 89：4285(1992)；Presta et al., J.Immunol.151：2623(1993)).

What is more important, antibody keeps the high binding affinity and other favourable biological characteristicses to antigen after humanization.In order to realize this target, according to a kind of preferred method, analyze the method for parental array and various conceptual humanized products to prepare humanized antibody by using the threedimensional model of parent and humanized sequence.Three dimensional immunoglobulin model is typically available, and is familiar with by those skilled in the art.It can be illustrated and be shown the computer program of the possible three-dimensional conformation structure of selected candidate immunoglobulin sequences sequence.Check that these display images allow to analyze possibility effect of the residue in candidate immunoglobulin sequences sequence functions, i.e. analyzing influence candidate immunoglobulin sequences combine the residue of the ability of its antigen.So, FR residues can be selected from acceptor and list entries and are combined, so as to realize desired antibody characteristic, such as the affinity to target antigen is improved.In general, some hypervariable region residues are direct and the most substantial influence being related to antigen binding.

Humanized antibody can be antibody fragment, such as Fab, and it is optionally with one or more cytotoxic agent couplings to generate immune conjugate.Or, humanized antibody can be full length antibody, such as total length IgG1 antibody.

Human antibody and phage display method

As the alternative of humanization, human antibody can be generated.For example, it is now possible to the such transgenic animals (such as mouse) of generation, they can generate human antibody full repertoire in the case where lacking endogenous immunoglobulin generation after immune.For example, having described antibody heavy chain joining region (J in chimeric and germ line mutant mice_H) gene homozygosis delete cause endogenous antibody generate complete inhibition.A large amount of human germline immunoglobulin's gene transfers will be caused into such germ line mutant mice to generate human antibody after antigen is attacked.See, for example, Jakobovits et al., Proc.Natl.Acad.Sci.USA 90：2551(1993)；Jakobovits et al., Nature 362：255-258(1993)；Bruggemann et al., Year inImmuno.7：33(1993)；United States Patent (USP) No.5,545,806,5,569,825,5,591,669 (belonging to GenPharm)；5,545,807；WO 97/17852.

Or, display technique of bacteriophage (McCafferty et al., Nature 348：552-553 (1990)) it can be used in vitro from immunoglobulin variable domain (V) gene complete or collected works generation human antibody and antibody fragment from epidemic disease donor rather.According to this technology, antibody variable domain gene is cloned into the way of meeting reading frame in filobactivirus such as M13 or fd main or secondary coat protein gene, and functional antibody fragment is shown as on phage particle surface.Because filamentous phage particle includes the single-stranded DNA copy of phage genome, the selection carried out based on the functional characteristic of antibody also causes coding to show that the gene of the antibody of those characteristics is selected.In this way, bacteriophage simulates some characteristics of B cell.Phage display can be carried out in a variety of forms, be summarized see, for example, Johnson, Kevin S.and Chiswell, David J., Current Opinion in Structural Biology 3：564-571(1993).The Several sources of V constant gene segment Cs can be used for phage display.Clackson et al., Nature 352：624-628 (1991) isolates a large amount of different anti- oxazolones antibody from the derivative small-sized V genes random combinatorial libraries for hanging oneself immune mice spleen.Marks et al., J.Mol.Biol.222 can substantially be followed：581-597 (1991) or Griffith et al., EMBO are J.12：Technology described in 725-734 (1993), V genes complete or collected works and Separated pin are built to the largely not antibody of synantigen (including autoantigen) by people donor is not immunized.Referring also to United States Patent (USP) No.5,565,332 and 5,573,905.

As described above, can also pass through vitro activated B cells next life human antibodies (referring to United States Patent (USP) No.5,567,610 and 5,229,275).

Antibody fragment

In some cases, it is advantageous using antibody fragment, rather than complete antibody.The reduced size of fragment allows quick removing, and can cause to be more easily accessible to solid tumor.

The multiple technologies for generating antibody fragment are developed.Traditionally, these fragments are derived by proteolytic digestion complete antibody (see, for example, Morimoto et al., Journal of Biochemicaland Biophysical Methods 24：107-117(1992)；Brennan et al., Science 229：81(1985)).However, these fragments directly can be generated by recombinant host cell now.Fab, Fv and scFv antibody fragment all can so be allowed readily to generate these substantial amounts of fragments in expression in escherichia coli and by E. coli secretion.Can from phage antibody library discussed above isolated antibody fragment.Or, directly it can reclaim Fab '-SH fragments and chemical coupling to form F (ab ') from Escherichia coli₂Fragment (Carter et al., Bio/Technology 10：163-167(1992))., can be directly from recombinant host cell culture separation F (ab ') according to another method₂Fragment.The Fab and F (ab ') of Half-life in vivo comprising salvage receptor binding epitope residue, with extension₂Fragment is recorded in United States Patent (USP) No.5,869,046.Other technologies for generating antibody fragment will be apparent for skilled practitioner.In other embodiments, the antibody of selection is Single-Chain Fv Fragment of Murine (scFv).Referring to WO 93/16185；United States Patent (USP) No.5,571,894；And United States Patent (USP) No.5,587,458.Fv and sFv are with entire binding site, lack the unique type of constant region；In this way, they are suitable to reduce non-specific binding when using in vivo.SFv fusion proteins can be built to generate fusion of the effector protein in sFv amino or carboxyl terminal.Compiled referring to Antibody Engineering, Borrebaeck, supra.Antibody fragment can also be " linear antibodies ", such as such as United States Patent (USP) No.5, described in 641,870.Such linear antibody fragments can be monospecific or bispecific.

Bispecific antibody

Bispecific antibody refers to the antibody for having binding specificity at least two different epitopes.Exemplary bispecific antibody can combine two kinds of different epitopes of CD20 protein.The binding site of CD20 binding sites and another protein can be united by this other antibody-like.Or, the arm of anti-CD20 arms and triggering molecule such as φt cell receptor molecule (such as CD3) or IgG Fc acceptors (Fc γ R) such as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16) or NKG2D or other NK cell activation parts on combination leucocyte can be united so that cellular defence mechanisms focus on and be positioned at CD20 expression cells.Bispecific antibody can be additionally used in the cell that cytotoxic agent is positioned to expression CD20.These antibody possess CD20 combination arms and combine the arm of cytotoxic agent (such as saporin, anti-interferon-α, vinca alkaloids, ricin A chains, methotrexate (MTX) or radioactive isotope hapten).Bispecific antibody can be prepared into full length antibody or antibody fragment (such as F (ab ')₂Bispecific antibody).

WO 96/16673 describes a kind of bispecific anti-ErbB/anti- Fc γ RIII antibody, United States Patent (USP) No.5, and 837,234 disclose a kind of bispecific anti-ErbB/anti- Fc γ RI antibody.WO 98/02463 shows a kind of bispecific anti-ErbB/Fc Alpha antibodies.United States Patent (USP) No.5,821,337 has taught a kind of bispecific anti-ErbB/CD3 antibody.

Method for building bispecific antibody is known in the art.Coexpression of the traditional mode of production of total length bispecific antibody based on two pairs of heavy chain immunoglobulin-light chains, two of which chain has different specificity (Millstein et al., Nature 305：537-539(1983)).Due to being randomly assigned for heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of 10 kinds of different antibodies molecules, wherein only a kind of have correct bispecific structure.The purifying of the correct molecule generally carried out by affinity chromatography step is fairly cumbersome and Product yields are low.J.10 similar code is disclosed in WO93/08829 and Traunecker et al., EMBO：3655-3659(1991).

According to a kind of different method, there will be the antibody variable domains for expecting binding specificity (antibody-antigen binding site) to be merged with immunoglobulin constant domain sequence.Preferably, with including at least part hinge, C _H2 and C_HThe heavy chain immunoglobulin constant domain in 3rd area is merged.It is preferred that there is the first heavy chain constant region (C that necessary site is combined comprising light chain at least one fusions _H1).By encoding immune immunoglobulin heavy chain fusions thing and, when needed, the DNA of light chain immunoglobulin inserts separated expression vector, and cotransfection is into suitable host cell.There is provided when not waited for the three of structure kind polypeptide chain ratio in the embodiment for the optimum point of production for expecting bispecific antibody, this provides greater flexibility to adjust the mutual ratio of three kinds of polypeptide fragments.However, when at least two polypeptide chains cause high yield with same ratio expression or when the ratio is to it is expected that the yield of chain combination has no significant effect, it is possible to which the coded sequence of two kinds or all three polypeptide chains is inserted into single expression vector.

In a preferred embodiment of this method, bispecific antibody has the hybrid immunoglobulin heavy chain of the first binding specificity on an arm, and hybrid immunoglobulin heavy chain-light chain on another arm is constituted to (providing the second binding specificity).Due to the separating pathway that presence of the light chain immunoglobulin only in half of bispecific molecule is provided convenience, consequently found that this dissymmetrical structure is easy to separate desired bispecific compound with undesired immunoglobulin chain combinations.This method is disclosed in WO94/04690.On generating the further details of bispecific antibody see, for example, Suresh et al., Methods in Enzymology 121：210(1986).

According to United States Patent (USP) No.5, another method described in 731,168 can transform the interface between a pair of antibody molecules, by the percent maximum of the heterodimer reclaimed from recombinant cell culture thing.It is preferred that interface include at least part C _H3 domains.In the method, one or more small amino acid side chains of first antibody molecular interface are replaced with larger side chain (such as tyrosine or tryptophan).By the way that big amino acid side chains are replaced with compared with small amino acid side chains (such as alanine or threonine), compensatory " cavity " with the same or similar size of bulky side chain is produced on the interface of secondary antibody molecule.This provides the mechanism that heterodimer yield is improved than other undesired end-products such as homodimer.

Bispecific antibody includes crosslinking or " Heteroconjugate " antibody.For example, a kind of antibody in Heteroconjugate thing can be coupled with avidin, another antibody is coupled with biotin.For example, this antibody-like by immune system cell it has been proposed that for targetting undesired cell (United States Patent (USP) No.4,676,980), and for treating HIV (WO 91/00360, WO 92/200373 and EP 03089).Any easily cross-linking method can be used to prepare Heteroconjugate antibodies.Suitable crosslinking agent is well-known in the art, and United States Patent (USP) No.4,676,980 are disclosed in together with many crosslinking technologicals.

The technology that bispecific antibody is generated by antibody fragment is also described in document.It is connected chemically to prepare bispecific antibody for example, can be used.Brennan et al, Science 229：81 (1985) are described cuts complete antibody to generate F (ab ') by proteolysis₂The code of fragment.These fragments are reduced in the case where there are two mercaptan complexing agent sodium arsenites, to stablize two neighbouring mercaptan and prevent the formation of intermolecular disulfide bond.Then Fab ' the fragments of generation are changed into thionitrobenzoate ester (TNB) derivative.Then one of Fab '-TNB derivatives are reverted into Fab '-mercaptan by the reduction of mercaptoethylmaine again, and mixed with another Fab '-TNB derivatives of equimolar amounts, to form bispecific antibody.The bispecific antibody of generation can be used as the selective immobilized reagent of enzyme.

Most new progress is easy to directly reclaim Fab '-SH fragments from Escherichia coli, and these fragments are chemically coupled to form bispecific antibody.Shalaby et al., J.Exp.Med.175：217-225 (1992) describes the bispecific antibody F (ab ') of full-length human₂The generation of molecule.Every kind of Fab ' fragments are separately secreted by Escherichia coli, and are oriented chemical coupling in vitro to form bispecific antibody.The bispecific antibody being thusly-formed can combine cell and the normal human T cells for being overexpressed ErbB2 acceptors, and triggering people's cell Cytotoxic Lymphocytes for the dissolving activity of people's lacteal tumor target.

Also describe the multiple technologies for directly generating and separating bispecific antibody fragment from recombinant cell culture thing.For example, generating bispecific antibody using leucine zipper.Kostelny et al., J.Immunol.148 (5)：1547-1553(1992).Leucine zipper peptide from Fos and Jun albumen is connected by Gene Fusion with the Fab ' parts of two kinds of different antibodies.Antibody homodimer is reduced to form monomer in hinge area, then reoxidized to form antibody heterodimer.This method can also be used for generating antibody homodimer.

Hollinger et al., Proc.Natl.Acad.Sci.USA 90：" double antibody " technology that 6444-6448 (1993) is recorded provides the replacement mechanism for building bispecific antibody fragment.The fragment includes the V being connected by joint_HAnd V_L, the joint too it is short cause same chain on two domains between can not match.Therefore, the V in a fragment is forced_HAnd V_LDomain and the complementary V in another fragment_LAnd V_HDomain is matched, and is consequently formed two antigen binding sites.It is also reported that building another strategy of bispecific antibody fragment by using scFv (sFv) dimer.Referring to Gruber e tal., J.Immunol.152：5368(1994).

Contemplate the antibody with more than two potency.For example, three-specific antibody can be prepared.Tutt et al, J.Immunol.147：60(1991).

Multivalent antibody

Multivalent antibody can the internalization (and/or alienation) by the cell for expressing the antibody combination antigen more faster than bivalent antibody.The present invention antibody can be readily can be generated by the recombination expression of the nucleic acid of encoding antibody polypeptide chain, the multivalent antibody with three or more antigen binding sites (such as tetravalent antibody) (beyond IgM classifications).Multivalent antibody can include dimerization domain and three or more antigen binding sites.It is preferred that dimerization domain include (or being made from it) Fc areas or hinge area.In this case, antibody is by three or more antigen binding sites comprising Fc areas and Fc areas amino terminal.Multivalent antibody preferred herein includes (or being made from it) three to about eight, but preferably four antigen binding sites.Multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains.For example, polypeptide chain can include VD1- (X1)_n-VD2-(X2)_n- Fc, wherein VD1 are the first variable domains, and VD2 is the second variable domain, and Fc is a polypeptide chain in Fc areas, X1 and X2 represented amino acids or polypeptide, and n is 0 or 1.For example, polypeptide chain can be included：VH-CH1- flexible joint-VH-CH1-Fc areas chain；Or VH-CH1-VH-CH1-Fc areas chain.Multivalent antibody herein is preferably further comprising at least two (and preferably four) light chain variable domain polypeptides.Multivalent antibody herein can include e.g., from about two to about eight light chain variable domain polypeptides.The light chain variable domain polypeptide contemplated herein includes light-chain variable domain, and optionally further includes CL domains.

Other amino acid sequence modifications

Contemplate the amino acid sequence modifications of CD20 binding antibodies described herein.For example, it may be desirable to improve the binding affinity and/or other biological characteristicses of antibody.By the way that suitable nucleotides change is introduced into anti-CD 20 antibodies nucleic acid or the amino acid sequence variation of anti-CD 20 antibodies is prepared by peptide symthesis.The residue that such modification is included in such as anti-CD 20 antibodies amino acid sequence is deleted and/or insertion and/or replacement.Only 4 want final construction that there is desired characteristic, any combination that can be deleted, inserted and be substituted is to obtain final construction.Amino acid change can also change the post translational processing of anti-CD 20 antibodies, such as change number or the position of glycosylation site.

Available for as some residues of preferred mutagenesis position or a kind of method in region being referred to as " alanine scanning mutagenesis " in identification anti-CD 20 antibodies, such as Cunningham and Wells, Science 244：Described in 1081-1085 (1989).Herein, identify a residue or one group of target residue (such as electrically charged residue, such as arginine, aspartic acid, histidine, lysine and glutamic acid), and replaced with neutral or negatively charged amino acid (most preferably alanine or polyalanine), to influence the interaction of amino acid and CD20 antigens.Then by introducing more or other variants or to alternate site, those amino acid positions that function sensitive is shown to replacement are weighed.So, although it is pre-determined to introduce the site of variant amino acid sequence, but the property of mutation itself need not be predetermined.For example, the consequence in order to analyze the mutation to anchor point, carries out Alanine-scanning or random mutagenesis, and expect activity to expressed anti-CD 20 antibodies variant screening in target codon or region.

Amino acid sequence insertion includes the fusion of amino and/or carboxyl terminal, length range from a residue to the polypeptide for including up to a hundred or more residues, and the sequence of single or multiple amino acid residues in insertion.The example of end insertion includes the anti-CD 20 antibodies with N- terminal methionyl residues or the antibody merged with cytotoxic polypeptide.Other insertion variants of anti-CD 20 antibodies molecule are included in N- the or C- terminal fusions enzyme (such as ADEPT) of anti-CD 20 antibodies or extend the polypeptide of antibody serum half-life period.

Another kind of variant is amino acid substitution variant.These variants have at least one amino acid residue to be replaced with different residues in anti-CD 20 antibodies molecule.The most interested site for substitute mutagenesis includes hypervariable region, but also contemplates FR changes.Conservative replacement is shown under title " preferably substituting " in the following table.If such replacement causes the change of biological activity, then can be introduced into table and be referred to as further describing on amino acid classes in the more substantial variation, or following article of " illustrate and substitute ", and screen product.

Amino acid replacement table

Original Residue	Illustrate and substitute	It is preferred that substituting
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg

Ile(I)	Leu；Val；Met；Ala；Phe；Nor-leucine	Leu
			Leu(L)	Nor-leucine；Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala；Nor-leucine	Leu

To the substantive sex modification of antibody biological characteristics by selecting dramatically different replacement in the effect for keeping following aspect to complete：(a) structure of the polypeptide backbone of replacement area, such as pleated sheet or helical conformation, (b) target site punishment son electric charge or hydrophobicity, or (c) side chain volume.Based on common side chain properties, naturally occurring residue is grouped as follows：

(1) it is hydrophobic：Nor-leucine, Met, Ala, Val, Leu, Ile；

(2) it is neutral, hydrophilic：Cys、Ser、Thr；

(3) it is acid：Asp、Glu；

(4) it is alkaline：Asn、Gln、His、Lys、Arg；

(5) residue of chain orientation is influenceed：Gly、Pro；With

(6) it is aromatic：Trp、Tyr、Phe.

Non-conservative replacement will need the member with one of these classifications to exchange another classification.

