CN1420129A - Humanized anti-CD 20 monoclonal antibody - Google Patents
Humanized anti-CD 20 monoclonal antibody Download PDFInfo
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Abstract
A humanized monoclonic antibody of CD20 for treating lymphocytoma B, and its associated gene sequence and expression system are disclosed. Its advantages are high bioactivity and high expression in cells of mammal.
Description
Invention field
The present invention relates to monoclonal antibody technique, antibody humanization's technology, and gene recombination technology.Especially the Humanized monoclonal antibodies of anti-CD20.
Background of invention
In recent years, the mab treatment of tumour is a field that research is very active, has obtained gratifying progress.Monoclonal antibody targeted therapy to non-Hodgkin lymphoma has also been obtained crucial progress.Existing discovering, what clinical therapeutic efficacy was best is the monoclonal antibody of CD20.
Studies have shown that combining of CD20 and antibody can activate Tyrosylprotein kinase and L-Cysteine HCL Anhydrous under proper condition, thus the apoptosis of mediated cell.Anti-CD20 antibodies mainly is to come killing tumor cell by mechanism such as the cytotoxicity of the cytotoxicity of antibody dependence, complement dependence, inducing apoptosis of tumour cell.CD20 has regulating effect at the bone-marrow-derived lymphocyte mitotic cycle from G0 different combination when G1 changes, anti-CD20 antibodies combines with CD20, changed the function of CD20, certainly will have influence to the bone-marrow-derived lymphocyte growth, different anti-CD20 antibodies is because the binding site difference, can cause different from effect.
At present, obtained mouse source and the Humanized monoclonal antibodies of anti-CD20 both at home and abroad, the good result of acquisition in clinical study who has has shown great application prospect.But all need people's immunogenicity forr a short time, expression amount is higher, and the polyclonal antibody with other good characteristics all the time.
Summary of the invention
The object of the present invention is to provide CD20 Humanized monoclonal antibodies, its genes involved sequence and expression system with potential medical science and pharmacy value.
To achieve these goals, the invention process following technical scheme.
The invention provides a kind of CD20 Humanized monoclonal antibodies, comprise people source Heng Qu, its variable region of heavy chain is selected from SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:13; Its variable region of light chain is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 and SEQ ID NO:15; When described variable region of light chain was SEQ ID NO:3, variable region of heavy chain was not SEQ ID NO:4.
The present invention also provides a kind of dna molecular, the variable region of heavy chain or the variable region of light chain of coding CD20 Humanized monoclonal antibodies.The dna sequence dna of variable region of heavy chain is selected from: SEQ ID NO:5, SEQ ID NO:9 and SEQ ID NO:14.The dna sequence dna of variable region of light chain is selected from: SEQ ID NO:6, SEQ ID NO:10 and SEQ ID NO:16.
The present invention also provides a kind of carrier, comprises described dna molecular.
The present invention also provides a kind of host cell, comprises the expression system of described humanized antibody.Described host cell can be for example to the various suitable cells of mammalian cells such as CHO from prokaryotic cell prokaryocytes such as intestinal bacteria.Its selection is the ordinary skill in the art.
In one of preferred implementation, host cell comprises consistency and expresses carrier, comprises dna molecular of the present invention in this carrier.
The present invention has also pointed out the purposes of antibody of the present invention in preparation diagnosis and/or treatment CD20 relative disease.
CD20 Humanized monoclonal antibodies of the present invention has been realized the high expression level in Chinese hamster ovary celI, and its product biological activity also obviously improves.With regard to being applied to treat clinically the bone-marrow-derived lymphocyte knurl, has great potential.
The accompanying drawing summary
The sequence of Fig. 1: monoclonal antibody 9D3,7D3,4E5 and 2H7 relatively.Wherein, panel A is that their weight chain variable region amino acid sequences compare; Component B is that their light chain variable region amino acid sequences compare; Component C is the comparison of 9D3 and 2H7 variable region of heavy chain dna sequence dna; Component D is the comparison of 9D3 and 2H7 variable region of light chain encoding sequence.
Fig. 2: the collection of illustrative plates of expression vector pMG18-3K.Wherein, Ck represents the constant region gene of people's antibody kappa light chain; IgG1 constant represents the weight chain constant area gene (GenBank AccessionNumber P01857, the N-terminal sequence of selected carrier is Ser-Gla-Ala-Cys-Cys) of people's IgG antibody 1; PA represents that SV40 adds poly (A) site.
Fig. 3: the result of study that detects each expression of cell lines intensity with Western trace method.
Fig. 4: polyclonal antibody specificity fluorescent dyeing competitive assay.Wherein panel A is to redye with the being at war with property of 9D3 of Texas Red mark after 7D3 handles bone-marrow-derived lymphocyte again; The component B 4E5 that to be 7D3 handle with Texas Red mark bone-marrow-derived lymphocyte again redyes; Component C after to be 7D3 to bone-marrow-derived lymphocyte handle again the Rituximab with Texas Red mark redye; Component D after to be 9D3 to bone-marrow-derived lymphocyte handle again the 7D3 with Texas Red mark redye; Component E after to be 9D3 to bone-marrow-derived lymphocyte handle again the 4E5 with Texas Red mark redye; Component F after to be 9D3 to bone-marrow-derived lymphocyte handle again the Rituximab with Texas Red mark redye; Component G after to be 4E5 to bone-marrow-derived lymphocyte handle again the 7D3 with Texas Red mark redye; Component H after to be 4E5 to bone-marrow-derived lymphocyte handle again the 9D3 with Texas Red mark redye; Component I after to be 4E5 to bone-marrow-derived lymphocyte handle again the Rituximab with Texas Red mark redye.
Preferred implementation one .CD20 MONOCLONAL ANTIBODIES SPECIFIC FOR
Adopt people CD20+B lymphocyte tumor cell strain DHL-4 (Demidem A, Lam T, Alas S, Hariharan K, Hanna N, Bonavida B., Chimeric anti-CD20 (IDEC-C2B8) monoclonalantibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs.Cancer Biother Radiopharm 1997 Jun; 12 (3): 177-86) as immunogen.With total amount is 5 * 10
7The ripe bone-marrow-derived lymphocyte of fresh culture adds complete Freund's adjuvant, fully is ground to conventional subcutaneous multi-point injection 10 age in days Balb/c mouse after the complete emulsification, injection 50 microlitres, totally 6 points at every.Then with 1 weekly interval totally 5 duplicate injections react with booster immunization, and the serum antibody level is monitored.Last intravenous injection 5 * 10
6New fresh cell/PBS mixed solution killed mouse after 3 days, took out spleen.