Any cysteine residues for not involving the holding correct conformation of anti-CD 20 antibodies are also alternative, generally with serine, to improve the oxidation stability of molecule and prevent abnormal crosslinking.On the contrary, cysteine key can be added into antibody to improve its stability (particularly when antibody is antibody fragment such as Fv fragments).

Particularly preferred class alternative variations involve the one or more some hypervariable region residues for substituting parental antibody (such as humanization or human antibody).It is typically chosen for the gained variant further developed relative to producing their parental antibody by the biological characteristics with improvement.A kind of facilitated method for producing such alternative variations involves the affinity maturation for using phage display.Briefly, several hypervariable region sites (such as 6-7 site) are mutated, all possible amino acid replacement is produced in each site.The antibody variants so produced are illustrated on filamentous phage particle with monovalent fashion, are used as the fusions with the M13 gene III products of each particle inner packing.Then its biological activity (such as binding affinity) is screened to the variant of phage display as disclosed herein.In order to identify the candidate hypervariable region site for modification, alanine scanning mutagenesis can be carried out to identify some hypervariable region residues that there is antigen binding significant contribution.Or the crystal structure of analysis antigen-antibody complex is probably beneficial with the contact point identified between antibody and h CD20.Such contact residues and neighbouring residue are the candidate locus substituted according to technology detailed in this article.Once producing such variant, this group of variant is screened with regard to as described herein, the antibody with good characteristic in one or more relevant assays, which may be selected, to be used to further develop.

The another kind of amino acid variant of antibody changes the original glycosylation pattern of antibody.Changing means to delete non-existent one or more glycosylation sites in the one or more carbohydrate moieties found in antibody, and/or addition antibody.

The typical N- connections of the glycosylation or O- connections of antibody.N- connections refer to the side chain that carbohydrate moiety is attached to asparagine residue.Tripeptide sequence asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid in addition to proline) are the recognition sequences that carbohydrate moiety enzymatic is attached to asparagine side chain.In this way, any presence of the tripeptide sequence of both in polypeptide generates potential glycosylation site.The glycosylation of O- connections refers to is attached to hydroxy-amino-acid, most commonly serine or threonine by one of sugars N-aceylgalactosamine, galactolipin or xylose, but 5-OxoPro or 5- hydroxylysines can also be used.

It is by changing (glycosylation site for being used for N- connections) that amino acid sequence makes it easily be completed comprising one or more above-mentioned tripeptide sequences that glycosylation site is added into antibody.It can be also changed by the way that one or more serines or threonine residues are added or substituted in the sequence to original antibodies (glycosylation site for being used for O- connections).

It is prepared by a variety of methods that encoding the nucleic acid molecules of the amino acid sequence variation of anti-CD 20 antibodies can be known by this area.These methods include but is not limited to from natural origin separation (in the case of naturally occurring amino acid sequence variation), or carry out oligonucleotide mediated (or fixed point) mutagenesis, PCR mutagenesis and cassette mutagenesis to prepare by the variant or the anti-CD 20 antibodies of non-variant pattern that prepare early stage.

It may want to modify the antibody of the present invention in terms of effector functions, for example, strengthen the cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody dependent cellular mediation of antibody.This can be realized by introducing one or more amino acid replacements in antibody Fc district.Or cysteine residues are introduced in Ke Fc areas, so as to allow to form interchain disulfide bond in this zone.The homodimer antibody so produced can have the cell killing and the cytotoxicity (ADCC) of antibody dependent cellular of the internalization ability of improvement and/or the complement-mediated of raising.Referring to Caron et al., J.Exp.Med.176：1191-1195 (1992) and Shopes, B., J.Immunol.148：2918-2922(1992).Homodimer antibody with enhanced antitumor activity can also be such as Wolff et al., Cancer Research 53：In 2560-2565 (1993) prepared by described use heterobifunctional crosslinker.Or, antibody can be transformed into dual Fc areas, thus can have dissolving and the ADCC abilities of enhanced complement-mediated.Referring to Stevenson et al., Anti-Cancer Drug Design 3：219-230(1989).

, can be as described in 739,277 that salvage receptor binding epitope is mixed into antibody (especially antibody fragment) such as such as United States Patent (USP) No.5 in order to extend the serum half-life of antibody.As used herein, term " salvage receptor binding epitope " refers to IgG molecules (such as IgG₁、IgG₂、IgG₃Or IgG₄) Fc areas in be responsible for extension IgG molecule bodies in serum half-life epitope.

Other antibody modifications

Other modifications of antibody have been contemplated herein.For example, one of antibody and a variety of non-proteinaceous polymers can be connected, such as the copolymer of polyethylene glycol, polypropylene glycol, polyoxyalkylene or polyethylene glycol and polypropylene glycol.Antibody can also be contained in the microcapsules prepared for example by condensation technique or by interfacial polymerization (being for example hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules respectively), in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule) or in macro emulsion.Such technology is disclosed in Remington ' sPharmaceutical Sciences, and the 16th edition, Osol, A. is compiled, and 1980.

Antibody of the screening with desired characteristic

The antibody with some biological characteristicses can be selected as described in EXPERIMENTAL EXAMPLESThe.

Method that the growth inhibitory effect of anti-CD 20 antibodies of the present invention can be known by this area is assessed, such as using endogenic or express after being transfected with CD20 genes CD20 cell.For example, tumor cell line and CD20 transfectional cells can be handled to a couple of days (such as 2-7 days) with the anti-CD-20 monoclonal antibody of the present invention of a variety of concentration, and dyed, or analyzed by some other colorimetric methods with crystal violet or MTT.Another method for measuring propagation will be by comparing the cell handled when existing or lacking anti-CD 20 antibodies of the present invention³H- thymidines are absorbed.After antibody processing, harvesting is simultaneously quantified in scintillation counter to the radioactive amount for mixing DNA.Suitable positive control includes handling the cell line with the known growth inhibiting antibody for suppressing selected cell line growth.

In order to select the antibody of inducing cell death, it can assess and be lost for example, by propidium iodide (PI), trypan blue or the 7AAD film integrality for absorbing display relative to control.PI intakes determination method can be carried out when lacking complement and immune effector cell.Single culture medium or containing concentration for the e.g., from about culture medium of 10 μ g/ml Suitable monoclonal antibodies in incubate expression CD20 tumour cell.By the cell culture time limit of 3 days.After per treatment, cell is cleaned and is distributed to 35mm and is stamped in 12 × 75 pipes of filter screen (strainer-capped) (often pipe 1ml, each treatment group 3 manage) to remove cell mass.Then PI (10 μ g/ml) is added into pipe.FACSCAN can be used^TMFlow cytometer and FACSCONVERT^TMCellQuest softwares (Becton Dickinson) analyze sample.The antibody of the cell death of statistical significant level can be induced to be elected to be the antibody of inducing cell death those measure absorbed according to PI.

In order to screen the antibody of the epitope combined with reference to purpose antibody on CD20, conventional cross can be carried out and block determination method, such as Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane, it is described in 1988.This determination method can be used for determine test antibody whether with the present invention anti-CD 20 antibodies combination same loci or epitope.Or the method that can be known by this area carries out epitope mapping.For example, mutagenesis can be carried out to antibody sequence, such as by Alanine-scanning, to identify contact residues.Test the combination of polyclonal antibody to Mutant Antibodies to ensure correct folding first.In a kind of different method, can by corresponding to the peptide of CD20 different zones and test antibody or with test antibody and with characterized or the antibody of known epitope together be used for competition assay.

Carrier, host cell and recombination method

Recombinant technique present invention also offers the nucleic acid of the separation of encoding humanized 2H7 variant antibodies, the carrier comprising the nucleic acid and host cell and for producing the antibody.

For recombinant production antibody, the nucleic acid of encoding antibody is separated, and inserts replicable vector, for further cloning (DNA cloning) or expressing.Encode the DNA routine protocols separation easy to use of monoclonal antibody and be sequenced (such as by using the oligonucleotide probe that can be combined with the gene specific of encoding antibody heavy and light chain).Many carriers can be obtained.Support element typically includes, but not limited to following one or more：Signal sequence, replication orgin, one or more marker gene, enhancer element, promoter and transcription terminator.

(i) signal sequence component

The present invention humanization 2H7 antibody not only can directly recombinant production, and can be as the fused polypeptide with heterologous polypeptide, the heterologous polypeptide is preferably the signal sequence of the N- ends of mature protein or polypeptide or other polypeptides with specific cleavage site.Selected Heterologous signal sequences are preferably by host cell recognizes and processes and (is cut by signal peptidase).For nonrecognition and the prokaryotic host cell of the natural CD20 binding antibodies signal sequence of processing, the signal sequence is replaced with the prokaryotic signal sequence for being selected from alkaline phosphatase, penicillase, 1pp or Thermostable α-amylase II targeting sequencings.For yeast secretary, signal sequences native can be replaced with the signal described in such as yeast invertase leader, α factor leaders (including saccharomyces and genus Kluyveromyces α factor leaders), acid phosphatase leader, Candida albicans glucoamylase leader or WO 90/13646.In mammalian cell expression, it is possible to use mammalian signal sequences and viral secretory leaders, such as herpes simplex gD signal.

DNA by the DNA of such prosoma (precursor region) with encoding humanized 2H7 antibody in the way of meeting reading frame is connected.

(ii) replication orgin component

Expression and cloning vector, which are all included, can make the nucleotide sequence that carrier is replicated in one or more selected host cells.Generally, in cloning vector, this sequence is the sequence that carrier can be made to be replicated independent of host chromosome DNA, including replication orgin or autonomously replicating sequence.It is known that such sequence of various bacteria, yeast and virus.Replication orgin from pBR322 plasmid is suitable for most of gramnegative bacteriums, 2 μ plasmid origins are suitable for yeast, and various viral origins (SV40, polyomavirus, adenovirus, VSV or BPV) are available for the cloning vector in mammalian cell.In general, mammalian expression vector does not need replication orgin component (use of SV40 starting points generally may be simply because it and include early promoter).

(iii) Select gene component

Expression and cloning vector can include Select gene, also referred to as selection marker.Typical Select gene encodes following protein：(a) resistance to antibiotic or other toxin, such as ampicillin, neomycin, methotrexate (MTX) or tetracycline are assigned；(b) auxotrophic defect is supplied；Or (c) provides the required nutrients that can not be obtained by complex medium, for example for bacillus encoding D-alanine racemase gene.

One example of selection scheme blocks the growth of host cell using medicine.The protein of drug resistance is assigned with those Hemapoiesis of heterologous gene successful conversion, thus survives selection scheme.The example of such dominant selection uses drug neomycin, mycophenolic acid and hygromycin.

Another example suitable for the selection marker of mammalian cell be those can identify the encoding humanized 2H7 antibody of intake of having the ability nucleic acid cell selection marker, DHFR, thymidine kinase, metallothionein-I and the preferred primate metallothionein's genes of-II, adenosine deaminase, ornithine decarboxylase etc..

For example, first by the way that all transformants are identified into the cell converted through DHFR Select genes containing methotrexate (MTX) (Mtx), being cultivated in a kind of culture medium of DHFR competitive antagonist.When using wild type DHFR, suitable host cell is Chinese hamster ovary (CHO) cell line (such as ATCC CRL-9096) of DHFR active defects.

Or, DNA sequence dna conversion or the host cell (wild-type host for particularly including endogenous DHFR) of cotransformation of encoded humanization 2H7 antibody, wild type DHFR protein matter and another selection marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by growth of the cell in containing such as aminoglycoside antibiotics of the selective agent for selection marker such as kanamycins, neomycin or G418 culture medium.Refering to United States Patent (USP) No.4,965,199.

It is trp1 genes (Stinchcomb the et al., Nature 282 that is present in yeast plasmid YRp7 suitable for the Select gene of yeast：39(1979)).Trp1 bases are in default of the yeast mutant of the growth ability in tryptophan, and such as ATCC No.44076 or PEP4-1 are there is provided selection marker.Jones, Genetics 85：12(1977).There are trp1 damages in yeast host cell genome to provide therewith for detecting the effective environment of conversion by the growth when lacking tryptophan.Similar, supply Leu2 defective yeasts bacterial strain (ATCC 20,622 or 38,626) with the known plasmid for carrying Leu2 genes.

In addition, the carrier derived from 1.6 μm of cyclic plasmid pKD1 can be used for conversion genus Kluyveromyces yeast.Or, it has been reported that the expression system for the large-scale production restructuring calf chymosin in Kluyveromyces lactis.Van den Berg, Bio/Technology 8：135(1990).Further disclose for the albuminised stable multicopy expression vector of the ripe recombinant human serum of industrial strain secretes by genus Kluyveromyces.Fleer et al., Bio/Technology 9：968-975(1991).

(iv) promoter component

Expression and cloning vector generally comprise the promoter recognized by host organisms, and the nucleic acid of it and encoding humanized 2H7 antibody is operatively connected.Promoter suitable for prokaryotic hosts includes phoA promoters, beta-lactamase and lactose promoter system, alkaline phosphatase promoter, tryptophan (trp) promoter systems and hybrid promoter such as tac promoters.However, other known promoters are also suitable.Promoter for bacterial system is also by comprising with encoding Shine-Dalgarno (S.D.) sequence that the DNA of CD20 binding antibodies is operatively connected.

The promoter sequence of known eukaryotic.In fact, all eukaryotic genes, which all have, is rich in AT areas, it is located at about 25 to 30 bases of site upstream of starting transcription.Another sequence found at the base of transcriptional start point upstream 70 to 80 of many genes is CNCAAT areas, and wherein N can be any nucleotides.It is AATAAA sequences at 3 ' ends of most of eukaryotic genes, it is probably the signal of the 3 ' end addition poly A tails to coded sequence.All these sequences suitably insert carrier for expression of eukaryon.

Include the promoter of glycerol 3-phosphate acid kinase or other glycolytic ferments, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, GPI, 3-phoshoglyceric acid mutase, pyruvate kinase, phosphotriose isomerase, glucose phosphate isomerase and glucokinase suitable for the example of the promoter sequence of yeast host.

It is the promoter region of the enzyme of alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, the digestive enzyme relevant with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and responsible maltose and galactose utilization as other Yeast promoters of the inducible promoter with the additional advantage that transcription is controlled by growth conditions.Further stated that suitable for the carrier and promoter of Yeast expression in EP 73,657.Yeast enhancers favorably can also be used together with Yeast promoter.

Humanization 2H7 antibody is transcribed by for example from viral such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B and most preferred simian virus 40 (SV40) genome by carrier in mammalian host cell, from heterologous mammal promoter such as actin promoter or immunoglobulin promoter, and the control of the promoter obtained from heat-shock promoters, if if such promoter is compatible with host cell systems.

The early and late promoter of SV40 viruses is easily obtained in the form of SV40 restriction fragments, the fragment also includes SV40 virus origin of replication.The immediate early promoter of human cytomegalovirus is easily obtained in the form of HindIII E restriction fragments.The system for using bovine papilloma virus as carrier DNA being expressed in mammalian hosts is disclosed in United States Patent (USP) No.4,419,446.A kind of improvement of the system has been recorded in United States Patent (USP) No.4,601,978.On in mouse cell the thymidine kinase promoter from herpes simplex virus control following table intelligent's beta-interferon cDNA referring also to Reyes et al., Nature 297：598-601(1982).Or, Rous sarcoma virus LTR can be used as promoter.

(v) enhancer element component

Transcription of the higher eucaryotic cells to the DNA of coding the present inventor source 2H7 antibody is improved often through by enhancer sequence insertion vector.Many enhancer sequences from mammalian genes (globulin, elastoser, albumin, alpha-fetoprotein and insulin) that it is now know that.However, usually using the enhancer from eukaryotic cell virus.Example includes enhancer (bp100-270), the sub- enhancer of cytomegalovirus early promoter, the enhancer and adenovirus cancers of polyomavirus replication orgin late period side of SV40 replication orgins late period side.On activating the reinforcing element of eukaryotic promoter referring also to Yaniv, Nature 297：17-18(1982).Can be by enhancer montage into carrier, positioned at 5 ' or 3 ' positions of CD20 binding antibody coded sequences, it is preferred that positioned at 5 ' sites of promoter.

(vi) tanscription termination component

Expression vector for eukaryotic host cell (yeast, fungi, insect, plant, animal, people or karyocyte from other multicellular organisms) will also be comprising terminating transcription and sequence necessary to stable mRNA.Such sequence can generally be obtained from 5 ' ends of eucaryon or viral DNA or cDNA non-translational regions with 3 ' ends once in a while.These regions are included in the nucleotide segment that polyadenylated fragments are transcribed into the mRNA of coding CD20 binding antibodies untranslated part.A kind of useful tanscription termination component is bovine growth hormone polyadenylation area.Referring to WO 94/11026 and the expression vector wherein disclosed.

(vii) selection and conversion of host cell

Host cell suitable for cloning or expressing the DNA in this paper carriers is above-described prokaryotes, yeast or higher eucaryotic cells.Prokaryotes suitable for this purpose include eubacteria, such as gram negative organism or gram-positive organism, such as enterobacteriaceae, such as Escherichia (Escherichia) such as ETEC (E.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonella (Salmonella) such as salmonella typhimurium (Salmonella typhimurium), Serratia (Serratia) such as serratia marcescens (Serratia marcescans), Shigella (Shigella), and bacillus (Bacilli) such as bacillus subtilis (B.subtilis) and bacillus licheniformis (the B.licheniformis) (DD 266 that on April 12nd, 1 announces, the bacillus licheniformis 41P disclosed in 710), pseudomonas (Pseudomonas) such as pseudomonas aeruginosa (P.aeruginosa), with streptomyces (Streptomyces).A kind of preferred escherichia coli cloning host is the (ATCC 31 of Escherichia coli 294,446), although other bacterial strains such as Escherichia coli B, Escherichia coli X1776 (ATCC 31,537) and Escherichia coli W3110 (ATCC27,325) are also suitable.These examples are exemplary, rather than restricted.

Beyond prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also the suitable clones or expressive host for the carrier for encoding CD20 binding antibodies.Saccharomyces cerevisiae (Saccharomyces cerevisiae) or conventional Saccharomyces cerevisiae are the most frequently used low eucaryon host microorganisms.However, can generally obtain many other genus and species and bacterial strain and available for the present invention, such as grain wine pombe (Schizosaccharomyces pombe)；Kluyveromyces (Kluyveromyces) host, such as Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.ffagilis) (ATCC 12, 424), Bulgarian kluyveromyces (K.bulgaricus) (ATCC 16, 045), Brunswick kluyveromyces (K.wickeramii) (ATCC 24, 178), K.waltii (ATCC 56, 500), drosophila kluyveromyces (K.drosophilarum) (ATCC 36, 906), Kluyveromyces thermotolerans (K.thermotolerans) and Kluyveromyces marxianus (K.marxianus)；Yarrow saccharomyces (Yarrowia) (EP 402,226)；Pichia pastoris phaff (Pichia pastoris) (EP 183,070)；Candida (Candida)；Filamentous fungi (Trichoderma reesia) (EP 244,234)；Neuraspora crassa (Neurospora crassa)；Perhaps prosperous saccharomyces (Schwanniomyces), all so prosperous yeast (Schwanniomyces occidentalis)；And filamentous fungi, such as Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (Tolypocladium) and aspergillus (Aspergillus) host such as aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Host cell suitable for expression glycosylation humanization 2H7 antibody is derived from multicellular organisms.The example of invertebral zooblast includes plant and insect cell.Many baculoviral strains and variant are identified and have allowed insect host cell accordingly, they are from hosts such as fall army worm Spodoptera frugiperda (caterpillar), Aedes aegypti Aedes aegypti (mosquito), aedes albopictus Aedesa albopictus (mosquito), Drosophila melanogaster Drosophila melanogaster (drosophila) and silkworm Bombyx mori.The public, which can obtain a variety of Strain, to be used to transfect, such as autographa california Autographa califomica NPV L-1 variants and silkworm Bombyx mori NPV Bm-5 strains, and this viroid can be used as virus herein according to the present invention, particularly for transfecting Spodopterafrugiperda cells.

The breeding of vertebrate cells has become routine protocols in culture (tissue cultures).The example of useful mammalian host cell line is the monkey kidney CV1 systems (COS-7, ATCC CRL1651) converted with SV40；Human embryonic kidney cell line (293 or in order to suspend culture in growth and be subcloned 293 cells, Grahamet al., J.Gen Virol.36：59(1977))；Baby hamster kidney cell (BHK, ATCC CCL 10)；Chinese hamster ovary cell/- DHFR (CHO, Urlaub et al., Proc.Natl.Acad.Sci.USA77：4216(1980))；Mouse Sai Tuoli (sertoli) cells (TM4, Mather, Biol.Reprod.23：243-251(1980))；MK cells (CV1, ATCC CCL 70)；African green monkey kidney cell (VERO-76, ATCC CRL-1587)；Human cervical carcinoma cell (HELA, ATCC CCL 2)；MDCK (MDCK, ATCC CCL 34)；Ox mouse (buffalo rat) liver cell (BRL 3A, ATCC CRL1442)；Human pneumonocyte (W138, ATCC CCL 75)；Human liver cell (Hep G2, HB 8065)；Mouse mammary tumor (MMT 060562, ATCC CCL 51)；TRI cells (Mather et al., Annals N.Y.Acad.Sci.383：44-68(1982)；MRC5 cells；FS4 cells；With people's hepatoma system (Hep G2).

Host cell is converted with the expression described above for being used to produce CD20 binding antibodies or cloning vector, and is cultivated in for evoked promoter, the conventional nutrient culture for selecting transformant or the gene of amplification coding expectation sequence and suitably changing.

(viii) host cell is cultivated

The host cell for producing CD20 binding antibodies of the present invention can be cultivated in a variety of culture mediums.Commercially available culture medium such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), the EagleShi culture mediums (DMEM, Sigma) of RPMI-1640 (Sigma) and DulbeccoShi improvement are suitable to culture host cell.Further, it is possible to use the culture medium of any culture medium described in following documents as host cell：Ham et al., Meth.Enz.58：44(1979)；Barnes et al., Anal.Biochem.102：255(1980)；United States Patent (USP) No.4,767,704；4,657,866；4,927,762；4,560,655；Or 5,122,469；WO 90/03430；WO 87/00195；Or United States Patent (USP) review 30,985.These any culture mediums can hormone supplemented and/or other growth factors (such as insulin, transferrin or EGF), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotic (such as GENTAMYCIN as needed^TMMedicine), trace element (being defined as the inorganic compound generally existed with the final concentration of micro-molar range) and glucose or the equivalent energy.Can also with suitable concentration include those skilled in the art will know that any other required supplement.Condition of culture, temperature, pH etc., previously selected for expression for host cell, this is obvious for those of ordinary skill.

(ix) purifying of antibody

When using recombinant technique, can in the cell, antibody is generated in periplasmic space, or be directly secreted into culture medium.If generating antibody in the cell, then as the first step, the particle debris of host cell or crack fragment is removed for example, by centrifugation or ultrafiltration.Carter et al., Bio/Technology10：163-167 (1992) describes the code of the antibody for being secreted into colibacillus periplasm space.Briefly, cell paste is made to melt when there is sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) about 30 minutes.Cell fragment can be removed by centrifugation.If by antibody-secreting into culture medium, then general first by commercialization protein concentration filter, such as supernatant of the Amicon or Millipore Pellicon ultra filtration units concentration from such expression system.In any above-mentioned steps, protease inhibitors such as PMSF can be included to suppress proteolysis, and antibiotic can be included to prevent the growth of external contaminant.

Such as hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography can be used to purify the antibody compositions prepared by cell, purification technique preferably is affinity chromatography.Albumin A depends on the species and isotype of any immunoglobulin fc region present in antibody as the suitability of affinity ligand.Albumin A can be used for antibody (Lindmark et al., J.Immunol.Meth.62 of the purifying based on people γ 1, γ 2 or the heavy chains of γ 4：1-13(1983)).Protein G recommends to be used for all mouse isotypes and people γ 3 that (Guss et al., EMBO are J.5：1567-1575(1986)).Matrix accompanying by affinity ligand is most commonly used that agarose, but can use other matrix.The matrix of physically stable such as controlled pore glass or poly- (styrene divinyl) benzene result in flow velocity more faster than agarose and shorter process time.If antibody includes C_H3 domains, then can be used Bakerbond ABX^TMResin (J.T.Baker, Phillipsburg, NJ) is purified.According to antibody to be recycled, it is possible to use classification, ethanol precipitation, reversed-phase HPLC on other oroteins purification technique, such as ion exchange column, the chromatography on tripoli, heparin SEPHAROSE^TMOn chromatography, anion or cationic ion-exchange resin (such as poly-aspartate post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preparation property purification step, the mixture comprising purpose antibody and pollutant can carry out low pH hydrophobic interaction chromatographies, using the elution buffer between pH about 2.5-4.5, preferably carry out (such as about 0-0.25M salt) in low salt concn.

Antibody coupling matter

Antibody can be coupled with cytotoxic agent such as toxin or radio isotope.In certain embodiments, the toxin is Calicheamicin (calicheamicin), maytansinoids (maytansinoid), dolastatin (dolastatin), auristatin E and the like or derivative.

It is preferred that medicine/toxin include the inhibitor and antimetabolite of DNA damage agent, microtubule polymerization or depolymerization.The preferred classes of cytotoxic agent include such as enzyme inhibitor such as dihydrofolate reductase inhibitor and thymidilate synthase inhibitors, DNA intercalators, DNA cutting agents, topoisomerase enzyme inhibitor, anthracycline antibiotic (anthracycline) medicine family, Changchun class (vinca) medicine, mitomycin (mitomycin), bleomycin (bleomycin), cytotoxic nucleoside, pteridine (pteridine) medicine family, two acetylenic antibiotic (diynenes), podophyllotoxin (podophyllotoxin) and differentiation inductor.The particularly useful member of those classifications includes such as methotrexate (MTX) (methotrexate), methopterin (methopterin), dichioromethotrexate (dichloromethotrexate), 5 FU 5 fluorouracil (fluorouracil), Ismipur (mercaptopurine), cytarabine (cytosine arabinoside), melphalan (melphalan), leurosine (leurosine), inrosidine (leurosidine), D actinomycin D (actinomycin), daunorubicin (daunorubicin), Doxorubicin (doxorubicin), N- (5, 5- diacetoxies amyl group) Doxorubicin, morpholino-Doxorubicin, 1- (2- chloroethyls) -1, 2- dimethyl methyls hydrazides (1- (2-choroehthyl) -1, 2-dimethanesulfonyl hydrazide), N⁸- acetyl spermidine (N⁸- acetyl spermidine), aminopterin-induced syndrome (aminopterin), methopterin (methopterin), ai sibo mycin (esperamicin), mitomycin C (mitomycin C), Mitomycin A (mitomycin A), D actinomycin D (actinomycin), bleomycin (bleomycin), carminomycin (carminomycin), aminopterin-induced syndrome (aminopterin), Talisomycin (tallysomycin), podophyllotoxin (podophyllotoxin) and podophyllotoxin derivative such as Etoposide (etoposide) or etoposide phosphate, vincaleukoblastinum (vinblastine), vincristine (vincristine), eldisine (vindesine), PTX (taxol), taxotere (taxotere), retinoic acid (retinoic acid), butyric acid (butyric acid), N⁸- acetyl spermidine (N⁸- acetyl spermidine), camptothecine (camptothecin), Calicheamicin (calicheamicin), bryostatin (bryostatin), cephalostatins, ansamitocin (ansamitocin), actosin, maytansinoids (maytansinoid) such as DM-1, maytansine (maytansine), maytansinol (maytansinol), N- demethyls -4, 5- removes epoxy maytansinol, C-19- dechlorination maytansinols, C-20- hydroxyl maytansinols, C-20- demethoxylation maytansinols, C-9-SH maytansinols, C-14- alkoxyl-methyl maytansinols, C-14- hydroxyls or acetyl-o-methyl maytansinol, C-15- hydroxyls/acetoxyl group maytansinol, C-15- methoxyl group maytansinols, C-18-N- demethylations maytansinol and 4, 5- deoxidation maytansinols；Auristatin, such as auristatin E, M, PHE and PE；Dolostatin, such as dolostatin A, dolostatin B, dolostatin C, dolostatin D, dolostatin E (20- tables and 11- tables), dolostatin G, dolostatin H, dolostatin I, dolostatin1, dolostatin 2, dolostatin 3, dolostatin 4, dolostatin 5, dolostatin 6, dolostatin7, dolostatin 8, dolostatin 9, dolostatin 10, deo-dolostatin 10, dolostatin 11, dolostatin 12, dolostatin 13, dolostatin 14, dolostatin 15, dolostatin 16, dolostatin17 and dolostatin 18；Cephalostatin, such as cephalostatin 1, cephalostatin 2, cephalostatin 3, cephalostatin 4, cephalostatin 5, cephalostatin 6, cephalostatin7, 25 '-table-cephalostatin 7, 20- tables-cephalostatin 7, cephalostatin 8, cephalostatin9, cephalostatin 10, cephalostatin 11, cephalostatin 12, cephalostatin 13, cephalostatin 14, cephalostatin 15, cephalostatin 16, cephalostatin 17, cephalostatin 18 and cephalostatin 19.

Maytansinoids are the mitotic inhibitors played a role by suppressing tubulin polymerization.Maytansine is initially from East Africa shrub tingia Caulis Mayteni (Maytenus serrata) isolated (United States Patent (USP) No.3,896,111).It is subsequently found certain micro-organisms and also generates maytansinoids, such as maytansinol and C-3 maytansinols ester (United States Patent (USP) No.4,151,042).The maytansinol and its derivative and analog of synthesis are disclosed in such as United States Patent (USP) No.4,137,230；4,248,870；4,256,746；4,260,608；4,265,814；4,294,757；4,307,016；4,308,268；4,308,269；4,309,428；4,313,946；4,315,929；4,317,821；4,322,348；4,331,598；4,361,650；4,364,866；4,424,219；4,450,254；4,362,663；And 4,371,533, the disclosure of which is clearly taken in herein is used as reference.

By maytansine and maytansinoids and the antibody coupling of specific binding tumor-cell antigen.Immune conjugate and its therapeutical uses comprising maytansinoids are disclosed in such as United States Patent (USP) No.5,208,020；5,416, the 064 and B1 of European patent EP 0 425 235, clearly takes in the disclosure of which and is used as reference herein.Liu et al., Proc.Natl.Acad.Sci.USA 93：8618-8623 (1996) describes the immune conjugate for including the maytansinoids for being referred to as DM1 being connected with the monoclonal antibody C242 for human colorectal cancer.It was found that the conjugate has the high cell toxicity of the colon cancer cell for culture, and show antitumor activity in tumour growth measurement method in vivo.Chari et al., Cancer Research 52：127-131 (1992) describes wherein maytansinoids through disulfde linker and the immune conjugate for combining another mouse monoclonal antibody TA.1 couplings of the mouse antibody A 7 of antigen or combination HER-2/neu oncogenes in human colon cancer cell line.

Know that many linking groups can be used for preparing antibody-maytansinoids conjugate, including such as United States Patent (USP) No.5,208,020 or B1 and Chari et al., the Cancer Research 52 of European patent 0 425 235 in this area：Disclosed in 127-131 (1992).Linking group includes disulphide group, sulfide group, acid-unstable group, photo-labile group, the unstable group of peptase or the unstable group of esterase, as disclosed in above-mentioned patent, preferably disulphide and sulfide group.

A variety of bifunctional protein coupling agents can be used to prepare the conjugate of antibody and maytansinoids, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), succinimide base -4- (N- Maleimidomethyls) hexamethylene -1- carboxylates, iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (to azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (to diazoniumbenzoyl) ethylenediamines), diisocyanate (such as toluene 2, 6- diisocyanate) and double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.Particularly preferred coupling agent includes N- succinimide bases -3- (2- pyridine radicals two is thio) propionic ester (SPDP) (Carlsson et al., Biochem.J.173：723-737 (1978)) and N- succinimide bases -4- (2- pyridylthios) valerate (SPP), thus disulfide bond is provided.

According to the type of connection, joint can be attached to multiple positions of maytansinoids molecule.For example, conventional coupling techniques can be used to pass through the reaction with hydroxyl to form ester bond.Reaction can occur in the C-3 positions with hydroxyl, C-14 positions, the C-15 positions through hydroxyl modified and the C-20 positions with hydroxyl modified through methylol.In a preferred embodiment, key connection is formed in the C-3 positions of maytansinol or maytansinol analog.

Calicheamicin

Another immune conjugate interested includes the CD20 binding antibodies being coupled with one or more calicheamicin molecules.Calicheamicin antibiotic family can produce double-strand DNA cleavage in sub- picomolar concentrations.Preparation on Calicheamicin family conjugate is referring to United States Patent (USP) No.5,712,374；5,714,586；5,739,116；5,767,285；5,770,701；5,770,710；5,773,001；5,877,296 (belonging to Cyanamid companies of the U.S.).Available Calicheamicin analogue includes but is not limited to γ₁ ^I、α₂ ^I、α₃ ^I, N- acetyl group-γ₁ ^I, PSAG and θ^I ₁(Hinman et al., Cancer Research53：3336-3342(1993)；Lode et al., Cancer Research 58：2925-2928(1998)；And the above-mentioned United States Patent (USP) for authorizing Cyanamid companies of the U.S.).Can be QFA with another antineoplastic of antibody coupling, it is a kind of antifolic thing.Calicheamicin and QFA have intracellular action site, and are difficult through plasma membrane.Therefore, these medicaments greatly strengthen their cytotoxic effect via the cellular uptake of antibody-mediated internalization.

Radio isotope

For selective destruction tumour, antibody can include high radioactive atom.A variety of radio isotopes can be used for the anti-CD 20 antibodies of generation radiation coupling.Example includes At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²With Lu radio isotope.When conjugate to be used to detect, it can be studied comprising radioactive atom for scitiphotograph, such as Tc^99mOr I¹²³, or being used for nuclear magnetic resonance (NMR) comprising spin label is imaged (also referred to as magnetic resonance imaging, mri), such as iodo- 123, iodine -131, indium -111, fluoro- 19, carbon -13, nitrogen -15, oxygen -17, gadolinium, manganese or iron.

Radioactively labelled substance or other labels can be mixed into conjugate in a known way.For example, can biosynthesis peptide, or by chemical amino acid synthetic method synthetic peptide, involve for example fluoro- 19 suitable amino group acid precursors for replacing hydrogen wherein using.Label, such as Tc can be adhered to through the cysteine residues in peptide^99mOr I¹²³、Re¹⁸⁶、Re¹⁸⁸And In¹¹¹.Yttrium-90 can be adhered to through lysine residue.IODOGEN methods (Fraker et al., Biochem.Biophys.Res.Commun.80：49-57 (1978)) it can be used for mixing iodo- 123.Monoclonal Antibodies in Immunoscintigraphy, Chatal, CRC Press, 1989 describe other methods in detail.

A variety of bifunctional protein coupling agents can be used to prepare the conjugate of antibody and cytotoxic agent, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), succinimide base -4- (N- Maleimidomethyls) hexamethylene -1- carboxylates, iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (to azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (to diazoniumbenzoyl)-ethylenediamines), diisothio-cyanate (such as toluene 2, 6- diisocyanate) and double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.For example, can be such as Vitetta et al., Science 238：It is described in 1098 (1987) to prepare ricin immunotoxin.The 1- isothiocyanic acid benzyl -3- methyl diethylene-triamine pentaacetic acids (MX-DTPA) of carbon-14 mark are for by the exemplary chelating agent of radioactive nucleotides and antibody coupling.Referring to WO94/11026.Joint can be easy for discharging " the cleavable joint " of cell toxicity medicament in cell.For example, sour unstable joint, peptidase-sensitive linker, photo-labile joint, dimethyl linker or containing disulfde linker (Chari et al., Cancer Research 52 can be used：127-131(1992)；United States Patent (USP) No.5,208,020).