The ordinary method separating spleen is unicellular, and definite cytoactive＞90%.Spleen cell is mixed centrifugal co-precipitation with the SP2/0 murine myeloma cell at 10: 1, add PEG (1400MW) and enter cytogamy in 37 ℃ of water-baths, time of fusion is 1 minute, 2 minutes, 5 minutes and 10 minutes.Added at interval 1 milliliter of fresh serum free medium, 2 milliliters, 5 milliliters and 10 milliliters in 5 minutes.After the centrifugation cell is joined in the 20%FCSRPMI-1640 nutrient solution, change 96 orifice plates over to and cultivate.Add 1 * HAT after 24 hours and select nutrient solution.Changed fresh culture 1 time in per 3 days, to growing the clone.
Select mono-clonal to form the hole, took out supernatant liquor, carry out ripe bone-marrow-derived lymphocyte fluorescent dye at the 14th day.In brief, supernatant and bone-marrow-derived lymphocyte 37 degree incubations 30 minutes, 1XPBS washing 10 times, the rabbit anti-mouse antibody that adds the FITC mark is anti-as two, 1XPBS washing 10 times, the 1XPBS that contains 1%Tween-20 washs 10 times, and the 1XPBS that contains 0.5%Triton X-100 washs 10 times, reads the fluorescence intensity at 650nm place on microplate reader.Positive hole keeps.Select 3 high expressing cell strains, expression amount＞1mg/L under 96 hole culture condition, stable clones to the 5th generation.Test kit with Gibco company carries out the monoclonal antibody type identification.As a result, according to the position that is cloned on 96 orifice plates, 3 clones' title and type are: 7D3,9D3 and 4E5 (are IgG (2a, k)).
Known, the CD20 monoclonal antibody can external evoked apoptosis.DHL-4 carries out the monoclonal antibody determination of activity with CD20+B lymphocyte tumor cell strain.Negative control be anti-people CD3 chimeric antibody (OKT3) (Genentech).Positive control is Rituximab (Genentech) and 2H7 (for example Genentech).Add behind cell and the monoclonal antibody and to carry out cell counting and bromination 3-[4,5-dimethylthiazole-2-thiazol-2-yl on the 5th～7 day]-2,5-phenylbenzene tetrazolium (MTT) detects (referring to for example: Sun Weimin, Wang Huiqin writes, " cytokine research method ", 1999, p65～67).
Biological activity test result (survivaling cell %) AC negative control positive control monoclonal antibody to be measured (μ g/ml) the OKT3 Rituximab 2H7 9D3 7D3 4,E51 100.0 66.7 94.3 93.2 99.6 87.910 100.0 32.1 38.4 32.2 46.5 33.7100 100.0 19.4 29.1 30.7 56.7 22.8 of antibody falls in CD20 Dan Ke
From The above results as can be seen, add antibody after survivaling cell quantity obviously reduce, illustrate that said monoclonal antibody has significantly to kill and wound CD20
+Bone-marrow-derived lymphocyte.Wherein, the kill capability of 9D3 pair cell is similar to positive control.Two. the variable region of mab order-checking
Because the constant region sequence in antibody gene downstream is known, so obtain monoclonal antibody 9D3, the variable region gene of 7D3 and 4E5 with the method for 5 ' RACE (the terminal rapid amplifying of cDNA).
Synthetic following primer:
GSP1-H??5＇-AGCTGGGAAG?GTGTGCACAC?CACT-3’(SEQ?ID?NO：17)
GSP2-H??5＇-CAGAGT?TCC?AGG?TCA?AGG?TCA-3’(SEQ?ID?NO：18)
GSP3-H??5＇-CTT?GAC?CAG?GCA?TCC?TAG?AGT-3’(SEQ?ID?NO：19)
GSP1-L??5＇-TTG?CTG?TCC?TGA?TCA?GTC?CAA?CT-3’(SEQ?ID?NO：20)
GSP2-L??5＇-TGT?CGT?TCA?CTG?CCA?TCA?ATC?TT-3’(SEQ?ID?NO：21)
GSP3-L??5＇-TTG?TTC?AAG?AAG?CAC?ACG?ACT?GA-3’(SEQ?ID?NO：22)
AAP?????5＇-GGC?CAC?GCG?TCG?ACT?AGT?ACG?GGI?IGG?GII?GGG?IIG-3’(SEQ?ID?NO：23)
AUAP????5＇-GGC?CAC?GCG?TCG?ACT?AGT?AC-3’(SEQ?ID?NO：24)
Wherein, GSP represents gene-specific primer, and AAP represents 5 '-RACE brachymemma anchor primer, and AUAP represents brachymemma universal amplification primer.GSP1 distance variable district gene is used for reverse transcription reaction farthest.GSP2 is used for nested PCR with GSP3, and wherein GSP3 is in GSP2.
Trizol Reagent reagent with Gibco company extracts 5 * 10 respectively
6Hybridoma 9D3, total RNA of 7D3 and 4E5.According to 5 '-RACE test kit specification sheets is that primer becomes cDNA with total RNA reverse transcription with GSP1.Adding poly (C) tail for then 3 ' the end of the first chain cDNA, is that primer carries out pcr amplification with GSP2 and AAP behind the tailing, and it is that primer carries out nested pcr amplification with AUAP and GSP3 again that amplified production is diluted 100 times.Warm start, reaction conditions are all adopted in twice PCR reaction: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 1 minute 10 seconds, 30 circulations; 72 ℃ 7 minutes.Nested PCR product reclaims purifying purpose fragment (light chain length 321bp, heavy chain length 366bp) after 1% agarose gel electrophoresis separates.Be cloned in pGEM-T (Promega) carrier, at the dull and stereotyped enterprising row filter of IPTG/X-gal, get 8 white bacterial plaques and be inoculated in the LB liquid nutrient medium that contains penbritin and increase behind the transformed into escherichia coli TG1 cell.Screening positive clone with the plasmid extraction test kit extracting plasmid of QIAGEN and check order, has been determined 9D3, the heavy chain of 7D3 and 4E5 and light chain variable region sequence.