Therapeutical uses

The humanization 2H7 CD20 binding antibodies of the present invention include CD20 positive cancers such as B cell lymphoma and leukaemia, and autoimmunity disease available for many pernicious and nonmalignant disease is treated.Stem cell (B cell precursor) in marrow lacks CD20 antigens so that healthy B cell can after treatment regenerate and normal level is returned within the several months.Hu2H7.v511 is by with the preferred antibody for the treatment of method in this article.

CD20 positive cancers refer to such cancer, wherein expressing CD20 abnormal cell proliferation on cell surface.CD20 positive B-cells tumour includes CD20 positive He Jiejinshi (Hodgkin) diseases, includes the Hodgkin's disease (LPHD) of lymphocytic predominance；Non_hodgkin lymphoma (NHL)；Follicular center cells (FCC) lymthoma；Acute lymphatic leukemia (ALL)；Chronic lymphocytic leukemia (CLL)；Hairy cell.

Term " non-Hodgkin's (Hodgkin) lymthoma " or " NHL " refer to the lymphatic system cancer beyond He Jiejin lymphomas as used herein.Typically can by exist in He Jiejin lymphomas it is inner-apply (Reed-Sternberg) cell and make a distinction He Jiejin lymphomas and non_hodgkin lymphoma in the absence of the cell in non_hodgkin lymphoma.The example that non_hodgkin lymphoma is covered when the term is used for this paper includes those skilled in the art (such as oncologist or virologist) will be accredited as such any lymthoma according to classification chart known in the art, such as Color Atlas of ClinicalHematology, 3rd edition, Victor A.Hoffbrand and John E.Pettit are compiled, Harcourt PublishersLtd., Revised European-American Lymphoma (REAL) scheme (American-European lymthoma correction chart) described in 2000.Referring specifically to the table in Figure 11 .57,11.58 and 11.59.More specific example includes but is not limited to recurrent or intractable NHL,Front (front line) rudimentary NHL,Stage III/IV NHL,Chemotherapy tolerance NHL,Precursor B lymphoblastic leukemias and/or lymthoma,SLL,B cell chronic lymphocytic leukemia and/or pre-lymphocytic leukemia and/or SLL,B cell prolymphocyte lymthoma,Immune cell tumor and/or lympho-plasmacytic (lymphoplasmacytic) lymthoma,Lymphoma lymphoplasmacytic,Marginal zone B-cell lymphoma,Splenic marginal zone lymthoma,Save outer edge area (extranodal marginal zone)-MALT lymthomas,Save marginal zone (nodal marginal zone) lymthoma,Hairy cell,Plasmacytoma and/or plasma cell myeloma,Rudimentary/follicular lymphoma,Middle rank/folliculus NHL,Lymphoma mantle cell,Follicle center lymphoma (folliculus),Intermediate diffusivity NHL,Diffusivity large B cell lymphoid tumor,Aggressiveness (agressive) NHL (including aggressive front NHL and aggressiveness recurrent NHL),Recurrent or intractable NHL after autologous stem cell transplantation,Primary Mediastinal large B cell lymphoid tumor,Lymphoma primary effusion,Senior immunoblast NHL,Senior lymphoblast NHL,Senior small non-cleaved cell NHL,Thesaurismosis (bulkydisease) NHL,Bai Jiteshi (Burkitt) lymthoma,The big granular lymphocytic leukemia of precursor (periphery),Mycosis fungoides and/or Sai Zhali (Sezary) syndrome,Skin lymphoma,Primary cutaneous type,Angiocentric lymphoma.

Painless lymthoma is a kind of slow-growing, incurable disease, wherein patient Average Survival 6 to 10 years after the multiple state of an illness disappears and recurs.In one embodiment, humanization CD20 binding antibodies or its functional fragment are used to treat painless NHL.

The humanization 2H7 antibody or its functional fragment of the present invention can be used for such as recurrent or refractory low grade or follicularis CD20 positive B-cells NHL as single pharmaceutical treatment, or drug scheme can be unified into other medicines be applied to patient.

In specific embodiments, humanization CD20 binding antibodies and its functional fragment are used to treat non_hodgkin lymphoma (NHL), the Hodgkin's disease (LPHD) of lymphocytic predominance, SLL (SLL) and chronic lymphocytic leukemia (CLL).

" autoimmunity disease " herein refer to caused by individual autologous tissue and for the disease or illness of individual autologous tissue or its isolate (co-segregate) or performance or resulting illness.The example of autoimmune disease or illness includes but is not limited to arthritis (rheumatoid arthritis such as acute arthritis,Chronic rheumatoid arthritis,Urarthritis,Acute gouty arthritis,Chronic inflammatory arthritis,Degenerative arthritis,Infectional arthritis,Lime (Lyme) arthritis,Hypertrophic arthritis,Psoriasis arthropathica,Arthritis vertebralis and young hair style rheumatoid arthritis,Osteoarthritis,Chronic progressive arthritis (arthritis chronica progrediente),Arthritis deformans,Chronic primary panarthritis,Adjuvant arthritis,And ankylosing spondylitis),Inflammatory hyperproliferative skin disease,Psoriasis such as plaque psoriasis,Psoriasis guttata,Pustular psoriasis and nail psoriasis,Idiocrasy includes atopic diseases such as hay fever and Qiao Bu Shi (Job) syndrome,Dermatitis includes contact dermatitis,Chronic contact dermatitis,Allergic dermatitis,Allergic contact dermatitis,Dermatitis herpetiformis and atopic dermatitis,High IgM syndromes chain x,Such as chronic allergic urticaria of nettle rash and chronic idiopathic urticaria include chronic auto-immune nettle rash,Polymyositis/dermatomyositis,Adolescent dermatomyositis,Toxic epidermal necrolysis,Chorionitis (including systemic scleroderma),Harden such as systemic sclerosis,Multiple sclerosis (MS) such as spinal cord-eye (spino-optical) MS,Primary progressive MS (PPMS) and recurrent regression (relapsing remitting) MS (RRMS),Progressive systemic sclerosis,Atherosclerosis,Artery sclerosis,Disseminated sclerosis (sclerosis disseminata) and incoordination (ataxic) hardening,Inflammatory bowel disease (IBD) (such as Chron (Crohn) disease,The gastrointestinal disease of autoimmunity mediation,Colitis such as ulcerative colitis (colitis ulcerosa),Microcosmic (microscopic) colitis,Collagenous colitis,Colitis polyposa,Necrotizing enterocolitis and transmural colitis,With autoimmune inflammatory enteropathy),Gangrenous pyaphysia,Erythema nodosum,Primary sclerotic cholangitis,Episcleritis,Respiratory Distress Syndrome(RDS) includes adult type or ARDS (ARDS),Meningitis,The inflammation of uvea all or in part,Iritis,Choroiditis,Autoimmune hematological illness,Rheumatoid,Sudden hearing loss,The disease such as allergic reaction and allergia of IgE mediations and atopic rhinitis,Silent Sen Shi (Rasmussen) encephalitis in encephalitis such as Lars and edge system and/or BBE,Uveitis such as anterior uveitis,Acute anterior uveitis,Granulomatous uveitis,Nongranulomatous uveitis,Phacoantigenic uveitis,Posterior uveitis or Autoimmune uveitis,With and without the glomerulonephritis (GN) of nephrotic syndrome is such as chronic or acute glomerulonephritis such as primary GN,Immune-mediated GN,Film GN (membranous nephropathy),Idiopathic film GN or idiopathic membranous nephropathy,Film proliferative or film proliferative GN (MPGN) include I types and II types,With radical property GN,Allergia illness and response,Allergic reaction,Eczema includes allergia or atopic eczema,Asthma such as bronchial astehma (asthma bronchiale) and autoimmune asthma,It is related to the illness of T cell infiltration and chronic inflammatory response,For the immune response of exotic antigen such as gestation fetus A-B-O blood groups,Chronic pulmonary inflammatory disease,Autoimmune myocarditis,Leukocyte adhesion deficiency,Systemic loupus erythematosus (SLE) (systemiclupus erythematodes) such as skin SLE,Subacute cutaneous lupus erythema tosus,Neonatal lupus syndrome (NLE),Lupus erythematosus disseminatus,Lupus (including lupus nephritis,Lupus encephalitis,Paediatrics lupus,Non- kidney lupus,The outer lupus of kidney,Discoid lupus,Lupus alopecia),Young hair style (I types) diabetes include paediatrics insulin-dependent diabetes mellitus (IDDM),The diabetes (type ii diabetes) of adult onset,Autoimmune diabetes,Idiopathic diabetes insipidus,With cell factor and the acute immune response relevant with delayed hypersensitivity of T- cell mediateds,Tuberculosis,Sarcoidosis,Granulomatosis includes lymphomatoid granulomatosis,Wei Genashi (Wegener) granulomatosis,Agranulocytosis,Vasculitides includes vasculitis (including big vessel vasculitis (including polymyalgia rheumatica and giant cell (high iS-One (Takayasu)) arteritis),Medium vessels vasculitis (including Chuan Qishi (Kawasaki) diseases and PAN/periarteritis nodosa),Microcosmic (microscopic) panarteritis,CNS vasculitises,Gangrenosum acne,Cutaneous or allergic angiitis,Systemic necrotizing vasculitis,With ANCA relevant blood vessels inflammation such as Qiu-apply Er Shi (Churg-Strauss) vasculitises or syndrome (CSS)),Temporal arteritis,Alpastic anemia,LADA alpastic anemia,Claire (Coombs) positive anemia,Dai-cloth Er Shi (DiamondBlackfan) anaemia,Hemolytic anemia or immune hemolytic anemia include autoimmune hemolytic anemia (AIHA),Pernicious anaemia (anemia pemiciosa),A Disenshi (Addison) diseases,Simple erythrocyte anemia or aregeneratory (PRCA),Factor IX lacks,Hemophilia A,LADA Neutropenia,Pancytopenia,Leukopenia,It is related to the disease of leukocyte infiltration,CNS inflammatory conditions,Multiple organ injury's syndrome such as those septicemia,Wound or bleeding are secondary,The disease of antigen-antibody complex mediation,Anti-GBM disease,Antiphospholipid antibody syndrome,Allergic neuritis,Bei Qieteshi (Bechet or Behcet) diseases,Ka Siermanshi (Castleman) syndrome,Gu Depasiqiushi (Goodpasture) syndrome,Lei Nuoshi (Reynaud) syndrome,Siogren (Sjogren) syndrome,Shi-about Er Shi (Stevens-Johnson) syndrome,Pemphigoid such as bullous pemphigoid and skin pemphigoid,Pemphigus (including pemphigus vulgaris,Pemphigus foliaceus,Mucosal pemphigus type pemphigus and pemphigus erythematosus),Autoimmune polyendocrinopathy,Lay Te Shi (Reiter) diseases or syndrome,Immune complex nephritis,Antibody-mediated ephritis,Neuromyelitis optica,Polyneuropathy,Chronic neuropathic such as IgM polyneuropathies or the neuropathy of IgM mediations,Thrombopenia (for example myocardial infarction patient occurs) includes thrombotic thrombocytopenic purpura (TTP),Post-transfusion purpura (PTP),The thrombopenia that heparin induces,Include chronic or acute ITP with autoimmunity or immune-mediated thrombopenia such as ITP (ITP),The autoimmunity disease of testis and ovary includes LADA orchitis and oaritis,Primary hypothyroidism,Hypoparathyroidism,Autoimmune endocrinopathy includes thyroiditis such as autoimmune thyroiditis,Hashimoto (Hashimoto) disease,Chronic thyroiditis (Hashimoto (Hashimoto) thyroiditis) or subacute thyroiditis,AITD,Idiopathic hypothyroidism,Graves (Graves) disease,Polyglandular syndrome such as autoimmune polyglandular syndrome (or pluriglandular endocrine disease syndrome),Paraneoplastic syndrome includes neurology paraneoplastic syndrome such as Lambert-Eton (Lambert-Eaton) myasthenic syndrome or Eaton-Lambert (Lambert-Eaton) syndrome,Stiff body or stiff man syndrome,Encephalomyelitis such as allergic encephalitis (encephalomyelitis allergica) and experimental allergic encephalomyelitis (EAE),Myasthenia gravis such as thymoma associated myasthenia gravis,Cerebellar degeneration,Neuromyotonia,Opsoclonus or opsoclonus myoclonic syndrome (OMS),And esthesioneurosis,Many focus motor neuropathies,Seat Han Shi (Sheehan) syndrome,Oneself immunity hepatitis,Chronic hepatitis,Lupoid hepatitis,Giant cell hepatitis,CAH or autoimmune chronic active hepatitis,Lymphoid interstitial pneumonia (LIP),Bronchiolitis obliterans (Nonimplantation) is to NSIP,Ge-bar Er Shi (Guillain-Barr é) syndrome,Bei Geershi (Berger) diseases (IgA nephrosis),Idiopathic IgA nephrosis,Linear IgA skin diseases,PBC,Pneumonocirrhosis,Auto immune enteropathy syndrome,Chylous diarrhea,Abdominal disease,Sprue (gluten enteropathy),Intractable sprue,Idiopathic sprue,Cryoglobulinemia,ALS (ALS) (Lu Geli kirschner (Lou Gehrig) disease),Coronary artery disease,LADA otopathy such as Autoimmune Inner Ear Disease (AIED),LADA anaudia,Opsoclonus myoclonic syndrome (OMS),Polychondritis such as intractable or relapsing polychondritis,Pulmonary alveolar proteinosis,Amyloidosis,Sclerotitis,Non-cancerous lymphocytosis,Primary lymphocytosis includes monoclonal B cell lymphocytosis (the undetermined monoclonal gamma globulin disease of such as benign monoclonal gammopathy and property (MGUS)),Peripheral nerve disease,Paraneoplastic syndrome,Passage disease such as epilepsy,Antimigraine,Arrhythmia cordis,Disorder of muscle,Become deaf,Blindness,Periodic paralysis and CNS passage disease,Autism,Inflammatory myopathy,Focal segmental glomerulosclerosis (FSGS),Endocrine ophthalmopathy,Uveoretinitis,Choroidoretinitis,LADA hepatology illness,Fibromyalgia,Multiple Endocrine exhaustion,Shi Miteshi (Schmidt) syndrome,Paranephritis,Lipogastry,Alzheimer's disease,Demyelinating disease such as LADA demyelinating disease and chronic inflammatory demyelinating polyneuropathy,Diabetic nephropathy,De Leisileshi (Dressler) syndrome,Alopecia areata,CREST syndrome (calcinosises,Lei Nuoshi (Raynaud) phenomenon,Esophageal dysmotility,Sclerodactyly and capillarectasia),Masculinity and femininity LADA is infertile,MCTD,Just add Si Shi (Chagas) diseases,Rheumatic fever,Habitual abortion,Farmer lung,Erythema multiforme,Postcardiotomy syndrome,Ke Xing Shi (Cushing) syndrome,Bird breeders' lung,Allergic granulomatous angiitis,Benign lymphocytic vasculitis,A Erboteshi (Alport) syndrome,Pulmonary alveolitis such as allergic pulmonary alveolitis and FA,Interstitial lung disease,Transfusion reaction,Leprosy,Malaria,Leishmaniasis,Trypanosomiasis (kypanosomiasis),Snail fever,Roundworm disease,Aspergillosis,Sampter Cotards,Kapp Lan Shi (Caplan) syndrome,Dengue,Endocarditis,Endomyocardial fibrosis,Diffusivity pulmonary interstitial fibrosis,Interstitial pulmonary fibrosis,Pulmonary fibrosis,Idiopathic pulmonary fibrosis,Cystic fibrosis,Entophthamia,Erythema elevatum diutinum (erythema elevatum et diutinum),Fetal erythrocytosis,Eosinophilic fascitis (faciitis),Shu Er Man (Shulman) syndrome,Fil Ti Shi (Felty) syndrome,flariasis,Cyclitis such as chronic cyclitis,Heterochronia cyclitis,Iridocyclitis (acute or chronic) or FuchShi cyclitises,Heng Nuo-Xu Lan Er Shi (Henoch-Schonlein) purpura,Human immunodeficiency virus (HIV) infects,Echovirus infects,Cardiomyopathy,Alzheimers (Alzheimer) disease,Parvovirus infections,Rubella virus infection,Syndrome after vaccination,Congenital rubella infects,Epstein-Ba Er (Epstein-Barr) virus infection,Parotitis,Evans (Evans) syndrome,LADA gonadal failure,Xi Denghamushi (Sydenham) chorea,Ephritis after streptococcus,Buerger's disease (thromboangitis ubiterans),Thyrotoxicosis,Tabetic crisis,Choroiditis,Megaloblastic polymyalgia,Endocrine ophthalmopathy,Chronic hypersensitivity pneumonitis,Keratoconjunctivitis sicca,Epidemic keratoconjunctivities,Idiopathic nephritic syndrome,Minute nephropathy,Benign familial and ischemia reperfusion injury,Retina autoimmunity,Arthritis,Bronchitis,Chronic obstructive airway disease,Silicosis,Aphtha,Aphthous stomatitis,Arteriosclerotic illness,Without spermatogenesis (aspermiogenese),Autoimmune hemolytic anemia,Primary kirschner (Boeck) disease,Cryoglobulinemia,Dupp Yi Telunshi (Dupuytren) contracture,Phacoanaphylaxis entophthamia (endophthalmiaphacoanaphylactica),Allergia enteritis (enteritis allergica),Leprosy section erythema nodosum,Idiopathic facial palsy,Chronic Fatigue Syndrome,Rheumatic fever (febris rheumatica),Ha-inner Er Shi (Hamman-Rich) diseases,Sensory neural hearing loss,Paroxysmal hemoglobinuria (haemoglobinuriaparoxysmatica),Hypogonadism,Regional enteritis (ileitis regionalis),Leukopenia,Infectious mononucleosis,Transverse (traverse) myelitis,Primary essential myxoedema,Nephrosis,Sympathetic ophthalmia (ophthalmia symphatica),Granulomatous orchitis (orchitisgranulomatosa),Pancreatitis,Acute polyradiculitis,Gangrenous pyaphysia,Kui Erwanshi (Quervain) thyroiditis,Acquired splenatrophy,Due to the sterility of anti-spermatozoon antibody,Non-malignant thymoma,Leucoderma,SCID and Epstein-Ba Er (Epstein-Barr) virus associated-diseases,Acquired immunodeficiency syndrome (AIDS),Parasitic disease such as Leishmania,TSS,Food poisoning,It is related to the illness of T cell infiltration,Leukocyte adhesion deficiency,With cell factor and the acute immune response relevant with delayed hypersensitivity (DH) of T- cell mediateds,It is related to the disease of leukocyte infiltration,Multiple organ injury's syndrome,The disease of antigen-antibody complex mediation,Anti-GBM disease,Allergic neuritis,Autoimmune polyendocrinopathy,Oaritis,Primary myxedema,Autoimmune atrophic gastritis,Sympathetic ophthalmia,Rheumatism,MCTD,Nephrotic syndrome,Inflammation of pancreatic islet,Many endocrinasthenias,Peripheral nerve disease,Autoimmune polyglandular syndrome I types,The Idiopathic hypoparathyroidism (AOIH) of adult onset,Whole alopecia,Dilated cardiomyopathy,Epidermolysis bullosa acquisita (epidermolisis bullosa acquisita,EBA),Hematochromatosis,Myocarditis,Nephrotic syndrome,Primary sclerotic cholangitis,Suppurative or apyetous nasosinusitis,Acute or chronic nasosinusitis,Eso-ethmoiditis,Frontal sinusitis,Maxillary sinusitis or sphenoiditis,Eosinocyte associated conditions such as eosinophilia,Ensinophilosis infiltrates,Eosinophilia-myalgia syndrome,Lv Fuleshi (Loffler) syndrome,Chronic eosinophilic pneumonia,Tropical ensinophilosis,Bronchial aspergillosis,Aspergilloma,Or the granuloma containing eosinocyte,Allergic reaction,Seronegativity arthritis vertebralis disease,Polyendocrine autoimmune disease,Sclerosing cholangitis,Sclera,Episclera,Chronic mucocutaneous candidiasis,Bruton's (Bruton) syndrome,Infancy transient hypogammaglobulinemia,Neat (Wiskott-Aldrich) syndrome in prestige Scott-Ao Er Delhis,Incoordination capillarectasia,The autoimmune conditions relevant with the following：Collagen disease, rheumatism, neurological disease, lymphnoditis, ischemia-reperfusion is disorderly, blood pressure response reduces (reduction in blood pressureresponse), dysfunction of blood vessel, capillarectasia (antgiectasis), tissue damage, Cardiovascular ischemia, hyperalgia, cerebral ischemia and the disease with vascularization, allergia supersensitivity illness, glomerulonephritis disease, reperfusion injury, the reperfusion injury of myocardium or other tissues, skin disease with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory conditions, eye and socket of the eye inflammatory conditions, granulocyte Transfusion related syndromes, the poisoning that cell factor induces, acute severe inflammation, chronic and refractory inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic keratopathy main artery illness, intra-arterial hyperplasia, peptic ulcer, cardiovalvulitis, and endometriosis.

In specific embodiments, humanization 2H7 antibody and its functional fragment are used to treat rheumatoid arthritis and juvenile rheumatoid arthritis, systemic loupus erythematosus (SLE) includes lupus nephritis, Wei Genashi (Wegener) diseases, inflammatory bowel disease, ulcerative colitis, ITP (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephrosis, IgM polyneuropathies, myasthenia gravis, ANCA relevant blood vessels are scorching, diabetes, Lei Nuoshi (Reynaud) syndrome, Siogren (Sjogren) syndrome, neuromyelitis optica (NMO) and glomerulonephritis.

" processing " or " treatment " or " mitigation " refer to therapeutic treatment, and wherein target is the recurrence for slowing down (mitigation) targeted pathological condition or illness if it can not cure or preventing illness.If after the humanization CD20 binding antibodies of the present invention of therapeutic dose are received according to the method for the present invention, patient shows observable and/or measurable reduction or disappearance in one or more of the sign and symptom of specified disease, then subject success " treatment " autoimmunity disease or CD20 positive B-cells malignant tumours.For example, for cancer, significant cancer cell number is reduced or cancer cell disappears；Tumor mass reduction；Metastases are suppressed (i.e. a certain degree of to slow down, preferably to stop)；Tumour growth is suppressed by a certain degree of；Paracmasis extends；And/or the one or more symptoms relevant with particular cancers obtain a certain degree of mitigation；Morbidity and mortality are reduced；And quality of life is improved.The mitigation of disease diagnostic or symptom can also by patient perceptions to.Complete response can be achieved in treatment, and all signs for being defined as cancer disappear, or partial response, wherein tumor mass reduction, preferably greater than 50%, more preferably 75%.If the state of an illness of patient obtains stabilization, it is also considered as patient and obtains medical treatment.In preferred embodiments, the treatment carried out with antibody of the present invention effectively causes the cancer of cancer patient not develop within 4 months after the treatment, 6 months after preferred therapeutic, more preferably 1 year, even more preferably 2 years or more years.These parameters for assessing the successful treatment of disease and improving are easy to measure by routine protocols known to the competent internist in this area.

" therapeutically effective amount " refers to the amount of the disease or illness in antibody and medicine effectively " treatment " subject.For cancer, the therapeutically effective amount of medicine can reduce the number of cancer cell；Reduce the size of tumour；Suppress and (slow down to a certain extent, preferably prevent) cancer cell infiltration into peripheral organs；Suppress and (slow down to a certain extent and preferably prevent) metastases；Suppress tumour growth to a certain extent；And/or mitigate one or more symptoms relevant with cancer to a certain extent.Referring to the definition of foregoing " treatment ".For autoimmunity disease, the antibody or other medicines of therapeutically effective amount effectively mitigate disease diagnostic and symptom.

To be known to the internist that is competent in terms of the corresponding disease for assessing the therapeutic efficiency of knurl or the parameter of achievement.Generally, competent internist will expect the mitigation of the sign and symptom of specified disease.Parameter may include stable disease, regression time, the Median Time of disease development.

Lymthoma and CLL, their diagnosis, treatment and standard medical code for measuring therapeutic efficiency are described below with reference to document：Canellos GP, Lister, TA, Sklar JL, The Lymphomas, W.B.Saunders Company, Philadelphia, 1998；Van Besien K and Cabanillas, F, Clinical Manifestations, Staging and Treatment of Non-Hodgkin ' s Lymphoma, Chap.70, pp 1293-1338, in：Hematology, Basic Principles and Practice, the 3rd edition, Hoffman et al. (eds.), Churchill Livingstone, Philadelphia, 2000；And Rai, K andPatel, D, Chronic Lymphocytic Leukemia, Chap.72, pp 1350-1362, in：Hematology, Basic Principles and Practice, the 3rd edition, Hoffman et al. (eds.), Churchill Livingstone, Philadelphia, 2000.

To be known to the internist that is competent in terms of the corresponding disease for assessing the therapeutic efficiency of autoimmunity disease or autoimmune-associated diseases or the parameter of achievement.Generally, competent internist can expect the mitigation of the sign and symptom of specified disease.It is exemplified below.

In one embodiment, humanization 2H7 antibody and specific hu2H7.v511 and its functional fragment are used to treat rheumatoid arthritis.

RA is influence more than 2,000,000 Americans and the debilitating autoimmunity disease of obstruction victim's daily routines.Occurs RA when the improper attack joints tissue of the self immune system of body and when causing chronic inflammation and the intraarticular damage of destruction health tissues.Symptom includes Joint Inflammation, swelling, stiff and pain.Further, since RA is systemic disease, it may have an impact to other tissue such as lung, eye and marrow.Have no knowledge about healing.Treatment includes a variety of steroids and nonsteroid anti-inflammatory drugs, immunodepressant, the antirheumatic drug (DMARD) and biological agent for alleviating the state of an illness.However, many patients continue not enough to treatment response.

Antibody can be used as the gamma therapy in early stage RA (i.e. not used methotrexate (MTX) (MTX)) patient, be used as monotherapy or joint such as MTX or endoxan.Or, antibody can be used to treat DMARD and/or MTX resistant patients as second-line therapy, be used as monotherapy or joint such as MTX.Humanization CD20 binding antibodies can be used for preventing and controlling joint injury, delay structure damage, mitigate the pain with the inflammation-related in RA, and generally mitigate sign and symptom of the moderate into severe RA.RA patient can be before with other medicines (conjoint therapy that the see below) treatment used in treatment RA, afterwards or together with which with humanization CD20 Antybody therapies.In one embodiment, the antirheumatic drug for previously slowing down the solution state of an illness with the humanization CD20 binding antibodies treatment of the present invention have failed and/or single methotrexate (MTX) is responded not enough patient.In an embodiment of this treatment, patient received single humanization CD20 binding antibodies (the 1st day and the 15th day intravenous infusion 1g) at 17 days in therapeutic scheme；CD20 binding antibodies+endoxan (the 3rd day and the 17th day intravenous infusion 750mg)；Or CD20 binding antibodies+methotrexate (MTX).

Based on a kind of method of therapeutic efficiency in assessment RA is with rheumatology association of the U.S. (AmericanCollege of Rheumatology, ACR) standard, it measures improvement percentage of tenderness and swollen joint etc..Compared with (such as the baseline of before processing) or placebo treatment are handled without antibody, RA patient can score as such as ACR 20 (20% improves).Assessing the other manner of Antybody therapy effect includes the Sharp X-rays scoring that X-ray scores such as the scoring that narrows to structural damage such as bone erosion and articular cavity.Can also based on treatment during or after health evaluating questionnaire (Health AssessmentQuestionnaire, the HAQ) score in each period, AIMS scores, the SF-36 prevention or improvement that are disabled to patient evaluation.The standards of ACR 20 may include to touch a tender spot in (pain) joint number and 20% improving both swollen joint number+following 5 extra measurements and the 20% of at least 3 improve：

1. according to the Patient Pain Assessment (VAS) of intuitive analog scale,

2. patient's overall evaluation (VAS) of disease activity,

3. doctor's overall evaluation (VAS) of disease activity,

4. the patient's self-assessment measured by health evaluating questionnaire disables, and

5. acute phase reactant, CRP or ESR.

ACR 50 is similar with 70 definition.Preferably, a certain amount of CD20 binding antibodies of the present invention are applied to patient, it is enough to realize at least ACR 20 score, most preferably at least preferably at least ACR 30, more preferably at least ACR 50, even more desirably at least ACR 70, ACR 75 and Geng Gao.

Psoriatic arthritis has unique and unique radiograph feature.For psoriatic arthritis, also joint erosion can be assessed by Sharp scores and joint space narrows.The humanization CD20 binding antibodies of the present invention can be used for prevention joint injury and mitigate the disease sign and symptom of illness.

Another aspect of the present invention is come sanatory method by the humanization CD20 binding antibodies of the present invention to patient therapeuticallv's effective dose with SLE or lupus nephritis.SLEDAI scores provide the numerical quantization of disease activity.SLEDAI is the Weighted Index of known 24 clinics relevant with disease activity and laboratory parameters, number range 0-103.Referring to Bryan Gescuk ＆ John Davis, " Noveltherapeutic agent for systemic lupus erythematosus ", in：Current Opinion inRheumatology 2002,14：515-521.Think that the antibody for being directed to double-stranded DNA causes other performances of kidney rubescent (renalflares) and lupus.Kidney rubescent time (time to renal flare) can be reached to the patient-monitoring for receiving Antybody therapy, this is defined as significant, the reproducible rise of blood in serum creatinine, urine protein or urine.Or the level of antibody that can be to patient-monitoring antinuclear antibodies and for double-stranded DNA.SLE treatment includes the corticosteroid and/or endoxan (HDCC) of high dose.

Spondyloarthropathy is one group of disorder of joint, including ankylosing spondylitis, psoriatic arthritis and Chron (Crohn) disease.Treatment achievement can be determined by the patient by checking and doctor's total evaluation survey tool.

The therapeutic efficiency of psoriasis is assessed by the clinical sign and symptom of monitoring of diseases than the change of baseline condition, including doctor's overall evaluation (PGA) change and psoriasis area and severity index (PASI) score, psoriasis symptom assessment (PSA).Can the humanization CD20 binding antibodies such as hu2H7.v511 of the periodic measurement present invention is treated in the intuitive analog scale for indicating the degree of itching that particular point in time is subjected to during whole treatment psoriatic.

Patient may they first infusion of therapeutic with antibody when be subjected to infusion reaction or infusion related symptoms.The order of severity of these symptoms is different and can generally be reversed by medical intervention.These symptoms include but is not limited to the heating of influenza sample, shiver with cold/stiff, nauseous, nettle rash, headache, bronchial spasm, angioedema.Expect that infusion reaction is minimized for the methods for the treatment of diseases of the present invention.In order to mitigate or minimize the antibody of such adverse events, the acceptable initial adaptation of patient or tolerance dose, treatment effective dose is then only.Adapt to dosage (conditioning dose) and will be less than treatment effective dose so that patient adapts to and is resistant to higher doses.

Dosage is administered

The factor relevant with dosage administration according to known to indication to be treated and the skilled internist in this area, will apply antibody of the invention effectively to treat the indication dosage that Side effect is minimized simultaneously.Preferable dosage is likely to be dependent on the order of severity, the stage of disease, the desired B cell regulation and control level of disease and disease, and other factorses known to the skilled internist in this area.

For the treatment of autoimmunity disease, it may be desirable to regulate and control the degree of B cell abatement by adjusting the dosage of humanization 2H7 antibody according to the order of severity of illness in disease and/or individual patient.B cell abatement can with and it is nonessential be complete.Or, whole B cell abatements are may expect in initial treatment, but can adjust dosage to reach that only part is cut down in successive treatment.In one embodiment, B cell abatement is at least 20%, i.e., retain 80% or less CD20 positive B-cells compared with the baseline values before treatment.In other embodiments, B cell abatement is 25%, 30%, 40%, 50%, 60%, 70% or more.Preferably, B cell abatement is enough to prevent advancing of disease, more preferably mitigates the S＆S of the disease specific in treatment, even more preferably cures disease.

Genentech and Biogen Idec clinical investigation have evaluated using multi-agent scope from the low treatment effect for reaching one anti-CD20 (hu2H7.v16 and Rituximab) the treatment autoimmunity diseases of 10mg up to 1g (see the background parts studied on Rituximab；And WO 04/056312, embodiment 16).In general, applying two doses of antibody in these clinical investigations, it is spaced about two weeks.The example for the therapeutic scheme studied in clinical investigation includes：It is 2 × 10mg (2 doses, every dose of 10mg that humanization CD20 antibody 2H7.v16, which is used for during rheumatoid arthritis,；For body weight 70kg, the patient of 67 inches of height, accumulated dose~10.1mg/m²), 2 × 50mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose 55mg/m²), 2 × 200mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose 220mg/m²), 2 × 500mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose~550mg/m²) and 2 × 1000mg (for body weight 70kg, the patient of 67 inches of height, accumulated dose~1100mg/m²)；And Rituxan be 2 × 500mg (for body weight 70kg, the patient of 67 inches of height, accumulated dose~550mg/m²), 2 × 1000mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose~1100mg/m²).In these each dosage, bone-marrow-derived lymphocyte is cut down in substantial circulation is observed after applying first dose of antibody.

In the inventive method that autoimmunity disease and abatement B cell are treated in Serum of Patients With Autoimmune Diseases, in one embodiment, 1 dose of flat dosage by 0.1mg of scope to 1000mg applies humanization 2H7.v511 antibody.We have found that realizing substantial B cell abatement less than 300mg, or even 10mg flat dosage.In this way, in the B cell abatement and treatment method of the present invention, in different embodiments, hu2H7.v511 antibody is applied with 0.1,0.5,1,5,10,15,20,25,30,40,50,75,100,125,150,200 or 250mg dosage.If target is part or short-term B cell abatement, then can use relatively low-dose, such as 20mg, 10mg or lower.

For the treatment of CD20 positive cancers, it may be desirable to the abatement maximization of the B cell of anti-CD 20 antibodies target of the present invention will be used as.So, for the treatment of CD20 positive B-cells tumours, wish that B cell abatement is at least enough to prevent advancing of disease, this can be assessed by the skilled internist in this area, for example other S＆Ss by monitoring tumour growth (size), the propagation of cancerous cell type, transfer, specific cancer.Preferably, B cell abatement is enough in the time of at least two month to prevent advancing of disease, more preferably 3 months, even more preferably 4 months, more preferably 5 months, even more preferably 6 months or more months.In even more preferably embodiment, B cell abatement is enough regression time extending at least six month, more preferably 9 months, more preferably 1 year, more preferably 2 years, more preferably 3 years, even more preferably 5 years or more years.In a most preferred embodiment, B cell abatement is enough to cure disease.In preferred embodiments, the B cell abatement in cancer patient is at least about the 75% and more preferably 80%, 85%, 90%, 95%, 99% and even 100% of the preceding baseline values for the treatment of.

Hu2H7 antibody includes v16 and v511 and is recorded in hereafter EXPERIMENTAL EXAMPLESThe 18-20 for treating NHL the dosage therapeutic regimen of clinical test and the example of dosage.

Dosage 50 in terms of mg/ agent, 75,100,125,150,200,250,300,350mg/ agent can also be used for B cell malignant tumour such as NHL maintenance therapy.

Administration frequency can change with a number of factors.At least 2 doses humanization 2H7 CD20 binding antibodies will be applied to patient, 2-4 agent, 2-8 agent, 2-10 agent are subjected in different embodiments.Generally, 2 doses were applied in one month, 1 is typically spaced, 2 or 3 weeks.According to the level of amelioration of disease or recurrence, more multi-agent or the maintenance therapy as disease can be entirely being applied during one's sickness.

One or more Current Therapies are invalid, the autoimmunity disease or B cell malignant tumor patient of Intolerance or taboo can be treated with any dosage regimen of the present invention.For example, present invention contemplates be used to the treatment method of the present invention respond not enough RA patient to TNF (TNF) inhibitor therapy or to antirheumatic drug (DMARD) therapy for mitigating disease.

In another embodiment, low dosage 200mg/ agent or the treatment of lower dosage can be used for maintenance therapy.

In one embodiment, this dosage and dosage regimen are used when treating rheumatoid arthritis (RA).