The dna sequence dna of 9D3 variable region and amino acid sequence:monoclonal antibody 9D3 heavy chain variable region gene sequence (5 ＇ → 3 ＇; 366bp ) ( SEQ ID NO：1 ) ：CAGGCCTACCTGCAGCAGTCCGGCGCCGAGCTGGTGCGCCCCGGCGCCTCCGTGAAGATGTCCTGCAAGGCCTCCGGCTACACCTTCACCTCCTACAACATGCACTGGGTGAAGCAGACCCCCCGCCAGGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACCTCCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGTGGACAAGTCCTCCTCCACCGCCTACATGCAGCTGTCCTCCCTGACCTCCGAGGACTCCGCCGTGTACTTCTGCGCCCGCGTGGTGTACTACTCCAACTCCTACTGGTACTTCGACGTGTGGGGCACCGGCACCACCGTGACCGTGTCCTCC 3669D3 ( 5＇→3＇,321bp ) ( SEQ ID NO：2 ) ：CAGATCGTGCTGTCCCAGTCCCCCGCCATCCTGTCCGCCTCCCCCGGCGAGAAGGTGACCATGACCTGCCGCGCCTCCTCCTCCGTGTCCTACATGCACTGGTACCAGCAGAAGCCCGGCTCCTCCCCCAAGCCCTGGATCTACGCCCCCTCCAACCTGGCCTCCGGCGTGCCCGCCCGCTTCTCCGGCTCCGGCTCCGGCACCTCCTACTCCCTGACCATCTCCCGCGTGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGTCCTTCAACCCCCCCACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCGC 3219D3 ( 122 ) ( SEQ ID NO：3 ) ：QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWVKQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARVVYYSNSYWYFDVWGTGTTVTVSS9D3 ( 107 ) ( SEQ ID NO：4 ) ：QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGAGTKLELKR
The heavy chain variable region gene sequence of the dna sequence dna of 7D3 variable region and amino acid sequence monoclonal antibody 7D3 (5 ＇ → 3 ＇; 366bp ) ( SEQ ID NO：5 ) CAGGCTCAACTACAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCCTCAGTGAAGATGTCGTGCAAGGCTTCTGGCTACACATTTACCCGTTACAATATGCACTGGGTAAAGCAGACACCTAGACAGGGCCTGGAATGGATTGGATATATTTATCCAGGAAATGGTGATACTAATTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAAGACTCTGCGGTCTATTTCTGTGCAAGACGTACTTATTATAGTAACTCTTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCTTCG 3667D3 ( 5＇→3＇,321bp ) ( SEQ ID NO：6 ) 001 CAAATCGTTC TCACTCAGTC TCCAGCAATC CTGTCTGCAT CTCCAGGGGA051 GAAGGTCACA ATGACCTGCA GGGCCAGCTC AAGTGTAAGT TACATGAATT101 GGTACCAGCA GAAGCCAGGA TCCTCCCCCT TTACTTGGAT TTATGCCCCA151 TCCAACCTGG CTTCTGGAGT CCCTGCTCGC TTCAGTGGCA GTGGGTCTGG201 GACCTCTTAC TCTCTCACAA TCAGCAGAGT GGAGGCTGAA GATGCTGCCA251 CTTATTACTG CCAGCAGTGG AGTAATAACC CACCCACGTT CGGTGCTGGC301 ACCAAGCTGG AGCTGAAACG A7D3 ( 122 ) ( SEQ ID NO：7 ) QAQLQQSGAELVRPGASVKMSCKASGYTFTRYNMHWVKQTPRQGLEWIGYIYPGNGDTNYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARRTYYSNSYWYFDVWGTGTTVTVSS7D3 ( 107 ) ( SEQ ID NO：8 ) QIVLTQSPAILSASPGEKVTMTCRASSSVSYMNWYQQKPGSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSNNPPTFGAGTKLELKR
The heavy chain variable region gene sequence of the dna sequence dna of 4E5 variable region and aminoacid sequence monoclonal antibody 4E5 (5 ＇ → 3 ＇, 366bp) (SEQ ID NO:9)
001?CAGGTTCAAC?TACAGCAGTC?TGGGGCTGAG?CTGGTGAGGC?CTGGGGCCTC
051?AGTGAAGTCC?TCCTGCAAGG?CTTCTGGCTA?CACATTTACC?AGTTACACTA
101?TGCACTGGGT?AAAGCAGACA?CCTAGACAGG?GCCTGGAATG?GATTGGAGCT
151?ATTTATCCAG?GAAATGGTGA?TACTTCCTAC?AATCAGAAGT?TCAAGGGCAA
201?GGCCACACTG?ACTGTAGACA?AATCCTCCAG?CACAGCCTAC?ATGCAGCTCA
251?GCAGCCTGAC?ATCTGAAGAC?TCTGCGGTCT?ATTTCTGTGC?AAGATATTAC
301?CAATCCGATC?AATCTTACTG?GTACTTCGAT?GTCTGGGGCA?CAGGGACCAC
The chain variable region gene sequence of 351 GGTCACCGTC TCCTCT monoclonal antibody 4E5 (5 ＇ → 3 ＇, 321bp) (SEQ ID NO:10)
001?CAAATTGTTC?TCTCCCAGTC?TCCAGCAATC?CAGTCTGCAT?CTCCAGGGGA
051?GAAGGTCACA?ATGACTTGCA?GGGCCAGCTC?AAGTGTAAGT?TACATGCACT
101?GGTACCAGCA?GAAGCCAGGA?TCCTCCCCCA?AACCCTGGAT?TTATGCCCCA
151?TCCAACCTGG?CTTCTGGAGT?CCCTGCTCGC?TTCAGTGGCA?GTGGGTCTGG
201?GACCTCTTAC?TCTCTCACAA?TCAGCAGAGT?GGAGGCTGAA?GATGCTGCCA
251?CTTATTACTG?CCAGCAGTGG?AGTTTTAACC?CACCCACGTT?CGGTGGATGC
Deduction amino acid sequence (107 amino acid) (SEQ ID NO:12) the QIVLSQSPAIQSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYAPSNLASG VPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGDGTKLELKR of deduction amino acid sequence (122 amino acid) (SEQ ID NO:11) the QVQLQQSGAELVRPGASVKSSCKASGYTFTSYTMHWVKQTPRQGLEWIGAIYPGNG DTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARYYQSDQSYWYFDVW GTGTTVTVSS monoclonal antibody 4E5 variable region of light chain of 301 ACCAAGCTGG AGCTGAAACG A monoclonal antibody 4E5 variable region of heavy chain
Utilize DNAstar software with three kinds of new CD20 monoclonal antibodies: the sequence of 9D3,7D3 and 4E5 and known 2H7 has been carried out amino acid and dna sequence dna relatively.According to shown in Figure 1, find that aminoacid sequence and the dna sequence dna of 9D3,7D3 and 4E5 is all inequality, but the aminoacid sequence of 9D3 and 2H7 is identical, but their dna sequence dna is different.Three. the structure of humanization chimeric mAb expression vector
Adopt the PCR reaction to add suitable restriction enzyme site for respectively above antibody variable region.Wherein 5 ＇ of variable region of heavy chain end designs Xba I restriction enzyme site, and 3 ＇ end adds Nhe I site, and in chain variable region gene 5 ＇ end design HindIII site, 3 ＇ end adds the BsiWI site.