" long-term " apply refers to short term patterns on the contrary, medicament is applied in a continuous mode, so that initial treatment effect (activity) is maintained into longer period of time.It is not the treatment free of discontinuities being carried out continuously that " interval " administration, which refers to, but substantially periodic.

Administration route

Humanization 2H7 antibody is applied to human patientses according to known method, such as applied by intravenous, for example inject or by continuous pouring for a period of time, by in subcutaneous, intramuscular, intraperitoneal, myelencephalon, intra-articular, intrasynovial, it is intrathecal or suction path, generally by intravenously or subcutaneously applying.

In one embodiment, humanization 2H7 antibody is applied using 0.9% sodium chloride solution as infusion medium by intravenous infusion.

In another embodiment, humanization 2H7 antibody is applied by being subcutaneously injected.

Conjoint therapy

When stating B cell tumour in the treatment, patient can be treated with the humanization 2H7 antibody of the present invention with the drug scheme of one or more therapeutic agent such as chemotherapeutic agents.Humanization 2H7 antibody can with chemotherapeutics simultaneously, it is sequential or replace administration, or applied after other therapies are without response.Standard chemotherapeutic for lymphoma treating may include endoxan, cytarabine, melphalan (melphalan) and mitoxantrone (mitoxantrone)+melphalan.CHOP is one of the most frequently used chemotherapy regimen for treating non_hodgkin lymphoma.The following is the medicine for CHOP schemes：Endoxan (trade mark cytoxan, neosar)；Adriamycin (Doxorubicin/hydroxyl Doxorubicin)；Vincristine (Oncovin)；With prednisolone (sometimes referred to as Deltasone or Orasone).In specific embodiments, CD20 binding antibodies are applied to required patient with one or more following chemotherapeutic agents：Doxorubicin, endoxan, vincristine and prednisolone.In a specific embodiment, combined to treat lymthoma (such as non_hodgkin lymphoma) patient with CHOP (endoxan, Doxorubicin, vincristine and prednisolone) therapy with the humanization 2H7 antibody of the present invention.In another embodiment, cancer patient can combine CVP (endoxan, vincristine and prednisolone) chemotherapy to treat with the humanization 2H7 CD20 binding antibodies of the present invention.In a specific embodiment, CVP is combined with humanization 2H7.v511 to treat the positive NHL patients of CD20.In a treatment CLL specific embodiment, hu2H7.v511 antibody using the chemotherapy combined of one or both of fludarabine (fludarabine) and endoxan (cytoxan) with being applied.

" chemotherapeutics " refers to the chemical compound available for treating cancer.The example of chemotherapeutics includes alkylating agents (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and CYTOXAN  endoxan (cyclophosphamide)；Alkylsulfonates (alkyl sulfonates), such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan)；Aziridines (aziridines), such as Benzodepa (benzodepa), carboquone (carboquone), meturedepa (meturedepa) and uredepa (uredepa)；Ethylenimines (ethylenimines) and methylamelamines (methylamelamines), including hemel (altretamine), triethylenemelamine (triethylenemelamine), triethylphosphoramide (triethylenephosphoramide), triethylene thiophosphamide (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine)；TLK 286(TELCYTA^TM)；Annonaceousacetogenicompounds (acetogenins) (especially bullatacin (bullatacin) and bullatacinone (bullatacinone))；Delta-9-Tetrahydrocannabinol (tetrahydrocannabinol) (Dronabinol (dronabinol), MARINOL )；β-lapachol (lapachone)；Lapachol (lapachol)；Colchicines (colchicines)；Betulic acid (betulinic acid)；Camptothecine (camptothecin) (including synthetic analogues Hycamtin (topotecan) (HYCAMTIN ), CPT-11 (Irinotecan (irinotecan), CAMPTOSAR ), acetyl camptothecine, scopoletin (scopoletin) and 9-aminocamptothecin)；Bryostatin (bryostatin)；callystatin；CC-1065 (including its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues)；Podophyllotoxin (podophyllotoxin)；Podophyllic acid (podophyllinic acid)；Teniposide (teniposide)；Cryptophycins (cryptophycins) (particularly cryptophycin 1 and cryptophycin 8)；Dolastatin (dolastatin)；Duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1)；Eleutherobin (eleutherobin)；pancratistatin；sarcodictyin；Spongistatin (spongistatin)；Nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), cholophosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard)；Nitrosourea (nitrosoureas), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine)；Diphosphonates (bisphosphonates), such as clodronate (clodronate)；Antibioticses, such as Enediyne Antibiotic (enediyne) (such as Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin ω I1 are (see, for example, Agnew, Chem.Intl.Ed.Engl.33：183-186 (1994)) and anthracycline antibiotic (anthracyclines) such as annamycin,AD 32,alcarubicin,Daunorubicin (daunorubicin),Dexrazoxane (dexrazoxane),DX-52-1,Epirubicin (epirubicin),GPX-100,Idarubicin (idarubicin),KRN5500,Menogaril (menogaril),Dynemicin includes dynemicin A,Ai sibo mycin (esperamicin),Neocarzinostatin (neocarzinostatin) chromophore and related chromoprotein Enediyne Antibiotic chromophore,Aclacinomycin (aclacinomycin),D actinomycin D (actinomycin),Anthramycin (anthramycin),Azaserine (azaserine),Bleomycin (bleomycin),Act-C (cactinomycin),carabicin,Carminomycin (carminomycin),Cardinophyllin (carzinophilin),Chromomycin (chromomycin),Actinomycin D (dactinomycin),Detorubicin (detorubicin),6- phenodiazine -5- oxygen-L- nor-leucines,ADRIAMYCIN  Doxorubicins (doxorubicin) (including morpholino Doxorubicin,Cyanomorpholino Doxorubicin,2- pyrroles is for Doxorubicin,Mycocet and deoxydoxorubicin),Esorubicin (esorubicin),Marcellomycin (marcellomycin),Mitomycin (mitomycins) such as mitomycin C,Mycophenolic acid (mycophenolic acid),Nogalamycin (nogalamycin),Olivomycin (olivomycin),Peplomycin (peplomycin),potfiromycin,Puromycin (puromycin),Triferricdoxorubicin (quelamycin),Rodorubicin (rodorubicin),Streptonigrin (streptonigrin),Streptozotocin (streptozocin),Tubercidin (tubercidin),Ubenimex (ubenimex),Zinostatin (zinostatin) and zorubicin (zorubicin)；Folacin, such as denopterin (denopterin), pteropterin (pteropterin) and Trimetrexate (trimetrexate)；Purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), thiapurine (thiamiprine) and thioguanine (thioguanine)；Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6- azauridines (azauridine), Carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine) and floxuridine (floxuridine)；Androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane) and Testolactone (testolactone)；Anti- adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane) and Trilostane (trilostane)；Folic acid supplement, such as folinic acid (folinicacid) (leucovorin)；Aceglatone (aceglatone)；Anti- folic acid antitumor agent; such as ALIMTA , LY231514 pemetrexeds (pemetrexed), dihydrofolate reductase inhibitor such as methopterin (methotrexate), antimetabolic species; such as 5 FU 5 fluorouracil (fluorouracil) (5-FU) and its prodrug such as UFT, S-1 and capecitabine (capecitabine), and thymidilate synthase inhibitors and glycinamide ribonucleotide transformylase inhibitor such as Raltitrexed (raltitrexed) (TOMUDEx^TM, TDX)；Dihydropyrimidine dehydrogenase inhibitor such as eniluracil (eniluracil)；Aldophosphamideglycoside (aldophosphamide glycoside)；Amino-laevulic acid (aminolevulinic acid)；Amsacrine (amsacrine)；bestrabucil；Bisantrene (bisantrene)；Edatrexate (edatraxate)；Defosfamide (defosfamide)；Demecolcine (demecolcine)；Diaziquone (diaziquone)；elfornithine；Elliptinium Acetate (elliptinium acetate)；Epothilones (epothilone)；Ethoglucid (etoglucid)；Gallium nitrate；Hydroxyurea (hydroxyurea)；Lentinan (lentinan)；Lonidamine (lonidamine)；Maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin)；Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；Mopidamol (mopidamol)；C-283 (nitracrine)；Pentostatin (pentostatin)；Phenamet (phenamet)；THP (pirambicin)；Losoxantrone (Iosoxantrone)；2- ethylhydrazides (ethylhydrazide)；Procarbazine (procarbazine)；PSK  polysaccharide compounds (JHS NaturalProducts, Eugene, OR)；Razoxane (razoxane)；Rhizomycin (rhizoxin)；Sizofiran (sizofiran)；Spirogermanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2,2 ', 2 "-trichlorotriethylamines；Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and snake rhzomorph (anguidin))；Urethane (urethan)；Eldisine (vindesine) (ELDISINE , FILDESIN )；Dacarbazine (dacarbazine)；Mannomustine (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；gacytosine；Cytarabine (arabinoside) (" Ara-C ")；Endoxan (cyclophosphamide)；Phosphinothioylidynetrisaziridine (thiotepa)；Taxoids (taxoids) and taxanes (taxanes), such as TAXOL  taxols (paclitaxel) (Bristo1-MyersSquibb Oncology, Princeton, N.J.), ABRAXANE^TMWithout cremophor (Cremophor), nano particle formulation taxol (the American Pharmaceutical Partners of albumin transformation, Schaumberg, Illinois) and TAXOTERE  Taxoteres (docetaxel) (Rh

Ne-PoulencRorer, Antony, France)；Chlorambucil (chlorambucil)；Gemcitabine (gemcitabine) (GEMZAR )；6- thioguanines (thioguanine)；Purinethol (mercaptopurine)；Platinum (platinum)；Platinum analogs or the analog based on platinum, such as cis-platinum (cisplatin), oxaliplatin (oxaliplatin) and carboplatin (carboplatin)；Vincaleukoblastinum (vinblastine) (VELBAN )；Etoposide (etoposide) (VP-16)；Ifosfamide (ifosfamide)；Mitoxantrone (mitoxantrone)；Vincristine (vincristine) (ONCOVIN )；Vinca alkaloids (vinca alkaloid)；Vinorelbine (vinorelbine) (NAVELBINE )；NSC-279836 (novantrone)；Edatrexate (edatrexate)；Daunomycin (daunomycin)；Aminopterin (aminopterin)；Xeloda (xeloda)；Ibandronate (ibandronate)；Topoisomerase enzyme inhibitor RFS 2000；DFMO (DMFO)；Retinoic acid-like (retinoid), such as retinoic acid (retinoic acid)；Pharmacy acceptable salt, acid or the derivative of any of above material；And the combination of two or more above-mentioned substances, such as CHOP (endoxan, Doxorubicin, the abbreviation of vincristine and prednisolone conjoint therapy) and FOLFOX (oxaliplatin (ELOXATIN^TM) joint 5-FU and folinic acid therapeutic scheme abbreviation).

This definition also includes acting as the antihormone agent of regulation or inhibitory hormone to function of tumor, such as anti-estrogens and SERM class (SERM), including such as TAM (tamoxifen) (including NOLVADEX  TAMs), Raloxifene (raloxifene), Droloxifene (droloxifene), 4-hydroxytamoxifen, Trioxifene (trioxifene), that Lip river former times sweet smell (keoxifene), LY117018, Onapristone (onapristone) and FARESTON  Toremifenes (toremifene)；Suppress the aromatase inhibitor of the aromatase enzyme of regulation estrogen production in adrenal gland, such as 4 (5)-imidazoles, aminoglutethimide (aminoglutethimide), MEGASE  megestrol acetates (megestrolacetate), AROMASIN  Exemestanes (exemestane), formestane (formestane), Fadrozole (fadrozole), RIVISOR  Vorozoles (vorozole), FEMARA  Letrozoles (letrozole) and ARIMIDEX  Anastrozoles (anastrozole)；Anti-androgens, such as Drogenil (flutamide), Nilutamide (nilutamide), bicalutamide (bicalutamide), Leuprorelin (leuprolide) and Goserelin (goserelin)；And troxacitabine (troxacitabine) (DOX nucleosides analogue of cytosine)；ASON, particularly those suppression are related to gene expression of the signal of adherent cell propagation in, such as PKC- α, Raf, H-Ras and EGF-R ELISA (EGF-R)；Vaccine, such as gene therapy vaccine, such as ALLOVECTIN  vaccines, LEUVECTIN  vaccines and VAXID  vaccines；PROLEUKIN  rIL-2；The inhibitor of LURTOTECAN  topoisomerases 1；ABARELIX  rmRH；And pharmacy acceptable salt, acid or the derivative of any of above material.

In the treatment when the literary autoimmunity disease or autoimmunity related disorders, patient can combine second of therapeutic agent such as immunodepressant with one or more hu2H7 antibody such as hu2H7.v511, such as be treated with drug scheme.Hu2H7 antibody can with immunodepressant simultaneously, it is sequential or replace administration, or applied after other therapies are without response.Immunodepressant can be applied with the dosage identical or relatively low with listed by this area.It is preferred that skeptophylaxis inhibitor depend on many factors, including sanatory type and patient medical history.

" immunodepressant " is used to refer to the material for acting as suppressing or covering the immune system of patient during complementary therapy herein.Such medicament is by including suppressing cell factor generation, lowering or suppressing autoantigen expression or cover the material of MHC antigens.The example of such medicament includes steroids, such as glucocorticosteroid, such as metacortandracin (prednisone), methylprednisolone (methylprednisolone) and dexamethasone (dexamethasone)；The pyrimidine of 2- amino -6- aryl -5- substitutions (referring to United States Patent (USP) No.4,665,077)；Imuran (azathioprine) (or endoxan (cyclophosphamide), if having adverse reaction to imuran)；Bromocriptine (bromocryptine)；Glutaraldehyde (it covers MHC antigens, such as United States Patent (USP) No.4, described in 120,649)；For MHC antigens and the anti-idiotype of MHC fragments；Cyclosporin A；Cell factor or cytokine receptor antagonist, including anti-interferon-γ ,-β or-Alpha antibodies；Anti-tumor necrosis factor-Alpha antibodies；Anti-tumor necrosis factor-β antibody；Anti- proleulzin antibody and anti-IL-2 receptor antibodies；Anti- L3T4 antibody；Heterologous antilymphocyte globulin (ALG)；General (pan) T antibody, preferably AntiCD3 McAb or anti-CD4/CD4a antibody；Soluble peptide containing LFA-3 binding domain (WO 90/08187 is published on July 26th, 90)；Streptokinase；TGF-β；Dornase；RNA or DNA from host；FK506；RS-61443；Deoxyspergualin (deoxyspergualin)；Rapamycin (rapamycin)；φt cell receptor (United States Patent (USP) No.5,114,721)；φt cell receptor fragment (Offner et al., Science 251：430-432(1991)；WO 90/11294；WO 91/01133)；And φt cell receptor antibody (EP 340,109), such as T10B9.

For the treatment of rheumatoid arthritis, patient can be treated with the antibody combined one or more following medicines of hu2H7：DMARD (antirheumatic drug for mitigating disease) (such as methopterin), NSAI or NSAID (nonsteroid anti-inflammatory drugs), HUMIRA^TM(adalimumab, adalimumab；AbbottLaboratories), ARAVA  (leflunomide, leflunomide), REMICADE  (infliximab, infliximab；Centocor Inc., Malvern, Pa), ENBREL (Etanercept, etanercept；Immunex, WA), cox 2 inhibitor.The DMARD for being generally used for RA is HCQ (hydroxychloroquine), SASP (sulfasalazine), methopterin (methotrexate), leflunomide (leflunomide), Etanercept (etanercept), infliximab (infliximab), imuran (azathioprine), Beracilline, golden (Gold) (oral), golden (Gold) (intramuscular), minocycline (minocycline), cyclosporin (cyclosporine), staphylococcal protein A immuno absorbence.Adalimumab (Adalimumab) is the human monoclonal antibodies with reference to TNF α.Infliximab (Infliximab) is the chimeric mAb with reference to TNF α.Etanercept (Etanercept) is " immunoadhesin " fusion protein, is connected with the Fc parts of human IgG1 by the extracellular ligand binding moiety of people 75kD (p75) Tumor Necrosis Factor Receptors (TNFR) and is constituted.For RA conventional therapy, see, for example, " Guidelines for the management of rheumatoid arthritis ", Arthritis ＆Rheumatism 46 (2)：328-346 (February, 2002).In a specific embodiment, RA patient is treated with the antibody combined methopterins of hu2H7 CD20 (MTX) of the present invention.MTX Exemplary doses are about 7.5-25mg/kg/ weeks.MTX can pass through oral and subcutaneous administration.

For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's disease, such as Remicade  (infliximab infliximab can be combined with the CD20 binding antibodies of the present invention；CentocorInc., Malvern, Pa), ENBREL (Etanercept etanercept；Immunex, WA) treat patient.

SLE treatment includes high dose corticosteroid and/or endoxan (HDCC).

For the treatment of psoriasis, patient can administration of anti-cd 20 binding antibody joint local treatment, such as topical steroids, anthralin (anthralin), calcipotriene (calcipotriene), clobetasol (clobetasol) and tazarotene (tazarotene), or joint methopterin, retinoic acid-like, cyclosporin, PUVA and UVB therapies.In one embodiment, it is sequential or simultaneously use CD20 binding antibodies and cyclosporin therapy psoriatic.

In order to which toxicity is minimized, traditional systemic therapy can be implemented with samsara, sequential, united or interval therapeutic scheme, or hu2H7 CD20 binding antibodies composition uses the relatively low-dose scheme for combining of present dose.

Medicinal proportional preparation

The treatment preparaton of the hu2H7 CD20 binding antibodies used according to the present invention passes through with the antibody and the optional acceptable carrier of pharmacy, excipient or stabilizer (Remington ' sPharmaceutical Sciences for expecting purity, 16th edition, Osol, A. (1980) are compiled) mixing, prepared in the form of freeze-dried formulation or the aqueous solution.Acceptable carrier, excipient or stabilizer are nontoxic, including buffer, such as phosphate, citrate and other organic acids to recipient in the dosage and concentration used；Antioxidant, including ascorbic acid and methionine；Preservative (such as octadecyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride, benzethonium chloride；Phenol, butanol or phenmethylol；Alkyl paraben, such as methyl p-hydroxybenzoate or propyl ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；And metacresol)；Low molecule amount (less than about 10 residues) polypeptide；Protein, such as serum albumin, gelatin or immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Amino acid, such as glycine, glutamine, asparagine, histidine, arginine or lysine；Monose, disaccharides and other carbohydrate, including glucose, mannose or dextrin；Chelating agent, such as EDTA；Carbohydrate, such as sucrose, mannitol, trehalose or sorbierite；Into salt gegenion, such as sodium；Metal composite (such as Zn- protein complexes)；And/or nonionic surfactant, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Exemplary hu2H7 antibody formulations are recorded in WO 98/56418, and clearly income is used as reference herein.Another preparaton is liquid multi-agent preparaton, comprising 40mg/mL hu2H7 antibody, 25mM acetates, 150mM trehaloses, 0.9% phenmethylol, 0.02% polysorbate20 pH5.0, and minimum storage life is preserved 2 years at 2-8 DEG C.Another anti-CD 20 antibodies preparaton interested is in 9.0mg/mL sodium chloride, the citric acid monohydrate sodium of 7.35mg/mL bis-, and 10mg/mL antibody is included in 0.7mg/mL polysorbate80s, and Injectable sterile water pH6.5.Also a kind of aqueous medicinal proportional preparation includes 10-30mM sodium acetates, about pH4.8 to the preferred pH5.5 of about pH5.5, it is used as the polysorbate of the about 0.01-0.1%v/v amounts of surfactant, the trehalose of about 2-10%w/v amounts, with the phenmethylol (U.S.6 as preservative, 171,586).Freeze-dried formulation suitable for subcutaneous administration is recorded in WO 97/04801.Such freeze-dried formulation can use suitable solvent to rebuild to increased protein concentration, and the preparaton of reconstruction can subcutaneous administration mammal to be treated in this article.

A kind of preparaton of humanization 2H7.v511 variants is 10mM histidines, 6% sucrose, 0.02% poly- sorbitol ester 20, the 12-14mg/ml antibody in pH5.8.In a specific embodiment, 2H7 variants, particularly 2H7.v511 are configured to 10mM sulfuric acid histidines, 60mg/ml sucrose, the poly- sorbitol esters 20 of 0.2mg/ml, and the 20mg/ml antibody in Injectable sterile water pH5.8.

Preparaton herein can also contain have more than one kind treat reactive compound necessary to specific indication, preferably those complementary activities and the compound not adversely affected each other.For example, it may be desirable to further provide for cytotoxic agent, chemotherapeutics, cell factor or immunodepressant (such as acting on the immunodepressant of T cell, such as cyclosporin or the antibody of combination T cell, such as antibody with reference to LFA-1).The effective dose of such other medicaments depends on amount, disease or the illness of antibody or the type for the treatment of and other factorses discussed above present in preparaton.These be typically with identical dosage described herein, used with administration route described herein, or the dosage used so far about 1-99%.

Active component can be also contained in the microcapsules prepared for example by condensation technique or by interfacial polymerization (being for example hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules respectively), in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule), or in macro emulsion.Such technology is disclosed in such as Remington ' sPharmaceutical Sciences, and the 16th edition, Osol, A. compiles (1980).

Extended release preparation can be prepared.The suitable example of extended release preparation includes the solid hydrophobic polymers semipermeable matrices containing antagonist, and the matrix is the form of approved product, such as film or microcapsules.The example of sustained-release matrix includes polyester, hydrogel (such as poly- (2- ethoxys-methacrylate) or poly- (vinyl alcohol)), polyactide (United States Patent (USP) No.3,773,919), the copolymer of Pidolidone and Pidolidone γ ethyl esters, nondegradable ethene-vinyl acetate copolymer, degradable lactic acid-ethanol copolymer such as LUPRON DEPOT^TM(the Injectable microspheres body being made up of lactic acid-ethanol copolymer and leuprorelin acetate) and poly- D- (-) -3-hydroxybutyrate.

Preparaton for applying in vivo must be sterile.This readily can be realized by using sterilised membrane filter filtering.

Product and kit

Another embodiment of the invention is the product for including the material that can be used for treatment autoimmunity disease and related disorders and CD20 positive cancers such as non_hodgkin lymphoma.The product includes on container and container or the label or package insert related to container.Suitable container is included such as medicine bottle, pencil, syringe.The container can be made of multiple material, such as glass or plastics.The container is equipped with the composition for effectively treating the illness, can have sterile access port (such as described container can be the medicine bottle of intravenous solution bag or the plug that can pierce with hypodermic needle).At least one of composition active agents are hu2H7 antibody, hu2H7.v511 of the invention.The label or package insert indicate that said composition is used to treat the specific illness.The label or package insert further include the specification on applying the antibody compositions to patient.

Package insert refers to the specification being typically included in during treatment is packed with product ommercialization, it include used about such treatment with product indication, usage, dosage, using, avoid and/or warning message.In one embodiment, package insert indicates that said composition is used to treat non_hodgkin lymphoma.

In addition, the product can further comprise second container, wherein being subjected to buffer solution, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), woods grignard (Ringer) solution and dextrose solution equipped with pharmacy.It can further comprise the other materials from business and user's position needs, including other buffer solutions, diluent, filter, pin and syringe.

The kit available for various purposes is additionally provided, for example, kills determination method, the positive control as apoptosis determination method, for purifying or immunoprecipitation CD20 from cell for B cell.In order to separate and purify CD20, the kit can include the hu2H7.v511 antibody being coupled with pearl (such as sepharose pearls).It can provide comprising CD20 vitro detections and quantitative antibody is used for, for example, be carried out in ELISA or western blot.As product, the kit includes on container and container or the label or package insert related to container.The container is equipped with the composition for including at least one anti-CD 20 antibodies of the present invention.It may include such as diluent and buffer solution, other container of control antibodies are housed.The label or package insert can provide the description of said composition and be intended to the specification that external or diagnosis is used.

EXPERIMENTAL EXAMPLESThe

Embodiment 1：Transformation from existing cell line to fucosylated less cell line

In order to realize the non-fucosylated antibody of high yield in Chinese hamster ovary celI, the expression of low FUT8 genes is struck using RNAi methods.Using from Ambion companies (Austin, TX pSilencer3.1-H1-Puro plasmids) press from both sides siRNA to produce bob, consisting of thering is adopted siRNA sequence to be connected with its reverse complemental antisense siRNA sequence by one short spacer region (9nt hairpin loops) 19nt (nucleotides) of FUT8 gene specifics, the 5-6 U (Fig. 3) at 3 ' ends is followed by.The method of siRNA probes for designing targeting CHO FUT8 genes refers to Elbashir et al., 2002.Based on obtainable CHO FUT8DNA sequences Designs, five kinds of different siRNA probes (probe #1-5) target different zones (Fig. 4).Probe 1 (SEQ ID NO.3 and NO.4)；Probe 2 (SEQ ID NO.5 and NO.6)；Probe 3 (SEQ IDNO.7 and NO.37)；Probe 4 (SEQ ID NO.38 and NO.39)；Probe 5 (SEQ ID NO.40 and NO.41).By 19nt by adopted sequence by the siRNA coded sequences that spacer region is connected with antisense sequences and 5-6 U is constituted be probe #2 (7-59 in SEQ ID 5) in SEQ ID NO.42 and probe #4 in SEQ ID NO.43.The RNAi 1-5 that probe 1-5 corresponds in Fig. 5 B.5 kinds of siRNA probes are built by the way that the synthetic oligonucleotide independent cloning of annealing is entered into pSilencer 3.1-H1-Puro plasmids.

In order to test effect of these RNAi probes, the FUT8 fusion proteins of FLAG marks are built using the CHO FUT8 partial dna sequences (numbering P_AAC63891) from Genbank.3 ' end 0.98kb fragments of FUT8 coded sequences are cloned as reverse transcriptase polymerase chain reaction (RT-PCR) using the total serum IgE and FUT8 primers purified from Chinese hamster ovary celI and PCR fragment is merged with 5 ' end FLAG sequence labels by obtained by.The Flag labels (metAspTyrLysAspAspAspAspLys-SEQ ID NO._) of 8 amino acid are added to 5 ' ends of the Partial cDNA Sequence of separation.The FUT8 fragments of tape label are cloned into expression vector.RNAi probes plasmid and flag the FUT8 plasmid co-transfections marked are entered into Chinese hamster ovary celI.Extract product of cell lysis within 24 hours after transfection and pass through immunoblotting assay FUT8 fusion protein levels using anti-flag M2 antibody (Sigma, MO).When there is RNAi probes, the expressing fusion protein for having 4 in 5 cases is significantly inhibited (Fig. 5).

The ability that these probes cut FUT8 transcription products is tested by the way that every kind of siRNA expression plasmids and Flag the FUT8 plasmid transient cotransfections marked are entered into Chinese hamster ovary celI.24 hours cell lysis and pass through Western blot analysis product of cell lysis with anti-Flag M2 antibody (Sigma, MO) after transfection.

As expected, RNAi1 (probe 1) transfectional cell shows the strongly expressed of the FUT8 products of Flag marks, because the FUT8 fusion proteins of Flag marks do not include the sequence (Fig. 5 A, 5B) that this probe is targetted.On the contrary, siRNA probes 2 (RNAi2) have different degrees of inhibition (Fig. 5 B) to 5 to the Flag FUT8 expressing fusion proteins marked.Probe #2 and #4 show best inhibition and are selected for further assessment.

Embodiment 2：The fucose content for the antibody that operation stabilization is expressed is expressed by instantaneous siRNA

RNAi2 and RNAi4 plasmids are transiently transfected into the expression Humanized anti-cd 20 antibodies 2H7.v16 previously set up stable Chinese hamster ovary celI system (clone #60).Then serum free medium transfectional cell separate inoculation entered in 250ml revolving bottles is used to produce antibody.

Expression and the 2H7.v16 antibody secreted and such as Papac et al., pass through the fucose content that N- connection oligosaccharides is analyzed in substance assistant laser desorpted/ionisation-time of flight mass spectral analysis (MALDI-TOF) in the cell culture fluid harvested with protein A column purification described in 1998.Antibody is also determined in Fc γ R binding assays (hereafter on the books).There are three class human Fc gamma receptors：Fc γ RI, Fc γ RII and Fc γ RIII.Some of them have functional alleles polymorphism, produce allograft (Dijstelbloem et al., 1999 with different acceptor properties；Lehrnbecher et al., 1999).Fc γ RIII (F158) have phenylalanine at the 158th and with low binding affinity (the Shields et al., 2001 and 2002) to human IgG Fc areas of the Fc γ RIII (V158) with valine than the 158th.

RNAi transiently transfects the non-fucosylated 2H7 antibody that cell generates about 35-37%, as shown in Figure 6.Compared with the 2H7 control cell lines (with unrelated RNAi plasmid transfections) of the non-fucosylated antibody (levels typical of the antibody produced by i.e. conventional Chinese hamster ovary celI) with about 2-4%, shown respectively with 35% to 37% non-fucosylated 2H7 collection of antibodies and Fc γ RIII (F158 allele) and Fc γ RIII (V158 allele) binding affinity are improved 6 times and 4 times (Fig. 7 D, 7E).The influence to other Fc acceptors is not seen (such as Fc γ RI and Fc γ RII- refer to Fig. 7 A, 7B, 7C).The glycan that the antibody that both cells transfected with analogies from RNAi plasmid transfections are produced is separated has similar galactose content distribution when comparing no galactolipin (G0), a galactolipin (G1) and two galactolipin (G2).These data displays can be reduced from the fucose content of the antibody of steady production cell line secretes by instantaneous RNAi plasmid transfections and the effect does not change other main glycan compositions, including G0, G1 and G2 distribution.

In order to confirm that RNAi transfectional cells have less FUT8 rna expressions really, Northern traces are carried out with 24 hours after the transfection RNA samples from transfection cell extraction.To being purified and being hybridized with 300bp probe from the total serum IgE containing control plasmid (random Mouse DNA sequences do not have homology with any known murine protein matter) and the cell of 2 RNAi plasmids.As shown in figure 8, the mRNA level in-site in two kinds of RNAi plasmid-transfected cells has been struck low (swimming lane 2 and 3).This is consistent with detecting the Western blotting of relatively low FUT8 protein contents in two kinds of RNAi plasmid-transfected cells.CHO FUT8 mRNA size is similar in rat cell, about 3.5kb.Endogenous α 1,6- fucosyltransferases RNA low pass of striking are crossed quantitative PCR and further confirmed (data are not shown).

Because two kinds of constructs of RNAi2 and RNAi4 all can effectively strike low endogenous FUT8 gene RNAs level, only selection RNAi4 plasmids are used for further stable transfection.600nM methotrexate (MTX)s (MTX) resistance is in RNAi4 constructs stable transfection and yield exceedes 1.5g/L antibody cell system clone 60 in bioreactor, puromycin gene is eliminated from pSilencer plasmids and replaced with the hygromycin under the control of SV40 promoters in the construct, is selected with 500 μ g/ml hygromycin.Positive colony picking is entered into 96 hole tissue culturing plates and endogenous FUT8 mRNA level in-sites are screened by Taqman.4 clone's expansion scales of varying level FUT8 mRNA reductions will be shown, antibody is produced in 250ml revolving bottles.Protein A purification is carried out to the antibody in HCCF and fucose content measure and Fc γ RIII combination mensurations is carried out.Fig. 7 A-E result is shown in tested Fc γ acceptors only Fc γ RIII and combined to be influenceed by the less antibody containing fucose.Therefore, the antibody products from stable transfection are only carried out with Fc γ RIII combination mensurations.

Described 4 of fucose content analysis display is the non-fucosylated antibody of generation in the range of 45-70% or 80%.Combination to including they and Fc γ RIII of the fucosylated TPPA of 5 kinds of varying levels.Fc γ RIII combination mensurations show that the improvement with low-affinity Fc γ RIII (F158) has increase than Fc γ RIII (V158), as shown in table 1.When with raising multiple to each antibody samples non-fucosylated matter percentage square mapping when, linear relationship all observed to two kinds of Fc γ RIII variants.Complete human IgG1 is included in two heavy chains, every Tiao Fc areas CH2 domains has a N- glycosylation site at Asn297.Therefore, there are 3 kinds of possibilities in terms of the fucose occupation rate of core carbohydrate structure for Fc.One heavy chain is fucosylated and one is not；Two heavy chains are all fucosylated；Or two heavy chains are not all fucosylated.Rise multiple and non-fucosylated glycan percentage to Fc γ RIII affinity square between linear relationship indicate that all not fucosylated antibody molecules of two heavy chains if if so may play a major role to the raising of elevated Fc γ RIII binding affinities.

Further expand in large-scale antibody producing being cloned in two kinds of stable transfections in bioreactor, the analysis of fucose content shows that fucosylation level remains stable during the research of 79 days, about 80% it is non-fucosylated.%G0, G1 and G2 on antibody titer and antibody glycan at the end of bioreactor is operated are also in desired extent.Therefore, it is the approach that can be used for producing such host cell that RNAi plasmid transfections are entered to the protein production cell line (being antibody producing cells system in this case) set up, and the host cell produces commercially valuable quantity, non-fucosylated carbohydrate with controlled quatity treatment antibody.

Table 1：The binding affinity of Fc γ RIII and the antibody of different fucose contents

Clone	Non- fucosylated (%)	Fc γ RIII (V158) are combined (again)	Fc γ RIII (F158) are combined (again)
				Control	3	1	1
5B	45	7.5	24.2
				6C	60	10.1	34.0
5F	70	13.7	52.4
				7C	63	10.4	32.4
Parent	5	1	1

Embodiment 3

In this embodiment, we construct a kind of new RNAi plasmids, and it includes two RNAi transcriptional units, target two different zones of FUT8 genes.The Plasmid Type in one region of than previous target gene of this plasmid is more effective.

Embodiment 4：The generation of the new stable cell lines of synchronous metabolic engineering (striking for fucosylation level is low) with fucose content

Material and method

Cell culture and transfection

By Chinese hamster ovary (CHO) cell in 37 DEG C of cultures in the growth medium containing 5%FBS (hyclone) and 1X GHT (glycine, hypoxanthine and thymidine).For transiently transfecting, use DMRIE-C transfection reagents (Invitrogen).For stable transfection, use Lipofectamine 2000 (Invitrogen).

Selection

After transfection, cell is centrifuged to collect sediment.Then sediment is resuspended in methotrexate (MTX) containing 25nM (MTX) culture medium.Culture medium was changed per 3-4 days.About 2 weeks after transfection, picking is individually cloned and cultivated in 96 orifice plates.It is generally necessary to make cell be grown in 96 orifice plates and converge for about 1 week.

Equal inoculum density determination method

By 5 × 10⁴Individual cells/well is inoculated with into 96 orifice plates.Next day, remove growth medium and replaced with production medium.That day added after production medium, plate is incubated 5-6 days in 33 DEG C, ELISA measure then can be carried out.

ELISA determination methods

When cell confluency, remove growth medium and production medium is added into each hole.That day added after production medium, plate is incubated 5-6 days in 33 DEG C, ELISA measure then can be carried out.Generally ELISA is carried out with serial dilution.

RNA analysis

It is with Qiagen RNA Purification Kits total serum IgE and quantitative by Taqman with gene-specific primer and probe.