Used PCR primer is: VL-sense strand: 5 ＇-TCC
TCTAGACAAATTSTTCTCTHCCAGTC-3 ＇; (SEQ ID NO:25) VL-antisense strand: 5 ＇-GTT
GCTAGCTCGSTTCAGCTCWAGCTTGG-3 ＇; (SEQ ID NO:26) VH-sense strand: 5 ＇-AGG
AAGCTTEAGGCHTAACTACAGCAGTC-3 ＇; (SEQ ID NO:27) VH-antisense strand: 5 ＇-TGA
CGTACGTGAGSAGACGGTGDCCGTGRTC-3 ＇ (SEQ ID NO:28)
Wherein, underscore is represented the restriction enzyme site that adds; Preceding 3 bases represent to protect base; According to the sequence of aforementioned gained monoclonal antibody of the present invention, H=A, C, T; S=C or G; D=A, G or T; R=A or G; W=A or T.
Carry out regular-PCR.Gained PCR product cloning is carried out the sequencing conclusive evidence in the pGEM-T (Promega product) be required correct goal gene.With Xba I and Nhe I antibody heavy chain variable region gene is downcut from carrier pGEM-T then and be inserted into the corresponding site of expression vector pMG18-3K, with HindIII and BsiWI antibody chain variable region is downcut from carrier pGEM-T again and be inserted into the corresponding site of expression vector pMG18-3K (referring to DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORINGBASED ON INCP-9 PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, C.M.Thomas School of Biological Sciences, University of Birmingham, Edgbaston, BirminghamB15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University ScorinaAv.4, Minsk 220080 Belarus).Constituted the expression vector pC9D3 of chimeric antibody at last, pC7D3, pC4E5 contains chimeric antibody gene c9D3 respectively, c7D3 and c4E5.Four. the design of humanization recombinant monoclonal antibodies VL and VH
According to humanization require (referring to, humanization of the murine anti-human CD3monoclonal antibody OKT3.Hum Antibod Hybridoma for example, 1994,5 (1,2): 41), take into account the access to your password preferences of son of mammalian cell simultaneously, to the design of recombinating of the VL of 9D3 and VH gene:
The aminoacid sequence (122 amino acid) of reorganization 9D3 VH is (SEQ ID NO:13): QAYLQNSGAEKVRPGASVKMSCSASGYTFMSYNMYWVKQTTRQGLEWIGAIYPGNG DTSYNQKSGAGKATLTVDKSDSTAYMQLSKLTSEDSAVYFDARVVYYSNSYWYFDV WGTGTTVGASS
The dna sequence dna of restructuring 9D3 VH is (SEQ ID NO:14): CAGGCCTACCTGCAGAACTCCGGCGCCGAGAAGGTGCGCCCCGGCGCCTCCGTGAA GATGTCCTGCTCCGCCTCCGGCTACACCTTCATGTCCTACAACATGTACTGGGTGA AGCAGACCACCCGCCAGGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGC GACACCTCCTACAACCAGAAGTCCGGCGCCGGCAAGGCCACCCTGACCGTGGACAA GTCCGACTCCACCGCCTACATGCAGCTGTCCAAGCTGACCTCCGAGGACTCCGCCG TGTACTTCGACGCCCGCGTGGTGTACTACTCCAACTCCTACTGGTACTTCGACGTG TGGGGCACCGGCACCACCGTGGGCGCCTCCTCC
The aminoacid sequence (107 amino acid) of reorganization 9D3 VL is (SEQ ID NO:15): QIVLSKNPAILSASPGEHKSMTCRASSSVDYMHWLQQKPGDSPKPWIYAASNLNSG VPADHKGSGSGTSYSLTIGHVEAEDAATSYCQQWSFNKPTFGAGTLLELKR
The dna sequence dna of restructuring 9D3 VL is (SEQ ID NO:16): CAGATCGTGCTGTCCAAGAACCCCGCCATCCTGTCCGCCTCCCCCGGCGAGCACAA GTCCATGACCTGCCGCGCCTCCTCCTCCGTGGACTACATGCACTGGCTGCAGCAGA AGCCCGGCGACTCCCCCAAGCCCTGGATCTACGCCGCCTCCAACCTGAACTCCGGC GTGCCCGCCGACCACAAGGGCTCCGGCTCCGGCACCTCCTACTCCCTGACCATCGG CCACGTGGAGGCCGAGGACGCCGCCACCTCCTACTGCCAGCAGTGGTCCTTCAACA AGCCCACCTTCGGCGCCGGCACCCTGCTGGAGCTGAAGCGC 321 5. the preparation of humanization recombinant monoclonal antibodies VL and VH gene
Comparison according to restructuring 9D3 VH dna sequence dna and native sequences; Design, synthetic and isozygoty and obtain primer for rite-directed mutagenesis: T1:CAGGCCTACCTGCAGAACTCCGGCGCCGAGAAGGTGCGCC (SEQ ID NO:29) T2:GATGTCCTGCTCCGCCTCCGG (SEQ ID NO:30) T3:CACCTTCATGTCCTACAACATGTACTGGGTGAAGCAGACCACCCGCCAG (SEQ ID NO:31) T4:CAACCAGAAGTCCGGCGCCGGCAAGGCCAC (SEQ ID NO:32) T5:GACAAGTCCGACTCCACCG (SEQ ID NO:33) T6:GCTGTCCAAGCTGACCTCCG (SEQ ID NO:34) T7:GTGTACTTCGACGCCCGC (SEQ ID NO:35) T8:CCACCGTGGGCGCCTCCTCC (SEQ ID NO:36)
With above-mentioned T1-T8 primer (final concentration is 100nM) and 9D3 heavy chain dna profiling (separate the pEGM-T[9D3 from intestinal bacteria]) final concentration is 20nM) carry out co-variation and common renaturation (referring to " molecular cloning lab guide " according to ordinary method, Jin Dongyan etc., 1992, Science Press), use DNA point mutation test kit (Invitrogen product) to suddenly change then, all operations carries out according to shop instruction.In brief, above-mentioned all Oligonucleolide primers T1～T8 mixtures and template are carried out using T4 dna ligase (MBI product after sex change and the renaturation, operation is carried out with reference to producer's explanation) connect, use T7 archaeal dna polymerase (Promega product then, operation is carried out with reference to producer's explanation) extend, connect once more