Fc γ Receptor Binding Assays-ELISA

The hole microwell plates (Nunc, Roskilde, Denmark) of MaxiSorp 96 are used in the 2 anti-GST of μ g/ml (clone 8E2.1.1, Genentech) prepared in 50mM carbonate buffer solutions pH9.6 to stay overnight in 4 DEG C of coatings with 100 μ l/ holes.Plate is cleaned with the PBS pH7.4 (cleaning buffer solution) containing 0.05% polysorbate and closed with the PBS pH7.4 containing 0.5%BSA with 150 μ l/ holes.In after incubation at room temperature 1 hour, plate is cleaned with cleaning buffer solution.The people Fc γ RIII prepared in containing 0.5%BSA, the PBS pH7.4 (measure buffer solution) of 0.05% polysorbate20 are added in plate with 0.25 μ g/ml, 100 μ l/ holes.Plate is incubated 1 hour and then cleaned with cleaning buffer solution.By antibody and goat F (ab ')₂Anti- κ (Cappel, ICNPharmaceuticals, Inc., Aurora, Ohio) incubates 1 hour to form antibody complex together with 1: 2 (w/w) ratio.11 twice of serial dilutions that compound IgG antibody (0.85-50000ng/ml, with three times serial dilution) is prepared in buffer solution is determined are added in plate.After incubating 2 hours, plate is cleaned with cleaning buffer solution.By the goat F (ab ') that the peroxidase labelling prepared in buffer solution is determined is added with 100 μ l/ holes₂Anti-human igg F (ab ')₂(Jackson ImmunoResearch, West Grove, PA), detects combined IgG.After incubating 1 hour, plate is cleaned with cleaning buffer solution and substrate 3,3 ', 5,5 '-tetramethyl benzidine (TMB) (Kirkegaard ＆ Perry Laboratories) is added with 100 μ l/ holes.By adding 1M phosphoric acid, terminating reaction with 100 μ l/ holes.450nm absorbance is read on multiskan Ascent reader (ThermoLabsystems, Helsinki, Finland).Calculate the absorbance (mid-OD) of standard curve midpoint.With four parameter nonlinear regression curve fitting procedures (KaleidaGraph, Synergysoftware, Reading, PA) corresponding concentration of standard items and sample in this mid-OD is determined from titration curve.By by the mid-OD concentration of standard items divided by sample, calculating relative activity.

Cytotoxicity (ADCC) determination method of antibody dependent cellular

A kind of ADCC determination methods form is as follows.Substantially such as (Shields et al., J.Biol.Chem.276：6591-6604 (2001)) use lactic dehydrogenase (LDH) reading determines the abilities of 2H7 IgG variants mediation natural killer (NK) cell cracking WIL2-S cells (the lymphoblast sample B cell system of expression CD20 a kind of).NK cells are prepared from the 100mL heparinized bloods diluted with 100mL PBS (phosphate buffered saline (PBS)), the blood is obtained from normal human donor (Koene the et al., Blood 90 that isotype is identified for Fc γ RIII (also known as CD16)：1109-1114(1997)).NK cells may be from (F158/V158) that CD16 is heterozygosis or the non-human donor that V158 or F158 is homozygosis.Blood layer after dilution is overlayed on 15mL separation of lymphocytes medium (ICN Biochemical, Aurora, Ohio) and centrifuged 20 minutes with 2000RPM.Leucocyte at layer and interface layer is distributed into 4 clean 50mL to manage, the RPMI culture mediums containing 15% hyclone are filled in pipe.Pipe is centrifuged 5 minutes with 1400RPM, abandoning supernatant.Sediment is resuspended in MACS buffer solutions (0.5%BSA, 2mM EDTA), and scheme (Miltenyi Biotech) purified NK cells with pearl (NK cell separation kits, 130-046-502) according to manufacturer.NK cells are diluted to 2 × 10 with MACS buffer solutions⁶Individual/mL.

Antibody is being determined into medium (F12/DMEM 50：50, no glycine, 1mM HEPES buffer solution pH7.2, penicillin/streptomycin (100 units/mL；Gibco), glutamine, and 1% heat-inactivated fetal bovine serum) in serial dilution (0.05mL) add to 96 hole round bottom tissue culturing plates.It is 4 × 10 that WIL2-S cells are diluted into concentration in buffer solution is determined⁵Individual/mL.WIL2-S cells (per hole 0.05mL) are mixed in 96 orifice plates with the antibody of dilution and are incorporated in incubation at room temperature 30 minutes, antibody is combined with CD20 (opsonic action).

By adding 0.1mL NK cells into each hole, ADCC reactions are started.In control wells, 2%Triton X-100 are added.Then plate is incubated 4 hours in 37 DEG C.The specification for following manufacturer with cytotoxicity (LDH) detection kit (Kit#1644793, Roche Diagnostics, Indianapolis, Indiana) measures LDH emission levels.0.1mL LDH developers are added into each hole, then mixing 10 seconds.Then plate is covered with aluminium foil and incubated in the dark 15 minutes in room temperature.Then read the optical density at 490nm places and be used to calculate by divided by total LDH for measuring in control wells and crack percentage.Function construction of the percentage as antibody concentration will be cracked, and EC is determined with 4 parameter curves (KaleidaGraph)₅₀Concentration.

Substance assistant laser desorpted/ionization time of flight mass (MALDI-TOF) mass spectral analysis of the oligosaccharides of asparagine connection

Use Papac et al., Glycobiology 8：Code in 445-454 (1998) discharges the oligosaccharides of N- connections from recombinant glycoprotein.In brief, by making 100 μ l methanol flow through PDVF films to Millipore Multiscreen vacuum manifold applying vacuums and making the microtiter plate (Millipore of 96 hole PVDF linings, Bedford, MA) hole conditioning (condition).With the pvdf membrane of 3 × 250 μ l water cleaning conditions.Each hole is set to drain completely by applying gentle vacuum to manifold between all cleaning steps.Film, the buffer solution guanidine hydrochloride containing 6M, 360mM Tris, 2mMEDTA, pH 8.6 are cleaned with reduction and carboxy methylation buffer solution (RCM).Glycoprotein sample (50 μ g) is applied to each hole, pvdf membrane is flowed it through and with 2 × 50 each holes of μ l RCM buffer solution for cleaning again by gentle vacuum.By adding 50 μ l 0.1M dithiothreitol (DTT)s (DTT) solution into every hole and incubating micro titre plate 1 hour in 37 DEG C, reduce the sample fixed.By the way that DTT is removed in vacuum and each hole is cleaned with 4 × 250 μ l water.Fresh and 0.1M 0.1M iodoacetic acid (IAA) solution is diluted to RCM buffer solutions by adding 50 μ l in 1M NaOH, makes cysteine residues carboxy methylation.Carboxy methylation is completed by being incubated in the dark 30 minutes in environment temperature.To plate applying vacuum to remove IAA solution and clean each hole with 4 × 250 μ l purified waters.By adding 100 μ l 1%PVP360 (polyvinylpyrrolidone 360,000MW) (Sigma) solution and being incubated 30 minutes in ambient stable, pvdf membrane is closed.By the way that PVP-360 solution is gently removed in vacuum and each hole is cleaned with 4 × 250 μ l water.PNGase F (New England Biolabs, Beverly, MA) digestive juice, i.e., 25 units/ml solution that 25 μ l are prepared in 10mM Tris acetates, pH8.3 are added into each hole, and in 37 DEG C digest within 3 hours.After digestion, sample is transferred to 500 μ l Eppendorf pipes and 2.5 μ l1.5M acetic acid solutions are added into every part of sample.The sample of acidifying is incubated 2 hours in ambient stable, oligosaccharides is transformed into OH- form from glycosyl amine (glycosylamine).Before MALDI-TOF mass spectral analyses, with small-sized reaction tube (the US Biochemical of loading, Cleveland, OH cationic ion-exchange resin (Hydrogen AG50W-X8 resins) (Bio-Rad of 0.7ml bed volumes), Hercules, CA) slurry make the oligosaccharides desalination of release.

For the MALDI-TOF mass spectral analyses of sample in aggressive mode (positive mode), oligosaccharides (0.5 μ l aliquots) after desalination is applied to equipped with 0.5 μ l 2, in the stainless steel target pipe of 5- dihydroxybenzoic acid matrix (sDHB), the matrix is configured to by the way that 2mg DHBs are dissolved in together with 0.1mg 5- methoxysalicylic acids in 1ml NaCl containing 1mM 25% ethanol water.Sample/substrate mixture is vacuum dried.By sample/substrate mixture vacuum drying, it is then set to absorb atmospheric humidity before analysis.The oligosaccharides of release is analyzed by MALDI-TOF on PerSeptive BioSystems Voyager-ELITE mass spectrographs.In an active mode with linear configuration and operation mass spectrograph is extracted in 20kV using delay.With about 1100 laser power and data are obtained with data investigation pattern (240 times scanning) to improve signal to noise ratio.Instrument is calibrated with standard oligosaccharide before to mass and uses 19 point Savitsky-Golay algorithms to make data flat.The integration (integration) of mass spectrometric data is completed with the software for data analysis of Caesar 7.2 (SciBridge Software).

As a result and discuss

In previous embodiment, α -1,6- fucosyltransferases (FUT8) activity in having struck 2H7.v16 cell lines low using RNAi technology.Region in the RNAi targeting FUT8 genes open read frames (ORF).Thus higher than the higher fucosylated antibody binding affinity and Geng Gao ADCC activity to Fc γ RIII acceptors of the fucosylated less antibody display produced by cell line.Fig. 9 A show the process for developing fucosylated less 2H7.v16 cell lines.Said process is the two-step method for requiring to exist before RNAi plasmid transfections stable antibody producing cells system.

In order to shorten the time required for this process, a kind of new one-step method is explored, wherein siRNA units are included in the expression plasmid of express express target protein matter (such as antibody), as shown in Figure 9 B.First, whether the expression plasmid of test bag box containing antibody expression and RNAi units can the expression simultaneously in transient transfection to understand antibody and RNAi.The structure of the 5 sets of plasmids transiently transfected is as shown in Figure 10.Determine by the fucosylation level of the protein of that 5 sets of plasmid expressions.

In Table 2 below, v511 and v114 refer to the hu2H7 antibody variants described in table 3.As shown in table 2, antibody from the control plasmid without RNAi units have 9% it is non-fucosylated.By there is the non-fucosylated of 33%-49% scopes comprising a kind of antibody of the plasmid expression of RNAi units.There is the non-fucosylated of 62%-65% scopes by the antibody of the plasmid expression comprising two kinds of RNAi units.These results are shown, compared with the 33-49% that a kind of RNAi units have only is added on expression plasmid, two kinds of RNAi transcriptional units are added on expression plasmid causes produced antibody to have higher 62-65%'s non-fucosylated, indicates the additive effect of two kinds of siRNAi transcription products.The antibody expressed in this embodiment is Humanized anti-cd 20 antibodies 2H7.v511 (being also known as hu2H7.v511 herein), and its sequence is provided in CD20 binding antibodies part above.

Table 2

The non-fucosylated percentage of plasmid

rkHCv511+rkLCv114                  9

rkHCv511RNAi4+rkLCv114RNAi4        49

rkHCv511RNAi2.4+rkLCv114RNAi2.4    62

CMV.PD.v511.RNAi4                  33

CMV.PD.v511.RNAi2.4                65

The cell after transfection is selected with one of two kinds of plasmid CMV.PD.v511.RNAi4 or CMV.PD.v511.RNAi2.4 (Figure 10 C) stable transfected cells and with 25nM methotrexate (MTX)s (MTX).Picking 72 is cloned and screened for antibody expression from each transfection.Express titre as shown in figure 11.Clone from CMV.PD.v511.RNAi2.4 plasmid transfections seems with titre generally relatively low compared with other two kinds transfect.

In order to understand whether the clone with preferably expression titre also has relatively low fucosylation level, the clonal analysis FUT8 mRNA for having higher expression to about 20% by Taqman are expressed.As shown in figure 12, the clone from CMV.PD.v511.RNAi2.4 plasmid transfections is general has relatively low FUT8 mRNA level in-sites compared with the clone from CMV.PD.v511.RNAi4 plasmid transfections.

6 clones (two come from CMV.PD.v511.RNAi4 plasmid transfections, and four come from CMV.PD.v511.RNAi2.4 plasmid transfections) with minimum FUT8 mRNA expressions are further assessed into antibody expression using equal inoculum density determination method.As a result the titre of this 6 clones is shown with compareing 2H7 v511 clones (clone 18 and 63 from CMV.PD.v511 plasmid transfections) quite, as shown in figure 13.However, CMV.PD.v511.RNAi2.4 clones seem with than CMV.PD.v511.RNAi4 clones and the low titre of control clone.

The fucose content of the produced antibody of the clones of 2H7.v511 shown in Figure 14 is determined as described above by MALDI-TOF mass spectral analyses.It was found that a clone, RNAi2.4-3d has reached that 94-95%'s is non-fucosylated.

Fc γ RIII combination mensurations are carried out with the antibody 2H7.v511 containing 65% non-fucosylated (from transiently transfecting) or containing 94-95% non-fucosylated (coming from most stable of clone RNAi2.4-3d).As a result as shown in fig. 15 a and fig. 15b.Compared with about 5% non-fucosylated control antibodies set, 65% non-fucosylated material is shown to high-affinity (V158 allele, Figure 15 B) and low-affinity (F158 allele, Figure 15 A) affinity of acceptor has 4.8 and 6.2 times of appropriateness rise respectively, and 95% non-fucosylated material shows the rise for having 6.8 and 9.8 times to the affinity of two kinds of receptor isoforms.

Because non-fucosylated antibody seems preferably to be combined with Fc γ RIII, ADCC activity is tested to them.7F (60-70% scopes non-fucosylated) will be cloned from 2H7.v16 and will be determined from the 2H7.v511 materials for transiently transfecting (65% is non-fucosylated) collection for ADCC activity.As seen in Figure 16 A and 16B, the fucosylated less 2H7 of two kinds of forms shows the ADCC activity higher than their corresponding high fucosylated counterpart.

A kind of new simplification (streamline) method is we described herein, Chinese hamster ovary celI is transformed from metabolism to produce (up to 95%) non-fucosylated antibody of even more high, i.e., by the way that heavy chain of antibody and light chain transcription unit are mixed into same plasmid together with 1-2 siRNA transcriptional units.Two siRNA transcription products used in the method target the different coding area in FUT8 genes and instructed by different Pol type III promoters H1 and U6.

In a word, we have demonstrated that it is feasible that RNAi technology is added into the exploitations of humanization 2H7 cell lines to strike low fucosylation level.Existing antibody producing cells system is successfully transformed into fucosylated less cell line.Strike low fucosylated while new antibody producing cells system is produced in addition, also successfully realizing.

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Claims (31)

1. the antibody comprising IgG Fc is produced in mammalian host cell reduces the method for antibody fucose content simultaneously, including the second nucleic acid of the nucleic acid of at least one encoding antibody and at least two siRNA in coding targeting FUT8 gene order SEQ ID NO.1 different codings area is introduced into host cell simultaneously, siRNA therein suppresses FUT8 expression and reduces the fucosylation level of antibody.

2. the method for claim 1 wherein light (L) chain of the nucleic acid encoding antibody of encoding antibody and heavy (H) both chains.

3. the method for claim 2, the wherein nucleic acid of encoding antibody H and L chains are with encoding siRNA nucleic acid on same expression vector.

4. the method for claim 1 wherein the nucleic acid of the nucleic acid of coding H chains and coding L chains is on separated expression vector, wherein each the expression vector of coding H and L chains also includes at least two siRNA of coding nucleic acid.

5. the method for claim 1 wherein two kinds of siRNA expressed under the control of different promoters.

6. the method for claim 5, one of which siRNA is expressed under Pol III promoters H1 control and second of siRNA is expressed under Pol III promoters U6 control.

7. the method for claim 1 wherein the first and second of siRNA target FUT8 gene order SEQ ID NO.1 733-751 and 1056-1074 nucleotides respectively.

8. the method for claim 1 wherein host cell be Chinese hamster ovary (CHO) cell or derivatives thereof.

9. the method for claim 1 wherein antibody fucosylation level reduction at least 90%.

10. the method for claim 1 wherein antibody fucosylation level reduction at least 95%.

11. the method for claim 1 wherein antibody be treatment antibody.

12. the antibody produced with the method for claim 1.

13. produce the method for the IgG antibody with improved ADCC, including the second nucleic acid of the nucleic acid of at least one encoding antibody and at least two siRNA in coding targeting FUT8 gene order SEQ ID NO.1 different codings area is introduced into host cell simultaneously, wherein antibody and siRNA expresses to produce the antibody of the fucosylated and elevated ADCC activity compared with the antibody produced by the cell without siRNA with reduction in cell.

14. the method for claim 13, antibody therein is included in Fc areas improves antibody to Fc γ RIII combination and/or ADCC amino acid change at least one.

15. the method for claim 14, antibody therein includes Fc amino acid replacements S298A, E333A, K334A.

16. the method for claim 15, it further includes Fc amino acid replacements K326A.

17. the method for claim 13, antibody therein is combined with CD20.

18. the method for claim 17, antibody therein is combined with the CD20 of primate.

19. the method for claim 17, CD20 binding antibodies therein are human antibodies.

20. the method for claim 17, CD20 binding antibodies therein are chimeric antibodies.

21. the method for claim 20, chimeric antibody therein is rituximab.

22. the method for claim 17, CD20 binding antibodies therein are humanized antibodies.

23. the method for claim 22, humanization CD20 binding antibodies therein, which are included, is selected from following VL and VH areas：SEQ ID NO.2 VL and SEQ ID NO.8 VH；SEQ ID NO.25 VL and SEQ ID NO.22 VH；And SEQ ID NO.25 VL and SEQ ID NO.33 VH.

24. the method for claim 22, humanization CD20 binding antibodies therein include L the and H chains respectively with sequence SEQID NO.13 and 14.

25. the method for claim 22, humanization CD20 binding antibodies therein include L the and H chains respectively with sequence SEQID NO.26 and SEQ ID NO.27.

26. the method for claim 22, humanization CD20 binding antibodies therein include L the and H chains respectively with sequence SEQID NO.26 and SEQ ID NO.34.

27. the method for claim 13, antibody therein is combined with BR3.

28. the antibody produced with the method for claim 13.

29. include sequence SEQ ID NO.42 and SEQ ID NO.43 nucleic acid.

30. comprising humanization CD20 binding antibodies and the composition of carrier with Fc areas, at least 95% antibody lacks fucose wherein in composition.

31. the host cell of the nucleic acid comprising at least one encoding antibody and at least two siRNA in coding targeting FUT8 gene order SEQ ID NO.1 different codings area the second nucleic acid, antibody described in host cell expression therein and the siRNA.

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