with the T4 dna ligase.Use Dpn I (NEB product) that above-mentioned hybrid molecule is digested at last, remove template strand.In like manner, synthetic sudden change 9D3 VL chain.Transform (conventional Calcium Chloride Method) e.colistraindh5 respectively with the sudden change chain that is not digested.The process enzyme filters out the right-on clone of sequence after cutting screening and order-checking.Then, variable region of light chain and variable region of heavy chain are carried out double digestion with XbaI/NheI and HindIII/Bsi WI respectively, separate on 1.5% agarose electrophoresis, glue reclaims (the glue recovery test kit with Promega company carries out according to shop instruction).Variable region of heavy chain that reclaims and variable region of light chain are inserted on the pMG18-3K expression vector and obtain recombinant expression plasmid, and note is made pH9D3.Five. the expression of monoclonal antibody
The expression vector pC9D3 that has chimeric antibody of above-mentioned structure, pC7D3, the expression vector transfection Escherichia coli DH5a of pC4E5 and humanization pH9D3.Be inoculated in then in 100 milliliters of L B substratum and increase.Ultrapure plasmid DNA purification kit extracting and purifying plasmid DNA with Qiagen company.Liposome method test kit with Invitrogen company is adopted transfection CHO cell with the plasmid DNA of above-mentioned purifying, and operation is carried out with reference to the specification sheets of producer.
The Chinese hamster ovary celI that transforms is selecting the MTX that carries out continuous 9 weeks on the substratum to select, and carries out the extreme dilution at last and cultivate on 96 orifice plates, carries out continuously 3 times, carry out mono-clonalization.
The monoclonal cell of choosing ties up on the RPM1641 substratum and cultivates, and supernatant is carried out the Western Blot experiment, judges expression intensity according to staining reaction.Found that pC9D3, pC7D3, four Chinese hamster ovary celI cordings that pC4E5 and humanization pH9D3 transform have obviously higher expression intensity.As shown in Figure 3, swimming lane 3 is C7D3, and swimming lane 5 is C9D3, and the expression amount of all the other swimming lanes is very low.40 swimming lanes are C4E5, and the 41st swimming lane is humanized H9D3.The expression amount of all the other swimming lane associated antibodies is very low.Six. the bioactivity research Purification of Monoclonal Antibodies of Humanized monoclonal antibodies:
Adopt albumin A affinity column direct separation and purification mouse resource monoclonal antibody of the present invention 9D3,7D3,4E5, chimeric monoclonal antibody C9D3, C7D3, C4E5 and humanization monoclonal antibody h9D3 from cells and supernatant.The SDS-PAGE electrophoresis proves that products therefrom purity is greater than 90%.
The product of above affinity chromatography passes through sieve chromatography once more, through product purity＞98% of SE-HPLC method proof acquisition.These samples can be used for following further analysis and research.Specificity fluorescent dyeing (competition experiments)
Use antibody purified 9D3,7D3 and 4E5 dye to mature B cell, pre B cell.As shown in Figure 4, find that cell can be by specific stain, and control cells (inoblast, CD20 feminine gender) strain is reactionless to dyeing.And behind first kind of antibody treatment bone-marrow-derived lymphocyte, second kind of antibody still can combine with bone-marrow-derived lymphocyte, proves that above-mentioned 4 kinds of monoclonal antibody 4E5,9D3,7D3 and Rituximab do not exist each other in conjunction with competition.This illustrates that antibody institute bonded immunologic determinants that these 3 cell strains produce is different each other, and also is different with standard monoclonal antibody.Chimeric C 9D3 and positive control have competitiveness, illustrate that their bonded are same antigenic determinants.The test of precipitation experiment Western trace shows, the 9D3 among the present invention, and 7D3,4E5 and their chimeric and humanization variation antibody thereof are all the same with contrast Rituximab, can and combine with CD20 protein-specific precipitation.The vitro cytotoxicity test
Known, but the CD20 monoclonal antibody can external cell death inducing.Use CD20
+Bone-marrow-derived lymphocyte tumor cell strain DHL-4 carries out the monoclonal antibody determination of activity.Negative control behaviour CD3 humanization monoclonal antibody OKT3.Positive control is Rituximab and 2H7).Add behind cell and the monoclonal antibody and to carry out cell counting on the 5th～7 day and MTT detects.
The biological activity test result of anti-CD-20 monoclonal antibody (survivaling cell %)
| Antibody concentration (μ g/ml) | |||
| ????1 | ????10 | ????100 | |
| OKT3 (negative control) | ????100.0 | ????100.0 | ????98.1 |
| Rituximab (positive control) | ????50.3 | ????12.6 | ????29.4 |
| 2H7 (positive control) | ????67.6 | ????19.1 | ????39.6 |
| C9D3 (chimeric) | ????59.2 | ????10.3 | ????30.7 |
| C7D3 (chimeric) | ????110.1 | ????27.3 | ????39.4 |
| C4E5 (chimeric) | ????95.7 | ????10.8 | ????29.7 |
| H9D3 (humanization) | ????61.3 | ????12.7 | ????28.6 |
From The above results as can be seen, survivaling cell quantity obviously reduces behind the adding antibody, illustrates that said monoclonal antibody has the bone-marrow-derived lymphocyte that significantly kills and wounds CD20+.Wherein, the kill capability of C9D3 and H9D3 pair cell is similar to positive control.The cells in vivo toxicity test
The DHL-4 lymphoma cell is according to 5 * 10
6The amount subcutaneous injection immunodefiiciency mouse an of/mouse treats that tumour grows to 0.3 * 0.3 (cm) when above, injects following CD20 monoclonal antibody, injects for 5 milligrams according to every mouse at the 10th day.The size of injection back the 10th day, the 20th day and the 30th day measurement tumour, the result is as follows:
10 days 20 days 30 days OKT3 (negative control) 0.3 * 0.35 0.65 * 0.95 1.4 * 1.5Rituximab (positive control) 0.28 * 0.31 0.45 * 0.40 0.60 * 0.81C4E5 0.27 * 0.29 0.35 * 0.42 0.55 * 0.63C9D3 0.28 * 0.30 0.36 * 0.40 0.51 * 0.62H9D3 0.30 * 0.28 0.45 * 0.50 0.61 * 0.97
The The above results explanation is compared with negative control, and the growth of in-vivo tumour is subjected to obvious suppression after the injection of antibodies.
＜110〉＜120〉CD20＜130〉016013＜160〉36＜170〉PatentIn version 3.0＜210〉1＜211〉366＜212〉DNA＜213〉 ( Mus musculus )＜400〉1caggcctacc tgcagcagtc cggcgccgag ctggtgcgcc ccggcgcctc cgtgaagatg 60tcctgcaagg cctccggcta caccttcacc tcctacaaca tgcactgggt gaagcagacc 120ccccgccagg gcctggagtg gatcggcgcc atctaccccg gcaacggcga cacctcctac 180aaccagaagt tcaagggcaa ggccaccctg accgtggaca agtcctcctc caccgcctac 240atgcagctgt cctccctgac ctccgaggac tccgccgtgt acttctgcgc ccgcgtggtg 300tactactcca actcctactg gtacttcgac gtgtggggca ccggcaccac cgtgaccgtg 360tcctcc 366＜210〉2＜211〉321＜212〉DNA＜213〉 ( Mus musculus )＜400〉2cagatcgtgc tgtcccagtc ccccgccatc ctgtccgcct cccccggcga gaaggtgacc 60atgacctgcc gcgcctcctc ctccgtgtcc tacatgcact ggtaccagca gaagcccggc 120tcctccccca agccctggat ctacgccccc tccaacctgg cctccggcgt gcccgcccgc 180ttctccggct ccggctccgg cacctcctac tccctgacca tctcccgcgt ggaggccgag 240gacgccgcca cctactactg ccagcagtgg tccttcaacc cccccacctt cggcgccggc 300accaagctgg agctgaagcg c 321＜210〉3＜211〉122＜212〉PRT＜213〉 ( Mus musculus )＜400〉3Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20?????????????????25??????????????????30Asn?Met?His?Trp?Val?Lys?Gln?Thr?Pro?Arg?Gln?Gly?Leu?Glu?Trp?Ile
35??????????????????40??????????????????45Gly?Ala?Ile?Tyr?Pro?Gly?Asn?Gly?Asp?Thr?Ser?Tyr?Asn?Gln?Lys?Phe
50??????????????????55??????????????????60Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Phe?Cys
85??????????????????90??????????????????95Ala?Arg?Val?Val?Tyr?Tyr?Ser?Asn?Ser?Tyr?Trp?Tyr?Phe?Asp?Val?Trp
100?????????????????105?????????????????110Gly?Thr?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
115 120＜210〉4＜211〉107＜212〉PRT＜213〉mouse (Mus musculus)＜400〉4Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly1,5 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20??????????????????25??????????????????30His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr
35??????????????????40??????????????????45Ala?Pro?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser
50??????????????????55??????????????????60Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu65??????????????????70??????????????????75??????????????????80Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Phe?Asn?Pro?Pro?Thr
85??????????????????90??????????????????95Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Leu?Lys?Arg
100 105＜210〉5＜211〉366＜212〉DNA＜213〉 ( Mus musculus )＜400〉5caggctcaac tacagcagtc tggggctgag ctggtgaggc ctggggcctc agtgaagatg 60tcgtgcaagg cttctggcta cacatttacc cgttacaata tgcactgggt aaagcagaca 120cctagacagg gcctggaatg gattggatat atttatccag gaaatggtga tactaattac 180aatcagaagt tcaagggcaa ggccacactg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaagac tctgcggtct atttctgtgc aagacgtact 300tattatagta actcttactg gtacttcgat gtctggggca cagggaccac ggtcaccgtc 360tcttcg 366＜210〉6＜211〉321＜212〉DNA＜213〉 ( Mus musculus )＜400〉6caaatcgttc tcactcagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcaca 60atgacctgca gggccagctc aagtgtaagt tacatgaatt ggtaccagca gaagccagga 120tcctccccct ttacttggat ttatgcccca tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 240gatgctgcca cttattactg ccagcagtgg agtaataacc cacccacgtt cggtgctggc 300accaagctgg agctgaaacg a 321＜210〉7＜211〉122＜212〉PRT＜213〉 ( Mus musculus )＜400〉7Gln Ala Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
20??????????????????25??????????????????30Asn?Met?His?Trp?Val?Lys?Gln?Thr?Pro?Arg?Gln?Gly?Leu?Glu?Trp?Ile
35??????????????????40??????????????????45Gly?Tyr?Ile?Tyr?Pro?Gly?Asn?Gly?Asp?Thr?Asn?Tyr?Asn?Gln?Lys?Phe
50??????????????????55??????????????????60Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Phe?Cys
85??????????????????90??????????????????95Ala?Arg?Arg?Thr?Tyr?Tyr?Ser?Asn?Ser?Tyr?Trp?Tyr?Phe?Asp?Val?Trp
100?????????????????105?????????????????110Gly?Thr?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
115 120＜210〉8＜211〉107＜212〉PRT＜213〉mouse (Mus musculus)＜400〉8Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly1,5 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20??????????????????25??????????????????30Ash?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr
35??????????????????40??????????????????45Ala?Pro?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser
50??????????????????55??????????????????60Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu65??????????????????70??????????????????75??????????????????80Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Ash?Ash?Pro?Pro?Thr
85??????????????????90??????????????????95Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Leu?Lys?Arg
100 105＜210〉9＜211〉366＜212〉DNA＜213〉 ( Mus musculus )＜400〉9caggttcaac tacagcagtc tggggctgag ctggtgaggc ctggggcctc agtgaagtcc 60tcctgcaagg cttctggcta cacatttacc agttacacta tgcactgggt aaagcagaca 120cctagacagg gcctggaatg gattggagct atttatccag gaaatggtga tacttcctac 180aatcagaagt tcaagggcaa ggccacactg actgtagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac atctgaagac tctgcggtct atttctgtgc aagatattac 300caatccgatc aatcttactg gtacttcgat gtctggggca cagggaccac ggtcaccgtc 360tcctct 366＜210〉10＜211〉321＜212〉DNA＜213〉 ( Mus musculus )＜400〉10caaattgttc tctcccagtc tccagcaatc cagtctgcat ctccagggga gaaggtcaca 60atgacttgca gggccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga 120tcctccccca aaccctggat ttatgcccca tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 240gatgctgcca cttattactg ccagcagtgg agttttaacc cacccacgtt cggtggatgc 300accaagctgg agctgaaacg a 321＜210〉11＜211〉122＜212〉PRT＜213〉 ( Mus musculus )＜400〉11Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Ser Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20??????????????????25??????????????????30Thr?Met?His?Trp?Val?Lys?Gln?Thr?Pro?Arg?Gln?Gly?Leu?Glu?Trp?Ile
35??????????????????40??????????????????45Gly?Ala?Ile?Tyr?Pro?Gly?Ash?Gly?Asp?Thr?Ser?Tyr?Asn?Gln?Lys?Phe
50??????????????????55??????????????????60Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Phe?Cys
85??????????????????90??????????????????95Ala?Arg?Tyr?Tyr?Gln?Ser?Asp?Gln?Ser?Tyr?Trp?Tyr?Phe?Asp?Val?Trp
100?????????????????105?????????????????110Gly?Thr?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
115 120＜210〉12＜211〉107＜212〉PRT＜213〉mouse (Mus muscuhs)＜400〉12Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Gln Ser Ala Ser Pro Gly, 15 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20??????????????????25??????????????????30His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Ser?Ser?Pro?Lys?Pro?Trp?Ile?Tyr
35??????????????????40??????????????????45Ala?Pro?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser
50??????????????????55??????????????????60Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Arg?Val?Glu?Ala?Glu65??????????????????70??????????????????75??????????????????80Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Ser?Phe?Ash?Pro?Pro?Thr
85??????????????????90??????????????????95Phe?Gly?Asp?Gly?Thr?Lys?Leu?Glu?Leu?Lys?Arg
100 105＜210〉13＜211〉123＜212〉PRT＜213〉artificial sequence＜400〉13Gln Ala Tyr Leu Gln Ash Ser Gly Ala Glu Lys Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Ser Ala Ser Gly Tyr Thr Phe Met Ser Tyr
20??????????????????25??????????????????30Asn?Met?Tyr?Trp?Val?Lys?Gln?Thr?Thr?Arg?Gln?Gly?Leu?Glu?Trp?Ile
35??????????????????40??????????????????45Gly?Ala?Ile?Tyr?Pro?Gly?Asn?Gly?Asp?Thr?Ser?Tyr?Ash?Gln?Lys?Ser
50??????????????????55??????????????????60Gly?Ala?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Asp?Ser?Thr?Ala65??????????????????70??????????????????75??????????????????80Tyr?Met?Gln?Leu?Ser?Lys?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Phe
85??????????????????90??????????????????95Asp?Ala?Arg?Val?Val?Tyr?Tyr?Ser?Asn?Ser?Tyr?Trp?Tyr?Phe?Asp?Val
100?????????????????105?????????????????110Trp?Gly?Thr?Gly?Thr?Thr?Val?Gly?Ala?Ser?Ser
115 120＜210〉14＜211〉369＜212〉DNA＜213〉＜400〉14caggcctacc tgcagaactc cggcgccgag aaggtgcgcc ccggcgcctc cgtgaagatg 60tcctgctccg cctccggcta caccttcatg tcctacaaca tgtactgggt gaagcagacc 120acccgccagg gcctggagtg gatcggcgcc atctaccccg gcaacggcga cacctcctac 180aaccagaagt ccggcgccgg caaggccacc ctgaccgtgg acaagtccga ctccaccgcc 240tacatgcagc tgtccaagct gacctccgag gactccgccg tgtacttcga cgcccgcgtg 300gtgtactact ccaactccta ctggtacttc gacgtgtggg gcaccggcac caccgtgggc 360gcctcctcc 369＜210〉15＜211〉107＜212〉PRT＜213〉＜400〉15Gln Ile Val Leu Ser Lys Asn Pro Ala Ile Leu Ser Ala Ser Pro Gly1 5 10 15Glu His Lys Ser Met Thr Cys Arg Ala Ser Ser Ser Val Asp Tyr Met
20??????????????????25??????????????????30His?Trp?Leu?Gln?Gln?Lys?Pro?Gly?Asp?Ser?Pro?Lys?Pro?Trp?Ile?Tyr
35??????????????????40??????????????????45Ala?Ala?Ser?Asa?Leu?Asn?Ser?Gly?Val?Pro?Ala?Asp?His?Lys?Gly?Ser
50??????????????????55??????????????????60Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Gly?His?Val?Glu?Ala?Glu65??????????????????70??????????????????75??????????????????80Asp?Ala?Ala?Thr?Ser?Tyr?Cys?Gln?Gln?Trp?Ser?Phe?Asn?Lys?Pro?Thr
85??????????????????90??????????????????95Phe?Gly?Ala?Gly?Thr?Leu?Leu?Glu?Leu?Lys?Arg
100 105＜210〉16＜211〉321＜212〉DNA＜213〉＜400〉16cagatcgtgc tgtccaagaa ccccgccatc ctgtccgcct cccccggcga gcacaagtcc 60atgacctgcc gcgcctcctc ctccgtggac tacatgcact ggctgcagca gaagcccggc 120gactccccca agccctggat ctacgccgcc tccaacctga actccggcgt gcccgccgac 180cacaagggct ccggctccgg cacctcctac tccctgacca tcggccacgt ggaggccgag 240gacgccgcca cctcctactg ccagcagtgg tccttcaaca agcccacctt cggcgccggc 300accctgctgg agctgaagcg c 321＜210〉17＜211〉24＜212〉DNA＜213〉＜400〉17agctgggaag gtgtgcacac cact 24＜210〉18＜211〉21＜212〉DNA＜213〉＜400〉18cagagttcca ggtcaaggtc a 21＜210〉19＜211〉21＜212〉DNA＜213〉＜400〉19cttgaccagg catcctagag t 21＜210〉20＜211〉23＜212〉DNA＜213〉＜400〉20ttgctgtcct gatcagtcca act 23＜210〉21＜211〉23＜212〉DNA＜213〉＜400〉21tgtcgttcac tgccatcaat ctt 23＜210〉22＜211〉23＜212〉DNA＜213〉＜400〉22ttgttcaaga agcacacgac tga 23＜210〉23＜211〉36＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 24； 25; 29; 30; 34; 35)＜223〉n=i＜400〉23ggccacgcgt cgactagtac gggnngggnn gggnng 36＜210〉24＜211〉20＜212〉DNA＜213〉primer＜400〉24ggccacgcgt cgactagtac 20＜210〉25＜211〉29＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜223〉h=a; C, t; S=c or g:＜400〉25tcctctagac aaattsttct cthccagtc 29＜210〉26＜211〉29＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜223〉s=c or g; W=a or t＜400〉26gttgctagct cgsttcagct cwagcttgg 29＜210〉27＜211〉29＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜223〉h=a, c, t;＜400〉27aggaagcttc aggchtaact acagcagtc 29＜210〉28＜211〉31＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜223〉s=c or g; D=a, g or t; R=a or g; w＝at＜400〉28tgacgtacgt gagsagacgg tgdccgtgrt c 31＜210〉29＜211〉40＜212〉DNA＜213〉＜400〉29caggcctacc tgcagaactc cggcgccgag aaggtgcgcc 40＜210〉30＜211〉21＜212〉DNA＜213〉＜400〉30gatgtcctgc tccgcctccg g 21＜210〉31＜211〉49＜212〉DNA＜213〉＜400〉31caccttcatg tcctacaaca tgtactgggt gaagcagacc acccgccag 49＜210〉32＜211〉30＜212〉DNA＜213〉＜400〉32caaccagaag tccggcgccg gcaaggccac 30＜210〉33＜211〉19＜212〉DNA＜213〉＜400〉33gacaagtccg actccaccg 19＜210〉34＜211〉20＜212〉DNA＜213〉＜400〉34gctgtccaag ctgacctccg 20＜210〉35＜211〉18＜212〉DNA＜213〉＜400〉35gtgtacttcg acgcccgc 18＜210〉36＜211〉20＜212〉DNA＜213〉＜400〉36ccaccgtggg cgcctcctcc 20
Claims (10)
1. a CD20 Humanized monoclonal antibodies comprises the variable region of heavy chain in inhuman source and other zones in variable region of light chain and people source, and described variable region of heavy chain is selected from SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:13; Described variable region of light chain is selected from SEQ ID NO:4, SEQ ID NO:8, SEQ IDNO:12 and SEQ ID NO:15; When described variable region of light chain was SEQ ID NO:3, variable region of heavy chain was not SEQ ID NO:4.
2. dna molecular, the described weight chain variabl area sequence SEQ of its coding claim 1 ID NO:7, SEQ IDNO:11 and SEQ ID NO:13.
3. dna molecular according to claim 2, it is selected from SEQ ID NO:5, SEQ ID NO:9 and SEQ ID NO:14.
4. dna molecular, the described light chain variable region sequence of its coding claim 1 SEQ ID NO:8, SEQ IDNO:12 and SEQ ID NO:15.
5. dna molecular according to claim 4, it is selected from SEQ ID NO:6, SEQ ID NO:10 and SEQ ID NO:16.
6. a carrier comprises claim 2 and/or 4 described dna moleculars.
7. host cell comprises the expression system of the described antibody of claim 1.
8. according to the described host cell of claim 5, contain the described carrier of claim 6.
9. according to the described host cell of claim 6, described host cell is selected from intestinal bacteria and CHO.
10. the purposes of the described antibody of claim 1 in the preparation of preparation diagnosis and/or treatment CD20 relative disease.
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| CNB011322268A CN1210307C (en) | 2001-11-16 | 2001-11-16 | Humanized anti-CD 20 monoclonal antibody |
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| WO2011100403A1 (en) | 2010-02-10 | 2011-08-18 | Immunogen, Inc | Cd20 antibodies and uses thereof |
| CN102167744A (en) * | 2010-02-25 | 2011-08-31 | 百迈博药业有限公司 | Human anti-CD20 monoclonal antibody and preparation method and application thereof |
| WO2011113308A1 (en) * | 2010-03-17 | 2011-09-22 | 永卓博济(上海)生物医药技术有限公司 | New humanized anti-cd20 monoclonal antibody |
| CN101203607B (en) * | 2005-03-31 | 2012-09-05 | 生物医药股份有限公司 | Anti-CD-20 monoclonal antibody |
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| CN101203607B (en) * | 2005-03-31 | 2012-09-05 | 生物医药股份有限公司 | Anti-CD-20 monoclonal antibody |
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